Experimental Study On Bone Protective Effect And Mechanism Of Guizhi-shaoyao-zhimu Decoction In Rheumatoid Arthritis

Guizhi-Shaoyao-Zhimu Decoction(GSZD),as a classic decoction of the "Golden Chamber",has a good effect on the treatment of rheumatoid arthritis,but there is no systematic study on its mechanism and material basis.This topic takes GSZD as the entry point of anti-RA bone destruction,and explores the specific biological mechanism and material basis of the bone protective effect of this decoction,so as to provide laboratory basis for the precise clinical application of GSZD in the treatment of RA.Methods:(1)To evaluate the osteoprotective effect of GSZD on RA.Collagen-induced arthritis model was established,and the improvement of joint swelling symptoms was evaluated by arthritis index and paw volume.The protection degree of GSZD against joint bone destruction was shown by HE staining,saffron solid green staining and Micro CT.The effect of this prescription on osteoclasts was determined by TRAP staining,ELISA detection of proinflammatory factor,RANKL and OPG levels.Combined with network pharmacology,build GSZD effective chemical composition and targets of database,combined with rheumatoid arthritis disease targets,to establish "drug-candidate compounds-targets-disease" network,choose the node degrees higher to analysis protein to protein interaction network,GO and KEGG enrichment,and screened the osteoclast differentiation related targets and pathways.Through computer aided technology,and the role in screening GSZD targets(TRAF6,c-Jun,I ? B-?,p65,p38 lightning,ERK and JNK)with high combination of value composition,by explaining the GSZD virtual screening technology from molecular level of bone protection material basis.CCK8 method was used to detect the effect of GSZD on RAW264.7 cells,and the dose which had no obvious effect on cell activity was selected.RAW264.7 cells were induced by RANKL to differentiate into osteoclasts.TRAP staining,bone absorption staining,and ELISA were used to detect Trap5 b,Cathepsin K,and MMP-9to assess bone resorption function.NF-?B signaling pathway related proteins were detected by WB.(2)To evaluate the protective effect of GSZD on systemic bone loss and explore the specific target and mechanism of the protective effect of this decoction.GSZD was used to intervene the rat model of systemic osteoporosis.The improvement effect of GSZD on osteoporosis was evaluated by HE,Masson and Micro CT,and the effect of TRAP staining on the number of osteoclasts was shown.ALP,Trap5 b,Cathepsin K and MMP-9 in bone homogenate were detected by ELISA,and protein and gene expressions of ALP,RANKL and OPG were detected by WB and QPCR.The osteoblasts from rat skull were cultured,and the appropriate dose was screened by CCK8 method to intervene the induction of bone nodules of osteoblasts.The expression level of ALP in cell culture medium was detected by ELISA to evaluate the effect of GSZD on osteoblasts.Rat bone marrow-derived mononuclear macrophages were cultured and induced to differentiate into osteoclasts by RANKL and M-CSF.CCK8 method to screen out the suitable drug concentration,intervention with cells at different concentrations,using the TRAP staining,bone absorption staining to assess bone resorption function,with ELISA and QPCR to detect TRAP5 b,Cathepsin K,the protein and gene expression of MMP-9,WB testing TRAF6,c-fos,NFATc1 and I?B-?,p65 /p-p65,p38 /p-p38,ERK/p-ERK,JNK/p-JNK protein expression level.Results:(1)GSZD can reduce the damage of bone and joint in CIA rats,and its mechanism of bone protection may be to inhibit the differentiation and maturation of osteoclasts through multi-target and multi-pathway.GSZD can significantly reduce joint swelling,arthritis score and toe volume in CIA model rats.At the same time,this formula can reduce the destruction of bone,synovium and cartilage in the ankle of CIA model rats,and reduce the number of osteoclasts in the ankle joint.Serum indexes of CIA rats showed that GSZD could reduce the expression of TNF-?,M-CSF,IL-1?,IL-6,IL-8,IL-17 A and other pro-inflammatory factors,and it was found that GSZD could reduce the level of RANKL in synovium and serum of CIA model rats,and regulate the ratio of OPG/RANKL.RAW264.7 cells were treated with 200,400 and 800?g/ m L doses by CCK8 assay.Interventions of RANKL-induced RAW264.7 cells with different doses of GSZD reduced the number of mature osteoclasts,the area of bone resorption depression,and the levels of bone resorption-specific cytokines Trap5 b,MMP-9,and Cathepsin K.WB results showed that GSZD inhibited the degradation of I?B? and reduced the nuclear translocation and transcriptional activity of p65.The network pharmacological results showed that 180 chemical active components of GSZD and 4604 targets related to RA disease were collected and collated.The PPI,GO and KEGG analysis showed that GSZD could play a therapeutic role on RA through multiple targets and pathways,such as regulating immunity,inhibiting inflammation,and promoting synovial cell apoptosis.This decoction can directly inhibit osteoclast differentiation to achieve the effect of improving bone destruction,mainly related to TNF,Jun,PPARG,Rela,IKBKB,Akt1,MAPK8,STAT1,MAPK14 and other targets,involved in PI3K-Akt,NF-?B,MAPK,JAK-STAT and other signaling pathways.Through virtual screening,99 compounds with high degree of binding between GSZD and 7 action targets(TRAF6,c-Jun,I?B-?,p65,p38,ERK,JNK)were screened out,which indicated that GSZD could directly inhibit the differentiation of OC.(2)GSZD can improve systemic osteoporosis in OP rats.It can inhibit the initiation of osteoclast differentiation in vitro,and reduce the expression of downstream bone resorption-specific factors by inhibiting the phosphorylated proteins of NF-?B and MAPK signaling pathways.High dose GSZD group can improve the bone density of osteoporosis rats,inhibit the thinning of growth plate in the distal femoral epiphysis,reduce the number and thickness of bone trabeculae,and improve the connection of bone trabeculae.It can also reduce the amount of OC.GSZD increased the expression of ALP and decreased the expression of MMP-9,Trap5 b,and Cathepsin K.WB and QPCR results suggested that this decoction could regulate the balance of RANKL/OPG.After screening by CCK8 test,200,40 and 800ug/m L GSZD were administered to intervene osteoblasts.The experimental results showed that GSZD had no significant effect on the formation of bone nodules and ALP secretion of osteoblasts.CCK8 assay showed that GSZD inhibited osteoclast differentiation and bone resorption at doses of 200,400 and 800?g/ m L,and reduced levels of Trap5 b,MMP-9,and Cathepsin K in cell culture medium.GSZD can inhibit the activation of TRAF6,reduce the phosphorylated proteins of NF-?B and MAPK signaling pathways,and inhibit the expression of downstream c-fos and NFATC1.Conclusion:(1)In vivo studies have shown that GSZD can not only alleviate the bone destruction of local joints in the rats with rheumatoid arthritis,but also improve the bone loss in the rats with systemic osteoporosis,thus clarifying the biological effect of this method on bone protection.This provides a laboratory basis for the precise clinical application of GSZD in the treatment of RA.On the basis of syndrome differentiation and treatment,this decoction is suitable for RA patients with early clinical stage or existing bone joint destruction and osteoporosis.(2)In vitro experiments,the target and mechanism of bone protection of GSZD were further studied.The experimental results showed that GSZD could inhibit the activation of TRAF6,reduce the phosphorylation of key proteins in NF-?B and MAPK pathways,and then regulate the expression of c-fos and NFATC1,thereby reducing the secretion of bone resorption-specific proteins,thus achieving the effect of bone protection.In addition to the direct effect of this decoction on osteoclasts,the regulation of bone metabolism may also be related to the indirect intervention of inflammation and immune regulation on bone metabolism,which provides research ideas for subsequent experiments.Shortcomings and Prospects:(1)Due to the short modeling time of CIA model,there had no experimental study on systemic bone loss of such model,so the osteoporosis model was selected in the second chapter of this study.Although the experimental results suggest that this prescription has an inhibitory effect on systemic bone loss,only the high-dose group has a significant therapeutic effect,which does not indicate the therapeutic effect of GSZDn on systemic bone loss of RA.Whether elderly rats or rats with idiopathic arthritis are more suitable for this study remains to be explored.(2)This study only researched the downstream targets and pathways that affect the differentiation and maturation of osteoclasts,and only conducted a preliminary exploration on one aspect of the mechanism of GSZD against RA bone loss,lacking of depth.In addition,this formula may also regulate bone metabolism from the upstream by regulating immunity and reducing pro-inflammatory factors,inhibiting osteoclasts and alleviating bone loss,which can be further studied in subsequent experiments.(3)After virtual screening of the material basis of bone protection of GSZD,due to lack of time,we did not extract the screened compounds and carry out relevant experimental verification,and lack of in-depth exploration of the bone protection effect of GSZD.