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目的建立体外肝细胞脂肪变性模型,探讨白介素-22(IL-22)调控的磷酸化酪氨酸蛋白激酶-1(pJAK1)/磷酸化信号转导激活转录因子-3(p-STAT3)信号通路在蓝莓益生菌血清改善肝细胞脂肪变性实验中的作用机制。方法制备蓝莓益生菌原液,并制备大鼠10%蓝莓益生菌低、中、高剂量血清及生理盐水血清。建立游离脂肪酸(FFA)诱导的正常肝细胞株L-02细胞脂肪变性模型,设立正常组,模型组,蓝莓益生菌低、中、高剂量血清组。定量检测细胞内三酰甘油(TG)含量;油红O染色检测细胞内的脂质沉积;RT-PCR和实时荧光定量PCR(qRT-PCR)检测各组IL-22、p-JAK1、p-STAT3、胆固醇调节元件蛋白-1c(SREBP-1c)的基因表达;免疫荧光及Western blot检测各组IL-22、p-JAK1、p-STAT3、SREBP-1c的蛋白表达。结果 FFA诱导刺激24h后,模型组细胞TG含量高于正常组(P<0.01),细胞内可见大量脂质沉积,且IL-22、p-JAK1、p-STAT3基因表达及蛋白表达均较正常组减弱(P均<0.01)、SREBP-1c基因及蛋白表达较正常组升高(P均<0.01);与模型组相比,蓝莓益生菌低、中、高剂量血清组的TG逐渐降低(P均<0.01),细胞内的脂肪沉积逐渐减轻,高剂量血清组IL-22、pJAK1、p-STAT3的基因表达和蛋白表达较模型组、低剂量血清组、中剂量血清组增强(P均<0.01),SREBP-1c的表达降低(P<0.01)。结论蓝莓益生菌通过对p-JAK1/p-STAT3信号通路的调节,参与拮抗肝细胞脂肪变性的作用。
OBJECTIVE: To establish an in vitro hepatic steatosis model to investigate the effect of interleukin-22 (IL-22) -controlled phosphorylation tyrosine kinase-1 (pJAK1) / phosphorylation signal transducer and activator transcription factor-3 The Mechanism of Action of Blueberry Probiotic Serum in Improving Hepatic Steatosis. Methods The blueberry probiotic liquid was prepared, and the low, middle and high dose of 10% blueberry probiotics and the serum of physiological saline were prepared. A model of steatosis induced by free fatty acid (FFA) in normal liver cell line L-02 cells was established. The normal group, model group and low, medium and high dose blueberry probiotic serum groups were established. Quantitative detection of intracellular triglyceride (TG) content; Lipid O staining was used to detect intracellular lipid deposition; RT-PCR and qRT-PCR were used to detect the expression of IL-22, p-JAK1, STAT3 and SREBP-1c were detected by Western blot. The protein expressions of IL-22, p-JAK1, p-STAT3 and SREBP-1c in each group were detected by immunofluorescence and Western blot. Results After 24 h stimulation with FFA, the content of TG in the model group was higher than that in the normal group (P <0.01), and a large amount of lipid deposition was observed in the model group. The gene expression and protein expression of IL-22, p-JAK1 and p- (P <0.01). The gene and protein expression of SREBP-1c were significantly higher than those in the normal group (P <0.01). Compared with the model group, the TG of the low, middle and high doses of probiotics in the blueberry decreased gradually P <0.01). The levels of IL-22, pJAK1 and p-STAT3 in high-dose group were significantly lower than those in model group, low-dose group and middle-dose group <0.01), SREBP-1c expression decreased (P <0.01). Conclusion Blueberry probiotics are involved in the antagonism of hepatic steatosis through the regulation of p-JAK1 / p-STAT3 signaling pathway.