| Purpose:To provide a new experimental basis for prevention and treatment of postmenopausal osteoporosis for Zuogui pill and Yougui pill,this study researches the prevention and treatment of Zuogui pill,Yougui pill and their disassembled prescriptions in ovariectomized rats by TGF-?1/Smads signaling transduction pathway,and the correlation analysis between TGF-?1/Smads signaling transduction pathway and bone mineral density to study the regulatory mechanism of Zuogui pill,Yougui pill and their disassembled prescriptions on ovariectomized rats.All those practice have been made on basis of the theory called “the interaction of the two opposing principles in nature”.Material and method: Dividing 90 SPF female rats at 2 months and weight control 200~220g into 3 groups randomly:Normal group with 10 rats,Sham group with 10 rats,for the rest 70 rats were ovariectomized model group.The model group established ovariectomized model of postmenopausal osteoporosis,and sham group only excised ovarian tissue surrounding fat.One week later,the survivors(70)were randomly divided into 7 groups,and 90 rats were divided into 9 groups at last:Control group,Sham group,OVX group,BJL group,ZGW group,YGW group,GTY group,ZYY group,BYY group,each group of 10 rats.The start of treatment after grouping,rats for per kilogram of body weight dose was 6.3 times of calculation and converted into a daily dose of each treatment group,traditional Chinese medicine was made into the decoction by 1g?ml-1,the positive control drug progynova was made into the mixed suspension with distilled water,each the treatment group was given corresponding drugs,control group,sham group and model group were gavaged with distilled water of equal volume(2ml).According to the weight adjusted dosage,rats in each group were weighed 1 times a month,during the period of intragastric administration.After 12 weeks drugs treatment,all the rats were drawing materials,remove the cause of death due to surgery,gavaged and so on,a total of 84 rats were included in the test.Before the study,the rats were fasted but no water 24 hours,after 2 hours with the last treatment,the rats were weighed and recorded.After intraperitoneal injection of 10% chloral hydrate,the rat uterus were removed and taked wet weight for the calculation of uterine index;The rat lumbar vertebrae were removed and got rid of the attached muscle fascia,and was wrapped with deionized water soaked gauze and stored at-80 ?frozen for dual-energy X-ray absorptiometry detection;The 1/3 part of the proximal tibia of the rats right hind limb was taken,got rid of the muscle,fascia and connective tissue and fixed in 4% paraformaldehyde fixative,Paraffin sections were prepared after several weeks of decalcification for morphological HE detection;The left hind limbs of the rat femurs were removed and got rid of the attached muscles,fascia and connective tissues and stored at-80 frozen for western ?blot detecting the protein expression of TGF-?1,T?R?,T?R?,Smad2,Smad3,Smad4,Smad7.Results: 1.Effects of Zuogui pill,Yougui pill and their disassembled prescriptions on uterine index in ovariectomized rats The statistical results of uterine index of rats in each group were shown as follows:After treatment for 12 weeks,compared with the Control group,the uterine index of the Sham group was similar to that of the Control group,no significant difference(P>0.05),except the Sham group,the uterine index of rats in each group were decreased,there were significant differences(P < 0.01),the uterine index of OVX group was the most significant down-regulated(P<0.01);Compared with OVX group,the uterine index of the GTY group was similar to that of the OVX group,no significant difference(P>0.05),the uterine index of rats in each group except the GTY group were rised,there were significant differences(P <0.01),among the uterine index of BJL and ZGW group were the most significant improvement(P<0.01);compared with the BJL group,the uterine index of the ZGW group was similar to that of the BJL group,no significant difference(P>0.05),the uterine index of the other group YGW?GTY?ZYY?BYY groups increased less than the BJL group,there were significant differences(P<0.01 or P<0.05);compared with the ZGW group,the uterine index of the sham group was similar to that of the ZGW group,no significant difference(P>0.05),the uterine index of the YGW?GTY?ZYY?BYY groups increased less than the ZGW group,there were significant differences(P<0.01 or P<0.05).2.The morphological effects on bone tissue ovariectomized rats for Zuogui pill,Yougui pill and their disassembled prescriptions The observation results of 1/3 in the proximal femur of right hind limb of rats in each group were as follows:Control group and the Sham group showed trabecular bone tightly packed,which morphological structure was complete and trabecular bone was connected by reticular,bone marrow cavity was relatively small;While the total number of trabecular bone in the OVX group was significantly reduced,the structure was sparse,the connectivity was poor and irregular,showing a large number of blind side in trabecular bone,and marrow cavity increased.Each treatment group after treatment for 12 weeks,the BJL?ZGW and YGW groups compared with GTY?BYY and ZYY groups,the therapeutic effect was obviously improved,compared with model group,the trabecular bone became thicker and widened,the number of trabecular bone was increased,the connectivity between the trabeculae was improved and the arrangement was compact.3.The impact for ovariectomized rats on section 2?3?4 lumbar spine bone mineral density for Zuogui pill,Yougui pill and their disassembled prescriptions The bone mineral density of the section 2?3?4 lumbar vertebrae lumbar vertebrae in each group were detected as follows:After administration of 12 weeks,compared with the Control group,the bone mineral density of the Sham?ZGW?YGW group were similar to that of the Control group,no significant difference(P>0.05),the bone mineral density of OVX group decreased,there were significant differences(P<0.01);compared with the OVX group,the bone mineral density of each treatment group was improved,there were significant differences(P<0.01);compared with the BJL group,the bone mineral density of the ZGW group was similar to that of the BJL group,no significant difference(P>0.05),the bone density of other medication groups didn't increase as well as the BJL group,there were significant differences(P<0.01);compared with the ZGW group,the bone mineral density of the YGW?GTY?ZYY or BYY group didn't increase as well as the ZGW group,there were significant differences(P<0.01).In general,Zuogui pill,Yougui pill and their disassembled prescriptions could effectively prevent the decline of bone density caused by ovariectomy and significant effect,the effect of Zuogui pill was better than Yougui pill and their disassembled prescriptions.4.Effects of Zuogui pill,Yougui pill and their disassembled prescriptions on TGF-?1/Smads signal transduction pathway in bone tissue of ovariectomized rats 4.1 Effects of Zuogui pill,Yougui pill and their disassembled prescriptions on the protein expression of TGF-?1 and its receptor protein in bone tissue of ovariectomized rats The detection results of TGF-?1 and its receptor protein in bone tissue of rats in each group were as follows:After administration of 12 weeks,compared with the Control group,the protein expression of TGF-?1?T?R??T?R?in the Sham group were similar to that of the Control group,no significant difference(P>0.05),the protein expression of TGF-?1?T?R??T?R?in the OVX group were decreased,there were significant differences(P<0.05 or P<0.01);compared with the OVX group,the protein expression of TGF-?1?T?R??T?R?in the BJL?ZGW?YGW group increased slightly,there were significant differences(P<0.05 or P<0.01);compared with the BJL group,the protein expression of TGF-?1?T?R??T?R?in the ZGW group and the protein expression of T?R??T?R?in the YGW group were similar to that of the BJL group,no significant difference(P>0.05),the protein expression of TGF-?1 in the YGW group and the protein expression of TGF-?1?T?R??T?R?in the GTY?ZYY?BYY group didn't increase as well as the BJL group,there were significant differences(P<0.01);compared with the ZGW group,the protein expression of T?R??T?R?in the YGW group were similar to that of the ZGW group,no significant difference(P>0.05),the protein expression of TGF-?1 in the YGW group and the protein expression of TGF-?1?T?R??T?R?in the GTY?ZYY?BYY group didn't increase as well as the ZGW group,there were significant differences(P<0.05 or P<0.01).4.2 Effects of Zuogui pill,Yougui pill and their disassembled prescriptions on the protein expression of transduction protein Smad2?Smad3?Smad4?Smad7 in bone tissue of ovariectomized rats The results of Smad2?Smad3 protein in bone tissue of rats in each group were as follows:After administration of 12 weeks,compared with the Control group,the protein expression of Smad2?Smad3 in the Sham group were similar to that of the Control group,no significant difference(P>0.05),the protein expression of Smad2?Smad3 in the OVX group were decreased,there were significant differences(P<0.05 or P<0.01);compared with the OVX group,the protein expression of Smad2?Smad3 in the BJL?ZGW?YGW group increased slightly,there were significant differences(P<0.05 or P<0.01);compared with the BJL group,the protein expression of Smad2?Smad3 in the ZGW?YGW group were similar to that of the BJL group,no significant difference(P>0.05),the protein expression of Smad2?Smad3 in the GTY?ZYY?BYY group didn't increase as well as the BJL group,there were significant differences(P<0.05 or P<0.01);compared with the ZGW group,the protein expression of Smad2?Smad3 in the YGW group and the protein expression of Smad3 in the GTY?ZYY?BYY group were similar to that of the ZGW group,no significant difference(P>0.05),the protein expression of Smad2 in the GTY?ZYY?BYY group didn't increase as well as the ZGW group,there were significant differences(P<0.01).The results of Smad4 protein in bone tissue of rats in each group were as follows:After administration of 12 weeks,compared with the Control group,the protein expression of Smad4 in the Sham group was similar to that of the Control group,no significant difference(P>0.05),the protein expression of Smad4 in the OVX group were decreased,there were significant differences(P<0.01);compared with the OVX group,the protein expression of Smad4 in the BJL?ZGW?YGW group increased slightly,there were significant differences(P<0.01);compared with the BJL group,the protein expression of Smad4 in the ZGW?YGW group were similar to that of the BJL group,no significant difference(P>0.05),the protein expression of Smad4 in the GTY?ZYY?BYY group didn't increase as well as the BJL group,there were significant differences(P<0.01);compared with the ZGW group,the protein expression of Smad4 in the YGW/ZYY group were similar to that of the ZGW group,no significant difference(P>0.05),the protein expression of Smad4 in the GTY?BYY group didn't increase as well as the ZGW group,there were significant differences(P<0.05).The results of Smad7 protein in bone tissue of rats in each group were as follows:After administration of 12 weeks,compared with the Control group,the protein expression of Smad7 in the Sham group was similar to that of the Control group,no significant difference(P>0.05),the protein expression of Smad7 in the OVX group rose,there were significant differences(P<0.01);compared with the OVX group,the expression of Smad7 protein was decreased in all groups there were significant differences(P<0.01);compared with the BJL group,the protein expression of Smad7 in the ZGW?YGW group were similar to that of the BJL group,no significant difference(P>0.05),the protein expression of Smad7 in the GTY?ZYY?BYY group didn't reduce as well as the BJL group,there were significant differences(P<0.01);compared with the ZGW group,the protein expression of Smad7 in the YGW?ZYY group were similar to that of the ZGW group,no significant difference(P>0.05),the protein expression of Smad7 in the GTY group didn't increase as well as the ZGW group,there were significant differences(P<0.05).5.Correlation analysis of TGF-?1/Smads signal transduction pathway and lumbar spine average bone mineral density Simple correlation analysis showed:The TGF-?1?T?R??T?R??Smad2 protein were respectively correlated with the mean BMD of the lumbar vertebrae in ovariectomized rats(P<0.05);there was a significant positive correlation between Smad3 ?Smad4 protein and mean BMD of lumbar spine(P<0.01);there was a significant negative correlation between Smad7 protein and lumbar spine bone mineral density(P<0.01).Conclusion: 1.After the rats were ovariectomized,bone mineral density decreased,bone microstructure damaged,after treatment with Zuogui pill,Yougui pill and their disassembled prescriptions 12 weeks,bone density increased in different degrees,the treatment effect of Zuogui pill and Yougui pill were better than common drugs of two prescriptions?pure nourishing Yin drugs?pure tonifying Yang drugs,Zuogui pill and Yougui pill have a certain preventive and therapeutic effect on postmenopausal osteoporosis.2.After the rats were ovariectomized,the expression level of TGF-?1 and its receptor T?R??T?R? and transduction protein Smad2?Smad3?Smad4 in bone tissue were decreased,the expression level of suppressor protein Smad7 was increased,it may be the one of the important mechanisms of postmenopausal osteoporosis,the mechanism of postmenopausal osteoporosis may be realized by TGF-?1/Smads signal transduction pathway.3.The expression of TGF-?1 and its receptor T?R??T?R ?and its downstream signal transduction molecules Smad2?Smad3?Smad4 protein were promoted by Zuogui pill,Yougui pill and their disassembled prescriptions,the expression of suppressor Smad7 protein was down regulated.Further confirmed,the occurrence mechanism of postmenopausal osteoporosis by Zuogui pill,Yougui pill and their disassembled prescriptions may be mediated by TGF-?1/Smads signal transduction pathway,and TGF-?1/Smads signal transduction pathway has a certain correlation with bone mineral density(BMD). |
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