Panax notoginseng saponins inhibit the formation of atherosclerotic foam cells in ApoE knockout mice by regulating TLR4/SYK signals
Cardiovascular disease is the leading cause of death worldwide. The root cause of most cardiovascular diseases is atherosclerosis. Vascular wall is accumulated by lipids, especially oxidized low density lipoprotein (ox LDL), which leads to atherosclerosis. Although many cells are involved in the development and progression of atherosclerosis, macrophages are the main source of foam cells and the key factor of atherosclerosis. Under the repeated stimulation of various pathological factors, monocytes gather at the lesion site, move to the lower endothelium, and differentiate into macrophages. They engulf the surrounding lipid components and other related substances to form foam cells, leading to the formation of vascular atherosclerotic plaques. Related studies have shown that the ability of macrophages to uptake lipoproteins is mainly regulated by TLR4/SYK signaling. Panax notoginseng saponins are effective active ingredients extracted from the roots of Panax notoginseng plants in the Araliaceae family. Their main functions are to promote blood circulation, remove blood stasis, unblock blood vessels and collaterals, and have anti-inflammatory, endothelial cell protective, and anti thrombotic effects. Previous studies have shown that Panax notoginseng saponins can effectively inhibit the thickening of carotid artery intima and the enlargement of atherosclerotic plaque, and its mechanism may be related to CD40-CD40L signal pathway, which is widely used in clinical cardiovascular and cerebrovascular diseases. However, the mechanism of its effect on atherosclerotic foam cells has not yet been studied. We observed the size of atherosclerotic plaque, the level of serum lipid expression, and the expression of TLR4, SYK, ERK1/2 mRNA and protein in each group from the in vivo experiment; In addition, in vitro experiments were carried out to evaluate the phagocytosis of macrophages in each group, and then to explore the influence and molecular mechanism of Panax notoginseng saponins (PNS) on the formation of atherosclerotic foam cells and the process of atherosclerosis in ApoE -/- mice, so as to provide an experimental basis for the prevention and treatment of atherosclerotic diseases.

AS is the main pathological basis of cardiovascular and cerebrovascular diseases, such as ischemic heart disease. Among them, the large amount of low-density lipoprotein (especially oxidized low-density lipoprotein) and the accumulation of cholesterol in macrophages are the main reasons for the formation of foam cells. At present, there is no effective treatment for AS, so seeking an effective therapeutic drug is an urgent problem to be solved in clinical practice.

PNS is a beneficial substance extracted from the roots of the plant Panax notoginseng. Its main functions are anti-inflammatory, inhibition of platelet aggregation, and protection of vascular endothelial cells. Relevant studies have shown that PNS has many effects such as neuroprotection, anti atherosclerosis and anti stroke after cerebral ischemia reperfusion. In this study, ApoE -/- mice with early AS plaque formation were treated with PNS by gavage. It was found that the carotid plaque area and foam cells in the plaque of PNS group mice were significantly reduced, which was consistent with the above related research results. Further research found that after PNS gavage treatment, the expression of TG, TC, and LDL in the serum of mice was significantly reduced, while the expression of HDL was significantly increased. This is consistent with previous studies showing that PNS has a significant ability to lower blood lipids. In view of previous studies above PNS and this experiment, PNS can inhibit the formation of vascular atherosclerotic plaque, but the mechanism of action has not been fully explored. This study explored how PNS inhibited the formation of atherosclerotic plaque in ApoE -/- mice by affecting the formation of foam cells, in order to obtain the effect of PNS on atherosclerosis and its related mechanisms.

Macrophages absorb a large amount of lipid substances and accumulate in the cells to form foam cells, which is the main factor in the formation of AS. Oxidized low density lipoproteins (oxLDL) are an important inducement of macrophage foam. Relevant studies have shown that LDL can also undergo weak oxidative modification, and its modified product is the least oxidized LDL (mm LDL), which also accelerates macrophages to phagocytosis lipid substances to form foam cells. TLR (especially TLR4) plays an important role in the formation of foam cells in atherosclerosis. Toll like receptors (TLRs) are part of the interleukin-1 receptor/toll like receptor superfamily, while TLR4 is a member of the TLR subfamily, primarily expressed on macrophages, neutrophils, and dendritic cells. In the early stage of atherosclerosis, LDL-C in the body can be oxidized or weakly oxidized to ox LDL and mm LDL. TLR4 on the surface of macrophages can recognize ox LDL, mm LDL and their active products, causing TLR4 to combine with SYK and phosphorylate, thus causing the activation of the downstream signal pathways Ras, Raf, and extracellular regulated protein kinases (ERK1/2), thereby enhancing the phagocytosis of macrophages on LDL, ox LDL and other lipid substances and increasing the aggregation in cells, producing foam cells, leading to the formation of atherosclerosis and the rupture of unstable plaque. To verify whether PNS can inhibit the formation of atherosclerosis through the above mechanisms. Therefore, this study detected the levels of TLR4, SYK, and ERK1/2 mRNA in mice by treating them in different ways. The results showed that the expression of TLR4, SYK, ERK1/2 mRNA, and p-ERK1/2 protein in PNS group mice was significantly decreased compared to the model group; At the same time, we treated RAW246.7 macrophages in different ways to observe the phagocytic function of RAW246.7 macrophages. The results showed that the phagocytic capacity of macrophages not treated with PNS was significantly stronger than that of macrophages treated with PNS, indicating that PNS could inhibit the ability of macrophages to phagocytose lipid substances, and ultimately prevent macrophages from transforming into foam cells. Previous studies have shown that the expression of TLR4 in atherosclerotic plaques is significantly increased, and the expression of CD36 in macrophages is correspondingly enhanced, which promotes macrophages to ingest ox LDL, and accelerates the rupture of foam cells and the instability of plaques. Based on the previous studies mentioned above, we further evaluated the effect of PNS on macrophages by detecting the CD36 content in RAW246.7 macrophages. Through research, it was found that the CD36 content in macrophages treated with PNS decreased significantly, indicating that PNS can inhibit the progression of atherosclerosis by suppressing the expression of CD36 in macrophages. In summary, PNS can inhibit the ability of macrophages to engulf lipids and the production of CD36 in macrophages, and its mechanism may be related to the inhibition of the TLR4/SYK signaling pathway.

Through the experiment, we found that after intragastric administration of PNS solution, the blood lipid of ApoE -/- mice decreased significantly, the scope of atherosclerotic lesions in the carotid artery became smaller, the lipid phagocytosis ability of macrophages and foam cells decreased significantly, and the expression of TLR4, SYK, ERK in the plaque was inhibited. Therefore, we confirmed that PNS can reduce blood lipid and inhibit the formation of atherosclerotic foam cells, and its mechanism may be related to the inhibition of TLR4/SYK signaling pathway in macrophages.