Corilagin Ameliorates Arteriosclerosis Obliterans Via Toll-like Receptor 4 Signal Pathway In Vitro And In Vivo

Peripheral artery disease,mainly manifested as arteriosclerosis obliterans(ASO)in lower extremity,is known as the third leading cause of atherosclerotic vascular morbidity after coronary heart disease and stroke.ASO was found in 6.6%(estimated 45.3million)of people over 35 years old in China.Factors such as advanced age,ethnic Han,smoking,hypertension,diabetes,hyperlipemia,low educational level and so on may increase the risk of ASO.Intermittent claudication,chronic limb-threatening ischemia,tissue loss,amputation and even death can be caused by ASO.Burden of atherosclerotic cardiovascular disease is rapidly increasing with economic growth,urbanization and population aging.Unfortunately,existing treatments including endovascular interventions,operative surgery therapy and pharmacotherapy were less effective.It is urgent to find a new way.Atherosclerosis has been studied for a long time and inflammation was recognized as a major and independent factor in atherogenesis in recent years.However,the molecular and cellular pathways haven't been clarified entirely.The cells involved in the innate immune response,especially monocyte/macrophage,promote the formation of atherosclerosis and regulate the stability of plaques by inducing foam cell formation,promoting cytokine release and interacting with endothelial cells and smooth muscle cells.Current studies have shown that Toll-like receptors(TLRs),vital components of innate immune system known for their ability in inducing inflammation response at the outset,play a key role in multiple atherosclerotic pathologic processes in which macrophages are involved.TLR4 is considered to be one of the key promoters of the atherosclerotic process.As an activator of a series of inflammatory cascades in nuclear factor kappa B(NK-?B)-dependent fashion,overexpression of TLR4 may lead excess release of proinflammatory cytokines and chemokines,including tumor necrosis factor-?(TNF-?),interleukin-1(IL-1),IL-6,type?interferon-?(IFN-?).Thus,it is a promising way to find a new medication to against atherosclerosis by regulating TLR4 signaling pathway.Corilagin,extracted from Phyllanthus urinaria or niruri,is one of the ellagitannin and has been found broad-spectrum pharmacological bioactivities in previous studies.It is noteworthy that corilagin can inhibit TLR4 signaling pathway in classical inflammatory model and herpes simplex encephalitis models.However,whether Corilagin could play an anti-inflammatory role in atherosclerosis and thus inhibit the progression of the disease has not been reported.Therefore,we intends to confirm the effect of Corilagin on TLR4signaling pathway and atherosclerosis in vitro and in vivo.Part?:Corilagin inhibit TLR4 signal pathway in Ana-1 cells and Mouse Peritoneal MacrophageObjective:Investigate the effect of Corilagin on the expression of molecules in TLR4signaling pathway in atherosclerotic macrophage model and its possible targets.Methods:CCK8 assay was used to evaluate the cytotoxicity of Corilagin.We stimulated Ana-1 cell lines or mouse peritoneal macrophage(MPMs)with ox-LDL as cell model for atherosclerosis.Small interfering RNA(si RNA)were used to knockdown TLR4 expression.Lentivirus were used to upregulate TLR4 expression.After 24h treatment with high-(100?g/ml),medium-(50?g/ml),low-(25?g/ml)concentration of corilagin,expression of TLR4,toll-interleukin 1 receptor domain-containing adaptor protein(TIRAP),myeloid differentiation factor 88(My D88),TNF receptor-associated factor 6(TRAF6),NF-?B essential modulator(NEMO),mitogen activated protein kinase p38(p38)and interferon regulatory factor 5(IRF5)were determined by q RT-PCR and Western bolt.Results:According to the result of CCK8 assay,we plotted the concentration-effect curve and calculated IC50=215.86?g/ml.We treat Ana-1 cells or MPMs with Corilagin at100?g/ml,50?g/ml and 25?g/ml for 24h which ensuring cell viability is at least 70%.Overexpression of TLR4,TIRAP,My D88,TRAF6,p38,NEMO,IRF5 induced by ox-LDL could be significantly inhibited by Corilagin.When TLR4 were down-or up-regulated,changes in expression levels of downstream molecules were consistent with TLR4 and Corilagin could still effectively restrain the ox-LDL-mediated overexpression of these molecules.Conclusion:Ox-LDL can activate the TLR4 pathway in macrophages and increase the expression levels of TLR4,TIRAP,My D88,TRAF6,p38,NEMO and IRF5.Meanwhile,TLR4 has a positive feedback regulation effect on downstream molecules.Corilagin can effectively inhibit the overexpression of molecules in the TLR4 pathway in the atherosclerotic cell model and obstruct the signaling of the pathway and its target may be TLR4.Part?:Effects of Corilagin on the TLR4 signaling pathway and plaque lesion in ASO ratsObjective:To confirm the effect of Corilagin on TLR4 signaling pathway of macrophages and its intervention on femoral artery plaque in ASO rat.Methods:42 male Sprague-Dawley rats were randomized divided into 7 groups including control group,sham-operation group,model group,high-(40 mg/kg�d),medium-(20mg/kg�d),low-(10 mg/kg�d)concentration Corilagin groups and aspirin group.Excepted control and sham-operation group,femoral artery of SD rats were separated and filled up with sterile water thus extracellular hypoosmosis could lead to the destruction of endothelial cells.After surgical treatment,they were fed with high fat and cholesterol fodder for 120 days.Since 91^th day,rats in drug intervention groups were administered intragastrically with Corilagin or aspirin for 30 days.When all these managements were finished,we collected peripheral blood samples and extracted mononuclear cells to detect the expression levels of TLR4,TIRAP,My D88,TRAF6,p38,NEMO and IRF5 by q RT-PCR,Western bolt and flow cytometry assay.Abundance of IFN-?,IL-1?,IL-6,TNF-?in plasma samples were detected by ELISA.Meanwhile,atheromatous plaques and the expression of TLR4 molecule in the lesion area were observed after Oil Red O staining or immunofluorescence staining.Results:After artificial damage for femoral artery intima by surgery and high-fat high cholesterol diet feeding,SD rats developed obvious atheromatous plaque in femoral artery with a high expression of TLR4.Molecules in TLR4 signaling pathway of peripheral blood macrophages were also overexpressed.Corilagin could significantly reduce the percentage plaque coverage for lumen area and the expression of molecules in TLR4 signaling pathway in lesion area or in peripheral blood macrophages.In corresponding to variation of TLR4 signaling pathway,abundance of IFN-?,IL-1?,IL-6 and TNF-?in plasma was significantly increased in model group and prominently degraded in Corilagin groups.Conclusion:Activation and overexpression of TLR4 signaling pathway in macrophage fueled releasing of pro-atherogenic cytokines such as IFN-?,IL-1?,IL-6 and TNF-?and finally leaded to the atherosclerosis in SD rats.Corilagin can effectively inhibit the TLR4signaling pathway and significantly reduce the release of the pro-atherogenic factors,thus slowing down the process of atherosclerosis.