Discussion On The Difference Of Function As Well As Its Mechanism Of Zuogui Pill And Yougui Pill On Osteoporosis Induced By Ovariectomy And Orchiectomy In Rats Based On Osteoblastic And Osteoclastic Regulatory Pathway

ObjectivesFrom the perspective of OPG/RANKL osteoclast regulatory pathway and Wnt/LRP5/β-catenin osteoblast regulatory pathway, this paper aims to reveal the difference of function as well as its mechanism of Zuogui pill with nourishing kidney and Yougui pill with warming kidney to castrated rat with osteoporosis but different genders so as to explore whether the "kidney governing bones" exist gender difference and its mechanism, then further clarifies the scientific connotation of kidney governing bones" and finally provides experimental foundation for clinical meditation of adopting Chinese meditation to treat osteoporosis.MethodsSelect160SD rats with the same weeks, SPF class and half of females and half of males, then divided them into six groups randomly:12ones in male and female blank control group respectively,12ones in male and female sham operation group respectively and56ones in female and male model group respectively. Before castration operation, at first, adopt Osteocore3Digital2D bone mineral density (dual-energy X-ray) scan all the rats to obtain scanned images for further analysis. Then conduct castration operation to the rats in both model groups, namely remove the ovaries of female model group and remove the testicle of male model group. After operation, divide the rats in both model groups into8groups in terms of weight:13ones in ovariectomized group (OVX group),14ones in orchiectomy (ORX group),14ones in estradiol valerate group (E2group),13ones in methyltestosterone group (T group), female and male Zuohui pill group, female and male Yougui pill group respectively(of which one in male sham operation group and6ones in model group died from surgical anesthesia).The OP model making of OVX rats:adopt pentobarbital sodium to conduct intraperitoneal injection for general anesthesia of female rats, then remove the bilateral ovaries; carry out abdominal operation on female sham operation group without removing ovaries but removing few adipose tissues around the ovaries. The OP model making of ORX rats:adopt pentobarbital sodium to conduct intraperitoneal injection for general anesthesia of male rats, then separate the bilateral testicles together with epididymis and remove the bilateral testicles; in the male sham operation group, same method is adopted to find and separate literal testicles but with testicles.Ten days after operation, conduct intragastric administration on rats in various medicated groups irrigate Zuoguipill decoction to the rats in female and male Zuogui pill group with the medicated concentration of0.968g/ml; irrigate Yougui pill decoction to the rats in female and male Yougui pill group with the medicated concentration of1.024g/ml; irrigate estradiol valerate solution to the rats in estradiol valerate group with the concentration of0.0092mg/ml; irrigate methyltestosterone solution to the rats in methyltestosterone group with the concentration of0.092mg/ml. The above mentioned medicated volume is1ml/100g per weight with one weighting every week. The medicated volume shall be adjusted according to the weight. Offer medicine at fixed time everyday (2’clock pm) for6days continuously,then offer medicine for another6days after one day’s rest. Further, keep this circulation for three months. The rats in male and female blank control group, the female and male sham operation group, OVX group, ORX group are irrigated with the equivoluminal purified water as above method (one rat in female Yougui pill group and male Zuogui pill group died respectively due to the careless intragastric administration).16days and3days before execution, the rats in various groups are injected with tetracycline hydrochloride by intraperitoneal injection (dose of30mg/kg weight), then conduct double immuhistochemical fluorescence staining on bone tissue. After medicine administration, adopt pentobarbital sodium to conduct intraperitoneal injection for general anesthesia and bone mineral density to scan the rats to obtain scanned images for further analysis. Then execute the rats in various groups immediately and take1/3right proximal shin bone to make undecalcified bone sections for the detection of index of bone histomorphometry; take1/3left proximal shin bone to make demineralized bone frozen sections for the detection of functional regulatory pathway of OPG/RANKL/RANK osteoclast and the relevant factor proteins and mRNA by immunohistochemistry and in situ hybridization.The experimental results data is expressed by mean±standard deviation (x±s) by adopting SPSS20.0statistical software. If the data is normal distribution, the ANOVA shall be adopted to conduct data processing. The LSD test shall be adopted for the comparison between groups at the time of homogeneity of variance; the Dunett’s T3(3) test shall be adopted at the time of heterogeneity of variance; if the data is not normal distribution, the non-parametric test shall be adopted on Kruskal-Wallis single factor ANOVA (K sample)(W); match data comparison and adopt matched samples for T test.Results1. The effect of Zuogui and Yougui pills on lumbar vertebra BMD as well as bone loss rate of castrated rats with different gendersExcept for T group, there appeared significant difference on lumber vertebra BMD of rats in various groups after medicine administration compared with that before castration (P<0.05or P<0.01).The group comparisons of limber vertebra BMD of rats with same genders after medicine administration:the model group (OVX group and ORX group) shares more obvious decline than that in sham operation group; the limber vertebra BMD of rats in E2group, female Zuogui pill group, female Yougui pill group enjoy sharper increase than that in OVX group, though still lower than that in sham operation group; compared with ORX group, the lumber vertebra BMD of rats in T group and male Yougui pill group increased significantly while the male Zuogui pill group showed no significant difference, though still lower than that in sham operation group; besides, there exist significant difference between female Zuogui pill group, female Yougui pill group and male Zuogui pill group and male Yougui pill group with the latter higher than the former.The group comparisons of lumber vertebra bone loss rate of rats with same genders after medicine administration, the results indicated that the rats in model group (OVX group and ORX group) showed obvious increase on lumber vertebra bone loss than that in sham operation group; the rats in E2group, female Zuogui pill group and female Yougui pill group shared obvious decline on lumber vertebra bone loss than that in OVX group, though still more than that in sham operation group; compared with ORX group, the rats in T group and male Yougui pill group had obvious decline on lumber vertebra bone loss while the male Zuogui pill group showed no significant difference, though still more than that in sham operation group; besides, there exist significant difference on bone loss rate between female Zuogui pill group, female Yougui pill group and male Zuogui pill group and male Yougui pill group with the latter higher than the former.The group comparisons of lumber vertebra bone loss rate of rats with different genders after medicine administration, the results indicated that there appeared significant difference on bone loss rate between female and male sham operation group, OVX group and ORX group, female and male Zuogui pill group, female and male Yougui pill group, of which the bone loss in OVX group was much more than that in ORX group (P<0.01) while the male Zuogui pill group was obviously more than that in female Zuogui pill group (P<0.05) and the male Yougui pill group was much more than that in female Yougui pill group (P<0.01), but the bone increase in female sham operation group was much lower than that in male sham operation group (P<0.05).2. The effect of Zuogui and Yougui pill on the index of bone his to morphology of castrated rats with different genders2.1The effect of Zuogui andd Yougui pill on shin bone TBV%and bone loss rate of castrated rats with different gendersThe group comparisons of shin bone TBV%of rats with same genders:the model group (OVX group and ORX group) shared more significant decline than that in sham operation group; the male Zuogui pill group showed no statistical difference compared with ORX group (P>0.05) while the rest of medicated groups shared sharper increase that that in model group with same genders, though still much lower than that in sham operation group with same gender. Besides, the female Yougui pill group was higher than that in female Zuogui pill group while the male Yougui pill group was higher than that in female Yougui pill group, whose difference shares statistical significance.The group comparisons of shin bone TBV%of rats with different genders:there appeared significant difference between female sham operation group and male sham operation group, OVX group and ORX group with the latter higher than the former.The group comparisons of shin bone loss rate of rats with same genders, the results indicated that the bone loss in model group (OVX group and ORX group) was much more than that in sham operation group; the bone loss in male Zuogui pill group showed no statistical significance compared with ORX goup(P>0.05) while the rest of medicated groups had obvious decline on bone loss compared with model group with same gender, though still much more than that in sham operation group. Besides, the bone loss in female Yougui pill group was much less than that in female Zuogui pill group (P<0.01) while the male Yougui pill group was much less than of that in male Zuogui pill group (P<0.05).The group comparisons of shin bone loss rate of rats with different genders, the results indicated that there exist significant difference on bone loss between female and male sham operation group, OVX group and ORX group, female and male Zuogui pill group and female and male Yougui pill group. Besides, the bone loss in OVX group was much more than than that in ORX group (P<0.05) while the male Zuogui pill group was much more than that in female Zuogui pill group (P<0.05) and the male Yougui pill group was much more than that in female Yougui pill group (P<0.05), but the bone increase in female sham operation group was much lower than that in male sham operation group (P<0.05).2.2The effect of Zuogui and Yougui pill on shin bone TRS%, TFS%and TRS/TFS ratio of rats with different gendersThe comparisons between groups with same gender the shin bone TRS%, TFS%and TRS/TFS ratio in model group (OVX group and ORX group) increased obviously that that in sham operation group with same genders. In the various medicated groups, the above parameters of E2group, T group and female Yougui pill group decreased obviously compared with that in model group with same gender and shared no statistical difference with sham operation group with same gender; the above parameter on rat shin bones in female Zuogui pill group was also much lower than that in ORX group, though still much higher than that in male sham operation group. In the two Chinese medicine treatment groups, there exist statistical difference on the above parameters on rat shin bone between female Yougui pill group and female Zuogui pill group, male Yougui pill group and male Zuogui pill group with the former lower than the latter.The group comparisons of shin bone TRS%, TFS%and TRS/TFS ratio of rats with different gender, the results showed that there appeared statistical difference on the above parameters between female and male sham operation groups, model groups (OVX group and ORX group) and Zuogui pill group and Yougui pill group, of which the TFS%of female sham operation was lower than that in male sham operation group and OVX group was higher than that in ORX group, while the female Zuogui pill was lower than male Zuogui pill and the female Yougui pill was lower that male Yougui pill; on the contrary, the TRS%of female sham operation group was higher than that in male Yougui pill group; the TRS/TFS ratio of female sham operation group was higher than that in male sham operation group and the OVX group was higher than ORX group, while the female Zuogui pill group was lower that that in male Zuogui pill group and the female Yougui pill group was lower than that in male Yougui pill group.2.3The effect of Zuogui and Yougui pill on shin bone OSW, MAR and mAR of rats with different genderThe comparison between groups with same gender:the shin bone OSW, MAR and mAR of rats in model group (OVX group and ORX group) increased obviously compared with sham operation with same gender. In various medicated groups, compared with model groups with same gender (OVX group and ORX group), the above parameters of E2group, T group and female Yougui pill group decreased significantly and shared no statistical difference with sham operation group with same gender; compared with OVX group, the above shin bone parameters in female Zuogui pill groups decreased obviously, though still higher than that in female sham operation group; compared with ORX group, the above parameters of rat shin bone in female Zuogui pill group shared no statistical difference while the male Yougui pill group decreased obviously, though still much higher than that in male sham operation group; compared with Zuogui pill group with same gender, the MAR and mAR in female and male Yougui pill decreased obviously while the OSW in male Yougui pill group decreased obviously compared with that in male Yougui pill group and the OSW n female Yougui pill group showed decreased tendency compared with male Zuogui pill group (P=0.054).The comparisons between groups of rats with different genders, the results indicated that there exist difference on the above parameters between two genders in model group (OVX group and ORX group), Zuogui pill group and Youzui pill group (P<0.05or P<0.01). Besides, the OVX group was higher than ORX group, while the female Zuogui pill group was lower than that in male Zuogui pill group and the female Yougui pill group was lower than that in male Yougui pill group; compared among sham operation groups, the OSW female showed no statistical difference compared with that of male (P>0.05) with decreased tendency, the MAR and mAR female was much lower that male (P<0.05or P<0.01).3. The effect of Zuogui and Yougui pill on OPG,RANKL protein, mRNA expression as well as RANKL/OPG and RANKL mRNA/OPG mRNA ratio in shin-bone marrow of castrated rats with different genders3.1The effect of Zuogui and Yougui pill on OPG protein and mRNA expression of shin-bone marrow of castrated rats with different gendersThe comparisons between groups with same gender, the result showed that compared with sham operation with same gender, the positive density of OPG protein and mRNA expression in shin bone marrow of rats in model group (OVX group and ORX group) decreased obviously; compared with model group with same gender (OVX group and ORX group), the positive density in E2group, T group and female Yougui pill group increased obviously and shared no statistical difference with sham operation group with same gender; compared with OVX group, the positive density of female Zuogui pill group increased obviously, though still much lower than that in female sham operation group; compared with ORX group, the positive density of OPG protein and mRNA expression in female Zuogui and Yougui pill group showed no statistical difference (P>0.05).The group comparisons of the positive density of OPG protein and mRNA of shin bone marrow of rats with different genders, the results indicated that the female sham operation group was lower than male sham operation group and the OVX group was lower than ORX group. The female Zuogui pill group was higher than male Zuogui pill group while the female Yougui pill group was higher than male Yougui pill group, between which showed statistical difference (P<0.05or P<0.01).3.2The effect of Zuogui and Yougui pill on RANKL protein and mRNA expression of shin-bone marrow of castrated rats with different gendersThe comparison between groups with the same genders, the results showed that compared with sham operation group with the same genders, the positive density of RANKL protein and mRNA expression of shin-bone marrow of rats in model group (OVX group and PRX group) increased obviously; compared with model group (OVX group and ORX group) with the same gender, the positive density in E2group, T group and female Yougui pill group decreased obviously. Besides, there appeared no statistical difference with sham operation group with the same gender; compared with OVX group, the positive density in female Zuogui pill group declined significantly, though still higher than that in sham operation group; there exist no statistical difference on positive density between ORX group and female Zuogui pill group, while the positive density in male Yougui pill group decreased obviously, though still higher than that in male sham operation group.The group comparison of the positive density of RANKL protein and mRNA expression of shin-bone marrow of rats with different genders, the result showed that the female sham operation was higher than male sham operation group and the OVX group was higher than ORX group. The female Zuogui pill group was lower than male Zuogui pill group while the male Yougui pill group was lower than male Yougui pill group, between which showed statistical difference (P<0.05or P<0.01).3.3The effect of Zuogui and Yougui pill on RANKL/OPG and RANKL mRNA/OPG mRNA ratio of shin-bone marrow of castrated rats with different gendersThe comparisons between groups with same genders, the results showed that compared with sham operation with same gender, the RANKL/OPG and RANKL mRNA/OPG mRNA ratio of shin-bone marrow of rats in model group (OVX group and ORX group) increased obviously; compared with model group (OVX group and ORX group) with same gender, the positive density in E2group, T group and female Yougui pill group decreased obviously and showed no statistical difference with sham operation group with same gender; the positive density in female Zuogui pill group decreased obviously and showed no statistical difference with ORX group, while positive density decreased obviously compared with that in male Yougui pill group, though still much higher than that in male sham operation group.The group comparisons of RANKL/OPG and RANKL mRNA/OPG mRNA ratio of shin-bone marrow of rats with different genders, the results showed that the female sham operation group was higher than male sham operation group and the OVX group was higher than ORX group, while the female Zuogui pill group was lower than male Zuogui pill group and the female Yougui pill group was lower than male Yougui pill group, between which showed no statistical difference (P<0.05或P<0.01).4. The effect of Zuogui, Yougui pill on Wntl, LRP5and β-catenin protein and mRNA expression in shin-bone marrow of castrated rats with different gendersThe comparison between groups with same gender, the result showed that compared with sham operation with same gender, the positive density of Wntl protein and mRNA expression in shin-bone marrow of rats in model group(OVX group and ORX group) increased significantly; compared with model group with same gender (OVX group and ORX group), the positive density in E2group, T group and female Yougui pill group decreased obviously and showed no statistical difference with sham operation group with same gender; compared with OVX group, the positive density in female Zuogui pill group declined obviously, though still much higher than that in female sham operation; the positive density in male Zuogui pill group showed no statistical difference compared with ORX group, while the positive density decreased obviously compared with male Yougui pill group, though still much higher than that in male sham operation.The group comparisons on the posotive density betwen Wntl protein and mRNA expression in shin-bone marrow of rats with different gender, the results indicated that the female sham operation group was lower than male sham operation group and the OVX group was higher than ORX group, while the female Zuogui pill was lower than male Zuogui pill group and the female Yougui pill group was lower than male Yougui pill group, between which showed no statistical difference(P<0.05或P<0.01).4.2The effect of Zuogui and Yougui pill on LRP5protein and mRNA expression in shin-bone marrow of castrated rats with different gendersThe comparisons between groups with same genders, the results showed that compared with sham operation with same gender, the positive density of LRP5protein and mRNA expression of shin-bone marrow of rats in model group (OVX group and ORX group) increased obviously; compared with model group with same gender (OVX group and ORX group), the positive density in E2group, T group and female Yougui pill group decreased obviously and showed no statistical difference with sham operation group with same gender; compared with OVX group, the positive density in female Zuogui pill group decreased significantly, though still much higher than that in female sham operation; the positive density in male Zuogui pill group showed no statistical difference with ORX group, while the positive density in male Yougui pill group decreased obviously compared with that in male Zuogui pill group, though still much higher than that n male sham operation group.The group comparisons on the positive density of LRP5protein and mRNA expression in shin-bone marrow of rats with different genders, the results showed that the female sham operation group was lower than male sham operation group and the OVX group was higher than ORX group, while the female Zuogui pill group was lower than male Zuogui pill group and the female Yougui pill group was lower than male Yougui pill group, between which showed no statistical difference (P<0.05或P<0.01).4.3The effect of Zuogui, Yougui pill on β—catenin protein and mRNA expression in shin-bone marrow of castrated rats with different gendersThe comparisons between groups with same gender, the results showed that compared with sham operation group with same gender, the positive density of β-catenin and mRNA expression in shin-bone marrow of rats in model group (OVX group and ORX group) increased obviously; compared with model group with same gender (OVX group and ORX group), the positive density in E2group, T group and female Yougui pill group decreased significantly and showed no statistical difference with sham operation with same gender; compared with OVX group, the positive density in female Yougui pill group decreased obviously, though still much higher than male sham operation group; the positive density of male Zuogui pill group showed no statistical difference with ORX group, while the positive density of male Yougui pill group decreased obviously compared with mate Zuogui pill, though still much higher than that in male sham operation group.The group comparisons on positive density of β-atenin protein and mRNA expression of rats with different genders, the results showed that the female sham operation group was lower than male sham operation group and the OVX group was higher than ORX group, while the female Zuogui pill group was lower than male Zuogui pill group and the female Yougui pill group was lower than male Yougui pill group, between which showed no statistical difference (P<0.05或(P<0.01).Conclusion1. The castration will cause rats with different gender but same weeks high-convention osteoporosis. Namely, both of the bone absorption and bone formation increased, but the former one was greater than the latter one, which leads to bone loss eventually. Differently, the bone turnover rate as well as the bone loss rate of OVX rats was much higher than that of ORX rats. The RANKL expression in marrow of OVX and ORX rats raised significantly, while the OPG expression declined significantly and the ratio (RANKL/OPG) also increased significantly, which then enhanced the osteoclast activity and increased the bone absorption at the same time; meanwhile, the obvious increase on Wnt1, LRP5and β-catenin expression enhanced the osteoclast activity together with bone absorption. However, the enhanced extent of osteoclast activity was greater than that of osteoblast activity, which caused the occurrence of osteoporosis; the above change of female castrated rats was much stronger than that of male castrated rats, because the castration will cause both female and male rats high-convention osteoporosis, but the bone turnover rate and bone loss rate of female rats was much higher than that of male rats.2. The effect of Zuogui and Yougui pill on castrated rats with osteoporosis but different genders exists distinct difference:Zuogui pill has obvious curative effect on OVX rats with osteoporosis, while does not have curative effect on ORX rats with osteoporosis; Yougui pill has obvious curative effect on OVX and ORX rats with osteoporosis, but its effect on OVX rats was much greater than that of ORX rats.3. The difference mechanism of Zuogui and Yougui pill on castrated rats with osteoporosis but different genders:(1) the Zuogui pill mechanism causing the difference lies in the fact that Zuogui pill enables to decline the RANKL expression in marrow of OVX rats with osteoporosis obviously, raise the OPG expression significantly and decrease the Wnt1, LRP5and β-catenin expression obviously at the same time, which then causes decline on bone absorption and bone formation, restrains bone’s high convention to reach a relative equilibrium state between bone absorption and bone formation so as to prevent bone loss, while the Zuogui pill does not have obvious effect on the above key factors in the marrow of ORX rats with osteoporosis.(2) the Yougui pill mechanism causing the difference lies in the fact that Yougui pill has the same effect with Zuogui pill on OPQ RANKL and Wnt1, LRP5and β-catenin in the marrow of OVX rats with osteoporosis, though the strength of Yougui pill is much greater than that of Zuogui pill. Yougui pill will obviously decrease the RANKL and Wnt1, LRP5and β-catenin expression in marrow of ORX rats with osteoporosis, but Yougui pill does not have obvious effect on OPG expression and its effect on RANKL, Wntl, LRP5and β-catenin is weaker than that on OVX rats with osteoporosis, which then cause the situation that the inhibiting effect on Yougui pill on bone high convention of OVX rats with osteoporosis is much stronger than that on ORX rats with osteoporosis so as to further cause its curative effect on osteoporosis more superior to the latter.4. The above results indicated that the "kidney governing bones" exists gender difference. The different changes of OPG/RANKL osteoclast regulatory pathway and Wnt/LRP5/β-catenin osteoblast are one of the mechanisms causing this difference.5. For OVX rats with osteoporosis, the curative effect of Yougui pill was more superior than Zuogui pill; For ORX rats with osteoporosis, the Yougui pill is in effect while the Zuogui pill is not in effect. Tips:kidney-yang deficiency as well as Yang-function insufficiency of kidneyzang maybe the main reason to cause osteoporosis after castration.