The Molecular Mechanism Of Hypothermic Circulatory Arrest On The Regulation Of TLR4 Mediated MyD88/TRIF Pathway In Hippocampal Neurons Autophagy

Objective:A rat model of hypothermic and selective hypothermic in vitro circulatory arrest was established to make rats suffer from ischemia and hypoxia at different temperatures.The MyD88 pathway was treated with inhibitor MIP and TRIF via inhibitor Resveratrol.Whether TLR4 mediated MyD88 or TRIF signal transduction pathway is involved in regulating the mechanism of autophagy in hippocampal neurons at different temperatures.Methods:A total of forty healthy male SD rats,,and four SD rats were used as sham operation group.four rats in each group of thirty-six SD rats were randomly divided into nine groups:the normal temperature stopping circulation group,hypothermic circulatory group,deep hypothermic stop circulation group,MIP treated normal temperature stop circulation group,MIP treated mild hypothermia cycle group,MIP treated deep hypothermia cycle group,RV treated normal temperature stop circulation group,RV treated mild hypothermia cycle group,RV treated deep hypothermic stop circulation group.The model of extracorporeal circulation in rats was established by external intubation.The sham operation group did not cool down or stop circulation,and circulate for twenty minutes.In the normal temperature group(37 degrees),after the CPB was established,the circulatory arrest lasted for ten minutes.The deep hypothermia group(20 degrees),after the establishment of cardiopulmonary bypass,stopped circulation for ten minutes.Mild hypothermia(28 degrees),after the establishment of extracorporeal circulation,stopped circulation for ten minutes.Group MIP was injected with MYD88 inhibitor MIP in the rat ventricle.Group RV was injected with TRIF inhibitor Resveratrol in rat ventricle.After stopping circulation,three rats were randomly taken out of each experimental group to take out the hippocampus quickly,and stored in the refrigerator at-81 degrees.The protein expression of TLR4,MYD88,TRIF,Beclin1 and TLR4,MYD88,TRIF and Beclinl of TLR4,MYD88,TRIF and Beclinl were analyzed by the immunoglobulin analysis.In each experimental group,one rats were randomly selected to take the whole brain and store it in formaldehyde.HE staining was used to analyze cell morphology.Results:The results of TLR4 protein expression and real-time fluorescence quantitative PCR:The expression of protein in the normal temperature group was significantly higher than that in the sham operation group(P<0.001).The expression of histone in hypothermia group was significantly lower than that at normal temperature(P<0.05).The expression of histone in hypothermia group was significantly lower than that in low temperature group(P<0.05).In the normal temperature group,mild hypothermia group and deep hypothermia group,there was no significant difference between untreated.MIP treated and RV treated(P>0.05).The trend of TLR4mRNA expression is roughly the same as that of TLR4 protein expression.MYD88 protein expression and real-time fluorescence quantitative PCR results:The expression of protein in the normal temperature group was significantly higher than that in the sham operation group(P<0.001).The expression of protein in hypothermia group was significantly lower than that at normal temperature(P<0.001).The expression of protein in hypothermia group was significantly lower than that in low temperature group(P<0.001).In the normal temperature group,mild hypothermia group and deep hypothermia group,the protein expression of MIP in each group was significantly decreased compared with untreated and RV treated group(P<0.001).The trend of MYD88mRNA expression is roughly the same as that of MYD88 protein expression.The results of TRIF protein expression and real-time fluorescence quantitative PCR:The expression of protein in the normal temperature group was significantly higher than that in the sham operation group(P<0.001).The expression of histone in hypothermia group was significantly lower than that at normal temperature(P<0.001).The expression of histone in hypothermia group was significantly lower than that in low temperature group(P<0.001).In the normal temperature group,mild hypothermia group and deep hypothermia group,the protein expression of RV in each group was significantly decreased compared with the untreated and MIP treated(P<0.05).The trend of TRIFmRNA expression is roughly the same as that of TRIF protein expression.Beclinl protein expression and real-time fluorescence quantitative PCR results:The expression of protein in the normal temperature group was significantly higher than that in the sham operation group(P<0.001).The expression of histone in hypothermia group was significantly lower than that at normal temperature(P<0.001).The expression of histone in hypothermia group was significantly lower than that in low temperature group(P<0.001).In the normal temperature group,mild hypothermia group and deep hypothermia group,the protein expression of MIP in each group was significantly decreased compared with untreated and RV treated(P<0.05).The trend of Beclinl mRNAy expression is roughly the same as that of Beclinl protein expression.MYD88 and Beclinl immunofluorescence results:not seen in the control group did not see the green fluorescence,more green fluorescence in the normal temperature group,mild hypothermia group of green fluorescence from room temperature to reduce,deep cryogenic group green fluorescence in the low temperature reduction;Compared with the RV treatment group and the untreated group,the fluorescence of the MIP treatment group was less than that in the normal temperature group,sublow temperature group and deep cryogenic group.TRIF immunofluorescence results:not seen in the control group did not see the green fluorescence,more green fluorescence in the normal temperature group,mild hypothermia group of green fluorescence from room temperature to reduce,deep cryogenic group green fluorescence in the low temperature reduction;Compared with the MIP treatment group and the untreated group,the fluorescence of the RV treatment group was lower than that in the normal temperature group,sublow temperature group and deep cryogenic group.Conclusions:1.Hypoxia can induce autophagy in hippocampal tissue of rats,and low temperature can inhibit autophagy.To some extent,as the temperature decreases,the inhibition of autophagy can be enhanced.2.Under the condition of normal temperature,mild hypothermia and deep hypothermia ischemia and hypoxia,the induction of autophagy in rat hippocampus is possibly related to TLR4\MYD88 signaling pathway.