Screening Of Active Ingredients And Mechanism Of Action Of Formula Jiangu Capsule In Improving Osteoblast Function

Objective:Based on the traditional Chinese medicine theory of tonifying Qi of the kidney,and dominating bone,generating marrow,Formula Jiangu Capsule（FJC）has been developed for the treatment of osteoporosis.It has significant clinical effects and minimal toxic side effects,but its material basis and mechanism of action have not been reported.The aim of this study is to establish an oxidative damage model to evaluate the efficacy of FJC in improving osteoblast function.The chemical composition and plasma components of the FJC are identified and analyzed,and the pharmacological substance basis that can improve osteoblast activity is screened.The molecular mechanism by which it improves osteoblast mineralization function is elucidated at both in vivo and in vitro.Methods:（1）HPLC fingerprint analysis was performed on FJC.Bone mesenchymal stem cells were extracted from the femurs of SD rats,which were induced differentiation into osteoblasts.Establish an oxidative damage model of osteoblasts using H₂O₂,different concentrations of FJC were applied to the model,and the cell viability and morphological changes were detected through MTT and optical microscopy;Flow cytometry and enzyme-linked immunosorbent assay were used to detect ROS production and changes in antioxidant enzyme activity in osteoblasts;Flow cytometry and Western blot were used to detect the expression of cell apoptosis and mitochondrial apoptosis pathway related proteins;Alkaline phosphatase（ALP）staining,Alizarin Red S staining,RT-PCR,Western blot were used to detect degree of osteoblasts early-stage differentiation and late-stage mineralization,gene and protein expression of osteogenic marker factors.（2）The chemical composition of FJC was detected by liquid chromatogen-tandem mass spectrometry（LC-MS/MS）on C18column with 0.1%formic acid water（A）-acetonitrile（B）as mobile phase system,electrospray ion source,and positive and negative ion scanning modes.The chemical composition of plasma after the FJC was given by intragastric administration was analyzed,and the prototype components of the formula were screened.ROS production,total antioxidant capacity（T-AOC）,and ALP activity of osteoblasts were detected to identify the chemical components that could improve the activity of osteoblasts.Molecular docking was used to analyze the combination of the above chemical components and osteogenic transcription factor Runx2,and to clarify the material basis for subsequent FJC to improve the molecular mechanism of osteoblast function.（3）MTT and release of LDH are used to detect the effect of active ingredients on osteoblasts activity induced by oxidative damage;Flow cytometry and Western blot were used to detect changes in apoptosis and related protein expression;ALP staining,Alizarin Red S staining,and RT-PCR were used to detect the early-stage and early-stage differentiation levels of osteoblasts,as well as changes in the mRNA expression of osteogenic markers,to determine their regulatory effects on osteoblast activity and function.Western blot and immunofluorescence were used to analyze the expression of autophagy related proteins in osteoblasts;the mRNA expression related to autophagy initiation and extension was detected by RT-PCR;autophagy activator Rapa and inhibitor 3-MA were used to detect apoptosis and mineralization;Western blot was used to detect the protein expression level of the autophagy related signaling pathway AMPK-mTOR-ULK,to clarify the regulatory mechanism of active ingredients on autophagy.In vivo,bone tissue microstructure,blood biochemical indicators,bone tissue growth,bone formation,and autophagy protein expression were detected by Micro-CT,ELISA,HE staining,and immunohistochemistry based on D-galactose induced osteoporosis mice,so as to systematically elucidate the effect of active ingredients in FJC on improving osteogenic function.Result:（1）Clarify the main components and stability of FJC.It was found that FJC significantly inhibit the decrease cell viability induced by H₂O₂,restore cell morphology,significantly reduce intracellular ROS production,increase the activity of antioxidant enzymes,inhibit apoptosis,reduce the expression of CytC and Cleaved Caspase3 proteins,and increase the Bcl-2/Bax ratio.In terms of osteoblast differentiation ability,FJC significantly increased the expression and activity of ALP induced by H₂O₂in early-stage differentiation of osteoblasts,and promoted the accumulation of calcium nodules in late differentiation.It also significantly increased the mRNA and protein levels of Runx2 and Osx,as well as downstream regulatory factors Col Ⅰ and BMP2.（2）A total of 33 chemical components were identified based on accurate relative molecular mass,combined with reference products and database analysis,and 25 chemical components were identified in plasma samples,including 15 prototype components.The effects of stilbene glycoside（TSG）,pinoresinol diglucoside（PDG）,emodin（EMO）and ecatechin（EC）on H₂O₂-induced ROS production,T-AOC changes and ALP activity of osteoblasts were detected.Comprehensive evaluation and screening determined that TSG had the best effect on resisting oxidative damage and enhancing osteogenic differentiation.The molecular docking of TSG,PDG,EMO,EC with Runx2 showed that all bind to Runx2,and TSG has the strongest binding ability with Runx2,at-9.0 KJ/mol.（3）TSG enhance cell viability and reduce LDH release induced by H₂O₂;apoptosis and related protein expression were inhibited;The expression of ALP and calcium nodules in osteoblast differentiation were significantly increased,and the mRNA expression of osteogenic markers was positively regulated by TSG.In addition,TSG upregulated the expression of LC3 and BECN1,downregulates the expression of p62,and increases the mRNA expression of FIP200,VPS34,ATG5,and ATG7,which were key factors of autophagy,to resist oxidative damage induced by H₂O₂.After 3-MA pretreatment,TSG was found to reduce the ability of inhibiting apoptosis and promoting calcium nodules deposition,and TSG exhibited a similar effect to Rapa,which were regulated by the AMPK-mTOR ULK signaling pathway.In addition,TSG significantly improved the microstructure of bone tissue induced by D-galactose,increased serum calcium and phosphorus levels,and decreasedβ-CTX,enhanced the expression of Col Ⅰ and OPN bone forming proteins,as well as the autophagy marker LC3,clarifying the role of TSG as the main material basis for FJC in improving osteoblast function.Conclusion:This study found that FJC could inhibit apoptosis of osteoblasts,increase the expression of osteogenic differentiation markers,and improve osteoblast dysfunction induced by oxidative damage.After the chemical components and plasma components of this formula were identified,TSG was the most effective active ingredient that could resist oxidative damage and enhance the differentiation of osteoblasts.TSG activates the autophagy pathway through the AMPK-mTOR-ULK signaling pathway,increases the expression of autophagy related genes,inhibits apoptosis of osteoblasts,enhances the expression and differentiation of cellular functional factors,and improves the mineralization function of osteoblasts induced by oxidative damage.In vivo,TSG can increase bone density,improve bone microstructure,and increase bone forming protein expression in bone loss model mice,which has potential preventive and therapeutic effects on osteoporosis.