Study On The Effect Of P38MAPK Signal Pathway On High Glucose-Induced The Apoptosis Of Osteoblast Mc3t3-e1 Cell

Objective: To construct lentiviral vectors expressing small-hairpin RNA (shRNA) targeting mouse p38MAPK gene.Methods: Three complementary DNA sequences targeting mouse p38- MAPK gene were selected according to principles of siRNA design. After phosphorylation and annealing, these double strands DNA were subcloned into Hpa1/Xho1 Sites of the siRNA expression vector PLL3.7. Sequence identification was taken after screening and restriction enzymes digestion. 293T cells were co-transfected with lentiviral vector and recombinant plasmids. Flow cytometry (FCM) was employed to detect the titers of virus . The recombinant lentivirus vector systems were used to transfect the MC3T3-E1 cells. The efficiency of transfection was tested according to expression level of GFP observed under fluorescence microscope 72 hours later. The mRNA levels of p38MAPK were analyzed by Real- time PCR .Results: The lentiviral vectors containing shRNAs targeting p38MAPK gene were successfully constructed and transfected into MC3T3-E1 cells. Recombinant plasmids were named p38MAPK-shRNA1, p38MAPK- shRNA2 and p38MAPK-shRNA3 respectively. Viral titers determined by Flow cytometry were2.4�10~8 TU/ml?2.8�10~8 TU/ml and 2.5�10~8 TU/ml. After p38MAPK-shRNAs transfected MC3T3-E1 osteoblast cells, observ- ed by Fluorescence microscopy, lentiviral vector infection efficiency achieved over 74%. Quantitative PCR results showed the interferencing effects of three shRNAs, compared with the normal control group and negative virus control group. p38MAPK-shRNA1, p38MAPK-shRNA2 and p38MAPK-shRNA3 transfected MC3T3-E1 osteoblasts and decrea- sed p38MAPK mRNA expression levels by 78.8%, 84.3% and 60.2% (p <0.01). p38MAPK-shRNA2 had the highest interfering efficiency.Conclusion: Three recombinant lentivirus vectors of shRNA targeting p38MAPK gene had been successfully constructed and transfected MC3T3-E1 osteoblast cells. Transfection of p38MAPK-specific shRNAs (particularly p38MAPK-shRNA2) significantly reduced p38MAPK mRNA levels in MC3T3-E1 osteoblast cells Objective: To examine the role of p38MAPK in high glucose-induced apoptosis of osteoblast MC3T3-E1 cell and investigate its effect on the expressions of apoptosis-related molecules including Capase3, bax and bcl-2.Methods: The lentiviral vector containing shRNA targeting p38MAPK was constructed and transfected MC3T3-E1 osteoblast cells. The cultured osteoblast MC3T3-E1 cells were divided into 5 groups: normal control group,high glucose group,p38MAPK-shRNA transfection group,p38MAPK signal transduction inhibitor group,negative control siRNA transfection group . Cells were collected after 7 days cultured in?-MEM. RT-PCR was used to determine the p38MAPK mRNA levels in MC3T3-E1 cells. TUNEL and Flow cytometry (FCM) was employed to detect the cell apoptotic percentage. The ultrastructural alternations of MC3T3-E1 osteoblast cells were observed under transmission electron microscopy (TEM). The protein levels of p38MAPK,p-p38MAPK, Caspase-3,bax and bcl-2 were assayed by Western blot. Activity of AP-1 in MC3T3-E1 osteoblast was examined by EMSA assay .Results: The lentiviral vector containing shRNA targeting p38MAPK was successfully constructed and transfected into MC3T3-E1 cells. Compared with the normal MC3T3-E1 osteoblasts, MC3T3-E1 osteoblast cells of the high glucose group and negative control shRNA group, changed in morphology under TEM as follows: microvilli on cell surface reduced, cell membrane incomplete, endoplasmic reticulum enlarged, mitochondria swelling,chromatin aggregated and concentrated. Compared with the cells of high glucose group, cells in p38MAPK-shRNA transfection group were greatly improved in morphology. Apoptosis of MC3T3-E1 osteoblast was significantly increased in high glucose group and negative control shRNA group, compared with normal control group(p<0.01). Compared with that of high glucose group, the apoptosis of MC3T3-E1 osteoblast cells of p38MAPK-shRNA transfection group and p38MAPK signal transduction inhibitor treatment group was significantly reduced, with a decrease of 50.8% and 37.5% (p<0.01, p<0.05) respectively. RT-PCR results suggested that in high glucose group and negative shRNA control group, p38MAPK mRNA expression of MC3T3-E1 osteoblast cells significantly increased compared with that of the normal control group (p<0.01); p38MAPK-shRNA transfection significantly reduced high glucose-induced MC3T3-E1 osteoblast activation of p38MAPK. Western Blot analysis showed that the protein levels of p38MAPK and phosphorlated p38MAPK were significantly increased in high glucose-induced MC3T3-E1 osteoblasts(p<0.01). After the p38MAPK-shRNA transfection and the intervention of p38 MAPK signal inhibitor, the protein levels of p38MAPK and phosphorlated p38MAPK in MC3T3-E1 osteoblasts were significantly decreased compared with that of the high glucose group and negative shRNA transfection group,the difference was statistically significant (p< 0.01, p<0.05). And compared with the normal control group, the expression of Caspase-3 and bax of MC3T3-E1 osteoblast in high glucose group and negative control shRNA transfection group was upregulated, while the expression of bcl-2 was significantly reduced (p<0.01). After p38MAPK-shRNA transfection and p38MAPK signal transduction inhibitor treatment, Caspase-3 and bax protein levels of MC3T3-E1 osteoblasts were significantly lower than that of high glucose group and negative control siRNA transfection group, while bcl-2 protein expression was significantly increased. The difference was statistically significant (p <0.01, p <0.05). EMSA assay showed that AP-1 activity of MC3T3-E1 osteoblast was significantly increased in high glucose group(p <0.01), while p38MAPK-shRNA transfection and p38MAPK signal transduction inhibitors treatment could significantly inhibit the AP- 1 activity of MC3T3-E1 osteoblasts, decreased 57.9% and 45.6% respectively compared with the high glucose group (p <0.01, p <0.05).Conclusion: p38MAPK may play an important role in high glucose-induced apoptosis of MC3T3-E1 osteoblasts. high glucose can induce apoptosis of MC3T3-E1 osteoblasts through activation of p38MAPK, activation of AP-1, upregulation of bax and Caspase-3 gene expression, reduced expression of bcl-2, activation of mitochondrial apoptosis pathway. p38MAPK-shRNA lentiviral vector can effectively inhibit the expression of p38MAPK, and reduce high glucose-induced MC3T3-E1 osteoblast apoptosis. This study provides a new theoretical foundation for prevention and treatment of diabetic osteoporosis.