Shenqi compound ameliorates renal fibrosis in diabetic kidney disease by inhibiting LRG1/TGF-β/Smad signaling pathway

Abstract

Background: Diabetic kidney disease (DKD) is one of the most common microvascular complications of diabetes. The progression of DKD to end-stage renal disease (ESRD) is characterized by glomerulosclerosis and tubulointerstitial fibrosis. Under hyperglycemic conditions, renal tubular epithelial cells undergo extracellular matrix (ECM) deposition, which ultimately leads to renal fibrosis. Current therapeutic strategies for DKD are confronted with tough challenges such as lack of specific therapy and multiple adverse effects. Shenqi Compound (SQ), a traditional Chinese medicine compound formula, has demonstrated favorable treatment effect in diabetes. However, the potential therapeutic effects of SQ on DKD and ECM deposition remain insufficiently explored.

Purpose: This study aimed to investigate the role of SQ in alleviating DKD and elucidated its underlying mechanisms in ECM deposition associated with DKD.

Methods: In vivo experiments, a DKD rat model was established by high-fat diet feeding and intraperitoneal injection of streptozotocin. Different concentrations of SQ were administered by oral gavage for 10 weeks, with irbesartan (Avapro) used as a positive control. Renal pathological injuries were assessed using HE, PAS and Masson staining. Disease indicators and levels of pathway-associated proteins were detected via IHC, WB and ELISA. The active ingredients of SQ were identified using LC-MS/MS. Network pharmacology analysis, bioinformatics analysis, molecular docking, and molecular dynamics simulation were employed to validate the interaction between the active component of SQ, Tanshinone IIA (TAN IIA), and leucine-rich alpha-2-glycoprotein 1 (LRG1). In vitro experiments, human renal tubular epithelial cells (HK-2) were stimulated with TGF-β1 to induce ECM deposition. The effects of TAN IIA on LRG1/TGF-β/Smad signaling pathway-driven renal ECM deposition were evaluated using CCK-8 assay, wound healing assay, WB, and cell immunofluorescence.

Results: Animal experiments results demonstrated that SQ ameliorated blood glucose, body weight, and renal pathological injury in DKD rats. SQ suppressed the activation of the LRG1/TGF-β/Smad signaling pathway, thereby effectively attenuated renal ECM deposition and kidney fibrosis. The active ingredient TAN IIA and DKD-associated pathway target LRG1 were identified through LC-MS/MS and database analysis. Molecular docking, molecular dynamics simulations, and small molecule-protein binding experiments confirmed that TAN IIA stably bind to LRG1 and inhibited LRG1 protein activity. Cell experiments demonstrated that TAN IIA exhibited no significant cytotoxicity to HK-2, suppressed cell migration, and inhibited the activation of LRG1/TGF-β/Smad signaling pathway.

Conclusion: Our study demonstrated that SQ may ameliorate ECM-related renal fibrosis in DKD by inhibiting the LRG1/TGF-β/Smad signaling pathway. The active ingredient of SQ, TAN IIA, bind to the LRG1 in a targeted manner, suppressing LRG1 activity and exerting anti-fibrotic effects. These findings provide a more sufficient theoretical foundation for the clinical application of SQ in DKD treatment.

Keywords: Diabetic kidney disease; Extracellular matrix deposition; LRG1/TGF-β/Smad Signaling Pathway; Shenqi Compound.