The Relationship Between TLR-2 And MMP-2 And Its Mechanism In Brain Ischemic Injury After Cerebral Ischemia And Reperfusion

Objective:To explore the changes and possible relationship between TLR-2 (toll-like receptor 2, TLR-2) and MMP-2 (matrix metalloproteinase-2, MMP-2) after brain cerebral ischemia and reperfusion. Using TLR-2 antagonist T2.5 (MAb-mTLR2, which can inhibit TLR-2 signaling pathway) to futher explore the possible role of TLR-2 and MMP-2 in brain injury formation after brain cerebral ischemia and reperfusion.Methods:Rats(250-280 g) underwent an 1 h right brain middle cerebral artery occlusion (MCAo) followed by reperfusion. Rats in this research were divided into three groups randomly:the sham group、the MCAo group、the T2.5-treated group (T2.5 antagonist was injected through jugular vein when rats began to reperfusion after 1 h-occlusion,0.1212 μg/g).To explore the relationship between TLR-2 and MMP-2 as well as the possible mechanism of the two on brain injury formation after cerebral ischemia and reperfusion. We chose infracted brain tissue from these three groups(the sham group、 the MCAo group、the T2.5-treated group) at the different reperfusion time points to conduct a series of experiments as follows:We firstly used Western Blot to observe the expression changes of TLR-2、 MMP-2 and neuron at 1h、2h、6h、12h、24 h after brain ischemia and reperfusion in infracted cortex of brain tissue in sham group and MCAo group(every time points in each group repeated 3 times, n=3).We secondly used TLR-2 antagonist T2.5 via jugular vein injection and Western Blot to observe the expression changes of TLR-2、MMP-2 and neuron at 2 h after brain ischemia and reperfusion in infracted cortex of brain tissue in MCAo group and T2.5-treated group(2 h in each group repeated 3 times, n=3).Lastly, we tested the BBB permeability (Evan’s blue)、brain cerebral edema (wet-dry weighting method)、infarcted volume (TTC staining) and neurological function score at 24 h after brain ischemia and reperfusion in three groups (sham group、MCAo group and T2.5-treated group) (every method repeated 3 times, n=3).Results:1、Western Blot showed, the expression level of MMP-2 began to increase at 1 h after brain ischemia and reperfusion and the differences were statistically significant compared to the sham group (p<0.05). The expression level of MMP-2 at 2 h、6h、12h、24 h after reperfusion was higher compared to the sham group and the differences were statistically significant (p<0.05).2、Western Blot showed, the expression level of neuron began to decrease at 2 h after brain ischemia and reperfusion compared to the sham group and the differences were statistically significant (p<0.05). At the 12 h after brain ischemia and reperfusion, the expression level of neuron dropped to lowest, and the differences between this two groups were statistically significant (p<0.05). At the 24 h after brain ischemia and reperfusion, the expression level of neuron seemed rise again, but the differences were still statistically significant when compared to the sham group (p<0.05).3、Western Blot showed, the expression level of TLR-2 began to decrease at 1h after brain ischemia and reperfusion. This trend maintained at 2h、6h after brain ischemia and reperfusion. but the differences between MCAo group and sham group were not statistically significant at these time points (1h、2h、6h)(P>0.05). At the 12h、24h after brain ischemia and reperfusion, the decreased extent compared to the sham group were statistically significant (p<0.05).4、At 24 h after brain ischemia and reperfusion, we tested BBB permeability、brain cerebral edema、infracted volume and neurological score. All of these were higher compared to the sham group and the differences were statistically significant (p<0.01、 p<0.05, p<0.01、p<0.001).5、To further explored the possible relationship between TLR-2 and MMP-2 as well as the possible role of TLR-2 and MMP-2 in BBB disruption and neuron death. we treated rats,which have been induced MCAo, TLR-2 antagonist T2.5 via jugular vein injection. Western Blot showed, compared to MCAo group, the expression level of TLR-2 and MMP-2 were lower in T2.5-treated group at 2 h after brain ischemia and reperfusion and the differences were statistically significant (p<0.05、p<0.001). The expression level of neuron in T2.5-treated group were higher than MCAo group and the differences were statistically significant (p<0.05). At 24 h after brain ischemia and reperfusion in T2.5-treated group, the BBB permeability、infracted volume and neurological score were lower than MCAo group and the differences were statistically significant (p<0.01、p<0.05、p<0.001). Although the brain cerebral edema in T2.5-treated group seemed lower than in MCAo group, the difference of the brain cerebral edema between MCAo group and T2.5-treated group were not statistically significant.Conclusion:1、In super acute period, the expression level of MMP-2 increases immediately which may be involved in neuron death and BBB disruption, resulting a series of reperfusion injury.2、In super acute period, the expression level of TLR-2 begins to decrease. The possible role of TLR-2 in brain ischemia and reperfusion injury remains to be pursued deeply.3、The TLR-2 antagonist T2.5 could alleviate the BBB disruption、neuron death and improve brain ischemia and reperfusion injury by decreasing the expression of MMP-2 whose expression may be inhibited through TLR-2 signaling pahway.