| Objective: To investigate the role of mi R-125 b on inflammation in post-MI together with the regulation function of miR-125 b on three main cell types,including myocardial cells,macrophages and fibroblasts,which regulated myocardial infarction.Methods:To establish the mouse model with myocardial infarction,then the expression of mi R-125 b and inflammatory cytokines including IL-1?,IL-6,IL-12,TNF-? were detected at the third and fifth day of post-MI(after myocardial infarction),meanwhile,the expression of endogenous HMGB1 in post-MI compared with the control group was detected by immunohistochemistry and immunofluorescence methods.To construct of overexpression-miR-125 b adenovirus or vector control adenovirus for infecting heart tissues in post-MI,the impact of mi R-125 b on cardiac function was observed by the cardiac color ultrasound in post-MI.Furthermore recombinant mouse HMGB1(rmHMGB1)proteins were compounded in vitro experiments.Overexpression-miR-125 b adenovirus and control adenovirus vector were compounded to infect H9C2 and 10T1/2 cells in vitro as well as heart tissue after MI in vito;miR-125 b mimic or control mimic were compounded to transfect primary-isolated macrophages from mice,The levels of cytokines IL-1?,IL-6,IL-12,TNF-? were detected via the RT-PCR and ELISA.The phosphorylation levels of NF-?B P65,MAPK JNK,MAPK P38 and MAPK ERK in inflammatory response(of macrophages)were detected through western blot.CD11b+ cells magnetic bead was performed to select macrophages cells from the heart tissue after MI-adenovirus infection,the levels of cytokines IL-1?,IL-6,IL-12,TNF-? were detected via the RT-PCR.Results:1.The expression of miR-125 b was increased in the myocardial tissue of mice during the inflammatory phase.At the third day of post-MI(after MI),the expression level of mi R-125b(6.10±0.11)from the peripheral tissues of infarcted area in the experimental group(group MI)increased remarkably compared with the sham operation group(group SHAM)(1.00±0.00)(P<0.05);at the fifth day,the expression level of miR-125b(2.42±0.06)increased remarkably compared with the sham group(1.00±0.00)(p<0.05).LVEF and LVFS values reflecting the cardiac function of those mice with overexpression-miR-125 b at the two and four weeks of post-MI in the experimental group were significantly lower than those in the control group(p < 0.05),2 w(after operation),LVEF(60.33±9.07)% vs(68.67±1.52)%,LVFS(29.02±0.71)% vs(33.26±4.24)%.4 w(after operation),LVEF(56.23±1.04)% vs(64.02±4.58)%,LVFS(20.50±2.12)% vs(24.63±2.83)%,the difference was statistically significant(all p < 0.05).2.At the same time,the expression of HMGB1,the endogenous risk molecular,and a large number of inflammatory cytokines increased in the peripheral tissues of infraction area.At the fifth day of post-MI,immunohistochemical displayed that HMGB1 protein level increased in the peripheral tissues of infraction.(p< 0.05),meanwhile in the third day,the immunofluorescence from the peripheral tissues of infraction area showed that the positive signals of HMGB1 marked red fluorescence increased obviously.IL-1?,IL-6,IL-12,TNF-? mRNA levels,from the peripheral tissues of infarcted area,increased in the MI-group at the third and fifth day of post-MI,vs SHAM group(p<0.05 or p<0.01).3.Three cell types including myocardial cells,fibroblasts and macrophages were transfected with overexpression-miR-125 b in vitro,PCR and ELISA detection results,and HMGB1 induced the expression of inflammatory cytokines,the results of PCR and ELISA detection: Overexpression-miR-125 b promoted the production of inflammatory cytokines IL-1?,IL-6,IL-12,TNF-? from the original generation of macrophages;for 3 hour stimulated with HMGB1 IL-1?,IL-6,IL-12,TNF-? mRNA expression levels in the overexpression-miR-125 b experimental group than in the control group were increased,IL-1?[(2815.44 ± 199.02)vs(1087.5 ± 67.18),(p < 0.05)];IL-6 [(5.55 ± 2.12)vs(3.05 ±0.78),(p>0.05)];IL-12 [(260.51 ±52.32)vs(147.08 ±26.87),(p <0.01)];TNF-?[(54.00±10.68)vs(40.62±8.83),(p>0.05)];And for 6 hour stimulated with HMGB1,IL-1?,IL-6,IL-12,TNF-? mRNA levels in the overexpression-miR-125 b experimental group than the control group were significantly higher relatively,IL-1?[(11093.48 ± 2637.83)vs(5313.987 ±1022.94),(p < 0.01)];IL-6 [(20.235 ± 3.91)vs(6.16 ± 1.19,(p < 0.01)];IL-12 [(484.56±36.14)vs(245.5±30.41),(p<0.01)];TNF-?[(84.22±22.65)vs(60.3± 9.28),(p < 0.01)].The production of inflammatory cytokines from the supernatant of macrophage was detected by ELISA,then the production of IL-1?,IL-6,IL-12 and TNF-? in overexpression-mi R-125 b group stimulated with HMGB1 for 3h increased than those of control group,IL-1? [(178±48.08)vs(159±41.01),(p>0.05)].IL-6 [(245±53.74)vs(216.5±47.37),(p>0.05)].IL-12[(13.592.12)vs(10.43±1.45),(p>0.05)].TNF-?[(1322.50±204.35)vs(8 89.74±125.87),(p>0.05)].Then for 6h stimulated with HMGB1,the production of IL-1?,IL-6,IL-12,TNF-? in overexpression-miR-125-b group increased than those of control group,IL-1? [(480.32 ± 113.14)vs(330.87 ± 70.71),(p <0.05)].IL-6[(688.09±72.12)vs(405.71±42.43),(p<0.01).IL-12 [(32.55±4.95)vs(19.50±3.54),(p<0.05)].TNF-? [(2264.5±161.93)vs(1393.18±193.74),(p<0.05)].In myocardial cells and fibroblasts,IL-1,IL-6,IL-12,TNF-? mRNA levels and the production of supernatant on extracellular protein levels also had varying change,but there was no statistically significant difference(p > 0.05).4.Macrophages were isolated from infarcted heart tissue which were infected by overexpression-miR-125 b or control adenovirus,the results of pcr showed that IL-1,IL-6,IL-12 and TNF-? mRNA levels in the experimental group increased compaired with the control(p?0.05).5.In the results of Western Blot,while rmHMGB1 induced the original generation of macrophage to inflammatory reaction,overexpression-mi R-125 b selectively promoted that inflammation.Only the phosphorylation of P65 was more remarkable in the overexpression-miR-125 b experimental group than the control group,the phosphorylation of P38 JNK,ERK had no obvious change.Conclusion:The expression of HMGB1 and inflammatory cytokines such as IL-1??IL-6?IL-12?TNF-? together with miR-125 b increased in the peripheral tissues of infraction area.mi R-125 b positively regulated the inflammatory response induced by HMGB1 in macrophages in post-MI by promoting the phosphorylation of P65 after activating NF-kB,but it didn't perform significance in myocardial cells and fibroblasts. |
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