Effect Of Jiaweiyanghe Decoction Th17/Treg Balance In Osteoporosis Model Mice

Objective:To explore the potential mechanism of Jiaweiyanghe decoction in the prevention and treatment of postmenopausal osteoporosis by studying the effect of Jiaweiyanghe decoction on helper T cells（Th17）and regulatory T cells（Treg）in a mouse model of ovariectomized osteoporosis.Methods:Forty-eight 8-week-old SPF mice were randomly divided into normal group（SHAM）,model group（OVX）,estradiol valerate group（EV）,and Jiawei Yanghe decoction high-dose,medium-dose,and low-dose groups（JH,JM,JL）,with 8 mice in each group.Except for the SHAM group,the ovaries of other groups were harvested to establish the osteoporosis mouse model.According to the conversion of human and mouse body surface area and the preliminary experimental dose exploration,the dose of each group was determined as estradiol valerate group(0.13g·kg^-1),Jiaweiyanghe decoction high-dose group(0.26g·kg^-1),medium-dose group(0.13g·kg^-1),low-dose group(0.065g·kg^-1),respectively.The model group and normal group were given the same volume of normal saline,and the samples were taken 90 days after intervention.The expressions of retinoic acid associated nuclear receptorγt（RORγt）,forkhead box protein p3（Foxp3）,bone-specific alkaline phosphatase（BALP）,osteocalcin（BGP）and interleukin-10（IL-10）in serum of mice were detected by enzyme-linked immunosorbent assay（ELISA）.The bone mineral density and mineral content of femur and tibia were detected by dual energy X-ray absorptiometry.Western blotting and q RT-PCR were used to detect the expression levels of Foxp3,RORγt,interleukin-10（IL-10）and their m RNA in bone tissue.HE staining was used to observe the pathological morphology of bone tissue.The percentage of positive cells expressing RORγt and Foxp3 was detected by immunohistochemistry.In order to investigate its mechanism of action,the group used in vitro assays to observe the proliferation and differentiation of mouse osteoblast precursor cells（MC3T3-E1）after intervention with different concentrations of drug-containing serum by alizarin red staining;the effect of each drug-containing serum on the viability of osteoblasts was detected by cell proliferation toxicity assay（CCK-8）;the expression levels of target proteins and osteogenic-related proteins were detected by Westernblot.The expression levels of target proteins and osteogenic related proteins were detected by Westernblot.Results:（1）ELISA showed that The serum levels of RORγt decreased and the levels of Foxp3,BALP,BGP and IL-10 increased in the mice in the model group intervened by Jiaweiyanghe decoction,among which,the changes were significant in the middle and high concentration groups（P<0.05）,but the increase was not significant in the low concentration group（P>0.05）.（2）Dual-energy X-ray bone densitometry showed that the BMD and bone mineral content of mice in the model group decreased compared with the normal group,and the PMOP mouse model was successfully established,and BMD and BMC in every groups were elevated significantly（P<0.05）.（3）Westernblot and q RT-PCR showed（in vivo）that compared with the model group,the expression of Foxp3,IL-10 and their m RNAs were significantly higher（P<0.01）and the expression of RORγt and their m RNAs were significantly lower（P<0.01）in bone tissues of the medium and high concentration groups of Jiaweiyanghe decoction;the expression of Foxp3,RORγt and their m RNAs in the low dose group of Jiaweiyanghe decoction Foxp3,RORγt,IL-10 and their m RNA expressions in bone tissue were not significantly changed in the low-dose group plus Jiaweiyanghe decoction group（P>0.05）.（4）HE staining showed that compared with the model group,the trabecular bone in Jiaweiyanghe decoction medium and high groups was thickened,the connectivity was increased,the spacing was smaller,the holes were reduced,the empty bone lacunae were reduced,and the structure was relatively intact.（5）Immunohistochemistry showed that the medium and high concentration groups of Jiaweiyanghe decoction could significantly up-regulate the Treg rate and down-regulate the Th17 rate in bone tissue（P<0.01）,but the changes of Th17 and Treg rate in bone tissue of Jiaweiyanghe decoction group in the low dose group were not significant compared with the model group（P>0.05）.（6）Cell proliferation toxicity assay（CCK-8）showed that the drug-containing serum of mice in medium and high concentration groups had a significant promoting effect on osteogenic proliferation and differentiation（P<0.05）,which was positively correlated with dose and time,and no cytotoxic reaction was observed in each group.（7）Alizarin red staining showed that compared with the model group,mineralized deposition increased in Jiaweiyanghe decoction groups,and increased significantly in medium-high concentration group（P<0.01）;There was no significant increase of mineralization area in low concentration group（P>0.01）.（8）Westernblot（in vitro）:compared with the model group,the expression of OPG and osteoblast-specific proteins Runx2 and RANKL were significantly increased after serum intervention in the medium and high concentration groups of Jiaweiyanghe decoction（P<0.01）,while the expression of OPG and osteoblast-specific proteins Runx2 and RANKL did not change significantly after serum intervention in the medium and high concentration groups of Jiaweiyanghe decoction（P>0.05）.Conclusion:（1）The possible way to prevent and treat PMOP by adding Jiaweiyanghe decoctionis by up-regulating the expression of Foxp3 and down-regulating the expression of RORγt,thus correcting the imbalance between helper T cells（Th17）and regulatory T cells（Treg）in the PMOP model,thus achieving the goal of preventing and treating PMOP.（2）One of the possible ways to prevent and treat PMOP by adding Jiaweiyanghe decoction is by promoting the secretion of cytokines such as IL-10 by regulatory T cells（Treg）,which can promote the proliferation and differentiation of osteoblasts.