Effects And Mechanisms Of Icariin Against Atherosclerosis Via MAPK Signaling Pathway

This research was supported by Jilin Natural Science Foundation and by Jilin Education Department Program.Atherosclerosis(AS) is a common disease which is harmful to human health, and it is the main pathological basis of cardiovascular diseases. In recent years, with the improvement of people’s living standard and the change of eating habits, the incidence and mortality of coronary heart disease and stroke caused by AS have increased. Therefore, inhibiting the occurrence and development of AS is an important link in the treatment of cardiovascular diseases, and it is great significance to reduce the mortality risk of cardiovascular diseases.Icariin(ICA), a flavonoid isolated from Epimedium brevicornum Maxim, is considered as the main pharmacological active constituent, and has been reported to possess various pharmacological effects, including anti-inflammatory, anti-osteoporosis, anti-tumor and immunoregulation. With the deepening research of icariin, its prevention and treatment on the diseases of cardiovascular system have becomed a research hotspot. However, little is known about the effects and mechanisms of icariin on AS, and the research is not deep enough.In this study, morphology, biochemistry, immunohistochemistry, immunofluorescence, flow experimental technology cytometry, Western blotting, Real time-PCR and other techniques were applied through the experiment of in vivo(animal) and in vitro(cells). The effects and molecular mechanisms of icariin against atherosclerosis were systematically studied by MAPK signal pathway as the breakthrough point, in order to provide apply the theoretical basis of icariin in the treatment of AS in clinic.In vivo experiments, we used rat model of AS established by our laboratory, that intraperitoneal injection with VD3(700 thousand U/kg) combined with high fat diet, successfully induced rat model of AS. After treatment with icariin for 4 weeks, various serum biochemical assays such as TC, TG, LDL-C, HDL-C, ox-LDL, SOD, MDA, TNF-α and IL-6 were detected, and the expression of ICAM-1, VCAM-1, E-selectin, PCNA in the thoracic aorta were detected, as well as the phosphorylation levels of ERK1/2, p38 and JNK in the thoracic aorta. The results showed that the levels of blood lipids including TC, TG, LDL-C and ox-LDL, increased markedly in the model group compared with those in the control group. However, icariin treatment decreased all these variables which was induced. In contrast, icariin recovered the decreased level of HDL-C in the AS rats. In addition, a significant decrease in the activity of SOD, an increase in the content of MDA as well as increases in the expression of ICAM-1, VCAM-1, E-selectin, PCNA and phosphorylation levels of ERK1/2, p38 and JNK was observed in the AS rats, but icariin treatment restored them. The above results showed that icariin has a significant effect on the AS rats.In vitro studies, to demonstrate whether the antiatherogenic mechanism of icariin was associated with the proliferation of vascular smooth muscle cell(VSMCs), ox-LDL(50 μg/ml) was used to induce proliferation of VSMCs). To clarify the effect of icariin on cell-cycle regulation, we analysed the phases of cell cycle using flow cytometry, and to demonstrate whether the effect of icariin on the cell cycle was associated with the expression of PCNA, Western blotting analysis was used. The results showed that treatment with ox-LDL markedly increased the synthesis of PCNA cells, markedly increased the percentage of VSMCs in S and G2/M phase, and correspondingly decreased the percentage in G0/G1 phase. However, pretreatment with icariin significantly reversed these effects. It shows that icariin inhibits ox-LDL induced VSMCs proliferation by blocking cell cycle progression.To demonstrate whether the antiatherogenic mechanism of icariin was associated with the injury of vascular endothelial cells, ox-LDL(100 μg/ml) was used to induce injury of human umbilical vein endothelial cells(HUVECs). In this experiment, the expression of ICAM-1 VCAM-1 and E-selectin in HUVECs induced by ox-LDL were detected using Real-time PCR. The results showed that ox-LDL could promote NF-κB p65 protein into the cell nucleus of HUVECs, and induced the overexpression of ICAM-1, VCAM-1 and E-selectin. Icariin can significantly reversed these effects. It is suggested that icariin inhibited the injury and apoptosis of HUVECs induced by ox-LDL.To further study the degree of oxidative damage in HUVECs induced by ox-LDL, in this experiment, the apoptosis rate of HUVECs was detected using flow cytometry, and the protein expression levels of apoptotic protease Caspase-3 and anti apoptosis gene Bcl-2 were detected by Western blotting. The results showed that compared with the normal cells, the apoptosis of HUVECs was induced significantly by ox-LDL, icariin could significantly improve the early apoptosis rate of HUVECs, significantly down-regulated the protein expression of Caspase-3 and up-regulated the protein expression of Bcl-2.To demonstrate whether icariin inhibited ox-LDL-induced VSMCs proliferation and HUVECs injury by inhibiting the activation of ERK1/2, p38 and JNK, we examined the phosphorylation levels of ERK1/2, p38 and JNK in smooth muscle cells and HUVECs. But icariin only could significantly inhibit ox-LDL induced ERK1/2, p38 phosphorylation in VSMCs, and only could inhibit ox-LDL induced p38, JNK phosphorylation in HUVECs. The results showed that icariin could only significantly inhibit ERK1/2, p38 phosphorylation in VSMCs induced by ox-LDL; and only inhibited significantly inhibit JNK, p38 phosphorylation in HUVECs induced by ox-LDL. The above results showed that MAPK signaling pathway mediated proliferation of VSMCs and endothelial cell injury induced by ox-LDL, and icariin improved VSMCs proliferation induced by ox-LDL via inhibiting the activation of ERK1/2 and p38 MAPK signaling pathway, and improved vascular endothelial cells injury induced by ox-LDL via inhibiting the activation of JNK and p38 MAPK signaling pathway.In summary, icariin can adjust to the levels of serum lipids and reduce the amount of ox-LDL generated in atherosclerosis rats, it can inhibit atherosclerosis in rats induced by high fat diet. The underlying mechanism may be related to improving VSMCs proliferation via inhibiting the activation of ERK 1/2 and p38 MAPK signaling pathway, and improving HUVECs injury via inhibiting the activation of JNK and p38 MAPK signaling pathway.