Publications by authors named "Shuan Zhao"

20 Publications

  • Page 1 of 1

Novel predictive biomarkers for acute injury superimposed on chronic kidney disease.

Nefrologia 2021 Mar-Apr;41(2):165-173. Epub 2020 Nov 4.

Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China; Shanghai Medical Center of Kidney, Shanghai, China; Shanghai Key Laboratory of Kidney and Blood Purification, Shanghai, China. Electronic address:

Introduction And Objectives: Chronic kidney disease (CKD) is a risk factor for the development of acute kidney injury (AKI). Recent studies have revealed numerous biomarkers eligible for AKI prediction. However, the expression and performance of AKI biomarkers in acute injury superimposed on preexisting CKD (AonC) remain elusive. The aim of this study was to evaluate whether biomarkers which robustly expressed in acute kidney injury could predict acute injury based on CKD.

Materials And Methods: Mice were classified into cohorts: AKI, CKD, AonC and sham. The AonC model mice were subjected to renal bilateral ischemia/reperfusion (I/R) injury fourteen days after intraperitoneally administrated with 20mg/kg aristolochic acid. Severity of acute ischemic injury was stratified by clamping the dissected bilateral renal arteries with non-traumatic microvascular clips for 20 or 35min. The AKI mice were induced with renal bilateral I/R injury and CKD mice were crafted with 20mg/kg aristolochic acid administrated intraperitoneally. Histology, genetic and protein expression of biomarkers were measured in three cohorts.

Results: We found that serum creatinine dramatically increased in severe (sAonC) but not in moderate (mAonC) injury mice. Upregulation of Kidney injury molecule-1 (KIM-1) mRNA, tissue inhibitor of metalloproteinase-2 (TIMP-2), Syndecan-1 (SDC-1) mRNA and insulin-like growth factor binding protein-7 (IGFBP7) protein indicated the onset of mAonC. An increase in neutrophil gelatinase-associated lipocalin (NGAL), rhomboid-like protein 2 (RHBDL2), Syndecan-1 (SDC-1) mRNA and protein, and a decrease in IGFBP7 protein were associated with sAonC.

Conclusions: Our study revealed the variational expression of AKI biomarkers in AonC kidneys, and uncovered IGFBP7 protein can be used as a sensitive biomarker to predict and differentiate AonC severity. The performance of RHBDL2 and SDC-1 in predicting severe AonC was promising, providing new biomarkers for predicting AonC.
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http://dx.doi.org/10.1016/j.nefro.2020.06.007DOI Listing
November 2020

Upregulation of miR-382 contributes to renal fibrosis secondary to aristolochic acid-induced kidney injury via PTEN signaling pathway.

Cell Death Dis 2020 08 14;11(8):620. Epub 2020 Aug 14.

Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China.

Acute kidney injury (AKI) has a critical role in the development of chronic kidney disease (CKD). Building on our previous findings, we explored the role of miR-382 in facilitating the transition of AKI to CKD using the Aristolochic acid (AA) nephropathy model, which was induced by intraperitoneal injection of aristolochic acid I salt (10 or 20 mg/kg). The effects of genetic depletion, pharmacologic inhibition, or overexpression of miR-382 on the PTEN/AKT signaling pathway were examined in vivo and in vitro. Changes in renal pathology and renal epithelial polarity were evaluated. A luciferase reporter assay was performed to investigate the reciprocal suppression relationship between miR-382 and PTEN. Renal fibrosis developed 14 d after AA exposure in a dose- and time-dependent manner. Renal abundance of miR-382 was upregulated following AA treatment, while genetic depletion or pharmacological inhibition of miR-382 partially reversed renal tubulointerstitial fibrosis. Expression of PTEN, a target of miR-382, was downregulated and subsequently its downstream AKT signaling pathway was activated during AKI to CKD transition induced by AA. Inhibition of PTEN in vitro resulted in the acquisition of the EMT phenotypes. Furthermore, upregulation of miR-382 in renal epithelial cells was partially mediated by the activation of NF-kB signaling, with a substantial elevation of proinflammatory cytokines. An in vivo study revealed that either miR-382 knockdown or miR-382 knockout was pivotal for inflammatory suppression, while an in vitro experiment confirmed that upregulation of miR-382 in cultured MTEC cells under AA exposure was remarkably reversed by NF-kB siRNA. These data indicated a novel role for the NF-κB/miR-382/PTEN/AKT axis in the pathogenesis of tubulointerstitial fibrosis following AA-induced acute renal tubular epithelial injury. Targeting miR-382 may lead to a potential novel therapeutic approach for retarding the AKT to CKD transition.
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http://dx.doi.org/10.1038/s41419-020-02876-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429500PMC
August 2020

RacGAP1 ameliorates acute kidney injury by promoting proliferation and suppressing apoptosis of renal tubular cells.

Biochem Biophys Res Commun 2020 06 15;527(3):624-630. Epub 2020 May 15.

Division of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China; Shanghai Medical Center of Kidney Disease, Shanghai, China; Shanghai Institute of Kidney and Dialysis, Shanghai, China; Shanghai Key Laboratory of Kidney and Blood Purification, Shanghai, China; Hemodialysis Quality Control Center of Shanghai, Shanghai, China. Electronic address:

Background: Acute kidney injury (AKI) remains correlated with high mortality. Novel therapeutic strategies are urgently needed for AKI patients. Rac GTPase-activating protein 1 (RacGAP1) regulates the activity of RhoGTPase and acts as a predictive biomarker in several types of malignant tumor but the role of RacGAP1 in AKI has not been revealed.

Methods: Animal models of AKI induced by renal ischemia-reperfusion (I/R) and cisplatin treatment were generated in C57BL/6 mice. Hypoxia/reoxygenation (H/R) and cisplatin treatment were practiced in human renal tubular epithelial (HK-2) and renal tubular duct epithelial cells of rat (NRK-52E) cells. The role of RacGAP1 in cell proliferation and apoptosis was estimated using western bolting, immunocytochemistry and flow cytometry. Verteporfin was used to activate the Hippo pathway to show whether the protective effects of RacGAP1 on cell growth and survival in renal tubular cells were dependent on the activation of YAP.

Results: The expression of RacGAP1 was significantly increased in mice kidneys after I/R or cisplatin treatment, combined with increased expression of RacGAP1 in H/R or cisplatin challenged cells. Overexpression of RacGAP1 protected HK2 and NRK-52E cells by promoting proliferation and decreasing apoptosis. We also disclosed that RacGAP1 exerted its function through activation of YAP.

Conclusion: The present study provides evidence that RacGAP1 is involved in AKI. It promotes proliferation and limits apoptosis of tubular epithelial cells via stimulating activation and nuclear translocation of YAP. Consequently, RacGAP1 may be a novel therapeutic target for AKI.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.140DOI Listing
June 2020

Erythrocyte transfusion limits the role of elevated red cell distribution width on predicting cardiac surgery associated acute kidney injury.

Cardiol J 2021 18;28(2):255-261. Epub 2020 May 18.

Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China, No 180 Fenglin Road, Shanghai 200032, China, 200032 Shanghai, China.

Background: Acute kidney injury (AKI) is one of the more serious complications after cardiac surgery. Elevated red cell distribution width (RDW) was reported as a predictor for cardiac surgery associated acute kidney injury (CSAKI). However, the increment of RDW by erythrocyte transfusion makes its prognostic role doubtful. The aim of this study is to elucidate the impact of erythrocyte transfusion on the prognostic role of elevated RDW for predicting CSAKI.

Methods: A total of 3207 eligible patients who underwent cardiac surgery during 2016-2017 were enrolled. Changes of RDW was defined as the difference between preoperative RDW and RDW measured 24 h after cardiac surgery. The primary outcome was CSAKI which was defined by the Kidney Disease: Improving Global Outcomes Definition and Staging (KDIGO) criteria. Univariate and multivariate analysis were performed to identify predictors for CSAKI.

Results: The incidence of CSAKI was 38.07% and the mortality was 1.18%. CSAKI patients had higher elevated RDW than those without CSAKI (0.65% vs. 0.39%, p < 0.001). Multivariate regression showed that male, age, New York Heat Association classification 3-4, elevated RDW, estimated glomerular filtration rate < 60 mL/min/1.73 m2, cardiopulmonary bypass time > 120 min and erythrocyte transfusion were associated with CSAKI. Subgroup analysis showed elevated RDW was an independent predictor for CSAKI in the non-transfused subset (adjusted odds ratio: 1.616, p < 0.001) whereas no significant association between elevated RDW and CSAKI was found in the transfused patients (odds ratio: 1.040, p = 0.497).

Conclusions: Elevated RDW is one of the independent predictors of CSAKI in the absence of erythrocyte transfusion, which limits the prognostic role of the former on predicting CSAKI.
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http://dx.doi.org/10.5603/CJ.a2020.0070DOI Listing
May 2020

LncRNA GAS5 promotes apoptosis as a competing endogenous RNA for miR-21 via thrombospondin 1 in ischemic AKI.

Cell Death Discov 2020 2;6:19. Epub 2020 Apr 2.

1Department of Nephrology, Zhongshan Hospital, Fudan University; Shanghai Institute of Kidney and Dialysis; Shanghai Key Laboratory of Kidney and Blood Purification; Shanghai Medical Center of Kidney Disease, Shanghai, China.

Mounting evidence has indicated that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) played important roles in renal ischemia/reperfusion (I/R) injury. However, the involvement of lncRNA growth arrest specific 5 (GAS5) in acute kidney injury (AKI) remained largely unexplored. This study aimed to determine possible mechanisms of GAS5 in the renal I/R process. We found that GAS5, noticeably upregulated by renal I/R injury, was further suppressed by delayed IPC while knockdown of miR-21 in vivo before IPC could significantly increased the GAS5 levels. Concurrently, TSP-1 was negatively regulated by miR-21 in vivo and vitro. Additionally, Reciprocal repression of GAS5 and miR-21 was identified. Knockdown of miR-21 in H6R0.5 treated HK-2 cells promoted apoptosis. Co-transfection of miR-21 mimic and pcDNA-GAS5 or pcDNA-Vector were performed, results of which showed that inhibition of miR-21 on TSP-1 could be rescued by overexpression of GAS5. This study suggested that GAS5 facilitated apoptosis by competitively sponging miR-21, which negatively regulated TSP-1 in renal I/R injury. This novel regulatory axis could act as a therapeutic target for AKI in the future.
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http://dx.doi.org/10.1038/s41420-020-0253-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118150PMC
April 2020

Transforming growth factor-β1-overexpressing mesenchymal stromal cells induced local tolerance in rat renal ischemia/reperfusion injury.

Cytotherapy 2019 05 23;21(5):535-545. Epub 2019 Jan 23.

Department of Nephrology, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China; Shanghai Key Laboratory of Kidney and Blood Purification, Shanghai, China; Shanghai Medical Center for Kidney, Shanghai, China; Shanghai Institute of Kidney and Dialysis, Shanghai, China. Electronic address:

Background: Regulatory T cells (Tregs) suppress excessive immune responses and play a crucial protective role in acute kidney injury (AKI). The aim of this study was to examine the therapeutic potential of transforming growth factor (TGF)-β1-overexpressing mesenchymal stromal cells (MSCs) in inducing local generation of Tregs in the kidney after ischemia/reperfusion (I/R) injury.

Methods: MSCs were transduced with a lentiviral vector expressing the TGF-β1 gene; TGF-β1-overexpressing MSCs (designated TGF-β1/MSCs) were then transfused into the I/R-injured kidney via the renal artery.

Results: MSCs genetically modified with TGF-β1 achieved overexpression of TGF-β1. Compared with green fluorescent protein (GFP)/MSCs, TGF-β1/MSCs markedly improved renal function after I/R injury and reduced epithelial apoptosis and subsequent inflammation. The enhanced immunosuppressive and therapeutic abilities of TGF-β1/MSCs were associated with increased generation of induced Tregs and improved intrarenal migration of the injected cells. Futhermore, the mechanism of TGF-β1/MSCs in attenuating renal I/R injury was not through a direct canonical TGF-β1/Smad pathway.

Conclusion: TGF-β1/MSCs can induce a local immunosuppressive effect in the I/R-injured kidney. The immunomodulatory activity of TGF-β1-modified MSCs appears to be a gateway to new therapeutic approaches to prevent renal I/R injury.
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http://dx.doi.org/10.1016/j.jcyt.2018.12.003DOI Listing
May 2019

Remote Ischemic Preconditioning Ameliorates Acute Kidney Injury due to Contrast Exposure in Rats through Augmented O-GlcNAcylation.

Oxid Med Cell Longev 2018 13;2018:4895913. Epub 2018 Aug 13.

Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

Remote ischemic preconditioning (RIPC) is an adaptive response, manifesting when local short-term ischemic preconditioning reduces damage to adjacent or distant tissues or organs. O-linked -N-acetylglucosamine (O-GlcNAc) glycosylation of intracellular proteins denotes a type of posttranslational modification that influences multiple cytoplasmic and nuclear protein functions. Growing evidence indicates that stress can induce an acute increase in O-GlcNAc levels, which can be cytoprotective. The current study aimed to determine whether RIPC can provide renoprotection against contrast-induced acute kidney injury (CI-AKI) by augmenting O-GlcNAc signaling. We established a stable model of CI-AKI using 5/6 nephrectomized rats exposed to dehydration followed by iohexol injection via the tail vein. We found that RIPC increased UDP-GlcNAc levels through the hexosamine biosynthetic pathway as well as global renal O-GlcNAcylation. RIPC-induced elevation of O-GlcNAc signaling ameliorated CI-AKI based on the presence of less tubular damage and apoptosis and the amount of reactive oxygen species. In addition, the use of alloxan, an O-GlcNAc transferase inhibitor, and azaserine, a glutamine fructose-6-phosphate amidotransferase inhibitor, neutralized the protective effect of RIPC against oxidative stress and tubular apoptosis. In conclusion, RIPC attenuates local oxidative stress and tubular apoptosis induced by contrast exposure by enhancing O-GlcNAc glycosylation levels; this can be a potentially useful approach for lowering the risk of CI-AKI.
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http://dx.doi.org/10.1155/2018/4895913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112094PMC
January 2019

Effect of long non-coding RNA growth arrest-specific 5 on apoptosis in renal ischaemia/reperfusion injury.

Nephrology (Carlton) 2019 Apr;24(4):405-413

Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China.

Aim: Long non-coding RNA (lncRNAs) have been shown to play a critical role in a variety of pathophysiological processes, such as cell proliferation, apoptosis and migration. However, there were few studies addressing the function of lncRNAs in renal ischaemia/reperfusion (I/R) injury. Apoptosis is an important pathogenesis during I/R injury. Here, we identified the effect of hypoxia-responsive lncRNA growth arrest-specific 5 (GAS5) on apoptosis in renal I/R injury.

Methods: Ischaemia/reperfusion injury in mice or hypoxia/re-oxygenation (H/R) in human proximal renal tubular epithelial cells (HK-2) was practiced to induce apoptosis. The kidneys and blood were collected at 24 h after reperfusion. The GAS5 messenger RNA (mRNA) expression and apoptosis-related gene mRNA and protein levels, including p53, cellular inhibitor of apoptosis protein 2 (cIAP2) and thrombospondin-1 (TSP-1), were analysed. GAS5 small-interfering RNA was transfected with H/R induced cells. Over-expression of GAS5 was performed by plasmid transfection.

Results: Apoptotic cells significantly increased in I/R-injured kidneys. GAS5 could be up-regulated in kidneys at 24 h after reperfusion and 3 h after re-oxygenation, combined with increased expression of its downstream apoptosis-related proteins p53 and cIAP2. GAS5 small-interfering RNA treatment down-regulated the mRNA and protein levels of p53 and TSP-1, and attenuated apoptosis induced by H/R in HK-2 cells. Conversely, over-expression of GAS5 up-regulated the mRNA and protein levels of p53 and TSP-1, and promoted apoptosis in HK-2 cells.

Conclusion: Long non-coding RNA GAS5 induced by I/R injury could promote apoptosis in kidney. TSP-1 might be one of the downstream effectors of GAS5, which will be explored in the future.
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http://dx.doi.org/10.1111/nep.13476DOI Listing
April 2019

Evaluation of three hormonal protocols for anovulatory lactating cows under regulations restricting the use of estrogenic compounds.

Anim Sci J 2018 Apr 9;89(4):640-647. Epub 2018 Jan 9.

College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

When European Union regulations restricted the use of estrogenic compounds in food-producing animals, refined hormonal protocols were no longer applicable for anovulatory cows. However, Ovsynch and its adaptations are routinely and uniformly applied to all cows regardless of ovarian function. To evaluate their efficacy on anovulatory cows, 143, 147 and 144 anovulatory cows received Ovsynch, Presynch and G6G protocols, respectively. In comparison, 150 cyclic cows were bred without using a synchronized protocol. Results showed that cows in the Presynch group had luteolysis responding to the last prostaglandin F (PGF ) injection greater than the Ovsynch group. The serous progesterone levels at the first gonadotropin-releasing hormone of Ovsych and the last PGF injection was greater in the G6G group than the other two hormonal treatment groups. Concentrations of Ca and total protein in cervical mucus in all three hormone-treated groups before artificial insemination (AI) were significantly different from the controls. The G6G group obtained a greater pregnancy rate compared with Ovsynch and Presynch, but significantly less than the controls. For open cows in the Ovsynch group, estrus rate within 24 days after the first AI was significantly less than the controls. In conclusion, the G6G treatment resulted to better reproductive performance in anovulatory cows.
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http://dx.doi.org/10.1111/asj.12953DOI Listing
April 2018

Acetylation promotes TyrRS nuclear translocation to prevent oxidative damage.

Proc Natl Acad Sci U S A 2017 01 9;114(4):687-692. Epub 2017 Jan 9.

State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai 200438, People's Republic of China

Tyrosyl-tRNA synthetase (TyrRS) is well known for its essential aminoacylation function in protein synthesis. Recently, TyrRS has been shown to translocate to the nucleus and protect against DNA damage due to oxidative stress. However, the mechanism of TyrRS nuclear localization has not yet been determined. Herein, we report that TyrRS becomes highly acetylated in response to oxidative stress, which promotes nuclear translocation. Moreover, p300/CBP-associated factor (PCAF), an acetyltransferase, and sirtuin 1 (SIRT1), a NAD-dependent deacetylase, regulate the nuclear localization of TyrRS in an acetylation-dependent manner. Oxidative stress increases the level of PCAF and decreases the level of SIRT1 and deacetylase activity, all of which promote the nuclear translocation of hyperacetylated TyrRS. Furthermore, TyrRS is primarily acetylated on the K244 residue near the nuclear localization signal (NLS), and acetylation inhibits the aminoacylation activity of TyrRS. Molecular dynamics simulations have shown that the in silico acetylation of K244 induces conformational changes in TyrRS near the NLS, which may promote the nuclear translocation of acetylated TyrRS. Herein, we show that the acetylated K244 residue of TyrRS protects against DNA damage in mammalian cells and zebrafish by activating DNA repair genes downstream of transcription factor E2F1. Our study reveals a previously unknown mechanism by which acetylation regulates an aminoacyl-tRNA synthetase, thus affecting the repair pathways for damaged DNA.
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http://dx.doi.org/10.1073/pnas.1608488114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278485PMC
January 2017

Roles of interferon-stimulated gene 15 protein in bovine embryo development.

Reprod Fertil Dev 2017 Jun;29(6):1209-1216

College of Animal Science and Technology, Yangzhou University, 225009, Jiangsu, China.

Interferon (IFN)-stimulated gene 15 (ISG15) is one of several proteins induced by conceptus-derived Type I or II IFNs in the uterus, and is implicated as an important factor in determining uterine receptivity to embryos in ruminants. But little is known about the role the ISG15 gene or gene product plays during embryo development. In the present study, both the expression profile and function of ISG15 were investigated in early bovine embryos in vitro. ISG15 mRNA was detectable in Day 0, 2, 6 and 8 bovine embryos, but IFN-τ (IFNT) mRNA only appeared from Day 6. This means that embryonic expression of ISG15 on Days 0 and 2 was not induced by embryonic IFNT. However, ISG15 mRNA expression paralleled the expression of IFNT mRNA in Day 6 and 8 embryos. ISG15-lentivirus interference plasmid (ISG15i) was injected into 2-cell embryos to knockdown ISG15 expression. This resulted in decreases in the proportion of hatching blastocysts, the diameter of blastocysts and cell number per diameter of blastocysts compared with control embryos. In addition, ISG15i inhibited IFNT, Ets2 (E26 oncogene homolog 2) mRNA and connexion 43 protein expression in Day 8 blastocysts, whereas exogenous IFNT treatment (100ngmL, from Day 4 to Day 8) improved ISG15 mRNA and connexion 43 protein expression. In conclusion, it appears that ISG15 is involved in early bovine embryo development and that it regulates IFNT expression in the blastocyst.
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http://dx.doi.org/10.1071/RD15209DOI Listing
June 2017

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

Theriogenology 2015 Sep 28;84(5):811-7. Epub 2015 May 28.

Laboratory of Animal Reproductive Science, College of Animal Science and Technology, Northeast Agricultural University, Harbin, China. Electronic address:

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.
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http://dx.doi.org/10.1016/j.theriogenology.2015.05.016DOI Listing
September 2015

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

Theriogenology 2015 Jul 18;84(2):184-92. Epub 2015 Mar 18.

National Key Laboratory of Animal Nutrition, Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing, China; National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China; Key Laboratory of Animal Genetics, Breeding, and Reproduction, Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China. Electronic address:

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation.
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http://dx.doi.org/10.1016/j.theriogenology.2015.03.011DOI Listing
July 2015

Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca(2+) influx.

Theriogenology 2015 Jun 7;83(9):1493-501. Epub 2015 Feb 7.

Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture, National Engineering laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China; Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing, China. Electronic address:

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca(2+)) concentration ([Ca(2+)]i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca(2+)]ivia blocking the influx of extracellular Ca(2+) or chelating ooplasmic free Ca(2+). After IVM, cumulus-oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca(2+)]i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus-oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca(2+)]i was significantly decreased. When oocytes were cultured in Ca(2+)-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca(2+)]i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca(2+)]i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca(2+) or reducing ooplasmic free Ca(2+). 1-Octanol, BAPTA-AM, or Ca(2+)-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.
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http://dx.doi.org/10.1016/j.theriogenology.2015.01.034DOI Listing
June 2015

Imidazole inhibits autophagy flux by blocking autophagic degradation and triggers apoptosis via increasing FoxO3a-Bim expression.

Int J Oncol 2015 Feb 21;46(2):721-31. Epub 2014 Nov 21.

Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R. China.

Imidazole, an organic alkaloid, is an important pharmacophore in drug discovery. Anti-neoplastic potential of imidazole derivatives has been documented in several studies; however, mechanisms by which tumor cells respond to these stimuli remain to be elucidated. Autophagy and apoptosis have key roles in tumorigenesis and tumor treatment. In this study, we systematically examined autophagic events induced by imidazole in HEC-1B cells. Accumulation of autophagic vacuoles in imidazole-treated cells was verified by conversion of LC3 protein, as well as confocal and transmission electron microscopy. Furthermore, imidazole blocked autophagic degradation by impairing maturation of autophagosomes into autolysosomes. Concurrently, imidazole treatment induced apoptosis in HEC-1B cells, accompanied by activation of caspase 9 and 3. The proapoptotic effect was mediated by increased Bim expression. Moreover, imidazole upregulated the protein level of Foxo3a and induced its increased nuclear localisation. In addition, siRNA-mediated silencing of FoxO3a effectively attenuated imidazole-induced Bim upregulation and cell death, indicating direct involvement of this pathway in the imidazole-induced apoptosis. Taken together, our data provided a molecular link between imidazoles and anticancer therapies; understanding of these properties of imidazole is essential for development of effective cancer therapeutics using imidazoles.
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http://dx.doi.org/10.3892/ijo.2014.2771DOI Listing
February 2015

Recombinant bovine interferon-τ enhances in vitro development of bovine embryos by upregulating expression of connexin 43 and E-cadherin.

J Dairy Sci 2014 Nov 18;97(11):6917-25. Epub 2014 Sep 18.

National Key Laboratory of Animal Nutrition, Laboratory of Animal Embryonic Biotechnology; National Engineering Laboratory for Animal Breeding; Key Laboratory of Animal Genetics, Breeding, and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China. Electronic address:

Interferon-τ (IFNT), produced in ruminants by embryonic trophoblastic cells before implantation, is involved in the maternal recognition of pregnancy. It is a pleiotropic molecule that alters the synthesis of endometrial proteins and inhibits the proliferation of some cells. The present study investigated the effects of recombinant bovine IFNT on the development of early-stage bovine embryos and the molecular mechanism underlying this effect. This study demonstrated that expression of mRNA encoding type I IFN receptor subunits was detectable from d 4 to 8 in in vitro fertilized (IVF) bovine embryos. A considerable number of IVF (n = 1,941) and parthenogenetic activated (n = 1,552) bovine embryos demonstrated that supplementing the culture medium with IFNT (100 ng/mL) produced a greater percentage of blastocysts, and the total cell number within the resulting blastocysts was higher. In addition, IFNT upregulated the expression levels of both mRNA and protein for connexin 43 (GJA1) and E-cadherin (CDH1) and expression levels for granulocyte-macrophage colony-stimulating factor and insulin-like growth factor 2 mRNA but not for their proteins in d 8 embryos. However, IFNT inhibited mRNA expression for leukemia inhibitory factor (LIF), LIF receptor α, and the sodium/potassium-transporting ATPase subunit β-1. We concluded that IFNT promoted the development of bovine embryos by upregulating the expression of GJA1 and CDH1. Thus, supplementing embryo cultures or transfer medium with IFNT may stimulate embryo development and improve embryo transfer efficiency.
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http://dx.doi.org/10.3168/jds.2014-8106DOI Listing
November 2014

A novel role of transient receptor potential mucolipin1 (TRPML1) in protecting against imidazole-induced cytotoxicity.

Biochem Cell Biol 2014 Aug 28;92(4):279-86. Epub 2014 May 28.

a Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R. China.

Lysosomotropic amines cause serious side effects such as cytoplasmic vacuolation and cell death. TRPML1 (also known as mucolipin1), a member of the transient receptor potential (TRP) protein family, may regulate fusion/fission of vesicles along the endocytic pathway and some aspects of lysosomal ion homeostasis. Nevertheless, it is still unknown whether TRPML1 is involved in death of mammalian cells induced by lysosomotropic agents. In this study, imidazole was used as a model to investigate the role of TRPML1 in the cytotoxicity of lysosomotropic agents. Overexpression of wild-type TRPML1 inhibited imidazole-induced vacuole formation and cell death in human endometrial adenocarcinoma (HEC-1B) cells. In contrast, siRNA-mediated TRPML1 knockdown increased the cell death induced by imidazole. Bafilomycin A1 raises the pH of acidic organelles and therefore suppresses accumulation of weak bases in them. Similarly, lysosomal pH was raised in TRPML1-overexpressing cells; therefore, we inferred that TRPML1 protected against imidazole toxicity by regulating the pH of acidic organelles. We concluded that TRPML1 had a novel role in protecting against lysosomotropic amine toxicity.
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http://dx.doi.org/10.1139/bcb-2014-0044DOI Listing
August 2014

The disturbances of endoplasmic reticulum calcium homeostasis caused by increased intracellular reactive oxygen species contributes to fragmentation in aged porcine oocytes.

Biol Reprod 2013 Nov 27;89(5):124. Epub 2013 Nov 27.

National Key Laboratory of Animal Nutrition, Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, National Engineering Laboratory for Animal Breeding, China Agricultural University, Beijing, China.

Accumulating evidence indicates that cellular and molecular abnormalities occur during oocyte aging, including fragmentation, increases in intracellular reactive oxygen species (ROS), and abnormal Ca(2+) oscillations. The objective of the present study was to characterize the relationships between intracellular ROS, Ca(2+) homeostasis of endoplasmic reticulum (ER), and fragmentation in aged porcine MII oocytes. Prolonged culture (36 h) of porcine oocytes resulted in elevated intracellular ROS level, impaired ER Ca(2+) homeostasis (i.e., Ca(2+) storage, Ca(2+) rising patterns after electroactivation, and the cluster distribution of ER), and increased fragmentation rates. However, when the porcine oocytes were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester), an intracellular Ca(2+) chelator, the fragmentation was significantly inhibited during in vitro aging. In order to pursue the underlying mechanisms, H2O2 and cycloheximide (CHX) were used to artificially increase or inhibit, respectively, the intracellular ROS levels in aged porcine oocytes during in vitro culture. The results demonstrated that incubation of porcine MII oocytes with H2O2 damaged the ER clusters and the Ca(2+) regulation of ER, leading to a high proportion of fragmented oocytes. In contrast, CHX, an intracellular inhibitor of ROS generation, prevented both increase of ROS level and damage of the ER Ca(2+) homeostasis in porcine oocytes during aging, resulting in low fragmentation rate. We conclude that the increased intracellular ROS damaged the ER clusters and ER Ca(2+) homeostasis, resulting in a disorder in ooplasmic free Ca(2+), which caused the fragmentations seen in porcine MII oocytes during aging.
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http://dx.doi.org/10.1095/biolreprod.113.111302DOI Listing
November 2013

Oocyte-secreted growth differentiation factor 9 inhibits BCL-2-interacting mediator of cell death-extra long expression in porcine cumulus cell.

Biol Reprod 2013 Sep 19;89(3):56. Epub 2013 Sep 19.

Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing, China.

Oocyte-secreted factors (OSFs) maintain the low incidence of cumulus cell apoptosis. In this report, we described that the presence of oocytes suppressed the expression of proapoptotic protein BCL-2-interacting mediator of cell death-extra long (BIMEL) in porcine cumulus cells. Atretic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cumulus cells strongly expressed BIMEL protein. The healthy cumulus- oocyte complex exhibited a low BIMEL expression in cumulus cell while the removal of oocyte led to an about 2.5-fold (P < 0.5) increased expression in oocytectomized complex (OOX). Coculturing OOXs with denuded oocytes decreased BIMEL expression to the normal level. The similar expression pattern could also be achieved in OOXs treated with exogenous recombinant mouse growth differentiation factor 9 (GDF9), a well-characterized OSF. This inhibitory action of GDF9 was prevented by the addition of a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Luciferase assay further demonstrated that BIM gene expression was forkhead box O3a (FOXO3a)-dependent because mutation of FOXO3a-binding site on the BIM promoter inhibited luciferase activities. Moreover, the activity of BIM promoter encompassing the FOXO3a-binding site could be regulated by GDF9. Additionally, we found that GDF9 elevated the levels of phosphorylated AKT and FOXO3a, and this process was independent of the SMAD signal pathway. Taken together, we concluded that OSFs, particularly GDF9, maintained the low level of BIMEL expression in cumulus cell through activation of the PI3K/FOXO3a pathway.
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http://dx.doi.org/10.1095/biolreprod.113.108365DOI Listing
September 2013

A novel polymorphism of GDF5 gene and its association with body measurement traits in Bos taurus and Bos indicus breeds.

Mol Biol Rep 2010 Jan 10;37(1):429-34. Epub 2009 Jul 10.

College of Animal Science and Technology, Northwest A & F University, Shaanxi, People's Republic of China.

Body measurement traits, influenced by genes and environmental factors, play numerous important roles in the value assessment of productivity and economy. Growth differentiate factor 5 (GDF5), involved in the development and maintenance of bone and cartilage, is an important candidate gene for body measurement traits selection through marker-assisted selection (MAS). In this study, based on the PCR-RFLP technology, we discovered and evaluated the potential association of the single nucleotide polymorphism (SNP) (T586C in exon 1) of the bovine GDF5 gene with body measurement traits in 985 Bos taurus breed, 42 Bos indicus breed and 76 Bos indicus x Bos taurus individuals. As the SNP marker, there were the significant effects on the Body length (BL) in the Bos taurus (BT) and Bos indicus x Bos taurus (BMY) populations (P < 0.05). In BT population, animals with the genotype TT had lower mean values for BL and Hip width (HW) than these with the TC and CC genotype (P < 0.01). In BMY population, animals with the genotype TC had lower mean values for BL than these with the genotype CC (P < 0.05). These results suggest that the SNP of the GDF5 gene could be a very useful genetic marker for body measurement traits in the bovine reproduction and breeding.
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http://dx.doi.org/10.1007/s11033-009-9604-5DOI Listing
January 2010