Spectrum-Effect Relationship and Component Knock-Out or Knock-In in Total Flavones of Abelmoschus manihot

Abstract

Background Diabetic kidney disease(DKD) is one of the microvascular complications of diabetes. The total flavones of Abelmoschus Manihot (TFA) has been widely used in China to reduce podocyte injury in DKD, however, not each flavone monomer can play the same effect. In a specific disease or pharmacological model, there may also be a group of chemical components with clear composition and content ratio that can play the same role as Abelmoschus Manihot extract. Methods The composition of the seven flavone monomers was investigated by spectrum-effect relationship and component knock-out and knock-in methods. We used HPLC to establish the chemical fingerprints, and assayed the protective effect and anti-inflammatory activity of podocytes in vitro. The protein expression level of synaptopodin and nephrin were measured by immunofluorescence staining, and the release of inflammatory factors of TNF-α and IL-1β were determined by enzyme-linked immunosorbent assay (ELISA) or real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Partial least squares method and multi-objective optimization were used to examine the spectrum-effect relationships. Then, we also determined the expression of TRPC6 in podocytes by RT-qPCR and western blot. Results When the compatibility proportion of rutin, hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, and quercetin was 0, 1.2, 0.88, 0.88, 0.48, 2.08, 0.4, the protein expression of synaptopodin and nephrin may be equivalent to TFA. And when the compatibility proportionof 7 active components was 0, 2.08, 1.74, 1.50, 0.12, 0.54, 0.1, which decreased the mRNA expression of TNF-α. Further, we found that B (hibifolin/hyperoside), C (hyperoside/quercetin-3'-o-glucoside), and E (isoquercetin/quercetin-3'-o-glucoside) have significant effects on synaptopodin, nephrin, and TNF- α, IL-1β. Collectively, our data indicated that TFA could ameliorate cytoskeleton rearrangement and inflammatory injury in podocytes under hign glucose conditions, and the proportion of hyperoside, isoquercitrin, hibifolin and quercetin-3'-o-glucoside had the similar efficacy, they may be the main active components of TFA. What’s more, the best combination of hyperoside, isoquercetin, hibifolin and quercetin-3'-o-glucoside decreased the expression of TRPC6. Conclusion The combination of hyperoside, isoquercetin, hibifolin, and quercetin-3'-o-glucoside in TFA was optimized by spectral efficiency relationship, and flavone monomer combination may play a protective role in podocyte cytoskeleton through TRPC6 pathway.