Yiqi Huoxue Recipe Ameliorates Diabetic Nephropathy by Mediating VAPB-PTPIP51 Complex to Activate Autophagy and Regulate MAM Contact

Linghao Dai¹Mengyi Wang²Bo Wang²Haowei Liang³Yang Guan²Zhongyan Du^2*Hui Wang^1*

• ¹Jinhua Academy of Zhejiang Chinese Medical University, Jinhua, China
• ²Zhejiang Chinese Medical University, Hangzhou, China
• ³Fenghua District Traditional Chinese Medicine Hospital of Ningbo, Ningbo, China

Background: Diabetic nephropathy (DN), a major complication of DM, poses a high mortality and health burden globally. Mitochondria-associated endoplasmic reticulum membranes (MAM) and mediated autophagy are regarded as the crucial factors for DN. Yiqi Huoxue recipe (YHR), a traditional Chinese medicine formula, was reported to treat DN and regulate autophagy, while the mechanism remains unclear. Methods: Firstly, UPLC-MS/MS analysis was performed to identify chemicals in YHR. Then, C57BL/6J mice were injected with streptozotocin and feed with high-fat diet to induce DN, then YHR (7.8, 15.6g/kg/d) were intragastric gavaged for 8 weeks. Biochemical and oxidative stress indicators were determined, H&E, PAS, and immunohistochemistry staining of nephrin were performed. Mitochondrial Ca2+ was detected by flow cytometry, autophagosomes and MAM were observed by TEM. The expression of VAPB, PTPIP51, LC3 II/I, P62 were detected with Western blot. Podocytes overexpressing PTPIP51 or VAPB were analyzed for cell activity with CCK-8 assay, autophagy flux by TEM, and the expression of LC3 II/I and P62 through Western blot. In si-PTPIP51 transfected and high glucose (HG) stimulated podocytes, CCK8, PCR, TEM and immunofluorescence staining were performed to detect the YHR-containing serum on cell activity, mtDNA, MAM, autophagosomes and LC3 expression. Results: The chemical fingerprint of YHR was constructed and composed chemicals. In DN mice, YHR treatment decreased the elevated fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), blood urea nitrogen (BUN), serum creatinine (Scr), urinary albuminuria (ALB), and microscale albuminuria (MAU) levels, alleviated kidney and glomerulus damages, mitochondrial Ca2+, oxidative stress, MAM abnormal contact, and activated the autophagy. The enhanced expression of MAM complex, VAPB-PTPIP51, were also inhibited in YHR groups. The cell activity and autophagosome formation were significantly inhibited in oe-PTPIP51 group and oe-VAPB group. In contrast, in HG-podocytes, si-PTPIP51 promoted the cell activity, mtDNA copy number, MAM contact, autophagosomes formation and LC3 expression. More importantly, the addition of YHR-containing serum enhanced this effect. Conclusion: YHR may protect kidneys in DN by regulating the MAM complex VAPB-PTPIP51 to trigger autophagy, providing insights into TCM's clinical application and DN drug development.