温阳消癥方调控TFEB介导的自噬流稳态缓解腺嘌呤诱导的肾间质纤维化OA

[Objective]To investigate the effect of Wenyang Xiaozheng Decoction on adenine-induced renal interstitial fibrosis(RIF)associated with chronic renal failure(CRF),and clarify its molecular mechanism.[Methods]Sixty male SD rats were randomly divided into blank control group,model group,Wenyang Xiaozheng Decoction group,autophagy inhibitor 3-methyladenine(3-MA)group,autophagy inducer rapamycin group and autophagy inducer trehalose group,with 10 rats in each group.Except for blank control group,the other groups were given 250 mg/(kg·d)adenine suspension by gavage for 4 weeks to establish the RIF model.After successful modeling,blank control group and model group were given 20 mL·kg-1 distilled water by gavage,once a day;Wenyang Xiaozheng Decoction group was given 2.4 g·mL-1 Wenyang Xiaozheng Decoction by gavage,once a day;3-MA group was given 15 mg·kg-1 3-MA intraperitoneal injection,once every other day;rapamycin group was given 1.5 mg·kg-1 rapamycin diluted with 0.9%sodium chloride injection by gavage,once a day;trehalose group was given 5%trehalose solution 20 mL·kg-1 by gavage,once a day,all groups were intervened for 4 weeks.At the end of 4th week(post-modeling)and 8th week(post-intervention),Scr,BUN,24-hour urinary protein levels and renal reactive oxygen species(ROS)content were detected.Real-time PCR was used to detect the mRNA expressions of microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),Beclin-1,p62,mammalian target of rapamycin(mTOR)and transcription factor EB(TFEB)in renal tissues.Western blot was used to detect the protein expressions of the above genes.HE staining and Masson staining were used to observe renal pathological morphology and quantify the degree of fibrosis.[Results]Compared with blank control group,the model group showed symptoms such as polydipsia,polyuria,decreased appetite and listlessness,with significant weight loss(P<0.01),significantly increased Scr,BUN,24-hour urinary protein and renal ROS content(P<0.01);the mRNA and protein expressions of LC3-Ⅱ,Beclin-1 in renal tissues were significantly up-regulated(P<0.05,P<0.01),while the protein expressions of p62 and mTOR were significantly down-regulated(P<0.05);severe renal pathological damage,massive collagen fiber deposition and significantly aggravated fibrosis were observed.Compared with model group,after 4 weeks,Wenyang Xiaozheng Decoction group showed significantly increased body weight(P<0.01).Both the Wenyang Xiaozheng Decoction and 3-MA groups demonstrated significantly decreased levels of Scr,BUN,24-hour urinary protein and renal ROS content(P<0.01,P<0.05).Concurrently,both treatments significantly down-regulated LC3-Ⅱ and Beclin-1 expression at both mRNA and protein levels(P<0.01,P<0.05),while significantly up-regulating p62 mRNA and protein expression(P<0.01,P<0.05).Additionally,TFEB mRNA expression was significantly reduced in 3-MA group(P<0.05);renal pathological damage was alleviated,collagen fiber deposition was reduced,and fibrosis was significantly alleviated.Compared with model group,there was no significant improvement in the above biochemical indicators,autophagy and TFEB-related indicators in rapamycin group and trehalose groups;among them,the 24 hour urinary protein level,LC3-Ⅱ and Beclin-1 expressions in rapamycin group were even further increased,only TFEB expression in trehalose group was significantly increased,and the pathological damage and fibrosis degree in both groups were not significantly relieved.No significant differences were found in all indicators between Wenyang Xiaozheng Decoction group and 3-MA group(P>0.05),and both were superior to rapamycin group and trehalose group(P<0.05,P<0.01).[Conclusion]Wenyang Xiaozheng Decoction alleviates adenine-induced RIF through TFEB-dependent restoration of autophagic flux and suppression of oxidative stress,implicating the TFEB-mediated autophagic pathway as a critical therapeutic target.