| Purpose:This study from the medical theories of "Yin Yang interaction",Zuogui Pill,a representative formula for nourishing kidney yin,and Yougui Pill,a representative formula for warming kidney yang,were selected.The effects of Zuogui Pill and Yougui Pill on glucose,lipid metabolism and energy metabolism in PMOP model rats were studied.Zuogui Pill and Yougui Pill were based on AMPK / m TOR The effect of signal pathway on the prevention and treatment of glucose,lipid and energy metabolism disorder in PMOP model rats can provide experimental basis for the prevention and treatment of postmenopausal metabolism disorder in PMOP women under the guidance of the theory of "Yin Yang interaction".Materials and Methods: In this study,60 two-month-old female Sprague Dawley rats of SPF grade were randomly divided into 8 blank rats(KB),8 sham rats(sham)and 44 ovariectomized rats.In the ovariectomized model group,bilateral back oophorectomy was used to prepare the model.In the sham-operated group(SHAM),only a part of fat tissue around the ovary and large ovary was removed,while in the blank group(KB),no treatment was performed.One week after the operation,4 rats died of bilateral back oophorectomy.40 rats were randomly divided into ovariectomized model group(OVX),Zuogui Pill group(ZGW),Yougui Pill group(YGW)and bujiale group(BJL)according to their weight.There were 6 groups in total compared with the original blank group(KB)and sham-operated group(SHAM).Each treatment group was given the corresponding drugs by gavage,KB group,sham group and OVX group were given distilled water by gavage.After 12 weeks of administration,the dead rats and 8 rats that did not meet the inclusion criteria were excluded.A total of 48 rats were included in this experiment,8 in each group.The bone mineral density of the left femur was measured under anesthesia,and the glucose tolerance was measured by cutting the tail and taking blood.The rats were not allowed to eat but not limited to drink water within 24 hours before taking the material,weighed 2 hours after the last administration,and anesthetized by intraperitoneal injection of 10% chloral hydrate solution for each group according to the standard of 0.3ml · 100g-1.After anesthetizing the rats,take the blood of abdominal aorta,and the blood was centrifuged according to 2000 r /min * 20 min after 2 hours of standing.The serum extracted by centrifugation was divided into aseptic EP tubes and frozen at 80 ℃,so that the total serum cholesterol,triglyceride,low-density lipoprotein cholesterol,high-density lipoprotein cholesterol,alanine aminotransferase(ALT)and glutamic oxaloacetic acid could be detected by automatic biochemical analyzer Ammonia enzyme(AST);One third of the proximal tibia of the left hind limb of the rat was taken,the muscle fascia and connective tissue attached to the tibia were carefully peeled off,fixed(decalcified)for several weeks,and then paraffin sections were made for morphological he detection;fresh liver of the rat was taken,then the wet weight was weighed,and the liver index of the rat was calculated,and then the anterior lobe of the liver was put into aseptic EP tube and frozen at 80 ℃ for morphological HE detection;The brown adipose tissue of the scapula was fixed in 4% paraformaldehyde for morphological HE detection.The adipose tissue of the abdominal posterior wall was frozen in the sterile EP tube at 80 ℃.Western blot(WB)was used to detect the phosphorylation of AMPK 1,m-TORC1,p70S6k1,and the protein expression of C/EBP,C/EBP line,TSC2,Rheb,and4EBP1 in the adipose tissue of the posterior wall of the rat abdomen.The expression of AMPK α 1 and m-torc1 in the adipose tissue of the rat’s abdominal posterior wall was detected by real-time fluorescence quantitative PCR,and the content of ATP in the adipose tissue of the rat’s abdominal posterior wall was detected by HPLC.Result:1.Effect of Zuogui Pill and Yougui Pill on bone mineral density of ovariectomized rats Compared with KB group,bone mineral density index of left femur of OVX group decreased(P < 0.05).Compared with the OVX group,the bone mineral density index of the left femur of the rats in the treatment group was increased(P< 0.05 or P < 0.01);Compared with the BJL group,the bone mineral density of the rats in the ZGW group and the YGW group was similar with no significant difference(P>.05),and the bone mineral density index of the rats in the ZGW group was higher than that in the YGW group.2.Effect of Zuogui pill and Yougui Pill on bone histomorphology of ovariectomized rats The experimental results showed that there were more trabeculae in the bone marrow cavity near the tibia of the left hind limb in the KB group and the sham group.The trabeculae were thick and well distributed.Although they were arranged irregularly,the trabeculae were connected with each other to form a complex network structure,and the bone marrow cavity between the trabeculae was relatively small;However,the morphology of bone trabeculae in the proximal tibia of the left hind limb of OVX group was significantly different from that of KB group.The number of bone trabeculae was significantly reduced,the structure was sparse,the distribution was uneven,the bone trabeculae became thin,the surface was relatively smooth,there was fracture,there was lack of mutual connection between them,and even there was a cavity in the local areai.After 12 weeks of medical treatment,the number of trabeculae in ZGW group,YGW group and BJL group increased in varying degrees,the structure arrangement and tissue morphology gradually approached KB group,the connection between trabeculae improved,and the marrow cavity decreased.Among them,the effect of ZGW group is obvious.3.Effect of Zuogui pill and Yougui Pill on liver function of ovariectomized rats Compared with KB group,the serum ALT and AST of OVX group were significantly higher(P< 0.01);compared with OVX group,ZGW group,YGW group and BJL group could reduce the serum ALT and AST(P< 0.01);compared with BJL group,ZGW group and YGW group had no significant difference(P> 0.05).Although YGW group could also reduce the serum ALT and AST,there was no significant difference.4.Effects of Zuogui and Yougui pills on liver index and histomorphology in ovariectomized rats Compared with the KB group,liver index in OVX group was significantly increased(P< 0.05 or P< 0.01),and that in ZGW group,YGW group and BJL group was also increased,but the difference was not statistically significant(P>0.05).Compared with OVX group,liver index of ZGW group,YGW group and BJL group were significantly decreased(P<0.01).Compared with the BJL group,there was no significant difference between the ZGW group and the YGW group(P>0.05),but the liver index of the ZGW group was lower than that of the YGW group.The results showed that the liver tissue structure of KB group and sham group was normal,the arrangement of hepatocytes was regular and clear,while the liver tissue of OVX group was swollen,the cell morphology was not clear,there were more fat vacuoles in it,the structure of hepatocytes was deformed,and the arrangement level was slightly disordered;the morphology of liver tissue of ZGW group,YGW group and BJL group was clear after 12 weeks of treatment To improve,the shape of hepatocytes became clear,and the arrangement became regular.5.Effect of Zuogui pill and Yougui Pill on glucose tolerance in ovariectomized rats The results showed that there was no significant difference in fasting blood glucose between the groups.Compared with KB group,the blood glucose level of OVX group increased significantly 30 minutes after administration of 50% glucose solution,decreased significantly 60 minutes after administration,and failed to return to normal level 120 minutes after administration.The changes of blood glucose in ZGW group,YGW group and BJL group were basically the same as those in KB group.Only OVX group showed significant abnormal glucose tolerance.Compared with KB,AUC level of OVX group increased significantly(P< 0.01);compared with OVX group,AUC level of ZGW group,YGW group and BJL group decreased significantly(P< 0.01);compared with BJL group,AUC level of ZGW group and YGW group had no significant difference(P> 0.05),but AUC level of ZGW group was lower than that of YGW group.6.Effect of Zuogui pill and Yougui Pill on blood lipid in ovariectomized rats Compared with KB group,the serum TC,TG and LDL-C of OVX group were significantly higher(P< 0.01),while the serum HDL-C of OVX group was significantly lower(P< 0.01);compared with OVX group,ZGW group,YGW group and BJL group,the serum TC,TG(P< 0.05 or P< 0.01)and LDL-C(P< 0.05)were all decreased There was no significant difference among ZGW group,YGW group and BJL group(P> 0.05),but serum TC of ZGW group was lower than that of YGW group,while serum TG,LDL-C and HDL-C of ZGW group were higher than that of YGW group.7.Effect of Zuogui pill and Yougui Pill on fat distribution in ovariectomized rats Compared with KB group,the fat index of OVX group was significantly higher(P<0.01);compared with OVX group,the fat index of ZGW group,YGW group and BJL group was significantly lower(P< 0.01);compared with BJL group,the fat index of OVX group was significantly higher(P< 0.01);there was no significant difference between ZGW group and YGW group(P> 0.05),but there was no significant difference between ZGW group The fat index of abdominal posterior wall of rats was higher than that of YGW group.There was no significant difference in brown fat index of scapula in each group(P > 0.05).8.Effect of Zuogui pill and Yougui Pill on brown adipose tissue morphology in ovariectomized rats The results showed that the brown adipose tissue structure of the scapula of KB group and sham group was normal,the volume of adipocytes was uniform and small,the shape was clear,the arrangement was regular and centralized,the color was dark and brown,while the volume of adipocytes in OVX group was significantly increased,the arrangement was slightly disordered,the color was evacuation,the color was obviously light,and the majority was white;ZGW group,YGW group and BJL group were treated with drug 1 After 2 weeks,the brown adipose tissue structure of the scapula of the rats was obviously improved.It was found that the arrangement of adipocytes was more regular and concentrated,and the color of adipocytes became darker and brown.9.Effect of Zuogui pill and Yougui Pill on the expression of C / EBP α and C / EBP βprotein in the adipose tissue of the abdominal posterior wall of ovariectomized rats Compared with KB group,the expression of C / EBP α and C / EBP β protein in the abdominal fat of OVX group was significantly higher(P< 0.01);compared with OVX group,the expression of C / EBP α and C / EBP β protein in the abdominal fat of ZGW group,YGW group and BJL group was significantly lower(P< 0.01);compared with BJL group,the expression of C / EBP α and C / EBP β protein in the abdominal fat of ZGW group and YGW group was not significantly different(P> 0.05),but the expression of C / EBP α and C / EBP β protein in the abdominal fat of ZGW group was not significantly different(P> 0.05)The down-regulation was more obvious in group YGW than in group YGW.10 Effects of Zuogui pill and Yougui pill on ATP content in adipose tissue of posterior abdominal wall of ovariectomized rats Compared with the KB group,the ATP content of fat in the posterior wall of the OVX group decreased significantly(P < 0.01).Compared with the OVX group,the fat ATP content in the posterior abdominal wall of rats in ZGW group,YGW group and BJL group increased significantly(P < 0.01).Compared with the BJL group,there was no statistically significant difference in the fat ATP content in the posterior abdominal wall of rats in the ZGW group(P > 0.05),and no significant difference in the fat ATP content in the posterior abdominal wall of rats in the YGW group(P < 0.05).The difference between ZGW group and YGW group was not statistically significant(P > 0.05),but the increase of ATP in ZGW group was more significant than that in YGW group.11.Effect of Zuogui pill and Yougui Pill on the expression of AMPK α 1 gene and protein phosphorylation in the adipose tissue of the abdominal posterior wall of ovariectomized rats Compared with KB group,there was no significant difference in AMPK α 1 m RNA expression between OVX group and OVX group(P> 0.05);compared with OVX group,there was no significant difference in AMPK α 1 m RNA expression between ZGW,YGW and BJL group(P > 0.05);compared with BJL group,there was no significant difference in AMPK α 1m RNA expression between ZGW and YGW group(P> 0.05).Compared with KB group,AMPK α 1 phosphorylation level of abdominal posterior wall fat in OVX group was significantly lower(P< 0.01);compared with OVX group,AMPK α 1phosphorylation level of abdominal posterior wall fat in ZGW,YGW and BJL group was significantly higher(P< 0.01);compared with BJL group,AMPK α 1 phosphorylation level of abdominal posterior wall fat in ZGW group was not significantly different(P> 0.05),and AMPK α 1 phosphorylation level of abdominal posterior wall fat in YGW group was significantly higher(P< 0.01)The phosphorylation level was not significantly increased(P< 0.05).12.Effect of Zuogui pill and Yougui Pill on the phosphorylation expression of m-torc1 gene and protein in the adipose tissue of the abdominal posterior wall of ovariectomized rats Compared with KB group,there was no significant difference in m-torc1 m RNA expression of abdominal posterior wall fat in OVX group(P> 0.05);compared with OVX group,there was no significant difference in m-torc1 m RNA expression of abdominal posterior wall fat in ZGW,YGW and BJL group(P > 0.05);compared with BJL group,there was no significant difference in m-torc1 m RNA expression of abdominal posterior wall fat in ZGW and YGW group(P > 0.05).Compared with KB group,the phosphorylation level of m-torc1 in the abdominal posterior wall fat of OVX group was significantly higher(P< 0.01);compared with OVX group,the phosphorylation level of m-torc1 in the abdominal posterior wall fat of ZGW,YGW and BJL group was significantly lower(P< 0.01);compared with BJL group,the phosphorylation level of m-torc1 in the abdominal posterior wall fat of YGW group was not statistically significant(P> 0.05),and the phosphorylation level of m-torc1 in the abdominal posterior wall fat of ZGW group was significantly lower(P< 0.01)The phosphorylation level was not significantly reduced(P< 0.05).13.Effect of Zuogui pill and Yougui Pill on protein phosphorylation expression of P70S6K1 in fat tissue of abdominal posterior wall of ovariectomized rats Compared with KB group,the phosphorylation level of P70S6K1 in the abdominal posterior wall fat of OVX group was significantly higher(P< 0.01);compared with OVX group,the phosphorylation level of P70S6K1 in the abdominal posterior wall fat of ZGW,YGW and BJL group was significantly lower(P< 0.01);compared with BJL group,the phosphorylation level of P70S6K1 in the abdominal posterior wall fat of ZGW group was not statistically significant(P> 0.05)The phosphorylation level was not significantly reduced(P< 0.05).14.The effect of Zuogui pill and Yougui Pill on the expression of TSC2,rheb and 4ebp1 in the adipose tissue of the abdominal posterior wall of ovariectomized rats Compared with KB group,the expression of TSC2 protein in the abdominal posterior wall fat of OVX group was significantly lower(P< 0.01);compared with OVX group,the expression of TSC2 protein in the abdominal posterior wall fat of ZGW,YGW and BJL group was significantly higher(P< 0.01);compared with BJL group,the expression of TSC2 protein in the abdominal posterior wall fat of ZGW and YGW group had no significant difference(P> 0.05),but the expression of TSC2 protein in the ZGW group was significantly higher than that of YGW group.Compared with KB group,there was no significant difference in rheb protein expression of abdominal posterior wall fat in OVX group(P> 0.05);compared with OVX group,there was no significant difference in rheb protein expression of abdominal posterior wall fat in ZGW,YGW and BJL group(P > 0.05);compared with BJL group,there was no significant difference in TSC2 protein expression of abdominal posterior wall fat in ZGW and YGW group(P> 0.05).Compared with KB group,there was no significant difference in the expression of 4ebp1 protein in abdominal posterior wall fat of OVX group(P> 0.05);compared with OVX group,there was no significant difference in the expression of 4ebp1 protein in abdominal posterior wall fat of ZGW,YGW and BJL group(P> 0.05);compared with BJL group,there was no significant difference in the expression of TSC2 protein in abdominal posterior wall fat of ZGW and YGW group(P> 0.05).Conclusion:1.After ovariectomy,bone mass was lost,bone tissue and structure were destroyed,and bone metabolism of high conversion type appeared in the rats.At the same time,postmenopausal osteoporosis rats showed abnormal liver function,abnormal glucose tolerance,weight gain,changes in body fat distribution,and significantly increased blood lipids.2.Zuogui pill and Yougui Pill can effectively prevent and treat postmenopausal osteoporosis,improve bone loss to a certain extent,and slow down bone tissue destruction.Among them,Zuogui pill is slightly better than Yougui Pill and bujiale.3.Zuogui pill and Yougui Pill can effectively prevent and treat the disorder of glucose and lipid metabolism in postmenopausal women,alleviate the abnormal situation of glucose tolerance to a certain extent,and improve the liver function and blood lipid level;among them,Zuogui pill and Yougui Pill have no significant difference.4.Zuogui pill and Yougui Pill can regulate the disorder of glucose,lipid metabolism and energy metabolism after menopause by intervening AMPK / m TOR signal pathway.There is no significant difference between Zuogui pill and Yougui Pill.5."Yang transforming Qi and Yin forming" is the theoretical basis of energy and material metabolism of the body,and the treatment of "Yin Yang mutual aid" affects the metabolic balance by regulating "Yin Yang balance". |
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