Effects Of Triptolide Intervention On Endothelial Cell And Renal Fibrosis Related Genes

Objective We aim to investigate the effects of triptolide(TP)on the expression of hypoxia inducible factor 1 alpha(HIF1?)and vascular endothelial growth factor(VEGF)in the human umbilical vein endothelial cells(HUVECs)under hypoxia.Meanwhile,in the renal cortical proximal convoluted tubule epithelial cells(Human kidney proximal tubular epithelial cell,HK-2)induced by TGF-?1,we want to investigate the effect of TP on the epithelial-mesenchymal transition(EMT).Futhermore,explore the plausible molecular mechanism of TP on renal fibrosis.Methods To investigate the effects of TP on the expression of HIF1? and VEGF in HUVECs under hypoxia,we selected primary human umbilical vein endothelial cells and cultured it in a special hypoxia culture chamber.The experiments were grouped and processed as follows:(1)HUVECs were treated with 0,40,80,160,and 320 nmol/L(named with hypoxia group,TP40 group,TP80 group,TP160 group,and TP320 group,respectively)under a hypoxic condition(37?,5%CO2,1%O2,94%N2)for culturing 12 hours.Meanwhile,cells were cultured under normoxia condition(without TP added)which was set as the normoxia group.Western blot assay was uesd to detect the expression of HIF1? in each group.(2)The cells were divided into normal control group,hypoxia group and TP80 group,the immunofluorescence was performed to detect the localization of HIF1? in cells.(3)Expressions of VEGF were detected by Western blot in the TP80 group and hypoxia group.(4)The cells were divided into hypoxia group,TP80 group,TP80 +KF20 group(80 nmol/L TP and 20 ?mol/L KC7F2),and the TP80+ KF30 group(80 nmol/L TP and 30 ?mol/L KC7F2).After 12 hours culturing,Western blot was used to detect the expressions of HIF1? and VEGF in each group.In order to investigate the role of TP in the epithelial-mesenchymal transition(EMT)induced by TGF-?1,the HK-2 cells were divided as follows:(1)1Normal control group,where cells were cultured under routine condition 48 h.2TGF-?1 group,10ng/ml TGF-?1 was used to stimulate the cultured cell 48 h.3TP group,20?mol/l TP and 10ng/ml TGF-?1 were used to costimulate the cells 48 h.Each experiment was repeated once,and the total RNA was extracted from the cells.After purification,the RNA was sequenced by Illumina Hiseq platform.(2)The sequence information of the transcriptome was analyzed and compared with the reference genome.After that,the expression of single genes in each treatment group was calculated by the FPKM method.(3)The differences of gene expression between the treatment groups were analyzed and found as the up-regulated and down-regulated genes.Meanwhile the number of differentially expressed genes was statistically counted.(4)KEGG pathway enrichment was used to analyze the differentially expressed genes.(5)Base on the results of KEGG pathway enrichment analysis,we verified protein expressions in the TGF-?-smad signal pathway.The grouping and processing conditions of the experiment were as same as in(1).A phase contrast microscope was used to observe the changes of cell morphology.(6)Various expressions of TGF-?-smad signaling pathway markers(such as smad2/3,TGF-?R? and TGF-?R??)and related EMT markers(such as E-cadherin,vimentin and N-cadherin)were detected by Western blot.Results(1)No HIF1? expression was detected in HUVECs under the normoxia condition.While in hypoxia condition,TP could induce the expression of HIF1?.When the TP concentration was 80 nmol/L,the expression level of HIF1? was significantly increased in the TP80 group than in the hypoxia group(P<0.05),while the TP160 group and the TP320 group had no significant change in expression of HIF1? compared with the hypoxic group.(2)Results of immunofluorescence showed that HIF1? was mainly expressed in the nucleus.(3)The expression of VEGF was significantly increased in the TP80 group than in the hypoxia group(P<0.05).(4)After the TP intervention,the expression level of HIF1? and VEGF in the TP80 group was higher than that in the hypoxia group.But after the KC7F2 intervention,HIF1? and VEGF expression levels were significantly decreased in the TP80+KF20 group and the TP80+ KF30 group than in the hypoxia group(P<0.05).(5)In the NC group,the TGF-?1 group,and the TGF-?1+TP group,FPKM expressions of 14903 genes in different treatment groups were calculated.(6)When we compared the NC group with the TGF-?1 group,161 genes were up-regulated and 48 genes were down-regulated;When we compared the NC group with the TGF-?1+TP group,2997 genes were up-regulated and 1954 were down-regulated;Between the TGF-?1 group and the TGF-?1+TP group,2982 genes were up-regulated and 1827 were down-regulated.We compaired regulated genes between the TGF-?1 group and the TP+TGF-?1 group,a volcano figure shows that the TP affected more up-regulated and down regulated genes than the TGF-?1 did.(7)The different expression of genes in the KEGG pathway enrichment analysis shows that the first 20 pathways have the most significant Q values.In pairwise comparisons,“pathway in cancer” yielded the most significant enrichment score,indicating that this may be involved in the regulation of tumors.(8)In the KEGG enrichment analysis,we compared the TGF-?1 group with the TP+TGF-?1 group,TP had impacted on many pathways that related to cell adhesion,including the TGF-beta and the Hippo signaling pathway.In the TGF-beta signaling pathway,TP mainly down regulated the expressions of smad2/3,THBS1,Noggin,Rbx1,SARA,ROCK1 and c-Myc,while the BABM1 and Smad5/8 expression were up-regulated.In the Hippo signaling pathway,TP also down regulated the expression of Smad2/3,but had no effect on the expression of E-cadherin.Futhermore,TP also affected the expression of many genes in the pancreatic cancer pathway.(9)When HK-2 cells were stimulated by 10ng/ml TGF-?1,cell morphology began to change from epithelial cobblestone-like to fusiform,with the mesenchymal related marker up-regulated in TGF-?-smad signaling pathway,such as the expression of N-cadherin,Smad2/3,TGF-?R?,TGF-?R?? and vimentin,but the epithelial properties related marker of E-cadherin was down-regulated.When TP interventions were compared in the TGF-?1 group with the TP+TGF-?1 group,those mesenchymal markers were all down regulated,but the expression of E-cadherin was not significantly increased.The cell morphology also had not changed to a cobblestone-like,but some cells began to turn a round shape.Conclusions TP can induce VEGF expression in endothelial cells by increasing the HIF1? expession under hypoxia.Meanwhile,TP also affects the expression of TGF-?-smad signaling pathway related markers,and has a certain inhibitory effect on EMT that induced by TGF-?1.So TP may be involved in the treatment of renal fibrosis.