基于AMPK通路研究葛根素对猪前体脂肪细胞成脂分化的影响

【目的】 探究葛根素是否通过AMPK通路促进松辽黑猪前体脂肪细胞分化，以期为在饲粮中添加葛根素改善猪肉肉质提供参考。【方法】 待松辽黑猪前体脂肪细胞汇合度达90%时进行为期4 d的诱导分化。诱导分化时，将细胞分为对照组（Con）、葛根素组（Pue）、葛根素+AMPK激活剂AICAR组（AICAR）及葛根素+AMPK抑制剂CompoundC组（CompoundC），对照组诱导液中不添加葛根素，Pue组添加40 μmol/L葛根素，AICAR组添加40 μmol/L葛根素+500 μmol/L AICAR，CompoundC组添加40 μmol/L葛根素+20 μmol/L CompoundC。诱导分化结束，收集各组细胞，用甘油三酯（TG）酶法检测细胞中甘油三酯浓度；实时荧光定量PCR检测AMPK各亚基以及成脂分化相关基因的表达水平；用Western blotting检测过氧化物酶体增殖物激活受体γ（PPARγ）、AMP依赖的蛋白激酶（AMPK）、p-AMPK （Thr-172）、乙酰辅酶A羧化酶（ACC）的蛋白表达水平；通过Autodock Vina 1.20对葛根素、AMPKα亚基做分子对接预测。【结果】 甘油三酯测定结果显示，与Con组相比，Pue组甘油三酯浓度显著增加（P<0.05）；与Pue组相比，AICAR组甘油三酯浓度显著降低（P<0.05）。实时荧光定量PCR结果显示，与Con组相比，Pue组PPKAG2、PPKAB1、PPKAB2、PPKAA1、PGC-1α表达量均显著降低（P<0.05），Pue组C/EBPα、PPARγ、ACC表达量均显著增加（P<0.05）；与Pue组相比，AICAR组PPKAG1、PPKAG2、PPKAB1、PPKAB2、PPKAA1表达量显著增加（P<0.05），AICAR组C/EBPα、PPARγ、ACC表达量均显著降低（P<0.05），CompoundC组PPKAG1、PPKAG2、PPKAB2表达量均显著降低（P<0.05），CompoundC组C/EBPα、ACC表达量均显著增加（P<0.05）。Western blotting结果显示，与Con组相比，Pue组PPARγ、ACC蛋白表达量显著增加（P<0.05），AMPK、p-AMPK蛋白表达量显著下降（P<0.05），且p-AMPK/AMPK显著下降（P<0.05）；与Pue组相比，AICAR组PPARγ、ACC蛋白表达量显著下降（P<0.05），AMPK、p-AMPK蛋白表达量显著增加（P<0.05），CompoundC组PPARγ、ACC蛋白表达量显著增加（P<0.05），AMPK、p-AMPK蛋白表达量及p-AMPK/AMPK显著下降（P<0.05）。【结论】 40 μmol/L葛根素能够显著增加脂肪细胞中甘油三酯含量，显著上调成脂分化基因PPARγ、C/EBPα、ACC的表达量，显著下调AMPK各亚基的表达量，AMPK激活剂AIRCR可显著抑制葛根素的作用，说明葛根素可通过抑制AMPK通路调节松辽黑猪前体脂肪细胞成脂分化。

Effect of Puerarin on Adipogenic Differentiation of Porcine Preadipocytes Based on AMPK Pathway

【Objective】 This study was aimed to explore whether puerarin could promote the differentiation of Songliao Black pig precursor adipocytes through the AMPK pathway, in order to provide a reference for adding puerarin to diet to improve pork meat quality.【Method】 When the confluence degree of precursor adipocytes of Songliao Black pig reached 90%, the differentiation was induced for 4 days.When inducing differentiation, the cells were divided into control group (Con), puerarin group (Pue), puerarin+AMPK activator AICAR group (AICAR) and puerarin+AMPK inhibitor CompoundC group (CompoundC).Puerarin was not added to the induction solution in the control group, while 40 μmol/L puerarin was added in the Pue group, 40 μmol/L puerarin +500 μmol/L AICAR were added in AICAR group, 40 μmol/L puerarin +20 μmol/L CompoundC were added in CompoundC group.After induction and differentiation, the cells of each group were collected and the triglyceride (TG)concentration was detected by triglyceride enzyme method, the expression levels of AMPK subunits and adipogenic differentiation related genes were detected by Real-time quantitative PCR, peroxisome proliferator activated receptor γ (PPARγ) AMP-dependent protein kinase (AMPK), p-AMPK (Thr-172), acetyl-CoA carboxylase (ACC) were detected by Western blotting.Autodock Vina 1.20 was used to predict the molecular docking of puerarin and AMPKα subunits.【Result】 The results of triglyceride determination showed that compared with Con group, the triglyceride concentration of Pue group was significantly up-regulated (P<0.05);Compared with Pue group, the triglyceride concentration of AICAR group was significantly decreased (P<0.05).The results of Real-time quantitative PCR showed that compared with Con group, the expressions of PPKAG2, PPKAB1, PPKAB2, PPKAA1 and PGC-1α in Pue group were significantly decreased (P<0.05), the expressions of C/EBPα, PPARγ and ACC in Pue group were significantly increased (P<0.05);Compared with Pue group, the expressions of PPKAG1, PPKAG2, PPKAB1, PPKAB2 and PPKAA1 in AICAR group were significantly increased (P<0.05), the expressions of C/EBPα, PPARγ and ACC in AICAR group were significantly decreased (P<0.05), the expressions of PPKAG1, PPKAG2 and PPKAB2 in CompoundC group were significantly decreased (P<0.05), the expressions of C/EBPα and ACC in CompoundC group were significantly increased (P<0.05).Western blotting results showed that compared with Con group, the expressions of PPARγ and ACC protein in Pue group were significantly up-regulated(P<0.05), the expressions of AMPK, p-AMPK protein were significantly down-regulated (P<0.05), and p-AMPK/AMPK was significantly decreased (P<0.05);Compared with Pue group, the expressions of PPARγ and ACC protein in AICAR group were significantly decreased (P<0.05), while AMPK and p-AMPK protein were significantly up-regulated (P<0.05), the expressions of PPARγ and ACC protein in CompoundC group were significantly increased (P<0.05), while the expressions of AMPK, p-AMPK protein and p-AMPK/AMPK were significantly decreased (P<0.05).【Conclusion】 40 μmol/L puerarin could significantly increase the content of triglycerides in adipocytes and significantly up-regulate the expressions of the adipogenic differentiation genes PPARγ, C/EBPα and ACC, significantly down-regulate the expressions of AMPK subunits.AMPK activator AIRCR could significantly inhibit the effect of puerarin, indicating that puerarin could regulate the adipogenic differentiation of Songliao Black pig precursor adipocytes by inhibiting AMPK pathway.