| Purpose:From this experiment Jingyue the "yin and yang Freemasons’ theory, selected Chinese classic recipe Bushentianjing Zuogui Wan and Yougui Wan and split side to side for a total of two common drug side groups to the yin Zuoguiwan medicine for Yin Fang group to Yougui Wan in medicine for Yang Yang Fang group to estradiol tablets pentanone positive control group, with postmenopausal osteoporosis(PMOP) rat BMSCs for the study, Emphasis was placed on the osteogenic differentiation potential left, and split side Yougui Wan decoction PMOP metabolism in rats after extraction of BMSCs and explore the left from the ERK1 / 2 signaling pathway pathway Yougui Wan its separate prescriptions on PMOP possible mechanisms of bone induced BMSCs into.Material and method: With bilateral ovariectomy was established postmenopausal osteoporosis model, SPF level 2-month-old female Sd nulliparous rats 90, 200-220 g, after adaptive feeding week, according to figures randomly randomly divided into normal group 10, sham group 10 and the surgery group 70, control group continued regular feeding, surgery group to implement bilateral ovariectomy surgery modeling deal. After three days, after the bilateral ovariectomized rats survived by weight again stratified randomly divided into 7 groups. The final group results for the normal group 10, 10 sham group, model group 8, progynova group 8, group 8 Zuoguiwan Yougui Wan group 8 common prescription group 8, Yin prescription group 8, yang prescription group 8.The dose per kg body weight of rats to calculate human 6.3 times, and every time the amount of rats fed a normal dosage of 2 times, orally once daily, blank control group, sham operation group, model group were fed with distilled water, the rest of the group fed with the appropriate liquid in each group were prepared. Were extracted from rat BMSCs 12 weeks after administration of each group, Then, to test the alkaline phosphatase(ALP) activity by the means of modified calcium-combat staining;To count the mineralization nodules with alizarin red staining, to detect the expressing of Cbfα1 and ColⅠm RNA by Q-PCR, to show the expression of core binding factor alpha 1( Cbfα1)and typeⅠcollagen( ColⅠ) protein with the method of Western blot.Then using ERK1 / 2, MAPK signal blockers way to PD98059(ERK1 / 2-specific blocker) intervention rat BMSCs PMOP, and Cbfα1, ColⅠm RNA express Quantitative real-time PCR assay, using Western blot assay Cbfα1, ColⅠ, ERK1 / 2, p-ERK1 / 2 protein expression.Results:1. Identification of BMSCs by flow cytometryBy flow cytometry, each group BMSCs of CD29, CD90 expression was positive, and CD29-positive rate of over 94%, CD90 positive rate was over 50%. CD45 expression was detected, wherein the negative rate BJL OVX group and 30% or less, the remaining groups in a negative rate of 6.4% or less.2. Zuogui Wan and Yougui Wan its separate prescriptions on the proliferation of rat BMSCs PMOP2.1 BMSCs growth curve drawingBMSCs a generally S-shaped growth curve, 1 ~ 2d after inoculation, incubation period, the cells begin to proliferate from the 3d and enter the logarithmic growth phase, the first 5 ~ 6d peaked after entering the plateau.2.2 Zuogui Wan and Yougui Wan its separate prescriptions on PMOP BMSCs proliferation of ratsThe results showed that the left side of its decomposed Yougui Wan effect on the proliferation of rat BMSCs PMOP for a time correlation. In the first 1d, compared with the Control group, OVX, ZGW, ZYY, BJL group had a significant difference(P <0.05), whereas no significant difference each group and OVX group than in promoting proliferation of( P >0.05). The first 3d when compared with the Control group, ZGW, YGW, ZYY BJL group with a significant difference(P <0.05), there was a significant difference(P <0.05) only BJL group compared with the OVX group. The first 5d, OVX, ZGW, YGW, GTY with BJL group and Control group were statistically significant(P <0.05), than there is a significant difference(P <0.05) with the same first and only 3d BJL group and OVX group. Section 7d, the comparison with the Control group and OVX group, ZGW, YGW, BJL groups were significantly different(P <0.05), compared with ZGW group, Sham, GTY, ZYY, BYY group was statistically significant(P <0.05).3. Left, the impact Yougui Wan its separate prescriptions on BMSCs and osteogenic differentiation of rat PMOP3.1 Effect of the left side of its decomposed Yougui Wan PMOP expression of rat BMSCs ALPPNPP method to detect PMOP rat BMSCs ALP activity, it is clear that when ZGW group 7d ALP activity in the highest, and the dosing groups in 7d when ALP activity was higher than other times. ALP activity group which ZGW four time periods and Control, OVX group were significantly improved(P <0.05); YGW group ALP activity in 1d, 5d, 7d when compared with the Control, OVX group significant increase(P <0.05); and BJL group 1d, 3d when compared with the Control, OVX group significant increase(P <0.05), and only when the 5d Control group had significantly increased(P <0.05), while the 7d and Control, OVX groups there was no significant difference(P> 0.05); GTY, ZYY, BYY group and Control, OVX group, ALP activity at different times sometimes different degree(P <0.05).To improve calcium cobalt assay ALP expression in rat BMSCs PMOP, BMSCs in the ALP after modified by calcium, cobalt staining, brownish black granular and together, the negative control without brown-black particles. The results showed that compared with the Control group, OVX, Sham cells have synapses retraction trend brownish-black sedimentary particles decreased, other groups increased cell density, cell synapses are many and long, brown-black particles increases, and does not difficult to see especially in the more prominent ZGW and YGW group, while brown-black particles ZGW group had significantly more YGW group.3.2 Effect of the left side of its decomposed Yougui Wan rat BMSCs PMOP mineralized nodulesBMSCs calcified nodules formed by Alizarin Red After stained red. The results showed that compared with the Control group, OVX, Sham, GTY and BYY group calcified nodules was significantly reduced, while ZGW, YGW, ZYY and calcified nodules BJL group increased significantly. Compared with BJL group, ZGW, YGW group calcified nodules increased significantlym RNA and protein expression3.3.1 Zuogui Wan and Yougui Wan affect its separate prescriptions on m RNA and protein expression in rat BMSCs Cbfα1 of PMOPThe results show that: Compared with the Control group: OVX group BMSCs significantly reduced(P <0.05) Cbfα1 m RNA expression; ZGW and BJL could significantly increase BMSCs in Cbfα1 m RNA expression(P <0.05), and ZGW group than BJL group; YGW group can significantly upregulate the expression of Cbfα1 m RNA(P <0.05). Compared with the OVX group: ZGW, YGW, BJL group can significantly improve BMSCs in Cbfα1 m RNA expression(P <0.05). Compared with ZGW groups: group BMSCs express YGW ColⅠ m RNA in less than normal(P <0.05), while GTY, ZYY, BYY group BMSCs Cbfα1 m RNA expression of varying degrees of reduction(P <0.05). Compared with the Control group: Sham, ZGW, YGW, BJL group BMSCs in Cbfα1 protein expression was no significant difference(P> 0.05), whereas the expression of Cbfα1 OVX, GTY, ZYY, BYY group BMSCs significantly lower(P <0.05); Compared with the OVX group, ZGW and Sham group were significantly increased in BMSCs Cbfα1 protein(P <0.05), the rest of the group had no significant difference(P> 0.05). Compared with ZGW group, YGW, GTY, ZYY, BYY, BJL group BMSCs significantly reduce Cbfα1 protein expression(P <0.05).3.3.2 The impact Zuogui Wan and Yougui Wan its separate prescriptions on PMOP rat BMSCs ColⅠm RNA and protein expressionThe results show that: Compared with the Control group: a significant reduction in ColⅠm RNA and protein expression in OVX group BMSCs(P <0.05), ZGW and BJL could significantly increase the expression of BMSCs in ColⅠ m RNA’s(P <0.05), and ZGW group than BJL Group; while ZGW, YGW, GTY, ZYY could significantly increase the expression of BMSCs in ColⅠ protein(P <0.05), and ZGW group than YGW group; compared with OVX group: ZGW, YGW, BJL group can significantly improve BMSCs in ColⅠ m RNA expression(P <0.05); ZGW, YGW, GTY, ZYY, BYY group can significantly increase the expression of BMSCs in ColⅠ protein(P <0.05). Compared with ZGW groups: group BMSCs express YGW ColⅠ m RNA in less than normal(P <0.05), whereas the expression GTY, ZYY, BYY group BMSCs ColⅠ m RNA’s are different degrees of reduction(P <0.05); BYY and BJL Group BMSCs in ColⅠ protein expression was significantly lower(P <0.05).4. ERK1 / 2 pathway in the left side of its decomposed Yougui Wan rat osteoblasts induced BMSCs PMOP4.1 ERK1 / 2 signaling pathway on the Zuogui Wan and Yougui Wan its demolition party intervention on PMOP rat BMSCs Cbfα1 m RNA and protein expressionThe results showed that compared with the Control group: OVX group Cbfα1m RNA expression in BMSCs significantly reduced(P <0.05); ZGW, YGW, GTY and BYY could significantly increase the expression Cbfα1m RNA of BMSCs(P <0.05). Compared with the OVX group: ZGW, YGW, GTY, ZYY, BYY, BJL group can significantly improve BMSCs in Cbfα1, m RNA expression(P <0.05), consistent with the two previous results. Join PD98059 blockers, can be seen from the figure, ZGW, YGW, GTY, ZYY group showed no significant difference(P> 0.05) after the addition of PD98059 blockers Cbfα1m RNA expression in BMSCs Control group, and Control, BYY and BJL group after joining PD98059 blockers compared with the Control group Cbfα1m RNA expression in BMSCs has been significantly reduced(P <0.05). And ERK1 / 2 signaling pathway on the Zuogui Wan and Yougui Wan its demolition party intervention on Cbfα1 protein expression in rat BMSCs PMOP, the results show that compared with the Control group: ZGW, YGW, BYY, BJL Cbfα1 protein expression in BMSCs group was not significant difference(P> 0.05), and OVX, Sham, GTY, ZYY group BMSCs significantly reduce Cbfα1 protein expression(P <0.05); compared with the OVX group, ZGW and BJL could significantly increase Cbfα1 protein expression in BMSCs(P <0.05), the rest of the group was no significant difference(P> 0.05), this is consistent with the previous two experiments. After joining PD98059 blockers, Control, group had significantly lower(P <0.05) protein expression in BMSCs Cbfα1 blockers compared with no increase, and ZGW, YGW and BYY groups joined PD98059 blockers BMSCs in Cbfα1 protein expression and Control group also decreased(P <0.05).4.2 ERK1 / 2 signaling pathway on the Zuogui Wan and Yougui Wan its demolition party intervention on rat BMSCs ColⅠm RNA PMOP and protein expressionThe results showed that compared with the Control group: OVX group ColⅠm RNA expression in BMSCs significantly reduced(P <0.05); ZGW, YGW, GTY and BJL could significantly increase the expression of BMSCs in ColⅠm RNA(P <0.05). Compared with the OVX group: ZGW, GTY, BJL group can significantly increase the expression of BMSCs in ColⅠm RNA(P <0.05), consistent with the two previous results. Join PD98059 blockers, can be seen from the figure, ZGW, YGW, GTY, ZYY group showed no significant difference(P> 0.05) after the addition of PD98059 blockers ColⅠm RNA expression in BMSCs Control group, and Control, BYY and BJL group after joining PD98059 blockers compared with the Control group ColⅠm RNA expression in BMSCs has been significantly reduced(P <0.05).The results showed that compared with the Control group: OVX, Sham, ZYY, BYY, BJL group BMSCs in ColⅠ no significant difference(P> 0.05) protein expression, while ZGW with YGW group significantly increased in BMSCs ColⅠ protein expression(P <0.05). Compared with the OVX group, ZGW, YGW, BYY and BJL group were significantly increased in BMSCs ColⅠ protein expression(P <0.05), GTY and ZYY was no significant difference(P> 0.05), which is also consistent with previous experimental results. Join PD98059 blockers, except Sham, the comparison with the rest of the group Control group after joining the blockade were significantly reduced in ColⅠ BMSCs protein expression(P <0.05).4.3 ERK1 / 2 signaling pathway on the Zuogui Wan and Yougui Wan its demolition party intervention in rat BMSCs PMOP ERK1 / 2 protein phosphorylationCompared with the Control group, obviously ZGW, YGW, BYY and BJL group induced ERK1 / 2 phosphorylation stronger role(P <0.05). Compared with the OVX group, ZGW, YGW and BJL Group phosphorylation of ERK1 / 2 on enhanced(P <0.05). And after adding blocker PD98059, Control group compared with the OVX group did not join when the Control group blocker induced phosphorylation of ERK1 / 2 protein was significantly decreased(P <0.05), while the addition of PD98059 resistance ZGW group lag comparisons with non-induced ERK1 / 2 phosphorylation turned into a weakened but did not increase with the Control group blocker induced ERK1 / 2 phosphorylation before protein significantly enhanced(P <0.05)Conclusion:1. Osteoblast proliferation and differentiation of rat BMSCs PMOP decline.2. Zuogui Wan and Yougui Wan its demolition party PMOP promote the proliferation and differentiation of rat BMSCs and left Yougui Wan promote osteogenic differentiation of BMSCs better than its demolition party, stating "seek yin yang" and "yin Yang from " meaning the Zuoguiwan and Yougui Wan promote osteogenesis significantly higher than the common single yang or yin side, Yin Fang, Yang Fang.3. Zuogui Wan and Yougui Wan its demolition PMOP promote osteogenic differentiation of rat BMSCs may be by adjusting or improving the ERK, JNK, P38 MAPK signaling pathway is completed, which may be medicine or TCM decoction prevention of osteoporosis one of the mechanisms. |
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