Study On The Effect And Mechanism Of ZhijingSan In The Treatment Of Bone Erosion In CIA Mice

Background:Rheumatoid arthritis(RA)is a typical inflammatory joint disease,which shows great harmfulness and high disability rate in RA patients.Joint bone erosion is the most difficult part of RA treatment,and various factors are combined to regulate the progress of bone erosion,in which the over-differentiation of osteoclasts is the central link,and activation of RANKL/NF-κB signaling pathway has been proved to be crucial in osteoclastogenesis.Traditional Chinese medicine has become popular as an alternative intervention to treat the serious chronic disease.It is of great significance to seek innovative drugs for RA treatment from traditional Chinese medicine,which is also a part of current new drug development.ZhiJingSan(ZJS),a traditional Chinese prescription composed of scolopendra(dried body of Scolopendra subspinipes mutilans L.Koch,scolopendridae)and scorpion(dried body of Buthus martensii Karsch,Buthus),which has been used to alleviate pain and rheumatism.It has been reported that a powder mixture of scolopendra and scorpion could improve joint swelling in collagen-induced arthritis(CIA)rats.However,the bone protection effect and possible mechanism of ZJS in RA have not been clearly defined.In this study,we investigated the bone protection effect and explored the underlying mechanism of ZJS in CIA mice in order to provide a new therapy for the treatment of RA.Research Methods:In vivo study,CIA murine model was established by bovine type Ⅱ collagen immunization.The oneset of arthritis,arthritic score,hind paw swelling,joint pathology,cytokine levels and bone erosion in CIA mice were determined to validate the therapeutic effect of ZJS on arthritis severity and bone erosion in CIA mice.In vitro study,osteoclast differentiation model was established,the effect of ZJS on osteoclastogenesis was characterized by TRAP staining and the expression levels of genes and proteins related to osteoclast differentiation as well as RANKL/NF-κB signaling pathway were detected by Western Blot and RT-qPCR,so as to explore the mechanism of ZJS in preventing bone erosion.In toxicity study,the acute toxicity and the long-term toxicity of human clinical equivalent dose of ZJS were preliminarily carried out to evaluate the toxicity of ZJS on ICR mice.Results:In vivo results,ZJS delayed the onset of arthritis and ameliorated arthritis severity.Whats more,ZJS inhibited joint bone erosion in CIA mice after continuous administration for one month and three months.Additionally,ZJS decreased the number of osteoclasts and the expression of cathepsin K in the ankle joints of CIA mice.In vitro study results also revealed that ZJS inhibited osteoclast differentiation and the expression levels of marker genes and proteins(cathepsin K,MMP9).Further research found that ZJS inhibited osteoclast differentiation by suppressing the RANKL/NF-κB signaling pathway.In vivo toxicity study,there was no toxicity in ICR mice after the maximum daily dose of ZJS(16 g/kg).In the long-term toxicity of human clinical equivalent dose study,the continuous administration of ZJS for three months significantly slowed down the rate of the weight gain of mice;increased the liver function indexes ALT,AST,y-GT and enal function index BUN;several hematology indexes were abnormal.Conclusion:ZJS prevented bone erosion in CIA mice by inhibting RANKL/NF-κB-mediated osteoclast differentiation.The daily oral administration at a maximal dose of ZJS which is equivalent to 960 times the clinical dose of human had no significant toxicity in ICR mice;long-term oral administration of ZJS(0.18 g/kg/d)for three months had a significant toxic effect on ICR mice.