Contribution Of TLR4/NF-κB Signaling In Intermittent Hypoxia-mediated Atherosclerosis Progression

Background Vulnerable atherosclerotic plaque is a common pathological change in most patients with acute coronary syndrome and stroke.Obstructive sleep apnea(OSA)is a type of sleep-related breathing disordered disease.Studies have shown that OSA patients accompanying atherosclerosis(AS)will make the prognosis of AS plaque even worse.Furthermore,it was found that Toll-like receptor 4(TLR4)and Nuclear factor κappa B(NF-κB)plays a key role in the pathogenesis of OSA and AS.However,it is still unclear whether OSA accelerates the progression of AS and the specific molecular mechanism of TLR4/NF-κB signaling involved in that progress.Objective In this study,OSA is typically characterized by intermittent hypoxia(IH),so we are able to simulate the pathophysiology of OSA by creating an IH environment.An early AS model was constructed by using Apo E-/-mice as a carrier,and then exposure to IH.Furthermore,the effects of IH on aortic plaque area,vulnerability and the expression of TLR4,NF-κB and its downstream inflammatory molecules were observed.The mechanism of TLR4/NF-κB signaling in this process was further elaborated by TLR4 interference.So to provide an experimental basis for clinical prevention and treatment of OSA accompanying unstable plaque.Methods1.Synthesis of TLR4 interference lentivirus Through the screening of si RNA,the highest interference effect of TLR4-si RNA sequence was be selected and recombined into lentivirus(LV)carrier.After transfection in vivo and the expression of enhanced green fluorescent protein(EGFP)was observed.2.Cell grouping and processing in vitro In cell experiments,we first screened out the hypoxia/reoxygenation time point which can significantly up-regulate the expression of TLR4,then cells were divided into Control group,hypoxia/reoxygenation group,hypoxia/reoxygenation plus blank lentivirus group,hypoxia/reoxygenation plus TLR4interference-lentivirus group.3.Animal experiment grouping and processing The Apo E-/-mice were randomly divided into the sham intermittent hypoxia group(n=15)(Sham),intermittent hypoxia group(n=15)(IH),intermittent hypoxia plus blank lentivirus group(n=15)(IH+LV-EGFP)and intermittent hypoxia plus TLR4 interference lentivirus group(n=15)(IH+LV-TLR4i).The early stage of AS plaque lesions was induced in Apo E-/-mice,then performed virus transfection and treated with intermittent hypoxia.4.Body weight and blood pressure Pre-or after-IH,body weight and tail arterial blood pressure were measured.5.Serum inflammatory factors and blood biochemical test ELISA was used to detect the content of serum tumor necrosis factor alpha(TNF-α)and interleukin-6(IL-6);blood biochemical test of blood lipids and blood glucose.6.Pathological examination General Oil Red O staining was performed on the artery trunk plaque lesions.Continuous frozen sections of aortic sinus were used to H&E staining,Oil Red O staining,Sirius red staining,immunohistochemical staining,specifically as follows:(1)The effect of aortic lentivirus transfection: the expression of EGFP in aortic sinus was observed by fluorescence microscopy;(2)Aortic plaque lesions: general oil red O staining was used to observe the size of the plaque lesion in the aortic wall;(3)Plaque area: the plaque area in aortic sinus sections was observed by H&E staining;(4)The expression of TLR4 and NF-κB in the plaque of aortic sinus wereanalyzed by immunohistochemistry;(5)Plaque instability: the expression of smooth muscle actin(α-SMA),monocytes¯ophages(MOMA-2)in the plaque of aortic sinus were analyzed by immunohistochemistry,Oil Red O staining was performed to detected the deposition of lipids,and Picro-Sirius Red staining was performed to detected the content of collagen I.The standard plaque stabilization score formula:(Oil Red O+area plus MOMA-2+ area)/(α-SMA+ area plus collagen I+ area).7.Molecular biology analysis(1)Western blotting was performed to detected the protein expression of TLR4,p65,p-p65,IL-1β,HIF-1α,Nox-2,p38,p-p38 in aortas and it also be used to detect the TLR4,p65,p-p65 levels in endothelial cells.(2)Real-time PCR was used to detecte the m RNA level of TLR4 in aortas and in endothelial cells.Results1.Compared with the Control group,the level of TLR4 protein was highest in the H6/R12(Reoxygenation for 12 hours after hypoxia 6 hours)group(p<0.05).2.The expression level of TLR4 m RNA,protein and p-p65 in H6/R12 group were significantly higher than those in the Control group(p<0.05).3.After H6/R12 treatment,the level of TLR4 m RNA,protein expression and phosphorylation of p65 in the TLR4 interference group were significantly lower than those in the blank lentivirus group(p<0.05).4.The fluorescence intensity of lentivirus transfected aortic plaque was significantly increased.5.There was no significant difference in the body weight of the four group mice before IH exposure.After IH treatment,the body weight of the IH group was significantly lower than that of the Sham group(p<0.05);But compared with the IH+LV-EGFP group,there was no significant decrease in body weight of the IH+LV-TLR4 i group(p<0.05).6.There was no significant difference in SBP and DBP between the four groupsbefore IH exposure;After IH exposure,compared with Sham group,the SBP and DBP of mice treated with IH were significantly increased(p<0.05),and there was no significant difference in blood pressure between the IH+LV-TLR4 i group and the IH+LV-EGFP group.7.The serum levels of cholesterol,LDL-C,HDL-C and glucose in the IH group were significantly higher than those in the Sham group(p<0.05),while IH exposure had no significant effect on serum TG level;Compared with the IH+LV-EGFP group,the levels of serum cholesterol and LDL-C were significantly decreased in the IH+LV-TLR4 i group(p<0.05),but there was no significant difference in the levels of HDL-C,TG and glucose.8.Compared with the Sham group,IH exposure increased the expression levels of TLR4,p-p65,IL-1β,HIF-1α,Nox-2,p-p38 in the aortic AS plaques.At the same time,it also increased the intranuclear aggregation of NF-κB and the content of serum IL-6 and TNF-α.By reducing the expression of endogenous TLR4,the effect of IH was significantly reduced(p<0.05).9.Compared with the Sham group,the lesion area of aortic plaque in the IH group was significantly increased(p<0.05),and the plaque burden of the IH+LV-TLR4 i group was 3 times lower than that of the IH+LV-EGFP group(p<0.05).10.Compared with the Sham group,the plaque instability index of the IH group was significantly increased(p<0.05);TLR4 interference reversed the effect of IH(p<0.05).Conclusions1.H6/R12 treatment induces activation of the proinflammatory transcription factor NF-κB via TLR4 signaling in endothelial cells;2.IH exposure amplifies the intensity of inflammatory response in the aortic plaques,induces the glycolipid disorders and hypertension,and lead to the increase of plaque burden and plaque instability;3.IH promoting the activation of TLR4/NF-κB signaling and its downstream inflammation in aortic plaques is the key molecular mechanism of IH acceleratingAS plaque progression,the results of this study provide a new way of thinking about the new pathogenesis and potential therapeutic targets of OSA accompanying unstable plaque.