TLR4-mediated MyD88-dependent Signaling Pathway In The Injury Of Perihematoma Tissus After Human Cerebral Hemorrhage.

Cerebral hemorrhage is one of critically illness of the nervous system, seriously affecting human health and quality of life of patients. But, at present, the lack of specific and effective treatment of cerebral hemorrhage means, mainly due to a mechanism of tissue damage after cerebral hemorrhage has not been fully elucidated. The pathological changes, including ischemia and hypoxia, of perihematoma tissue after Cerebral Hemorrhage are similar to cerebral ischemia reperfusion injury, there are also inflammatory response and apoptosis. In this study, using human brain hemorrhage tissue, we detected the qualitative and quantitative of many relevant indicators at gene and protein levels, in oder to discuss the role of TLR4-MyD88-NF-KB signaling pathway in the brain hemorrhage and molecular mechanism.In this study, we selected 24 patients with hypertensive cerebral hemorrhage who were all under craniotomy and hematoma removal, and took brain tissue from about 1cm near to the hematoma as a experimental group and brain tissue away from the hematoma as controls during the approach.All Specimens were divided into three sub-groups:≤6h group,6-24h group,24～72 h group and>72 h group according to the time interval between onset to taking samples.Study was divided into three parts:(1)Proofing the perihematoma tissue after Cerebral Hemorrhage existing inflammation and apoptosis.HE staining, TUNEL staining and immunohistochemical method were used to observe the pathological changes, apoptosis and detect the expression of TNF-a and Caspase-3. We found Significant difference between experimental group and control group from HE stain. TUNEL-positive cells in the experimental group in the cerebral hemorrhage appeared during 6-24 h, reached the peak during 24～72h, decreased when it was after 72h, but significantly higher than those in the control group (P<0.05). TNF-a, Caspase-3 expression of experim-ental group also began to increase during 6-24h, reached the peak during 24-72h, decreased when it was after 72h, but significantly higher than those in the control group (P<0.05).(2) The effect of TLR4 in the inflammatory response and neuronal apoptosis after human brain hemorrhage.We applied RT-PCR and immunohistochemical methods respectively, to detect the expression of TLR4 on gene and protein levels, and analysed correlation of TLR4 with TNF-a, Caspase-3 expression.Expression of TLR4mRNA and TLR4 protein in the experimental group began to increase during 6-24h, reached the peak during 24-72h, decreased when it was after 72h, but significantly higher than those in the control group (P<0.05).Correlation analysis showed that:TLR4mRNA and TNF-a, Caspase-3 protein expression were positively correlated (P<0.01);TLR4 protein expression and TNF-a, Caspase-3 expression always made a positive correlation (P<0.01).(3) The role of TLR4-mediated MyD88-dependent signaling pathway in human brain hemorrhage injury.We applied RT-PCR, Westen blot and immunohistochemistry methods to detect the expression of MyD88 on gene and protein levels, while detected the expression of NF-κB by immunohistochemical staining. MyD88's expression also began to increase during 6-24h, reached the peak during 24-72h, decreased when it was after 72h, while the level of MyD88 in the experimental group in each period were significantly higher than controls, both in protein expression and mRNA levels (P<0.05).The expression of NF-κB also began to increase during 6-24h, reached the peak during 24-72h,decreased when it was after 72h, the differences between two groups were significant (P<0.05). MyD88 and TNF-α, Caspase-3 expression were positively correlated (P<0.01)Conclusions:(1) perihematoma tissue after Cerebral Hemorrhage existed inflammation and apoptosis. TNF-a and Caspase-3 were important cytokines involved in brain injury of intracerebral hemorrhage. (2) Expression of TLR4 in human brain tissue in intracerebral hemorrhage increased significantly, and made a positive correlation with TNF-a and Caspase-3 expression. TLR4 played an important role in inflammatory reactions and apoptosis in perihematoma tissue after Cerebral Hemorrhage. (3) TLR4 possiblely activated NF-κB which was on the pathway downstream by upregulating the expression of MyD88 to aggravated the inflammatory response and apoptosis in perihematoma tissue after Cerebral Hemorrhage.In this study, we are based on the human brain tissue around the hematoma, discussing the real human cerebral hemorrhage injury mechanisms, to overcome the differences between heterogeneous. Through the various aspects and levels, we studied the role of TLR4 on brain injury after intracerebral hemorrhage and its mechanism. We found that TLR4-mediated MyD88 dependent pathway could activate downstream NF-κB, induced TNF-αand other inflammatory cytokine releasing, then start the inflammatory response. Meanwhile, it could induced nerve cells to produce pro-apoptotic factors, activated protease caspase system through cascade, then started caspase3, leaded to apoptosis.Further research can use the principles and methods of antigen-antibody reaction or imperfections gene transfer to block the TLR4-mediated MyD88-dependent pathway for opening up new roads for the treatment of cerebral hemorrhage.