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

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.