| Since liver lies at the heart of metabolism and detoxification, the integrity of its structure and function is the key for vital functions. Thus, apoptosis of liver cell may trigger a variety of injuries or diseases. For instance, liver is crucial for lipid digestion, absorption, transportation, catabolism and anabolism. SOCS3(Suppressor of cytokine signaling3), as a signal transduction cytokine, is involved in those lipid metabolism, as well as cell proliferation, differentiation, apoptosis and so on. This experiment is aimed to explore the effection of SOCS3on apoptosis and lipid metabolism in liver cells of kunming white mice by over-expressing SOCS3with hydrodynamic injection. We injected the ringer’s solution dissolving recombinant plasmid pEGFP-N1-SOCS3via hydrodynamic injection. Then,12h、24h and48h after the injection, orbital blood was got for the assessment of blood lipid, and samples from liver, kidney and leg muscle were used for the extraction of total RNA and protein, as well as frozen sections. Apoptosis of liver cell here was observed by Hoechst staining and TUNEL staining. We also studied the changes of body weight, serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), low density lipoprotein (LDL-C), transcriptional changes of SOCS3, Bax, Bel-2, Caspase3, C-myc, Mcl-1, HSL, ATGL and FAS with Real-time PCR. Besides, Western blotting was used to detect genes related to apoptosis like Bax, Bcl-2and Caspase3, involving lipid metabolism like HSL, ATGL and FAS, and JAK2/STAT3signaling pathways. We attempted to clarify what role does SOCS3participate in the process of apoptosis and lipid metabolism in liver cells of mice. The main results are as follows:1Recombinant plasmid pEGFP-N1-SOCS3was injected via the tail vein into mice, then we observed the liver tissue frozen sections under fluorescence microscope. We detect that the12h treatment group had obvious GFP expression, GFP expression of the24h treatment group was increased, and it was decreased in48h treatment group, proving successful transfection. Detecte the SOCS3mRNA expression by RT-PCR, we found that the mRNA expression of SOCS3were increased in the liver, kidney and leg muscle compared with normal control group, and it was the most significantly in liver(p<0.01).2Recombinant plasmid pEGFP-N1-SOCS3was injected via the tail vein into mice. Observe the mice liver tissue frozen sections after HE staining, we found that the central vein of hepatic lobule and sinus around expansed in12h treatment group. Besides, hepatic sinus had obvious passive congestion, but we didn’t find obvious necrosis foci;24h after injection, the liver sinus gap widened, hepatic sinus internal bleeding receded, and liver cell structure recovered; All returned to normal basicly48h after injection. After Hoechst33258and TUNEL staining, each group have the apoptosis cells and normal cells, the number of apoptotic cells in recombinant plasmid processing group increased obviously, and it has a tendency to increase with the extension of time.3After the recombinant plasmid pEGFP-Nl-SOCS3was injected via the tail vein into the mouse, compared with normal control group, the mRNA expression of Bax gene in the recombinant plasmid treats especially in the12h treatment group was significantly higher (p <0.01) but subsequently gradually reduced; mRNA of c-myc gene expression in the recombinant plasmid treatment groups was gradually increased,24h later, it reached a significant level (p<0.05); the mRNA expression of Bcl-2gene in12h treatment group was significantly reduced (p<0.05), while in24h treatment group, it reached a highly significant reduction (P<0.01), and in the48h treatment group it has been restored, but still significantly lower than the control group (P<0.05). Found by the Western blotting detection, the Bax protein level in the12h treatment group was also extremely significantly rising (P<0.01), then gradually decreased; the Bcl-2protein level in the12h treatment group was significantly reduced (P<0.05); Caspase-3protein level in the12h treatment group had significantly increased and reached an extremely significant level (P<0.01), followed by a rapid reduction or even lower than that in control group, but didn’t reach a significant level; When transfected with empty vector, the expression level of Bax, Bcl-2, and Caspase-3, these three proteins had no significant difference compared with the control group. p-STAT3protein level in12h treatment group was significantly reduced (P<0.05), but gradually increases in the24h and48h treatment groups, but was still lower than the control group (p>0.05). The protein level of p-STAT3in empty plasmid treatment groups didn’t change significantly (p>0.05). By inference, hydrodynamic injection of recombinant plasmid pEGFP-N1-SOCS3may promote liver cell apoptosis through the regulation of JAK2/STAT3signaling pathway genes and apoptosis-related gene expression.4After the mice were injected with recombinant plasmid pEGFP-Nl-SOCS3via the tail vein,12h,24h and48h later, the mouse is killed. Compared with the control group,12h and24h after the injection, the abdominal fat didn’t change significantly but increased significantly after48h. The concentration of TG, TC and LDL-C in the mice’s serum gradually increased with time and reached a significant level (p<0.05) in the48h treatment group; but the concentration of HDL-C decreased, though not significantly. According to the RT-PCR detection, the mRNA expression of HSL and ATGL had been significantly reduced. That of HSL reached a significant level in the24h treatment group (p<0.05), while ATGL in the48h treatment group had been significantly reduced (p<0.05); and the mRNA expression of FAS in24h and48h treatment groups had been significantly increaesed (p<0.05). Due to the Western blotting analysis, compared with the control group, the protein expression of FAS had been significantly decreased, in which the12h and24h treatment groups had reached a significant level (p<0.05); the protein expression of ATGL was significantly increasing, with the12h and24h treatment group achieving their significant levels (p<0.05). The results of the Western Blotting Detection and Real-time PCR are basically coincident with each other. In addition, the protein expressions of the PPAR-yand SREBP1had increased, with the12h and24h treatment groups both have significantly increased (p<0.05and p<0.05). These results suggest that transfection of SOCS3in vivo could impact the expression of key genes of lipid metabolism by regulating the transcription factor PPAR-yand SREBP1, and thus promote the lipid synthesis of liver cell.To sump up, this research implemented the over-expression of SOCS3gene in mice liver through the tail vein of high pressure injection. After intravenous recombinant plasmid pEGFP-Nl-SOCS3was injected, the activation of JAK2/STAT3signaling pathway was inhibited significantly, then promoted the liver cell apoptosis in mice; and increased expression of lipid metabolic regulation factor PPAR-yand SREBP1, then promoted the mice liver fat synthesis. |
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