Investigation Of The Mechanisms Underlying The Protective Effects Of Triptolide And Glucocorticoid On TGF-β-induced Podocyte Injury

Part1.Triptolide protects podocytes from TGF-β-induced injury by preventing miR-30 downregulationObjective:Triptolide has a strong anti-proteinuric effect by directly protecting the podocyte and is used to treat glomerular diseases.However,the mechanism underlying its protective effect on podocytes is largely unknown.MiR-30 family has recently been shown to be essential for podocytes but is downregulated by injurious factors,leading to the injury of podocytes.In this study,we explore whether Triptolide protects podocytes by preventing the downregulation of miR-30Methods:TGF-β is a critical mediator in various podocyte injuries and we previously found that TGF-β can downregulate miR-30S and induces podocyte injury,therefore we used TGF-β-induced podocyte injury model to address the issue.After 30 min of preincubation with triptolide,the podocytes were treated with 5 ng/mL TGF-β for 24 h,followed by Western blotting to examine pSmad2/3,MAPK pp38,NF-κB pp65,NFATC3 and synaptopodin.We used Annexin V staining and flow cytometry to detect apoptosis.Podocyte cytoskeletion injury was examined by phalloidin staining;and q-PCR was used to measure the expression of miR-30s.Additionally,we isolated mouse glomeruli and treated them with TGF-β in the presence or absence of triptolide to verify the effect of triptolide on miR-30s expression Meanwhile,we isolated glomeruli from rata and treated with TGF-β,followed by western blotting to examine the effect of triptolide on phosphorylation of Smad2/3.Results:We found that Triptolide can protect podocytes from TGF-β-induced cytoskeletal disruption and apoptosis.Consistently,Triptolide inhibited TGF-β-induced activation of MAPK p38,NF-kB and calcineurin/NFATC3.which are known to be injurious to podocytes Meanwhile,Triptolide could completely prevent TGF-β-induced miR-30 downregulation,indicating that Triptolide protects podocytes by sustaining miR-30 expression Mechanistically,we found that Triptolide can inhibit TGF-β-induced Smad2/3 phosphorylation/activation,which may explain the restoration of miR-30 by Triptolide.Ex vivo study found that Triptolide prevented TGF-β-induced miR-30 downregulation and the phosphorylation of Smad2/3 in the isolated glomeruli of mice or rats.Conclusion:These results demonstrate that 1)Triptolide prevents miR-30s downregulation to protect podocytes from TGF-β-induced injury;2)Mechanistically,Triptolide can block TGF-β-induced phosphorylation/activation of Smad2/3,thereby sustaining miR-30s expression in TGF-β-treated podocytes.Part 2.The interaction between glucocorticoid pathway and TGF-β-Smad2/3 pathway in podocyte injury.Objective:TGF-β-Smad2/3 signaling plays a crucial role in podocyte injury,while glucocorticoid is capable of directly acting on and protecting podocytes from injury in glomerular diseases.However,the potential relationship between them has not been reported In the present study,we aim to determine whether glucocorticoid signaling can interfere with TGF-β signaling,thereby protecting podocytes.Methods:In vitro,cultured podocytes were treated with or without glucocorticoid,and cytoplasmic and nuclear fractions were separated for western blotting analysis of glucocorticoid receptor.After TGF-β treatment in the presence or absence of glucocorticoid,nuclear translocation and phosphorylation of Smad2/3 were detected by immunofluorescence and western blotting,respectively.We alsoused Co-IP to determine whether Smad2/3 and glucocorticoid receptor could interact physically.Results:1)In the absence of GC,glucocorticoid receptor was found to be abundantly present in nuclei of podocytes;2)TGF-β induced phosphorylation and nuclear translocation of Smad2/3,leading to the binding of Smad2/3 with glucocorticoid receptor in nuclear of podocytes;3)glucocorticoid induced marked degradation of the glucocorticoid receptor and dephosphorylation of pSmad2/3;4)glucocorticoid did not affect TGF-β-induced phosphorylation and nuclear translocation of Smad2/3.Conclusions:glucocorticoid receptor is present in nuclei of podocytes and may function in the absence of the glucocorticoid;TGF-β induces phosphorylation and nuclear translocation of Smad2/3,and then pSmad3 binds to GR and is thereby protected from dephosphorization by nuclear phosphatases;glucocorticoid can directly enter nuclei to dissociate GR and pSmad3,resulting in GR degradation,pSmad3 dephosphorylation and inactivation,and thus protection for podocytes.