| Objective:Studies have indicated that epithelial-mesenchymal transition(EMT)is closely linked to the pathogenesis of diabetic kidney disease(DKD).Triptolide possesses anti-inflammatory and immunosuppressive effects,and can protect DKD as well as reduce the level of urinary protein.Recently,microRNAs(miRNAs)have emerged as critical regulators of DKD and become a research hotspot in the field of life medicine.However,any research that whether triptolide alleviates renal EMT by regulating miRNAs in DKD has not yet been covered at home and abroad.Therefore,the aim of this study was to determine the possible effect and mechanism of triptolide in DKD at animal and cellular levels and to explore whether it may further alter the activity of downstream signaling pathways by regulating the expression of miRNAs to mitigate renal EMT process in DKD.Methods:1.In vivo:60 SD rats were raised,and 15 were randomly chosen to receive a standard diet(NC group,n= 15),while the remaining rats were fed with high-fat diet combined with tail vein injection of streptozocin(STZ)to establish a model of type 2 diabetes mellitus(DM).The established DM rats were randomly divided into the DKD group(n= 15)and DKD group(n= 15).Rats were gavaged with triptolide for 12 weeks in the DKD +TP group,while the NC and DKD groups were gavaged with isometric normal saline.At the end of feeding,24 hours urine samples were obtained for the measurement of urinary microablumin(UMA).Measure the body weight and kidney weight and collect blood samples for the measurement of blood glucose,blood lipid,liver and kidney function.HE,PAS and Masson stainings were performed to analyze the histopathological changes of kidneys.Immunohistochemistry was used to analyze the expression of EMT markers.Western Blotting was adopted to analyze the protein expression of EMT markers,PTEN,and signal molecules of PI3K/AKT pathways from rat kidneys.RT-PCR were used to analyze the expression of miR-188-5p in rat kidneys.2.In vitro:Human proximal tubular cells(HK-2 cells)were the research objects and CCK 8 method was used to determine the optimal therapeutic concentration of triptolide intervention.The experiment was divided into 4 groups,including normal glucose group(NG group),mannitol group(MA group),high glucose(HG group)and triptolide group(HG+TP group).Morphological changes of HK-2 cells were observed through light microscope.Immunofluorescence and RT-PCR were used to analyze the mRNA and protein levels of EMT markers.Western Blotting was used to analyze the protein expression of EMT markers,PTEN,and signal molecules of PI3K/AKT pathways.LY294002,a PI3K inhibitor,was adopted to testify the activity of p-AKT and expression of EMT markers under HG.RT-PCR were used to analyze the expression of miR-188-5p and PTEN.The possible target gene interacting with miR-188-5p was predicted through TargetScan.The interaction between miR-188-5p and the 3’-UTR of PTEN was verified by Dual-Luciferase reporter assay.Cell transfection experiments were divided into NG group,HG group,HG+control group(miR-iNC),HG+miR-188-5p inhibitor group(miR-188-5pi),HG+TP,HG+TP+control group(miR-188-5pm),HG+TP+miR-188-5p mimics group(miR-188-5pm).miR-188-5p expression after transfection was detected using RT-PCR.Western Blotting was used to examine the protein expression of EMT markers,PTEN,and signal molecules of PI3K/AKT pathways.Results:1.Compared with the NC group,body weight was obviously reduced,while blood glucose and lipid levels,Kidney weight/Body weight and UMA levels were significantly elevated in the DKD group(P<0.05).Triptolide markedly decreased Kidney weight/Body weight and UMA levels(P<0.05),but had no significant effects on their body weight,blood glucose and lipid levels(P<0.05).No significant differences were observed in the markers of liver and kidney function between groups(P>0.05).2.Compared with the NC group,HE staining showed that the glomerular area enlarged,mesangial matrix expanded,renal tubules swelled and deformed in the DKD group.The accumulation of glycogen exhibited by PAS staining was increased in the DKD group relative to the NC group,especially in glomeruli.The collagen fibers indicated by Masson staining was also increased and tubulointerstitial fibrosis was obvious in the DKD group.However,all above renal pathological changes were all improved after triptolide intervention.3.Both immunohistochemistry and Western blotting revealed that compared with the NC group,the renal tubular EMT in rat kidneys was exacerbated in the DKD group,PTEN expression was significantly decreased,and the activity of PI3K/AKT pathway was significantly increased(P<0.05).Compared with the DKD group,EMT in rat kidneys was partly recovered in the DKD+TP group,PTEN expression was increased and the activity of PI3K/AKT pathway was decreased(P<0.05).4.Compared with the control group,low concentrations of triptolide(≤5ng/mL)had no marked influences on the viability of HK-2 cells(P>0.05),but if the concentrations of triptolide were ≥7.5ng/mL,Cells survival was significantly impaired(P<0.05).5.NG-treated cells appeared a cobblestone-like epithelial shape,while HG-treated cells transformed into long spindle-like mesenchymal shape.Triptolide-treated cells regained the appearance of epithelial cell.Immunofluorescence,RT-PCR and Western blotting all showed that relative to the NG group,EMT was upregulated in the HG group,PTEN expression was downregulated and the activity of PI3K/AKT pathway was upregulated(P<0.05).Triptolide downregulated EMT,upregulated PTEN expression and downregulated the activity of PI3K/AKT pathway(P<0.05).Mannitol control made no significant differences in the above changes relative to the NG group(P>0.05).6.Compared with the HG group,LY294002 inhibited the activation of p-AKT and decreased EMT in HK-2 cells(P<0.05).7.miR-188-5p expression in the kidneys of the DKD group was higher than the NC group(P<0.05),while miR-188-5p expression in the DKD+TP group was significantly lower than the DKD group(P<0.05).Compared with the NG group,miR-188-5p expression was significantly increased while PTEN mRNA level was obviously decreased in the HG group(P<0.05).Relative to the HG group,miR-188-5p expression was significantly reduced while PTEN mRNA level was obviously increased in the HG+TP group(P<0.05).8.The predicted results of TargetScan showed that the 3’-UTR of PTEN from 762-768 sites could be combined with the miR-188-5p by complementary sequence.As shown in Dual luciferase reporter assay,miR-188-5p overexpression reduced the level of luciferase activity in the wild-type PTEN-3’-UTR reporter(P<0.05)but had no significant effects on the mutant PTEN-3’-UTR reporter(P>0.05).9.miR-188-5p expression was reduced by almost 60%after transfected with miR-188-5p inhibitor(P<0.05).PTEN expression was increased but the activity of PI3K/AKT pathway and EMT was decreased(P<0.05)when miR-188-5p deletion.10.miR-188-5p expression was approximately 45 times higher after transfected with miR-188-5p mimics(P<0.05).miR-188-5p overexpression decreased PTEN expression but increased the activity of PI3K/AKT pathway and EMT(P<0.05).Conclusions:1.Triptolide decreased UMA levels,improved renal pathological changes in the DKD rats,and protected renal tubular cells from EMT through PI3K/AKT pathway both in vivo and in vitro.2.Triptolide reduced miR-188-5p expression in the kidneys of DKD rats and HG-induced HK-2 cells.miR-188-5p inhibited PTEN expression via directly interacting its 3’-UTR to affect the activity of PI3K/AKT pathway.3.miR-188-5p abrogation rescued the HG-induced EMT through inactivating PI3K/AKT pathway.miR-188-5p overexpression reversed the anti-EMT effect of triptolide through activating PI3K/AKT pathway. |
PDF Full Text Request