The Mechanism Of HMGB1/TLR4 Regulating Neuronal Apoptosis And Autophagy In T2DM With OSA

Objective This study was to reveal the phenomenon of hippocampal neuronal apoptosis and autophagy damage in T2 DM combined with OSA,and to elucidate the role of HMGB1-TLR4 in it.Methods(1)The 12-week-old T2 DM model KKAy mice and normal C57 BL / 6J mice(C57 mice)were randomly divided into four groups: C57 group,C57+IH group,KKAy group,KKAy+IH group,C57+IH group.The KKAy+IH group received intermittent hypoxia(IH)exposure for 4 weeks.The oxygen concentration in the cycle were reduced from 21% to 5% for 30 seconds,then recovered to 21% for 90 seconds.The C57 group and C57+IH group received intermittent atmospheric oxygen(control)exposure to maintain a 21% oxygen concentration for 4 weeks.The mice were sacrificed and the brain tissues were taken for immunofluorescence and western blot after the exposure.(2)The HT22 cells were divided into four groups: the NC group,the IH group,the HG group and the HG + IH group.The NC group and the IH group were cultured with 25 m M(control)high glucose medium,the HG group and the HG + IH group were incubated in 50 m M high glucose medium for 16 h.The NC group and the HG group were subjected to intermittent normal oxygen circulation for 8 hours,and the oxygen concentration was maintained at 21%.The IH group and the HG + IH group were exposed to IH for 8 h,each cycle including 1.5% O2 5min and 21% O2 5min.After the treatment,immunofluorescence experiments and western blot experiments were performed.(3)HMGB1 si RNA was transfected into HT22 cells,and treated with HG and IH after 6 h of transfection,followed by western blot.Results:(1)The results of tissue immunofluorescence assay showed that compared with C57 group,the expression of LC3?Beclin1 were decreased and the expression of P62 ?cleaved-caspase3 was increased in the C57 + IH group,the KKAy group and the KKAy + IH group.Compared with the C57+IH group and the KKAy group,the above indicators were most significantly changed in the KKAy+IH group.There was no difference between the KKAy group and the C57+IH group.The expression of LC3 and Beclin1 were the lowest,and the expression of P62?cleaved-caspase3 and Bax/Bcl2 protein were significantly increased in KKAy+IH group by western blot.The protein expression levels of HMGB1 and TLR4 in the C57+IH group,the KKAy group and the KKAy+IH group were higher than those in the C57 group.HMGB1 and TLR4 protein expression were most pronounced in KKAy+IH group compared to the C57+IH group and the KKAy group.There was no difference between the C57+IH group and the KKAy group.(2)Cellular immunofluorescence experiments showed that compared with the NC group,the expression of LC3 and Beclin1 was decreased in the HG group and the IH group,and the expression of P62 and cleaved-caspase3 was increased,especially in the HG+IH group.The results of western blot showed that the expression of LC3 and Beclin1 protein was significantly decreased in the HG+IH group,and the expression of P62?cleaved-caspase3 and Bax/Bcl2 protein were significantly increased.And the expression of HMGB1 and TLR4 were obvious in the HG+IH group.(3)HT22 cells transfected with si HMGB1 reversed the effects of apoptosis and autophagy under the stimulation of HG and IH.The results of western blot analysis were as follows.Compared with the HG+IH+NC-si group,the expression of HMGB1 and TLR4 was decreased in the HG+IH+HMGB1-si group,the expression of LC3 and Beclin1 was increased,and the protein expressions of P62,cleaved-caspase3 and Bax/Bcl2 were decreased.Conclusion Combined with the results of previous experiments,the T2 DM model KKAy mice showed a tendency to enhanced apoptosis and weakened autophagy in hippocampal neurons after IH exposure,leading to serious cognitive and behavioral disorders.Under HG+IH stimulation,HT22 hippocampal neurons exhibit enhanced apoptosis and impaired autophagy.At the same time,HMGB1 was released in large amounts,activated and bound to TLR4,and HMGB1 si RNA was able to reverse the effects of HG+IH-induced apoptosis and autophagy damage.In conclusion,apoptosis and autophagy damage in T2 DM combined with OSA hippocampal neurons were achieved through the HMGB1-TLR4 signaling pathway.We propose that HMGB1 can be used as a therapeutic target to prevent or delay the occurrence of apoptosis and autophagy in hippocampal neurons caused by T2 DM combined with OSA,and may eventually improve cognitive dysfunction.