Publications by authors named "Huai Huang"

36 Publications

Astragaloside IV Protects 6-Hydroxydopamine-Induced SH-SY5Y Cell Model of Parkinson's Disease via Activating the JAK2/STAT3 Pathway.

Front Neurosci 2021 23;15:631501. Epub 2021 Mar 23.

Department of Neurology, Hebei PetroChina Central Hospital, Langfang, China.

Objectives: Astragaloside IV (AS-IV), the main active component of Astragalus membranaceus, bears anti-inflammatory, antioxidant, and neuroprotective activity. Parkinson's disease (PD) is a common neurodegenerative disease. This study explored the protective effect of AS-IV on the cell model of PD.

Materials And Methods: SH-SY5Y cells were incubated with different concentrations (10, 50, 100, 150, and 200 μM) of 6-hydroxydopamine (6-OHDA) for 0, 3, 6, 12, 24, and 48 h to establish the PD cell model. Different concentrations (0, 25, 50, 100, 150, and 200 μM) of AS-IV or 15 mM JAK2/STAT3 pathway inhibitor SC99 was added for intervention 2 h before 6-OHDA treatment. The viability and morphological damage of 6-OHDA-treated SH-SY5Y cells were measured using MTT assay and Hoechst 33258 staining. The expression of microtubule associated protein 2 (MAP2) was detected by immunofluorescence staining. The levels of inflammation and oxidative stress were measured using ELISA. Apoptosis of 6-OHDA-treated SH-SY5Y cells was detected using flow cytometry, and phosphorylation level of JAK2 and STAT3 were detected using Western blot analysis.

Results: The survival rate of SH-SY5Y cells treated with 100 μM 6-OHDA for 24 h was about 50%. AS-IV (25-100 μM) significantly improved the viability (all < 0.01), increased MAP2 expression, and repaired the morphological damage induced by 6-OHDA. AS-IV inhibited IL-1β, IL-6, and TNF-α level (all < 0.05), reduced MDA and ROS content and increased SOD concentration, thereby reducing inflammation and oxidative stress (all < 0.01) in 6-OHDA-treated SH-SY5Y cells. Moreover, AS-IV decreased apoptosis rate and Bax/Bcl-2 ratio induced by 6-OHDA (all < 0.05). Mechanically, AS-IV significantly increased the phosphorylation of JAK2 and STAT3 ( < 0.01); the addition of SC99 decreased the cell viability, increased the apoptosis rate, enhanced the levels of inflammatory factors and oxidative stress.

Conclusion: AS-IV enhanced the cell viability, and inhibited apoptosis, inflammation and oxidative stress of 6-OHDA-treated SH-SY5Y cells via activating the JAK2/STAT3 signaling pathway. This study may confer novel insights for the management of PD.
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http://dx.doi.org/10.3389/fnins.2021.631501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021720PMC
March 2021

Novel therapeutic evaluation biomarkers of lipid metabolism targets in uncomplicated pulmonary tuberculosis patients.

Signal Transduct Target Ther 2021 Jan 18;6(1):22. Epub 2021 Jan 18.

Institute of Cell Biology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, 310058, Hangzhou, China.

Currently, the management of pulmonary tuberculosis (TB) lacks potent medications and accurate efficacy evaluation biomarkers. In view of the fact that the host lipids are the important energy source of Mycobacterium tuberculosis (Mtb), UPLC-MS/MS based on lipid metabolism was used to monitor the plasma lipid spectrum of TB patients from the initial diagnosis to cured. The analysis showed that TB patients presented aberrant metabolism of phospholipids, glycerides, and sphingolipids. Upon the treatment, the abnormal expression of Cer (d18:1/24:0), CerP (d18:1/20:3), LPE (0:0/22:0), LPA (0:0/16:0), and LPA (0:0/18:0) in TB patients were gradually normalized, indicating that the intervention of lipid metabolism could block energy metabolism and inhibit the cell wall synthesis of Mtb. Furthermore, the increase in ceramide (Cer) levels could promote autophagosome-lysosome fusion. LPA (0:0/16:0) and LPA (0:0/18:0) had a great potential in the early diagnosis (both sensitivity and specificity were 100%) and efficacy evaluation (both sensitivity and specificity were 100%) of TB, indicating that the above lipid metabolites could be used as potential biomarkers for TB.
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http://dx.doi.org/10.1038/s41392-020-00427-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814055PMC
January 2021

Identification of potential lipid biomarkers for active pulmonary tuberculosis using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Exp Biol Med (Maywood) 2021 Feb 11;246(4):387-399. Epub 2020 Nov 11.

Institute of Cell Biology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China.

Early diagnosis of active pulmonary tuberculosis (TB) is the key to controlling the disease. Host lipids are nutrient sources for the metabolism of . In this research work, we used ultra-high-performance liquid chromatography-tandem mass spectrometry to screen plasma lipids in TB patients, lung cancer patients, community-acquired pneumonia patients, and normal healthy controls. Principal component analysis, orthogonal partial least squares discriminant analysis, and K-means clustering algorithm analysis were used to identify lipids with differential abundance. A total of 22 differential lipids were filtered out among all subjects. The plasma phospholipid levels were decreased, while the cholesterol ester levels were increased in patients with TB. We speculate that the infection of may regulate the lipid metabolism of TB patients and may promote host-assisted bacterial degradation of phospholipids and accumulation of cholesterol esters. This may be related to the formation of lung cavities with caseous necrosis. The results of receiver operating characteristic curve analysis revealed four lipids such as phosphatidylcholine (PC, 12:0/22:2), PC (16:0/18:2), cholesteryl ester (20:3), and sphingomyelin (d18:0/18:1) as potential biomarkers for early diagnosis of TB. The diagnostic model was fitted by using logistic regression analysis and combining the above four lipids with a sensitivity of 92.9%, a specificity of 82.4%, and the area under the curve (AUC) value of 0.934 (95% CI 0.873 - 0.971). The machine learning method (10-fold cross-validation) demonstrated that the model had good accuracy (0.908 AUC, 85.3% sensitivity, and 85.9% specificity). The lipids identified in this study may serve as novel biomarkers in TB diagnosis. Our research may pave the foundation for understanding the pathogenesis of TB.
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http://dx.doi.org/10.1177/1535370220968058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885049PMC
February 2021

Serum sCD14, PGLYRP2 and FGA as potential biomarkers for multidrug-resistant tuberculosis based on data-independent acquisition and targeted proteomics.

J Cell Mol Med 2020 11 23;24(21):12537-12549. Epub 2020 Sep 23.

Institute of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China.

Multidrug-resistant tuberculosis (MDR-TB), defined as tuberculosis (TB) resistant to at least isoniazid and rifampicin, is a major concern of TB control worldwide. However, the diagnosis of MDR-TB remains a huge challenge to its prevention and control. To identify new diagnostic methods for MDR-TB, a mass spectrometry strategy of data-independent acquisition and parallel reaction monitoring was used to detect and validate differential serum proteins. The bioinformatic analysis showed that the functions of differential serum proteins between the MDR-TB group and the drug-sensitive tuberculosis group were significantly correlated to the complement coagulation cascade, surface adhesion and extracellular matrix receptor interaction, suggesting a disorder of coagulation in TB. Here, we identified three potential candidate biomarkers such as sCD14, PGLYRP2 and FGA, and established a diagnostic model using these three candidate biomarkers with a sensitivity of 81.2%, a specificity of 90% and the area under the curve value of 0.934 in receiver operation characteristics curve to diagnose MDR-TB. Our study has paved the way for a novel method to diagnose MDR-TB and may contribute to elucidate the mechanisms underlying MDR-TB.
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http://dx.doi.org/10.1111/jcmm.15796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686995PMC
November 2020

The novel potential biomarkers for multidrug-resistance tuberculosis using UPLC-Q-TOF-MS.

Exp Biol Med (Maywood) 2020 03 11;245(6):501-511. Epub 2020 Feb 11.

Medical Research Center, Yue Bei People's Hospital, Shaoguan 512025, China.

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http://dx.doi.org/10.1177/1535370220903464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158596PMC
March 2020

Screening and identification of plasma lncRNAs uc.48+ and NR_105053 as potential novel biomarkers for cured pulmonary tuberculosis.

Int J Infect Dis 2020 Mar 11;92:141-150. Epub 2020 Jan 11.

Institute of Cell Biology, Zhejiang University School of Medicine, Hangzhou, 310058, China; Medical Research Center, Yubei People's Hospital, Shaoguan 512026, China. Electronic address:

Background: Tuberculosis (TB) treatment takes a long time, and a gold standard test to define TB cure is lacking. This may lead to early discharge of TB patients, resulting in an increased risk of disease transmission and drug resistance. Plasma lncRNAs might act as potential biomarkers to evaluate TB cure in an efficient and precise manner.

Methods: A lncRNA microarray assay was used to screen differentially expressed plasma lncRNAs in untreated TB and cured TB subjects. The expression levels of lncRNAs were verified by qPCR. Target genes of lncRNAs were predicted using a coding-non-coding gene co-expression network and mRNA-lncRNA-miRNA interaction network analysis.

Results: The expression levels of lncRNAs uc.48+ (p < 0.001) and NR_105053 (p = 0.03) were found to differ significantly between the untreated TB group and the cured TB group. The predicted target genes of uc.48+ were EP300, BAI1 and NR_105053 were TLR9, MYD88, BAI1, respectively. A predictive model for cured TB was established by the combination of uc.48+ and NR_105053 expression, with a sensitivity of 90.00% and specificity of 86.36%, and an area under the curve (AUC) value of 0.945.

Conclusions: lncRNAs uc.48+ and NR_105053 may serve as potential biomarkers to distinguish between untreated TB patients and cured TB subjects. This study provides an experimental basis to evaluate the effect of TB treatment and may also provide new clues to the pathological mechanisms of TB.
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http://dx.doi.org/10.1016/j.ijid.2020.01.005DOI Listing
March 2020

A group of serum proteins as potential diagnostic biomarkers for Yin-deficiency-heat syndrome.

Anat Rec (Hoboken) 2020 08 10;303(8):2086-2094. Epub 2020 Jan 10.

Department of Anatomy and Embryology, School of Medicine, Zhejiang University, Hangzhou, China.

Yin-deficiency-heat (YDH) syndrome is a very common subhealth status in Traditional Chinese Medicine. However, currently, there is no unified standard for diagnosing YDH syndrome. We applied the iTRAQ-2D LC-MS/MS method to explore the potential of serum protein profiles as biomarker for YDH syndrome. A total of 120 differentially expressed proteins (79 downregulated and 41 upregulated) were identified by the proteomic profiling. The results of KEGG pathway analysis showed that the functions of the differentially expressed proteins were mainly involved in complement and coagulation cascades. The clinical data showed that YDH syndrome was closely related to inflammation and coagulation, compared with the healthy controls. The ELISA validation results indicated that the expression levels of ALB, CFI, and KLKB1 were downregulated in the YDH syndrome group (p < .05). Moreover, we established a decision tree model based on the combination of these three proteins and achieved a sensitivity of 87.5%, a specificity of 84.4%, and AUC of 0.891. The results indicated that the combination of ALB, CFI, and KLKB1 may serve as potential biomarkers for diagnosing YDH syndrome. Our study can provide a new method for YDH syndrome diagnosis, and may also provide an experimental basis to understand the molecular mechanism of YDH syndrome.
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http://dx.doi.org/10.1002/ar.24351DOI Listing
August 2020

Study on potential biomarkers of energy metabolism-related to early-stage Yin-deficiency-heat syndrome based on metabolomics and transcriptomics.

Anat Rec (Hoboken) 2020 08 7;303(8):2109-2120. Epub 2020 Jan 7.

Department of Anatomy and Embryology, Zhejiang University, Hangzhou, China.

Yin-deficiency-heat (YDH) syndrome is a common sub-health state of the human body in traditional Chinese medicine (TCM). However, due to the lack of objective quantitative diagnostic indicators, patients with early-stage YDH syndrome cannot be treated in time and can develop a pathological (disease) state. Therefore, it is necessary to apply modern diagnostic techniques in order to identify the biological markers for the diagnosis of early-stage YDH syndrome. In the present study, we performed Solexa sequencing and non-targeted metabolomics analysis using high-performance liquid chromatography coupled with mass spectrometry to screen differentially expressed mRNAs and differential metabolites in individuals with early-stage YDH syndrome and healthy controls. Bioinformatics methods were used to perform enrichment analysis of differentially expressed mRNAs and differential metabolites for biological functions and signaling pathways. Furthermore, we found that differentially expressed mRNAs and differential metabolites were related to energy metabolism. Real-time PCR was used to validate the mRNA expression in the serum of subjects with early-stage YDH syndrome. We found that the mitochondrially encoded NADH dehydrogenase 2 (MT-ND2) mRNA was differentially expressed in the serum of individuals with early-stage YDH syndrome. Receiver operating characteristic (ROC) curve and logistic regression analysis were used to evaluate the efficacy of the diagnostic model based on eight differential metabolites. We combined the three metabolites such as Glycine, Sphingomyelin, and Isocitrate to establish the diagnostic model with a sensitivity of 0.853 and a specificity of 0.800. The combination of the above three metabolites may serve as a potential biomarker for the diagnosis of early-stage YDH syndrome. Our study reveals potential biomarker for the diagnosis of early-stage YDH syndrome and also provides a new method for the quantification and objectification of TCM syndromes.
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http://dx.doi.org/10.1002/ar.24355DOI Listing
August 2020

Serum proteins TGFBI, PCSK9, and CCL14 are potential biomarkers for different traditional Chinese medicine syndromes of multidrug-resistant tuberculosis.

Anat Rec (Hoboken) 2020 08 7;303(8):2131-2143. Epub 2020 Jan 7.

Medical Research Center, Yuebei People's Hospital, Shaoguan, China.

Patients with multidrug-resistant tuberculosis (MDR-TB) tend to have a long course of anti-TB treatment and severe side effects. Traditional Chinese Medicine (TCM) has a synergistic effect in attenuation of MDR-TB. However, the lack of objective biological standards to classify and diagnose MDR-TB TCM syndromes could result in less effective TCM treatment. Therefore, in this study, we identified differentially expressed proteins (DEPs) in serum of individuals with MDR-TB TCM syndromes by applying isobaric tags for relative and absolute quantification coupled with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) method and bioinformatics analysis. The functional analysis of DEPs was also performed. Additionally, DEPs among three different TCM syndromes of MDR-TB were validated by enzyme-linked immunosorbent assay (ELISA). Finally, a receiver operating characteristic (ROC) curve was performed to estimate the diagnostic ability of DEPs. A total of 71 DEPs were identified in the three different MDR-TB TCM syndrome groups such as the pulmonary Yin deficiency (PYD) syndrome group, the Hyperactivity of Fire due to Yin deficiency (HFYD) syndrome group, and the deficiency of Qi and Yin (DQY) syndrome group. The results showed that the expression level of transforming growth factor-beta-induced protein ig-h3 (TGFBI) was lower in the PYD syndrome group (p = .002), the proprotein convertase subtilisin/kexin type 9 (PCSK9) was overexpressed in the HFYD syndrome group (p < .0001), and the C-C motif chemokine ligand 14 (CCL14) expression level was reduced in the DQY syndrome group (p = .004). Our study demonstrated that serum TGFBI, PCSK9, and CCL14 may serve as potential novel biomarkers for PYD syndrome, HFYD syndrome and DQY syndrome of MDR-TB, respectively. The study provides a biological basis for MDR-TB TCM syndromes classification and can be of great significance for the treatment of different TCM syndromes.
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http://dx.doi.org/10.1002/ar.24353DOI Listing
August 2020

Screening of potential biomarkers for Yin-deficiency-heat syndrome based on UHPLC-MS method and the mechanism of Zhibai Dihuang granule therapeutic effect.

Anat Rec (Hoboken) 2020 08 7;303(8):2095-2108. Epub 2020 Jan 7.

Medical Research Center, Yuebei People's Hospital, Shaoguan, China.

Background: Yin-deficiency-heat (YDH) syndrome is a subhealth state of the individual, mainly manifested as oral ulcers, dry mouth, constipation, and other symptoms. Zhibai Dihuang granule (ZDG), as a classic traditional Chinese medicine, is effective in treating YDH syndrome. We screened the potential biomarkers for diagnosing YDH syndrome, and explored the mechanisms of the therapeutic effect of ZDG.

Methods: Plasma samples from the Pinghe (PH, healthy control) group, the Shanghuo (SH, YDH syndrome) group, and the ZDG treated group (therapeutic group) were analyzed by using metabolomics profiling. The data were analyzed by multivariate statistical and bioinformatics analyses.

Results: We screened four differential metabolites such as, decanoylcarnitine, dodecanoylcarnitine, phosphatidylcholine (PC), and Aspartate (Asp) Arginine (Arg) Proline (Pro) in the SH group and the PH group. The results showed that the combination of above four metabolites could serve as a potential biomarker for the early diagnosis of YDH syndrome. The metabolites decanoylcarnitine and glucose were found to be differentially expressed in the YDH syndrome group and tended to be normalized after ZDG treatment.

Conclusion: The increased levels of four differential metabolites (decanoylcarnitine, dodecanoylcarnitine, PC, and Asp Arg Pro) revealed that individuals with YDH syndrome may have increased energy metabolism in the body, which could lead to disorders of fatty acids β-oxidation and immune function. The levels of two differential metabolites including decanoylcarnitine and glucose returned to normal after ZDG treatment, indicating that ZDG could treat YDH syndrome by regulating glucose metabolism and fatty acids β-oxidation. Our study provides a new method for the diagnosis of YDH syndrome, and may provide theoretical basis for novel therapeutic strategies of YDH syndrome.
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http://dx.doi.org/10.1002/ar.24352DOI Listing
August 2020

Astragaloside IV Alleviates Cerebral Ischemia-Reperfusion Injury by Activating the Janus Kinase 2 and Signal Transducer and Activator of Transcription 3 Signaling Pathway.

Pharmacology 2020 11;105(3-4):181-189. Epub 2019 Dec 11.

Department of Neurosurgery, Chinese National Petroleum Corporation Central Hospital, Langfang, China.

Astragaloside IV (AS-IV) is an active component extracted from the traditional Chinese herbal medicine. AS-IV is a neuroprotective component in cerebral ischemic models. However, roles of AS-IV in cerebral ischemia-reperfusion (I/R) injury and the underlying mechanisms are rarely investigated. The role of AS-IV in oxygen - glucose deprivation reoxygenation (OGD/R)-induced cell proliferation and apoptosis assays were analyzed by Cell Counting Kit-8 and Flow cytometric. Western Blot assays were performed to measure the related expression levels in SH-SY5Y cells. Meanwhile, activities of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) in OGD/R-induced cells were determined by relative commercial kits. AS-IV was also used in cerebral I/R rat model, aimed to investigate the effects on cerebral infarct. The results indicated that OGD/R suppressed viability, enhanced apoptosis, which could be reversed by AS-IV treatment. Compared with the control group, the expression of p-JAK2 and p-STAT3 was significantly increased by AS-IV (60 μg/mL) under the OGD/R condition. Furthermore, AS-IV (60 μg/mL) treatment markedly increased SOD activity, whereas significantly decreased MDA activity and production of ROS in OGD/R-induced cells. The protective effects of AS-IV mentioned above were weaken or abolished while adding JAK2 inhibitor AG490. In addition, the effects of AS-IV on Janus kinase 2 and signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in cerebral I/R injury were also verified in vivo. AS-IV protected against cerebral I/R injury and reversed by AG490. Therefore, in vitro and in vivo analyses suggested that AS-IV may protect against cerebral I/R injury partly mediated by JAK2/STAT3 signaling pathway and antioxidative effects. AS-IV may serve as a novel therapeutic regimen for cerebral I/R injury.
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http://dx.doi.org/10.1159/000503361DOI Listing
October 2020

Plasma metabolites Xanthine, 4-Pyridoxate, and d-glutamic acid as novel potential biomarkers for pulmonary tuberculosis.

Clin Chim Acta 2019 Nov 20;498:135-142. Epub 2019 Aug 20.

School of Medicine, South China University of Technology, Guangzhou 510006, PR China; Institute of Cell Biology, Zhejiang University, Hangzhou 310058, PR China. Electronic address:

Background: The lack of rapid and efficient diagnostic methods has been one of the most frustrating challenges in controlling the pulmonary tuberculosis (TB) epidemic. This study was aimed to identify novel non-invasive biomarkers for pulmonary TB.

Methods: The subjects in this study were divided into four groups: the pulmonary TB group, the community-acquired pneumonia (CAP) group, the lung cancer (LC) group, and the normal control (NC) group. Plasma small molecule metabolites were investigated in each group by using ultra-high performance liquid chromatography coupled with Q Exactive mass spectrometry. Multivariate statistical methods and bioinformatics were used to analyze the data.

Results: We identified three differential plasma metabolites such as, Xanthine, 4-Pyridoxate and d-glutamic acid in the pulmonary TB group, compared to the other groups (CAP, LC and NC). The pathway enrichment analysis indicated that the energy source in pulmonary TB was multi-center, which might be involved in maintaining the reproductive ability and virulence of Mycobacterium tuberculosis.

Conclusion: The results suggested that Xanthine, 4-Pyridoxate, and d-glutamic acid may serve as potential biomarkers for pulmonary TB. The present study provides experimental basis for developing potential biomarkers of pulmonary TB.
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http://dx.doi.org/10.1016/j.cca.2019.08.017DOI Listing
November 2019

Human Urinary Kallidinogenase Reduces Lipopolysaccharide-Induced Neuroinflammation and Oxidative Stress in BV-2 Cells.

Pain Res Manag 2019 24;2019:6393150. Epub 2019 Jul 24.

Department of Neurology, The Affiliated Houjie Hospital, Guangdong Medical University, Dongguan 523945, China.

Migraine is one of the most common neurological disorders which poses significant socioeconomic burden worldwide. Neuroinflammation and oxidative stress both play important roles in the pathogenesis of migraine. Human urinary kallidinogenase (UK) is a tissue kallikrein derived from human urine. Increasing evidence suggests that UK may protect against ischemic stroke, but UK's treatment potential against migraine remains to be explored. Immortal BV-2 murine microglial cells were treated with UK (125 nM, 250 nM, and 500 nM) and then given lipopolysaccharides (LPS, 1000 ng/mL). Cell viability of BV-2 cells was tested by the CCK-8 assay. Expressions of tumor necrosis factor- (TNF), prostaglandin E2 (PGE2), interleukin-6 (IL-6), and interleukin-1 (IL-1) were examined with the ELISA method and western blot. Intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to determine oxidative stress. Our results showed that LPS administration increased the levels of proinflammatory cytokines (TNF, PGE2, IL-6, and IL-1) and oxidative stress (ROS and MDA) when compared with the control group and decreased significantly upon introduction with UK. Taken together, UK treatment reduced LPS-induced neuroinflammation and oxidative stress in a dose-dependent manner, which might be a potential treatment of migraine.
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http://dx.doi.org/10.1155/2019/6393150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681601PMC
December 2019

Disease-associated mutations in human TUBB3 disturb netrin repulsive signaling.

PLoS One 2019 21;14(6):e0218811. Epub 2019 Jun 21.

Department of Biological Sciences, University of Toledo, Toledo, OH, United States of America.

Missense mutations in the human TUBB3 gene cause a variety of neurological disorders associated with defects in axon guidance and neuronal migration, but the underlying molecular mechanisms are not well understood. Recent studies have shown that direct coupling of dynamic TUBB3 in microtubules with netrin receptors is required for netrin-1-mediated axon guidance, and the interaction of netrin-1 repulsive receptor UNC5C with TUBB3 is involved in netrin-1 mediated axonal repulsion. Here, we report that TUBB3 mutations perturb netrin-1/UNC5C repulsive signaling in the developing nervous system. Among twelve mutants reported in previous studies, five of them show significantly reduced interaction with UNC5C in comparison to the wild-type TUBB3. TUBB3 mutants R262C and R62Q exhibit decreased subcellular colocalization with UNC5C in the peripheral area of the growth cone of primary mouse neurons. Netrin-1 reduces the colocalization of UNC5C with wild-type TUBB3, but not TUBB3 mutants R262C or R62Q, in the growth cone. Results from the in vitro cosedimentation assay indicate that netrin-1 inhibits cosedimentation of UNC5C with polymerized microtubules in primary mouse neurons expressing the wild-type TUBB3, but not R262C or R62Q. Expression of either R262C or R62Q not only blocks netrin-1-induced growth cone collapse and axonal repulsion of primary EGL cells in vitro, but also results in axon projections defects of chicken dorsal root ganglion neurons in ovo. Our study reveals that human TUBB3 mutations specifically perturb netrin-1/UNC5C-mediated repulsion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218811PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588280PMC
March 2020

MiR-21b-3p protects NS2OY cells against oxygen-glucose deprivation/reperfusion-induced injury by down-regulating cyclooxygenase-2.

Am J Transl Res 2019 15;11(5):3007-3017. Epub 2019 May 15.

Department of Orthopaedics, General Hospital of Southern Theater Command, PLA Guangzhou, Guangdong, People's Republic of China.

Recent studies have shown abnormal expression levels of cyclooxygenase-2 (COX-2) and miR-21b-3p in cerebral ischemia-reperfusion (I/R) rat models. Decreased COX-2 expression could reduce brain injury and thus could be a target of miR-21b-3p according to the miRNA databases (miRDB) analysis. However, its functions and underlying mechanisms in I/R injury remain unclear. In our study, we have established an oxygen/glucose deprivation and reperfusion (OGD/R) model by using NS2OY cells. The expression of miR-21b-3p and COX-2 was determined by quantitative real-time PCR or Western blot, and the fluorescence intensities were detected by fluorescence in situ hybridization (FISH) or immunofluorescence. After transfection and OGD/R treatments, the functions of miR-21b-3p and COX-2 on cell viability and apoptosis were detected using cell-counting kit 8, Edu staining, flow cytometry and Hoechst staining, respectively. Finally, dual-luciferase reporter assay was used to explore the relationship between miR-21-b-3p and COX-2. The results have showed that COX-2 mRNA and protein expression were significantly increased; however, the expression of miR-21b-3p was remarkably reduced in NS2OY cells after OGD/R treatment. The changes were most remarkable in OGD 2 h/R24 group. Function analysis has showed that when NS2OY cells were exposed to OGD/R injury, overexpressed miR-21b-3p significantly downregulated COX-2 expression, increased cell viability and decreased apoptosis. In addition, knocking down the expression of COX-2 could also increase cell viability and decrease apoptosis. Dual-luciferase reporter assays showed miR-21b-3p as the target of 3'-UTR of COX-2. Therefore, we concluded that OGD/R-induced injury by down-regulating COX-2.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556624PMC
May 2019

l-Histidine, arachidonic acid, biliverdin, and l-cysteine-glutathione disulfide as potential biomarkers for cured pulmonary tuberculosis.

Biomed Pharmacother 2019 Aug 21;116:108980. Epub 2019 May 21.

Institute of Cell Biology, Zhejiang University School of Medicine, Hangzhou, 310058, China. Electronic address:

Lack of laboratory standards for cured tuberculosis (TB) can lead to early discharge of untreated TB patients from the hospital, resulting in increased risk of TB spread and of developing drug resistant Mycobacterium tuberculosis (Mtb). We used ultra-high performance liquid chromatography coupled with mass spectrometry (LC-MS) to detect heparin anticoagulant in plasma of untreated TB patients, two-month treated TB patients, cured TB subjects, and healthy controls. Screening of differentially expressed metabolites resulted in identification of four differentially expressed metabolites such as, l-Histidine, Arachidonic acid (AA), Biliverdin, and l-Cysteine-glutathione disulfide after 6 months of TB treatment. Among them, l-Cysteine-glutathione disulfide and AA could be identified after 2 months of TB treatment. We established a cured TB model with an area under the curve (AUC) of 0.909 (95% CI, 0.802-0.970), 86.2% sensitivity, and 85.2% specificity. The diagnostic model fitted from the four differential metabolites in combination (l-Histidine, AA, Biliverdin, and l-Cysteine-glutathione disulfide) can be used as potential biomarkers for cured TB. Our study provided laboratory standards for hospital discharge of TB patients, as well as experimental basis for evaluating the efficacy of anti-TB drugs.
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http://dx.doi.org/10.1016/j.biopha.2019.108980DOI Listing
August 2019

Elevated pulmonary tuberculosis biomarker miR-423-5p plays critical role in the occurrence of active TB by inhibiting autophagosome-lysosome fusion.

Emerg Microbes Infect 2019 ;8(1):448-460

a Institute of Cell Biology , Zhejiang University School of Medicine , Hangzhou , People's Republic of China.

Rapid diagnosis of pulmonary tuberculosis is an effective measure to prevent the spread of tuberculosis. However, the grim fact is that the new, rapid, and safe methods for clinical diagnosis are lacking. Moreover, although auto-lysosome is critical in clearing Mycobacterium tuberculosis, the pathological significance of microRNAs, as biomarkers of tuberculosis, in autophagosome maturation is unclear. Here, these microRNAs were investigated by Solexa sequencing and qPCR validation, and a potential diagnostic model was established by logistic regression. Besides that, the mechanism of one of the microRNAs involved in the occurrence of tuberculosis was studied. The results showed that the expression of miR-423-5p, miR-17-5p, and miR-20b-5p were significantly increased in the serum of patients with tuberculosis. The combination of these three microRNAs established a model to diagnose tuberculosis with an accuracy of 78.18%, and an area under the curve value of 0.908. Bioinformatics analysis unveiled miR-423-5p as the most likely candidate in regulating autophagosome maturation. The up-regulation of miR-423-5p could inhibit autophagosome maturation through suppressing autophagosome-lysosome fusion in macrophages. Further investigations showed that VPS33A was the direct target of miR-423-5p, and the two CUGCCCCUC domains in VPS33A 3'-UTR were the direct regulatory sites for miR-423-5p. In addition, an inverse correlation between VPS33A and miR-423-5p was found in peripheral blood mononuclear cells of patients with tuberculosis. Since the inhibition of autolysosome formation plays a critical role in tuberculosis occurrence, our findings suggests that miR-423-5p could suppress autophagosome-lysosome fusion by post-transcriptional regulation of VPS33A, which might be important for the occurrence of active tuberculosis.
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http://dx.doi.org/10.1080/22221751.2019.1590129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455132PMC
August 2019

Effects of major ozonated autoheamotherapy on functional recovery, ischemic brain tissue apoptosis and oxygen free radical damage in the rat model of cerebral ischemia.

J Cell Biochem 2019 04 2;120(4):6772-6780. Epub 2018 Nov 2.

Department of Orthopaedics, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, P.R., China.

Stroke is the second leading cause of death and disability in the world, with a heavy burden on patients, their families, and society. At present, a major focus of cerebrovascular disease research is to find a safe and effective new method to promote early functional recovery in the acute phase of cerebral infarction. Major ozonated autohemotherapy (MOAH) can maintain ATP and energy metabolism in cerebral ischemia and hypoxia, and reduce cell apoptosis. In the current study, the model of middle cerebral artery occlusion in the Sprague Dawley rat was established and evaluated by the clinical functional score, Hoechst staining, immunohistochemistry, Western blot analysis, and biochemical detection. Then, the effects of MOAH on neurological function, apoptosis, and oxygen free radical damage after acute ischemia in middle cerebral artery were evaluated. Moreover, the potential two mechanisms have been illustrated for MOAH effects. This study would lay a theoretical foundation for the application of MOAH and find an effective and early treatment method for the cerebral infarction.
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http://dx.doi.org/10.1002/jcb.27978DOI Listing
April 2019

miR-92 Suppresses Robo1 Translation to Modulate Slit Sensitivity in Commissural Axon Guidance.

Cell Rep 2018 09;24(10):2694-2708.e6

Department of Biological Sciences, University of Toledo, M.S. 601, 2801 W. Bancroft St., Toledo, OH 43606, USA. Electronic address:

Temporospatial regulation of guidance signaling is essential for axon outgrowth and pathfinding in the developing nervous system. Regulation of Robo1 levels in commissural neurons modulates Slit sensitivity facilitating proper axon guidance. The mechanisms underlying this regulation in the vertebrate nervous system are not well understood. Here, we report that miR-92, a highly conserved microRNA (miRNA), regulates chicken Robo1 expression in commissural neurons by binding to the 3' untranslated region (3' UTR) of Robo1 mRNA. miR-92 and Robo1 are differentially expressed in the developing spinal cord. miR-92 interacts with the Robo1 3'UTR to cause translational repression, but not mRNA degradation. Disruption of the miR-92/Robo1 3' UTR interaction induces premature responsiveness to Slit2 repulsion of precrossing commissural axons (CAs) in vitro and causes CA projection defects in vivo. These results indicate that miR-92 represses Robo1 expression thereby regulating Slit sensitivity to control CA projection and midline crossing.
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http://dx.doi.org/10.1016/j.celrep.2018.08.021DOI Listing
September 2018

Human TUBB3 Mutations Disrupt Netrin Attractive Signaling.

Neuroscience 2018 03 31;374:155-171. Epub 2018 Jan 31.

Department of Biological Sciences, University of Toledo, 2801 West Bancroft St., Toledo, OH 43606, USA. Electronic address:

Heterozygous missense mutations in human TUBB3 gene result in a spectrum of brain malformations associated with defects in axon guidance, neuronal migration and differentiation. However, the molecular mechanisms underlying mutation-related axon guidance abnormalities are unclear. Recent studies have shown that netrin-1, a canonical guidance cue, induced the interaction of TUBB3 with the netrin receptor deleted in colorectal cancer (DCC). Furthermore, TUBB3 is required for netrin-1-induced axon outgrowth, branching and pathfinding. Here, we provide evidence that TUBB3 mutations impair netrin/DCC signaling in the developing nervous system. The interaction of DCC with most TUBB3 mutants (eight out of twelve) is significantly reduced compared to the wild-type TUBB3. TUBB3 mutants R262C and A302V exhibit decreased subcellular colocalization with DCC in the growth cones of primary neurons. Netrin-1 increases the interaction of endogenous DCC with wild-type human TUBB3, but not R262C or A302V, in primary neurons. Netrin-1 also increases co-sedimentation of DCC with polymerized microtubules (MTs) in primary neurons expressing the wild-type TUBB3, but not R262C or A302V. Expression of either R262C or A302V not only suppresses netrin-1-induced neurite outgrowth, branching and attraction in vitro, but also causes defects in spinal cord commissural axon (CA) projection and pathfinding in ovo. Our study reveals that missense TUBB3 mutations specifically disrupt netrin/DCC-mediated attractive signaling.
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http://dx.doi.org/10.1016/j.neuroscience.2018.01.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841466PMC
March 2018

Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion.

J Neurosci 2017 06 8;37(23):5620-5633. Epub 2017 May 8.

Department of Biological Sciences, University of Toledo, Toledo, Ohio 43606

Modulation of microtubule (MT) dynamics is a key event of cytoskeleton remodeling in the growth cone (GC) during axon outgrowth and pathfinding. Our previous studies have shown that the direct interaction of netrin receptor DCC and DSCAM with polymerized TUBB3, a neuron-specific MT subunit in the brain, is required for netrin-1-mediated axon outgrowth, branching, and attraction. Here, we show that uncoupling of polymerized TUBB3 with netrin-1-repulsive receptor UNC5C is involved in netrin-1-mediated axonal repulsion. TUBB3 directly interacted with UNC5C and partially colocalized with UNC5C in the peripheral area of the GC of primary neurons from the cerebellar external granule layer of P2 mouse pups of both sexes. Netrin-1 reduced this interaction as well as the colocalization of UNC5C and TUBB3 in the GC. Results from the cosedimentation assay indicated that UNC5C interacted with polymerized TUBB3 in MTs and netrin-1 decreased this interaction. Knockdown of either TUBB3 or UNC5C blocked netrin-1-promoted axon repulsion and caused defects in axon projection of DRG toward the spinal cord Furthermore, live-cell imaging of end-binding protein 3 tagged with EGFP (EB3-GFP) in primary external granule layer cells showed that netrin-1 differentially increased MT dynamics in the GC with more MT growth in the distal than the proximal region of the GC during repulsion, and knockdown of either UNC5C or TUBB3 abolished the netrin-1 effect. Together, these data indicate that the disengagement of UNC5C with polymerized TUBB3 plays an essential role in netrin-1/UNC5C-mediated axon repulsion. Proper regulation of microtubule (MT) dynamics in the growth cone plays an important role in axon guidance. However, whether guidance cues modulate MT dynamics directly or indirectly is unclear. Here, we report that dissociation of UNC5C and polymerized TUBB3, the highly dynamic β-tubulin isoform in neurons, is essential for netrin-1/UNC5C-promoted axon repulsion. These results not only provide a working model of direct modulation of MTs by guidance cues in growth cone navigation but also help us to understand molecular mechanisms underlying developmental brain disorders associated with TUBB3 mutations.
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http://dx.doi.org/10.1523/JNEUROSCI.2617-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469302PMC
June 2017

ERβ up-regulation was involved in silibinin-induced growth inhibition of human breast cancer MCF-7 cells.

Arch Biochem Biophys 2016 Feb 6;591:141-9. Epub 2016 Jan 6.

China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China. Electronic address:

We previously reported that silibinin induced a loss of cell viability in breast cancer (MCF-7) cells by ERα down-regulation. But whether this cytotoxicity depends on another estrogen receptor, ERβ, has yet to be elucidated. Therefore, we sought to explore the effects of ERβ modulation on cell viability by using an ERβ-selective agonist (Diarylprepionitrile, DPN) and an antagonist (PHTPP). Our data demonstrated that ERβ served as a growth suppressor in MCF-7 cells, and the incubation of silibinin, elevated ERβ expression, resulting in the tumor growth inhibition. The cytotoxic effect of silibinin was diminished by PHTPP and enhanced by DPN. Silencing of ERβ by siRNA confirmed these results. Apoptotic cascades, including the sequential activation of caspase-9 and -6, and finally the cleavage of caspase substrates, PARP and ICAD, caused by treatment with silibinin, were all repressed by PHTPP pre-treatment but exacerbated by DPN. Unlike ERα, ERβ did not involve autophagic process in the regulation, since neither autophagic inhibitor (3-MA) nor the inducer (rapamycin) affected the cell survival rates regardless ERβ activity. Taken together, silibinin induced apoptosis through mitochondrial pathway by up-regulating ERβ pathways in MCF-7 cells without the involvement of autophagy.
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http://dx.doi.org/10.1016/j.abb.2016.01.002DOI Listing
February 2016

Therapeutic efficacy of intensified walk training under the electrocardiogram telemetry in stroke induced lower limb dysfunction patients with heart failure.

Int J Clin Exp Med 2015 15;8(9):16599-605. Epub 2015 Sep 15.

The Second Department of Neurological Rehabilitation, Guangzhou General Hospital of Guangzhou Military Command Guangzhou 510010, Guangdong Province, China.

Objectives: This study aimed to explore the therapeutic efficacy of intensified walk training under the electrocardiogram (ECG) telemetry in stroke induced lower limb dysfunction patients with heart failure.

Material And Methods: A total of 40 patients with stroke induced lower limb dysfunction and heart failure were randomized into control group and walk training group (n=20 per group). Besides comprehensive rehabilitation, patients in walk training group received intensified walk training under the ECG telemetry and patients in control group received traditional training. After 5-week treatment, the FMA score of lower limbs, ADL score, 6-min walking distance and left ventricular ejection fraction (EF) by heart ultrasonography were determined.

Results: There were no marked differences in the demographics between two groups at baseline, and no severe complications were observed during training in the walk training group. In control group, 6 patients developed lung edema which required further therapy. After 5-week training, the FMA score of lower limbs, ADL score and 6-min walk distance were improved to different extents, but the improvement was more obvious in walk training group (P<0.05). The left ventricular EF remained unchanged in both groups.

Conclusions: In patients with stroke induced lower limb dysfunction and heart failure, routine rehabilitation in combination with additional walk training under the ECG telemetry is helpful to increase the training efficiency and training intensity and improve the low limb function and walk distance when the safety is assured.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659078PMC
December 2015

ERα down-regulation plays a key role in silibinin-induced autophagy and apoptosis in human breast cancer MCF-7 cells.

J Pharmacol Sci 2015 Jul 12;128(3):97-107. Epub 2015 May 12.

China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China. Electronic address:

The estrogen receptor alpha (ERα) has been proven to be one of the most important therapeutic targets in breast cancer over the last 30 years. Previous studies pointed out that a natural flavonoid, silibinin, induced apoptosis in human breast cancer MCF-7 cells. In the present study we report that exposure of MCF-7 cells to silibinin led to cell death through the down-regulation of ERα expression. Silibinin-induced apoptosis of MCF-7 cells through up-regulation of caspase 6 due to ERα signalling repression was further boosted by ERα antagonist. Moreover, up-regulation of autophagy induced by silibinin accounted for apoptotic exacerbation, being further enhanced by ERα inhibition. Upon ERα activation, series of downstream signalling pathways can be activated. We found that silibinin reduced the expressions of Akt/mTOR and extracellular-signal-related kinase (ERK), which respectively accounted for the induction of autophagy and apoptosis. These effects were further augmented by co-treatment with ERα inhibitor. We conclude that the treatment with silibinin of ERα-positive MCF-7 cells down-regulates the expression of ERα, and subsequently mTOR and ERK signaling pathways, ERα downstream, finally resulting in induction of autophagy and apoptosis.
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http://dx.doi.org/10.1016/j.jphs.2015.05.001DOI Listing
July 2015

Clinical treatment of orthostatic hypotension after spinal cord injury with training based on electric uprise bed coupled with remote ECG and BP monitor.

Med Sci Monit 2014 Dec 22;20:2767-75. Epub 2014 Dec 22.

Department of Geriatrics, Guangzhou General Hospital of Guangzhou Military Command, Guangdong, China (mainland).

Background: The treatment for orthostatic hypotension (OH) after spinal cord injury (SCI) is an important part of rehabilitation in late-stage SCI. Electric uprise bed training is a relatively commonly used method in treating OH, and how to carry out uprise bed training safely and effectively is an urgent problem. In the early stage of SCI, we used a remote monitoring system to monitor the whole process of uprise bed training, and we explored a safe and efficient method of electric uprise bed training.

Material And Methods: The experimental group consisted of 36 patients diagnosed with orthostatic hypotension (OH) after SCI and who received training with an electric uprise bed coupled with remote monitoring system, and the control group of 18 subjects who used a traditional training method.

Results: There were no differences in baseline data between the 2 groups. There were no severe symptoms during training in the experimental group, but 3 patients had severe symptoms in the control group. Among the 32 enrolled subjects reaching upright training status within 30 days (17 subjects in the experimental group and 15 subjects in the control group), time interval of training from horizontal position to erect position in the experimental group was 18.00±3.12 days and 21.40±4.95 days in the control group. Time interval in the experimental group was significantly less than in the control group. However, among all 36 subjects, by combining results of follow-up, there was no significant difference of time interval of training from horizontal position to erect position between the experimental group and the control group. In the experimental group 90.52% of patients finished training compared to 78.19% in the control group (P<0.01). After training, values of OCs and OCd of the experimental group were lower than in the control group. There was no significant difference between groups in number of re-diagnosed OH.

Conclusions: Implementation of training with electric uprise bed coupled with remote monitoring system is generally safe for patients with OH after SCI. For patients who could reach standing training status within 30 days, implementation can improve efficiency of training by shortening time interval of training from horizontal position to erect position. It can increase orthostatic blood pressure change during position change.
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http://dx.doi.org/10.12659/MSM.891137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280052PMC
December 2014

Effect of qilongtoutong granule on calcitonin gene-related peptide, beta-endorphin, serotonin, dopamine, and noradrenalin in migraine model rats and mice.

J Tradit Chin Med 2014 Apr;34(2):188-93

Objective: To study the effect of Qilongtoutong granule (QLTT) on plasma calcitonin gene-related peptide (CGRP), beta-endorphin (beta-EP), 5-HT, dopamine (DA), noradrenalin (NE), and blood viscosity in migraine model rats and mice.

Methods: Both the acute blood stasis model group and nitroglycerin-induced migraine model group included 60 Sprague-Dawley rats. The reserpine-reduced model group had 60 Kunming mice. Rats from each test were grouped into normal control group, model group, Zhengtian pill (ZTP) group, and high, moderate, or low-dose QLTT groups. In the acute blood stasis model test, after gavage for 7 days, rats were given 0.8 mL/kg adrenaline hydrochloride subcutaneously twice, and kept in ice water for 5 min. After fasting for 12 h, rats were anesthetized and blood samples were collected for detection of blood viscosity. In the nitroglycerin-induced migraine group, after gavage for 7 days, rats were intraperitoneally injected nitroglycerin (10 mg/kg), and 4 h later, blood samples were collected from postcava for measuring the plasma CGRP and beta-EP levels. In the reserpine-reduced model test, except the normal control group, mice were administered reserpine (0.25 mg/ kg, i.h.) for 9 days. Mice received intragastric administration from the third day to the ninth day. One hour after the last gavage, blood and brain tissue samples were obtained. Then, blood clotting time and the contents of neurotransmitters were determined.

Results: QLTT- (3.6, 1.8, and 0.9 g/kg) and ZTP-treated rats had lower blood viscosity than that in model rats under different shear rates (P < 0.01). QLTT- (3.6, 1.8 g/kg) and ZTP-treated rats had significantly lower plasma CGRP levels and higher plasma beta-EP levels than those in model rats (P < 0.01). QLTT treatment at dose of 0.9 g/kg had lower plasma CGRP levels as well (P < 0.05). QLTT- (5.2, 2.6 g/kg) and ZTP-treated mice had longer blood clotting time than that in model mice (P < 0.01). QLTT- (2.6 g/kg) and ZTP-treated mice had higher plasma serotonin (5-HT) levels than those in model mice (P < 0.05).

Conclusion: QLTT-treated animals had lower plasma CGRP level, higher plasma beta-EP, 5-HT, higher brain tissue 5-HT, NE, DA levels, and lower blood viscosity than those in the migraine model animals.
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http://dx.doi.org/10.1016/s0254-6272(14)60077-7DOI Listing
April 2014

Evaluation of analgesic, sedative effects and antimigraine mechanism of Qilong Toutong Granule () in rodents.

Chin J Integr Med 2014 Apr 16. Epub 2014 Apr 16.

Department of Encephalopathy, The First Hospital of Hunan University of Chinese Medicine, Hunan University of Chinese Medicine, Changsha, 410208, China.

Objective: To evaluate the analgesic and sedative effects of Qilong Toutong Granule (, QTG) and explore its possible mechanisms.

Methods: Kunming mice were randomly divided into 6 groups: normal control group, Zhengtian Pill (, ZTP) group, Western medicine group, and high-dose (5.2 g/kg), medium-dose (2.6 g/kg) and low-dose (1.3 g/kg) of QTG groups. After completing the prophylactic treatment for 3 days, hot-plate test and acetic acid-induced writhing test were used to assess the analgesic effect, and spontaneous locomotor test and sodium pentobarbital-induced hypnosis activity were adopted to estimate the sedative effect. Sprague-Dawley rats were grouped into normal control group, model group, ZTP group, rizatriptan group, and high-dose (3.6 g/kg), medium-dose (1.8 g/kg), and low-dose (0.9 g/kg) of QTG groups. After gavage for continuous 7 days, rats were intraperitoneally injected nitroglycerin, and 4 h later, blood samples were collected from postcava for measuring the levels of plasma calcitonin gene-related peptide (CGRP) and beta-endorphin (β-EP) by radioimmunoassay. Subsequently, rats were perfused transcardially and the brain tissues containing the trigeminal nucleus caudalis (TNC) were achieved for detecting the number of Fos-immunoreactive cells by immunohistochemical method.

Results: In the mice experiments, compared with the normal control group, high- and medium-dose of QTG groups significantly raised the pain threshold (P<0.01), reduced the number of writhing response (P<0.01) and spontaneous activity (P<0.01), but had no influence on the sleeping rate of mice (P>0.05), and low-dose of QTG group also raised the pain threshold at 120 min (P=0.007), as well as lowered locomotor activity of mice at 2 h (P=0.003). On the study of migraine model rats, high- and medium-dose of QTG groups remarkably down-regulated the levels of plasma CGRP (P<0.01), up-regulated the levels of plasma β-EP (P<0.01) and inhibited the expression of Fos protein in TNC (P<0.01), compared with the model group.

Conclusions: QTG has obvious analgesic and sedative action and its mechanism on relieving migraine may be through regulating the levels of neurotransmitters and/or neuropeptides, and inhibiting the activation of Fos pathway.
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http://dx.doi.org/10.1007/s11655-014-1811-9DOI Listing
April 2014

Hyperbaric oxygen therapy provides neuroprotection following spinal cord injury in a rat model.

Int J Clin Exp Pathol 2013 15;6(7):1337-42. Epub 2013 Jun 15.

Neurological Rehabilitation, Division II. Neurology Specialty Hospital, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, Guangdong Province, China.

Objective: To investigate the effect of hyperbaric oxygen therapy (HBOT) on the iNOS mRNA-iNOS-NO signaling pathway and neurofunction protected in a rat spinal cord injury model.

Methods: A total of 36 Sprague-Dawley rats were randomly divided into 3 groups: control group (n=12), SCI group (n=12) and SCI + HBOT group (n=12). SCI + HBOT group In the SCI group and SCI + HBOT groups, SCI was performed on rats. In the SCI + HBOT group, rats with SCI underwent HBO treatment 30 min after SCI for 24 sessions. After HBO therapy, measurement of motor evoked potential (MEP), Basso, Beattie, Bresnahan (BBB) scoring and pathological examination were done. RT-PCR and immunohistochemistry were employed to detect the mRNA and protein expression of iNOS, respectively. Diazo colorimetry was performed to detect the serum NO content.

Results: The mRNA and protein expression of iNOS in the spinal cord and the serum NO content were markedly increased in the SCI group as compared to the control group (P<0.05). However, the mRNA and protein expression of iNOS and the serum NO content were dramatically reduced in the SCI + HBOT group as compared to the SCI group (P<0.05).

Conclusion: HBO therapy can promote the neuroprotection following SCI, which may be related to the effect of HBO on the iNOS mRNA-iNOS-NO signaling pathway.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3693198PMC
February 2014

Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance.

J Cell Sci 2013 Jul 2;126(Pt 14):3070-81. Epub 2013 May 2.

Department of Biological Sciences, University of Toledo, M.S. 601, 2801 W. Bancroft Street, Toledo, OH 43606, USA.

The coupling of axon guidance cues, such as netrin-1, to microtubule (MT) dynamics is essential for growth cone navigation in the developing nervous system. However, whether axon guidance signaling regulates MT dynamics directly or indirectly is unclear. Here, we report that TUBB3, the most dynamic β-tubulin isoform in neurons, directly interacts with the netrin receptor DCC, and that netrin-1 induces this interaction in primary neurons. TUBB3 colocalizes with DCC in the growth cones of primary neurons and MT dynamics is required for netrin-1-promoted association of TUBB3 with DCC. Netrin-1 not only increases co-sedimentation of DCC with polymerized MT, but also promotes MT dynamics in the growth cone. Knocking down TUBB3 inhibits netrin-1-induced MT dynamics, axon outgrowth and attraction in vitro and causes defects in commissural axon projection in the embryo. These results indicate that TUBB3 directly links netrin signaling pathways to MT dynamics and plays an important role in guiding commissural axons in vivo.
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http://dx.doi.org/10.1242/jcs.122184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711200PMC
July 2013

c-Jun N-terminal kinase 1 (JNK1) is required for coordination of netrin signaling in axon guidance.

J Biol Chem 2013 Jan 6;288(3):1883-95. Epub 2012 Dec 6.

Department of Biological Sciences, University of Toledo, Toledo, Ohio 43606, USA.

The JNK family of MAPKs is involved in a large variety of physiological and pathological processes in brain development, such as neural survival, migration, and polarity as well as axon regeneration. However, whether JNK activation is involved in axon guidance remains unknown. Here, we provide evidence indicating the JNK pathway is required for Netrin signaling in the developing nervous system. Netrin-1 increased JNK1, not JNK2 or JNK3, activity in the presence of deleted in colorectal cancer (DCC) or Down syndrome cell adhesion molecule (DSCAM), and expression of both of them further enhanced Netrin-1-induced JNK1 activity in vitro. Inhibition of JNK signaling either by a JNK inhibitor, SP600125, or expression of a dominant negative form of MKK4, a JNK upstream activator, blocked Netrin-1-induced JNK1 activation in HEK293 cells. Netrin-1 increased endogenous JNK activity in primary neurons. Netrin-1-induced JNK activation was inhibited either by the JNK inhibitor or an anti-DCC function-blocking antibody. Combination of the anti-DCC function-blocking antibody with expression of DSCAM shRNA in primary neurons totally abolished Netrin-1-induced JNK activation, whereas knockdown of DSCAM partially inhibited the Netrin-1 effect. In the developing spinal cord, phospho-JNK was strongly expressed in commissural axons before and as they crossed the floor plate, and Netrin-1 stimulation dramatically increased the level of endogenous phospho-JNK in commissural axon growth cones. Inhibition of JNK signaling either by JNK1 RNA interference (RNAi) or the JNK inhibitor suppressed Netrin-1-induced neurite outgrowth and axon attraction. Knockdown of JNK1 in ovo caused defects in spinal cord commissural axon projection and pathfinding. Our study reveals that JNK1 is important in the coordination of DCC and DSCAM in Netrin-mediated attractive signaling.
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http://dx.doi.org/10.1074/jbc.M112.417881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548497PMC
January 2013