The Mechanism Of Tongxinluo Acting In Protecting Hypoxia-induced Brain Injury

Object: Chinese medicine Tongxinluo(TXL) has obvious protective effects on hypoxic brain injury. However, the molecular mechanism whereby TXL improves brain injury remains unclear. In the present study, we studied the effects of TXL on hypoxic brain injury and its mechanism with animal model and cultured microvascular endothelial cells.Methods: Male C57BL/6J mice received chronic intermittent hypobaric hypoxia treatment in a hypobaric chamber simulating 5000-meter altitude for 28 days. TXL power was administered by intragastric administration. Brain sections were prepared and stained with antibody against tight junction proteins and CD31. Cultured microvascular endothelial cells were pre-incubated with TXL, and then were cultured in a sealed canister for hypoxia, and stained with antibody against tight junction proteins. Total proteins were prepared from the cultured microvascular endothelial cells and brain tissues, and Western blot analysis was performed for detecting tight junction proteins.Results:1 TXL can improve hypoxia-induced brain damage by increasing expressions of vascular endothelial tight junction proteins VE-cadherin, β-catenin and ZO-1.Compared with control, tight junction proteins VE-cadherin, β-catenin, ZO-1 and CD31 in brain tissues of TXL-treated mice markedly increased, as evidenced by a more continuous and linear labeling of VE-cadherin, β-catenin or ZO-1 along the vessel. The microvascular endothelium of mice treated without TXL showed a discontinuous, less regular distribution, indicating that the microvessels are disrupted by hypoxia. The results suggest that TXL can promote expressions of the tight junction proteins in brain microvessels.Western blot analysis and immunofluorescence staining showed that the tight junction proteins VE-cadherin, β-catenin and ZO-1 obviously increased after treatment with TXL, accompanied with upregulation of KLF4 expression. These findings suggest that TXL can protect the hypoxia-induced brain damage by increasing vascular endothelial tight junction proteins VE-cadherin, β-catenin and ZO-1.2 TXL promotes the expressions of tight junction proteins VE-cadherin, β-catenin and ZO-1 in microvascular endothelial cells under hypoxia conditions.The confocal images of immunofluoresecence staining show that compared with control group, the tight junctions of TXL group was more intact, indicating that TXL can improve the tight junctions in the brain microvessels. Conversely, the tight junction proteins of the hypoxia group were not intact.Western blot analysis showed that mice treated with TXL exhibited a increased tight junction proteins VE-cadherin, β-catenin and ZO-1, with KLF4 upregulated consistently.3 TXL increases the expressions of VE-cadherin, β-catenin and ZO-1 via inducing KLF4 phosphorylation.To determine the relationship among TXL, KLF4 and tight junction proteins, we examined KLF4 phosphorylation with co-immunoprecipitation. The results show a higher level of KLF4 phosphorylation in microvascular endothelial cells treated with TXL, accompanied by an increase in tight junction protein expression, which indicates that TXL increases the expressions of VE-cadherin, β-catenin and ZO-1 via promoting KLF4 phosphorylation.4 TXL induces the phosphorylation of S444 and S415 sites of KLF4.Microvascular endothelial cells were transfected with different phosphorylation-deficient mutants of KLF4. The Western blot analysis indicates that the expressions of VE-cadherin, β-catenin and ZO-1 were decreased in microvascular endothelial cells transfected with the GFP-KLF4, GFP-S444 A and GFP-S415 A mutants, without changes in the cells transfected with GFP-S470 A.After microvascular endothelial cells were pre-incubated with TXL for 24 hours, then transfected with different phosphorylation-deficient mutants of KLF4, and received hypoxia treatment for 12 hours. Western blot analysis reveals that, compared with the control group, the expressions of KLF4 and tight junction proteins were markedly increased in microvascular endothelial cells pre-incubated with TXL. The transfection of GFP-S470 A mutant had no effect on the expression of these proteins. The GFP-S444 A and GFP-S415 A mutants decreased the expression of tight junction proteins.5 TXL induces KLF4 phosphorylation through AKT signaling pathway.Western blot analysis showed that AKT phosphorylation was markedly increased in TXL treated mice and microvascular endothelial cells.In addition, we treated microvascular endothelial cells with AKT inhibitor, and then examined the KLF4 phosphorylation. Compared with control group, AKT inhibitor reduced significantly KLF4 phosphorylation induced by TXL.Conclusions:1 TXL can improve the hypoxia-induced brain damage.2 TXL increases the expressions of the tight junction proteins VE-cadherin, β-catenin and ZO-1 in microvascular endothelial cells.3 TXL increases the expressions of VE-cadherin, β-catenin and ZO-1 via inducing KLF4 phosphorylation.4 TXL induces the phosphorylation of S444 and S415 sites of KLF4.5 TXL induces KLF4 phosphorylation through AKT signaling pathway.