MicroRNA-155 Activates NLRP3 Inflammasome By Modulating MEK/ERK/NF-κB Pathway In Carotid Atherosclerotic Plaques Of ApoE^-/- Mice

Objective:The formation of atherosclerotic（AS）plaques is one of the main causes of ischemic stroke.MicroRNA-155（miR-155）can regulate a variety of inflammation related cells,thus affecting the formation and development of AS.As an important member of pattern recognition receptor（PRR）,NLRP3 inflammasome participates in the innate immune process and activates a variety of inflammatory factors such as IL-1βand IL-18,which plays an important role in the formation of AS.However,the specific role and mechanism of miR-155 and NLRP3inflammasome in the process of AS are still unclear.In this study,ApoE^-/-mice were used to establish the model of carotid atherosclerosis,to study the effect of miR-155 on the expression of NLRP3 inflammasome,inflammatory factors and MEK/ERK/NF-κB signal pathway related proteins,to explore the possible mechanism of miR-155 involved in the regulation of NLRP3 inflammasome to mediate the formation of carotid atherosclerotic plaques,and to find a new way to prevent and treat atherosclerosis.Materials and Methods:Sixty 6-week-old male ApoE^-/-mice were randomly divided into 5 groups（12 in each group）:the blank group（high-fat diet+right carotid cannula+non transfected lentivirus）,the negative control（NC）group（high-fat diet+right carotid cannula+empty lentivirus）,the miR-155 mimic group（high-fat diet+right carotid cannula+LV-miR-155）,miR-155 inhibitor group（high-fat diet+right carotid cannula+LV-miR-155-RNAi）,the miR-155 mimic and ERK inhibitor group（high-fat diet+right carotid cannula+LV-miR-155+ERK inhibitor）.All ApoE^-/-mice were fed for 8 weeks.Afterwards,a silicone ring was placed in the right common carotid artery of all mice so that the formation of carotid atherosclerotic plaques could be induced of each mouse.Forty-eight hours after the insertion of the silicone ring,the mice in each group were injected with lentivirus through the tail vein to establish the necessary model.An ERK inhibitor was injected once every two days at a dose of 5 mg/kg.After 8 weeks,we obtained blood samples from the inner canthus vein of the mice.The mice were sacrificed by pentobarbitalsodium（15 mg/100 g）.And the carotid artery specimens were collected surgically.The blood lipid levels were measured by the enzyme method.Oil red O,HE and immunohistochemical staining were used to observe the degree of carotid plaque formation.PCR was used to measure the expression of miR-155.The blood lipid levels were measured by the enzyme method.Western blotting was used to measure the expression of NLRP3inflammasome-related proteins,IL-1β,IL-18 and MEK/ERK/NF-κB signalling pathway-related proteins.We used Prism 5 and SPSS 22.0 statistical software to process the data.The measurement data are expressed as X±S,and the mean differences among groups were compared by one-way ANOVA.We compared the mean values of two groups using t-tests.The difference of P<0.05was deemed statistically significant.Rusults:There was no significant difference between NC group and blank group in plasma lipid level,carotid atherosclerotic plaque formation degree,the expression of miR-155,NLRP3inflammasome-related proteins,IL-1β,IL-18,and MEK/ERK/NF-κB signalling pathway-related proteins.Compared with those of the NC group,the increases in TC and LDL in the miR-155 mimic group were statistically significant（P<0.05）,and the TG concentration increased,while HDL decreased,carotid intima thickening was obvious,the degree of carotid plaque formation increased,the expression levels of miR-155,NLRP3inflammasome-related proteins,IL-1β,IL-18,and MEK/ERK/NF-κB signalling pathway-related proteins were also significantly increased in the miR-155mimic group（P<0.05）.The miR-155 inhibitor group had the opposite effect.Injection of ERK inhibitors into miR-155 mimic mice reduced the levels of TC and LDL,plaque formation,and the expression levels of p-NF-κB,NLRP3 inflammasome-related proteins and IL-1β,IL-18.Conclusion:In ApoE^-/-mice,miR-155 can regulate MEK/ERK pathway,promote the phosphorylation of NF-κB to activate NLRP3 inflammasome,enhance the inflammatory response,and promote the formation and development of AS plaque.Mi R-155 may become a new target for the prevention and treatment of AS.