| Objective:to observe the occurrence and development of early brain injury(EBI)after SAH and the expression of inflammatory factors(MyD88?TRIF?IL-6?IL-1??TNF-?)in Toll-like receptor 4(TLR4)signaling pathway by establishing a rat model of spontaneous subarachnoid hemorrhage(SAH),to investigate the mechanism of inhibition of inflammatory response and the protective effect of nerve damage during the EBI of SAH by the method of giving TAK-242 or placebo.Methods:70 healthy adult Sprague-Dawley rats were randomly allocated into 7 groups,normal controlgroup(n=10),SAH 24hgroup(n=10),SAH 72h group(n=10),SAH+TAK-242 24h group(n=10),SAH+TAK-242 72h group(n=10),SAH+vehicle24hgroup(n=10),SAH+vehicle 72h group(n=10).Except normal group,all SAH rats in the other groups were subjected to injecting the blood into the prechiasmatic cistern,and the rats in the SAH+TAK-242 groups were giving the treatment of TAK-242 after SAH,the rats in the SAH+vehicle groups were giving the treatment of palcebo after SAH.ALL the rats were killed at corresponding time points(24h or 72h),the brain tissue were used to detected the MyD88 and the TRIF levels by the method of Western blot and RT-PCR,the blood serum was used to estimated the levels of IL-6,IL-1? and TNF-? by the method of ELISA,Tunel and Nissl methods were used to evaluate the nerve cells apoptosis and survival.All the results were statistically analyzed by the software of IBM SPSS Statistics 26.Results:1.The mNSS score was higher in SAH 24h group(12.90±1.79)and SAH 72h group(8.70±0.95)compared with normal control group(1.20±0.42)(P<0.01);after SAH 24hs,the mNSS score was higher in SAH 24h group and SAH+vehicle 24h group(13.50±1.58)compared with SAH+TAK-242 24h group(9.50±0.85)(P<0.01);after SAH 72hs,the mNSS score was higher in SAH 72h group and SAH+vehicle 72h group(9.33±0.95)compared with SAH+TAK-242 72h group(6.70±0.95)(P<0.01).TAK-242 can improve the mNSS score of rats after SAH.2.MyD88 expression in normal control group(0.70±0.01)was low(P<0.01),the expression of MyD88 in the brain tissue of SAH rats increased,the expression of MyD88 was lower in normal control group compared with SAH 24h group(2.09±0.04,P<0.01)and SAH 72h group(1.43±0.11,P<0.05);after SAH 24hs,the expression of MyD88 was lower in SAH+TAK-242 24h group(1.65±0.26)compared with SAH 24h group and SAH+vehicle 24h group(2.01±0.17)(P<0.01,P<0.05);after SAH 72hs,the expression of MyD88 was lower in SAH+TAK-242 72h group(1.07±0.12)compared with SAH 72h group and SAH+vehicle 72h group(1.45±0.29)(P<0.01,P<0.05).TAK-242 can reduce MyD88 expression in rats after SAH.3.The TRIF expression of rat brain tissue after SAH was significantly higher than that of normal rats,the expression of TRIF was lower in normal control group(1.00±0.02)compared with SAH 24h group(4.79±0.16)and SAH 72h group(3.56±0.28,P<0.01);after SAH 24hs,the expression of TRIF was lower in SAH+TAK-242 24h group(2.76±0.21)compared with SAH 24h group(P<0.05)and SAH+vehicle 24h group(4.70±0.23,P<0.01);after SAH 72hs,the expression of TRIF was lower in SAH+TAK-242 72h group(1.92±0.28)compared with SAH 72h group and SAH+vehicle 72h group(3.52±0.29,P<0.01).TAK-242 can reduce TRIF expression in rats after SAH.4.The contents of serum IL-1?,IL-6,TNF-? were lower in normal control group compared with SAH 24h group and SAH 72h group(P<0.01);after SAH 24hs,the contents of serum IL-1?,IL-6,TNF-? were lower in SAH+TAK-24224h group compared with SAH 24h group and SAH+vehicle 24h group(P<0.01);after SAH 72hs,the contents of serum IL-1?,IL-6,TNF-? were lower in SAH+TAK-242 24h group compared with SAH 72h group and SAH+vehicle 72h group(P<0.01).TAK-242 can reduce the contents of serum IL-1?,IL-6,TNF-? in rats after SAH. normal control group(IL-1?:1.71±0.05,IL-6:14.91±0.14,TNF-?:14.44±0.95,pg/ml),SAH 24h group(IL-1?:4.54±0.03,IL-6:55.15±0.68,TNF-?:55.19±1.07,pg/ml),SAH 72h group(IL-1?:3.62±0.03,IL-6:32.04±1.85,TNF-?:28.27±0.79,pg/ml),SAH+TAK-242 24h group(IL-1?:2.69±0.03,IL-6:27.88±0.14,TNF-?:22.76±1.01,pg/ml),SAH+TAK-242 72h group(IL-1?:2.11±0.04,IL-6:24.29±1.26,TNF-?:18.08±0.68,pg/ml),SAH+vehicle 24h group(IL-1?:4.57±0.01,IL-6:54.96±1.31,TNF-?:55.87±0.72,pg/ml),SAH+vehicle 72h group(IL-1?:3.61±0.06,IL-6:31.68±1.14,TNF-?:27.93±0.72,pg/ml).5.After SAH,the apoptosis of neurons in rat brain tissue increased,and the damage of neurons was more obvious than that in normal rats,the difference of apoptosis rate per field between normal control group(2.8%)and SAH 24h group(16.94%)and SAH 72h group(21.32%)was statistically significant(P<0.05);after SAH 24hs,the apoptosis rate was lower in SAH+TAK-242 24h group(9.67%)compared with SAH 24h group and SAH+vehicle 24h group(17.29%)(P<0.05);after SAH 72hs,the apoptosis rate was lower in SAH+TAK-242 72h group(11.11%)compared with SAH 72h group and SAH+vehicle 72h group(21.75%)(P<0.05).TAK-242 can reduce apoptosis level of rats nerve cell after SAH.6.More Nissl bodies were observed in rats brain tissue in normal control group,Nissl bodies were decreased in brain tissue after SAH,in normal control group(49.20±1.79)Nissl bodies were more than those in SAH 24h group(11.50±0.85)and SAH 72h group(5.90±0.74)(P<0.01);after SAH 24hs,in SAH 24h group and SAH+vehicle 24h group(10.80±0.92)Nissl bodies were lower compared with SAH+TAK-242 24h group(31.10±1.79)(P<0.01);after SAH 72hs,in SAH 72h group and SAH+vehicle 72h group(4.80±1.14)Nissl bodies were lower compared with SAH+TAK-242 72h group(26.00±1.41)(P<0.01).TAK-242 can improve neuronal survival in rats brain tissue after SAH.Conclusion:TAK-242 reduces the expression of inflammatory factors and the inflammatory effect of EBI after SAH by inhibiting TLR4,indirectly improving the neuron damage caused by EBI,and finally realizing the early brain protection after SAH. |
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