| Adipocyte can not only reserve or release energy, but also secrete many cytokines to play an important role in regulating itself and other cells. TNF-αis one of the cytokines, it can produce a variety of effects to adipocyte, such as inhibit the growth of adipocyte, inducing the apoptosis of preadipocytes and adipocytes. However, owing to the evidently ability of adipocyte to resist apoptosis, TNF-αinduced adipocyte apoptosis was not well studied and poorly understood. SOCS is a negative feedback regulator of the JAK/STAT signaling pathway. JAK2/STAT3 signaling pathway participate in many signal transduction systems, influencing cell growth, survival, differentiation and apoptosis functions.In this study, we used siRNA interfere technique to silence SOCS3 gene in 3T3-L1 preadipocytes and mouse preadipocytes. Study the effect of SOCS3 siRNA on TNF-αinduced apoptosis in both cells. The main results were summarized as following:1. 3T3-L1 preadipocytes were cultured in vitro and treated with TNF-αat the concentrations of 20,40,60,80,100,150,200 ng/mL for 24 h, respectively. Optical microscope to observe morphological changes during apoptosis. TNF-αinduced 3T3-L1 preadipocytes apoptosis showed a dose dependent manner. After treated with 100 ng/mL TNF-αfor 24h, primary preadipocyte apoptosis was apparent, accompanied by reduced cell volume, chromatin condensation, and nuclear shrinkage.2. Using siRNA interfere technique, this study successfully interfere the expression of SOCS3 gene. The gene silencing effect of SOCS3 siRNA1 is the most obvious. SOCS3 mRNA expression were suppressed by 57% and 50% by SOCS3 siRNA1 in 3T3-L1 preadipocytes and mouse preadipocytes, it may lay solid foundation for the further research on the function of SOCS3.3. After SOCS3 expression was inhibited by SOCS3 siRNA infection, 3T3-L1 preadipocytes were treated with TNF-αat 100 ng/mL for 24 h. Morphological changes during apoptosis were observed by fluorescence microscope after staining by Hoechst 33258 and PI fluorescent dyes respectively. RT-PCR and Western blotting to measure the expression of apoptosis-associated gene c-myc,survivin,mcl-1,bcl-2,bax,NF-κB, and JAK2/STAT3 pathway key gene SOCS1, SOCS2, JAK2, STAT3. Compared with control group, in SOCS3 siRNA group the number of cells apoptosis was decreased remarkably; the expression level of survivin and NF-κB mRNA increased significantly (P<0.05), the expression level of c-myc and bcl-2 mRNA increased extremely significantly (P<0.01), the expression level of bax and SOCS1 mRNA decreased significantly (P<0.05), no changes were found for the expression of mcl-1 (P>0.05); Western blotting analysis showed that inhibition of SOCS3 also upregulated the expression of Bcl-2,NF-κB and p-STAT3 (P<0.05), but downgulated bax expression (P<0.05). Here, no distinct change was detected in LipofectineTM2000 group or negative control group. Taken together, our data established that knocking down SOCS3 can regulate the expression of apoptosis-associated gene by JAK2/STAT3 pathway, then effectively inhibit TNF-αinduced apoptosis in 3T3-L1 preadipocytes.4. After SOCS3 expression was inhibited by SOCS3 siRNA infection, mouse preadipocytes were treated with TNF-αat 100 ng/mL for 24 h. Hoechst 33258 and PI staining showed that the number of cells apoptosis was decreased remarkably; RT-PCR analysis showed that, the expression level of NF-κB mRNA increased significantly (P<0.05), the expression level of mcl-1 and bcl-2 mRNA increased extremely significantly (P<0.01), the expression level of bax and SOCS1 mRNA decreased extremely significantly (P<0.01), no changes were found for the expression of bax,c-myc and survivin (P>0.05); Western blotting analysis showed that inhibition of SOCS3 also upregulated the expression of p-STAT3,Bcl-2 and NF-κB (P<0.01), but downgulated bax expression (P<0.05). Our data established that knocking down SOCS3 can regulate the expression of apoptosis-associated gene by JAK2/STAT3 pathway, then effectively inhibit TNF-αinduced apoptosis in mouse preadipocytes. |
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