The Relationship Between Toll-like Receptor 4 Induced Autophagy And Atherosclerosis Plaque And Intervention Treatment

Stoke is the leading cause of death and disability in China. Atherosclerosis is usually complicated by acute thrombosis, which is triggered by the rupture or erosion of atherosclerotic plaque, which leads to acute ischemic events. It is important to recognize the heterogeneity of atherosclerosis plaque and that various pathophysiological processes. PART Ⅰ The effect of Toll-like receptor 4 on atherosclerotic plaque formation and progressionObjection: To observe the change of Toll-like receptor 4 expression in the formatin and progression of atherosclerotic plaque, and investigate its mechanism.Method: 20 Apo E(-/-) mice of 8 weeks, after a week of adjustable feeding, were divided into four groups: control group, normal diet group, 10 weeks model group and 20 weeks model group. Then control group was killed. Normal diet group was fed with common fodders for 10 weeks, and then killed. 10 weeks model group and 20 weeks model group were fed with high fat fodders for 10 weeks and 20 weeks respectively, and then killed. Total cholesterol(TC), triglyceride(TG) and oxidized low density lipid protein(ox LDL) were measured. The specimens were stained by HE, Oil Red, Masson and Sirius Red. MOMA-2 and α-actin and TLR4, Interleukin-1β(IL-1β), Interleukin-6(IL-6) and Tumor necrosis factor-α(TNF-α) were measured by Immunohistochemical staining.Results:1. Levels of TC, TG, ox LDL in normal diet group were higher than those in the control group. 10 weeks model group had higher levels of TC, TG and ox LDL than the normal diet group, accompany with atherosclerotic plaque formation.2. The levels of TC, TG and ox LDL in 20 weeks model group were higher than those in the 10 weeks model group. The atherosclerotic plaques found in 20 weeks model group had increased lipid content, decreased plastic fiber and collagen content, as well as more microphages and less vascular smooth muscles.3. The levels of TLR4, IL-1β, IL-6 and TNF-α expressed in the atherosclerotic plaque in were higher 20 weeks model group were higher than those in the 10 weeks model group.Conclusion:1. The atherosclerotic plaque models could be established by Apo E(-/-) mice fed with high fat fodders. The longer high fat diet, the severer and more vulnerable the atherosclerotic plaque had.2. The atherosclerotic plaque models established by high fat diet had higher levels of inflammation and oxidized stress, leading to macrophages increased, vascular smooth muscle cells apoptosis, and collagen synthesis decreased, thus promoting the progress of atherosclerotic plaque, as well as reducing the stability of the atherosclerotic plaque.3. As the progression of the atherosclerotic plaque, the increased TLR4 and inflammatory factors might be involved in the progression of atherosclerotic plaque. PART Ⅱ The relationship between Toll-like receptor 4 mediated autophagy and atherosclerosisObjection: To observe the relationship between TLR4 expressed in atherosclerotic plaque and autophagy, and investigate the effect of TLR4 induced autophagy on atherosclerosis plaque stability.Method: 24 Apo E(-/-) mice of 8 weeks, after a week of adjustable feeding and 10 weeks of high fat fodders, were divided into three groups: LPS group, control group and TAK group. LPS group was received intraperitoneal injection of LPS(1mg/kg) twice a week; TAK group was received intraperitoneal injection of TAK(0.3mg/kg) twice a week; the Control group was received an injection of same amount of saline twice a week. Continued with high fat fodders for 10 weeks, the mice were killed, and carotid artery and aorta were obtained. Serum TC, TG, ox LDL was measured. The specimens were stained by HE, Oil Red, Masson and Sirius Red. MOMA-2 and α-actin and TLR4, Interleukin-1β(IL-1β), Interleukin-6(IL-6) and Tumor necrosis factor-α(TNF-α) were measured by Immunohistochemical staining. LC3Ⅱ/Ⅰ,Beclin-1,P62 were measured by Western Blot.Results:1. Levels of TC, TG, ox LDL in LPS group were higher than those in the control group and TAK group(P<0.05).2. The atherosclerotic plaques found in LPS group had more microphages, less vascular smooth muscles and higher vulnerable index than the control group(P<0.05). The atherosclerotic plaques found in TAK group had decreased lipid content, increased collagen content, less microphages, more vascular smooth muscles and lower vulnerable index(P<0.05).3. The levels of TLR4, IL-1β, IL-6 and TNF-α expressed in the atherosclerotic plaque in LPS group were higher than those in the control group and TAK group(P<0.05), as well as those were lower in TAK group than those in control group(P<0.05).4. The expression of LC3Ⅱ/Ⅰ in LPS was higher than those in control group and TAK group(P<0.001). LPS group had higher expression of Beclin-1, compared with control group and TAK group, while it was decreased in TAK group(P<0.001). The expression of P62 in LPS group was lower than those in control group and TAK group(P<0.01).Conclusion:1. The TLR4 in atherosclerotic plaque was up-regulated by LPS, while down-regulated by TAK-242.2. LPS induced significantly increased autophagy, while TAK-242 inhibited the activity of autophagy.3. The TLR4 activator LPS could promote the secretion of inflammatory factors, aggravate dyslipidemia and increase the vulnerability of the atherosclerotic plaque. PART Ⅲ The effect of atorvastatin and combination with probucol on TLR4 induced autophagyObjection: To observe the effect of atorvastatin and combination with probucol on atherosclerotic plaque, and the association with TLR4 induced autophagy, and investigate its antiatherosclerotic mechanism.Method: 24 Apo E(-/-) mice of 8 weeks, after a week of adjustable feeding and 10 weeks of high fat fodders, were divided into three groups: control group, statin group and combination group. Statin group was received gavage of atorvastatin(10mg/kg/d) daily; combination group was received gavage of atorvastatin and probucol fodders; the control group was received gavage of same amount of high purity water daily. After 10 weeks the mice were killed, and carotid artery and aorta were obtained. Serum TC, TG, ox LDL was measured. The specimens were stained by HE, Oil Red, Masson and Sirius Red. MOMA-2 and α-actin and TLR4, IL-1β, IL-6 and TNF-α were measured by Immunohistochemical staining. LC3Ⅱ/Ⅰ,Beclin-1,P62 were measured by Western Blot.Results:1. The mice in statin and combination group had lower levels of TC and ox LDL, and levels of ox LDL were lower in the combination group than those in the statin group(P<0.05).2. Compared with control group, the atherosclerotic plaque in the statin and combination group had decreased lipid content, increased collagen conten, decreased expression of TLR4, IL-1β, IL-6 and TNF-α, more VSMCs and lower Vulnerable Index(P<0.05). The combination group had less macrophages and lower Vulnerable Index in atherosclerotic plaque than the statin group(P<0.05).3. The expression of Beclin-1 in statin and combination group was higher than those in control group(P<0.05). The combination group had higher expression of LC3Ⅱ/Ⅰand lower expression of P62 than the control group and statin group(P<0.05).Conclusion:1. Atorvastatin down-regulated the expression of TLR4, inhibited oxidative stress and inflammatory reaction, increased autophagy, promoted the stability of the atherosclerotic plaque.2. The combination of atorvastatin and probucol had more effective inhibition of oxidative stress and macrophages, as well as increased activity of autophagy, which contributed to stable the atherosclerotic plaque more effectively.