| Purpose:This study is based on the theory of"yin and yang mutual aid"in traditional Chinese medicine,the following thalamus-pituitary-adrenal axis as the entry point.To detect the effect of Zuogui/Yougui Pill(?????)and its separated prescription on the expression of TGF-?1 and PKA in the hypothalamus and adrenal of ovariectomized rats and to analyze the imbalance of HPA axis in postmenopausal osteoporosis model,Seek the Experimental Basis of Prevention and treatment of PMOP by Zuogui/Yougui Pill from Endocrinology.Material and method:A total of 95 SPF female,two-month-old SD rats weighing 200-220 g,according to the weight,stratified randomization method was used to divide into 10 normal Control group,10 SHAM operation group and 75ovariectomized group.Model group using bilateral ovariectomy to prepare postmenopausal osteoporosis model,the SHAM operation group only removed part of the adipose tissue around the ovary.After modeling 7 days,Postoperative survivors were randomly divided into 7 groups according to body weight,the last 90 rats were divided into 9 groups,the Control group,SHAM group,OVX group,BJL group,ZGW group,YGW group,GTY group,ZYY group,BYY group,Each group of 10.After the group began drug treatment,Calculated as 6.3 times the human dose per kilogram of rats,herbal medicine amount of 1g.ml~-11 decoction,Positive Control drug to make good music with distilled water suspension,each treatment group fed the corresponding drugs,Control group,SHAM group and OVX group were fed an equal volume of distilled water.During gavage,rats in each group were weighed once a month,and the dosage was adjusted according to body weight.After treatment with12w material drawn to remove due to surgery,gavage and other causes of death,a total of 84 rats were included in the index test.24h before of drawning the rats can not eat but drink,2h after the last administration weighed and recorded,weighed and recorded two hours after the last dose,after intraperitoneal injection of 10%chloral hydrate anesthesia,abdominal aorta blood,after standing for 2 hours,centrifugation at1000 rpm for 10 minutes,taking the upper serum,frozen at-80°C,radioimmunoassay was used to detect serum ACTH and COR contents.Remove rat lumbar spine,remove attached muscle fascia,wrapped in gauze soaked with deionized water and placed in-80?frozen,BMD was measured by dual energy X-ray absorptiometry.Remove rat hypothalamus and adrenal gland,frozen at-80?,the expression of TGF-?1 and PKA protein were detected by Western blotting.Results:1.Effect of Zuogui/Yougui Pill and its separated prescription on the lumbar vertebral bone mineral density in the second,third and fourth ovariectomized ratsThe results of lumbar BMD of the second,third and fourth sections of rats in each group were as follows:After 12w's treatment,Compared with Control group,BMD in SHAM group,BJL group and ZGW group was similar(P>0.01),and BMD in OVX group,YGW group and the disassembled group decreased significantly(P<0.01).Compared with the OVX group,the BMD of each drug group increased,with significant difference(P<0.01).Compared with BJL group,the bone mineral density in ZGW group was similar,there was no significant difference(P>0.01).The bone mineral density in YGW group and its disassembled group decreased significantly(P<0.01).Compared with the ZGW group,the bone mineral density in the YGW group and the disassembled group was less than that in Zuoguiwan group(P<0.01).2.Effect of Zuogui/Yougui Pill and its separated prescription on serum ACTH and COR in ovariectomized rats.The results of serum ACTH in each group were as follows:Compared with Control group,serum ACTH levels in SHAM group,YGW group and BYY group were similar(P>0.05),and the levels of ACTH in OVX,BJL,ZGW,GTY and ZYY groups were significantly decreased(P<0.05);Compared with OVX group,there was no significant difference in ACTH content in BJL,GTY and ZYY groups(P>0.05).The levels of ACTH in ZGW,YGW and BYY groups were significantly increased(P<0.05).Compared with BJL group,there was no significant difference in ACTH content among the intervention groups(P>0.05).Compared with ZGW group,the level of serum ACTH in GTY group was significantly lower(P<0.05),while there was no significant difference between YGW group,ZYY group and BYY group(P>0.05).The serum COR test results of the rats in each group are as follows:Compared with Control group,COR levels in SHAM group,YGW group and BYY group were similar(P>0.05),COR content in OVX group,BJL group,ZGW group,GTY group and ZYY group were significantly decreased(P<0.05);Compared with OVX group,the content of serum COR in each Intervention group increased significantly(P<0.05).Compared with BJL group,serum COR levels in YGW group and BYY group were significantly higher than those in BJL group(P<0.05).There was no significant difference between ZGW group,GTY group and ZYY group(P>0.05).Compared with ZGW group,the levels of serum COR in YGW group and BYY group were significantly higher than those in ZGW group(P<0.05).There was no significant difference between ZGW group,GTY group and ZYY group(P>0.05).3.Effect of Zuogui/Yougui Pill and its separated prescription on expression of TGF-?1 and PKA in adrenal gland of ovariectomized ratsThe results of TGF-?1 protein expression in adrenal tissue in each group were as follows:Compared with the Control group,there was no difference in the expression of TGF-?1 in SHAM,BJL,ZGW,YGW,ZYY and BYY groups(P>0.05),OVX group,GTY group elevated expression,the difference was significant(P<0.05);Compared with OVX group,there was no difference in GTY group(P>0.05),while the other groups were significantly lower(P<0.05).Compared with BJL group,GTY group was significantly increased(P<0.05),while the other groups had no significant difference(P>0.05).Compared with the ZGW group,the GTY group was significantly increased(P<0.05),and the other groups were not significantly different(P>0.05).The results of PKA protein expression in adrenal tissue in each group were as follows:Compared with Control group,there was no significant difference between SHAM group and each administration group(P>0.05).The expression of PKA protein in adrenal gland of OVX group was significantly decreased(P<0.05).Compared with OVX group,the expression of PKA in adrenal gland of each treatment group was significantly increased(P<0.05).Compared with BJL group,there was no significant difference between each TCM treatment group(P>0.05).Compared with ZGW group,there was no difference between YGW group and each separated prescription group(P>0.05).4.Effect of Zuogui/Yougui Pill and its separated prescription on the expression of TGF-?1 and PKA in hypothalamus of ovariectomized ratsThe hypothalamic tissue TGF-?1 protein expression in each group of test results are as follows:Compared with the Control group,there was no difference in the expression of TGF-?1 in SHAM,BJL,ZGW,YGW,ZYY and BYY groups(P>0.05),OVX group and GTY group elevated expression,the difference was significant(P<0.05);Compared with OVX group,there was no difference in GTY group(P>0.05),while the other groups were significantly lower(P<0.05).Compared with BJL group,GTY group was significantly increased(P<0.05),while the other groups had no significant difference(P>0.05).Compared with the ZGW group,the GTY group was significantly increased(P<0.05),while the other groups were not significantly different(P>0.05).PKA protein expression in hypothalamus of rats in each group had no significant difference(P>0.05).Conclusion:1.After bilateral ovaries were excised,the BMD was significantly reduced,which could better simulate the postmenopausal osteoporosis disease model,the bone loss can be prevented after the treatment of Zuogui/Yougui Pill and its separated prescription.The treatment effect of Zuogui/Yougui Pill is better than its separated prescription.Zuogui/Yougui Pill has some preventive and therapeutic effects on postmenopausal osteoporosis.2.After bilateral ovaries were excised,the contents of ACTH and COR decreased,and the steady state of HPA axis was destroyed.This is one of the pathological states of postmenopausal osteoporosis model,may also be a pathological secondary to the body during bone loss,Zuogui/Yougui Pill and its separated prescription can fight this pathological change,prescription of supplement Yang is more prominent in improving serum ACTH,COR content of PMOP,the regulation of Yougui Pill which is based on the theory from Yin cite Yang in this area is closest to the normal group.3.Zuogui/Yougui Pill and its separated prescription can inhibit overexpression of TGF-?1 in the hypothalamus and adrenal tissue of ovariectomized rats,improve Adrenal PKA Protein Inhibitory State.This may be one of the mechanisms by which Zuogui/Yougui Pill and its separated prescription improves the HPA axis function in PMOP rats. |
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