Atherosclerosis is a chronic inflammatory disease related to many risk factors for cardiovascular and cerebrovascular diseases, often involving multiple organs. Its pathological features are the formation of lipid plaques on the inner wall of blood vessels and the proliferation of smooth muscle cells, leading to narrowing and hardening of lumen, which in turn affects the blood supply to the tissues. High-fat-fed ApoE-/- mice have been widely used in the study of hyperlipidemia, atherosclerosis and its complications [13]. In this study, high-fat fed ApoE-/- mice was used to induce the establishment of atherosclerosis model, and different drugs were continuously intervened for 12 weeks. The results showed that compared with the normal diet control group, the serum TG, TC and LDL-C levels of high-fat fed ApoE-/- mice were significantly increased, HDL-C was decreased, and atherosclerotic plaque formation in the intima of the thoracic aorta and massive proliferation of the smooth muscle layer of the media. The occurrence of atherosclerosis in high-fat ApoE-/- mice is not only related to dyslipidemia, but also the proliferation of smooth muscle layer cells in the vascular media plays an important role in its formation and development [14, 15]. Abnormally elevated blood lipids, vascular intimal lipid deposition and vascular media smooth muscle cell proliferation are the common pathological basis for the occurrence and development of atherogenesis [16]. In this study, after high-fat fed ApoE-/- mice were treated with Ruanmailing oral liquid, the results showed that Ruanmailing oral liquid can reduce serum lipids in a dose-dependent manner, inhibit the formation of lipid plaques in the intima of the thoracic aorta and the proliferation of smooth muscle cells in the media to reduce the area of atherosclerotic plaques in high-fat fed ApoE-/- mice. It shows that Ruanmailing oral liquid can inhibit the formation of atherosclerotic plaque and the proliferation of smooth muscle cells by regulating serum lipid levels, thereby delaying the process of atherosclerosis.
The pathogenesis of atherosclerosis is very complicated, in which the proliferation of arterial smooth muscle cells runs through the formation and development of atherosclerosis. Inhibiting cell proliferation and reducing migration and differentiation are important ideas and directions for preventing and treating atherosclerosis [13]. TGF-β1 has the highest proportion (>90%), the strongest activity, the most functions, and the widest distribution in somatic cell lines. It has become a hot spot in clinical and experimental research [17]. As a multifunctional cytokine, TGF-β1 has a two-way regulatory effect. The change of TGF-β1 can promote the remodeling of blood vessel wall, the growth of damaged arteries and the transcriptional differentiation of vascular cells. On the other hand, TGF-β1 can also be used as an anti-inflammatory and anti-atherogenic factor to prevent the occurrence of atherosclerotic complications [18]. SMAD4 protein is an important signal molecule that mediates the transfer of TGF-β1 signal from the cell membrane to the nucleus. TGF-β1/SMAD4 is an important regulatory pathway for the differentiation and proliferation of arterial smooth muscle cells and the process of atherosclerosis [9]. In this study, we observed that compared with the control group, the levels of TGF-β1 and SMAD4 protein in high-fat fed ApoE-/- mice were significantly decreased, and large raised plaques were also observed in the lumen of thoracic aorta, and proliferative cells and thick fibrous cap were visible in the outer layer of the cavity. It shows that the arterial proliferation response is one of the important mechanisms of atherosclerotic plaque formation in high-fat fed ApoE-/- mice, and it is related to the inhibition of the TGF-β1/SMAD4 signaling pathway. Compared with the high-fat model group, the serum TGF-β1 levels and the SMAD4 mRNA and protein levels of the thoracic aorta in the low- and high-dose Ruanmailing group were significantly increased, and there was no significant difference from the Lipitor group. It indicates that the dose-dependent Ruanmailing oral liquid may increase the expression of TGF-β1 and downstream SMAD4 protein, enhance the transduction of the TGF-β1/SMAD4 signaling pathway, and inhibit the proliferation of arterial smooth muscle cells and the progression of atherosclerosis.
On the basis that TGF-β1 mediates the intracellular signal transduction of SMAD4 protein, whether it can regulate the proliferation of smooth muscle cells by intervening in the signal transduction process of the TGF-β1/SMAD4 signaling pathway in the cytoplasm or nucleus may be a new approach for anti-atherosclerosis research. GATA2 is an important zinc-finger transcription factor. In addition to being an important regulatory factor in the differentiation of pluripotent hematopoietic stem cells into various mature lineages, it also participates in the regulation process of various organs such as early neurodevelopment and bone metabolism [19, 20]. GATA2 can negatively regulate SMAD4, thereby promoting smooth muscle cell differentiation and proliferation in the process of atherogenesis. Studies have found that GATA2 can inhibit the transcriptional activity of endogenous SMAD4, and has a quantity-dependent effect. As the transfection dose of GATA2 increases, the inhibitory effect on SMAD4 activity increases. This study shows that compared with the high-fat model group, Ruanmailing oral liquid can significantly decreased GATA2 levels and increased TGF-β1 and SMAD4 levels in high-fat ApoE-/- mice, suggesting that GATA2 acts as a negative regulator of the TGF-β1/SMAD4 pathway may play an important role in reducing the differentiation and proliferation of smooth muscle cells and slowing down the process of atherosclerosis.
Compared with the use of statin drugs such as Lipitor to prevent and treat atherosclerosis, traditional Chinese medicine has the characteristics of multiple targets. It has synergistic effects such as lipid-lowering, inhibiting the proliferation and migration of vascular smooth muscle cells, and anti-oxidation, and has fewer side effects, providing a new coping strategy for the prevention and treatment of atherosclerosis. The main ingredients of Ruanmailing oral liquid are Chinese herbal medicine Shudi, Wuweizi, Gouqi, Fuling, Baiziren, Yuanzhi, Renshen, Danggui, Huangqi and so on. Taking a comprehensive view of the whole prescription, it has the effects of invigorating the kidney, promoting blood circulation and reducing phlegm. Elderly patients with atherosclerosis have pathological characteristics of deficiency of kidney essence, excessive phlegm and excessive blood stasis, and the pharmacological effects of Ruanmailing oral liquid are in line with this pathophysiological characteristic. This study confirmed from an experimental point of view that Ruanmailing oral liquid has significant lipid-lowering and anti-atherosclerotic effects.