| Objective1.To investigate the role of zuogui pill in promoting BMSCs proliferation and osteogenic differentiation in mice in vitro and the mechanism of FoxJ1 regulation of BMP/Smad signaling pathway in promoting BMSCs osteogenic differentiation in zuogui pill;2.In vivo study to investigate the role of FoxJ1 in regulating the BMP/Smad pathway in the treatment of osteoporosis in mice with zuogui pill;Methods1.Cell experimental study(1)Zuogui pill regulates the FoxJ1/BMP/Smad signal axis to promote osteogenic differentiation of BMSCs in mice BMSCs were isolated and cultured from 8-week-old mice,and their cell morphology was observed.BMSCs surface markers CD29,CD44,sca-1 and CD117 were detected by flow cytometry.CCK8 method was used to detect zuogui pills with different concentrations in 1d,2d,3d and 5d.The proliferation effect of 7d on BMSCs in mice was screened for the optimal concentration.Experimental groups:control group(CON group),osteogenic induction group(OI group),zuoguiwan group(ZGW group),and zuoguiwan+osteogenic induction group(ZGW+OI group).BMSCs osteogenic differentiation was induced by osteogenic induction solution,BMSCs osteogenic ability was detected by ALP staining,and bone mineralization was detected by alizarin red staining.The osteogenic parameters of BMSCs in mice were ALP,OCN and runt-related transcription factor 2(Runx2)and the expression of osteoblast related transcription factor(Osx)and forkhead box J1(FoxJ1)mRNA,and the expression of FoxJ1,bone morphogenetic protein 2(BMP2),Smad1 and p-smad1/5 by western-blot analysis.(2)Zuogui pill promotes osteogenic differentiation of BMSCs by regulating the BMP/Smad pathway through FoxJ1 Mouse BMSCs transfected with siRNA were divided into three groups:empty vector(siCtrl)group,FoxJ1 transfection(siFoxJ1)group,ZGW+siCtrl group and ZGW+siFoxJ1 group.The expression of FoxJ1 mRNA was detected by PCR,and the expression of FoxJ1 protein was detected by western-blot.For osteogenic induction in the above groups,ALP staining was used to detect osteogenic differentiation,alizarin red staining was used to detect bone mineralization,and western-blot was used to detect the expression of proteins related to the BMP/Smad pathway.2.Animal experimental research(1)Establishment of osteoporosis model and its influence on FoxJ1 expressionThe 6-week-old female mice were ovariectomized to construct a mouse model of osteoporosis,micro-ct was used to detect the fine structure of vertebral bone,HE staining was used to detect the vertebral bone histomorphology,and western-blot was used to detect the expression of FoxJ1 protein(2)The anti-osteoporosis effect of zuogui pill by regulating the BMP/Smad pathway through FoxJ1The above ovariectomized mice were injected by tail vein of SiRNA and gavage of zuogui pill.Experimental groups:siCtrl group,siFoxJ1 group,ZGW+siCtrl group,ZGW+si FoxJ1 group.Micro-ct was used to detect the fine structure of vertebral bone,HE staining was used to detect the histology of vertebral bone,and western-blot was used to detect the expression of FoxJ1,BMP2,Smad1 and p-smad1/5 proteins.Results1.Cell experimental study(1)Zuogui pill regulates the FoxJ1/BMP/Smad signal axis to promote osteogenic differentiation of BMSCs in mice BMSCs were spindle-shaped and vortex shaped.Flow cytometry results showed that the CD29 positive rate was 98.1%,CD44 positive rate was 98.6%,sca-1 positive rate was 97.3%,and CD117 positive rate was 8.5%.CCK8 test results using two factors of repetitive measure analysis of variance,and suggests that different concentration left to pill intervention time significant differences on cell proliferation,and the influence of different concentration left to pill on cell proliferation changes over time,there are interaction,with 10 mu g/ml 5 days left to pill intervention to promote cell proliferation effect the most significant;ALP staining results showed that the ALP staining effect increased with the increase of osteogenic induction time.As a whole,on the 7th and 14th days of osteogenic induction,the color of the petri dish in OI group was significantly darker than that in CON group.On the 14th day,the color of the petri dish in OI group was significantly darker than that in CON group.Microscopically,compared with CON group,the ALP stained cells in OI group were significantly increased and the color deepened.On the 7th and 14th days of osteogenesis induction,there was no significant color difference between the ZGW group and the CON group.Compared with the OI group,on the 7th day of osteogenesis induction,there was no significant color difference between the ZGW+OI group and the OI group,while on the 14th day of osteogenesis induction,compared with the OI group,the ALP stained cells of the ZGW+OI group were significantly increased and the color deepened.Microscopically,on the 7th and 14th days of osteogenesis induction,alp-stained cells in the ZGW+OI group were significantly increased and their color deepened compared with that in the OI group.Alizarin red staining results showed that,on the 7th day of osteogenic induction,compared with the CON group,the red staining range of cells in the OI group was significantly larger when viewed as a whole.Microscopically,the red staining of cells in the OI group was significantly deeper and the number of stained cells increased.Compared with CON group,ZGW group had no significant change in overall view and under mirror view.Compared with the OI group,the staining degree of the ZGW+OI group did not change significantly when viewed as a whole.Microscopically,the alizarin red staining of the ZGW+OI group was darker and the number of stained cells increased.On the 14th day of osteogenesis induction,compared with the CON group,the alizarin red staining in OI group was significantly deepened and mineralized nodules were observed.Microscopically,the mineralized nodules in OI group increased,the color of red dye deepened,and the number of stained cells increased.Compared with the CON group,there was no significant change in the whole view of ZGW group.Compared with the OI group,the staining range of the ZGW+OI group increased significantly when viewed as a whole,while that of the ZGW+OI group increased and deepened under the microscope.Fluorescence quantitative PCR results showed that mRNA expressions of OI bone-related genes ALP,OCN,Runx2 and Osx were significantly increased compared with CON group(P<0.05,P<0.01).The mRNA expressions of Runx2 and Osx in ZGW group were significantly increased(P<0.01,P<0.05).Compared with the OI group,the expressions of ALP,OCN and Runx2 mRNA in the ZGW+OI group were significantly increased,and the differences were statistically significant(P<0.05,P<0.01).There was no statistical difference in the expression of Osx mRNA in the ZGW+OI group compared with the OI group,but there was an upward trend.The results of fluorescence quantitative PCR showed that the expression of FoxJ1 mRNA in OI group was lower than that in CON group on 1d,3d and 7d after osteogenesis induction(P<0.01).FoxJ1 mRNA expression in the ZGW group was significantly lower than that in the CON group at 1d and 3d,and the difference was statistically significant(P<0.01,P<0.05).On the 7th day of osteogenic induction,FoxJ1 mRNA expression in the ZGW group was not significantly different from that in the CON group,but there was still a decreasing trend.Compared with OI group,FoxJ1 mRNA expression in ZGW+OI group was significantly decreased on the 7th day of intervention(P<0.01).There was no statistical difference in the amount of FoxJ1 mRNA decreased on the 1st and 3rd days of intervention,but there was still a downward trend.Western blot results showed that compared with CON group,FoxJ1 protein expression in OI and ZGW groups was significantly decreased,and the difference was statistically significant(P<0.05).Compared with the OI group,the expression of FoxJ1 protein in the ZGW+OI group was significantly decreased,and the difference was statistically significant(P<0.01).Compared with CON group,the expression of BMP2 protein in OI and ZGW groups was significantly increased,and the difference was statistically significant(P<0.05).P-smad1/5/Smad1 was significantly increased,and the difference was statistically significant(P<0.01).Compared with the OI group,the expression of BMP2 protein in the ZGW+OI group was significantly increased,with statistically significant differences(P<0.05),and p-smadl/5/Smad1 was significantly increased,with statistically significant differences(P<0.01).2.Animal research(1)Establishment of osteoporosis model and its influence on FoxJ1 expressionMicroCT results showed that bone microstructural parameters BV/TV(%),Tb.N(mm),Tb.Th(mm)and Tb.Sp(mm)in the OVX group were significantly decreased compared with Sham group(P<0.01).In addition,the 3d reconstruction shows that compared with the Sham group,the trabeculae in the OVX group are sparse and discontinuous,the number of trabeculae is significantly reduced,the width is narrowed,and the gap between trabeculae and trabeculae is widened.HE staining results showed that compared with Sham group,trabeculae in OVX group were seriously damaged,fractured and discontinuous,with trabeculae narrowed and gaps widened.Western-blot results showed that FoxJ1 protein expression was significantly increased in OVX group compared with Sham group(P<0.05).(2)The anti-osteoporosis effect of zuogui pill by regulating the BMP/Smad pathway through FoxJ1MicroCT results showed that compared with siCtrl group,the BV/TV of siFoxJ1 and ZGW+siCtrl group significantly increased,and Tb.Sp significantly decreased,with statistically significant differences(P<0.01).Although there was no statistical difference between Tb.Compared with the siFoxJ1 group,both BV/TV and Tb.Sp in the ZGW+siFoxJ1 group were significantly increased(P<0.01,P<0.05),and Tb.The 3d reconstruction showed that compared with siCtrl group,the trabeculae of siFoxJ1 group became dense and continuous,the number of trabeculae increased,and the gap between trabeculae and trabeculae narrowed.The bone trabecular continuity of ZGW+siCtrl group was good.Compared with the siFoxJ1 group,the number of trabeculae in the ZGW+siFoxJ1 group increased and the gap between trabeculae narrowed.HE staining results showed that the bone trabeculae in siCtrl group were fractured,arranged irregularly,and the number of trabeculae was reduced.Compared with the siCtrl group,the trabecular arrangement of the siFoxJ1 group was improved and orderly and continuous,while the trabecular arrangement of the ZGW+siCtrl group was orderly and the number of trabeculae increased.Compared with the siFoxJ1 group,the trabeculae of the ZGW+siFoxJ1 group were slightly wider,while the others showed no significant improvement.Western-blot results showed that compared with the siCtrl group,FoxJ1 protein expression in the siFoxJ1 group and the ZGW+siCtrl group significantly decreased,with statistically significant differences(P<0.01),and BMP2 protein expression and p-smadl/5/Smad1 significantly increased,with statistically significant differences(P<0.01,P<0.05).Compared with the siFoxJ1 group,FoxJ1 protein expression increased significantly(P<0.05),while the expression of BMP2 protein and p-smad1/5/Smad1 showed a trend of change,but there was no statistical difference.Conclusion1.In vitro studies have demonstrated that silencing foxJ1 can promote osteogenic differentiation of BMSCs in mice.Zuogui pill inhibited foxJ1 to activate the BMP/Smad signaling pathway and promoted osteogenic differentiation of BMSCs in mice.2.In vivo studies have confirmed that high expression of foxJ1 is an important mechanism leading to bone microstructure and bone histomorphological damage in ovariectomized mice.Zuogui pill activated the BMP/Smad signaling pathway by preparing foxJ1 and improved osteoporosis in mice. |
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