Effects Of Ischemic Post-conditioning On Toll-like Receptor 4-TRIF Signal Transduction Pathways In Rat Models Of Focal Cerebral Ischemia/Reperfusion

Objective: Ischemic post-conditioning(IPO)can stimulate endogenous protective effect and inhibit the inflammatory response associated with ischemia reperfusion(I/R)injury,while the underlying mechanisms are not well understood.This study investigated the effects of IPO on the expression of toll-like receptor 4(TLR4)-Toll-interleukin 1 receptor domain-containing adapter-inducing interferon-b(TRIF)signaling pathway,further expound the neuroprotective mechanisms and provide theoretical basis of applying IPO to clinical application.Methods:Adult male Sprague-Dawley rats(n = 130)were randomized into sham(n = 30),model group(n = 50),and treatment(n = 50)groups;each of these was divided into five subgroups(sham,n = 6;I/R and IPO,n = 10)corresponding to different time points(6,12,24,48,and 72 h)after operation or reperfusion.Rats in the latter two groups were established to middle cerebral artery occlusion(MCAO)model by the Zea-longa suture-occluded method.Rats in the sham group were just exposed with common carotid artery and the bifurcation of vessels through surgery,and without insertion of the suture.Two hours after MCAO,rats in the treatment group underwent 6 cycles of IPO(reperfusion for 10 s/re-occlusion for 10 s)justat the beginning of reperfusion.The suture was removed 2 h after MCAO in the model group.At time points(6,12,24,48,and 72 h)after reperfusion,rats were scored for neurological deficits,infarct volume and apoptotic cells count.Real-time quantitative PCR(qPCR)tested m RNA expression of TLR4,TRIF,TRIF-related adaptor molecule(TRAM),interferon regulatory factor 3(IRF-3)and interferon-β(IFN-β)in ischemic cortex.Immunohistochemistry tested protein positive cells count of TLR4,TRIF,TRAM,IRF-3 and IFN-β in ischemic cortex.Western blotting tested protein expression of TLR4,TRIF,TRAM,IRF-3 and IFN-β in ischemic cortex.Results: Rats in the model and treatment groups showed neurological deficits and cerebral infarction.In constrast with model group,neurological deficits scores of treatment group were improved(t=2.963~5.262,P<0.05),infarct volume of treatment group was reduced(t=3.341~3.875,P<0.05),and apoptotic cells count of treatment group was decreased(t=2.332~3.643,P<0.05).TLR4,TRIF,TRAM,IRF-3 and IFN-β m RNA and protein levels increased 6h after reperfusion,reaching a peak at 24 h.The results of qPCR showed that TLR4,TRIF,TRAM,IRF-3 and IFN-β m RNA levels were lower in the treatment group than in the model groupat each time point(t=2.240~6.587,P<0.05).The results of immunohistochemistry showed that TLR4,TRIF,TRAM,IRF-3 and IFN-β protein positive cells count were lower in the treatment group than in the model group at each time point(t=2.256~8.180,P<0.05).The positive cells were mainly distributed among ischemic temporal frontal cortex and temporal cortex.The results of western blotting showed that TLR4,TRIF,TRAM,IRF-3 and IFN-β protein levels were lower in the treatment group than in the model group at each time point(t=2.943~8.227,P<0.05).Discussion: IPO plays neuroprotective effect and improves neurological function of MCAO rat through inhibiting apoptosis and reducing cerebral infarct volume.IPO exerts neuroprotection by blocking the inflammatory response induced by I/R,including inhibition of TLR4-TRIF signaling pathway and downstream inflammation-associated cytokines like IRF-3 and IFN-β expression.