Tongxinluo Exerts A Protective Effect On Ischemia-and Hypoxia-induced Injury Of Brain And BV2 Cells Through Modulating KLF5 Expression

Objective: Hypoxia-ischemia brain injury is a main cause of death and permanent disability worldwide,and think that oxidative stress and inflammation are the important causes of hypoxia-ischemia brain injury.Despite recent progress in prevention and treatment,hypoxia-ischemia brain injury still remains a large unmet medical need.Elucidation of cellular and molecular mechanism that influence the pathogenesis of ischemic injury could lead to the development of new therapeutic approachesand insight into disease pathophysiology.Chinese medicine tongxinluo(TXL)is a compound formulated according to the meridian theory of traditional Chinese medicine.TXL is widely used in clinical treatment of cardiovascular diseaseand the effect is remarkable.Clinical studies have demonstrated that TXL is also protective against cerebrovascular diseases,such as cerebral arteriosclerosis,headaches and cerebral infarction.However,the molecular mechanism whereby TXL improves ischemic brain injury remainsunclear.In the present study,we studied the effects of TXL on ischemia-hypoxia brain injuryand its mechanism with animal model and neural microglia BV2 cells.Methods: Male ApoE-/-mice were randomly assigned to one of the following three groups: control group,high-fat diet group and high-fat diet with TXL.7.5mg/10 g body weight of TXL was administered by intragastric administration.The control group and high-fat diet group received equal dose of saline.After three months,TTC staining and HE staining were used to test ischemic brain injury.Expression of HIF-1a,NRG-1,HO-1,KLF5 and KLF4 in ischemic brain tissues were detectedby Western blotting and RT-PCR analysis.At the cellular level,neural microglia(BV2)cells were treated withpalmitic acid(C16:0)for different times,Western blot and RT-PCR analysis were performed for HIF-1a,NRG-1,HO-1,KLF5 and KLF4.BV2 cells were also cultured in thethree gas incubator under hypoxia condition for different times,Westernblot analysis and RT-PCR were used to detect the expression of HIF-1a,NRG-1,HO-1 and KLF5.Results:1 Brain ischemia induces expression of HIF-1a,NRG-1 and HO-1,TXL can abrogate upregulation of HIF-1a,NRG-1 and HO-1 induced by hypoxia.TTC staining results can be seen,normal control group brain was evenly dyed red,and high-fat diet group was obviously the infarction area,TTC staining showed that the TXL treatment obviously reduced ischemia-induced cerebral infact volumes.HE staining showed that brain tissues of the normal control group had normal morphological structure,the brain tissues of high-fat diet group showed a large number of neuron edema,vacuolation and necrosis.Neuron damage of TXL group wasreduced.These results suggest that tongxinluo can greatly improve the brain damage caused by hypoxia.Western blot and RT-PCR analysis results showed that the cerebral ischemia induced the expression of HIF-1a,NRG-1 and HO-1,and TXL could abolish hypoxia-induced HIF-1a,NRG-1 and HO-1.2 Hypoxia induced the expression of HIF-1a,NRG-1 and HO-1 in BV2 cells,TXL treatment could abrogate hypoxia-induced expression of HIF-1a,NRG-1 and HO-1.In order to study the effect of hypoxia on nerve cells,we treated BV2 cells with palmitic acids(C16:0)and incubated cells under hypoxi-a condition.Western blot and RT-PCR showed that C16:0 induced the expression of HIF-1a,NRG-1 and HO-1.When BV2 cells were incubaed under hypoxia condition for different times.The expression of HIF-1a,NRG-1 and HO-1were upregulated in a time-dependent manner.BV-2 cells were preincubated with different concentrations of TXL for 24 hours,and then were cultured under hypoxic condition.Western blot and RT-PCR analysis showed that,with the increase of concentration of TX-L,the expression level of HIF-1a,NRG-1 and HO-1 were dose dependently decreased.Cell immunofluorescence staining also showed that compared with the control group,the immunofluorescence staining intensity of HIF-1a,NRG-1 and HO-1 was increased in hypoxia-induced BV2 cells,and cells was incubated with TXL,the immunofluorescence staining intensity of HIF-1a,NRG-1 and HO-1 was significantly reduced.3 Cerebral ischemia induced KLF5 expression,TXL treatment could suppress the ischemia-induced KLF5 upregulation.These results suggest that hypoxia-induced the expression of HIF-1a,NRG-1 and HO-1,then,whether the expression of HIF-1a,NRG-1 and HO-1was affected by the regulation of KLF5 and KLF4? Western blot and RT-PCR showed that the cerebral ischemia induced the expression of KLF5,after the TXL treatmented,the expression of KLF5 was significantly decreased,but KLF4 expression had no obvious change as showed by Western blot and RT-PCR.4 Hypoxia induced KLF5 expression in BV2 cells,TXL treatment could down-regulate hypoxia-induced KLF5 expression.In order to investigate hypoxia-induced upregulation of HIF-1a,NRG-1and HO-1 whether was due to KLF5 and KLF4 expression.The BV2 cells were treated with palmitic acid C16:0 or were incubated underhypoxia condition,Western blot and RT-PCR showed expression level of KLF5 was increased in hypoxia-induced BV2 cells,but the expression level of KLF4 did not be affected.When BV2 cells were preincubated with TXL and then were cultured under hypoxia condition was significantly down-regulated,but KLF4 expression had no obvious change.Cellimmunofluorescence staining of KLF5 showed that compared with the control group,KLF5 immunofluorescence staining was increased in hypoxia–incubated BV2 cells,after the TXL preincubation,the immunofluorescence staining inensityof KLF5 was significantly reduced.5 KLF5 mediated the expression of HIF-1a,NRG-1 and HO-1 induced by hypoxia.These results suggest that hypoxia–induced upregulation of HIF-1a,NRG-1 and HO-1 may be due to KLF5 of expression.Knockdown of KLF5 by si-KLF5 attenuated the upregulation of HIF-1a,NRG-1 and HO-1expression induced by hypoxia,indicating that KLF5 mediated hypoxia-induced expression of HIF-1a,NRG-1 and HO-1 genes.Conclusions:1 Brain ischemia induces expression of HIF-1a,NRG-1 and HO-1,TXL can abrogate upregulation of HIF-1a,NRG-1 and HO-1 induced by hypoxia.2 Hypoxia induced the expression of HIF-1a,NRG-1 and HO-1 in BV2 cells,TXL treatment could abrogate hypoxia-induced expression of HIF-1a,NRG-1 and HO-1.3 Cerebral ischemia induced KLF5 expression,TXL treatment could suppress the ischemia-induced KLF5 upregulation.4 Hypoxia induced KLF5 expression in BV2 cells,TXL treatment could down-regulate hypoxia-induced KLF5 expression.5 KLF5 mediated the expression of HIF-1a,NRG-1 and HO-1 induced by hypoxia.