Study On Mechanisms Of Radix Dipsaci, Herba Pyrolae And Herba Cynomorii In Treatment Of Ostecoporosis Induced By Ovariectomy In The Rats

ObjectiveIn order to provide further experimental evidences for Chinese medicines for treatment of osteoporosis and the mechanism of "the kidney dominating the bone", in the study, the action links and characteristics of single drugs Xu Duan(续断Radix Dipsaci), Lu Xian Cao(鹿衔草Herba Pyrolae) and Suo Yang(锁阳Herba Cynomorii) with the functions of warming and recuperating the kidney-yang in treatment of osteoporosis induced by ovariectomy in the rat were investigated by using bone histomorphometry from the angle of the osteoclast differentiation and regulatory factors OPG, RANKL, and the cytokines IL-1 and IL-6 closely related with bone metabolism.Methods81 Wistar female rats, weighing 250±20g, SPF grade, were randomly divided into 3 groups, a blank control group (n=12), a pseudo-operation group(n=12) and a modeling group(n=57). After the rat in the modeling group was anesthetized with pentobarbital sodium, bilateral ovaries were removed; in the pseudo-operation group only a little fatty tissue nearby the bilateral ovaries was removed. One month after the operation, the rats in the modeling group were divided again into six sub-groups, a model group(n=12), a stilbestrol group (positive control group, n=12), a Xu Duan group(n=11), a Lu Xian Cao group(n=11) and a Suo Yang group(n=11).The rats in the Xu Duan group, the Lu Xian Cao group and the Suo Yang group were intragastrically administrated respectively with Xu Duan decoction, Lu Xian Cao decoction and Suo Yang decoction,5.6ml/kg body weight, 1g crude drug/ml, and in the positive control group were intragasdrically administrated with stilbestrol, 5.6ml/kg body weigh,0.008mg/ml. The rats in the blank control group, the pseudo-operation group and the model group were intragastrically administrated with the equal volume of distilled water. The above treatments were given for 3 months, once a day,6 sessions a week with an one-day interval.At 15 days and 3 days before the rat was killed, tetracycline hydrochloride (30mg/kg body weight) was respectively injected intraperitoneally for fluorescence labeling of the bone. Then the rat was anesthetized with intraperitoneal injection of pentobarbital sodium(45mg/kg), and the blood was taken from femoral artery, and 2h later, the blood was centrifriged for 10min at 2000rpm, and the supernatant was taken and kept at-20℃for determination of IL-1, IL-6 contents. After the rat was killed, the right tibia of the rat was taken and fixed with 4% paraformaldehyde solution and the bone tissue section without decalcification was made for detection of bone histomorphometry; and the left tibia was fixed with 4% paraformaldehyde solution(prepared with 0.1M PBS(PH7.3) and the frozen tissue section with decalcification was made for detection of OPG, RANKL protein and mRNA expression.Statistical AnalysisThe experimental data were expressed as mean±standard deviation(x±s), and the data were processed with one way ANOVA and q test was used for comparison between groups.Results1. The tibial bone trabecula volume percentage(TBV%) in the model group was significantly lower than that in the pseudo-operation group; As compared with the model group, the TBV% in the Xu Duan group, the Lu Xian Cao group and the positive control group significantly increased, and did not change in the. Suo Yang group; Compared with the pseudo-operation group, the TBV% in the Xu Duan group, the Lu Xian Cao group and the Suo Yang group significantly decreased, and did not significantly change in the positive control group.2. Compared with the pseudo-operation group, tibial bone trabecula resorption surface percentage(TRS%) was significantly increased in the model group; Compared with the model group, the TRS% significantly decreased in the Xu Duan group, the Lu Xian Cao group and the positive control group, and did not change in the. Suo Yang group; Compared with the pseudo-operation group, the TRS% in the Xu Duan group and the Suo Yang group significantly increased, and did not significantly change in the Lu Xian Cao group and the positive control group.3. As compared with the pseudo-operation group, tibial bone trabecula formation surface percentage (TFS%) and bone trabecula mineralization rate(MAR) in the model group significantly increased. As compared with the model group, TFS% and MAR significantly decreased in the Xu Duan group and the positive control group, and did not change in the Lu Xian Cao group and the Suo Yang group; Compared with the pseudo-operation group, TFS% and MAR significantly increased in the Xu Duan group, the Lu Xian Cao group and the Suo Yang group, and did not significantly change in the positive control group.4. As compared with the pseudo-operation group, the tibial bone cortical mineralization rate (mAR) and para-cortex mean width (OSW) in the model group significantly increased. As compared with the model group, the mAR and OSW did not significantly change in the Xu Duan group, the Lu Xian Cao group and the Suo Yang group, but compared with the pseudo-operation group, they significantly increased in the Xu Duan group, the Lu Xian Cao group and the Suo Yang group. MAR and OSW in the positive control group were significantly lower than those in the model group with no significant difference between the positive control group and the pseudo-operation group.5. Compared with the pseudo-operation group, OPG protein expressions in both the tibial osteoblast and bone marrow matrix cells in the model group significantly decreased. As compared with the model group, the OPG protein expressions in both the tibial osteoblast and bone marrow matrix cells significantly increased in the Lu Xian Cao group and the positive control group, and did not significantly change in the Xu Duan group and the. Suo Yang group; Compared with the pseudo-operation group, OPG protein expressions in both the tibial osteoblast and bone marrow matrix cells significantly decreased in the Xu Duan group and the Suo Yang group, and did not significantly change in the Lu Xian Cao group and the positive control group.6. RANKL protein expression in the tibial osteoblast in the model group was significantly higher than that in the pseudo-operation group. As compared with the model group, RANKL protein expression in the tibial osteoblast in the Xu Duan group, the Lu Xian Cao group and the positive control group was significantly down-regulated, and did not change in the. Suo Yang group; Compared with the pseudo-operation group, the RANKL protein expression in the tibial osteoblas was significantly up-regulated in the Xu Duan group and the Suo Yang group, and did not significantly change in the Lu Xian Cao group and the positive control group.RANKL protein expression in the tibial bone marrow matrix in the model group was significantly higher than that in the pseudo-operation group. As compared with the model group, the RANKL protein expression was significantly down-regulated in the tibial marrow matrix cells in the Xu Duan group, the Lu Xian Cao group and the positive control group, and did not significantly change in the. Suo Yang group; RANKL protein expression in the tibial bone marrow matrix cells significantly increased in the Xu Duan group, the Lu Xian Cao group and the Suo Yang group was significantly higher than that in the pseudo-operation group, with no significantly difference between the pseudo-operation group and the positive control group.7. Compared with the pseudo-operation group, OPG mRNA expressions in both the tibial osteoblast and bone marrow matrix cells in the model group were significantly down-regulated. As compared with the model group, the OPG mRNA expressions in the tibial osteoblast and bone marrow matrix cells were significantly up-regulated in the Lu Xian Cao group and the positive control group, and did not significantly change in the Xu Duan group and the. Suo Yang group; Compared with the pseudo-operation group, OPG mRNA expressions in the tibial osteoblast and bone marrow matrix cells were significantly down-regulated in the Xu Duan group and the Suo Yang group, and did not significantly change in the Lu Xian Cao group and the positive control group.8. RANKL mRNA expression in tibial osteoblast in the model group was significantly higher than that in the pseudo-operation group. As compared with the model group, the RANKL mRNA expression in the tibial osteoblast was significantly decreased in the Xu Duan group, the Lu Xian Cao group and the positive control group, and did not significantly change in the Suo Yang group; Compared with the pseudo-operation group, RANKL mRNA expression in tibial osteoblast did not significantly change in the Xu Duan group, the Lu Xian Cao group, the Suo Yang group and the positive control group.RANKL mRNA expression in tibial marrow matrix cells in the model group was significantly higher than that in the pseudo-operation group. As compared with the model group, the RANKL mRNA expression in tibial marrow matrix cells was significantly down-regulated in the Xu Duan group, the Lu Xian Cao group and the positive control group, and did not significantly change in the Suo Yang group; Compared with the pseudo-operation group, RANKL mRNA expression in tibial marrow matrix cells significantly increased in the Xu Duan group and the Suo Yang group, and did not significantly change in the Lu Xian Cao group and the positive control group.9. Compared with the pseudo-operation group, peripheral serum IL-1 and IL-6 contents in the model group significantly increased. As compared with the model group, the serum IL-1 and IL-6 contents significantly decreased in the Lu Xian Cao group and the positive control group, and IL-6 content significantly decreased in the Xu Duan group, and did not significantly change in the. Suo Yang group; Compared with the pseudo-operation group, the serum IL-1 and IL-6 contents significantly increased in the Xu Duan group and the Suo Yang group, and IL-6 content in the Lu Xian Cao group significantly increased, and IL-1 content in the Lu Xian Cao group and IL-1 and IL-6 contents in the positive control group did not significantly change.There were no significant differences in the above indexes between the pseudo-operation group and the blank control group, indicating that the operation does not influence the results of the experiment.Conclusion1. Xu Duan(续断Radix Dipsaci) and Lu Xian Cao(鹿衔草Herba Pyrolae) have a certain therapeutic effects on the high-transformational model of osteoporosis induced by ovariectomy, while Suo Yang(锁阳Herba Cynomorii) does not have the effect.2. Lu Xian Cao(鹿衔草Herba Pyrolae) not only can increase expressions of OPG protein and mRNA in osteoblast and marrow matrix cells and inhibit expressions of RANKL protein and mRNA, and also decrease IL-1 and IL-6 levels in peripheral blood, so as to treat osteoporosis;3. Xu Duan(续断Radix Dipsaci) can reduce expression of RKNAL protein and mRNA, so as reduce binding of RNAK with RANKL, and over-resorption of the bone; In addition, it also has a certain inhibitory action on IL-6.