Study On Kangshuqianggu Prescription Based On AKT/PPAR-γ Signaling Pathway Gufang Regulates Bone Fat Metabolism In Female Rats After Castration Mechanism

Objective:In order to explore the treatment mechanism of famous traditional Chinese medicine prescription-Kangshuqianggu prescription for postmenopausal osteoporosis（PMOP）,this study established a PMOP model by removing bilateral ovaries of female non-pregnant rats.To observe the effect of Kangshuqiang Gugan decoction on bone metabolism and bone microstructure in model rats,and to study the therapeutic effect of Kangshuqiang Gugan decoction on postmenopausal osteoporosis and the possible mechanism of action.The objective is to clarify whether Kangshuqianggu Fang regulates the changes of bone fat by activating AKT/PPAR-γsignaling pathway,and thus plays a role in improving postmenopausal osteoporosis.This study provides a new idea for exploring the mechanism of Kangshuqianggu prescription in the treatment of postmenopausal osteoporosis.Method:（1）Animal model construction:After 2 weeks of artificial feeding,6-week-old SPF grade female non-pregnant rats were randomly divided into sham operation group and model group according to body weight stratification.The model group was prepared by bilateral ovary removal,and the sham operation group was modeled by bilateral ovary removal,and the fourth lumbar vertebra（L4）was removed at 12 weeks after modeling.Micro-CT was used to observe the 3D reconstruction images of L4 cone and the changes of micro-structure parameters in two groups of rats,and to evaluate whether the PMOP model was established successfully.（2）Treatment experiment:After 2 weeks of artificial feeding,48 6-week-old SPF female non-pregnant rats were randomly divided into SHAM group（SHAM group）,model group（OVX group）,Kangshujianggufang high-dose group（HIGH group）,medium-dose group（MEDIUM group）,low-dose group（LOW group）and alendronate sodium group（ALLSN group）according to their body weight.Each group consisted of8 animals.In addition to the SHAM group,the isopagic fat around bilateral ovaries was removed,and the classical bilateral ovaries were removed in other groups to establish the model.After modeling,SHAM group and OVX group were intragastric with equal volume of normal saline（0.9%）.The concentrations of Kangshujianggufang decoction in HIGH,MEDIUM and LOW groups were 37.708g/kg,18.854g/kg and 9.427g/kg,respectively,and ALLSN group was intragastric with 1.042mg/kg.After 12 weeks of intervention,the patients were anesthetized by intraperitoneal injection of 10%pentobarbital sodium.After abdominal aorta blood was taken,all patients were euthanized.The lumbar vertebrae（L1-L4）were removed for further follow-up experiments.（3）Serum Bone gla protein（BGP）,Estradiol（E₂）andβ-Collagen degradation product of rats in each group were detected and compared by enzyme-linked immunosorbent assay（ELISA）.β-CTX)and Amino terminal propeptide of type I procollagen（PINP）.（4）Hematoxylin-eosin staining（HE staining）was used to detect pathological changes and differences of L4 vertebral bone trabecula in rats.（5）Micro-CT was used to observe the 3D reconstruction of L4 vertebrae and the changes of micro-structural parameters,including Bone mineral density（BMD）,Bone volume（BV）,Bone volume/Trabecular volume,bone density（BMD）and Bone volume（BV）.BV/TV),Trabecular thickness（Tb.Th）,Trabecular number（Tb.N）,Bone surface area and tissue volume（Bone surface/trabecular）volume,BS/TV),Trabecular separation/Spacing（Tb.Sp）;（6）Quantitative real-time PCR（qRT-PCR）was used to detect the expression of AKT mRNA and PPAR-γmRNA in lumbar vertebra（L1-L3）of each group,and the differences between the groups were analyzed.（7）Western Blot（WB）was used to detect the changes of AKT and PPAR-γprotein expression in lumbar vertebra（L1-L3）of each group.Results:（1）Compared with SHAM group,the bone trabecular distance of L4 vertebral body in OVX group was enlarged,the bone trabecular became thinner and thinner,and the continuity was interrupted obviously,showing the pathological state of osteoporosis.BMD,BV,BV/TV,Tb.Th,Tb.N,BS/TV of L4 cone were significantly decreased,and Tb.Sp was significantly increased（P<0.05）.The results indicate that the model of postmenopausal osteoporosis has been successfully constructed,which can be used for subsequent experimental research.（2）ELISA results showed that compared with SHAM group,serum E₂and PINP contents in OVX group were significantly decreased,while serum BGP andβ-CTX contents were significantly increased（P<0.05）.Compared with OVX group,the contents of E₂and PINP in serum in HIGH,MEDIUM and ALLSN groups were significantly increased,and the contents of BGP andβ-CTX were significantly decreased（P<0.05）,but there was no significant difference in PINP in LOW group（P>0.05）.The levels of E₂and PINP in MEDIUM group were the most increased.BGP andβ-CTX are reduced most.（3）HE staining results showed that L4 pyramidal bone trabeculae in the SHAM group were dense,plump and strong,and presented a complete and continuous network structure,while the trabeculae in the OVX group were sparse,the continuity part was interrupted,and the arrangement was irregular.After drug intervention,the trabeculae of the rats were significantly improved,and the continuity was good,and the structure was complete.（4）Micro-CT results showed that compared with SHAM group,BMD,BV,BV/TV,Tb.Th,Tb.N and BS/TV of L4 cone in OVX group were significantly decreased,while Tb.Sp was significantly increased（P<0.05）.Compared with OVX group,the contents of BMD,BV,BV/TV,Tb.Th,Tb.N and BS/TV in all administration groups were significantly increased,while the contents of Tb.Sp were significantly decreased（P<0.05）,but there was no significant difference in Tb.Th in LOW group（P>0.05）.In the MEDIUM group,BMD,BV,BV/TV,Tb.Th,Tb.N,BS/TV were increased,and Tb.Sp were decreased.（5）Compared with SHAM group,AKT mRNA in OVX group was significantly decreased and PPAR-γmRNA was significantly increased（P<0.05）.Compared with OVX group,AKT mRNA was significantly increased and PPAR-γmRNA was significantly decreased in all treatment groups（P<0.05）.（6）Compared with SHAM group,the expression of AKT and PPAR-γin OVX group was significantly decreased（P<0.05）;Compared with OVX group,AKT protein expression was significantly increased and PPAR-γprotein expression was significantly decreased（P<0.05）,and MEDIUM group had the best improvement effect.Conclusion:In this study,a PMOP model was successfully constructed using the classical method of bilateral ovaries removal in female non-pregnant rats.After treatment with Kangshuqianggu formula,bone metabolism can be significantly improved,and the occurrence of postmenopausal osteoporosis in rats can be delayed or reduced,including significantly increasing serum E₂,PINP,lumbar bone density and bone mass,and significantly reducing the levels of BGP andβ-CTX,and the medium-dose treatment effect of Kangshuqianggu formula is the best.Further mechanism study showed that Kangshuqianggu Fang may down-regulate PPAR-γmRNA and protein levels by up-regulating AKT mRNA and protein levels in lumbar vertebra of rats.The results of this study indicate that Kangshuqiang Gufang may exert its anti-PMOP effect by activating the AKT-PPAR-γsignaling pathway,which provides a new idea for exploring the mechanism of Kangshuqiang Gufang in treating PMOP.