腺相关病毒 9 介导的 TLR4 小干扰 RNA 通过抑制 NF-κB 和 MAPK 信号通路减轻大鼠心肌缺血再灌注损伤。
Adeno-associated Virus 9-mediated Small RNA Interference of TLR4 Alleviates Myocardial Ischemia and Reperfusion Injury by Inhibition of the NF-κB and MAPK Signaling Pathways in Rats.
机构信息
Harbin Medical University, Harbin, China.
Department of Cardiology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, China.
出版信息
Curr Mol Med. 2019;19(2):127-135. doi: 10.2174/1566524019666190311122521.
BACKGROUND
Despite intensive investigation, effective therapeutic procedures for myocardial I/R injury are still in demand.
OBJECTIVE
To explore the effect of adeno-associated virus 9 (AAV9)-mediated small interfering RNA targeting TLR4 in the treatment of myocardial ischemia and reperfusion (I/R) injury and its influence on the NF-κB and MAPK signaling pathways.
METHODS
Rats were divided into 3 groups, namely, the sham, AAV9-siRNA control, and AAV9-TLR4 siRNA groups. siRNA solution or normal saline was injected through the tail vain. The rat myocardial I/R injury model was then established. HE staining and TUNEL staining were applied to compare the pathological changes in cardiomyocytes in the three groups. Immunohistochemical staining and western blotting were utilized to detect TLR4 expression under siRNA interference. Serum inflammatory factor (IL-1β, TNF-α) expression was determined by an ELISA commercial kit. Key proteins in the MAPK (p38, JNK 1/2) and NF-κB (p65) signaling pathways were determined to identify the TLR4 siRNA functional mechanism.
RESULTS
Fluorescence microscopic images of the myocardium indicated that AAV9- mediated siRNA was efficiently transfected into the myocardium, and the infarcted size after I/R injury was decreased by AAV9-TLR4 siRNA when compared with negative control rats (P<0.05). TLR4 protein expression was significantly decreased by siRNA interference (P<0.001). Apoptosis-related factor BCL-2 expression was increased in the TLR4 gene silencing group, whereas Bax expression was decreased. The Bax/BCL-2 ratio was also decreased, demonstrating a protective effect for cardiomyocytes. Inflammatory factors were lower in the TLR4 gene silencing group than in the siRNA control group (P<0.001). The MAPK and NF-κB signaling pathways were activated in myocardial I/R injury; however, the primary proteins in these two signaling pathways were downregulated upon interference of TLR4 siRNA, with significant differences (P<0.05).
CONCLUSION
AAV9-TLR4 siRNA has a positive effect on myocardial I/R injury by inhibiting the MAPK and NF-κB signaling pathways and can be used as a potential therapeutic method for myocardial I/R injury.
背景
尽管进行了深入研究，但仍需要有效的治疗方法来治疗心肌缺血再灌注（I/R）损伤。
目的
探讨腺相关病毒 9（AAV9）介导的靶向 TLR4 的小干扰 RNA 在治疗心肌缺血再灌注损伤中的作用及其对 NF-κB 和 MAPK 信号通路的影响。
方法
将大鼠分为 3 组，即假手术组、AAV9-siRNA 对照组和 AAV9-TLR4 siRNA 组。通过尾静脉注射 siRNA 溶液或生理盐水。然后建立大鼠心肌 I/R 损伤模型。通过 HE 染色和 TUNEL 染色比较三组心肌细胞的病理变化。免疫组化染色和 Western blot 检测 TLR4 在 siRNA 干扰下的表达。采用 ELISA 试剂盒检测血清炎症因子（IL-1β、TNF-α）的表达。检测 MAPK（p38、JNK1/2）和 NF-κB（p65）信号通路中的关键蛋白，以确定 TLR4 siRNA 的功能机制。
结果
心肌荧光显微镜图像表明，AAV9 介导的 siRNA 有效转染至心肌，与阴性对照组大鼠相比，AAV9-TLR4 siRNA 可减少 I/R 损伤后的梗死面积（P<0.05）。siRNA 干扰显著降低 TLR4 蛋白表达（P<0.001）。TLR4 基因沉默组凋亡相关因子 BCL-2 表达增加，Bax 表达减少。Bax/BCL-2 比值也降低，对心肌细胞有保护作用。TLR4 基因沉默组炎症因子低于 siRNA 对照组（P<0.001）。心肌 I/R 损伤激活了 MAPK 和 NF-κB 信号通路；然而，TLR4 siRNA 干扰后，这两个信号通路的主要蛋白下调，差异有统计学意义（P<0.05）。
结论
AAV9-TLR4 siRNA 通过抑制 MAPK 和 NF-κB 信号通路对心肌 I/R 损伤有积极作用，可作为心肌 I/R 损伤的潜在治疗方法。
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