[Effect of electroacupuncture at "Fenglong" (ST 40) on liver cholesterol metabolism in hyperlipidemia rats based on AMPK/mTOR pathway]

Abstract

Objective: To observe the effects of electroacupuncture (EA) at "Fenglong" (ST 40) on the expression of adenosine 5'-monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), and the expression of the downstream molecules related to cholesterol metabolism i.e. sterol regulatory element binding protein-2 (SREBP-2), recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and adenosine triphosphate binding cassette transporter G5/G8(ABCG5/G8) in the rats with hyperlipidemia (HPL) so as to explore the possible mechanism of EA in the intervention of HPL.

Methods: Thirty SPF-grade male SD rats were randomly divided into a blank group, a model group, an AMPK agonist group, an EA group, and an EA+AMPK inhibitor group, 6 rats in each group. The high-fat feeding method was adopted to establish HPL model. After successfully modeled, the rats in the EA group received EA intervention at bilateral "Fenglong" (ST 40), with disperse-dense wave, in the frequency of 2 Hz/100 Hz, the intensity of 1 mA. EA was given once daily, for 30 min in one intervention. In the AMPK agonist group, the intraperitoneal injection with AMPK agonist A-769662 was administered, 30 mg/kg, twice a day. In the EA+AMPK inhibitor group, the intraperitoneal injection of AMPK inhibitor Compound C was administered, 25 mg/kg, once a day, 30 min before EA intervention. In the intervention groups, the interventions were delivered continuously for 5 days a week and lasted 4 weeks. Using automated biochemical analyzer, the blood lipid-related indexes (serum total cholesterol [TC], triglycerides [TG], low-density lipoprotein cholesterol [LDL-C] and high-density lipoprotein cholesterol [HDL-C] as well as alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) were detected in the rats. HE staining and oil red O staining were used to observe the morphology of liver tissue. Liver index was calculated by the weight. Using ELISA, the contents of TC and TG of liver tissue and the contents of of TC and bile acid in feces were detected. The protein phosphorylation levels of AMPK and mTOR in the liver tissue were detected using Western blot; and the positive expression of SREBP-2, HMGCR and ACBG5/G8 was detected using immunohistochemical staining.

Results: After modeling, the levels of serum TC, TG and LDL-C of rats in the model group, the AMPK agonist group, the EA group and the EA+AMPK inhibitor group were all higher than those in the blank group (P<0.01); and there was no statistically difference in the levels of serum HDL-C among groups (P>0.05). After intervention, compared with the blank group, in the model group, the levels of serum TC, TG, LDL-C, ALT and AST, the liver index, the levels of TC and TG in liver tissue, the levels of TC and the bile acid in feces were increased (P<0.01); HE and oil red O staining showed that the hepatocytes were disordered, and there were macrovesicular lipid droplets in the cells and the obvious lipid accumulation; the protein expression of phosphorylated AMPK (p-AMPK) in liver tissue and the ratio of p-AMPK and AMPK were reduced (P<0.01), the protein expression of phosphorylated mTOR (p-mTOR) and the ratio of p-mTOR and mTOR were elevated (P<0.01); and the positive expression of SREBP-2, HMGCR, ABCG5 and ABCG8 in liver tissue was increased (P<0.01, P<0.05). Compared with the model group, in the AMPK agonist group and the EA group, the levels of serum TC, TG, LDL-C, ALT and AST, liver indexes, the levels of TC and TG in liver tissue were reduced (P<0.01), while the levels of TC and bile acid in feces were increased (P<0.05, P<0.01); HE staining and oil red O staining showed that the hepatocytes were in order, and lipid accumulation; the protein expression of p-AMPK and the ratio of p-AMPK and AMPK in liver tissue increased (P<0.01), while the protein expression of p-mTOR and the ratio of p-mTOR and mTOR decreased (P<0.01); the positive expression of SREBP-2 and HMGCR in liver tissue was reduced (P<0.01), while that of ABCG5 and ABCG8 up-regulated (P<0.05, P<0.01) . Compared with the EA group, in the EA+AMPK inhibitor group, the levels of serum TC, TG, LDL-C, ALT and AST, liver index, the levels of TC and TG in liver tissue were increased (P<0.05, P<0.01), while the levels of TC and bile acid in feces were reduced (P<0.01); lipid accumulation was aggravated; the protein expression of p-AMPK and the ratio of p-AMPK and AMPK in liver tissue were reduced (P<0.01, P<0.05), while the protein expression of p-mTOR and the ratio of p-mTOR and mTOR elevated (P<0.05, P<0.01); the positive expression of SREBP-2 and HMGCR in liver tissue was increased (P<0.01), while that of ABCG5 and ABCG8 was down-regulated (P<0.01).

Conclusion: EA at "Fenglong" (ST 40) can attenuate hyperlipidemia in HPL rats. It may be achieved by regulating the AMPK/mTOR pathway, inhibiting the expression of cholesterol synthesis related molecules, SREBP-2 and HMGCR, and up-regulating the expression of cholesterol excretion molecules, ABCG5 and ABCG8, thereby reducing liver cholesterol accumulation and increasing cholesterol excretion.

Keywords: Point ST 40 (Fenglong); adenosine 5'-monophosphate-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR); cholesterol metabolism; electroacupuncture; hyperlipidemia.