Publications by authors named "Letian Shan"

52 Publications

Human Umbilical Cord-Derived Mesenchymal Stem Cells Ameliorate Skin Aging of Nude Mice Through Autophagy-Mediated Anti-Senescent Mechanism.

Stem Cell Rev Rep 2022 Jul 21. Epub 2022 Jul 21.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China.

Skin aging is a currently irreversible process, affected by increased oxidative stress, activated cellular senescence, and lacked regeneration of the dermal layer. Mesenchymal stem cells (MSCs), such as human umbilical cord-derived MSCs (hucMSCs), have pro-regeneration and anti-aging potencies. To explore whether hucMSCs can be used to treat skin aging, this study employed skin-aging model of nude mice to conduct in vivo assays, including biochemical analysis of superoxide dismutase (SOD) and malondialdehyde (MDA), gross observation, histopathological observation, and immunohistochemical analysis. To clarify how hucMSCs work on skin aging, this study employed skin-aging model of human dermal fibroblasts (HDFs) to conduct in vitro assays by applying conditional medium of hucMSCs (CMM), including wound healing assay, senescence staining, flow cytometric oxidative detection, real time PCR, and western blot analysis. The in vivo data demonstrated that hucMSCs dose-dependently removed wrinkles, smoothed skin texture, and increased dermal thickness and collagen production of aged skin by reversing SOD and MDA levels and up-regulating Col-1 and VEGF expressions, indicating anti-oxidative and pro-regenerative effects against skin aging. The in vitro data revealed that hucMSCs significantly reversed the senescence of HDFs by promoting cell migration, inhibiting ROS production, and restoring the overexpressions of oxidative and senescent markers through paracrine mode of action, and the paracrine mechanism was mediated by the inhibition of autophagy. This study provided novel knowledge regarding the anti-aging efficacy and paracrine mechanism of hucMSCs on skin, making hucMSCs-based therapy a promising regime for skin aging treatment.
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http://dx.doi.org/10.1007/s12015-022-10418-9DOI Listing
July 2022

Onset of p53/NF-κB signaling crosstalk in human melanoma cells in response to anti-cancer theabrownin.

FASEB J 2022 Aug;36(8):e22426

Department of Pharmaceutical Biology, Johannes Gutenberg University, Mainz, Germany.

As a major tea component, theabrownin represents a promising anti-cancer candidate. However, its effect on the melanoma is unknown. To evaluate the in vitro and in vivo anti-melanoma efficacy of TB, we conducted cell viability, immunostaining, comet, and TUNEL assays on human A375 melanoma cells, and employed a zebrafish xenograft model of A375 cells. Real-time PCR (qPCR) and western blot were conducted to explore the molecular mechanisms of TB. In vitro, TB significantly inhibited the proliferation of A375 cells, and A375 cells showed the highest inhibitory rate among the other melanoma cell line (A875) and human dermal fibroblasts. TB triggered DNA damage and induced apoptosis of A375 cells and significantly inhibited the growth of A375 xenograft tumors in zebrafishes. Several key molecular events were activated by TB, including DNA damage-associated p53 and NF-κB pathways, through up-regulation of GADD45α, γ-H2A.X, phospho-ATM(p-ATM), phospho-ATR (p-ATR), phospho-p53 (p-p53), phospho-IKKα/β (p-IKKα/β), phospho-p65 (p-p65), etc. However, the TB-activated molecular events were counteracted by either knockdown of p53 or p65, and only dual knockdown of both p53 and p65 completed counteracted the anti-melanoma efficacy of TB. In conclusion, TB triggered DNA damage and thereby inhibited proliferation and induced cellular senescence and apoptosis of melanoma cells through mechanisms mediated by p53/NF-κB signaling crosstalk. This is the first report on the efficacy and mechanisms of TB on melanoma cells, making TB a promising candidate for anti-melanoma agent development.
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http://dx.doi.org/10.1096/fj.202200261RDOI Listing
August 2022

Human umbilical cord-derived mesenchymal stem cells not only ameliorate blood glucose but also protect vascular endothelium from diabetic damage through a paracrine mechanism mediated by MAPK/ERK signaling.

Stem Cell Res Ther 2022 06 17;13(1):258. Epub 2022 Jun 17.

School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, China.

Background: Endothelial damage is an initial step of macro- and micro-vasculature dysfunctions in diabetic patients, accounting for a high incidence of diabetic vascular complications, such as atherosclerosis, nephropathy, retinopathy, and neuropathy. However, clinic lacks effective therapeutics targeting diabetic vascular complications. In field of regenerative medicine, mesenchymal stem cells, such as human umbilical cord-derived MSCs (hucMSCs), have great potential in treating tissue damage.

Methods: To determine whether hucMSCs infusion could repair diabetic vascular endothelial damage and how it works, this study conducted in vivo experiment on streptozotocin-induced diabetic rat model to test body weight, fasting blood glucose (FBG), serum ICAM-1 and VCAM-1 levels, histopathology and immunohistochemical staining of aorta segments. In vitro experiment was further conducted to determine the effects of hucMSCs on diabetic vascular endothelial damage, applying assays of resazurin staining, MTT cell viability, wound healing, transwell migration, and matrigel tube formation on human umbilical vein endothelial cells (HUVECs). RNA sequencing (RNAseq) and molecular experiment were conducted to clarify the mechanism of hucMSCs.

Results: The in vivo data revealed that hucMSCs partially restore the alterations of body weight, FBG, serum ICAM-1 and VCAM-1 levels, histopathology of aorta and reversed the abnormal phosphorylation of ERK in diabetic rats. By using the conditioned medium of hucMSCs (MSC-CM), the in vitro data revealed that hucMSCs improved cell viability, wound healing, migration and angiogenesis of the high glucose-damaged HUVECs through a paracrine action mode, and the altered gene expressions of IL-6, TNF-α, ICAM-1, VCAM-1, BAX, P16, P53 and ET-1 were significantly restored by MSC-CM. RNAseq incorporated with real-time PCR and Western blot results clarified that high glucose activated MAPK/ERK signaling in HUVECs, while MSC-CM reversed the abnormal phosphorylation of ERK and overexpressions of MKNK2, ERBB3, MYC and DUSP5 in MAPK/ERK signaling pathway.

Conclusions: HucMSCs not only ameliorated blood glucose but also protected vascular endothelium from diabetic damage, in which MAPK/ERK signaling mediated its molecular mechanism of paracrine action. Our findings provided novel knowledge of hucMSCs in the treatment of diabetes and suggested a prospective strategy for the clinical treatment of diabetic vascular complications.
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http://dx.doi.org/10.1186/s13287-022-02927-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9205155PMC
June 2022

Intra-Articular Injection of Adipose-Derived Stem Cells Ameliorates Pain and Cartilage Anabolism/Catabolism in Osteoarthritis: Preclinical and Clinical Evidences.

Front Pharmacol 2022 21;13:854025. Epub 2022 Mar 21.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China.

Osteoarthritis (OA) is the most common joint disorder, lacking disease-modifying treatments. Adipose-derived mesenchymal stem cells (ADSCs) are adult multipotent stromal cells obtained from fat tissue, which holds great potential in treating OA. This study aimed to evaluate the anti-OA efficacy of ADSCs from preclinical and clinical facets and explore the underlying mechanism of action. , a single dose of 5 × 10 ADSCs was injected into the knee joints of monoiodoacetate-induced OA rat model. The levels of metabolic and hypertrophic molecules (MMP13, Collagen II, Collagen X) of chondrocytes were measured by immunohistochemistry. , cell viability assay was conducted to detect the proliferation ability of chondrocytes treated with ADSCs conditioned medium (ADSCs-CM). Quantitative real-time polymerase chain reaction and Western blot assays were applied to explore the mechanism of action of ADSCs. Moreover, a retrospective analysis was conducted to determine the clinical efficacy and safety of ADSCs on OA patients. The animal study showed that ADSCs significantly alleviated OA cartilage lesions in rats, as was confirmed by downregulation of the MMP13 and Collagen X and upregulation of the Collagen II. data showed that ADSCs-CM promoted the proliferation of chondrocytes, and significantly restored the IL-1β-induced abnormal expressions of molecular markers IL-6, Aggrecan, MMP3, MMP13, Collagen II, Collagen X, ADAMTS5, ADAMTS9, SOX6, and SOX9 in chondrocytes. Such regulatory effects of ADSCs-CM on the proliferation and these anabolic, catabolic, and hypertrophic markers of chondrocytes suggested a paracrine-based mode of action of ADSCs. Furthermore, the clinical data showed that ADSCs reduced pain and repaired cartilage damage in OA patients, with no adverse events. This study demonstrated the anti-OA efficacy, safety, and a paracrine-based mechanism of ADSCs, providing a promising cell-based therapeutic option for OA treatment.
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http://dx.doi.org/10.3389/fphar.2022.854025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8978713PMC
March 2022

Fuzi decoction ameliorates pain and cartilage degeneration of osteoarthritic rats through PI3K-Akt signaling pathway and its clinical retrospective evidence.

Phytomedicine 2022 Jun 20;100:154071. Epub 2022 Mar 20.

Department of Pharmaceutical Biology, Johannes Gutenberg University, Mainz, 55128, Germany.

Background: Osteoarthritis (OA) is a difficult disease but the clinic lacks effective therapy. As a classic formula of traditional Chinese medicine (TCM), Fuzi decoction (FZD) has been clinically applied for treating OA-related syndromes, but its anti-OA efficacy and mechanism remain unclear.

Purpose: To experimentally and clinically determine the anti-OA efficacy of FZD and clarify the underlying mechanism.

Methods: UPLC/MS/MS was applied to identify the main components of FZD. A monoiodoacetate (MIA)-induced OA rat model was employed to evaluate the in vivo efficacy of FZD against OA, by using pain behavior assessment, histopathological observation, and immunohistochemical analysis. Primary rat chondrocytes were isolated to determine the in vitro effects of FZD by using cell viability assay, wound healing assay, and real-time PCR (qPCR) analysis on anabolic/catabolic mRNA expressions. RNA sequencing (RNA-seq) and network pharmacology analysis were conducted and the overlapping data were used to predict the mechanism of FZD, followed by verification with qPCR and Western blot assays. Finally, a retrospective analysis was performed to confirm FZD's efficacy and safety in OA patients.

Results: The UPLC/MS/MS result showed that FZD contained atractylenolide I, benzoylhypaconitine, benzoylmesaconitine, benzoylaconitine, hypaconitine, mesaconitine, aconitine, lobetyolin, paeoniflorin, and pachymic acid. The in vivo data showed that FZD restored the cartilage degeneration in MIA-induced OA rats by ameliorating pain behavior parameters, recovering histopathological alterations, benefitting cartilage anabolism (up-regulating Col2 expression), and suppressing catabolism (down-regulating MMP13 and Col10 expressions). The in vitro data showed that FZD increased cell viability and wound healing capacity of chondrocytes, and restored the altered expressions of anabolic and catabolic genes of chondrocytes. The overlapping results of RNA-seq and network pharmacology analysis suggested that PI3K/Akt signaling mediated the anti-OA mechanism of FZD, which was verified by qPCR and Western blot experiments. Clinically, the anti-OA efficacy and safety of FZD were confirmed by the retrospective analysis on OA patients.

Conclusion: The scientific innovation of this study was the determination of anti-OA efficacy of FZD by experimental and clinical evidence and the discovery of its mechanism by integrated RNA-seq, network pharmacology, and molecular experiments, which suggests FZD as a promising TCM agency for OA treatment.
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http://dx.doi.org/10.1016/j.phymed.2022.154071DOI Listing
June 2022

Tanshinol suppresses osteosarcoma by specifically inducing apoptosis of U2-OS cells through p53-mediated mechanism.

J Ethnopharmacol 2022 Jun 21;292:115214. Epub 2022 Mar 21.

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany.

Ethnopharmacological Relevance: Radix Salviae miltiorrhizae (also called Danshen in traditional Chinese medicine) is a famous herbal medicine, which has been frequently used to treat blood stasis syndrome including osteosarcoma (OS) in traditional Chinese medicine. Main components of Danshen have been assumed to exhibit anti-OS capacity. Nevertheless, tanshinol (TS, main component of Danshen)'s efficacy and mechanism in OS hasn't been clearly described ever since. This drew our attention, since OS is the most frequent primary bone carcinomas in children and adolescents, with a high incidence and fatality rate. Unfortunately, chemotherapy for OS has faced many clinical challenges due to the increasing chemoresistance and recurrence. This study was then designed to deeply explore TS's role in OS therapy.

Aim Of The Study: To explore the anti-OS efficacy and mechanism of TS, we conducted in vivo and in vitro experiments by using a zebrafish xenograft model and U2-OS cells.

Materials And Methods: CCK-8 assay, DAPI and γ-H2A.X immunofluorescence staining, and flow cytometry (apoptosis verification) were employed to determine the anti-proliferative and pro-apoptotic effects of TS. qPCR and Western blot were used to examine TS's molecular actions and mechanism on apoptosis of U2-OS cells.

Results: The in vivo data showed that TS significantly inhibited U2-OS tumor growth in larval zebrafish from 2 to 20 ng/mL. In vitro data indicated that TS exerted significant anti-proliferative and pro-apoptotic effects on U2-OS cells in a dose-dependent manner. Moreover, TS has no inhibitory effect on bMSCs, suggesting its safety on normal bone-forming cells. Molecular data illustrated that TS obviously activated the p53 signaling-related proteins (p-p53, Bax, CASP3, CASP9) and its upstream JNK (p-JNK, p-c-JUN) and ATM (p-ATM) signaling molecules through phosphorylation and cleavage, followed by up-regulation of the pro-apoptotic genes, NOXA, PUMA, TP53, BAX, and BIM, and down-regulation of Bcl-2 protein.

Conclusion: In sum, TS specifically induced apoptosis of U2-OS cells by activating p53 signaling pathways, indicating TS as a promising candidate for OS treatment.
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http://dx.doi.org/10.1016/j.jep.2022.115214DOI Listing
June 2022

Growth factors-based platelet lysate rejuvenates skin against ageing through NF-κB signalling pathway: In vitro and in vivo mechanistic and clinical studies.

Cell Prolif 2022 Apr 11;55(4):e13212. Epub 2022 Mar 11.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China.

Introduction: Platelets benefit tissue regeneration by secreting growth factors, and platelet products, for example, platelet lysate (PL), have been clinically applied for tissue rejuvenation. To determine the anti-ageing efficacy and mechanism of human PL (hPL) on skin, this study conducted clinical retrospective analysis, nude mice-based in vivo study and human dermal fibroblasts (HDFs)-based in vitro study.

Methods: Flow cytometry was employed for quality control of hPL, and ELISA was used for quantification of growth factors (EGF, IGF-1, PDGF and TGF-β) in hPL. After d-galactose modelling, skin texture grading, histopathological observation, immunofluorescence analysis and oxidative stress assays were conducted on nude mice, while SA-β-gal staining, CCK-8 and wound healing assays were conducted on HDFs. qPCR and western blot were conducted to clarify hPL's mechanism.

Results: The clinical retrospective data showed that hPL obviously rejuvenated human skin appearances without adverse events. The animal data showed that hPL exerted rejuvenative effects on skin, and the cellular data showed that hPL significantly promoted the proliferation and migration of HDFs and suppressed senescence-associated secretory protein secretion and senescence state of senescent HDFs by suppressing NF-κB pathway. The NF-κB-dependent mechanism was verified positively by using P65 siRNA and negatively by using prostratin. Furthermore, EGF, IGF-1, PDGF and TGF-β were found as the main ingredients in hPL, which contributed to the efficacy and mechanism of hPL.

Conclusion: This study provided novel knowledge of hPL, making it ideal for skin rejuvenation.
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http://dx.doi.org/10.1111/cpr.13212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9055903PMC
April 2022

Green tea-derived theabrownin suppresses human non-small cell lung carcinoma in xenograft model through activation of not only p53 signaling but also MAPK/JNK signaling pathway.

J Ethnopharmacol 2022 Jun 7;291:115167. Epub 2022 Mar 7.

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany.

Ethnopharmacological Relevance: According to the theory and practice of traditional Chinese medicine (TCM), the pathogenesis of lung carcinoma is associated with many syndromes, such as "sputum stasis", "cough", "lung fever", "lung toxin", and "hemoptysis", which should be removed for therapeutic purpose. Tea is not only a world-wide beverage, but also a TCM herb, possessing activities against the above syndromes. Recently, green tea extract exerted inhibitory effects on a variety of tumor cells. As a pigment active substance of green tea, theabrownin (TB) has been found to inhibit many cancer cells.

Aim Of The Study: This study focused on the efficacy and mechanism of TB on non-small cell lung cancer (NSCLC) cell lines. The in vivo efficacy of TB on p53-deficient NSCLC (H1299) cells and p53-wild type NSCLC (A549) cells NSCLC cells were determined, and its mechanism of action was explored.

Materials And Methods: In vivo, two lung cancer cell lines, H1299 (p53-deficient) and A549 (p53-wild type) were selected to establish xenograft models of larval zebrafish, respectively. For in vitro experiments, wound healing assay, DAPI staining, TUNEL assay, immunofluorescence assay, and flow cytometry were conducted in these two cell lines. RNA sequencing (RNAseq), real time PCR (qPCR) and Western blot (WB) were performed for the mechanism study.

Results: The in vivo results showed that TB significantly inhibited the H1299 and the A549 xenograft tumor growth in larval zebrafish (dosage ranged from 2.13 to 21.3 μg/ml). Wound healing assay results showed that TB suppressed the migration of H1299 cells. DAPI staining, TUNEL assay, and immunofluorescence assay results showed that TB inhibited the growth of H1299 cells by inducing apoptosis. RNAseq, qPCR and WB data showed that TB significantly up-regulated the MAPK/JNK pathway-related proteins (ASK-1, JNK and c-JUN) through phosphorylation activation, accompanying with down-regulation of the epithelial-mesenchymal transition (EMT)-associated genes (N-CADHERIN, SLUG, FIBROWNECTIN and ZEB1) and anti-apoptotic molecules (BCL-2), and up-regulation of the metastasis-related gene HSPA6 and the pro-apoptotic molecules (BIM, BAX, PARP, c-PARP, γ-H2A.X, c-CASP3, c-CASP8, c-CASP9, DDIT3 and DUSP8).

Conclusion: This study determined the in vivo efficacy of green tea-derived TB on p53-deficient NSCLC (H1299) cells and p53-wild type NSCLC (A549) cells and clarified its p53-independent mechanism mediated by the activation of MAPK/JNK signaling pathway.
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http://dx.doi.org/10.1016/j.jep.2022.115167DOI Listing
June 2022

Green tea-derived theabrownin induces cellular senescence and apoptosis of hepatocellular carcinoma through p53 signaling activation and bypassed JNK signaling suppression.

Cancer Cell Int 2022 Jan 25;22(1):39. Epub 2022 Jan 25.

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany.

Background: Theabrownin (TB) is a bioactive component of tea and has been reported to exert effects against many human cancers, but its efficacy and mechanism on hepatocellular carcinoma (HCC) with different p53 genotypes remains unclarified.

Methods: MTT assay, DAPI staining, flow cytometry and SA-β-gal staining were applied to evaluate the effects of TB on HCC cells. Quantitative real time PCR (qPCR) and Western blot (WB) were conducted to explore the molecular mechanism of TB. A xenograft model of zebrafish was established to evaluate the anti-tumor effect of TB.

Results: MTT assays showed that TB significantly inhibited the proliferation of SK-Hep-1, HepG2, and Huh7 cells in a dose-dependent manner, of which SK-Hep-1 was the most sensitive one with the lowest IC values. The animal data showed that TB remarkably suppressed SK-Hep-1 tumor growth in xenograft model of zebrafish. The cellular data showed TB's pro-apoptotic and pro-senescent effect on SK-Hep-1 cells. The molecular results revealed the mechanism of TB that p53 signaling pathway (p-ATM, p-ATR, γ-H2AX, p-Chk2, and p-p53) was activated with up-regulation of downstream senescent genes (P16, P21, IL-6 and IL-8) as well as apoptotic genes (Bim, Bax and PUMA) and proteins (Bax, c-Casp9 and c-PARP). The p53-mediated mechanism was verified by using p53-siRNA. Moreover, by using JNK-siRNA, we found JNK as a bypass regulator in TB's mechanism.

Conclusions: To sum up, TB exerted tumor-inhibitory, pro-senescent and pro-apoptotic effects on SK-Hep-1 cells through ATM-Chk2-p53 signaling axis in accompany with JNK bypass regulation. This is the first report on the pro-senescent effect and multi-target (p53 and JNK) mechanism of TB on HCC cells, providing new insights into the underlying mechanisms of TB's anti-HCC efficacy.
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http://dx.doi.org/10.1186/s12935-022-02468-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8788116PMC
January 2022

A Novel Mutation of the KLK6 Gene in a Family With Knee Osteoarthritis.

Front Genet 2021 11;12:784176. Epub 2021 Nov 11.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China.

To investigate the correlation between gene mutation and knee osteoarthritis (KOA), a whole-exome sequencing (WES) was applied to analyze blood samples of four KOA patients and two normal subjects in a family. Gene mutations were identified by gene-trapping and high-throughput sequencing analysis across the differences between the patients and normal subjects. The interactive gene network analysis on the retrieval of interacting genes (STRING) database and the KOA-related genes expression data sets was performed. A possibly detrimental and nonsynonymous mutation at the kallikrein-related peptidase 6 (KLK6) gene (rs201586262, c. C80A, P27H) was identified and attracted our attention. KLK6 belongs to the kallikrein family of serine proteases and its serum level is known as a prevalent biomarker in inflammatory and malignant diseases. KLK6 expresses in the extracellular compartment for matrix degradation, highlighting that KLK6 plays a role in the pathogenesis of KOA. By using the gene databases, the KOA-related genes were mined after de-duplication and IL6 was selected as the most relevant gene through interactive analysis of protein-protein interaction (PPI) network. The data suggested that KLK6 gene mutation and the related expression alteration of IL6 gene might determine the occurrence of hereditary KOA. The is the first study discovering the gene mutation of KLK6 as a factor of pathogenesis of KOA, especially the hereditary KOA.
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http://dx.doi.org/10.3389/fgene.2021.784176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8631809PMC
November 2021

Identification and validation of hub genes of synovial tissue for patients with osteoarthritis and rheumatoid arthritis.

Hereditas 2021 Sep 28;158(1):37. Epub 2021 Sep 28.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, P. R. China.

Background: Osteoarthritis (OA) and rheumatoid arthritis (RA) were two major joint diseases with similar clinical phenotypes. This study aimed to determine the mechanistic similarities and differences between OA and RA by integrated analysis of multiple gene expression data sets.

Methods: Microarray data sets of OA and RA were obtained from the Gene Expression Omnibus (GEO). By integrating multiple gene data sets, specific differentially expressed genes (DEGs) were identified. The Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein-protein interaction (PPI) network analysis of DEGs were conducted to determine hub genes and pathways. The "Cell Type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT)" algorithm was employed to evaluate the immune infiltration cells (IICs) profiles in OA and RA. Moreover, mouse models of RA and OA were established, and selected hub genes were verified in synovial tissues with quantitative polymerase chain reaction (qPCR).

Results: A total of 1116 DEGs were identified between OA and RA. GO functional enrichment analysis showed that DEGs were enriched in regulation of cell morphogenesis involved in differentiation, positive regulation of neuron differentiation, nuclear speck, RNA polymerase II transcription factor complex, protein serine/threonine kinase activity and proximal promoter sequence-specific DNA binding. KEGG pathway analysis showed that DEGs were enriched in EGFR tyrosine kinase inhibitor resistance, ubiquitin mediated proteolysis, FoxO signaling pathway and TGF-beta signaling pathway. Immune cell infiltration analysis identified 9 IICs with significantly different distributions between OA and RA samples. qPCR results showed that the expression levels of the hub genes (RPS6, RPS14, RPS25, RPL11, RPL27, SNRPE, EEF2 and RPL19) were significantly increased in OA samples compared to their counterparts in RA samples (P < 0.05).

Conclusion: This large-scale gene analyses provided new insights for disease-associated genes, molecular mechanisms as well as IICs profiles in OA and RA, which may offer a new direction for distinguishing diagnosis and treatment between OA and RA.
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http://dx.doi.org/10.1186/s41065-021-00201-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8480049PMC
September 2021

Cytotoxicity of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide in multidrug-resistant cancer cells through activation of PERK/eIF2α/ATF4 pathway.

Biochem Pharmacol 2021 11 25;193:114788. Epub 2021 Sep 25.

Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany. Electronic address:

After decades of research, multidrug resistance (MDR) remains a huge challenge in cancer treatment. In this study, the cytotoxic of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide (MCC1734) has been investigated towards multidrug-resistant cancer cell lines. MCC1734 exerted cytotoxicity on cell lines expressing different mechanisms of drug resistance (P-glycoprotein, BCRP, ABCB5, EGFR, p53 knockout) to a different extent. Interestingly, sensitive CCRF-CEM cells and multidrug-resistant P-gp-overexpressing CEM/ADR5000 cells represented similar sensitivity towards MCC1734, indicating MCC1734 can bypass P-gp-mediated resistance. Microarray-based mRNA expression revealed that MCC1734 affected cells by multiple pathways, including cell cycle regulation, mitochondrial dysfunction, apoptosis signaling, and EIF2 signaling. MCC1734 stimulated the generation of excessive reactive oxygen species and the collapse of mitochondria membrane potential in CCRF-CEM cells, companied by the arrest of the cell cycle in the GM phase and apoptosis induction as determined by flow cytometry. In addition, our immunoblotting analysis highlighted that MCC1734 triggered endoplasmic reticulum (ER) stress, evidenced by the activation of p-PERK, p-eIF2α, ATF4 and CHOP. The anti-cancer effects of MCC1734 were further observed in vivo using human xenograft tumors transplanted to zebrafish, providing further support for MCC1734 as a promising new candidate for cancer drug development.
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http://dx.doi.org/10.1016/j.bcp.2021.114788DOI Listing
November 2021

Pain relief and cartilage repair by Nanofat against osteoarthritis: preclinical and clinical evidence.

Stem Cell Res Ther 2021 08 26;12(1):477. Epub 2021 Aug 26.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, People's Republic of China.

Background: Osteoarthritis (OA) is the most common joint degenerative disorder, with little effective therapy to date. Nanofat is a cocktail of cells obtained from fat tissue, which possesses regenerative capacity and has a potential in treating OA. This study aimed to determine the anti-OA efficacy of Nanofat from basic and clinical aspects and explore its action mode.

Methods: Flow cytometry was performed to characterize Nanofat. A monoiodoacetate-induced OA rat model was employed for in vivo study. Cell viability and wound healing assays were conducted for in vitro study. Real-time PCR and Western blot assays were applied to explore the molecular action mode of Nanofat. Moreover, a retrospective analysis was conducted to determine the clinical efficacy and safety of Nanofat on knee OA patients.

Results: The in vivo results showed that Nanofat significantly attenuated pain symptoms and protected cartilage ECM (Col2) from damage, and its effects were not significantly differed with adipose tissue-derived stem cells (both P > 0.05). The in vitro results showed that Nanofat promoted the cell viability and migration of chondrocytes and significantly restored the IL-1β-induced abnormal gene expressions of Col2, Aggrecan, Sox9, Adamts5, Mmp3, Mmp9 Mmp13, IL-6 and Col10 and protein expressions of Col2, MMP9, MMP13, and Sox9 of chondrocytes. The regulatory actions of Nanofat on these anabolic, catabolic, and hypertrophic molecules of chondrocytes were similar between two treatment routes: co-culture and conditioned medium, suggesting a paracrine-based mode of action of Nanofat. Moreover, the clinical data showed that Nanofat relieved pain and repaired damaged cartilage of OA patients, with no adverse events.

Conclusion: In sum, this study demonstrated the anti-OA efficacy as well as a paracrine-based action mode of Nanofat, providing novel knowledge of Nanofat and suggesting it as a promising and practical cell therapy for clinical treatment of OA.
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http://dx.doi.org/10.1186/s13287-021-02538-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390235PMC
August 2021

A Systematic Review and Meta-Analysis of the SuperPATH Approach in Hip Arthroplasty.

Biomed Res Int 2021 21;2021:5056291. Epub 2021 Jul 21.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, 310053 Zhejiang, China.

Objective: To compare the clinical and radiographic results of the supercapsular percutaneously assisted total hip (SuperPATH) approach and the conventional approach in hip arthroplasty.

Design: Based on a prepublished protocol (PROSPERO: CRD42020177717), we searched PubMed, Embase, and Cochrane for relevant literatures up to January 30, 2021. The methodological qualities were assessed using the guidelines provided by the Cochrane Collaboration for Systematic Reviews. Randomized- or fixed-effect models were used to calculate the weighted mean difference (WMD) or odds ratio (OR), respectively, for continuous and dichotomous variables.

Results: 6 articles were included in the study, and 526 patients were selected, which included 233 cases in the SuperPATH groups and 279 cases in the conventional groups, and 4 cases performed two surgeries in succession. The SuperPATH group demonstrated shorter incision length (WMD = -7.87, 95% CI (-10.05, -5.69), < 0.00001), decreased blood transfusion rate (OR = 0.48, 95% CI (0.25, 0.89), = 0.02), decreased visual analogue scale (VAS) (WMD = -0.40, 95% CI (-0.72, -0.08), = 0.02), and higher Harris hip score (HHS) (WMD = 1.98, 95% CI (0.18, 3.77), = 0.03) than the conventional group. However, there was no difference in VAS ( = 0.14) and HHS ( = 0.86) between the two groups 3 months later, nor in the acetabular abduction angle ( = 0.32) in either group.

Conclusions: SuperPATH, as a minimally invasive approach with its reduced tissue damage, quick postoperative recovery, and early rehabilitation, demonstrates the short-term advantages of hip arthroplasty. As the evidences in favor of the SuperPATH technique were limited in a small number of studies and short duration of follow-up, more research is required to further analyze its long-term effect.
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http://dx.doi.org/10.1155/2021/5056291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8321717PMC
September 2021

A novel moniliformin derivative as pan-inhibitor of histone deacetylases triggering apoptosis of leukemia cells.

Biochem Pharmacol 2021 12 13;194:114677. Epub 2021 Jul 13.

Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany. Electronic address:

New and potent agents that evade multidrug resistance (MDR) and inhibit epigenetic modifications are of great interest in cancer drug development. Here, we describe that a moniliformin derivative (IUPAC name: 3-(naphthalen-2-ylsulfanyl)-4-{[(2Z)-1,3,3-trimethyl-2,3-dihydro-1H-indol-2-ylidene]methyl}cyclobut-3-ene-1,2-dione; code: MCC1381) bypasses P-gp-mediated MDR. Using transcriptomics, we identified a large number of genes significantly regulated in response to MCC1381, which affected the cell cycle and disturbed cellular death and survival. The potential targets of MCC1381 might be histone deacetylases (HDACs) as predicted by SwissTargetPrediction. In silico studies confirmed that MCC1381 presented comparable affinity with HDAC1, 2, 3, 6, 8 and 11. Besides, the inhibition activity of HDACs was dose-dependently inhibited by MCC1381. Particularly, a strong binding affinity was observed between MCC1381 and HDAC6 by microscale thermophoresis analysis. MCC1381 decreased the expression of HDAC6, inversely correlated with the increase of acetylated HDAC6 substrates, acetylation p53 and α-tubulin. Furthermore, MCC1381 arrested the cell cycle at the G/M phase, induced the generation of reactive oxygen species and collapse of the mitochondrial membrane potential. MCC1381 exhibited in vivo anti-cancer activity in xenografted zebrafish. Collectively, MCC1381 extended cytotoxicity towards P-gp-resistant leukemia cancer cells and may act as a pan-HDACs inhibitor, indicating that MCC1381 is a novel candidate for cancer therapy.
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http://dx.doi.org/10.1016/j.bcp.2021.114677DOI Listing
December 2021

Urine Metabolomics Profiling of Lumbar Disc Herniation and its Traditional Chinese Medicine Subtypes in Patients Through Gas Chromatography Coupled With Mass Spectrometry.

Front Mol Biosci 2021 9;8:648823. Epub 2021 Jun 9.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China.

Lumbar disc herniation (LDH) possesses complex pathogenesis, which has not been well elucidated yet. To date, specific or early diagnosis of LDH remains unavailable, resulting in missed opportunity for effective treatment. According to Traditional Chinese medicine (TCM) theory, LDH can be divided into two subtypes (reality syndrome and deficiency syndrome). The purpose of this study was to analyze the metabolic disorders of LDH and its TCM subtypes and screen out potential biomarkers for LDH diagnosis. Gas chromatography coupled with mass spectrometry (GC-MS) was applied to test the urine samples from 66 participants (30 healthy volunteers, 18 LDH patients with deficiency syndrome and 18 patients with reality syndrome). PCA analysis showed a distinct separation tendency between the healthy subjects and LDH patients but no obvious separation between the different syndromes (reality syndrome and deficiency syndrome) of LDH patients. As a result, 23 metabolites were identified significantly altered in the LDH patients, as compared with the healthy subjects. The altered metabolites belong to amino acid metabolism, nucleic acid metabolism, carbohydrate metabolism, and vitamin metabolism, which are related to osteoporosis and inflammation. Our results indicate metabolic disorders of LDH and thereby propose a group of metabolic biomarkers for potential application in early diagnosis of LDH in clinic, which provide a reasonable explanation for the pathogenesis of LDH.
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http://dx.doi.org/10.3389/fmolb.2021.648823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8220151PMC
June 2021

Selection of safe artemisinin derivatives using a machine learning-based cardiotoxicity platform and in vitro and in vivo validation.

Arch Toxicol 2021 07 22;95(7):2485-2495. Epub 2021 May 22.

Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, 55128, Mainz, Germany.

The majority of drug candidates fails the approval phase due to unwanted toxicities and side effects. Establishment of an effective toxicity prediction platform is of utmost importance, to increase the efficiency of the drug discovery process. For this purpose, we developed a toxicity prediction platform with machine-learning strategies. Cardiotoxicity prediction was performed by establishing a model with five parameters (arrhythmia, cardiac failure, heart block, hypertension, myocardial infarction) and additional toxicity predictions such as hepatotoxicity, reproductive toxicity, mutagenicity, and tumorigenicity are performed by using Data Warrior and Pro-Tox-II software. As a case study, we selected artemisinin derivatives to evaluate the platform and to provide a list of safe artemisinin derivatives. Artemisinin from Artemisia annua was described first as an anti-malarial compound and later its anticancer properties were discovered. Here, random forest feature selection algorithm was used for the establishment of cardiotoxicity models. High AUC scores above 0.830 were achieved for all five cardiotoxicity indications. Using a chemical library of 374 artemisinin derivatives as a case study, 7 compounds (deoxydihydro-artemisinin, 3-hydroxy-deoxy-dihydroartemisinin, 3-desoxy-dihydroartemisinin, dihydroartemisinin-furano acetate-d3, deoxyartemisinin, artemisinin G, artemisinin B) passed the toxicity filtering process for hepatotoxicity, mutagenicity, tumorigenicity, and reproductive toxicity in addition to cardiotoxicity. Experimental validation with the cardiomyocyte cell line AC16 supported the findings from the in silico cardiotoxicity model predictions. Transcriptomic profiling of AC16 cells upon artemisinin B treatment revealed a similar gene expression profile as that of the control compound, dexrazoxane. In vivo experiments with a Zebrafish model further substantiated the in silico and in vitro data, as only slight cardiotoxicity in picomolar range was observed. In conclusion, our machine-learning approach combined with in vitro and in vivo experimentation represents a suitable method to predict cardiotoxicity of drug candidates.
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http://dx.doi.org/10.1007/s00204-021-03058-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8241674PMC
July 2021

Protective effects of total flavonoids from Qu Zhi Qiao (fruit of Citrus paradisi cv. Changshanhuyou) on OVA-induced allergic airway inflammation and remodeling through MAPKs and Smad2/3 signaling pathway.

Biomed Pharmacother 2021 Jun 19;138:111421. Epub 2021 Mar 19.

Zhejiang You-du Biotech Limited Company, Quzhou 324200, China; Department of Pharmacy, School of Medicine, Zhejiang University City College, 310015 China. Electronic address:

Allergic asthma is one of the inflammatory diseases, which has become a major public health problem. Qu zhi qiao (QZQ), a dry and immature fruit of Citrus paradisi cv. Changshanhuyou, has various flavonoids with pharmacological properties. However, there is a knowledge gap on the pharmacological properties of QZQ on allergic asthma. Therefore, here, we explored the efficacy and mechanism of total flavonoids from QZQ (TFCH) on allergic asthma. We extracted and purified TFCH and conducted animal experiments using an Ovalbumin (OVA)-induced mice model. Bronchoalveolar lavage fluid and Swiss-Giemsa staining were used to count different inflammatory cells in allergic asthma mice. We conducted histopathology and immunohistochemistry to evaluate the changes in the lungs of allergic asthma mice. Moreover, we used ELISA assays to analyze chemokines and inflammatory cytokines. Furthermore, western blot analyses were conducted to elucidate the mechanism of TFCH on allergic asthma. We established that TFCH has anti-inflammatory effects and inhibits airway remodeling, providing a potential therapeutic strategy for allergic asthma.
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http://dx.doi.org/10.1016/j.biopha.2021.111421DOI Listing
June 2021

Anti-Proliferative, Pro-Apoptotic, Anti-Migrative and Tumor-Inhibitory Effects and Pleiotropic Mechanism of Theaflavin on B16F10 Melanoma Cells.

Onco Targets Ther 2021 25;14:1291-1304. Epub 2021 Feb 25.

School of Basic Medicine, Zhejiang Chinese Medical University, Hangzhou, People's Republic of China.

Purpose: Theaflavin (TF) is a primary pigment of tea, exhibiting anti-proliferative, pro-apoptotic and anti-metastatic activities on cancer cell lines. However, it is unknown whether TF is effective in treating melanoma cells.

Methods: To determine the effects of TF on melanoma cells, we conducted in vitro assays of cell viability, DAPI staining, wound healing, transwell, and flow cytometry as well as in vivo experiments on B16F10-bearing mouse model. Real-time PCR (qPCR) and Western blot (WB) were conducted to explore the molecular actions of TF.

Results: The cell viability assay showed that TF exerted inhibitory effect on B16F10 cells in a dose-dependent manner from 40 to 400 μg/mL, with IC values ranging from 223.8±7.1 to 103.7±7.0 μg/mL. Moreover, TF induced early and late apoptosis and inhibited migration/invasion of B16F10 cells in a dose-dependent manner, indicating its pro-apoptotic and anti-migrative effects. In vivo, TF significantly inhibited B16F10 tumor size in mice model from 40 to 120 mg/kg, which exerted higher effect than that of cisplatin. The molecular data showed that TF significantly up-regulated the mRNA expressions of pro-apoptotic genes (, and ), up-regulated the protein expressions of apoptosis-related p53 and JNK signaling molecules (ASK1, phosphorylated Chk1/2, cleaved caspase 3, phosphorylated JNK, c-JUN, cleaved PARP, and phosphorylated p53), and down-regulated the protein expressions of proliferation-related MEK/ERK and PI3K/AKT signaling molecules (phosphorylated MEK1/2, phosphorylated ERK1/2, phosphorylated PI3K, and phosphorylated AKT) as well as the expressions of MMP2 and MMP9.

Conclusion: It can be concluded that TB exhibited anti-proliferative, pro-apoptotic, anti-migrative, and tumor-inhibitory effects on melanoma cells through pleiotropic actions on the above pathways. This study provides new evidence of anti-melanoma efficacy and mechanism of TF, contributing to the development of TF-derived natural products for melanoma therapy.
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http://dx.doi.org/10.2147/OTT.S286350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920628PMC
February 2021

Identification of differentially expressed genes, signaling pathways and immune infiltration in rheumatoid arthritis by integrated bioinformatics analysis.

Hereditas 2021 Jan 4;158(1). Epub 2021 Jan 4.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, PR China.

Background: The disability rate associated with rheumatoid arthritis (RA) ranks high among inflammatory joint diseases. However, the cause and potential molecular events are as yet not clear. Here, we aimed to identify differentially expressed genes (DEGs), pathways and immune infiltration involved in RA utilizing integrated bioinformatics analysis and investigating potential molecular mechanisms.

Materials And Methods: The expression profiles of GSE55235, GSE55457, GSE55584 and GSE77298 were downloaded from the Gene Expression Omnibus database, which contained 76 synovial membrane samples, including 49 RA samples and 27 normal controls. The microarray datasets were consolidated and DEGs were acquired and further analyzed by bioinformatics techniques. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were performed using R (version 3.6.1) software, respectively. The protein-protein interaction (PPI) network of DEGs were developed utilizing the STRING database. Finally, the CIBERSORT was used to evaluate the infiltration of immune cells in RA.

Results: A total of 828 DEGs were recognized, with 758 up-regulated and 70 down-regulated. GO and KEGG pathway analyses demonstrated that these DEGs focused primarily on cytokine receptor activity and relevant signaling pathways. The 30 most firmly related genes among DEGs were identified from the PPI network. The principal component analysis showed that there was a significant difference between the two tissues in infiltration immune.

Conclusion: This study shows that screening for DEGs, pathways and immune infiltration utilizing integrated bioinformatics analyses could aid in the comprehension of the molecular mechanisms involved in RA development. Besides, our study provides valuable data related to DEGs, pathways and immune infiltration of RA and may provide new insight into the understanding of molecular mechanisms.
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http://dx.doi.org/10.1186/s41065-020-00169-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784358PMC
January 2021

Theabrownin inhibits the cytoskeleton‑dependent cell cycle, migration and invasion of human osteosarcoma cells through NF‑κB pathway‑related mechanisms.

Oncol Rep 2020 12 12;44(6):2621-2633. Epub 2020 Oct 12.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, P.R. China.

Considering the high metastatic potential of osteosarcoma, not only pro‑apoptosis, but also anti‑metastasis is important for anti‑osteosarcoma therapy. Previously, the authors reported the pro‑apoptotic and tumor‑inhibitory effects of theabrownin (TB) on osteosarcoma cells; however, its effects on the metastasis‑related migration and invasion of osteosarcoma cells remain unknown. The present study conducted RNA sequencing (RNA‑seq) on xenograft zebrafish samples and performed in vitro experiments, including RT‑qPCR, cell viability analysis, clone formation assay, cell cycle analysis, immunofluorescence, cell migration assay, cell invasion assay, wound healing assay and western blot (WB) analysis to evaluate the anti‑metastatic effects and mechanism of TB against osteosarcoma cells. The RNA‑seq data revealed that TB significantly downregulated the expression of genes involved in the microtubule bundle formation of U2OS cells, which was verified by RT‑qPCR. The cell viability and clone formation data indicated that TB significantly inhibited U2OS cell viability and colony numbers. The results of cell cycle analysis revealed the blocked cell cycle progression of U2OS by TB. The immunofluorescent data revealed an evident cytoskeleton‑inhibitory effect of TB against the microfilament and microtubule formation of U2OS cells. The results of cell migration and invasion demonstrated that TB significantly inhibited U2OS cell migration and invasion. The results of WB analysis revealed that TB significantly regulated key molecules of epithelial‑mesenchymal transition [EMT; e.g., E‑cadherin, vimentin, Snail‑1, Slug and zinc finger E‑box‑binding homeobox 1 (ZEB‑1)] and those of the nuclear factor (NF)‑κB pathway (e.g., NF‑κB, phospho‑IKKα and phospho‑IKKβ), indicating that NF‑κB pathway‑related EMT suppression may mediate the mechanisms underlying the anti‑migratory and anti‑invasive effects of TB against osteosarcoma. To the best of our knowledge, this is the first study on the inhibitory effects and mechanisms of TB on the cytoskeleton‑dependent cell cycle, migration and invasion of human osteosarcoma cells. The findings presented herein suggest that TB may be a promising anti‑metastatic candidate for anti‑osteosarcoma therapy.
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http://dx.doi.org/10.3892/or.2020.7801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640368PMC
December 2020

Growth factors-based beneficial effects of platelet lysate on umbilical cord-derived stem cells and their synergistic use in osteoarthritis treatment.

Cell Death Dis 2020 10 14;11(10):857. Epub 2020 Oct 14.

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany.

Poor viability of mesenchymal stem cells (MSCs) at the transplanted site often hinders the efficacy of MSCs-based therapy. Platelet lysate (PL) contains rich amounts of growth factors, which benefits cell growth. This study aimed to explore how human PL benefits umbilical cord-derived MSCs (huc-MSCs), and whether they have synergistic potential in osteoarthritis (OA) treatment. As quality control, flow cytometry and specific staining were performed to identify huc-MSCs, and ELISA was used to quantify growth factors in PL. CCK-8 and flow cytometry assays were performed to evaluate the effects of PL on the cell viability and cell cycle progression of huc-MSCs. Wound healing and transwell assays were conducted to assess the migration of huc-MSCs. RNA sequencing, real time PCR, and Western blot assays were conducted to explore the growth factors-based mechanism of PL. The in vitro results showed that PL significantly promoted the proliferation, cell cycle, and migration of huc-MSCs by upregulating relevant genes/proteins and activating beclin1-dependent autophagy via the AMPK/mTOR signaling pathway. The main growth factors (PDGF-AA, IGF-1, TGF-β, EGF, and FGF) contributed to the effects of PL in varying degrees. The in vivo data showed that combined PL and huc-MSCs exerted significant synergistic effect against OA. The overall study determined the beneficial effects and mechanism of PL on huc-MSCs and indicated PL as an adjuvant for huc-MSCs in treating OA. This is the first report on the growth factors-based mechanism of PL on huc-MSCs and their synergistic application. It provides novel knowledge of PL's roles and offers a promising strategy for stem cell-based OA therapy by combining PL and huc-MSCs.
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http://dx.doi.org/10.1038/s41419-020-03045-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560841PMC
October 2020

Theaflavin Induces Apoptosis of A375 Human Melanoma Cells and Inhibits Tumor Growth in Xenograft Zebrafishes Through P53- and JNK-Related Mechanism.

Front Pharmacol 2020 31;11:1317. Epub 2020 Aug 31.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China.

Theaflavin (TF) is a major active pigment and polyphenol of tea, possessing anti-cancer activities. However, little is known about its activity and mechanism on melanoma cells. To fill this gap, we conducted experiments (cell viability assay, morphology observation, DAPI staining, and flow cytometry) and experiment by using a xenograft model of larval zebrafishes. Real-time PCR (qPCR) and Western blot (WB) analyses were conducted to explore the mechanism of TF. The data showed that TF exerted significant anti-proliferative and pro-apoptotic effects on A375 cells in a concentration-dependent manner. , TF significantly inhibited A375 tumor growth in larval zebrafishes at 0.67 and 2.0 μg/ml (1.3 to 3.9 μM). qPCR and WB data showed that TF significantly activated the P53 pathway-related proteins (ATM, CHK1/2, P53, and CASP8/3) and the JNK pathway-related proteins (ASK1, JNK, and C-JUN) through phosphorylation and cleavage, followed by activation of pro-apoptotic molecules (PARP, , , , and ). In sum, TF possessed cytotoxic pro-apoptotic and tumor-inhibitory effects on A375 cells through activations of P53 and JNK pathways. This is the first report on TF regarding its effects and mechanism on A375 cells, making it a promising candidate of natural products for clinical treatment of melanoma.
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http://dx.doi.org/10.3389/fphar.2020.01317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490558PMC
August 2020

Theabrownin Induces Apoptosis and Tumor Inhibition of Hepatocellular Carcinoma Huh7 Cells Through ASK1-JNK-c-Jun Pathway.

Onco Targets Ther 2020 9;13:8977-8987. Epub 2020 Sep 9.

The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, People's Republic of China.

Purpose: Theabrownin (TB), a main pigment and bioactive component of tea, has been shown anti-tumor activities against carcinomas, but its effects on hepatocellular carcinoma (HCC) remain unclear.

Methods: Hepatocellular carcinoma Huh7 cells were used for analyses. Cell viability assay was performed to determine TB's anti-proliferative effect, and flow cytometry with annexin V-FITC/PI double staining and DAPI staining were performed to determine its pro-apoptotic effect. Real-time PCR and Western blot assays were conducted to detect the molecular actions of TB. And a xenograft model of zebrafishes was established to evaluate the in vivo effect of TB. SP600125 (JNK inhibitor) was in vivo and in vitro used to verify the regulatory role of the JNK signaling pathway in the anti-hepatic carcinoma mechanism of TB.

Results: TB exerted significant anti-proliferative and pro-apoptotic effects on Huh7 cells in a dose-dependent manner. The molecular data showed that TB up-regulated the gene expressions of and and up-regulated the protein expressions of ASK-1, Bax, phosphorylated JNK, and phosphorylated c-Jun with down-regulation of Bcl-2. The in vivo data showed that TB exerted significant tumor-inhibitory effect which was even stronger than that of cis-platinum. Furthermore, the JNK inhibitor significantly weakened TB's effects both in vivo and in vitro and blocked the related molecular pathway.

Conclusion: TB exerts anti-proliferative, pro-apoptotic, and tumor-inhibitory effects on Huh7 cells through activation of the JNK signaling pathway. For the first time, this study provides new evidence of anti-HCC effects and mechanism of TB.
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http://dx.doi.org/10.2147/OTT.S254693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490432PMC
September 2020

Evaluation of Long-Time Decoction-Detoxicated (Processed Debeaux Lateral Root With Peel) for Its Acute Toxicity and Therapeutic Effect on Mono-Iodoacetate Induced Osteoarthritis.

Front Pharmacol 2020 24;11:1053. Epub 2020 Jul 24.

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany.

Background: As a degenerative joint disease with severe cartilage destruction and pain, osteoarthritis (OA) has no satisfactory therapy to date. In traditional Chinese medicine (TCM), Debeaux derived () has been developed for joint pain treatment. However, it causes adverse events in OA patients. Long-time decoction has been traditionally applied to reduce the aconite toxicity of and other aconite herbs, but its detoxifying effect is uncertain.

Methods: was extracted with dilute decoction times (30, 60, and 120 min) and evaluated by toxicological, chemical, pharmacological assays. Acute toxicity assay and chemical analysis were employed to determine the toxicity and chemoprofile of extracts, respectively. Since the detoxified (d) was defined, its therapeutic effect was evaluated by using an OA rat model induced by monosodium iodoacetate. d at 14 g/kg was orally administered for 28 days, and the pain assessments (mechanical withdrawal threshold and thermal withdrawal latency) and histopathological analyses (HE and safranin-O staining) were performed. Real-time PCR (qPCR) was applied to determine the molecular actions of d on cartilage tissue and on chondrocytes. MTT assay was conducted to evaluate the effect of d on the cell viability of chondrocytes. The cellular and molecular assays were also conducted to analyze the functions of chemical components in d.

Results: The chemoprofile result showed that the contents of toxic alkaloids (aconitine, mesaconitine, and hypaconitine) were decreased but that of non-toxic alkaloids (benzoylaconitine, benzoylmesaconitine, and benzoylhypaconitine) were increased with increasing decoction time. Acute toxicity assay showed that only extract with 120 min decoction was non-toxic within the therapeutic dose range. Thus, it was defined as d for further experiment. In OA experiment, d significantly attenuated joint pain and prevented articular degeneration from MIA attack. qPCR data showed that d restored the abnormal expressions of , , , , and up-regulated expression in rat cartilage. , dcontaining serum significantly proliferated rat chondrocytes and regulated the gene expressions of , , , and in a similar way as the data. Moreover, aconitine, mesaconitine, and hypaconitine exerted cytotoxic effects on chondrocytes, while benzoylaconitine and benzoylhypaconitine except benzoylmesaconitine exhibited similar molecular actions to d, indicating contributions of benzoylaconitine and benzoylhypaconitine to d.

Conclusions: This study defined d and demonstrated d as a potential analgesic and disease modifying agent against OA with molecular actions on the suppression of chondrocyte hypertrophy and extracellular matrix degradation, providing a promising TCM candidate for OA therapy.
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http://dx.doi.org/10.3389/fphar.2020.01053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396609PMC
July 2020

Vitamin K chloro derivative (VKT-2) inhibits HDAC6, activates autophagy and apoptosis, and inhibits aggresome formation in hepatocellular carcinoma cells.

Biochem Pharmacol 2020 10 25;180:114176. Epub 2020 Jul 25.

Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Mainz, Germany. Electronic address:

Epigenetics plays a vital role in regulating gene expression and determining the specific phenotypes of eukaryotic cells. Histone deacetylases (HDACs) are important epigenetic regulatory proteins effecting multiple biological functions. Particularly, HDAC6 has become a promising anti-cancer drug target because of its regulation of cell mobility, protein trafficking, degradation of misfolded proteins, cell growth, apoptosis, and metastasis. In this study, we identified one out of six vitamin K derivatives, VKT-2, as HDAC6 inhibitor using molecular docking and cell viability assays in HDAC6-overexpressing HuH-7 cancer cells. Microscale thermophoresis and HDAC6 enzymatic assays revealed that VKT-2 bound to HDAC6 and inhibited its function. We further identified its cytotoxic activity. VKT-2 hyperacetylated HDAC6 substrates and disturbed tubulin integrity leading to significant inhibition of tumor migration in both HuH-7 spheroids and U2OS-GFP-α-tubulin cells. Moreover, VKT-2 induced autophagic and apoptotic cell death in HuH-7, while aggresome formation was restrained after VKT-2 treatment. A HuH-7 cell-xenograft model in zebrafish larvae provided evidence that VKT-2 inhibited the tumor growth in vivo. To best of our knowledge, it is the first time to demonstrate that vitamin k derivatives (VKT-2) inhibits HDAC6 in solid tumor cells. These unique findings suggested that VKT-2 is a promising anti-cancer agent targeting HDAC6.
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http://dx.doi.org/10.1016/j.bcp.2020.114176DOI Listing
October 2020

CircAGFG1 drives metastasis and stemness in colorectal cancer by modulating YY1/CTNNB1.

Cell Death Dis 2020 07 17;11(7):542. Epub 2020 Jul 17.

the First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China.

Colorectal cancer (CRC) is a common malignancy with high occurrence and mortality worldwide. In recent years, the overall survival rate of CRC patients has been improved because of the advances in early diagnosis and therapy. However, the prognosis of CRC patients at the advanced stage is still poor due to high recurrence rate and metastasis. The function of circular RNA (circRNA) ArfGAP with FG repeats 1 (circAGFG1) has been explored in non-small-cell lung cancer and triple-negative breast cancer. Nevertheless, its role in CRC is not clear. In this study, circAGFG1 was upregulated in CRC cell lines. CircAGFG1 silencing significantly suppressed cell proliferation, migration, invasion, and stemness, while promoted cell apoptosis in CRC. Meanwhile, we found that circAGFG1 also accelerated CRC tumor growth and metastasis in vivo. Importantly, circAGFG1 activated Wnt/β-catenin pathway through regulating CTNNB1. Afterwards, YY1 was found to transcriptionally activate CTNNB1. Furthermore, circAGFG1 directly sponged miR-4262 and miR-185-5p to upregulate YY1 expression. Eventually, rescue assays demonstrated that the effect of circAGFG1 silencing on CRC cell functions was observably reversed by upregulating YY1 or CTNNB1. In brief, our findings uncovered that circAGFG1 modulated YY1/CTNNB1 axis to drive metastasis and stemness in CRC by sponging miR-4262 and miR-185-5p.
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http://dx.doi.org/10.1038/s41419-020-2707-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367849PMC
July 2020

Inhibition of cell migration and induction of apoptosis by a novel class II histone deacetylase inhibitor, MCC2344.

Pharmacol Res 2020 10 10;160:105076. Epub 2020 Jul 10.

Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Mainz, Germany. Electronic address:

Epigenetic modifiers provide a new target for the development of anti-cancer drugs. The eraser histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase that targets various non-histone proteins such as transcription factors, nuclear receptors, cytoskeletal proteins, DNA repair proteins, and molecular chaperones. Therefore, it became an attractive target for cancer treatment. In this study, virtual screening was applied to the MicroCombiChem database with 1162 drug-like compounds to identify new HDAC6 inhibitors. Five compounds were tested in silico and in vitro as HDAC6 inhibitors. Both analyses revealed 1-cyclohexene-1-carboxamide, 2-hydroxy-4,4-dimethyl-N-1-naphthalenyl-6-oxo- (MCC2344) as the best HDAC6 inhibitor among the five ligands. The binding affinity of MCC2344 to HDAC6 was further confirmed by microscale thermophoresis. Additionally, the anti-cancer activity of MCC2344 was tested in several tumor cell lines. Leukemia cells were the most sensitive cells towards MCC2344, particularly the P-glycoprotein-overexpressing multidrug-resistant cell line CEM/ADR5000 exhibited remarkable collateral sensitivity towards MCC2344. Transcriptome analysis using microarray hybridization was performed for investigating downstream mechanisms of action of MCC2344 in leukemia cells. MCC2344 affected microtubule dynamics and suppressed cell migration in the wound healing assay as well as in a spheroid model by hyper-acetylation of tubulin and HSP-90. MCC2344 induced cell death in CEM/ADR5000 cells by activation of PARP, caspase-3, and p21 in addition to the downregulation of p62. MCC2344 significantly inhibited tumor growth in vivo in zebrafish larvae without mortality until 20 pM. We propose MCC2344 as a novel HDAC6 inhibitor for cancer treatment.
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http://dx.doi.org/10.1016/j.phrs.2020.105076DOI Listing
October 2020

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats.

J Vis Exp 2020 05 20(159). Epub 2020 May 20.

The First Affiliated Hospital, Zhejiang Chinese Medical University;

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients' daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 β and TNF α) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.
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http://dx.doi.org/10.3791/60649DOI Listing
May 2020

Hepatoprotective and Anti-Oxidative Effects of Total Flavonoids From Qu Zhi Qiao (Fruit of Citrus Paradisi cv.Changshanhuyou) on Nonalcoholic Steatohepatitis and Through Nrf2-ARE Signaling Pathway.

Front Pharmacol 2020 22;11:483. Epub 2020 Apr 22.

Institute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou, China.

Nonalcoholic steatohepatitis (NASH) is a liver disease defined as the dynamic condition of hepatocellular injury during the progress of nonalcoholic fatty liver disease (NAFLD). Total flavonoids from the dry and immature fruits of cv. (accepted species name: Citrus × aurantium L) () are purified and named TFCH. This study was purposed to investigate and analyze the effect of TFCH on NASH model through Nuclear factor erythroid 2-related factor 2 (Nrf2)- antioxidant response elements pathway and . study was performed using male C57BL/6 mice fed with high fat diet 16 weeks for NASH model. After 7-week modeling, mice in TFCH-treated group were daily treated with intragastric administration of TFCH at 25 mg/kg, 50 mg/kg, 200 mg/kg, respectively, for successive 8 weeks. Histopathological and immunohistochemical analyses were conducted for evaluating severity of NASH-mice model and the effect of TFCH treatment. experiment was performed by using human LX-2 cells and cultured with Free fatty acid (FFA) (Oleic acid: palmitic: l: 0.5 mmol/L) for 24 h and then treated with TFCH at different concentrations (0, 25, 50, 100, 200 mg/ml) for 6 h,12 h, and 24 h. Anti-apoptosis effect of TFCH on LX-2 cells cultured with FFA was revealed by the CCK-8 assay. Lipid parameters and oxidative stress markers were measured and , results showed that TFCH dose-dependently and greatly increased the antioxidant ability and reduced the oxidative damage in NASH model. The protein expression of Nrf2 and the downstream target genes in mice liver and human LX-2 cells were tested by Western blot analysis to investigate the possible molecular mechanisms of TFCH. Our results indicated that TFCH up-regulated protein expression of these genes and have the significant influence in activating the Nrf2-ARE signaling pathway. This study shows Nrf2-ARE signaling pathway may provide novel therapeutic opportunities for NASH therapy in the future.
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http://dx.doi.org/10.3389/fphar.2020.00483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189874PMC
April 2020
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