RES regulated serum lipids and ameliorated AS in ApoE-/- mice
The level of serum lipids including TC, TG, LDL-C, and HDL-C in the 0th, 10th and 20th weeks of the experiment were detected while the content of non-HDL-C was calculated (Fig.1A-C). The level of TC, TG, LDL-C, and non-HDL-C significantly increased after ten weeks of HFD. At the 20th week, compared to mice of the control group, the concentration of TC, TG, LDL-C as well as non-HDL-C all increased while HDL-C level decreased. Compared to mice of HFD+LPS group, 5mg/kg (BW)/day of RES decreased content of TC, TG and non-HDL-C at 10th week, and decreased content of TC, TG, LDL-C and non-HDL-C, meanwhile increased content of HDL-C at 20th week, suggesting that the ability of RES on regulating lipids in atherosclerotic mice induced by HFD and LPS will increase with prolonged use.
Both HFD and LPS are harmful to the mammal blood vessels. After twenty weeks of HFD together with five times of LPS injection, thickened coronary wall was observed (Fig.1D), and large area of atherosclerotic plaque was detected on aortas (Fig.1E-F). Simvastatin or RES treatment ameliorated the pathological change of coronary wall slightly. In addition, simvastatin or RES treatment decreased the infiltrated lesion of the aorta and the plaque area ratio significantly. These results indicated that RES treatment can inhibit the progression of AS.
Serum of mice was collected at 0th, 10th and 20th respectively and diluted by a quarter in saline and the lipids were detected according to the operating instruction of the kits. (A) The level of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), and non-HDL-C of serum in the 0th week (n=8). (B) The level of TC, TG, LDL-C, HDL-C, and non-HDL-C in the 10th week (n=8). (C) The level of TC, TG, LDL-C, HDL-C, and non-HDL-C in the 20th week (n=8). (D) Mice were killed at the end of the 20th week and the hearts were preserved. The pathologic condition of the coronary artery was analyzed by H&E. (E) The aortas were separated from the aortic arch to the left and right common iliac artery and were stained by Oil Red O. (F) The areas of aortic plaque were measured by MapInfo 7.0 (n=5).
Data are represented with mean ± SD, #P<0.05, ##P<0.01, ###P<0.001 versus the control group; *P<0.05, **P<0.01 versus the HFD+LPS group.
RES inhibited the activation of CD4+ T cells in atherosclerotic mice
In order to observe the effects of RES on CD4+ T cells in atherosclerotic mice, the frequency of CD4+ T cells in peripheral blood mononuclear cells (PBMC) was detected. It was observed that the frequency of CD4+ T cells was increased with combinational stimulation of HFD and LPS while RES decreased the ratio of CD4+ T cells in PBMC (Fig.2A-B), which suggested RES improved the inflammatory status in AS. CD25, CD44 and CD62L were detected to analyze the activation of CD4+ T cells (Fig.2C-D). It was found that HFD and LPS injection increased the expression of CD25 and CD44, and decreased CD62L expression in CD4+ T cells in peripheral blood. Intragastric administration of RES decreased CD25 and CD44 expression, but CD62L expression was not changed. The expression change of activation markers suggested that RES can reduce the expression of CD25 and CD44, but does not affect CD62L expression in AS mice with combinational stimulation of HFD and LPS.
Peripheral blood of mice was collected at the end of the 20th week and lymphocyte isolate medium was used to obtain peripheral blood mononuclear cells (PBMC). (A-B) CD4 FITC antibody was used and the frequency of CD4+ T cells in PBMC was detected by flow cytometry (n=4). (C-D) CD4 FITC, CD25 APC, CD44 PerCP and CD62L PE antibody were used to stain the cells and the frequency of CD25+, CD44+ and CD62L+ cells in CD4+ T cells were analyzed by flow cytometry (n=4).
Data are represented with mean ± SD, #P<0.05, ##P<0.01, ###P<0.001 versus the control group; *P<0.05, **P<0.01 versus the HFD+LPS group.
RES inhibited the activation of CD4+ T cells in vitro
We further studied the effects of RES on the activation of CD4+ T cells in vitro. To access the proliferation of CD4+ T cells, Ki67, which is specifically expressed in proliferating cells, was detected (Fig.3A-B). TCR and LPS treatment increased the expression of Ki67 in CD4+ T cells while 20μM and 40μM of RES decreased Ki67 expression under TCR and LPS treatment. Surprisingly, increased Ki67 expression was detected under 80μM of RES treatment.
In addition, TCR and LPS stimulation significantly increased the expression of CD25 and CD44 and decreased CD62L expression in CD4+ T cells. 20, 40, and 80μM of RES decreased the expression of CD25 in CD4+ T cells while 20 and 40μM of RES decreased CD44 expression, the influence of 80μM of RES on CD44 expression was not detected. It was notably that 20 and 40μM of RES have no influence on CD62L expression, but 80μM of RES unexpectedly decreased CD62L (Fig.3C-D).
Cytokines secreted by CD4+ T cells were detected (Fig.3E). 80μM of RES increased IL-6, IL-10 and TGF-β1 level in supernatant but had no effect on IL-2 level. 40μM of RES increased IL-2 and TGF-β1 but did not influence IL-6 and IL-10 secretion. 20μM of RES increased the TGF-β1 level but did not change the secretion of IL-2, IL-6, and IL-10. RES was not powerful in regulating IL-2, IL-6, and IL-10 secretion, but promotes TGF-β1 secretion. In summary, different concentrations of RES has different effects on the expression of markers of proliferation and activation in CD4+ T cells as well as cytokines secretion.
CD4+ T cells were stimulated by 1μg/mL anti-CD3 and 1μg/mL anti-CD28 and cultured in a cell incubator. 0.1μg/mL LPS and 20, 40, 80μM of RES were added and cells were collected 24 hours later.
(A-B) The expression of proliferation marker, Ki67 was observed by flow cytometry (n=4). (C-D) The expression of activation markers including CD25, CD44, and CD62L of CD4+ T cells were observed by flow cytometry (n=4). (E) The cytokines of IL-2, IL-6, IL-10, and TGF-β1 in the culture supernatant were detected by ELISA (n=5).
Data are represented with mean ± SD, #P<0.05, ##P<0.01, ###P<0.001 versus the control group; *P<0.05, **P<0.01, ***P<0.01 versus the TCR+LPS group.
RES inhibited the expression of Dnmt1 and Dnmt3b in CD4+ T cells
To observe the expression of Dnmt in CD4+ T cells, the level of mRNA and protein of Dnmt were detected (Fig.4A-C). mRNA expression of Dnmt1 and Dnmt3b were increased in CD4+ T cells treated by TCR and LPS, and protein expression of Dnmt1 was also increased. 20μM of RES decreased the expression of Dnmt1 and Dnmt3b in both mRNA and protein levels. 40 and 80μM of RES decreased protein expression of Dnmt1 and decreased mRNA and protein expression of Dnmt3b. In order to confirm whether these expression changes of Dnmt related to the activities of CD4+ T cells, 5-Aza, an inhibitor for Dnmt was used. It was verified that 5-Aza significantly inhibited the expression of Dnmt3b in both mRNA and protein levels, but Dnmt1 is not influenced by 5-Aza (Fig.4D-F). 40μM of RES was similar to 5-Aza in inhibiting Dnmt3b and besides, 40μM of RES decreased Dnmt1 protein expression, but mRNA expression of Dnmt1 has not been detected. Both 5-Aza and 40μM of RES inhibited the expression of Ki67 (Fig.4G-H), CD25 and CD44 (Fig.4I-J). In addition, the combination of 5-Aza and 40μM of RES reduced Dnmt1 protein level and Dnmt3b mRNA and protein level, and decreased the expression of Ki67, CD25, and CD44.
CD4+ T cells were stimulated by 1μg/mL anti-CD3 and 1μg/mL anti-CD28 and cultured in a cell incubator. 0.1μg/mL LPS and 20, 40, 80μM of RES were added and cells were collected 24 hours later. (A) mRNA level of Dnmt in CD4+ T cells detected by qRT-PCR (n=4). (B-C) The protein level of Dnmt in CD4+ T cells detected by WB (n=4).
5-Aza were pretreated for 36 hours, and cells were collected after the treatment of LPS and 40μM of RES. (D) mRNA level of Dnmt in CD4+ T cells (n=4). (E-F) The protein level of Dnmt in CD4+ T cells (n=4). (G-H) The expression of proliferation marker, Ki67 was observed by flow cytometry (n=4). (I-J) The expression of activation markers including CD25 and CD44 of CD4+ T cells were observed by flow cytometry (n=4).
Data are represented with mean ± SD, #P<0.05, ##P<0.01, ###P<0.001 versus the control group; *P<0.05, **P<0.01, ***P<0.001 versus the TCR+LPS group.