Apigenin Suppresses Mouse Peritoneal Fibrosis By Down-regulating Mir34a Expression

Part 1 Apigenin suppresses hyperglycemia-induced peritoneal fibrosis by down-regulating miR34a expressionObjective:Peritoneal dialysis(PD)is one of the commonly used treatment methods for patients with end-stage renal disease.It is accepted by a large number of patients because of its remarkable convenience and quality of life benefits.However,the hypertonicity and low histocompatibility of peritoneal dialysate will lead to fibrosis of peritoneal mesothelial cells(PMCs).Peritoneal mesothelial cells will gradually lose their semi-permeable membrane properties,resulting in decreased ultrafiltration capacity or even failure.Patients have to undergo more complicated hemodialysis treatment,which seriously affects their quality of life and increases treatment costs.Epithelial-mesenchymal transition(EMT)is one of the important pathological changes in peritoneal fibrosis(PF).Several studies have shown that miRNA are involved in the EMT process,but the role of miR34a in EMT is still unclear.Apigenin is the most common natural active ingredient in Chinese herbal medicine.It has anti-inflammatory,anti-oxidation and other pathological activities,but its protective effect in peritoneal fibrosis is rarely reported.In this study,mouse peritoneal mesothelial cells(MPMCs)were treated with high glucose to construct a PF model,to investigate the role of miR34a in PF and to explore the effect of apigenin on miR34a and its therapeutic effect on PF.Methods:In this study,MPMCs were isolated and fibrosis was induced by high glucose to explore the role of miR34a in PF in mice,and apigenin was given to explore its effect on miR34a and its therapeutic effect on PF.The survival rate of mouse peritoneal mesothelial cells and the half inhibitory concentration of apigenin on MPMCs were detected by CCK8 method.qRT-PCR and Western Blot were used to detect the mRNA and protein level changes of the EMT epithelial markers E-cadherin and TTF,and interstitial markers N-cadherin and Vimentin.Result:1.High glucose can successfully induce EMT in MPMCs,and the mRNA and protein levels of EMT-specific interstitial markers N-cadherin and Vimentin increase with the increase of high glucose concentration and the prolongation of action time.While the mRNA and protein levels of epithelial markers including E-cadherin and TTF decrease with the increase of high glucose concentration and the prolongation of action time.2.miRNA are involved in regulating the EMT process of MPMCs.miR34a has the most obvious effect,and its expression can be inhibited by apigenin.3.Apigenin can reverse the inhibition of high glucose on the proliferation of MPMCs.4.Apigenin can reduce the high glucose-induced EMT of MPMCs.Conclusion:In summary,the MPMCs were isolated and induced by high glucose to establish a PF model.miR34a was involved in the process of mouse PF.Apigenin promoted cell proliferation and significantly reduced the EMT by down-regulating miR34a expression,confirming its promising therapeutic effect on PF.The results of this study provide a theoretical basis for the treatment of PF in PD patients with apigenin by down-regulating the expression of miR34a.Part 2 Apigenin suppresses autophagy and apoptosis during peritoneal fibrosis in mice by down-regulating miR34a expression Objective:Peritoneal dialysis(PD)is a common renal replacement therapy,and about 10%-15%of patients with end-stage renal disease worldwide are receiving PD.The technique survival of peritoneal dialysis is limited due to peritoneal fibrosis(PF)induced by peritoneal dialysate.Exploring effective treatment of PF is an important issue that needs to be solved urgently.Autophagy is a process for cells to remove misfolded proteins and damaged senescent cells,and it is at a low level of activity under physiological conditions.Under stress,autophagy is significantly activated to maintain internal environment stability and provide energy for cells by degrading its own components.Studies have shown that autophagy and apoptosis of peritoneal mesothelial cells(PMCs)are initial reversible processes of PF,and inhibiting autophagy and apoptosis of PMCs can reduce their fibrosis.In this study,by constructing a PF mouse model,we studied the role of miR34a in PF in vivo,and explored the effect of apigenin on miR34a and its effect on autophagy and apoptosis in the process of PF.Method:In this study,the mouse PF model was induced by intraperitoneal injection of 4.25%peritoneal dialysate,and then intervened by intragastric administration of apigenin and/or tail vein injection of miR34a inhibitor.HE staining was used to observe the peritoneal histological changes.Masson’s trichrome stain was used to detect the degree of PF.TUNEL assay was used to detect the apoptosis of MPMCs.The number of peritoneal apoptotic bodies was observed by electron microscope.Hoechst and flow cytometry detected the occurrence of high glucose-induced apoptosis autophagy in MPMCs.And Western Blot detected the protein expression levels of MPMCs apoptosis and autophagy biomarkers.Result:1.4.25%peritoneal dialysate can successfully induce mouse PF model.2.Apigenin can significantly improve peritoneal thickening,inflammatory cell infiltration and collagen fiber accumulation in mice.3.Apigenin can significantly inhibit the apoptosis of mouse peritoneal cells.4.Apigenin can significantly inhibit the autophagy of mouse peritoneal cells.5.Apigenin can reverse the pro-apoptotic effect of high glucose on MPMCs.6.Apigenin can affect the expression of apoptosis and autophagy-related proteins in MPMCs during EMT.Conclusion:In conclusion,4.25%peritoneal dialysate was used to construct a mouse PF model,and oral administration of apigenin and/or tail vein injection of miR34a inhibitor could inhibit the process of PF in mice by inhibiting apoptosis and autophagy.The results of this study further confirmed the therapeutic effects of apigenin in PF by down-regulating miR34a in animals.