In our research, we demonstrated that RES effectively ameliorated AS induced by HFD companied with LPS in ApoE−/− mice. In addition, our results indicated that RES treatment significantly inhibited the accumulation of lipids on vessel wall, inhibited the proliferation and activation of CD4+ T cells, regulated the secretion of cytokines and decreased the expression of Dnmt. Our research clarified the mechanism of RES in preventing AS through regulating activation of CD4+ T cells and Dnmt expression.
AS is a chronic inflammatory disease. High level of serum lipids and the inflammatory status are risk factors of AS[2, 28]. Feeding ApoE−/− mice with HFD can induce AS and LPS injection can accelerate the progression of disease. In our study, HFD companied with LPS induced dysfunction of serum lipids and formation of atherosclerotic plaque. Administration of RES counteracts thickened coronary artery, decreased the accumulation of lipids on the aortic wall, and regulated the level of serum lipids, indicated the positive function of RES on HFD and LPS induced AS in ApoE−/− mice.
Dyslipidemia is a recognized risk factor of AS. In our study, RES decreased the high level of TC and TG in 10th week of the experiment and inhibited the dysfunction of TC, TG, LDL-C as well as HDL-C in 20th week. RES has been widely reported to regulate serum lipids[30, 31], promote the degradation of ox-LDL, suppress adipocyte differentiation and TC accumulation, and stimulate lipolysis. RES powerfully regulated the serum lipids and our result further verified this efficiency and hinted that RES play this function in a long time.
Rather than regulated serum lipids, RES attenuated Trimethylamine-N-Oxide (TMAO)-induced AS in ApoE−/− mice, ameliorated HFD induced AS in LDLR−/− mice, countered systemic lupus erythematosus-associated AS and alleviated AS in atherosclerotic mice whose left carotid artery was partial ligated. Our result is consistent with previous studies that RES possess beneficial effects on AS and the influences of RES on AS induced by HFD and LPS was firstly observed in our research. LPS is an inflammatory risk factor. The infection of bacteria like Mycobacterium tuberculosis can increases the level of LPS. Elevated LPS is easily to induce a low-grade inflammation state, which is another risk factor for AS in addition to a high level of serum lipids.
RES ameliorates the symptoms of AS through multiple mechanisms. RES regulates the production of vasodilator and vasoconstrictor, inhibits the generation of oxidative stress/reactive oxygen species and ameliorates inflammation. But the precise mechanism of how RES alleviate AS still complex and incompletely clarified. Our study verified a mechanism of RES in ameliorating HFD and LPS induced AS by regulating Dnmt expression, inhibiting the activation of CD4+ T cells.
CD4+ T cells are the most abundant T cells population in atherosclerotic plaque and play important roles throughout all stages of AS. CD62L is critical for migration of naïve CD4+ T cells into lymphoid tissue and highly expressed on naïve CD4+ T cells and downregulated upon the activation. Besides, CD4+ T cells increase the expression of CD25 and CD44 after activation[41, 42, 43, 44].
Activated CD4+ T cells response to the antigen signal, secret inflammatory cytokines and began to proliferation. Increased and activated CD4+ T cells accelerate the progression of inflammation and induce the progression of AS. RES inhibited the proliferation and activation of CD4+ T cells both in atherosclerotic mice and in cultured splenic CD4+ T cells in vitro, which remained us that RES ameliorated AS by inhibiting the proliferation and activation of CD4+ T cells.
CD4+ T cells are constituents of the adaptive immune response. Activated CD4+ T cells secrete proinflammatory cytokines and inflammation mediated by activated CD4+ T cells plays an important role in the initiation and progression of AS. RES decreased the secretion of IL-6, increased IL-10 and TGF-β1. IL-6 contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. Continual secretion of IL-6 plays a pathological effect on chronic inflammation. It has been reported that low content of IL-10-producing CD4+ T cells is a risk factor for progression of coronary AS and evoking TGF-β1-mediated anti-inflammatory response inhibited AS progression. Besides, RES increased the secretion of IL-2 in CD4+ T cells. IL-2 is a proinflammatory cytokine and the level of IL-2 may be close correlated with the development and progression of CVD[51, 52]. But IL-2 can induce the development of Tregs, which protect against AS. The effects of increased IL-2 in AS still under discussion. RES promoted lymphocyte proliferation and IL-2 production and we confirmed the same function of RES on CD4+ T cells. RES inhibited the activation and proliferation of CD4+ T cells, regulated the secretion of cytokines. We concluded that RES restrained the immune response of CD4+ T cells.
According to our results, the effects of different concentrations of RES on CD4+ T cells are inconsistent. Marco Craveiro et al. reported that 20 and 100 µM of RES possess different influences on human CD4+ T cells stimulated by TCR. Both 20 and 100 µM of RES decreased the expression of CD25 and CD71 but did not affect CD69 expression. In addition, 20 µM of RES inhibited the proliferation of human CD4+ T cells while 100 µM of RES had no effects. These results indicated that the different concentration of RES can cause different or even opposite effects on activation and proliferation of CD4+ T cells, which remained us that the dose of RES in application should pay enough attention.
Dnmt1 maintain the methylation and Dnmt3b mediate the de novo methylation. Aberrant or abnormal expression of Dnmt is associated with a variety of human diseases including AS. Therefore, Dnmt are promising therapeutic epigenetic targets as specific inhibitors might regulate the expression of Dnmt and stop or even reverse aberrant cellular processes[56, 57]. RES has been described as a regulator of Dnmt expression and majority of the research concluded that RES function as inhibitors of Dnmt in many kind of cells. In our research, RES inhibited the increased Dnmt1 and Dnmt3b induced by anti-CD3/CD28 companied with LPS in CD4+ T cells, which is consistent with the research that RES inhibited Dnmt expression.
Inhibited expression of Dnmt induced the regulation of cellular activities and we investigated in CD4+ T cells that decreased expression of Dnmt1 and Dnmt3b induced the inhibition of proliferation and activation, suggested that RES induced inhibition of proliferation and activation of CD4+ T cells is partly through the inhibition of Dnmt1 and Dnmt3b.
There are some new sights in this research that our study suggested RES ameliorated AS induced by HFD together with LPS in ApoE−/− mice. In addition, RES function like an inhibitor of Dnmt1 and Dnmt3b in CD4+ T cells. Finally, RES inhibited the proliferation and activation as well as regulated the secretion of cytokines in CD4+ T cells. This research clarified much about the mechanism of RES in AS treatment.
Limitations also existed in this research. Although RES can inhibit the expression of Dnmt1 and Dnmt3b in CD4+ T cells, whether there existed dysfunctional methylation of DNA calls for further examined. Other probable mechanisms may exist besides the Dnmt1 and Dnmt3b inhibition that underlies the RES-mediated inhibition of the proliferation and activation of CD4+ T cells.