| Purpose:Based on the theroy of“Yin and Yang Mutual Aid ” in traditional Chinese medicine,this paper studies the different effects of Zuogui Pill of nourishing kidney yin and Yougui Pill of warming kidney yang on adipogenic differentiation of bone/bone marrow in PMOP rats,and to preliminarily explores the differences of the effects of Zuogui Pill and Yougui Pill on the adipogenic of PMOP rats through the AMPK/m TOR pathway,and tries to clarify the regulatory mechanism of Zuogui Pill and Yougui Pill on the adipogenic of PMOP rats.Material and method:60 SPF-grade SD female nulliparous rats were randomly divided into Control group(Control)8 rats,SHAM group(SHAM)8 rats and ovariectomized group 44 rats.The ovariectomized group estabilshed postmenopausal osteoporosis model(postmenopausal osteoporosis,PMOP)by Bilateral ovariectomized method.SHAM group only removed the same amount of fat around ovaries.The surviving rats were randomly divided into model group(OVX),Zuogui pill group(OVX+ZGW),Yougui pill group(OVX+YGW)and Bujiale group(OVX+BJL).After 7 days of modeling,the rats were administrated by gavage.In addition to rat model-making,gavage and other causes of death and failure to meet the inclusion criteria of 12 rats,a total of 48 rats were included.After 12 weeks,the bone mineral density of the right femur of rats was measured with digital whole body bone densitometer.Fasting but giving water before operation,weighing and anaesthetizing 2 hours after the last administraction,taking bloodfrom abdominal aorta,centrifuging serum,subpackaging in refrigerator at-80 ℃ for storage,and detecting estradiol content in rat serum by enzymelinked immunosorbent assay.The rats uterus was taken,weighed,and the rats uterus index was calculated,and then placed in 4% paraformaldehyde for uterine morphological examination.Take the left femur of the rat,remove the fascia muscle,place it in 4% paraformaldehyde,and decalcify it for 21 days for morphological examination of femur,and use HE method to detect the morphology of uterus and left femur of the rat.Take rat femur,cut off both ends of femur,wash marrow cavity with PBS buffer solution repeatedly until it is white and transparent,centrifuge the washed marrow,discard the upper PBS,and store it in a refrigerator at-80℃ for gene and protein detection;Take the epiphyseal end of rat femoral shaft and it is wrapped with wet gauze and frozen in a refrigerator at-80℃for gene and protein detecion.The expression of PPAR γ,LPL,AMPK,m TORC1 m RNA in rat bone and bone marrow tissue were detected by real-time fluorescence quantitative PCR.The expression of PPAR γ,LPL,C/EBP α,C/EBPβ,FABP4,TSC2,Rheb,4EBP1 proteins and the phosphorylation level of AMPKα,m TOR and p70s6k1 proteins in rat bone and bone marrow tissue were detected by western blot.Results:1 Effects of Zuogui pill and Yougui pill on PMOP model rats.1.1 Effects of Zuogui pill and Yougui pill on bone density and morphyology of PMOP rats.Bone mineral density test results:Compared with the Control group,BMD in the OVX group was significiantly decreased(P<0.01),PMOP model rats were successfully prepared.Compared with the OVX group,the BMD of the rats in Zuogui pill group,Yougui pill group and Bujiale group was significiantly increased(P<0.01).Compared with the Zuogui pill group,there was no difference in BMD between the Yougui pill group and Bujiale group(P>0.05).Results of bone morphology examination:In Control group and Sham group,the bone trabeculae were reticular,closely arranged and regular,with complete morphology and structure and small bone marrow space.In OVX group,the total amount of bone trabeculae decreased,and the arrangement was sparese and scattered,and the morphological structure became thinner and fractured,and the bone marrow space increased.After 12 weeks of treament,the bone morphology of each administration group was improved to different degrees.The total amount of bone trabeculae increased,and some of the bone trabeculae gradually arranged in a regular network,and the morphological structure widened.The connectivity was improved to different degrees,and the bone marrow space decreased.1.2 Effects of Zuogui pill and Yougui pill on serum E2 level in PMOP rats.Compared with the Control group,the serum E2 level in the OVX group was significiantly decreased(P<0.01),it is suggested that estrogen is significantly decreased after menopause in rats.Compared with the OVX group,the serum E2 level of the rats in Zuogui pill group,Yougui pill group and Bujiale group was significiantly increased(P<0.01 or P<0.05).Compared with the Zuogui pill group,there was no significiant difference in rats serum E2 level in the Yougui pill group and Bujiale group(P>0.05).1.3 Effects of Zuogui pill and Yougui pill on uterine index and morphology in PMOP rats.Uterine index test results:Compared with the Control group,the uterine index of rats in the OVX group significiantly decreased(P<0.01).Compared with the OVX group,Zuogui pill group,Yougui pill group and Bujiale group had significiantly higer uterine index(P<0.01 or P<0.05).Compared with the Zuogui pill group,Yougui pill and Bujiale group had no significiant difference in uterine index(P>0.05).Morphological results of uterus showed:In the Control group,the uterine structure was normal,and the endometrium contained simple columnar epithelium.The lamina propria had more blood vessels and uterine glands,and the muscular layer was composed of multiple closely arranged muscle fibers.In the Sham group,there were no significant pathological changes in the uterus.Compared with the Control group,the OVX group rats showed obvious atrophy of the uterus,obvious thinning of the endometrium,obvious shrinkage of the endometrium and muscular layer,and decreased glands in the lamina propria.Compared with the OVX group,the administration group can resist the above pathological changes and alleviate the uterine atrophy to a certain extent.2 Effects of Zuogui pill and Yougui pill on the expression of PPAR γ and LPL m RNA and Protein,and C/EBPα,C/EBPβ,FABP4 Protein in Bone/Bone marrow of PMOP rat.The results of m RNA detection are as follows:Compared with the control group,PPAR γ and LPL m RNA in bone/bone marrow of rats in model group are significantly increased(P<0.01).Compared with OVX group,PPAR γ and LPL m RNA in bone/bone marrow of rats in Zuogui pill group,Yougui pill group and Bujiale group were significantly decreased(P<0.01).Compared with Zuogui pill group,PPAR γ and LPL m RNA in bone/bone marrow of rats in Yuogui pill and Bujiale group increased significantly(P<0.01).The protein detection results are as follows: Compared with the Control group,the expression of PPAR γ,LPL,C/EBP α,C/EBP β,FABP4 protein in bone/bone marrow of rats in the OVX group is significantly increased(P<0.01).Compared with the OVX group,the expressions of PPAR γ,LPL,C/EBP α,C/EBPβ,FABP4 protein in bone/bone marrow of rats in Zuogui pill group,Yougui pill group and Bujiale group were significantly decreased(P<0.01 or P<0.05).Compared with Zuogui pill group,the expressions of PPARγ,LPL,FABP4 protein in bone/bone marrow,bone marrow tissue C/EBPα protein,and bone tissue C/EBPβ protein in rats of Yougui pill group were significantly increased(P<0.01),while the expressions of bone tissue C/EBPα protein and bone marrow tissue C/EBPβ protein had no significant difference(P>0.05).Compared with Zuogui pill group,the expressions of PPAR γ,LPL,C/EBP α,C/EBP β,FABP4 protein in bone/bone marrow of rats in Bujiale group had no significant difference(P>0.05).3 Effects of Zuogui pill and Yougui pill on AMPK/m TOR Signal Pathway in Bone/Bone Marrow of PMOP rats.The results of m RNA detection are as follows:Compared with the Control group,AMPK m RNA in bone/bone marrow of rats in the OVX group is significantly increased(P<0.01),and m TORC1 m RNA is significantly decreased(P<0.01).Compared with the OVX group,AMPK m RNA in bone/bone marrow of rats in Zuogui pill group,Yougui pill group and Bujiale group decreased significantly(P<0.01),and m TORC1 m RNA increased significantly(P<0.01).Compared with Zuogui pill group,AMPK m RNA in bone/bone marrow of rats in Yougui pill group and Bujiale group increased significantly(P<0.01),and m TORC1 m RNA decreased significantly(P<0.01). The protein detection results are as follows:Compared with the Control group,the phosphorylation level of AMPKα protein and the expression of TSC2 protein in bone /bone marrow of rats in the OVX group are significantly increased(P<0.01),while the phosphorylation level of m TOR and p70s6k1 protein and the expression of Rheb protein are significantly decreased(P<0.01);Compared with the OVX group,the phosphorylation level of AMPK α protein and the expression of TSC2 protein in bone /bone marrow of rats in Zuogui pill group,Yougui pill group and Bujiale group decreased(P<0.01 or P<0.05),while the phosphorylation level of m TOR,p70s6k1 protein and the expression of Rheb protein increased(P<0.01 or P<0.05).Compared with Zuogui pill group,there was no significant difference in phosphorylation level of AMPKα protein in bone/bone marrow of rats in Yougui pill group(P>0.05),and the expression of TSC2 protein increased(P<0.01),while the phosphorylation level of m TOR protein in bone tissue,the phosphorylation level of p70s6k1 protein in bone/bone marrow and the expression of Rheb protein in bone tissue decreased(P<0.01 or P<0.05),and there was no significant difference in phosphorylation level of m TOR protein in bone marrow tissue and the expression of Rheb protein(P>0.05).Compared with Zuogui pill group,the phosphorylation level of AMPKα and m TOR protein,the expression of TSC2,Rheb protein in bone/bone marrow of rats in Bujiale group had no signififcant difference(P>0.05),while the phosphorylation level of p70s6k1 protein in bone/bone marrow was significantly increased(P<0.01).The expression of 4EBP1 protein in bone/bone marrow of rats in each group had no statistical significance.Conclusion:1 PMOP model rats showed decreased bone density,damaged bone microstructure,decreased estrogen level,and obvious astrophy of uterine tissue.Both Zuogui pill and Yougui pill can improve the bone density and estrogen level of rats,improve the bone microstructure,alleviate the state of uterine atrophy rats,and effectively prevent and treament PMOP.2 The bone/bone marrow of the PMOP model rats showed lipid metabolism disorder and excessive lipid differentiation.Both Zuogui pill and Yougui pill can reduced the expression of bone/ bone marrow PPAR γ,LPL m RNA and protein and C/EBPα,C/EBPβ,FABP4 protein in PMOP model rats,and effectively improve the disorder of bone/bone marrow lipid metabolism in PMOP model rats,and reduce the level of lipid differentiation in PMOP model rats.3 The expressioms of AMPK m RNA expression and protein phosphorylation level and TSC2 protein expression in bone/bone marrow tissue of PMOP model rats were significantly increased,while the expressions of m TOR m RNA expression,m TOR and p70s6k1 protein phosphorylation level and Rheb protein expression were decreased.Both Zuogui pill and Yougui pill resist the changes of AMPK/m TOR related indexes to varying degrees,thus regulating AMPK/m TOR signaling pathway.4.Zuogui pill and Yougui pill can prevent and treament PMOP by regulating AMPK/m TOR signaling pathway to reduce lipid differentation in PMOP model rats. |
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