Effect of electroacupuncture on lipophagy in hepatocytes in a hyperlipidemia rat model

To explore the mechanism of electroacupuncture in alleviating hyperlipidemia in a rat model by modulating mammalian target of rapamycin complex 1 (mTORC1)-mediated lipophagy in hepatocytes.

A total of 30 SD rats were randomly divided into blank (n = 6) and modeling groups (n = 24) using the random number table method. A hyperlipidemic rat model was established by feeding rats a high-fat diet (feeding for 8 weeks). After successful modeling, the modeling group was randomly divided into the model, electroacupuncture, mTORC1 inhibitor, and electroacupuncture + mTORC1 agonist groups, with six rats in each group. Except for the blank group, all other rats were fed with high fat diet. Rats in the electroacupuncture and electroacupuncture + mTORC1 agonist groups received electroacupuncture intervention at bilateral "Fenglong" (ST40) acupoints (dilatational wave 2 Hz/100 Hz, current intensity 1 mA) for 30 min once daily. Rats in the mTORC1 inhibitor group received intraperitoneal injections of the mTORC1 inhibitor, rapamycin (2 mg/kg), once daily. Rats in the electroacupuncture + mTORC1 agonist group received intraperitoneal injections of the mTORC1 agonist MHY1485 (10 mg/kg) once daily. The interventions were administered for five consecutive days per week for 4 weeks. Upon completion of the intervention, the following analyses were performed: serum contents of total cholesterol (TC), triglycerides (TAG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), free fatty acids (FFA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using a fully automated biochemical analyzer. Hepatic histopathological changes and lipid deposition were observed using hematoxylin-eosin and oil red O staining. The liver condition was observed and the liver index was calculated. Hepatic TC and TAG levels were measured using an enzyme-linked immunosorbent assay. The ultrastructure of the liver tissue was observed using transmission electron microscopy, and the mean fluorescence intensity of perilipin 2 (PLIN2) and microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ in the liver tissue was detected using immunofluorescence. Protein expression of LC3-Ⅱ/LC3-Ⅰ, phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR, and mTORC1 in liver tissue was detected using Western blotting.

Compared to the blank group, the model group rats showed increased serum TC, TAG, LDL-C, ALT, AST, and FFA levels, along with decreased HDL-C levels (P < 0.05). The liver index and hepatic TC and TAG levels were also elevated (P < 0.05). Histological examination of liver tissue revealed substantial lipid accumulation, numerous lipid droplets within hepatocytes, abnormal mitochondrial morphology, and scarce autophagic vacuole. The mean fluorescence intensity of PLIN2 increased, whereas that of LC3-Ⅱ decreased (P < 0.05). Additionally, the LC3-Ⅱ/LC3-Ⅰ ratio was reduced, whereas the p-mTOR/mTOR ratio and mTORC1 protein expression were increased (P < 0.05). Compared to the model group, rats in the mTORC1 inhibitor and electroacupuncture groups exhibited decreased serum TC, TAG, LDL-C, ALT, AST, and FFA levels (P < 0.05), along with a reduced liver index and hepatic TC and TAG levels (P < 0.05). Histological examination showed markedly attenuated lipid accumulation and visible autophagic vacuole in the hepatocytes. The mean fluorescence intensity of PLIN2 decreased, whereas that of LC3-Ⅱ increased (P < 0.05). Moreover, the LC3-Ⅱ/LC3-Ⅰ ratio increased, whereas the p-mTOR/mTOR ratio and mTORC1 protein expression decreased (P < 0.05). In comparison with both the electroacupuncture and mTORC1 inhibitor groups, the electroacupuncture + mTORC1 agonist group demonstrated increased serum TAG, TC, LDL-C, ALT, AST, and FFA levels (P < 0.05) as well as elevated liver index and hepatic TC and TAG levels (P < 0.05). Liver tissues exhibited aggravated lipid deposition and absence of autophagic vacuole in liver cells. The mean fluorescence intensity of PLIN2 was enhanced, whereas that of LC3-Ⅱ was reduced (P < 0.05). Furthermore, the LC3-Ⅱ/LC3-Ⅰ ratio decreased, and the p-mTOR/mTOR ratio and mTORC1 protein expression increased (P < 0.05).

Electroacupuncture at "Fenglong" (ST40) may improve blood lipid levels in hyperlipidemic rats by inhibiting mTORC1 and promoting hepatocyte lipophagy.