The Mechanism Of Ginsenoside Rc In Osteoporotic Bone Formation

Osteoporosis is a metabolic disease characterized by systemic osteopenia and destruction of the microstructure of bone tissue.The basis of bone metabolism is the balance of osteoclasts and osteoblasts,and breaking the balance can lead to osteoporosis.Patients with osteoporosis have obvious alveolar bone resorption,loose teeth,and low implant survival rate,which seriously affects the oral health of patients with osteoporosis,and is also a difficult problem for dentists in clinical work.At present,the main clinical treatment for osteoporosis is a drug intervention,but the existing clinical drugs have side effects.Traditional Chinese medicine is known for its multiple pharmacological effects and mild adverse reactions.Ginseng is a precious traditional Chinese medicine with a long history of medicinal use and special effects.Its main active ingredient is ginsenoside.Among them,ginsenoside Rc belongs to panaxadiol-type saponins and is one of the main active components of many precious traditional Chinese medicines.Ginsenoside Rc possesses significant antiinflammatory and antioxidant effects.In recent years,some studies have found that ginsenosides can promote bone formation and inhibit bone resorption.In addition to Rc,protopanaxadiol-type ginsenosides also include Rb1,Rb2,Rb3,Rd,Rh2,and CK,etc.They have similar chemical structures,among which Rb2(Rc isomer),CK(a metabolite of Rc),and Rb1,Rd,and Rh2 all had high activity in promoting cell proliferation and osteogenesis.Therefore,it is predicted that ginsenoside Rc also can promote bone formation and treat osteoporosis.But at present,there is no report on the role of traditional Chinese medicine monomer ginsenoside Rc in osteoporotic bone formation.This article aims to explore the mechanism of ginsenoside Rc in osteoporotic bone formation.The research methods are as follows.First,the mechanism of ginsenosides in osteoporotic bone formation was explored based on network pharmacology.We selected 24 ginsenosides from the published literature,obtained the targets of ginsenosides from the Pubchem database,swisstargetpridiction database,etc.,and obtained the targets of osteoporosis from 4 disease databases such as Gene Cards.After taking the intersection of the two targets,a PPI network was constructed,and then GO and KEGG analysis were performed.Secondly,to explore the effect of ginsenoside Rc on osteoporosis mice,in the in vivo experimental part,the mouse OVX animal model was established,and 5 groups were set up,(1)sham operation group,(2)OVX + saline group,(3)OVX+25mg/kg ginsenoside Rc,(4)OVX+50mg/kg ginsenoside Rc,(5)OVX+1mg/kg estradiol,of which OVX+estradiol was a positive control.After 3 months of the administration,the samples were collected,and the effects of ginsenoside Rc on OVX mice were detected by Micro-CT,H&E staining,Masson staining,immunohistochemical staining,q RT-PCR,and Western Blot.Next,the effect of ginsenoside Rc on the proliferation and osteogenic differentiation of MC3T3-E1 cells was explored.By setting the concentration gradient of ginsenoside Rc in vitro,the effect of ginsenoside Rc on the proliferation and the osteogenic differentiation of MC3T3-E1 cells was detected by CCK-8,live-dead cell staining,ALP staining,alizarin red staining,q RT-PCR and Western Blot.Finally,when exploring the effect of ginsenoside Rc on the Wnt/β-Catenin signaling pathway in cells,the Wnt pathway inhibitor XAV-939 was used to group the cells as follows,(1)Ginsenoside Rc(-)XAV-939(-),(2)Ginsenoside Rc(+)XAV-939(-),(3)Ginsenoside Rc(+)XAV-939(+),(4)Ginsenoside Rc(-)XAV-939(+).After 14 days of culture,Western Blot detection was performed.The results of the study are as follows.288 ginsenoside-related targets,1391osteoporosis-related targets,and 74 common targets were obtained.The protein-protein interaction analysis found that SRC,AKT1,EGFR,VEGFA,STAT3,FGF2,MAPK1,etc.played a crucial role in the treatment of ginsenoside with osteoporosis.GO-BP analysis showed that the treatment of osteoporosis by ginsenosides may be related to the regulation of kinase activity and other biological processes.GO-CC showed that the cellular components involved were receptor complexes,etc.GO-MF showed that the molecular functions involved include multiple protein kinase activities.KEGG enrichment analysis showed that ginsenoside-osteoporosis-related pathways in bone remodeling include PI3K-Akt signaling pathway,Wnt signaling pathway,etc.Micro-CT showed that treatment of OVX mice with ginsenoside Rc significantly inhibited the sharp decrease of BMD,BV/TV,and Tb.N and the increase of Tb.Sp.Histological staining indicated that ginsenoside Rc could prevent osteopenia and improve bone structure in OVX mice.QRT-PCR and western blot showed that ginsenoside Rc prevented OVX-induced bone loss by promoting the m RNA and protein expression of bone formation-related genes.A low concentration of ginsenoside Rc can stimulate the proliferation of MC3T3-E1 cells.Live-dead cell staining and cytometry analyses showed that100-200 μmol/L ginsenoside Rc was not conducive to cell survival after day 5.ALP staining and viability assay,alizarin red staining,and quantitative analysis showed that ginsenoside Rc dose-dependently promoted the osteogenic differentiation of MC3T3-E1 cells.It was concluded that 50 μmol/L ginsenoside Rc was the optimum concentration to promote the osteogenic differentiation of MC3T3-E1 cells.50μmol/L ginsenoside Rc can promote the expression of m RNA and protein of osteogenesis-related genes.Ginsenoside Rc-induced increases in the expression ofβ-catenin,p-GSK-3β,COL-1,ALP,and RUNX-2 were significantly attenuated by 10μmol/L XAV-939 treatment,while p-β-catenin and the decrease in GSK-3β expression were significantly increased.The conclusions of the study are as follows.Ginsenosides may treat osteoporosis by activating SRC,AKT1,EGFR,VEGFA,STAT3,FGF2,MAPK1,etc.,and regulating the PI3K-Akt,MAPK,Wnt signaling pathway,etc.In vivo experiments show that ginsenoside Rc can prevent bone loss caused by OVX,enhance the microstructure of trabecular bone,and promote the m RNA and protein expression of bone formation-related genes.In vitro experiments show that 50 μmol/L ginsenoside Rc can promote cell proliferation,osteogenic differentiation,mineralization,and the m RNA and protein expression of osteogenic-related genes,including COL-1,ALP,RUNX-2,BMP-2,OCN.Ginsenoside Rc may promote the osteogenic differentiation of MC3T3-E1 cells by activating the canonical Wnt pathway.