The Effect Of Osteopractic Total Flavone On The Cathepsin K And OPG/RANKL/RANK System In Rat Model Of Osteoporosis

Object:To explore the rationale and mechanism for the treatment of osteoporosis with osteopractic total flavones,by observing the effect of osteopractic total flavone on the indexes of the Cathepsin K and OPG/RANKL/RANK system in the rat model of osteoporosis,measuring the bone mineral densities,estrogen levels before and after drug administration,as well as HE and toluidine blue staining.Methods:180 healthy SD rats that weigh 230～280g were used for this study.The rats were randomly divided into 7 groups:blank group,model group,sham group,treatment group 1(VOX+HIGH OTF),treatment group 2(VOX+MIDDLE OTF),treatment group 3(VOX+LOW OTF)&positive control group(estradiol valerate).Bilateral ovariectomy was performed on all the rats to build animal models of osteoporosis.One and a half month later,the modeling was finished,and intragastric administration of high,medium and low-dose OTF was performed on the rats in the treatment groups once daily,and estradiol valerate was given intragastrically to the rats in the control group once daily.At 1.5,3&6 months after the drug administration,the rats were euthanized,and their femurs and blood were collected.Then the indexes of the Cathepsin K and OPG/RANKL/RANK system,the bone mineral density and the estrogen level were determined,and HE and toluidine blue staining were done.SPSS 17.0 software was adopted for result analysis.Statistical analysis was done using t test for comparison between two samples,variance analysis for comparison among multiple samples,and F test.t=mean ± SD(x±s)was used in the t test,and p-value threshold of 0.05 and 95%confidence intervals were used in the one-way analysis of variance.Statistical graph were designed by using the graphpad prism 5.01 software.Results:The BMD measurements right after the modeling showed statistically significant difference(p<0.05)between the treatment groups and the estrogen group vs.the sham group and the blank group,suggesting the success of the modeling.At 3 months after the modeling,BMDs of the rats from the treatment groups and the estrogen group increased,statistically significant compared to those of the sham group and the blank group(p<0.05),and there was no significant difference in BMD between the treatment groups and the estrogen group(p>0.05),indicating that 3 months’ medication improved the BMDs of the osteoporotic rats and that OTF and estrogen had therapeutic effect on the osteoporotic rats.At 6 months after the treatment,the BMDs of the rats from the treatment groups and the estrogen group were significantly increased compared to immediately after the modeling(p<0.05),and the bone mineral contents were further increased significantly,though not to the normal level,and the comparison between the treatment groups and the estrogen group vs.the sham group and the blank group showed no statistically significant difference(p>0.05).The HE and toluidine blue staining of the bones of the osteoporotic rats demonstrated that OTF could reduce trabecular bone loss,increase trabecular width and improve bone microstructure.Expression of Cathepsin K mRNA in the tibial metaphysis of the osteoporotic rats measured immediately after the modeling showed higher Cathepsin K mRNA expression level in the treatment groups and the estrogen group than in the sham group and the blank group.The expression level in the treatment groups and the estrogen group decreased at 3 months,and further decreased at 6 months(p<0.05 in the treatment group 1 and the estrogen group).The decrease in the treatment group 1 was not so much as that in the estrogen group.The expression levels in the treatment groups were significantly different from those in the sham group and the control group(p<0.05).The test of the serum OPG/RANKL/RANK levels in the osteoporotic rats showed significantly decreased serum estradiol and OPG level and significantly increased RANKL and RANK level in the rats from the model group,and slightly decreased serum estradiol and OPG level and slightly increased RANKL and RANK level in the rats from the sham group.After 3 months,rats from the model group and the sham group showed no significant change in serum estradiol,OPG,RANKL and RANK level.At 3 months,the rats from the treatment groups showed increased serum estradiol and OPG level and decreased RANKL and RANK level statistically significant compared to those from the model group.Comparison between the treatment groups and the control group showed the highest efficacy in the treatment group 1,which was equal to that in the control group,followed by that in the treatment group 2,and then the treatment group 3.Conclusion:It is proved in this study that OTF can increase the estrogen level and BMD,improve the bone microstructure,increase the bone mineral content,reduce the Cathepsin K mRNA expression in the tibial metaphysis,and inhibit the bone resorption of the osteoclasts in the rat model of osteoporosis.Treatment group 1 showed slightly poorer,still satisfactory though,outcome compared to the estrogen group,which is significantly better than that of the treatment group 2&3.The results indicated that that estrogen is correlated to OPG,RANKL and RANK,that estrogen and OTF have an effect on the expression of OPG,RANKL and RANK,.and that the content of OPG,RANKL and RANK will change more and more significantly as the dose of OTF increases.