1.Recombinant Irisin Stimulates Migration And Angiogenesis Of Endothelial Cells And Its Mechanism 2.Preventive Effect Of Dihydromyricetin Against Cisplatin-Induced Nephrotoxicity And Its Mechanism

Background:Angiogenesis,the sprouting of pre-existing vasculature to form new vessels,requires many coordinated endothelial cell activities,such as proliferation,migration,and alignment to form vessel-like tube structures.This process,an essential step in tissue repair,occurs in vascular injuries caused by various disorders,such as cardiovascular disease and many other chronic metabolic diseases,especially diabetes.Hence,the factors that can restore injured endothelial cells and stimulate new collateral vessel growth may have important roles in the treatment of vascular damage caused by various diseases.A number of endogenous factors and hormones have been reported to participate in the regulation of angiogenesis.For a long time,a large plenty of studies have shown that exercise is the frontline defense for prevention of many cardiovascular and metabolic diseases.In 2012,Spiegelman lab at Harvard University reported for the first time that irisin,secreted by skeletal muscles in response to PGC-la during exercise,is a cleaved and secreted fragment of fibronectin type III domain containing protein 5(Fndc5),a type I transmembrane protein of skeletal muscles which can be up-regulated by PGC-la and exercise.As a link between exercise and metabolism,irisin is thought to be involved in increased total body energy expenditure,reduced body weight,and increased insulin sensitivity in mice.In our previous study,we demonstrated the effect of irisin in promoting HUVEC proliferation via the ERK signaling pathway and that it partially protects the cell from high glucose-induced apoptosis.These finding suggest that there is a link between irisn and the vascular endothelium.However,no previous studies have evaluated whether irisin directly regulates other functions of human ECs such as migration and angiogenesis.Objective:1.To investigate the influence of human recombinant irisin(r-irisin)on HUVEC migration and cord formation in vitro.And the molecular mechanism and signal pathways by which irisin may exert its effects.2.ISV formation of zebrafish was evaluated for the effects of irisin in vivo.Materials and methods:1.Expression and purification of human recombinant irisin protein.2.Cell isolation and culture of primary human umbilical vein endothelial cell.3.Measurement of the effect of irisin on migration of HUVEC by wound-healing migration assay and transwell chamber migration assay.4.Measurement of the effect of irisin on cord formation of HUVEC by Matrigel tube formation assay.5.Measurement of the effect of irisin on attenuating chemically-induced blood vessel damage in zebrafish by zebrafish angiogenesis assay.6.Real-time PCR analysis to measure the mRNA expression of related genes after irisin stimulation.7.Western blot analysis to measure the expression of related molecular mechanism and signal pathways after irisin stimulation.8.Gelatin zymography analysis to measure the activity of MMP-2 and MMP-9 after irisin stimulation.9.Statistical analysis:The data are presented as the mean±standard deviation(SD).Each experiment was repeated at least of 3 times.Student's t test was used for comparing differences of different groups.Comparisons among values of multiple groups were performed by one-way analysis of variance(ANOVA).Statistical significance was set at P<0.05.Results:1.Irisin promotes migration in HUVEC.(1)Compared with the control group,the wound healing of HUVEC was gradually accelerated in the groups treated with 10 nM and 20 nM irisin at both 12 and 24 h.(2)The Transwell chamber migration assay revealed that after treatment with irisin at concentrations of 10 nM and 20 nM for 24 h,HUVEC showed a significant increase in migratory ability.2.Irisin promotes angiogenesis in HUVEC.The results of Matrigel tube formation assay showed that the presence of irisin induced a significant increase in capillary-like tube formation after just 6 h compared with the control.3.The results of zebrafish angiogenesis assay showed that irisin could attenuate chemically-induced blockage of ISV formation in zebrafish.4.Irisin up-regulates expression and activity of MMP-2 and MMP-9.(1)The quantitative real-time PCR results showed that irisin significantly stimulated expression of MMP-2 and MMP-9 and slightly reduced expression of TIMP-1 both in vitro and in vivo.(2)Western blot showed that MMP-2 and MMP-9 protein levels were also highly up-regulated after treatment with irisin for 24 h.(3)Gelatin-zymography analysis showed that irisin potently increased the gelatinolytic activity of MMP-2 and MMP-9 secreted from HUVEC.5.Irisin mediates HUVEC migration and tube formation through the ERK signaling pathway.(1)Western blot showed that phosphorylated ERK(P-ERK)was significantly increased after treatment with irisin for 5 min and 10 min.(2)Irisin-induced cell migration and tube formation was significantly inhibited by 10 ?M U0126,a specific inhibitor of the ERK pathway.6.Effects of the ERK inhibitor on irisin-induced expression and activity of MMP-2 and MMP-9.(1)Western blot showed that after treatment with U0126 for 24 h,the irisin-induced increase in protein expression of MMP-2 and MMP-9 was significantly inhibited.(2)Gelatin zymography analysis showed that after treatment with U0126 for 24 h,the irisin-induced increase in gelatinolytic activity of MMP-2 and MMP-9 was also significantly inhibited.Conclusion:1.Human recombinant irisin protein promoted migration and angiogenesis in HUVEC in vitro.2.Human recombinant irisin protein attenuated chemically-induced ISV angiogenic impairment in zebrafish in vivo.3.Human recombinant irisin protein can up-regulate mRNA and protein expression and gelatinolytic activity of MMP-2/-9 through activation of the ERK signaling pathway in endothelial cells.Background:Cis-dichlorodiamineplatinum(?)(also called cisplatin in clinica),which belongs to platinum-based antineoplastic drugs,can work in synergy with a variety of antitumor drugs.And it is one of the first-line chemotherapy drugs for the treatment of several malignant solid tumors,such as head-neck carcinoma,small cell and non-small cell lung cancer,melanoma,bladder cancer,testicular cancer,ovarian cancer,endometrial cancer etc.However,nephrotoxicity is a frequent severe side effect of dose-dependent cisplatin chemotherapy,limiting its clinical use despite being one of the most potent chemotherapy drugs.Dihydromyricetin(DMY),also known as ampelopsin,is a natural flavonoid compound extracted from the leaves of ampelopsis grossedentata,which is commonly known as vine tea.Vine tea is considered to have numerous pharmacological functions,is used in traditional Chinese medicine to accelerate detoxification of ethanol and treat fever and cough,and possesses antioxidative and antimicrobial properties.Zhu et al.have demonstrated that DMY exerts a cardioprotective effect against adriamycin-induced injury in ICR mice.However,little is known about the effect of DMY in cisplatin-induced nephrotoxicity.According to the previous study,we investigated the renoprotective effect of DMY in a cisplatin-treated mouse model.And DMY mediated a protective effect against cisplatin-induced nephrotoxicity by ameliorating apoptotic cell death in HK-2 cells.Objective:1.To observe the protective effect of DMY on cisplatin-induced HK-2 apoptosis and necrosis.2.To establish cisplatin induced nephrotoxicity model in C57/BL6 mouse,and further investigate the possible protective mechanisms.Materials and methods:1.Cell viability and apoptotic cells measured by WST-1 and DAPI staining.2.Bcl-2 and Bax expression measured by Western blot.3.Establish cisplatin induced nephrotoxicity model in C57/BL6 mouse.4.Assessment of serum creatinine concentration and blood urea nitrogen.5.Measurement of Oxidative Stress Markers.6.PAS and HE staining for histological examination and evaluation of interstitial polymorphonuclear leukocyte infiltration.7.Immunohistochemical staining for 8-OHdG was performed using anti-8-OHdG antibody.8.Apoptotic cells were detected using the TUNEL assay.9.The mRNA expressions of inflammatory factor were detected by Q-PCR.10.Statistical analysis:The data are presented as the mean±standard deviation(SD).Each experiment was repeated at least of 3 times.Student's t test was used for comparing differences of different groups.Comparisons among values of multiple groups were performed by one-way analysis of variance(ANOVA).Statistical significance was set at P<0.05.Results:1.Effect of DMY on cisplatin-induced cell viability and apoptosis in HK-2.(1)Cell viability was inhibited by cisplatin in a concentration-dependent manner(10,20,30,or 50 ?M),while DMY effectively abated the inhibitory effects caused by cisplatin.(2)The results of DAPI staining shows that extensive nuclear fragmentation was clearly observed in the cisplatin-treated group.On the contrary,after pretreatment with DMY for 24 h,these nuclear changes were decreased.(3)Western blot analysis indicated that the decreased expression of Bcl-2 caused by cisplatin was partially upregulated by the pretreatment of DMY and Bax expression was downregulated in the DMY coadministration group.2.Effect of DMY on cisplatin-induced nephrotoxicity.(1)The cisplatin-treated group exhibited a significant increase in SCr and BUN compared with the control group.The enhanced levels of SCr and BUN were significantly decreased in the cisplatin with DMY groups in a dose-dependent manner.(2)The results of PAS staining show that the cisplatin-treated group showed severe loss of brush border and cast formation.On the contrary,DMY treatment significantly attenuated these pathological changes induced by cisplatin.(3)The results of TUNEL staining show that cisplatin-administered mice exhibited a significantly higher number of positive nuclei compared with control,while the DMY-treated group indicated a decrease in apoptotic cells.3.Effect of DMY on oxidative stress in kidney tissues.(1)Immunohistochemical staining for 8-OHdG shows that the numbers of 8-OHdG-positive cells were much greater in the cisplatintreated group when compared to the control mice.Also,a significant decrease of the positive cell numbers was observed in the cisplatin with Dihydromyricetin group.(2)Three days after cisplatin treatment,a significant increase in tissue MDA level and decrease of CAT and SOD activities occurred compared with the control group,while pretreatment of DMY partially blocked the increase of MDA level and attenuated the reduction of these antioxidant levels.4.Effect of Dihydromyricetin on inflammatory mediators in kidney tissues.(1)The Q-PCR results show that compare with cisplatin treatment group,gene expression of IL-1?,IL-6,TNF-a,and MCP-1 was significantly lower pretreatment with DMY.(2)With the semiquantitative measurement of PMN infiltration by HE staining,the cisplatin treated group had greater PMN infiltration when compared with the DMY coadministration group.Conclusion:1.DMY protected against cisplatin-induced apoptotic cell death of HK-2 cells.2.DMY protect against cisplatin-induced nephrotoxicity in mice.3.DMY ameliorated cisplatin-induced nephrotoxicity via attenuating cisplatin-induced oxidative stress and inflammatory response.