Role Of TLR4/NF-κB Signaling Pathway In Hydrogen-rich Water Ameliorating Radiation-induced Brain Injury In Rats

Objective:The role of TLR4/NF-κ B signaling pathway in the amelioration of radiation-induced brain injury induced by hydrogen-rich water was investigated by whole brain irradiation in rats.Methods:Thirty male SD(Sprague Dawley)rats were selected as the study subjects and randomly divided into control group,irradiation group(IR group),and hydrogen-rich water intervention group(IR+HRW group)according to their body weight,with 10 rats in each group.The control group was not irradiated and was given purified water(20ml/kg)by gavage every day.IR group and IR+HRW group received a single 20 Gy whole brain irradiation,3 days before irradiation,10 min before irradiation and 30 days after irradiation were given purified water/hydrogen-rich water(20 ml/kg)by continuous gavage.The general condition of the rats was observed every day,and the body weight were measured on the 7th,14 th,21st,and 30 th days after irradiation.Half of the animals were dissected on day 7 after irradiation,and the remaining animals were dissected after the Morris water maze to detect the learning and memory ability of rats on day 30 after irradiation,HE staining was used to detect the pathological changes of the hippocampus.The GSH activity,MDA,IL-1 β and hydroxyl radical content in brain tissue were detected by ELISA.q RT-PCR and Western blotting were used to detect m RNA and protein expression levels of TLR4,NF-κ B,NLRP3 and CASPASE 1 in the hippocampus of rats.Results:1.7 days after irradiation,the rats in the IR group had symptoms such as head hair removal and salivation,and the symptoms such as head hair removal and salivation of the rats in the IR+HRW group were alleviated compared with those in the IR group.Compared with the body weight on the day of irradiation,the body weight of rats in the IR group tended to decrease on the 7th day after irradiation,while that of rats in the IR+HRW group tended to increase,but none of the differences were statistically significant(P>0.05).HE staining showed that compared with the IR group,the cells in the IR+HRW group were relatively orderly arranged,with nuclear shrinkage,fragmentation and lysis,and the dark staining of cytoplasm was relatively alleviated.The results of ELISA experiment showed that the GSH activity in the brain tissue of the rats in the IR+HRW group was increased and the contents of MDA,hydroxyl radicals and IL-1β were decreased compared with those in the IR group,but the differences were not yet statistically significant(P>0.05).The results of q RT-PCR experiment showed that the expression levels of TLR4,NF-κB,NLRP3,Caspase 1 m RNA were decreased in the hippocampus of rats in the IR+HRW group compared with the IR group(P<0.05),and the results of Western blotting experiment showed that compared with the IR group,the expression levels of NF-κB p65,NLRP3,and CASPASE 1 protein had a tendency to decreased(P<0.05)and TLR4 protein expression levels had a tendency to decrease,but the differences were not statistically significant(P>0.05).2.30 days After irradiation,the rats in the IR group had symptoms such as head hair removal and salivation,while the symptoms of the rats in the IR+HRW group were milder.No animal died in the control and IR+HRW groups,while one rat died in the IR group.From day 14 to day 30 after irradiation,the body weight of the rats in the IR+HRW group tended to be higher than those in the IR group,but the differences were not statistically significant(P>0.05).The results of the Morris water maze showed that,except for day 3,there was a tendency to shorten the escape latency in the IR+HRW group relative to the IR group from day 1 to day 5,but the difference was not yet statistically significant(P>0.05),and the time taken to reach the original position of the platform after the platform was removed on day 6 decreased,and the number of crossing the platform and the time spent in the original platform quadrant increased(P<0.05).HE staining showed that the number of hippocampal cells in IR+HRW group was slightly reduced and arranged neatly,without obvious nuclear hyperchromatic and pyknotic phenomenon were observed.The results of ELISA experiments showed that the content of MDA and hydroxyl radicals in the brain tissue of rats in the IR+HRW group was reduced compared with that of the IR group(P<0.05),and the activity of GSH was increased and the content of IL-1 β was reduced,but the differences were not yet statistically significant(P>0.05).The results of q RT-PCR experiment showed that the expression levels of TLR4 and Caspase 1 m RNA in the hippocampus of rats in IR+HRW group decreased(P<0.05),and the expression levels of NF-κB and NLRP3 m RNA had a tendency to decrease compared with the IR group,but the differences were not statistically significant(P>0.05).The results of Western blotting experiments showed that the expression levels of TLR4 and CASPASE 1 proteins were decreased in the hippocampus of the IR+HRW group rats compared with the IR group(P<0.05),and the expression levels of NF-κ B p65 and NLRP3 proteins tended to decrease,but the differences were not yet statistically significant(P>0.05).Conclusions:1.Ionizing radiation can cause differences in TLR4,NF-κB,NLRP3,CASPASE 1m RNA,and protein expression in rats,suggesting that ionizing radiation may cause pyroptosis of brain cells through TLR4/NF-κB signaling pathway,leading to radiation-induced brain injury in rats.2.Hydrogen-rich water can improve radiation brain injury in rats induced by ionizing radiation,and its mechanism may be related to regulating TLR4/NF-κB signaling pathway,inhibiting inflammatory factors and weakening oxidative stress response.