MiR-145-5p Regulates Mesangial Autophagy In The Treatment Of CGN By Salvianolic Acid B

Chronic glomerulonephritis(CGN)is an immunoinflammatory disease caused by many factors.Its main pathological features are inflammatory infiltration of glomeruli and abnormal proliferation of mesangial cells.In recent years,it has been reported that autophagy is considered to be a physiological activity of entocyte degradation that can maintain cell homeostasis,which is closely related to the improvement of CGN.Traditional Chinese medicine is widely used in the treatment of CGN with definite curative effect.Our preliminary research had found that Salvianolic acid B(Sal B),the main active ingredient of Salvia miltiorrhiza,which is commonly used in promoting blood circulation and improving congestion,can protect renal function and improve CGN.Its mechanism is related to the regulation of autophagy.ObjectiveThe purpose of this research is to explore the internal relationship between miR-145-5p and autophagy in the improvement of CGN by Sal B.Explain the molecular mechanism of Sal B improving CGN from the aspect of miR regulating autophagy.So as to broaden a new vision for the CGN pathogenesis and provide new ideas for the prevention as well as the treatment of CGN.Methods1.In vivo,Cationic bovine serum albumin(C-BSA)was prepared and injected into SD rats every other day for 4 weeks,and the establishment of CGN model was evaluated by detection of 24 h proteinuria.After 4 weeks,the model was established by intragastric administration of Sal B at a dose of 200 mg/kg per day for 2 weeks.The rats in miR-145-5p inhibition group were injected with 140 nM miR-145-5p antagomir(hereinafter referred to as"Antagomir")via tail vein every other day for 2 weeks.The expression of miR-145-5p in renal tissue was detected by real-time fluorescence quantitative PCR(qPCR),the renal function was evaluated by serum creatinine(Scr)and serum urea nitrogen(Bun),and the renal pathology was observed by hematoxylin-eosin(HE)staining,Schiff periodate(PAS)staining and transmission electron microscope.Immunofluorescence staining of C3 complement and CD68 was used to observe renal immune deposition and inflammatory infiltration,and qPCR was used to detect IL-2,IL-6,TNF-αand NF-κB mRNA in renal tissue to evaluate renal inflammation.The level of renal autophagy was evaluated by Western blotting(WB).The expression and phosphorylation of PI3K/AKT/AS160 pathway,autophagy and Rab14 protein in renal tissue were detected by WB.2.In vitro,The model of cell inflammation and proliferation was established by human mesangial cell(HMC)treated with 20 μg/mL lipopoly sac charide(LPS)for 24 h,and cell proliferation was detected by CCK8.The administration group was treated with 20 μM Sal B,the miR-145-5p inhibition group was treated with 100 nM miR-145-5p inhibitor(hereinafter referred to as "Inhibitor").The miR-145-5p mimic group was transfected with 30 nM miR-145-5p mimic(hereinafter referred to as "Mimic"),and the pathway inhibitor group was treated with 40 nM LY294002.Cell cycle was detected by PI flow,and the autophagy flow was detected by MOI=300 transfection with autophagy double-labeled fluorescent adenovirus.Renal inflammation was evaluated through detecting IL-2,IL-6,TNF-α and NF-κB mRNA in renal tissue by qPCR,and the transfection effect of Mimic was evaluated by miR-145-5p level.The level of autophagy was evaluated by WB detection of LC3B and Beclinl proteins,and the expression and phosphorylation levels of PI3K/AKT/AS160 pathway and Rab14 were detected.The dual luciferase reporter vector was constructed and transfected into 293T cells to verify the targeted regulation relationship between miR-1455p and Rab14.Results1.Compared with model group,after Sal B intervention,24-hour proteinuria,Scr and Bun of CGN model rats was decreased.Renal pathology,renal immune deposition and inflammatory infiltration was significantly improved,and inflammatory factor secretion was decreased.The expression of miR-145-5p and the level of autophagy in kidney increased further after Sal B treatment in CGN rats.Compared with model group,after administration of Antagomir,the expression level of miR-145-5p in CGN model rats decreased significantly,and the level of renal autophagy decreased accordingly.Renal immune deposition and inflammatory infiltration were not improved and aggravated,but the difference was not statistically significant.2.Compared with Sal B group,after treatment with Sal B and Inhibitor at the same time,Sal B the effect of improving the proliferation and inflammation of HMC receded.Autophagy level decreased and autophagy activity was blocked in the early stage.Meanwhile,the expression of miR-145-5p was inhibited while the Rab14 protein and the phosphorylation level of PI3K/AKT/AS160 pathway increased.Compared with model group,Mimic and LY294002 could also improve the proliferation and inflammation of HMC,autophagy level increased and autophagy activity developed to the later stage,when the phosphorylation level of PI3K/AKT/AS160 pathway was inhibited and the expression of Rab14 decreased.3.Compared with the negative control,the transfection of miR-145-5p down-regulated wild-type Rab14,and the reported fluorescence of mutant Rab14 had no significant effect,but the down-regulation of mutant Rab14 did not reach the positive standard.It is likely that there is no obvious interaction between miR-145-5p and this site on the predicted Rab14 3’ UTR.ConclusionsTotally,Sal B promote the up-regulation of miR-145-5p and inhibit the expression of Rab14,promote the proliferation and autophagy of mesangial cells,and reduce the inflammatory response of mesangial cells,which is related to its effect on PI3K/AKT/AS160 signal pathway,and then improve the pathological progress of CGN.The results of this research clarify the mechanism of Sal B improving CGN and the molecular mechanism of miR-145-5p regulating renal autophagy.From the point of view of miR,it provides a new idea and treatment strategy for clinical prevention and treatment of CGN.