CD40小干扰RNA对自身免疫性心肌炎大鼠血白细胞介素-21与白细胞介素-35表达的影响

Effect of CD40 small interfering RNA on the expressions of peripheral blood interleukin -21 and interleukin - 35 in rats with autoimmune myocarditis

目的探讨CD40小干扰RNA（siRNA）对实验性自身免疫性心肌炎（EAM）大鼠外周血白细胞介素-21（IL-21）与白细胞介素-35（IL-35）表达的影响及意义。方法将6～8周20只雄性Lewis大鼠采用随机数字表法分为正常组、EAM组、CD40 siRNA组及siRNA组，每组5只。实验第0天及第7天，正常组大鼠双后肢足垫区皮下注射PBS缓冲液，其余3组双后肢足垫区皮下注射猪心肌球蛋白，建立EAM大鼠模型。第7天CD。siRNA组及siRNA组大鼠经尾静脉分别注射CD40 siRNA及siRNA慢病毒表达载体。第21天，行超声心动图检查后处死大鼠。光镜下观察心肌组织病理变化并计算心肌组织病理积分。采用酶联免疫吸附试验检测大鼠外周血IL-21与IL-35水平。结果1．除正常组大鼠，余3组大鼠EAM发病率均为100％，无一例死亡。2．CD40 siRNA组大鼠心脏质量／体质量比值明显低于EAM组，差异有统计学意义（3．13±0．21比3．80±0．29，F=0．332，P〈0．05）；其心肌组织病理积分亦明显低于EAM组，差异有统计学意义（2．22±0．43比3．32±0．51，F=0．456，P〈0．05）。3．超声心动图检测显示，EAM组有1例大鼠探及大量心包积液，CD40 siRNA组大鼠均未探及心包积液。EAM组、CD40 siRNA组及siRNA组大鼠室间隔和左心室后壁均增厚，心腔缩小，室壁运动减低。CD。siRNA组左心室短轴缩短率较EAM组明显升高，差异有统计学意义[（63．34±11．06）％比（38．56±6．98）％，F=16．080，P〈0．05]。4．CD。siRNA组大鼠外周血IL-21水平明显低于EAM组，差异有统计学意义[（141．19±17．46）ng／L比（157．81±17．58）ng／L，F=57．008，P〈0．05]；而其IL-35水平明显高于EAM组，差异有统计学意义[（195．96±18．26）ng／L比（174．78±13．91）ng／L，F=31．727，P〈0．05]。5．EAM组大鼠外周血IL-21水平与心肌组织病理积分呈正相关（r=0．69，P〈0．05），而其IL-35水平与心肌组织病理积分呈负相关（r=-0．64，P〈0．05）。结论cD4。siRNA可减轻EAM大鼠的心肌炎症，降低心肌损伤程度。IL-21与IL-35水平变化可在一定程度上反映心肌损伤程度。CD40 siRNA对EAM大鼠的治疗作用可能与下调IL-21、上调IL-35的表达有关。

Objective To explore the effect of CD40 small interfering RNA（siRNA） on the expressions of pe- ripheral blood interleukin（IL） -21 and IL- 35 in rats with experimental autoimmune myocarditis （EAM） and its sig- nificance. Methods Twenty 6 - 8 week male Lewis rats were divided into normal group, EAM group, CD40 siRNA group and siRNA group by using random number table, with 5 rats in each group. The normal rats were induced with phos- phate buffer saline in double foot pads on day 0 and day 7, while the rest 3 groups were induced with cardiac myosin protein to establish EAM models. The rats in CD40siRNA group and siRNA group were respectively injected with CD40 siRNA and siRNA slow virus expression vector through the tail vein of rats on day 7. The rats were executed on 21 day after echocardiogram examination was made. The histopathologic changes were observed by using light microscope and the myocardial histopathology scores were calculated. Enzyme - linked immunosorbent assay was used to determine the levels of IL - 21 and IL - 35 in peripheral blood. Results （ 1 ） Except the normal group, the total incidence rate of rats of each group was 100% ,and there was no rat death. （2）Compared with EAM group,the heart mass/body ratio and myocardial histopathology scores were lower in CD40 siRNA group, and the differences were significant （3.13 ± 0.21 vs 3.80±0.29,2.22 ±0.43 vs 3.32 ±0.51 ,F =0. 332,0. 456,all P 〈 0.05）. （3） The echocardiogram showed that there was only 1 rat in EAM group with massive pericardial effusion, and there was no pericardial effusion in CD40 siRNA group. EAM group, CD40 siRNA group and siRNA group displayed hypertrophy of the ventricular septum and left ventricular wall, narrow heart cavity and weakening of ventricular wall motion. The left ventrieular shortening rate in CD4o siRNA group was significantly higher than that in the EAM group [ （63.34 ± 11.06） % vs （ 38.56 ± 6.98 ） %, F = 16. 080 ,P 〈 0.05 ]. （4）The peripheral blood level of IL -21 in CD4osiRNA group was lower than that in EAM group [ （ 141.19 ± 17.46） ng/L vs （ 157.81 ± 17.58 ） ng/L, F = 57. 008, P 〈 0.05 ], while its level of IL - 35 was signifi- cantly higher than that in the EAM group [ （ 195.96 ± 18.26 ） ng/L vs （ 174.78±13.91 ） ng,/L, F = 31. 727, P 〈 0. 05]. （5）The level of IL -21 in peripheral blood was positively correlated with myocardial histopathology scores in EAM group （ r = 0.69, P 〈 0.05）, but IL - 35 was negatively correlated with myocardial histopathology scores （ r = - 0.64, P 〈 0.05 ）. Conclusions CD4o siRNA might relieve the myocardial inflammation and reduce the myocar- dial injury of EAM rats. The levels of IL - 21 and IL - 35 can partly reflect the degree of myocardial injury. The mecha- nism may be related to down- regulating the expression IL- 21 and up- regulating the expression of IL- 35.