THHta Attenuates Podocyte Injury In Diabetic Nephropathy By MicroRNA-29a/PTEN Signaling Pathway

Background and aim:Diabetic Nephropathy(DN)is the most common chronic microvascular complications of diabetes mellitus.It is also one of the major causes of death in diabetic patients.Thecurrent available treatment is ineffective for preventing the further development of DN and better therapeutic options are urgently needed.There is an increasing amount of evidence suggests that podocyte injury may be one of the causes of DN.Podocyte injury can be caused by a number of factors such as the inflammatory responses,oxidative stress and the activation of the renin-angiotensin-aldosterone system(RAAS).Investigation of the molecular mechanisms of podocyte injury is critical in the development of better therapeutic strategies for DN.Previous studies have shown that the total alkaloid content of the traditional Chinese medicinal herb Tripterygium hypoglaucum(levl.)Hutch(THHta),also known as Kunming Begonia,can reduce podocyte injury induced by high glucose during diabetic nephrapathy.However,the underlying molecular mechanisms of the nephroprotective activity of THHta remains unknown.Currently,there is no available in vitro model to investigate the molecular mechanisms of high glucose podocyte injury.In this study,we first aimed to measure the changes of several parameters,including cell proliferation,reactive oxygen species(ROS)levels and apoptosis,in podocytes treated by different levels of glucose to established a high glucose podocyte injury model.Furthermore,it is now widely understood that microRNAs are important regulators of nephropathy,notably in glomerulosclerosis,an important late stage physiopathological changes during DN.We then investigated the involvement of miRNA using microarray analysis.This study has identified the micro RNA miR-29a downstream of THHta signaling.miR-29a has been shown to downregulate expression of the gene PTEN,encoding for the phosphatase and tensin homolog(PTEN)protein,to contribute to the reduction of podocyte injury and the delaying of diabetic nephropathy.Elucidation of the target molecules and a potential mechanism of THHta in the podocyte injury model during our study has provided insights into the novel approaches for future research and drug development.Research methods:(1)To establish the high glucose-induced podocyte injury model,conditionally immortalized mouse podocytes cultured in vitro were randomly divided into 5 groups,including one normal glucose control group(5.5 mmol/L glucose)and four high glucose groups of different doses(10,20,30 and 40 mmol/L glucose).Cells were cultured for another 24 h,and then the proliferation level,represented by the changes in enzymatic activity of cellular NADP(H)oxidoreductase,was detected using the MTT assay.ROS level of cells was tested using the DCFH-DA fluorescence probe.Levels of cell death were detected by annexin V-FITC/PI staining followed by flow cytometry analysis.To verify the level of apoptosis,the cells were incubated with 10 and 30 mmol/L glucose for 24 hours,then the expressions of cleaved-caspase-3,cleaved-PARP and Bcl-2 were detected by Western blot.(2)Using the podocyte injury model,the podocytes were randomly divided into 6 groups:normal glucose control group(5.5 mmol/L glucose),high glucose(HG)group(30 mmol/L glucose)and high glucose group supplemented with different doses of THHta(1.25,2.5 and 5?g/mL)(HG + THHta).Cell proliferation,ROS and apoptosis levels were determined as previously described.In addition,the levels of tumor necrosis factor-a(TNF-?)and interleukin-6(IL-6)were detected by the enzyme-linked immunosorbent assay(FLISA)to measure the level of immune response.(3)The changes of microRNAs expression related to podocyte injury was screened by microRNA microarray.The expression of miR-29a,which has shown the most prominent changes during the microarray analysis,was detected by real-time fluorescence quantitative PCR(qRT-PCR)in cells treated with different concentrations of THHta(1.25,2.5 and 5?g/mL).Conditionally immortalized mouse podocytes cultured in vitro were randomly divided into HG group(30 mmol/L glucose)and HG+THHta group(5?g/mLTHHta),HG+THHta+miR-29a mimics group,HG+THHta+mimics negative control(NC)group,HG+THHta+miR-29a inhibitor group and HG+THHta+inhibitor negative control(NC)group.Cell proliferation and ROS levels were determined as previously described.Three potential targets of miR-29a were analyzed by bioinformatics and verified by luciferase assay.MiR-29a inhibitor and miR-29a mimics were transfected into podocytes and the cells were incubated for 24 hours.mRNA levels of PTEN were detected by qRT-PCR,and the expression of PTEN was checked by Western blot.(4)In order to verify the inhibition of PTEN expression by miR-29a,miR-29a mimics and pcDNA-PTEN plasmid encoding PTEN-luciferase fusion protein were co-transfected into podocytes,followed by treatment with THHta and high glucose.Expression of PTEN was verified by luciferase assay,mRNA measurement and ELISA.(5)To confirm that PTEN expression is essential to the high glucose-mediated injury,cell proliferation,ROS and apoptosis levels were determined as previously described on the miR-29a,miR-29a mimics and pcDNA-PTEN plasmid transfected cells treated with or without high glucose and THHta.Research result:(1)First,the high glucose-induced podocyte injury model was establishedand evaluated.As expected,mouse podocytes cultured with higher glucose concentrations suffered a reduction in their viability.Meanwhile,production of reactive oxygen species(ROS)in podocytes increased in response to the increase in glucose concentration.Both changes were observed in a concentration-dependent manner.Similarly,the apoptosis rate increased with the increase of the glucose concentration,as indicated by the annexin V/PI staining experiments.This is supported by the Western blot experiments showing that the expression of pro-apoptotic factor cleaved-caspase-3 and cleaved-PARP increased with the increase of glucose concentration,while the anti-apoptotic factor Bcl-2 level was down-regulated.The apoptosis induction ability of high glucose in the mouse podocytes is consistent with the previous findings in human.In summary,we have established a high glucose induced podocyte injury model,which will serve as a useful tool for the following studies.(2)We then proceeded to investigate the effect of THHta on the high glucose-induced podocyte injury using the in vitro podocyte injury model established in this study.Consistent with the previous findings,the proliferation activity of HG group(30 mmol/L glucose)was significantly lower than that of control group while it had an elevated ROS production level.Compared to the HG group,the proliferative activity of HG+THHta groups with different doses of THHta showed an ascending trend,among which the proliferation activity of HG+THHta group(5 pg/mL THHta)was the most obvious.With the increase of THHta concentration in the tested range(1.25-5 ?g/mL),its ability to alleviate the inhibition effect of high glucose on cell proliferation increased in a dose-dependent manner.It also proved that THHta does not affect cell viability at the tested concentrations.Furthermore,compared to the HG group,both the ROS and apoptosis levels in the cells of the HG+THHta groups showed a dose-dependent decreasing trend,and the decrease in the HG+THHta group(5 ?g/mL)was the most significant in both assays(p<0.01).In addition,we are also interested in the inflammatory responses elicited during high glucose treatment and the potential effect of THHta in immunomodulation.Detection of TNF-? and IL-1? in the cell supernatant by ELISA showed that the TNF-? and IL-1? increased in the HG group(p<0.01)while the expression of TNF-? and IL-1? in all HG+THHta groups showed a downward trend,in which the expression of TNF-? and IL-1(3 in the 5 g/mL group of HG+THHta had the most prominent and significant reduction(p<0.01).After the THHta concentration was increased,the extracellular levels of glucose-induced TNF-a and IL-1?gradually decreased,showing an inhibitory effect of THHta on the production of pro-inflammatory cytokines by the podocytes.In general,THHta is capable of subverting all high glucose-induced changes on podocytes tested in this experiment,including the inhibition of cell proliferation,increased apoptosis and the production of ROS and inflammatory cytokines.(3)After confirming the inhibition of high glucose-induced podocyte injury by THHta,it is necessary to investigate the molecular basis behind these effects.It is believed that miRNA regulates many aspects of the physiopathological development of DN.Hence,microRNA expression of the treated cells was determined by microarray.Compared with the HG group,the expression of 19 miRNAs was significantly increased in the HG+THHta group,and the expression of 21 miRNAs was significantly decreased.All of them are closely related to inflammation and/or apoptosis.Among them,miR-29a was previously determined to participate in the negative regulation of DN,while itself was inhibited downstream of high glucose treatment.In our microarray experiment,the reduction of miR-29a in the high glucose podocyte injury model was detected and it was reversed with the addition of THHta.The resulting increase in the miR-29a level due to THHta at 5 ?g/L is also the one of the most prominent compared toother miRNA candidates.Verification of the expression changes of miR-29a by qRT,PCR confirmed that the expression of miR-29a was downregulated under high glucose stimulation,while the expression of miR-29a was gradually up-regulated with the increased concentration of THHta.Next,we investigated the biological functions of miR-29a using gain-and loss-of-function assay.The results showed that miR-29a inhibitor could suppress the therapeutic effect of THHta,as evidenced by its inhibition of the THHta-promoted cell proliferation and apoptosis prevention.Oppositely,miR-29a mimics can enhance the therapeutic effect of THHta.Using bioinformatics analysis and luciferase reporter assay,we found that there is a conserved binding site in the PTEN gene with the miR-29a sequence,which encodes for the PTEN,a phosphoinositide phosphatase known to be involved in cell cycle regulation.Combined with luciferase assay,qRT-PCR and Western blot results showed that PTEN is a target gene of miR-29a.Further studies showed that miR-29a mimics could enhance the therapeutic effect of THHta,while overexpression of PTEN could reverse the therapeutic effect of THHta,suggesting that PTEN is critical to the high-glucose mediated podocyte injury and it may be exploited as a potential drug target.Conclusion:THHta inhibits the expression of miR-29a by upregulating the expression of PTEN,inhibiting apoptosis,promoting cell proliferation,and ultimately alleviate podocyte injury caused by a high glucose concentration.The results provided a new potential target for the future treatment of diabetic nephropathy.