| Objective: In view of the theory of "kidney essence and bone marrow",which is one of the core contents of TCM theory,according to the principle of yin and yang theory of yin and yang,To investigate the effects of Zuogui pill and Yougui pill on the osteogenesis and differentiation of bone marrow mesenchymal stem cells(BMSCs)in ovariectomized rats,and to explore the effects of Zuogui pill and Yougui pill on ovariectomized rats BMSCs autophagy.Material and method: SPF grade 65 2-month-old SD female non-fertile rats were randomly divided into 6 groups: blank control group(KB),model group(OVX),sham operation group(SHAM),Zuogui pill group(ZGW),Yougui pill group(YGW)and Progynova group(BJL).After 7 days of adaptive feeding,the postmenopausal osteoporosis(PMOP)model was established by bilateral ovariectomy.After 7 days of operation,the rats were fed with the same dose ratio The BMSCs were isolated from the femur and tibia of the ovariectomized rats by whole bone marrow adherence method.The BMSCs were identified by flow cytometry,and the cells were treated with MTT(thiazole Blue)method to detect the cell proliferation rate.The expression of autophagy-associated protein Beclin1 and P62 was detected by Wesrern blot method.The autophagy level of each group was detected by immunofluorescence.The best concentration and best time of autophagy induced by rapamycin(RAPA)and autophagy induced by BMSCs were screened by MTT(thiazolyl blue)method and the Wesrern blot method The expression of Beclin1 and ubiquitin-binding protein P62(Sequestosome-1,SQSTM1)was used to determine the concentration and duration of action of rapamycin.The autophagy level of autophagy-induced cells was detected by immunofluorescence.BMSCs were induced to osteogenesis and adipogenesis respectively.The cells were treated with ELISA and PNPP method.Alkaline phosphatase(ALP)activity was detected by immunohistochemistry.The intracellular ALP was observed by modified calcium-cobalt staining And the formation of intracellular mineralized nodules was observed by alizarin red staining.The expressions of Collagen I,COLI and Runx2 were detected by Wesrern blot method.The expression of autophagy-associated protein Beclin1,P62,microtubule-associated protein 1 The expression of autocrine in each group was detected by immunofluorescence assay.(Peroxisome proliferator-acticvated receptor,PPAR?)was detected by Wesrern blot method and the expression of lipopolysaccharide-stimulating enzyme(PPAR?)was detected by Wesrern blot method.The expression of lipophyllin lipase(LPL)was detected by immunofluorescence assay.The levels of autophagy were detected by immunofluorescence assay.Results: 1 Proliferation of BMSCs in Zuogui pill and Yougui pill and ovariectomized rats 1.1 Identification of BMSCs The positive cells were extracted and cultured on the P3 generation.Two kinds of positive cell surface factors CD29 and CD90 were detected by flow cytometry.The surface cells of the negative cells were CD11 b / c.The expression rates of CD29 and CD90 were 92.9%.The expression rate of CD 11 b / c was 34.0%.The cell morphology and cell flow identification were observed under inverted microscope to determine the BMSCs.1.2 Growth curve of BMSCs and the effect of Zuogui pill and Yougui pill proliferation of BMSCs in ovariectomized rats 1.2.1 Growth curve showed that the growth curve of BMSCs was S-type,the first 72 h after inoculation was incubation period,72 h after the cells began to enter the logarithmic phase,168 th cell growth reached the plateau.1.2.2 Proliferation results showed that compared with the KB group,SHAM group and KB group no significant difference(P>0.05),OVX group cell proliferation rate was significantly reduced(P<0.05).Compared with OVX group,three cell proliferation rate of the drug group were increased(P<0.05).The cell of ZGW group has the best proliferation ability.2 Study on osteogenesis and adipogenic differentiation of BMSCs in Zuogui pill and Yougui pill and ovariectomized rats 2.1 Effects of Zuogui pill and Yougui pill on osteogenesis induced by ovariectomized BMSCs in ovariectomized rats 2.1.1 Osteogenesis-related indicators ALP expression 2.1.1.1 PNPP method The results showed that compared with the KB group,SHAM group no significant difference(P>0.05),the ALP activity of OVX group was significantly lower(P<0.05).Compared with the OXV group,The ALP activity of BMSCs in ovariectomized rats was increased,and the ZGW group had the most significant effect.The ALP activity was the highest in 10 D group.The above results show that,Zuogui pill and Yougui pill are conducive to ovarian rats to direction differentiation of BMSCs,the Yougui pill is better than the Zuogui pill.2.1.1.2 ELISA method The results showed that there was no significant difference in the SHAM group compared with the KB group(P>0.05).The ALP expression in the OVX group was significantly decreased(P<0.05).Compared with OVX group,ALP expression of three drug groups were significantly higher(P<0.05),and most obvious ZGW group.The above results show that,Zuogui pill and Yougui pill are conducive to ovarian rats to direction differentiation of BMSCs,the Yougui pill is better than the Zuogui pill.2.1.1.3 Modified Calcium Cobalt Staining Method Results showed that there was no significant difference in SHAM group compared with KB group,the brownish yellow granules in the OVX group were observed in the microscopic particles decreased significantly.Compared with OVX,the brownish yellow granules in the three drug groups were observed in the microscopic particles increased obviously,and the most obvious is ZGW group.The above results show that,Zuogui pill and Yougui pill could enhance to the ovaries rat BMSCs osteogenetic differentiation after the expression of ALP,and the effect of Zuogui pill is better than Yougui pill.2.1.2 Effects of Zuogui pill and Yougui pill on the formation of mineralized nodules after osteogenesis of BMSCs in ovariectomized rats.Results showed that there was no significant difference in SHAM group compared with KB group,the brownish yellow granules in the OVX group were observed in the microscopic particles decreased significantly.Compared with OVX,the brownish yellow granules in the three drug groups were observed in the microscopic particles increased obviously,and the most obvious is ZGW group.It can be seen that Zuogui pill and Yougui pill could promote the osteogenic induction of BMSCs Of the formation,and the effect of Zuogui pill is better than Yougui pill.2.1.3 Effects of Zuogui pill and Yougui pill on expression of COL I and Runx2 proteins in osteoblast differentiation of ovariectomized rats.2.1.3.1 Expression of COL I protein The results showed that there was no significant difference in the SHAM group compared with the KB group(P>0.05).The expression of COLI protein in OVX group was lower(P<0.05).Compared with the OVX group,the COLI protein level in the three drup groups was significantly higher(P<0.05),and the most obvious is ZGW group.The above results show that,Zuogui pill and Yougui pill can through improve osteogenesis related index COL I expression promote differentiation of rat BMSCs development to the ovary,and the effect of Zuogui pill is better than Yougui pill.2.1.3.2 Runx2 protein expression The results showed that there was no significant difference between the OVX group and the KB group(P>0.05).Compared with the OVX group,the expression level of Runx2 protein in ZGW group and BJL group was significantly higher than that in OVX group(P<0.05).It can be seen that Zuogui pill can improve osteogenesis related indicators through the expression of Runx2 promote to the ovaries rat BMSCs differentiation.2.2 Effects of Zuogui pill and Yougui pill on adipogenic induction of BMSCs in ovariectomized rats.2.2.1 Effects of Zuogui pill and Yougui pill on the formation of lipid droplets in BMSCs induced by ovariectomized rats The results showed that there was no significant difference in SHAM group compared with KB group,the brownish yellow granules in the OVX group were observed in the microscopic particles increased significantly.Compared with OVX,the brownish yellow granules in YGW group were observed in the microscopic particles increased obviously,the brownish yellow granules in ZGW group and BJL group were decreased obviously.it can be seen,Zuogui pill can ovary differentiation of rat BMSCs into fat,Yougui pill can enhance the ability of ovary rat BMSCs into lipid differentiation.2.2.2 Effects of Zuogui pill and Yougui pill on PPAR? and LPL protein expression in BMSCs after ovariectomized rats 2.2.2.1 Expression of PPAR? protein The expression of PPAR? protein in OVX groupwas significantly higher than that in KB group(P<0.05),and the expression of PPAR? protein in SHAM group(P>0.05).ZGW group and BJL group was lower than that in OVX group(P<0.05),and the PPAR? protein in YGW group was the most obvious expression increased(P<0.05).2.2.2.2 Expression of LPL protein The expression of LPL protein in ZGW group,YGW group and BJL group was significantly decreased and the most obvious in the YGW group compared with KB group(P<0.05).There was no significant difference in SHAM group(P>0.05).The expression level of LPL protein in OVX group was significantly higher than that in KB group(P<0.05).The above results show that,the Yougui pill can be adjusted by lowering the expression of LPL protein to ovarian rats after differentiation of BMSCs into fat related lipid metabolism.3 Screening of autophagy induced by rapamycin in ovariectomized rats 3.1 MTT method to screen the best concentration of rapamycin and the role of time The results showed that rapamycin could induce autophagy of BMSCs in ovariectomized rats,and the optimal concentration was 2 * 10-6 mol · L-1 RAPA,the best time was 16 h.3.2 Wesrern blot was used to detect the expression of autophagy-related proteins 3.2.1 Expression of Beclin1 protein The results showed that the expression of Beclin1 protein in KB+3-MA group and RAPA+3-MA group was significantly lower than that in KB group(P<0.05),and the expression of Beclin1 protein in RAPA group was significantly enhanced(P<0.05).Compared with RAPA group,the expression of Beclin1 protein in RAPA+3-MA group was significantly decreased(P<0.05).Thus,RAPA can induce autophagy of BMSCs.3.2.2 Expression of P62 protein The expression of P62 protein in KB+3-MA group,RAPA group and RAPA+3-MA group was significantly higher than that in KB group(P<0.05),and the expression of Beclin1 protein in RAPA group was significantly higher(P<0.05).Compared with KB+3-MA,The expression of P62 protein in RAPA group and RAPA+3-MA group was significantly decreased(P<0.05).So,RAPA can induce autophagy of BMSCs.3.3 Immunofluorescence assay for the expression of autophagy-related indicator LC3 The results showed that the fluorescence point-like polymerization in the cytoplasm of the RAPA group was observed in the microscopic particles increased significantly(P<0.05).Compared with the RAPA group,the point-like polymerization in RAPA+3-MA group decreased significantly(P<0.05).So,RAPA can induce autophagy of BMSCs.4 Effects of Zuogui pill and Yougui pill on autophagy of BMSCs 4.1 Effects of Zuogui pill and Yougui pill on autophagy of BMSCs in ovariectomized rats 4.1.1Effects of Zuogui pill and Yougui pill on the expression of autophagy-related proteins in BMSCs of ovariectomized rats 4.1.1.1 Expression of P62 protein The results showed that compared with KB group,SHAM group had no significant difference(P>0.05),the expression of P62 protein in OVX group increased(P <0.05).Compared with OVX group,the expression of P62 protein in ZGW group,YGW group and BJL group decreased(P<0.05),and the decrease of ZGW group was the most obvious.So,Zuogui pill and Yougui pill could increase the autophagy of BMSCs in ovariectomized rats,and the effect of Zuogui pill is better than Yougui pill.4.1.1.2 Expression of Beclin1 protein The results showed that compared with KB group,SHAM group had no significant difference(P>0.05),the expression of Beclin1 protein in OVX group decreased(P<0.05).Compared with OVX group,the expression of Beclin1 protein in ZGW group,YGW group and BJL group increased(P<0.05),and the increase of ZGW group was the most obvious.So,Zuogui pill and Yougui pill could increase the autophagy of BMSCs in ovariectomized rats,and the effect of Zuogui pill is better than Yougui pill.4.1.2 Immunofluorescence assay for the effect of Zuogui pill and Yougui pill on BMSCs autophagy in ovariectomized rats The results showed that compared with KB group,SHAM group had no significant difference(P>0.05),the fluorescence point-like polymerization in the cytoplasm of the OVA group was observed in the microscopic particles decreased(P<0.05).Compared with OVX group,the fluorescence point-like polymerization in ZGW group,YGW group and BJL group increased(P<0.05),and the increase of ZGW group was the most obvious.So,Zuogui pill and Yougui pill could increase the autophagy of BMSCs in ovariectomized rats,and the effect of Zuogui pill is better than Yougui pill.4.2 Effects of Zuogui pill and Yougui pill on autophagy after ovariectomy in ovariectomized rats 4.2.1 Effects of Zuogui pill and Yougui pill on autophagy-related protein expression in ovariectomized BMSCs after osteogenesis differentiation in ovariectomized rats 4.2.1.1 Expression of P62 protein The results showed that compared with KB group,the expression of P62 protein in OVX group increased(P<0.05).Compared with OVX group,the expression of P62 protein in SHAM group,ZGW group,YGW group and BJL group decreased(P<0.05),and ZGW group was the most obvious.The results showed that Zuogui pill and Yougui pill could improve the autophagy of BMSCs after ovariectomy,and the effect of Zuogui pill was better.4.2.1.2 Expression of Beclin1 protein The results showed that compared with KB group,SHAM group,OVX group and GW group had no significant(P> 0.05).Compared with OVX group,the expression of Beclin1 protein in ZGW group and BJL group was significantly higher than that in OVX group(P<0.05),and ZGW group was the most obvious.The above results showed that Zuogui pill could promote autophagy after ovariectomy in ovariectomized BMSCs. 4.2.1.3 Expression of LC3 protein The results showed that compared with KB group,SHAM group had no significant difference(P>0.05),the expression of LC3? protein in OVX group decreased(P<0.05).Compared with OVX group,the expression of LC3? protein in ZGW group,YGW group and BJL group increased(P<0.05),and the increase of ZGW group was the most obvious.The results showed that Zuogui pill and Yougui pill could improve the autophagy of BMSCs after ovariectomy,and the effect of Zuogui pill was better.4.2.2 Immunofluorescence assay for the effect of Zuogui pill and Yougui pill on autophagy after osteoblast differentiation in ovariectomized rats The results showed that compared with KB group,SHAM group and OVA group had no significant difference(P>0.05).Compared with OVX group,the fluorescence point-like polymerization in the cytoplasm of ZGW group,YGW group and BJL group increased(P<0.05),and the increase of ZGW group was the most obvious.So,Zuogui pill and Yougui pill could improve the autophagy of BMSCs after ovariectomy,and the effect of Zuogui pill was better.4.3 Effects of Zuogui pill and Yougui pill on autophagy after BMSCs adipogenic differentiation in ovariectomized rats 4.3.1Effects of Zuogui pill and Yougui pill on the expression of autophagy-related proteins in BMSCs after ovariectomized rats 4.3.1.1 Expression of P62 protein The results showed that compared with KB group,SHAM group had no significant difference(P>0.05),the expression of P62 protein in OVX group decreased(P<0.05).Compared with OVX group,the expression of P62 protein in ZGW group and BJL group increased(P<0.05),YGW group and had no significant difference(P>0.05).The results showed that the level of autophagy was higher in BMSCs after ovariectomized rats,and Yougui pill can improve the autophagy of ovarian rats after BMSCs adipogenic differentiation.4.3.1.2 Expression of Beclin1 protein The results showed that compared with KB group,SHAM group had no significant difference(P>0.05),the expression of P62 protein in OVX group significantly increased(P<0.05).Compared with OVX group,the expression of P62 protein in ZGW group and BJL group decreased(P<0.05),YGW group had no significant difference(P>0.05).The results showed that the level of autophagy was higher in BMSCs after ovariectomized rats,and Yougui pill can improve the autophagy of ovarian rats after BMSCs adipogenic differentiation.4.3.1.3 Expression of LC3 protein The results showed that compared with KB group,SHAM group had no significant difference(P>0.05),the expression of LC3? rotein in OVX group significantly increased(P<0.05).Compared with OVX group,the expression of LC3? rotein in ZGW group and BJL group decreased(P<0.05),the expression of LC3? rotein in YGW group significantly increased(P<0.05).The results showed that the level of autophagy was higher in BMSCs after ovariectomized rats,and Yougui pill can improve the autophagy of ovarian rats after BMSCs adipogenic differentiation.4.3.2 Immunofluorescence assay for the effect of Zuogui pill and Yougui pill on autophagy after adipogenic differentiation of BMSCs in ovariectomized rats The results showed that compared with KB group,SHAM group had no significant difference(P>0.05),the fluorescence point-like polymerization in the cytoplasm of the OVA group was observed in the microscopic particles significantly increased(P<0.05).Compared with OVX group,the fluorescence point-like polymerization in ZGW group,BJL group significantly decreased(P<0.05),the fluorescence point-like polymerization in YGW group significantly increased(P<0.05).So,the level of autophagy was higher in BMSCs after ovariectomized rats,and Yougui pill can improve the autophagy of ovarian rats after BMSCs adipogenic differentiation.In conclusion: 1 Zuogui pill and Yougui pill can promote the proliferation of BMSCs in ovariectomized rats,and Zuogui pill promote the proliferation of BMSCs in ovariectomized rats.2 PMOP prevention and treatment mechanism with BMSCs osteogenesis,adipogenic differentiation is closely related.Zuogui pill and Yougui pill can regulate ovariectomy BMSCs osteogenesis,adipogenic differentiation of the relevant indicators.Among them,Zuogui pill promote the expression of osteoblastic related indexes and inhibit the expression of adipogenic indicators.Yougui pill can promote the expression of osteogenesis-related indicators and adipogenic-related indicators.By comparison,Zuogui pill to enhance the ability of osteoblast differentiation of BMSCs,Zuogui pill to improve the ability of BMSCs adipogenic differentiation.3 The autophagy classic inducer rapamycin can effectively induce autophagy of BMSCs in ovariectomized rats at a working concentration of 2 * 10-6 mol · L-1 for 16 h.4 Zuogui pill and Yougui pill can improve the autophagy level of BMSCs in ovariectomized rats,which may be one of the mechanisms of Zuogui pill and Yougui pill to promote the proliferation of BMSCs in ovariectomized rats.5 Zuogui Pill may enhance the osteogenic differentiation of BMSCs in ovariectomized rats by increasing the autophagy level of osteoblast differentiation in ovariectomized rats.Yougui pill may enhance the autophagy level of BMSCs by ovariectomized rats Osteoporosis ability of BMSCs in ovariectomized rats. |
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