| Purpose: Based on the TCM theory of kidney storage essence and bone marrow and Yin and Yang mutual aid,this study studied the effect of Zuogui and Yougui pills on bone formation in PMOP rats by regulating AMPK/m TOR signaling pathway,and explored the mechanism of Zuogui and Yougui pills on bone formation in the process of prevention and treatment of PMOP.Material and method: A total of 60 SPF grade 2-month-old SD female non-fertile rats were randomly divided into blank group(KB)(n=8),sham operation(SHAM)(n=8)and model group(n=44).The postmenopausal osteoporosis(PMOP)model was established by removing bilateral ovaries of rats.In the Sham group,adipose tissue about the size of an ovary around the ovary was removed.After operation,The living rats in model group were randomly divided into model group(OVX),Zuogui pill group(ZGW),Yougui pill group(YGW)and Bujiale group(BJL group).After 7 days of adaptive feeding,the drug was given by gavage once a day for 12 weeks.Twelve rats that did not meet the inclusion criteria due to modeling,intragastric and ministration and malnutrition were excluded,a total of 48 rats were included.After the last gavage,they fasted for 12 hours.Blood was collected from the abdo minal aorta,and the serum was stored at-80? after standing,centrifugation and separation.The left thigh femur of rats was taken,muscle and fascia tissues were removed,and both ends of the thigh were cut off.The bone marrow cavity was rinsed with PBS buffer,repeated several times until the backbone turned white.The PBS buffer containing bone marrow was centrifuged,packed separately,and stored at-80?.The right thigh femur was taken,muscle and fascial tissue were removed,wrapped in gauze with normal saline and stored at-80?.The L3-L5 lumbar vertebrae of rats were taken,muscle tissue was removed and fixed with 4%paraformaldehyde.Micro-CT was used to detect the bone mass parameters of L3-L5 lumbar vertebrae and to evaluate the bone microstructure of the lumbar vertebrae.The contents of BGP and BALP in serum were determined by enzyme-linked immunosorbent assay.The gene expression of COL I,Runx2,AMPK? and m TOR in femur and bone marrow were detected by Real-time PCR.The protein expressions of COL I,Runx2,ALP and BGP in femur and bone marrow and the protein phosphorylation levels of AMPK? and m TOR in bone marrow were detected by Western-blot.Results1 Effect of Zuo and Yougui pills on PMOP rat model and bone formation in bone and bone marrow tissue1.1 Effect of Zuo and Yougui pills on parameters and morphology of lumbar vertebra in PMOP rats Micro-CT detection results show that compared with KB group,BMD,BS/TV,BV/TV,Conn-Dens,Tb.Th in OVX group decreased(P<0.01),while Tb.Sp,BS/BV increased(P<0.01),trabecular bone from the slab structure to the rod structure change,sparse,small,more fracture,bone trabecular structure is arranged unevenly,bone microstructure damage obviously,successful preparation of PMOP rats model.Compared with OVX group,BMD,BV/TV,BS/TV,Tb.Th and Conn-Dens in BJL,ZGW and YGW groups increased(P<0.05 or P<0.01),while Tb.Sp,BS/BV decreased(P<0.05 or P<0.01).The slab-like structure of bone trabeculae increased,the structure of bone trabeculae was compact,thickened,fracture was reduced,and the arrangement was uniform.1.2 Effect of Zuo and Yougui Pills on serum BGP and BALP concentration in PMOP ratsThe results showed that compared with KB group,serum BGP and BALP content of OVX group increased(P<0.01).Compared with OVX group,serum BGP and BALP content of ZGW,YGW and BJL group decreased(P<0.01).Compared with BJL group,serum BGP content of ZGW and YGW groups had no significant difference(P(29)0.05).serum BALP content of YGW group increased(P<0.01),ZGW group had no significant difference(P(29)0.05).1.3 Effect of Zuo and Yougui Pills on COL I and Runx2 gene and protein expression in bone and bone marrow of PMOP rats1.3.1 Effect of Zuo and Yougui Pills on COL I and Runx2 gene expression in bone and bone marrow of PMOP ratsThe results showed that compared with KB group,the expression of COL I and Runx2 gene in bone and bone marrow of OVX group decreased(P<0.01).Compared with OVX group,the expression of COL I and Runx2 gene in bone and bone marrow of ZGW,YGW and BJL groups increased(P<0.05).Compared with BJL group.The expression of COL I gene in bone of ZGW group increased,and that in YGW group decreased(P<0.05),and that in bone marrow of ZGW and YGW groups had no significant difference(P(29)0.05),the expression of Runx2 gene in bone and bone marrow of ZGW and YGW groups had no significant difference(P(29)0.05).1.3.2 Effect of Zuo and Yougui Pills on COL I and Runx2 protein expression in bone and bone marrow of PMOP ratsThe results showed that compared with KB group,the expression of COL I and Runx2 protein in bone and bone marrow of OVX group decreased(P<0.01).Compared with OVX group,the expression of COL I and Runx2 protein in bone and bone marrow of ZGW,YGW and BJL groups increased(P<0.01).Compared with BJL group,the expression of COL I protein in bone,bone marrow of ZGW group increased(P<0.01),in YGW group,that in bone increased(P<0.01),and that in bone marrow had no significant difference(P(29)0.05),the expression of Runx2 protein in bone and bone marrow of YGW group decreased(P<0.05),in ZGW group,that in bone dreacased(P<0.01),and that in bone marrow had no significant difference(P(29)0.05)..1.4 Effect of Zuo and Yougui Pills on BGP and ALP protein expression in bone and bone marrow of PMOP ratsThe results showed that compared with KB group,the expression of BGP protein in bone and bone marrow of OVX group decreased(P<0.01).Compared with OVX group,the expression of BGP protein in bone and bone marrow of ZGW,YGW and BJL groups increased(P<0.01).Compared with BJL group,the expression of BGP protein in bone,bone marrow of ZGW group increased(P<0.01),in YGW group,and that in bone marrow increased(P<0.01),and that in bone had no significant difference(P(29)0.05)The results showed that compared with KB group,the expression of ALP protein in bone and bone marrow of OVX group increased(P<0.01).Compared with OVX group,the expression of ALP protein in bone and bone marrow of ZGW,YGW and BJL groups decreased(P<0.01).Compared with BJL group,the expression of ALP protein in bone and bone marrow of ZGW,YGW group had no significant difference(P(29)0.05).2 Effect of Zuo and Yougui Pills on AMPK/m TOR signaling pathway in bone marrow of PMOP rats2.1 Effect of Zuo and Yougui Pills on the expression of AMPK? gene and protein phosphorylation in bone marrow of PMOP rats2.1.1 Effect of Zuogui and Yougui Pills on the expression of AMPK? gene in bone marrow of PMOP ratsThe results showed that compared with KB group,the expression of AMPK? gene in bone marrow of OVX group increased(P<0.01).Compared with OVX group,the expression of AMPK? gene in bone marrow of ZGW,YGW and BJL groups decreased(P<0.01).Compared with BJL group,the expression of AMPK? gene in bone marrow of ZGW group was obviously lower(P<0.05),and that in YGW group was no significant difference(P(29)0.05)2.1.2 Effect of Zuogui and Yougui Pills on the protein phosphorylation of AMPK? in bone marrow of PMOP ratsThe results showed that compared with KB group,the protein phosphorylation level of AMPK? in bone marrow of OVX group increased(P<0.01).Compared with OVX group,the protein phosphorylation level of AMPK? in bone marrow of ZGW,YGW and BJL groups decreased(P<0.01).Compared with BJL group,the protein phosphorylation level of AMPK?in bone marrow of ZGW,YGW were no significant difference(P(29)0.05).2.2 Effect of Zuo and Yougui Pills on the expression of m TOR gene and protein phosphorylation in bone marrow of PMOP rats2.2.1 Effect of Zuo and Yougui Pills on the expression of m TOR gene in bone marrow of PMOP ratsThe results showed that compared with KB group,the expression of m TOR gene in bone marrow of OVX group decreased(P<0.01).Compared with OVX group,the expression of m TOR gene in bone marrow of ZGW,YGW and BJL groups increased(P<0.01).Compared with BJL group,the expression of m TOR gene in bone marrow of ZGW group increased(P<0.01),and that in YGW group decreased(P<0.01).2.2.2 Effect of Zuo and Yougui Pills on the protein phosphorylation of m TOR in bone marrow of PMOP ratsThe results showed that compared with KB group,the protein phosphorylation level of m TOR in bone marrow of OVX group decreased(P<0.01).Compared with OVX group,the protein phosphorylation level of m TOR in bone marrow of ZGW,YGW and BJL groups increased(P<0.01).Compared with BJL group,the protein phosphorylation level of m TOR in bone marrow of ZGW,YGW were no significant difference(P(29)0.05).Conclusion1 In PMOP rats,bone mass decreased and bone microstructure were destroyed,and bone admetabolism was disordered.Zuogui pill and Yougui pill can improve the bone mass and bone microstructure destruction in PMOP rats,correct the disorder of bone metabolism and prevent PMOP.2 The occurrence of PMOP is related to the down-regulated expression of osteogenic genes and proteins and insufficient bone formation.Both Zuogui pill and Yougui pill can promote the expression of osteogenic genes and proteins,promote bone formation,and prevent PMOP.The effect of Zuogui pill is better than Yougui pill by comparison.3 AMPK signaling pathway was activated and m TOR signaling pathway was inhibited in PMOP rats.Zuogui and Yougui pills can inhibit the activation of AMPK signaling pathway,relieve the inhibition of m TOR signaling pathway,and regulate the AMPK/m TOR signaling pathway in PMOP rats.By comparison,Zuogui pill and Yougui pill have no significant difference in regulating the phosphorylation level of AMPK/m TOR signaling pathway,but Zuogui pill is better than Yougui pill in regulating the gene expression of AMPK/m TOR signaling pathway.4 Zuogui and Yougui pills can regulate AMPK/m TOR signaling pathway to promote the expression of osteogenic indicators in PMOP rats,promote bone formation,and effectively prevent and cure PMOP. |
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