The Mechanism Study Of Prevention And Treatment Of Steroid-induced Osteonecrosis Of The Femoral Head With Chinese Medicine By Differential Expression Of LncRNA In Rat Osteoblasts

Objectives1.To establish the models of normal osteoblast and steroid-induced osteoblast in rat.2.To explore the differential expression of LncRNA in steroid-induced osteoblast,and to analyze the possible mechanism of action of differentially expressed LncRNA.3.To explore the mechanism of action of LncRNA MEG3 in steroid-induced osteoblast,and to analyze the mechanism of action of Yuan Shi Sheng Mai Cheng Gu tablets in the treatment of steroid-induced osteonecrosis of the femoral head(SONFH).Methods1.The skull was taken from 10 fetal rats which were born within 24 hours.Using trypsin and collagenase I in turn to digest osteoblasts.Alkaline phosphatase staining,alizarin red staining and collagen I immunofluorescence staining were used to identify the cultured cells.To achieve the steroid-induced osteoblasts,the normal osteoblasts were cultured with methylprednisolone solution at a concentration of 10^-6 mol/L for 72 hours,the cells were collected and stored for later use.2.Arraystar Rat LncRNA Microarray V2.0 was applied to analyze the differentially expressed genes of normal osteoblasts and steroid-induced osteoblasts.10 differentially expressed LncRNAs were verified by Real-time quantitative PCR.The GO and KEGG pathway were used to analyze the biological functions and signaling pathways of differentially expressed genes.3.To establish the overexpressed LncRNA MEG3 vector and transfected into osteoblasts.Divided into normal control group and MEG3 overexpression group,after which cell proliferation and apoptosis were detected by CCK8 kit and flow cytometry.P53 protein expression was detected by Western-Bolt.Results1.The cultured cells showed typical morphological characteristics of osteoblasts,alkaline phosphatase staining,alizarin red staining and collagen I immunofluorescence staining were positive.2.According to the microarray analysis,we found 8059 lnc RNAs expressed in the control group and 302 were upregulated and 151 were downregulated significantly when compared to the steroid group(fold change>2.0,P value<0.05).From the m RNA expression profiling data,20078 m RNAs were differentially expressed between the control group and the steroid group,among which 548 were upregulated and 565 were downregulated(fold change>2.0,P value<0.05).Real-time quantitative PCR confirmed that five significantly up-regulated LncRNAs(including ENSRNOT00000076045,NR?131064,uc.420+,XR?147168 and XR?596946)and two significantly down-regulated LncRNAs(including ENSRNOT00000061527 and XR?589471)expression results were consistent with the gene chip results.GO analysis showed that differentially expressed genes were related to hormone reaction,and KEGG signaling pathway showed that differentially expressed genes were enriched in multiple signaling pathways.3.The relative expression level of LncRNA MEG3 was significantly higher in the MEG3 overexpression group than in the normal control group(P<0.05).In addition,compared with the normal control group,the proliferation of osteoblasts in the MEG3 overexpression group was significantly decreased,the apoptosis was significantly enhanced,and the expression of P53 protein was significantly increased(P<0.05),but after the intervention of Chinese medicine,the proliferative capacity of osteoblasts was significantly increased,the apoptosis and the expression of P53 protein were significantly decreased(P<0.05).Conclusions1.Osteoblasts achieved from the skull of fetal rats by this method are high quality with large quantities,so this method can be used as a reliable and efficient way of primary culture.2.LncRNA MEG3 is up-regulated in steroid-induced osteoblast and inhibitis the proliferation of osteoblast.In addition,LncRNA MEG3 may play a role in inducing the apoptosis of osteoblast by activating the expression P53 protein,has the potential to be a novel biomarker for the diagnosis of SONFH and a new target for biological therapy.3.The Chinese medicine Yuan Shi Sheng Mai Cheng Gu tablets can effectively promote the proliferation of osteoblasts,inhibit the apoptosis of osteoblasts,and reduce the expression of P53 protein,and thereby play a role in the treatment of SONFH,which can provide a new reference for the discover of new drugs in the future.