Study On The Theoretical Basis And Therapeutic Mechanism Of Zuoguiwan In The Treatment Of PMOP Based On Sympathetic Inhibition

ObjectiveObserving the expression of ?2-adrenergic receptor(?2-AR)mRNA and protein in the tibia and hypothalamus of ovariectomized osteoporosis rats,exploring the possible pathogenesis of postmenopausal osteoporosis(PMOP),and finding the pharmacodynamics and possible mechanism of Zuoguiwan at the same time.Providing theoretical and experimental references for the clinical application of Zuoguiwan in the treatment of PMOPMethods50 female SD rats were randomly divided into sham operation group(10 rats)and model group(40 rats).The postmenopausal osteoporosis rat model was established by castration.The remaining 36 rats in the model group were randomly divided into model group,estradiol group,low dose Zuoguiwan group and high dose Zuoguiwan group,with 9 rats in each group.After 12 weeks of intragastric administration,the bone mineral density(BMD),cancellous bone three-dimensional morphology and bone parameters(Bone volume fraction(BV/TV),Trabecular Thickness(Tb.Th),Trabecular Number(Tb.N)and Trabecular Separation(Tb.Sp))were detected by micro-CT(u-CT).the morphological changes of bone tissue were observed by Hematoxylin and eosin(HE)staining.The markers of bone turnover in serum bone alkaline phosphatase(BALP),?cross-linked C-telopeptide of type 1 collagen(?-CTX)and btarte-resistant acid phosphatase(TRAP-5b)were mesured using enzyme-linked immunosorbent assay(ELISA).The mRNA expression of of ?2-AR,OPG and RANKL mRNA in the tibia and hypothalamus were measured by qPCR.The protein expression of tibia ?2-AR,OPG and RANKL and The protein expression of hypothalamus ?2-AR were measured by Western blot(WB).Result(1)BMD:Compared with the sham operation group,the BMD of the femur in the model group was significantly lower(P<0.01);compared with the model group,the BMD of the femur in the Low dose group of Zuoguiwan,High dose group of Zuoguiwan and the estradiol group was significantly increased(P<0.05;P<0.01),but the difference between the three groups was not statistically significant(P>0.05)(2)Bone histomorphology:?-CT three-dimensional image shows that compared with the sham operation group,the trabecular bone of the model group is obviously sparse,the number is reduced,the thickness is reduced,the fracture is widened,the spacing is widened,and the trabecular bone structure has a large blank.region.Compared with the model group,the trabecular bone of the low dose group of Zuoguiwan,high dose group of Zuoguiwan and the estradiol group became denser,the continuity increased,the void ratio decreased,and the microstructure improved significantly.HE staining showed that compared with the sham operation group,the trabecular bone of the model group was obviously sparse,thin,incomplete structure,loosely arranged,and there were a large number of empty bone lacuna,and the trabecular spacing was widened.Compared with the model group,the number of trabeculae in low dose group of Zuoguiwan,high dose group of Zuoguiwan and the estradiol group was significantly increased,the distribution was uniform,the structure was complete,the arrangement was acceptable,the trabecular empty bone lacuna was reduced,and the spacing was narrowed(P>0.05)(3)Cancellous bone parameters:Compared with the sham operation group,BV/TV,Tb.Th and Tb.N in the model group were significantly decreased(P<0.01)and Tb.Sp was significantly increased(P<0.01);Compared with the group,the BV/TV,Tb.Th,Tb.N in the low dose group of Zuoguiwan,high dose group of Zuoguiwan and the estradiol group were significantly increased(P<0.01)and Tb.Sp was significantly decreased(P<0.01);Compared with the estradiol group,there was no significant difference between the indicators of the low dose group of Zuoguiwan,high dose group of Zuoguiwan and no statistical significance(P>0.05)(4)Serum bone metabolism index:Compared with the sham operation group,the serum BALP of the model group was significantly decreased(P<0.01)and the ?-CTX and TRAP-5b were significantly increased(P<0.01).Compared with the model group,the Serum BALP was significantly increased in the low dose group of Zuoguiwan,high dose group of Zuoguiwan and the estradiol group(P<0.01),while ?-CTX and TRAP-5b were significantly decreased(P<0.01)Compared with estradiol group,the Serum TRAP-5b in the low dose group of Zuoguiwan was elevated(P<0.05)(5)mRNA expression:Compared with the sham operation group,the expression of OPG mRNA in the tibia and hypothalamus of the model group was significantly decreased(P<0.01);P<0.05)and the expression level of RANKL mRNA was significantly increased(P<0.01;P<0.05);Compared with the model group,the expression level of OPG mRNA in the tibia and hypothalamus of the low dose group of Zuoguiwan,high dose group of Zuoguiwan and the estradiol group was increased(P<0.05;P<0.01)and the expression levels of ?2-AR,RANKL mRNA were decreased to different extents(P<0.01;P<0.05).Compared with the estradiol group,the expression level of OPG mRNA in the hypothalamus of the low-dose group of Zuoguiwan and the expression level of ?2-AR mRNA in the hypothalamus of the high-dose group of Zuoguiwan was increased(P<0,05).(6)Protein expression:Compared with the sham operation group,the expression of ?2-AR protein in the tibia and hypothalamus of the model group was significantly increased(P<0.01),and the expression level of RANKL protein in the tibia was also significantly increased(P<0.01),while the expression level of OPG protein was significantly decreased(P<0.01).Compared with the model group,the expression levels of ?2-AR protein in the tibia and hypothalamus of the low dose group of Zuoguiwan,high dose group of Zuoguiwan and the estradiol group were different(P<0.05;P<0.01),the expression level of RANKL protein in tibia was also significantly decreased(P<0.01)and the expression level of OPG protein in tibia was increased(P<0.01;P<0.05).Compared with estradiol group,there were no significant differences in the low dose group of Zuoguiwan and high dose group of Zuoguiwan.Conclusion(1)The formation of PMOP may be associated with increased sympathetic excitability,activation the ?2-AR of the tibia and hypothalamus,which in turn affects the OPG/RANKL/RANK system.(2)Zuoguiwan can promote bone formation by inhibiting bone resorption,thereby reversing PMOP high conversion bone status,increasing BMD and delaying bone loss.(3)Zuoguiwan can affect OPG/RANKL/RANK system by lowering the level of beta 2-AR in bone tissue and hypothalamus and inhibiting the excitability of sympathetic nerve,thus affecting bone remodeling may be one of the mechanisms of Zuoguiwan in the treatment of PMOP.