The Role And Mechanism Of Melatonin On Cerebral Ischemia Reperfusion Injury

ObjectiveIn this study,a Middle cerebral artery occlusion(MCAO)model was established to investigate the effect of melatonin on brain Ischemia/reperfusion(I/R)injury rats and its possible mechanism.MethodsNinety adult male Sprague-Dawley(SD)rats with body weight of 250-280 g were randomly divided into three groups: Sham group,I/R group and MT group.In Sham group,only blood vessels were isolated without thrombus insertion.MCAO was prepared in I/R group and MT group.In MT group,thrombus was pulled out and reperfusion was performed after 2h ischemia,and melatonin was injected 10mg/kg intraperitoneally.After the completion of reperfusion,the degree of neurological impairment was assessed and scored.TTC staining was used to measure cerebral infarction volume.Hematoxylin-eosin(HE)staining and Nissl staining were used to observe the changes in brain morphology and Niss L corpuscle of rats in each group.The activity of superoxide dismutase and the content of malondialdehyde were detected.The apoptosis of neurons in each group was observed by TUNEL cell apoptosis staining.The expression of TLR4 and MyD88 proteins was observed by immunofluorescence staining.The protein expressions of TLR4,MyD88,Cleaved Caspase-3 and Bcl-2 were determined by Western blot.Results1、Neurological function score results showed that rats in the Sham group had normal neurological function without neurological function defect.The neurological function of rats in I/R group and MT group was damaged,and the neurological function of MT group was recovered to a certain extent compared with the I/R group,and the difference was statistically significant(P<0.01).2、TTC results showed that the brain tissue of the Sham group was normal without infarct area.I/R group showed obvious infarct area,and MT group significantly reduced the infarct area volume compared with I/R group,and the difference was statistically significant(P<0.01).3、SOD activity in I/R group was significantly decreased and MDA content was significantly increased compared with Sham group with statistical significance(P<0.01);Compared with I/R group,SOD activity was increased and MDA content was decreased in MT group with statistical significance(P<0.01).4、HE staining results showed that the tissue structure of the Sham group was complete and normal,no obvious edema was observed,the morphology of neurons was complete,and the nucleoli were clear.Compared with the Sham group,the number of neurons in I/R group was significantly reduced.The necrosis of nerve cells and the vacuoles left after the necrosis were observed.Compared with the I/R group,the number of neurons in MT group increased,the vacuoles decreased,and the morphology was relatively regular.5、The results of Nissl staining showed that the nerve cells in the Sham group were arranged neatly,with relatively high expression of Nissl bodies and deep staining.Compared with Sham group,the arrangement of neurons in I/R group was reduced,the number of Nishi bodies was reduced or disappeared,and the staining was lighter.Compared with I/R group,MT group had darker staining,more orderly arrangement of nerve cells and more compact cell layers,and the number of Nishi bodies was not significantly reduced.6、TUNEL kit test results showed that the apoptosis rate of brain tissue sections in I/R group was the highest,and the apoptosis rate of MT group was lower than that in I/R group,with statistical significance(P<0.01).7、Western blot results showed that compared with the Sham group,the expressions of TLR4,MyD88 and Cleaved Caspase-3 were significantly increased in the I/R group,and the expression of Bcl-2 was significantly decreased,with statistical significance(P<0.01).Compared with I/R group,TLR4 and MyD88 expression were significantly decreased in MT group,Cleaved Caspase-3 and protein expression was significantly increased with statistical significance(P<0.01)in Bcl-2 with statistical significance(P<0.05).Conclusions1、The mechanism of cerebral ischemia-reperfusion injury involves TLR4/MyD88 signaling pathway.2、Melatonin may play a role in anti-oxidative stress and anti-apoptosis by regulating TLR4/MyD88 signaling pathway,so as to play a protective role in cerebral ischemia reperfusion injury.