Song Changheng, Yan Qiqi, Ma Yujie, Li Pei, Yang Ying, Wang Yuhan, Li Wenjie, Wan Xinyu, Li Yubo, Zhu Ruyuan, Liu Haixia, Zhang Zhiguo
Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, People's Republic of China.
Department of Endocrinology, Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, People's Republic of China.
Drug Des Devel Ther. 2024 Dec 27;18:6359-6377. doi: 10.2147/DDDT.S482965. eCollection 2024.
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.
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.
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.
MZGW-H effectively ameliorates OVX-induced osteoporosis partially achieved by increasing SCFAs metabolism and modulating the SCFA-GPR41-p38MAPK signaling pathway.
加味左归丸（MZGW）是左归丸与红曲米的组合物，用于治疗骨质疏松症（OP），但其作用机制尚不清楚。我们旨在验证MZGW的抗骨质疏松作用并探讨其潜在机制。
建立体内去卵巢（OVX）大鼠模型和体外RANKL诱导的破骨细胞（OCs）模型。采用超高效液相色谱-串联质谱法（UPLC-MS/MS）检测MZGW高剂量组（MZGW-H）中的关键活性成分。对OVX大鼠进行显微CT扫描和组织形态学分析。进行16S rRNA基因测序以研究MZGW-H的抗骨质疏松作用与肠道菌群之间的关系。采用CCK-8法检测加味左归丸含药血清（MZGW-DS）对破骨细胞的最佳作用浓度。利用qRT-PCR和蛋白质印迹法探索MZGW潜在的抗骨质疏松途径，即短链脂肪酸-GPR41-p38丝裂原活化蛋白激酶信号通路。敲低GPR41以进一步反向验证该途径是否为MZGW-DS对破骨细胞发挥抑制作用的关键途径。
通过UPLC-MS/MS主要检测到三种主要血液成分，阿魏酸、L-抗坏血酸和核黄素。16S rRNA基因测序表明MZGW-H改变了短链脂肪酸的代谢。体内研究证实，MZGW-H通过调节短链脂肪酸-GPR41-p38丝裂原活化蛋白激酶信号通路改善了OVX大鼠的微观结构损伤，改善了组织学变化，并降低了抗酒石酸酸性磷酸酶（TRAP）、骨碱性磷酸酶（BALP）和骨钙素（BGP）水平。CCK-8结果显示5% MZGW-DS组是抑制破骨细胞分化的MZGW-DS最佳浓度。在体外，MZGW-DS在抑制破骨细胞方面优于外周血短链脂肪酸浓度。敲低GPR41后，MZGW-DS不能通过短链脂肪酸-GPR41-p38丝裂原活化蛋白激酶信号通路抑制破骨细胞相关蛋白（组织蛋白酶K和活化T细胞核因子1）的表达。
MZGW-H通过增加短链脂肪酸代谢和调节短链脂肪酸-GPR41-p38丝裂原活化蛋白激酶信号通路有效改善OVX诱导的骨质疏松症。
