Effect of “Wenyang Tongmai” moxibustion on endothelial function in ApoE^-/- atherosclerotic mice via PI3K/Akt/mTOR 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.