Promoting Effect Of Icariin On Osteogenic Differentiation And Mineralization Of BMSCs By Inducing Autophagy

Objective Icariin(ICA)was used as the starting point to explore the correlation between icariin,postmenopausal osteoporosis and autophagy in animal experiments.The roles of icariin and autophagy in osteoblastic differentiation and mineralization of BMSCs were studied at the cellular and molecular level,and the mechanism was studied,so as to provide new theoretical ideas for the prevention and treatment of postmenopausal osteoporosis.Methods1.Bilateral ovaries were removed to construct a female Babl/c mouse model of postmenopausal osteoporosis,and Micro-CT examination was performed 2 months after surgery,which confirmed the success of postmenopausal osteoporosis model.2.Femoral bone marrow cells from female Babl/c mice at 6-8 weeks were extracted and subcultured for 2 times.Specific cell membrane surface markers of bone marrow mesenchymal stem cells(BMSCs)were used.CD9,CD34,CD44 and CD45 were used for cell identification by flow cytometry and cellular immunofluorescence.In addition,osteogenic induction was conducted using the induction medium for bone marrow mesenchymal stem cells of Babl/c mice,and alizarin red staining was performed 21 days later.3.Femoral BMSCs from Sham group(Sham)and osteoporosis group(OVX)were extracted and cultured in BMSCs osteoblastic differentiation medium for 7 days and 21 days.After 7 days,alkaline phosphatase(ALP)activity was detected by BCIP/NBT alkaline phosphatase staining and p NPP assay.After 21 days,BMSCs mineralization levels in different groups were detected by alizarin red staining.BMSCs RNA and protein were extracted from the two groups,and BMP2 and RNUX2 m RNA levels were detected by fluorescence quantitative PCR,and the expressions of BMP2 and RNUX2 were detected by Western blot.4.BMSCs from femur of Babl/c mice at 6-8 weeks were extracted,and the 3rd generation BMSCs were divided into four groups on average.BMSCs were cultured in osteogenic induction medium,and treated with PBS,ICA(10 M),CQ(5 M),ICA+CQ(10 M+5 M)for 7 days and 21 days.After 7 days,alkaline phosphatase(ALP)activity was detected by BCIP/NBT alkaline phosphatase staining and p NPP assay.After 21 days,BMSCs mineralization levels in different groups were detected by alizarin red staining.BMSCs RNA and proteins were extracted from different treatment groups,and the expressions of autophagy related genes LC3,P62 and beclin-1 m RNA were detected by fluorescence quantitative PCR,and the expressions of autophagy related proteins LC3,P62 and beclin-1 were detected by Western blot.5.Confirmed in postmenopausal osteoporosis after the success of the building,the average in mice is divided into five groups: control group(Sham),osteoporosis group(OVX),osteoporosis + epimedium group(OVX + ICA),osteoporosis +chloroquine group(OVX + CQ),epimedium osteoporosis + + chloroquine group(OVX + ICA + CQ),were given epimedium,chloroquine,epimedium +chloroquine.One month after administration,the mice were put to death under anesthesia.The femur was examined by micro-ct for the number,thickness and cortical thickness of bone trabeculae,followed by HE staining and immunofluorescence detection for the expression of autophagy related marker protein(P62).6.BMSCs of femur of each group were extracted and cultured in BMSCs osteogenic differentiation induction medium for 21 days.The cell mineralization levels of different treatment groups were detected by alialiin red staining experiment,and the staining images were semi-quantitatively analyzed by image pro plus 6.0software.Proteins in BMSCs of each group were extracted,and the expressions of osteogenic related proteins BMP2,RUNX2 and autophagy related proteins LC3,P62 and beclin-1 were detected by Western blot.Results1.The model of postmenopausal osteoporosis in mice was established successfully.Two months after surgery,Micro-CT results showed that the number of bone trabeculae,the thickness of bone trabeculae and the bone cortex in the femur of mice with bilateral ovary excision decreased compared with the sham operation group,and the data analysis showed statistical differences,confirming the successful establishment of the model of postmenopausal osteoporosis in mice.2.The results of flow cytometry showed that the expressions of cell membrane markers extracted were CD9+,CD44+,CD34-and CD45-,which were consistent with the results of cellular immunity.It was proved that the extracted cells were BMSCs,not hematopoietic cells.The results of alizarin red staining showed that the extracted bone marrow cells could differentiate into osteoblasts,which proved that the extracted cells were BMSCs.3.ALP staining results showed that BMSCs in OVX group had lower bone capacity than Sham group,and the difference was statistically significant(P<0.05).Alizarin red staining showed that BMSCs in OVX group were less mineralized than those in Sham group,and the difference was statistically significant(P<0.05).Fluorescence quantitative PCR showed that BMSCs expression in OVX group was lower than that in Sham group.Western blot results showed that BMSCs expression in OVX group was lower than that in Sham group,and the differences were statistically significant(P<0.05).4.In vitro,ICA enhanced the autophagy activity and osteogenic differentiation and mineralization of BMSCs.ALP staining results showed that the BMSCs osteoblastic differentiation was enhanced in ICA group and decreased in CQ group.The results of alizarin red staining showed that BMSCs mineralization was enhanced in ICA group and decreased in CQ group.Western blot results of BMSCs protein showed that ICA could up-regulate the expression of autophagy related marker protein LC3 and down-regulate the expression of P62 protein.On the contrary,the expression of autophagy related marker protein LC3 was significantly decreased and the expression of P62 protein was increased in the CQ group.The results of fluorescence quantitative PCR were consistent with those of Western blot.5.Micro-CT and HE staining results showed that CQ increased the number of bone trabeculae,the thickness of bone trabeculae and bone cortex,while ICA increased the number of bone trabeculae,bone trabeculae and bone cortical thickness.Tissue immunofluorescence results suggested that ICA significantly reduced the expression of P62.6.In vivo,ICA enhanced autophagy activity and osteogenic differentiation of BMSCs in mice.The results of alizarin red staining showed that BMSCs mineralization was enhanced in ICA group and decreased in CQ group.Western blot results of BMSCs protein showed that ICA could up-regulate the expression of osteogenic related proteins RUNX2 and BMP2.Meanwhile,the expression of autophagy related marker protein LC3 was significantly increased,while the expression of P62 protein was significantly decreased.On the contrary,in the CQ group,the expression of osteogenic related protein was decreased,the expression of autophagy related marker protein LC3 was significantly decreased,and the expression of P62 protein was increased.Conclusion During the process of ovariectomized osteoporosis,the number,thickness and cortical thickness of bone trabeculae decreased significantly.BMSCs can be successfully extracted by whole bone marrow adherent method with simple operation and less cell loss.ICA promoted osteogenic differentiation and mineralization of BMSCs in vitro and in vivo.ICA could increase the number of bone trabeculae and increase the thickness of bone cortex in osteoporosis mice.During the process of ovariectomized osteoporosis,the level of autophagy decreased significantly.ICA can promote osteogenic differentiation and mineralization of BMSCs by enhancing autophagy.