The Mechanism Of TXL On Endothelial Cell Injury Induced By Hypoxia

Objective: Hypoxia is one of the common factors that lead to vascular endothelial damage. Most of cardiovascular diseases were caused by vascular endothelial damage. So preventing vascular endothelial cells injury will decrease the occurrence of cardiovascular disease.micro RNA-155 is a typical multi-functional micro RNA and plays a key role in the development and progression of cardiovascular disease. A recent study found that mi R-155 could influence some function of endothelial cells, monocyte macrophages and vascular smooth muscle cells related to cardiovascular diseases.KLF4 and KLF5 are important members of KLF family. KLF4 and KLF5 belong to zinc-finger containing transcription factor, which play important roles in the regulation of cell proliferation, differentiation and tissue formation. Sumoylation is one of translational modification which regulated the expression and activation of proteins. Sumoylation could affect the various properties of the target protein localization, activity and stability.Tongxinluo(TXL) is a compound traditional Chinese medicine, has been widely used in China to treat patients with cardiovascular and cerebrovascular disease. This study aims to clarify the protective effect that of Tongxinluo on endothelial cell tight junction proteins and its molecular mechanism.Methods: C57BL/6J mice were randomly assigned to one of the following three groups: control group, hypoxia group, and hypoxia with TXL pre-incubation group; mi R-155 knock-out(mi R-155-/-) mice were randomly assigned to one of the following two groups: hypoxia group and hypoxia with TXL pre-incubation group. Hypoxia group and hypoxia with TXL pre-incubation group were in a hypobaric chamber simulating an altitude of 5,000 m(6 h /day, 28 days). 7.5mg/10 g TXL was intragastrically administered 3d before hypoxia treatment in hypobaric chamber(TXL pre-incubation group). HIF-1α, UBC9, KLF4, KLF5 and tight junction proteins was detected after 28 days hypoxia treatment by immunofluorescence staining in aorta of mice; The expression levels of HIF-1α, UBC9, KLF4, KLF5 and tight junction proteins were detected by Western bolt in Cardiac microvascular endothelial cells(HCMECs) after incubation with TXL, transfected with mi R-155 inhibitor or p Ad-mi R-155, respectively. The interaction of UBC9 with KLF4 or KLF5 and sumoylation of KLF4 and KLF5 was detected by Co-immunoprecipitation(COIP). Luciferase reporter assay was used to analysis sumoylation of KLF4 and KLF5 on the promoter activity of mi R-155.Results:1 TXL reversed expression of tight junction proteins induced by hypoxiaImmunofluorescence staining showed that the expression of tight junction proteins in aorta of chronic hypoxia group is lower than that of control group. Compared with chronic hypoxia groups, positive cells of tight junction proteins in hypoxia with TXL pre-incubation group were increased. Meanwhile, western bolt got the similar results, which indicating TXL reversed the expression of tight junction proteins induced by hypoxia.2 TXL reduced the expression of mi R-155To detect the expression level of mi R-155 in aorta of hypoxic group mice, real-time PCR was used. These results showed that mi R-155 expression was significantly increased in aorta of hypoxic mice compared with the control group. However the mi R-155 expression was significantly lower in aorta of hypoxia with TXL pre-incubation group than that of hypoxia group mice. These data demonstrated that hypoxia promoted the expression of mi R-155 and TXL inhibited the expression of mi R-155, suggesting that TXL regulated the expression of tight junction proteins might possibly related to mi R-155.3 mi R-155 regulated the expression of tight junction proteinsIn order to verify the above hypothesis, we applied mi R-155-/- mice to examine the effect of mi R-155 on tight junction proteins. Interestingly, the expression of tight junction proteins in aorta of mi R-155-/- mice is higher than that of wild type mice. In addition, transfection with adenovirus of mi R-155 and mi R-155-inhibitor, respectively, in HCMEC got similar results. These results showed that mi R-155 could significantly inhibit the expression of tight junction proteins.4 TXL promoted the expression of HIF-1α, UBC9, KLF4 and KLF5In order to further clarify the molecular mechanisms of TXL on mi R-155 expression, we detect the expression of HIF-1α,UBC9,KLF4,KLF5 expression by immunofluorescence staining. The results showed that the expression of HIF-1α and KLF5 in aorta of chronic hypoxia group is higher than that of control group. On the contrary, the expression of KLF4 and UBC9 is lower than that of control group. Positive cells of HIF-1α, KLF4, KLF5 and UBC9 in hypoxia with TXL pre-incubation group were the most significant among three groups. These results showed that TXL promoted the expression of KLF4, KLF5 and UBC9.5 TXL regulated the interaction of UBC9 between KLF4 and KLF5HCMECs was incubation with TXL for 24 h and treated with hypoxia. The interaction of UBC9 between KLF4 and KLF5 was detected by co-immunoprecipitation(COIP) analysis. The results showed that hypoxia suppressed the interaction between UBC9 and KLF4, however, promoted the interaction between UBC9 and KLF5. However, TXL could reverse the interaction of UBC9 with KLF4 or KLF5, which indicating that TXL could change the interaction of UBC9 between KLF4 or KLF5.6 TXL regulated the sumoylation of KLF4 and KLF5UBC9 is a key enzyme for sumoylation. Next, we detect the effect of Tongxinluo on KLF4, KLF5 sumoylation by co-immunoprecipitation(COIP). Hypoxia decreased the sumoylation levels of KLF4, but increased the sumoylation levels of KLF5. However, TXL pre-incubation reversed the effect of hypoxia on the sumoylation of KLF4 and KLF5. Together, these results suggested that TXL promoted KLF4 sumoylation, but inhibited sumoylation of KLF5.7 Sumoylated KLF4 regulated the expression of mi R-155Since hypoxia could suppress the sumoylation of KLF4 and accelerate KLF5 sumoylation, whether sumoylation of KLF4 or KLF5 affect the expression of mi R-155? Then, reporter gene analysis was used to examine the effect of sumoylation of KLF4 or KLF5 on mi R-155 expression. The results showed that the expression of mi R-155 was decreased after co-transfection of KLF4 and UBC9 plasmid in 293 A cells. However, co-transfection of KLF5 and UBC9 plasmids had no effect on mi R-155 promoter, compared with KLF5 transfection group. These results suggested that sumoylated KLF4 could inhibit the activation of mi R-155 promoter.Conclusions:1 TXL promoted the expression of hypoxia-induced tight junction proteins.2 TXL could decrease the expression of mi R-155.3 mi R-155 could regulate tight junction proteins expression.4 TXL promoted the expression of HIF-1α, UBC9, KLF4, KLF5 induced by hypoxia.5 TXL regulated the interaction of UBC9 between KLF4 and KLF5.6 TXL regulated the sumoylation levels of KLF4 and KLF5.7 Sumoylated KLF4 could regulate the expression of mi R-155.