Effects Of αMSH And MC5R Antagonist On The Molecular Mechanisms Of Apoptosis And Inflammation Regulating In 3T3-L1 Adipocytes

αMSH is a kind of neuroendocrine hormone secreted by hypothalamic, can regulate expression of MC5 R receptor molecule, thereby to mediate adipocytes differentiation and lipid metabolism mechanism and so on. Studies have shown that αMSH through MC5 R can inhibit apoptosis and the cytokine expression with a certain extent in macrophages and C2C12 myoblasts, suggesting αMSH may have an impact on the adipocytes apoptosis and inflammation, however, the specific mechanism is unclear all the time. In recently years, the disscuss about MC5 R primarily focused on adipocytes differentiation and metabolism regulation, ect. So, the present research was to investigate the regulation of αMSH by MC5 R with molecular mechanisms of apoptosis and inflammation. JNJ10229570 was treated as MC5 R a competitive antagonist, but studies on adipocytes is less. The trial is the first time to explore the role of JNJ10229570 on adipocytes. Construction palmitate-induced apoptosis model and LPS-treatment of inflammation, hoping to reveal MC5 R in the role of physical activity, that possible in terms of prevention and treatment of obesity or other MC5 R related diseases provide the theoretical basis. In this experiment, we obtained the followings results:1. αMSH was hardly toxicity of 3T3-L1 adipocytes in a certain concentration, 300 nM, 500 nM, 700 nM and 900 nM of αMSH made the expression level significantly increased MC5R(P<0.05); 0.8μM of MC5 R antagonist enabled to decrease MC5 R expression levels significantly(P<0.05). 0.2mM palmitate impacted on adipocytes, respectively by CCK-8 kit to detect cell viability, when treated for 24 h, cell survival rate reached 78%, it was the best processing time. Stained Oil Red O and Bodipy showed that αMSH can promote lipid droplets; on the contrary, MC5 R antagonists may inhibit lipid droplets deposition in 3T3-L1 adipocytes.2. In the 3T3-L1 adipocytes, the cells were treated with palmitate in order to construct apoptosis models. Combined Hoechst 33258 staining with Annexin V-FITC/PI staining and Flow Cytometry and the results showed that αMSH can reduce the number of apoptosis, treatment of palmitate-incubated adipocytes with αMSH significantly attenuated palmitate-induced apoptosis(P<0.05); and MC5 R antagonist had the opposite function. With RT-PCR and Western Blotting to detect the cell apoptosis related factors,we found that αMSH significantly increased the expression of antiapoptotic factor Bcl-2, the expression of pro-apoptotic factor Bax was decreased significantly, the activity of cleaved Caspase-9, cleaved Caspase-3 significantly were lower in the interference group(P<0.05), the separate palmitate could promote the expression of Bax,and the expression of antiapoptotic factor Bcl-2 was decreased significantly, but the activity of cleaved Caspase-9, cleaved Caspase-3 significantly enhanced(P<0.05), the expression levels of palmitate-induced apoptotic with αMSH was between the palmitate group and the individual αMSH group(P<0.05); MC5 R antagonist increased the expression of apoptosis-related factor Bax, cleaved Caspase-9, cleaved Caspase-3 levels induced by palmitate(P<0.05).3. The inflammation model were treated with lipopolysaccharide to detect the expression levels of inflammatory cytokines on the transcription and translation level. MTT assay showed that LPS optimum concentration was 1μg/mL. αMSH could inhibit the expression of IL-1β, IL-6 and MCP-1(P<0.05); MC5 R antagonist promoted the expression of inflammatory factor IL-1β, IL-6 and MCP-1(P<0.05), both had role in the expression of TNF-α, but the difference was not significant.4. JNK signaling pathway participated in apoptosis and inflammation, αMSH inhibited JNK phosphorylation; MC5 R antagonists improved the level of JNK phosphorylation. When added JNK inhibitor(SP600125), JNK and its downstream c-Jun phosphorylation and protein expression levels were declined.In summary: αMSH inhibited 3T3-L1 adipocytes apoptosis, reduced the expression of proinflammatory cytokine levels, MC5 R antagonist was able to induce apoptosis and triggered the cell inflammatory response; and JNK signaling pathways may play important roles in the regulation of apoptosis and inflammation in 3T3-L1 adipocytes.