Modified Zuo Gui Wan Ameliorates Ovariectomy-Induced Osteoporosis in Rats by Regulating the SCFA-GPR41-p38MAPK Signaling Pathway.

Objective: Modified Zuo Gui Wan (MZGW) was a combination of Zuo Gui Wan and red yeast rice used for treating osteoporosis (OP), but its mechanism remains unclear. We aimed to validate the anti-OP effect of MZGW and explore its underlying mechanism.

Methods: An ovariectomy (OVX) rat model in vivo and a RANKL-induced osteoclasts (OCs) model in vitro were established. Key active ingredients in MZGW high dose (MZGW-H) group were detected by UPLC-MS/MS. Micro-CT scans and histomorphology analysis were performed in OVX rats. 16S rRNA gene sequencing was performed to investigate the relationship between the anti-OP effect of MZGW-H and intestinal flora. CCK-8 assay was applied to examine the optimal concentration of Modified Zuo Gui Wan drug serum (MZGW-DS) on osteoclasts. The qRT-PCR and Western blotting were utilized to explore the potential anti-OP pathway of MZGW, namely the SCFA-GPR41-p38MAPK signaling pathway. GPR41 was knocked down to further reverse to verify whether the pathway was the key pathway for MZGW-DS to exert its inhibitory effect on osteoclasts.

Results: The three main blood components, Ferulic acid, L-Ascorbic acid and Riboflavin, were examined mainly by UPLC-MS/MS. 16S rRNA gene sequencing showed that MZGW-H changed the metabolism of SCFAs. In vivo studies verified that MZGW-H ameliorated microstructure damage, improved histological changes and reduced TRAP, BALP, and BGP in OVX rats by regulating the SCFA-GPR41-p38MAPK signaling pathway. CCK-8 revealed that 5% MZGW-DS group was the most optimal concentration of MZGW-DS to inhibit osteoclast differentiation. In vitro, MZGW-DS was better than peripheral blood concentration of SCFAs in inhibiting osteoclasts. After the knockout of GPR41, MZGW-DS could not inhibit the expression of osteoclast-related protein (CTSK and NFATc1) via SCFA-GPR41-p38MAPK signaling pathway.

Conclusion: MZGW-H effectively ameliorates OVX-induced osteoporosis partially achieved by increasing SCFAs metabolism and modulating the SCFA-GPR41-p38MAPK signaling pathway.