Abstract: BACKGROUND: Atherosclerosis serves as the core pathological basis for cardiovascular diseases, closely linked to lipid metabolism disorders and endothelial dysfunction. Previous studies have indicated that moxibustion effectively protects mitochondrial morphology and function, inhibiting cell apoptosis. 
OBJECTIVE: To explore the potential mechanism of moxibustion in regulating fatty acid oxidation via mitochondrial autophagy for anti-atherosclerosis effects.
METHODS: Thirty male ApoE−/− mice were randomly divided into a model group, a moxibustion group, and a moxibustion+mitochondrial division inhibitor 1 (Mdivi-1) group, with 10 mice in each group. They were fed a high-fat diet for 12 weeks to replicate the atherosclerosis model. Ten male C57BL/6J mice served as the control group, receiving a regular diet. All groups received interventions on the first day of modeling. The control and model groups underwent only grasping and fixation, while the moxibustion group received moxibustion at “Danzhong,” “Shenque,” and bilateral “Neiguan” and “Xuehai” acupoints for 30 minutes, 5 times a week continuously. The moxibustion+Mdivi-1 group was administered Mdivi-1 (1.2 mg/kg/d) via intraperitoneal injection 30 minutes before moxibustion, also 5 times a week continuously. Both groups were treated for 12 weeks and samples were then collected. Enzyme determination was used to measure serum triglyceride and total cholesterol levels, while colorimetry determined low-density lipoprotein cholesterol and high-density lipoprotein cholesterol levels. Hematoxylin-eosin staining was used to observe the pathological morphology of the mouse thoracic aorta and liver. Oil red O staining was performed to assess liver lipid changes. Enzyme-linked immunosorbent assay was used to detect carnitine palmitoyl transferase-1α (CPT1α) and medium-chain acyl-CoA dehydrogenase (MCAD) levels in the liver and endothelin-1 levels in the serum. The nitric acid reductase method was conducted to measure serum nitric oxide levels. Western blot assay was adopted to analyze BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3), becline-1, thylakoid outer membrane translocase 20 (TOMM20) protein expression in liver tissue, as well as endothelial nitric oxide synthase and phosphorylated endothelial nitric oxide synthase in the thoracic aorta.
RESUITS AND CONCLUSION: (1) Compared with the control group, mice in the model group showed significantly increased levels of serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol (P < 0.01), while high-density lipoprotein cholesterol levels were significantly reduced (P < 0.05). The inner wall of the thoracic aorta was irregular, with intimal thickening and matrix dissolution. Hepatic tissue cells were swollen and rounded, showing spotty necrosis of hepatocytes, neutrophil infiltration, numerous fat vacuoles, and steatosis. Oil red O staining of the mouse liver also revealed a large accumulation of lipid droplets. The contents of CPT1α and MCAD and the expression levels of BNIP3 and Becline-1 proteins in liver tissue were significantly reduced (P < 0.05, P < 0.01), while the expression of TOMM20 protein was significantly increased (P < 0.05). The nitric oxide content in mouse serum was significantly reduced (P < 0.01), while the endothelin-1 concentration was significantly increased (P < 0.01). The expression of endothelial nitric oxide synthase and phosphorylated endothelial nitric oxide synthase proteins in the thoracic aorta tissue decreased (P < 0.05, P < 0.01). (2) Compared with the model group, the serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels in the moxibustion group were significantly reduced (P < 0.01). The thoracic aortic lumen was more regular, and the media was more neatly arranged. The swelling of liver tissue cells was significantly reduced, the texture was clear, and lipid droplet deposition was reduced. Oil red O staining showed a significant reduction in lipid droplet accumulation. The contents of CPT1α and MCAD and the expression levels of BNIP3 and Becline-1 proteins in liver tissue were significantly increased (P < 0.05, P < 0.01), while the expression of TOMM20 protein was significantly decreased (P < 0.05). The nitric oxide content in mouse serum was significantly increased (P < 0.01), while the endothelin-1 content was significantly decreased (P < 0.01). The expression of endothelial nitric oxide synthase and phosphorylated endothelial nitric oxide synthase proteins in the thoracic aorta tissue increased (P < 0.01). (3) Compared with the moxibustion group, the serum total cholesterol and low-density lipoprotein cholesterol levels in the moxibustion + Mdivi-1 group were significantly increased (P < 0.05, P < 0.01). The inner wall of the thoracic aorta was slightly irregular, with intimal thickening and a small number of foam cells. The swelling of liver tissue cells was aggravated. Oil red O staining of liver tissue showed increased lipid droplet accumulation. The contents of CPT1α and MCAD and the expression levels of BNIP3 and Becline-1 proteins in liver tissue were significantly reduced (P < 0.05, P < 0.01). The nitric oxide content in mouse serum decreased (P < 0.05), while the endothelin-1 content significantly increased (P < 0.01). The expression of endothelial nitric oxide synthase and phosphorylated endothelial nitric oxide synthase proteins in the thoracic aorta tissue decreased (P < 0.01). To conclude, moxibustion can improve lipid levels, fatty acid oxidation, lipid accumulation, restore endothelial damage, and reduce symptoms of atherosclerosis in ApoE−/− mice. The mechanism may be related to regulating mitochondrial autophagy, which affects fatty acid oxidation, reduces lipid accumulation in liver tissue, and improves endothelial dysfunction.

Key words: moxibustion, atherosclerosis, mitochondrial autophagy, fatty acid oxidation, lipid accumulation, endothelial function