Comparison Of Bone Microstructure And Biomechanical Properties Between Male And Female Castrated Rats And The Study Of Intervention Effect Of Yougui Pill

ObjectiveTo observe the similarities and differences of bone micro structure and biomechanical properties between Ovariectomized rats and testicular excision rats,and to observe the differences and similarities of the intervention of Yougui pill on bone microstructure and biomechanical properties of ovariectomized rats and testicular excision rats,and from the perspective of MAPK(Erkl/2)/c-Fos/AP-1 signaling pathway to reveal the mechanism of the difference in part.It will provide further experimental basis for the treatment of osteoporosis,and to enrich the scientific connotation of the "kidney main bone" theory.MethodsA total of 156 SD rats of the same age(3 months).SPF grade,half female and half male,were selected.The rats were randomly divided into 8 groups according to their weight:female basal group and male basal group,female blank control group and male blank control group,female sham-operated group and male sham-operated group with 12 rats in each group.Female model group and male model group,42 rats in each group.The female and male rats were anesthetized with 3%pentobarbital sodium according to the dose of 0.15ml/100g body weight,followed by bilateral ovariectomy or orchiectomy(castration).During the operation,the female model group died 5 rats and male model group died 4 rats,and the procedure of operation in female and male sham operation group was the same as that in female and male model group,but no ovaries or testes were removed,only a few adipose tissue around ovary or testis was removed.On the day before the operation,the rats in the female and male basal groups were killed after anesthesia,and bilateral femurs were taken for the detection of Micro-CT bone histomorphometry and bone biomechanical indexes of the electronic universal test machine.The measured data is used as the base value before other groups implement processing factors.After 3 months of operation.37 rats in the female model group were randomly divided into 3 groups according to their body weight:13 rats in ovariectomized group(OVX group).12 rats in OVX Plus estradiol valerate group(estrogen group,that is.positive control group).12 rats in OVX Plus Yougui pill group(female Yougui pill group)and 38 rats in male model group were randomly divided into 3 groups according to their weight:13 rats in testicular group(ORX group),13 rats in ORX Plus methyltestosterone group(androgen group,that is,positive control group),and 12 rats in ORX Plus Yougui pill group(male Yougui pill group).At the end of the group,the rats in each group were treated with different drugs.The female Yougui pill group and the male Yougui pill group were given the decoction of Yougui pill.The concentration and volume were 1.024g crude drug/ml and 1ml/100g body weight respectively,and the dosage was 10.24g crude drug/kg body weight(This dose is an effective dose that has been confirmed in previous studies,equivalent to the equivalent dose of adult clinical use,which is 6.5 times the adult clinical dose.).The estradiol valerate solution(E2)was given to rats in estrogen group.The concentration and volume of estradiol valerate(E2)were 0.0184mg/ml and 1ml/100g body weight,and the dosage of estradiol valerate(E2)was 0.184mg/kg body weight.Rats in androgen group were given methyltestosterone solution(T),Theconcentration and volume of methyltestosterone were 0.184mg/ml and 1ml/100g body weight,and the dosage of methyltestosterone was 1.84mg/kg body weight.All the above groups were given once a day for 6 consecutive days.After one day's rest,they were given again for 6 days,so that they could be given for 3 months.Female and male blank control group,female and male sham operation group,OVX group and ORX group were given the same volume of pure water by gastric gavage according to the above-mentioned method.During the gavage,the rats were weighed once a week,and the volume of the drug was adjusted according to the weight change.At the end of administration,the rats in each group were killed immediately after anesthesia,and bilateral femurs were taken for the detection of Micro CT bone histomorphometry and bone biomechanics index of electronic universal tester.The frozen sections of decalcified bone were made to detect the protein expression of MAPK(Erkl/2).c-Fos and AP-1.SPSS 20.0 software was used for statistical processing.The results were expressed as mean ±standard deviation(x±s).If the data are normal distribution.one-way ANOVA(ANOVA)is used to deal with the variance,LSD test is used to compare the variance between two groups.and Dunnett's T3(3)test is used when the variance is uneven.If the data are non-normal distribution,then the non-parameter test Kruskal-Wallis single factor ANOVA(k sample)(W)is used to process the data,and the pairwise comparison between the two groups is carried out by using the Stepwis step-down test under its termsResults1 Comparison of morphology and structure of cancellous bone of femur in ovariectomized rats and testicular excision rats and intervention effect of Yougui Pill1.1 Comparison of BV/TV change rate of femoral cancellous bone in ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillAt the end of the experiment,the BV/TV of femoral cancellous bone in female sham-operated group and male sham-operated group increased significantly.There was a significant difference when OVX group and ORX group compared with their corresponding sham-operation group,which showed a negative increase,that is,a significant decrease.The BV/TV of estrogen group,female Yougui pill group,androgen group and male Yougui pill group all showed negative growth,compared with their corresponding sham operation group,there was significant difference.However,the degree of decrease was significantly lower than that of their corresponding OVX and ORX groups.The decrease of BV/TV in OVX group was significantly higher than that in ORX group,while that in female Yougui pill group was significantly lower than that in male Yougui pill group1.2 Comparison of Conn.Dn change rate of femoral cancellous bone in ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillAt the end of the experiment,the Conn.Dn of femoral cancellous bone in female sham-operated group and male sham-operated group increased significantly.There was a significant difference when OVX group and ORX group compared with their corresponding sham-operation group,which showed a negative increase,that is.a significant decrease.The Conn.Dn of estrogen group,female Yougui pill group,androgen group and male Yougui pill group all showed negative growth,compared with their corresponding sham operation group,there was significant difference.However,the degree of decrease was significantly lower than that of their corresponding OVX and ORX groups.The decrease of Conn.Dn in OVX group was significantly higher than that in ORX group,while that in female Yougui pill group was significantly lower than that in male Yougui pill group.1.3 Comparison of Cb.C change rate of femoral cancellous bone in ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillAt the end of the experiment,the Cb.C of femoral cancellous bone in female sham-operation group and male sham-operation group showed negative increase,that is.decrease,while the increase of OVX group and ORX group showed significant difference compared with their corresponding sham-operation group.The Cb.C of estrogen group,female Yougui pill group,androgen group and male Yougui pill group were also increased,there was significant difference compared withtheir corresponding sham operation group,but the degree of increase was obviously less than that of their corresponding OVX group and ORX group.Compared with ORX group,Cb.C increased significantly in OVX group,while Cb.C in female Yougui pill group was lower than that in male Yougui pill group,but there was no significant difference between female Yougui pill group and male Yougui pill group.1.4 Comparison of DA of femora cancellous bone in ovariectomized rats and testicular excision rats and intervention effect of Yougui PillThe DA of femoral cancellous bone in OVX group,estrogen group and female Yougui pill group was significantly lower than that in female sham operation group,while the DA in estrogen group and female Yougui pill group was significantly higher than that in OVX group.Compared with the male sham operation group,the DA of ORX group,androgen group and male Yougui pill group was significantly lower than that of the male sham operation group,while the DA of androgen group and male Yougui pill group was significantly higher than that of the ORX group.The DA of ORX group was significantly higher than that of OVX group,but there was no significant difference between male Yougui pill group and female Yougui pill group.1.5 Comparison of Tb.Pf of femoral cancellous bone in ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillCompared with female sham-operated group,the Tb.Pf of femur cancellous bone was significantly increased in OVX group,estrogen group and female Yougui pill group,while the Tb.Pf in estrogen group and female Yougui pill group was significantly lower than that in OVX group.The Tb.Pf of ORX group,androgen group and male Yougui pill group was significantly higher than that of male sham operation group,while the Tb.Pf of androgen group and male Yougui pill group was significantly lower than that of ORX group.The Tb.Pf of ORX group was significantly lower than that of OVX group,but there was no significant difference between male Yougui pill group and female Yougui pill group.2 Comparison of biomechanical properties of femoral cortical bone in ovariectomized rats and testicular excision rats and intervention effect of Yougui PillThe changes of Maximum Load(ML).Breaking Strength(BS)and Elastic Modulus(EM)can reflect the biomechanical properties of femoral cortical bone in rats.2.1 Comparison of ML change rate of femoral cortical bone in ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillAt the end of the experiment,ML of femoral cortical bone in female and male sham-operated rats was significantly increased,while ML in OVX and ORX groups was also increased,but the degree of increase was significantly lower than that of their corresponding sham-operated groups.The increase of ML in estrogen group and female Yougui pill group was significantly higher than that in OVX group,but there was no significant difference between female sham operation group.The increasing degree of ML in androgen group and male Yougui pill group was significantly higher than that in ORX group,but significantly lower than that in male sham operation group.The increase of ML in OVX group was significantly lower than that in ORX group,and that in female Yougui pill group was significantly lower than that in male Yougui pill group.2.2 comparison of BS change rate of femoral cortical bone in ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillAt the end of the experiment,the BS of femoral cortical bone in female and male sham-operated rats increased significantly,while that in OVX and ORX groups showed a negative increase,that is,decreased,compared with their corresponding sham-operated groups,there was a significant difference between the two groups.The levels of BS in estrogen group,female Yougui pill group,androgen group and male Yougui pill group were all increased,compared with their corresponding sham-operated groups,there was no significant difference,but significantly higher than those in their corresponding OVX and ORX groups.The decrease of BS in OVX group was significantly higher than that in ORX group,but there was no significant difference between female Yougui pill group and male Yougui pill group.2.3 Comparison of EM change rate of femoral cortical bone in ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillAt the end of the experiment,the EM of femoral cortical bone in female sham-operation group and male sham-operation group increased significantly,while the EM in OVX group and ORX group showed a negative increase,that is.decreased.compared with their corresponding sham-operation group,there was a significant difference.The EM of estrogen group,female Yougui pill group,androgen group and male Yougui pill group were all increased,compared with their corresponding sham-operated groups,there was no significant difference,but significantly higher than those of their corresponding OVX and ORX groups.There was no significant difference in the decrease of EM between the OVX group and the ORX group,compared with the increase degree of EM,there was no significant difference between the female Yougui pill group and male Yougui pill group.3 comparison of the expression of MAPK(Erkl/2),c-Fos,AP-1 protein in tibia bone marrow of ovariectomized rats and testicular excision rats and the intervention effect of Yougui Pill3.1 Comparison of the expression of MAPK(Erkl/2)protein in tibia bone marrow of ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillThe positive density of MAPK(Erkl/2)protein expression in tibia bone marrow of OVX group was significantly higher than that of female sham operation group.The positive density of MAPK(Erkl/2)protein of estrogen group and female Yougui pill group was not significantly different from that of female sham operation group.Compared with OVX group,the positive density of MAPK(Erkl/2)protein expression in estrogen group and female Yougui pill group was significantly lower.The positive density of MAPK(Erkl/2)protein expression in ORX group was significantly higher than that in male sham operation group,but the positive density of MAPK(Erkl/2)protein of androgen group and male Yougui pill group was not significantly different from that of female sham operation group.Compared with ORX group,the positive density of MAPK(Erkl/2)protein expression in androgen group and male Yougui pill group was significantly lower.The expression density of MAPK(Erkl/2)protein in ORX group was significantly lower than that in OVX group.The positive expression density of MAPK(Erkl/2)protein in male Yougui pill group was significantly higher than that in female Yougui pill group.3.2 Comparison of c-Fos protein expression in tibia bone marrow of ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillCompared with female sham-operation group,the positive density of c-Fos protein expression in tibia bone marrow of OVX group was significantly higher,but there was no significant change in estrogen group and female Yougui pill group.Compared with OVX group,the positive density of c-Fos protein expression in estrogen group and female Yougui pill group was significantly lower.The positive density of c-Fos protein expression in ORX group and male Yougui pill group was significantly higher than that in male sham-operation group,but there was no significant difference between androgen group and male sham-operation group.Compared with ORX group,the positive density of c-Fos protein expression in male Yougui pill group and androgen group was significantly lower.The expression density of c-Fos protein in ORX group was significantly lower than that in OVX group.The male Yougui pill group was significantly higher than that of the female Yougui pill group.3.3 Comparison of the expression of AP-1 protein in tibia bone marrow of ovariectomized rats and testicular excision rats and the intervention effect of Yougui PillThe positive density of AP-1 protein expression in tibia bone marrow of OVX group was significantly higher than that of female sham operation group.The positive density of AP-1 protein of estrogen group and female Yougui pill group was not significantly different from that of female sham operation group.The positive density of AP-1 protein expression in estrogen group and female Yougui pill group was significantly lower than that in OVX group.Compared with the male sham-operated group.the positive density of AP-1 protein increased significantly in the ORX group and the male Yougui pill group,but there was no significant difference in the androgen group.Compared with ORX group,the positive density of AP-1 protein was significantly decreased in both androgen group and male Yougui pill group.The expression density of AP-1 protein in ORX group was significantly lower than that in OVX group.The male Yougui pill group was significantly higher than the female Yougui pill group.There was no significant difference between the female sham operation group and the female blank control group and there was also no significant difference between the male sham operation group and the male blank control group,so that the influence of the surgical factors on the experiment was excluded.Conclusions(1)Osteoporosis occurred in both male and female rats after castration.However,there were significant differences in micro structure between the two groups.The decrease of BV/TV and Conn.Dn and the increase of Cb.C in femora cancellous bone of female ovariectomized rats were significantly higher than those of male testicular excision rats.DA was significantly lower and Tb.Pf was significantly higher than that in male testicular excision rats.Yougui Pill has obvious therapeutic effect on osteoporosis induced by female ovariectomized rats and male testicular excision rats,but there are some differences in the improvement of microstructure,which is reflected in:the improvement degree of Yougui Pill on BV/TV and Conn.Dn in female ovariectomized rats was better than that of male testicular excision rats,but there was no significant difference in the improvement of DA.Cb.C and Tb.Pf between the two groups.(2)after castration,the biomechanical properties of cortical bone decreased significantly in female and male rats,but there were significant differences,which showed that the maximum load and breaking strength of female rats were significantly lower than that of male rats.There was no significant difference in elastic modulus between male and female.Yougui Pill has obvious important effect on maximum load,breaking strength and elastic modulus.The effect of Yougui Pill on maximum load by the male testicular excision rats was significantly higher than that of the female ovariectomized rats,but there were some differences in the effect of Yougui Pill on the maximal load.However,there was no significant difference in the increase of fracture strength and elastic modulus between the two groups.(3)MAPK(Erk1/2)/c-Fos/AP-1 signaling pathway plays an important role in osteoporosis.After castration,the expression of MAPK(Erkl/2),c-Fos and AP-1 protein in bone marrow(cancellous bone)of female and male rats was significantly increased,while the expression of these three key factors in female ovariectomized rats was significantly stronger than that in male testicular excision rats.This is one of the mechanisms of sex difference in the micro structure of cancellous bone induced by castration in female and male rats.Yougui Pill can inhibit the osteoporosis caused by castration by inhibiting this pathway.Yougui Pill inhibitory effect on female castration rats is significantly stronger than that of male castrated rats.This is one of the mechanisms of sex difference in the improvement of the microstructure of the cancellous bone of the female ovariectomized rats is better than that of the male testicular excision rats.