| ObjectiveBased on the previous studies of my research group,this study will be carried out from two aspects:theoretical discussion and animal experiment.The theoretical part combs the understanding of postmenopausal osteoporosis(PMOP)in traditional Chinese and Western medicine,and discusses the theoretical basis of "phlegm turbidity" as the pathogenic factor of PMOP,so as to provide theoretical support for subsequent experimental research.The experimental part is based on the high correlation between "phlegm turbidity"and lipid metabolism disorder,and between lipid metabolism disorder and PMOP.Taking the central and peripheral regulation effect of leptin(LPN)on lipid/bone metabolism as the starting point,the PMOP model is established by removing bilateral ovaries(Ovariectomized,OVX)of rats,and the intervention effect of Bushen Huatan Decoction on lipid/bone metabolism of model rats is observed,as well as its effect on LPN hypothalamic neuropeptide Y(NPY)axis,and the cellular and molecular mechanism of bone lipid balance and homeostasis,in order to preliminarily clarify the central and peripheral mechanisms of Bushen Huatan Decoction in reducing postmenopausal bone loss and regulating lipid metabolism,so as to provide experimental basis for "treating PMOP from phlegm".Methods1.Theoretical researchThrough the research methods of combing ancient books and literature retrieval,this paper summarizes the ancient and modern doctors'understanding of the clinical manifestations,etiology,pathogenesis,prevention and treatment of PMOP.From a large number of basic,clinical and epidemiological investigations at home and abroad,this paper summarizes the role of lipid/bone metabolism disorder caused by estrogen deficiency in the pathogenesis of PMOP,as well as the close relationship between "phlegm turbidity" and lipid metabolism disorder and PMOP,so as to lay a theoretical foundation for "treating PMOP from phlegm" and provide a theoretical evidence for experimental research.2.Experimental researchSeventy-five 6-month-old SPF-grade infertile female SD rats were divided into sham-operated group,model group,Bushen Huatan Decoction group,atorvastatin group and Livial group according to random number table method,with 15 rats in each group.In the sham-operated group,only the fat tissue of the same quantity around the ovary was removed,while in the other 4 groups,both ovaries were removed to establish PMOP model.At the 5th week after operation,the rats were given intragastric administration for 8 weeks:9.4 g/kg for Bushen Huatan Decoction group,0.92 mg/kg for atorvastatin group,0.23 mg/kg for Livial group,2 ml normal saline once a day for model group and sham-operated group.Experiment 1:Hematoxylin-eosin(HE)staining was used to observe the pathological morphology of bone tissues and relative area of bone marrow adipose tissue(BMAT)in each group.The tibia bone mass,microstructure and body fat content were detected by Micro computed tomography(Micro CT).Enzyme linked immunosorbent assay(ELISA)was used to detect serum LPN levels.Experiment 2:Real-time fluorescence quantitative PCR(RT-PCR)was used to detect the expression of LPN,leptin receptor(OBR),NPY and the Y2 receptor mRNA in hypothalamus.The expression and localization of LPN,leptin receptor(OBR),NPY and the Y2 receptor proteins in hypothalamus were detected by western blotting(WB)and immunohistochemistry.Experiment 3:Transcriptional levels of PI3K/Akt/FoxO1 signaling pathway genes in hypothalamus of rats were detected by RT-PCR.The protein expression and phosphorylation of PI3K/Akt/FoxO1 signaling pathway in hypothalamus of rats were detected by WB.Experiment 4:The mRNA relative expressions of Runx2,PPAR?,LPN and OBR in bone tissues were detected by RT-PCR.The expression and localization of LPN and OBR proteins in bone tissues were detected by WB and immunohistochemistry.Experiment 5:The mRNA relative expression levels of ERK1/2 and P38MAPK in bone tissues were detected by RT-PCR.The WB was used to detect the expression and phosphorylation of ERK1/2 and P38MAPK in bone tissues proteins.Results1.Theoretical researchThe kidney is a water organ and deficiency of the kidney can produce phlegm.The deficiency of kidney essence in postmenopausal women is the fundamental pathogenesis of PMOP and provides the pathological basis for the internal production of phlegm.There is an inextricable link between bone and lipid metabolism and the regulation of bone and fat is related and interdependent.Estrogen deficiency in postmenopausal females leads to a disruption in the homeostasis of lipid-bone metabolism which has a critical role in the development of PMOP.Patients with phlegm tend to suffer from lipid dysfunction,and lipid metabolism-related disorders such as hyperlipidemia are most commonly associated with phlegm-dampness,and treatment of these disorders often begins with phlegm-dampness.Additionally,many phlegm-reducing herbs have lipid-lowering effects,and "phlegm-dampness" is one of the most common pathogenic traits shared by obesity and other lipid metabolismrelated diseases and PMOP.In terms of treatment,as PMOP improves,the disorders of lipid metabolism are also improved to varying degrees,and statin lipid-lowering drugs have been shown to have anti-osteoporotic effects.All of these prove that "phlegm" is closely related to lipid metabolism disorders and PMOP.Hence,"phlegm-dampness" is an important factor in the pathogenesis of PMOP,and "kidney deficiency and phlegm production" is the pivotal point in the traditional Chinese medicine pathogenesis of lipid metabolism disorders and PMOP.2.Experimental researchExperiment 1:Bushen Huatan Decoction can effectively improve lipid/bone metabolism in OVX rats.(1)Effect on bone metabolismHE dyeing showed that compared with sham-operated group,model group showed typical pathological manifestations of osteoporosis,such as reduced number of trabecular bone,decreased continuity,irregular arrangement,widened spacing,and increased fat content.Additionally,compared with model group,the bone BMAT relative area of sham-operated group,atorvastatin group,Livial group and Bushen Huatan Decoction group was significantly improved(P<0.05),which suggested that Bushen Huatan Decoction can effectively alleviate the pathological changes of postmenopausal bone tissue.The detective results of Micro CT showed that compared with shamoperated group,decreased of BMD,BV/TV,BS/BV,Tb.N,Tb.Th were observed while increased for Tb.Sp,BS/BV in Model group(P<0.05).Compared with the model group,the BMD,BV/TV,Tb.N and Tb.Th of the rats in the Atorvastatin,Levitra and Kidney Tonic Phlegm groups were significantly higher(P<0.05);Tb.Sp and BS/BV were significantly lower(P<0.05).It indicates that OVX rats showed decreased bone mass and bone microstructure destruction,suggesting that the formula of tonifying the kidney and resolving phlegm can effectively reduce the pathological changes of postmenopausal bone tissue.(2)Effect on lipid metabolismMicro CT showed that the abdominal circumference,subcutaneous fat volume and visceral fat volume of the model group increased significantly compared with sham operation group(P<0.05);And compared with the model group,the abdominal circumference,subcutaneous fat volume and visceral fat volume of rats in atorvastatin group,Livial group and Bushen Huatan group decreased significantly(P<0.05).From the detection of serum indexes of rats in each group,it can be seen that LPN in the model group decreased significantly compared with the sham operation group(P<0.05);Compared with the model group,the levels of LPN in atorvastatin group,Livial group and Bushen Huatan group increased significantly(P<0.05),indicating that the distribution and content of fat in OVX rats were abnormal and Bushen Huatan Decoction can effectively improve the lipid metabolism of rats and increase the level of serum LPN.Experiment 2:Bushen Huatan Decoction plays a central regulatory role on lipid and bone metabolism in OVX rats through LPN-NPY axis of hypothalamus.(1)Compared with sham operated group,the expression of LPN and OBR decreased significantly in the Model group(P<0.05)(P<0.01)while increase for NPY and Y2 receptor(P<0.01);Compared with the model group,the expression of LPN and OBR increased significantly in theatorvastatin group,Livial group,Bushen Huatan Decoction group(P<0.05)(P<0.01),while the expression of NPY and Y2 receptor decreased importantly(P<0.05)(P<0.01),The LPN and OBR of Bushen Huatan Decoction group were lower than those of atorvastatin group and Livial group(P<0.05),while the levels of NPY and Y2 in Bushen Huatan Decoction group and Livial groups were lower than those in atorvastatin group(P<0.05),suggesting that Bushen Huatan Decoction can up-regulate the expressions of LPN and OBR in hypothalamus and inhibit the secretion of NPY and its Y2 receptor in hypothalamus to plays a central regulatory role in improving the lipid and bone metabolism in OVX rats.(2)Compared with the model group,the decrease of NPY and Y2 in the hypothalamus were more significant than the increase of LEP and OBR in Bushen Huatan Decoction group,suggesting that the Bushen Huatan Decoction may also inhibit the expression of NPY in the OVX rats through other ways thus plays a central regulatory role.Experiment 3:Bushen Huatan Decoction regulates NPY expression in hypothalamus by activating PI3K/Akt/FoxO1 signaling pathway through LPN.Compared with sham operated group,a significantly increased expression for FoxO1 and a decreased expression for p-PI3K/PI3K?p-Akt/Akt?pFoxO1/FoxO1 were observed in hypothalamus tissue of model group(P<0.05).Compared with the model group,FoxO1 expression decreased significantly while the expression of p-PI3K/PI3K?p-Akt/Akt?p-FoxO1/FoxO1 increased significantly in atorvastatin group,Livial group and Bushen Huatan Decoction group.It is suggested that Bushen Huatan Decoction can increase the phosphorylation level of proteins in PI3K/Akt/FoxO1 pathway and downregulate FoxO1 expression in bone tissue,indicating that the central regulation effect of Bushen Huatan Decoction on lipid and bone metabolism in rats is completed through THE PI3K/Akt/FoxO1 signaling pathway via LPNhypothalamus NPY pathway.Experiment 4:LPN mediated the peripheral regulation effect of Bushen Huatan Decoction on lipid and bone metabolism in OVX rats.Compared with sham-operated group,the expression of LPN,OBR,Runx2 were significantly decreased and PPARy was evidently increased in the model group(P<0.05);compared with model group,the levels of LPN,OBR,Runx2 were significantly increased and PPAR? was obviously decreased in the bone tissues of atorvastatin group,Lival group and Bushen Huatan Decoction group.These results suggested that Bushen Huatan Decoction could promote the expression of LPN and OBR in bone tissue,up-regulate the expression of Runx2 to promote osteogenic differentiation,down-regulate the expression of PPARy to inhibit lipid differentiation and reduce abnormal accumulation of bone marrow fat,which can maintain bone lipid balance and reduce postmenopausal bone loss in OVX rats.Experiment 5:Effect of LPN on lipid/bone metabolism in OVX rats mediated by MAPKs signal system.Compared with sham-operated group,the expressions of ERK1/2,PERK1/2,P38MAPK and P-P38MAPK were significantly decreased in the bone tissues of the model group;Compared with the model group,the expression levels of ERK1/2,P-ERK1/2,P38MAPK and P-P38MAPK were significantly increased in the bone tissues of rats in atorvastatin group,Livial group and Bushen Huatan Decoction group,suggesting that the expression of LPN in bone tissues,the MAPKs signaling system was activated after Bushen Huatan Decoction formula up-regulated.It maintained bone lipid balance in OVX rats play a peripheral regulatory role in lipid and bone metabolism in OVX rats.Conclusions1."Treating PMOP from phlegm" is reasonable and feasible in terms of both TCM theory,pathophysiology and clinical application.2.Bushen Huatan Decoction can effectively improve lipid/bone metabolism in OVX rats,and has a good effect on the prevention and treatment of PMOP,the mechanism of which is related to the regulation of LPN.In the center,Bushen Huatan Decoction promoted the activation of PI3K/Akt/FoxO1 signaling pathway through LPN,inhibited the synthesis and secretion of NPY in the hypothalamus,and then realized the central regulation of lipid/bone metabolism;In bone tissue,Bushen Huatan Decoction can upregulate the expressions of LPN and OBR,promote osteogenic differentiation and inhibit lipid differentiation through MAPKs signaling system,thus maintaining the balance and homeostasis of bone lipid and playing a role in peripheral regulation of lipid/bone metabolism. |
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