Study Of Pathological Mechanism And Intervention By Nourishing Kidney-yin Method On Kidney-yin Deficiency Syndrome In Postmenopausal Osteoporosis Rats

Objective:Based on the syndrome differentiation results of pre-clinical studies,the main disease location of postmenopausal osteoporosis is kidney,and the main disease is yin deficiency.This subject uses Erzhi pill as a probe.With the Sirtl/Runx2 signaling pathway which mediating bone metabolic disorders as the starting point,from the perspective of prescriptions and cross-references,the mechanism of action of nourishing kidney-yin method for postmenopausal osteoporosis was partly explored.Methods:60 female Sprague Dawley rats in 3 months old,All rats were randomly divided into three groups consisting of 20 rats into sham operation group and 40 rats into operating group.Bilateral ovariectomy was performed in 40 female rats in the operating group.Approximately the skin,the abdominal cavity and the muscles were incised,and the ovaries were exposed.The oviduct was ligated with a silk thread and the ovariectomy was performed bilaterally,while the remaining 20 animals underwent a sham surgery in which the bilateral ovaries were examined and returned to the original position under the same protocol.After 2 weeks,the 40 rats of operating group were randomly divided into two groups.as follows:ovariectomized(OVX)and OVX + EZP groups.Rats belonging to OVX + EZP group were administered 0.3 g/ml EZP(3 ml/kg)orally by gavage,whereas those belonging to the Sham and OVX groups were given saline(3 ml/kg)orally by gavage.Body weights of rats were monitored every two weeks.The rats underwent treatment for 12 weeks.The microarchitecture of trabecular bone in the L5 was analyzed by micro-CT.The same specimen was scanned to obtain different section images and the L5 scans were performed in three spatial dimensions.The biomechanical properties of tibia and femur were determined in a three-point bending test by a universal testing machine.Serum estra diol(E2),sex hormone-binding globulin(SHBG),receptor activator of NF-kB ligand(RANKL),osteoprotegeri’n(OPG)and bone alkaline phosphatase(BALP)levels were performed using an ELISA kit according to the manufacturer’s instructions.Real-time polymerase chain reaction(PCR)assay and western blot assay were used to measure the expressions of Sirtl/Foxo3a axis related biomarkers(mRNA and protein),including Sirtl,Runx2,Foxol,Foxo3a and PPAR-y.Expression of miR-132 was measure using a TaqMan microRNA assay,a real-time PCR.Results:1.Micro-CT.The images with micro-CT and results from HE staining of the L5 of the OVX group revealed a significantly decreased trabecular bone volume compared with the sham group.The BMD,BV/TV and Tb.Th of OVX group were lower than those of sham group,and the Tb.Sp displayed the contrary tendency.After administration of EZP,upregulated the BMD and Tb.Th,and downregulated the Tb.Sp compared with the OVX group(P<0.01,P<0.05).2.HE staining.HE staining of the L5 of the OVX group exhibited significantly reduced furcation and a relatively scant marrow space compared with those in the sham group.3.The biomechanical function of tibia and femur.Tibia break point and femur break point of the OVX group were lower than that of sham group(P<0.01),After administration of EZP,upregulated tibia break point and femur break point compared with the OVX group(P<0.05).4.Bone metabolism biomarkers serum test results.The serum E2 level of OVX group was lower than that of sham group,and the serum SHBG level displayed the contrary tendency(P<0.01,P<0.01);the serum OPG,OPG/RANKL and BALP of OVX group was lower than those of sham group(P<0.01),and the serum RANKL showed the contrary tendency(P<0.01).There were no difference in the serum E2 and SHBG levels after administration of EZP(P>0.05).However,after administration of EZP,the biomarkers of bone metabolism OPG,OPG/RANKL and BALP in serum were higher than those of OVX group(P<0.05),and the RANKL in serum was lower than that of OVX group(P<0.01).5.The expressions of biomarkers related to the Sirtl/Foxo3a axis.Both the results of mRNA and protein exhibited the same tendency.All the key regulators of the Sirtl/Foxo3a axis exhibited significant difference with the Sham group(P<0.01,P<0.05).The expressions of both mRNA and protein of Sirtl,Runx2,Foxol and Foxo3a were upregulated by administration of EZP(P<0.01,P<0.05).The expressions of both mRNA and protein of PPAR-γ were downregulated by administration of EZP(P<0.01,P<0.05).6.The expressions of miR-132 in results of TaqMan microRNA assay.The results of TaqMan microRNA assay showed that expressions of the miR-132 in the OVX group was significant higher than those of the Sham group(P<0.01),the administration of EZP significantly downregulated the expression of miR-132 in the ovariectomized rat model(P<0.05).Conclusions:1.Erzhi Pill improves bone mineral density and Trabecular number in ovariectomized rats and enhances biomechanical properties.2.Erzhi pill can inhibit osteoclast differentiation,reduce bone resorption,and reduce bone loss by activating Sirtl/Runx2 signaling pathway,thereby inhibiting the pathological process of osteoporosis in ovariectomized rats.