Effects Of TGF-β1 And Runx2 Expression Of Osteoblasts Osteogenic Differentiation After Application Of Adiponectin

Objective:To investigate the biological effect of adiponectin induced osteoblast from primary culture. To investigate the effects of adiponectin on TGF-(3 I and Runx2 expression in osteoblast, and explore the role and mechanism of adiponectinonbone remodeling.Methods:1. The effects of adiponectin on rat osteoblast osteogenic differentiationRat osteoblasts were primary cultured and proliferated by the twice-enzymedigestion. These cells were also identified with Alizarin red staining of calcium nodules after osteogenic induction cultured 14 days.② The 3-5 passage of osteoblastswas subjected to 4μg/ml of adiponectin for 1, 3,5,7,9 days.The MTT assay was performed to assess the changes in osteoblasts proliferative activity after application of adiponectin.③ The 3-5 passage of osteoblastswas subjected to 4μg/ml of adiponectin for 1, 3,5,7,9 days. The alkaline phosphatase staining was performed to assess alkaline phosphatase activities in osteoblasts after application of adiponectin.④ The 3-5 passage of osteoblastswas subjected to 0,1,2,4,8μg/ml of adiponectin for 7 days.The protein level of TGF-β1 andRunx2 were detected byELISA method.2. The mechanism of adiponectin on rat osteoblast osteogenic differentiation﹐steoblastswere subjected to pretreatment with 5μmol/L TGF-β1 inhibitor(SB431542) for 15min,30min, 1h,2h,4h,or to pretreatment with 0,1, 2.5,5,10,20μmol/L TGF-β1 inhibitor(SB431542) for 2h, and then were subjected to 4μg/ml of adiponectin for 7 days.TO identify the optimal concentration and the effective time of TGF-β1 inhibitor(SB431542), the MTT assay was performed to assess the changes in osteoblasts proliferative activity after application of TGF-β1 inhibitor(SB431542).② The protein level of TGF-β1 after application of TGF-β1 inhibitor(SB431542) were detected by ELISA method. Divided into five groups, control group: osteoblasts without adiponectin and SB431542; group APN:4μg/ml of adiponectin; group SB431542+APN:pretreatment with 5μmol/L TGF-β1 inhibitor(SB431542) for 2h and add 4μg/ml of adiponectin; group SB431542: pretreatment with 5μmol/L TGF-β1 inhibitor(SB431542) for 2h; group DMSO:10μmol/L DMSO culture. Osteoblasts were cultured for 7 days.The protein level of TGF-β1were detected by ELISA method.③The mRNA level of Runx2 after application of TGF-β1 inhibitor(SB431542) were investigated by RT-qPCR. Divided into five groups, control group: osteoblasts without adiponectin and SB431542; group APN:4μg/ml of adiponectin; group SB431542+APN:pretreatment with 5μmol/L TGF-β1 inhibitor(SB431542) for 2h and add 4μg/ml of adiponectin; group SB431542: pretreatment with 5μmol/L TGF-β1 inhibitor(SB431542) for 2h; group DMSO:10μmol/L DMSO culture. Osteoblasts were cultured for 7 days.The mRNAlevel of Runx2 wereinvestigated by RT-qPCR.Results:1. The effects of adiponectin on rat osteoblast osteogenic differentiation① The amplification cells showed representative morphological and biological characteristics of osteoblasts.Alizarin red staining presented positive results.② The result of MTT showed as follows:The OD value of all the groups was higher than control group(P<0.05). The peak of OD value after application of adiponectin was observed at 7 days. The OD value of 9days decreased, but still higher than control group(P<0.05).③ The result of ALP stainingshowed as follows:The OD value of all the groups was higher than control group(P<0.05). The peak of OD value after application of adiponectin was observed at 7 days. The OD value of 9days decreased, but still higher than control group(P<0.05).④ The result of ELISA showed:The expression of TGF-β1 and Runx2 increased after application of adiponectin. The peak of TGF-β1 andRunx2 expression after application of adiponectin was observed at 4μg/ml.There were significant differences in all groups (P<0.05)2.The mechanism of adiponectin on rat osteoblast osteogenic differentiation① The result of MTT showed:The OD value of all the groups was lower than control group(P<0.05). The nadir of OD value after application of TGF-β1 inhibitor(SB431542) was observed at 5μmol/L and 2h. There were significant differences in all groups(P<0.05).② The result of ELISA showed:Comparing with control group, the expression of TGF-pl increased in group APN(P<0.05). Comparing with groupAPN, the expression of TGF-β1 significantly decreased in group SB431542+APN(P< 0.05). There was no statistical significance in control group, group SB431542 and group DMSO(P>0.05).③ The result of RT-qPCR showed:Comparing with control group, the expression of Runx2 increased in group A(P<0.05). Comparing with control APN, the expression of Runx2 significantly decreased in group SB431542 (P< 0.05). There was no statistical significance incontrol group, group SB431542 and group DMSO(P>0.05).Conclusion:1.The expression of TGF-β1 and Runx2 in osteoblasts increases after application of adiponectin. Adiponectin plays an important role in promoting osteoblastsosteogenic differentiation during bone remodeling.2. As a downstreamsubstrate of TGF-β1, Runx2 participates in promotingosteoblastsosteogenic differentiation after application of adiponectin via the TGF-β1 signal pathway.