| Objectives:Acute phase serum amyloid protein A (A-SAA) is one of the frequently studied adipocytokines recently, which present mild increase in obesity and T2DM. Our previous studies showed that human recombinated A-SAA could significantly induce ECV304 endotheliocytes and human umbilical vein endothelial cells,(HUVECs)IL-6,IL-8 mRNA expression in a dose- and time-dependent manner.However,whether A-SAA will effect the expression of inflammatory cytokines in adipocytes is unclear. Our aims are to examine the roles of acute phase serum amyloid protein A in regulating adipocytokines and related kinases expression in 3T3-L1 adipocytes in this study.Methods:3T3-L1 preadipocytes cultured and differentiated in six-well plates containing high glucose DMEM plus 10% fetal calf serum according to Kolapo M Ajuwon were treated with 3 different concentrations of human recombinated A-SAA(1 ug/ml,10 ug/ml,20 ug/ml) for 3 time periods(8h,24h,48h)after preadipocytes fully differentiated. IL-6,TNF-a and adiponectin mRNA expressions were detected by Reverse transcription Polymerase Chain Reaction(RT-PCR), the concentrations of TNF-a,IL-6 and adiponectin in conditioned media were tested by Enzyme-linked Immunosorbent Assay( ELISA), and the phosphorylated protein expressions of JNK and ERK were detected by Western Blot.Results:Compared with control group, both 10μg/ml A-SAA and 20μg/ml A-SAA significantly induced the IL-6 mRNA expression respectively after incubated for 8h(P<0.05 and P<0.01)and compared with 1μg/ml A-SAA group, 20μg/ml A-SAA also significantly induced the IL-6 mRNA expression(P<0.05);compared with control group, 3 different concentrations of A-SAA (1 ug/ml,10 ug/ml,20 ug/ml) significantly induced the IL-6 mRNA expression respectively after incubated for 24h(P<0.01)and compared with 1μg/ml A-SAA group, 20μg/ml A-SAA also significantly induced the IL-6 mRNA expression(P<0.01);compared with control group,both 1μg/ml A-SAA and 10μg/ml A-SAA also significantly induced the IL-6 mRNA expression respectively after incubated for different times (8h,24h,48h)(P<0.05 and P<0.01);compared with control group,20μg/ml A-SAA also significantly induced the IL-6 mRNA expression after incubated for 8h and 24h ( P <0.01);compared with control group,the mRNA expression of adiponectin was significantly decreased after stimulated with 10μg/ml A-SAA for 24h(P<0.05), the mRNA expression of TNF-a was significantly increased after treated with 10μg/ml A-SAA for 24h(P<0.05);different concentrations of human recombinated A-SAA with different times did not alter the protein expressions of TNF-a,IL-6 and adiponectin(P﹥0.05)in conditioned media. A-SAA appeared to activate the kinases of JNK and ERK.Conclusions:A-SAA induced the mRNA expression of IL-6 in a dose-dependent manner, the mRNA expression of adiponectin was significantly decreased after stimulating with 10μg/ml A-SAA for 24h, meanwhile the mRNA expressions of TNF-a and IL-6 was significantly increased when stimulated with 10μg/ml A-SAA for 24h. Therefore,A-SAA did not induce insulin resistance of 3T3-L1 adipocytes through increasing the expression of IL-6 and TNF-a and/or decreasing the expression of adiponectin in this study , A-SAA seemed to participate in the insulin resistance of adioicytes through MAPK pathway. |
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