The Protective Mechanism Of Danshen Tongluo Jiedu Decoction On Anoxia/Reoxygenation Injury Of CMECs Based On TLR4/MYD88/NF-?B Pathway

Objective:1.To establish hypoxia / reoxygenation model of Cardiac microvascular endothelial cells,and to explore the protective effect and influence of Danshen Tongluo Jiedu decoction on hypoxia / reoxygenation injury of cardiac microvascular endothelial cells.2.To explore the protective mechanism of Danshen Tongluo Jiedu decoction on hypoxia / reoxygenation injury of cardiac microvascular endothelial cells.Method:1.Danshen Tongluo Jiedu decoction and blank control group serum were prepared,CCK8 method was used to detect the survival activity of CMECs in different concentration of serum(0%,5%,10%,15%,20%),and determine the best concentration of serum;2.CMECs was cultured in vitro and the cells were cultured in logarithmic phase.The hypoxia / reoxygenation model was established by hypoxia for 2hours and reoxygenation for 2 hours.The model group was randomly divided into normal group(C group),model group(H / R group),Danshen Tongluo Jiedu decoction containing serum group(DTJD group),MyD88 inhibitor group(ST-2825 group);3.Cardiac Microvascular Endothelial Cells damage were measured by LDH methods;4.The expression of autophagy related protein LC3 in CMECs was observed by immunofluorescence staining;5.TUNEL staining were used to detect the effect of apoptosis on hypoxia /reoxygenation injury of CMECs;6.Western blot was performed to detect the expression of TLR4,MyD88,NF-?B,Caspase-3.Result:1.The results of CCK8 showed that compared with 5%,10%,20%,25% medicated serum,15% medicated serum group had better CMECs cell activity,the difference was statistically significant(P < 0.01).2.LDH detection results showed that: compared with the normal group,LDH in CMECs culture medium of model group,Danshen Tongluo Jiedu decoction containing serum group and MyD88 inhibitor group increased(P< 0.01);compared with the model group,LDH in CMECs culture medium of Danshen Tongluo Jiedu decoction containing serum group and MyD88 inhibitor group decreased(P < 0.01).3.The results of TUNEL test and caspase-3 protein content showed that:compared with the normal group,the number of TUNEL positive cells and caspase-3 protein content in the model group increased significantly(P <0.01);compared with the model group,the number of TUNEL positive cells and caspase-3 protein content in the Danshen Tongluo Jiedu decoction containing serum group and MyD88 inhibitor group decreased(P < 0.01).4.The results of immunofluorescence showed that compared with the normal group,the LC3 positive cells of model group,Danshen Tongluo Jiedu decoction containing serum group and MyD88 inhibitor group increased,while compared with the model group,the LC3 positive cells of Danshen Tongluo Jiedu decoction containing serum group and MyD88 inhibitor group decreased.5.TLR4 / MyD88 / NF-? B pathway protein expression level: compared with the model group,the expression of TLR4 / MyD88 / NF-?B pathway protein in the model group increased(P < 0.01);compared with the model group,the expression of TLR4 / MyD88 / NF-? B pathway protein decreased(P < 0.01)after the treatment of the MyD88 inhibitor group.Conclusion:1.Danshen Tongluo Jiedu decoction has protective effect on hypoxia / reoxygenation cardial microvascular endothelial cells,which is related to the reduction of LDH and apoptosis.2.The protective effect of Danshen Tongluo Jiedu decoction on hypoxia /reoxygenation cardial microvascular endothelial cells is through inhibiting TLR4 / MyD88 / NF-? B molecular signal pathway and regulating autophagy protein LC3.