| Abjective:This study observed Treg/Th17 cells differentiation and the change of their related factors Foxp3 and RORγT through animal experiments,which explore the immune regulatory mechanism of Xuling Jiangu Formula on OP.Methods:Healthy female Sprague-Dawley(SD)rats of SPF grade,aged 6 months,were randomly divided into sham operation group,model group,and Xuling Jiangu Formula group,with 8 rats in each group.In addition to the sham operation group,the other two groups constructed ovariectomized OP model.One month after molding,the rats were continuously administered by gavage at a dose conversion multiple of 6.25 times for rats,at dose of 1ml/100 g per day for 16 weeks.In the end,the abdominal aorta blood,spleen,thymus,and bone tissue of the rats were collected.Then,the tibial bone density of the rats was measured.HE staining was used to observe pathological changes,Calculate spleen and thymus index,detect blood routine,and detect IL-10,TGF-β,IL-6 and IL-17 contentin serum by ELISA,and detect the m RNA and protein expression of OPG,RANKL,Foxp3,RORγT in bone tissue by RT-PCR,Western Blot,and flow cytometry was used to detect the number of Th17 and Treg cells.Software such as SPSS24.0,Image J,Graph Pad Prism 8,and Flow Jo10.8.1 were used to analyze the data.Results:(1)Dual energy X-ray: Compared with the sham operation group,the bone mineral density of the model group decreased(P<0.05);Compared with the model group,the bone mineral density in the Xuling Jiangu Formula group was significantly higher(P<0.05).(2)HE staining: Compared with Sham group,the number of bone trabeculae in OVX group was significantly reduced and incomplete,the bone trabeculae was sparse and not connected into a network,most of which were fractured,with a large amount of fibrous tissue,and a large number of vacuolar adipocytes in the medullary cavity.Compared with OVX group,the number of trabeculae in Xuling Jiangu Formula group increased,and the structure was significantly improved,similar to that in the sham group.The white adipocytes in the Xuling Jiangu Formula group also decreased.(3)ELISA: Compared with the sham operation group,the serum levels of IL-17 and IL-6 in the osteoporosis model group were significantly increased(P<0.05 or 0.01),the serum levels of IL-10 and TGF-βin the Xuling Jiangu Formula group were significantly decreased;Compared with the osteoporosis model group,the serum levels of IL-17 and IL-6 in the Xuling Jiangu Formula group were significantly decreased(P<0.05 or 0.01),the serum levels of IL-10 and TGF-βin the Xuling Jiangu Formula group were significantly increased.(4)RT-PCR: Compared with the sham operation group,the expression of Foxp3 and OPG m RNA in the osteoporosis model group decreased,the expression of RORγT and RANKL m RNA increased(P<0.05 or 0.01);Compared with the osteoporosis model group,the expression of Foxp3 and OPG m RNA in the Xuling Jiangu Formula group increased,the expression of RORγT m RNA decreased(P<0.05 or 0.01);The expression of RANKL m RNA tends to decrease.(5)Wstern Blot: Compared with the sham operation group,the expression of Foxp3 and OPG proteins in the osteoporosis model group decreased,The expression of RORγT and RANKL proteins increased(P<0.05 or 0.01);Compared with the osteoporosis model group,the expression of Foxp3 and OPG proteins in the Xuling Jiangu Formula group increased,and the expression of RORγT and RANKL proteins decreased(P<0.05 or 0.01).(6)Flow cytometry: Compared with the sham operation group,the number of Treg cells in the osteoporosis model group decreased,while the number of Th17 cells in the osteoporosis model group increased;Compared with the osteoporosis model group,the number of Treg cells in the Xuling Jiangu Formula group increased(P<0.05),the number of Th17 cells decreased.Conclusions:Xuling Jiangu Formula can regulate the differentiation of Treg and Th17 subsets of immune cells and their related factors,thereby regulating the OPG/RANKL pathway,inhibiting the differentiation of osteoclast,and improving the bone density of rats.This effect is one of the mechanisms of Xuling Jiangu Formula in treating OP. |
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