Identification of Rab7 as an autophagy marker: potential therapeutic approaches and the effect of Qi Teng Xiao Zhuo granule in chronic glomerulonephritis.

CONTEXT: Qi Teng Xiao Zhuo granule (QTXZG) is a traditional Chinese medicine (TCM) used for therapeutic effects on chronic glomerulonephritis (CGN). However, the underlying mechanism remains unclear. OBJECTIVE: To investigate the molecular mechanism of QTXZG on CGN by proteomics. MATERIALS AND METHODS: The CGN model was induced in Sprague-Dawley rats by injecting adriamycin (3.5 mg/kg, Day 1; 3.0 mg/kg, Day 14) twice through the tail vein. Urine samples were collected on the 21st day; and the rats divided randomly into control, adriamycin, QTXZG administration groups. Rats in the QTXZG group received QTXZG (10.8 g/kg); control and adriamycin groups were given physiological saline once per day for 30 days. Proteomics was applied to identify the candidate proteins combined with autophagy database and verified by immunofluorescence (IF) and western blots (WB). RESULTS: 278 differentially expressed proteins (DEPs) were identified based on proteomics and Rab7 was screened as an autophagy protein biomarker. In vitro cell experiments, we found that QTXZG (20%, IC50 = 23.47%) could decrease the expression of NLRP3, Caspase-1, IL-18, IL-1ß, while increasing the expression of Pink1, Parkin, Rab7, Podocalyxin. The cell apoptosis rate increased from 6.68 ± 0.07 to 11.03 ± 0.36%. Overexpression of Rab7 resulted in an increase in autophagy relevant protein expression. DISCUSSION AND CONCLUSION: TCM CGN-regulating herbs (QTXZG) can exert therapeutic effects by affecting the Rab7/Pink1/Parkin pathway to promote mitochondrial autophagy. New breakthroughs in targeted Rab7 may eventually enable such applications.

A Novel Method for Identifying Parkin Binding Agents in Complex Preparations of Herbal Medicines.

Parkin is a crucial E3 ubiquitin ligase for initiating mitophagy through the PINK1/Parkin pathway. Regulating the expression and activity of parkin can remedy mitophagy and human disease. We developed an efficient method to isolate natural parkin ligands from herbal medicines by combining centrifugal ultrafiltration and liquid chromatography/mass spectrometry. The heterologous expression technology identified functionally active and pure parkin proteins. After evaluating the reliability of the method using DL-selenomethionine and DL-dithiothreitol as positive controls, this method was successfully applied to capture parkin ligands from Polygoni Cuspidati Rhizoma et Radix and Sophorae Flavescentis Radix. LC/MS identified seven novel parkin-targeting compounds, namely, 7,4'-dihydroxy-5-methoxy-8-(γ, γ-dimethylallyl)-flavanone, kushenol I, kurarinone, sophoraflavanone G, torachrysone-8-O-glucoside, apigenin, and emodin, supported by the molecular docking analysis. Five of the seven novel compounds (kushenol I, kurarinone, sophoraflavanone G, apigenin, and emodin) can activate parkin in in vitro autoubiquitination assays. Meanwhile, kushenol I and kurarinone had antisteatosis activity in fat emulsion-damaged human hepatocytes. These results confirmed the effectiveness of the method for identifying parkin ligands from complex preparations, useful to advance drug discovery from medicinal herbs.