To observe the effect of moxibustion on the lipid metabolism, aortic arch and mitochondrial structure, PTEN-induced kinase 1 (PINK1)/Parkin signaling pathway, and the expressions of apoptosis-related proteins in atherosclerotic (AS) mice, so as to explore its potential mechanisms underlying prevention and treatment of AS.

Ten C57BL/6J mice were fed with normal chow and used as the control group. Thirty ApoE^-/- mice were fed with high-fat chow to establish the AS model, and randomly divided into model, moxibustion, and moxibustion+Mdivi-1 (mitochondrial fission inhibitor) groups, with 10 mice in each group. For mice in the moxibustion group, moxibustion was applied to “Danzhong” (CV17), “Shenque” (CV8), and bilateral “Neiguan” (PC6) and “Xuehai” (SP10) for 30 min. The mice in the moxibustion + Mdivi-1 group received intraperitoneal injection of Mdivi-1(1.2 mg·kg^-1·d^-1) 30 min before each session of moxibustion, and those of the control, model and moxibustion groups received equal volume of lysosomal injection. The intervention was conducted once daily, 5 d a week for 12 consecutive weeks. The contents of serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and triglyceride (TG) were measured by using an automatic biochemical analyzer. Histopathologial changes of the aorta arch were observed by H.E. staining, and the mitochondrial structure of the aorta was observed using a transmission electron microscopy. The expression levels of PINK1, Parkin, Bax, Bcl-2, and Caspase3 protein in the aortic tissue were detected using Western blot, and the immunoactivity of mitochondrial cytochrome C (Cyt C) in the aortic tissue was determined using immunofluorescence staining.

Compared with the control group, the contents of serum TC, TG and LDL-C, expression levels of PINK1, Parkin, Bax and Caspase3 protein, and the immunoactivity of Parkin and Cyt C were significantly increased (P<0.01, P<0.05), while the content of serum HDL-C level and the expression of Bcl-2 protein notably decreased (P<0.01) in the model group. In comparison with the model group, the moxibustion group showed a significant decrease in the contents of serum TC, TG and LDL-C, expression levels of Bax and Caspase3, and immunoactivity of Cyt C (P<0.01, P<0.05), and a striking increase in the expressions of PINK1, Parkin, and Bcl-2 protein and immunoactivity of Parkin (P<0.01). After intraperitoneal injection of Mdivi-1, the effects of moxibustion disappeared in lowering the levels of TC, TG and LDL-C, the expressions of Bax, Caspase3 and the immunoactivity of Cyt C and in up-regulating the expressions of PINK1, Parkin, Bcl-2 and the immunoactivity of Parkin. Morphological observation showed uneven intima of the aortic arch in the model group, with plaque proliferation, hyperplasia of fibrous tissue or smooth muscle tissue, swollen mitochondria with matrix dissolution, and reduction in the number of cristae accompanied by vacuoles, etc; while in the moxibustion group, morphological observation showed relatively regular lumen of the aortic arch, with less endometrial degeneration and fewer swelling endothelial cells, and a smaller amount of plaque formation and some foam cells, slightly swollen mitochondria with partially dissolved matrix and secondary lysosomes; and the morphological observation showed shedding of local endothelial cells of the aortic arch, with thickened intima, proliferated smooth muscle tissue, and foam cells within the plaque; slightly swollen mitochondria, reduction in the number of cristae, and partially dissolved matrix in the moxibustion + Mdivi-1 group.

Moxibustion can improve the lipid metabolism level, relieve pathological injury of the thoracic aorta, restore mitochondrial structure and function in ApoE^-/- AS mice, which may be related to its functions in reducing Cyt C metastasis, and inhibiting apoptosis by regulating PINK1/Parkin signaling pathway.

To observe the impact of “Wenyang Tongmai” (warming yang and unblocking vessels) moxibustion on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway and endothelial function-related factors in ApoE^-/- mice with atherosclerosis (AS), so as to explore its potential mechanism underlying prevention and treatment of AS.

Thirty male ApoE^-/- mice were randomly assigned to three groups: model, moxibustion, and moxibustion plus PI3K agonist (moxibustion + agonist) groups, with 10 mice in each group. The AS model was established by feeding the mice with high-fat diet for 12 weeks. An additional 10 male C57 BL/6J mice were used as the control group, and fed with a standard diet for 12 weeks. The interventions began on the first day of modeling. Mice of the control and model groups were only restrained for fixation. Moxibustion was applied to “Danzhong” (CV17), “Shenque” (CV8), bilateral “Neiguan” (PC6) and “Xuehai” (SP10) acupoints for 30 min per session. Mice of the moxibustion + agonist group received intraperitoneal injection of PI3K agonist 740Y-P (5 μmol·kg^-1·d^-1) 30 min before each moxibustion. The intervention was conducted 5 days per week for 12 consecutive weeks. General conditions and body weight of the mice were recorded. Biochemical analysis was performed to measure serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) contents. Histopathological changes in the thoracic aorta were observed via H. E. staining. The contents of serum nitric oxide (NO) and endothelin-1 (ET-1) were detected using ELISA. The reactive oxygen species (ROS) content in the aorta was detected by flow cytometry. The Western blot and qPCR were employed to analyze the expression of PI3K, Akt, mTOR, and eNOS proteins and mRNAs in the aortic tissue, respectively.

Compared to the control group, the model group showed a significant increase in the body weight at week 8 and 12 after modeling (P<0.05), serum TC, TG, and LDL-C and ET-1 contents, and aorta ROS content, expression level of mTOR mRNA, and p-PI3K/PI3K and p-mTOR/mTOR ratios in the aorta tissue (P<0.01), and a significant decrease in the serum HDL-C and NO contents, expression levels of eNOS mRNA and protein, and p-Akt/Akt ratio in the aorta (P<0.01, P<0.05). Compared with the model group, the moxibustion intervention obviously lowered the body weight at week 8 and 12, contents of serum TC, TG, LDL-C, ET-1 and aorta ROS, expression level of mTOR mRNA, and p-PI3K/PI3K and p-mTOR/mTOR ratios (P<0.01, P<0.05), and evidently elevated serum NO content, expressions of eNOS mRNA and protein, and p-Akt/Akt ratio (P<0.01). Compared to the moxibustion group, the moxibustion+agonist group exhibited an evident elevation in the serum TC, TG, and LDL-C contents (P<0.01, P<0.05), ET-1, ROS contents and the ratios of p-PI3K/PI3K and p-mTOR/mTOR (P<0.01, P<0.05), and a striking downregulation in the content of NO, expressions of eNOS mRNA and protein, and p-Akt/Akt ratio (P<0.05, P<0.01). Histopathological examination revealed uneven aortic inner walls, intimal thickening, and plaque proliferation in the model group, while minor intimal cell detachment in the aortic arch and no obvious lesions in the moxibustion group, and a small amount of “plaque” and hyperplasia in the aortic arch, and an occasional endothelial cell sloughing of the intima in the moxibustion+angonist group.

“Wenyang Tongmai” moxibustion can reduce body weight, alleviate dyslipidemia and mitigate AS symptoms in ApoE^-/- mice, which may be associated with its functions in inhibiting the PI3K/Akt/mTOR signaling pathway, reducing oxidative stress and protecting the endothelial function.