Inhibitory effect of melatonin on secondary brain injury in rats with cerebral haemorrhage and its mechanism
Objective To observe the inhibitory effect of melatonin on secondary brain injury(SBI)in rats with in-tracerebral haemorrhage(ICH)and its possible mechanisms.Methods Seventy-two SPF-grade healthy male rats were randomly divided into sham operation group,model group,melatonin group,and inhibitor group,with 18 rats in each group.In the model group,melatonin group and inhibitor group,we used the method of basal ganglia injection of collage-nase to establish the ICH models,and rats in the sham operation group were only inserted the needle after drilling;after the models were successfully established,rats in the melatonin group were injected with 30 mg/kg of melatonin intraperito-neally,rats in the inhibitor group were injected with 30 mg/kg of Luzindole intraperitoneally,and rats in the sham opera-tion and model groups were injected with equal volume of normal saline intraperitoneally,and the drugs were administered for 3 d.We evaluated the neurological functions of the rats in each group using the modified neurological deficit score(mNSS),cornering test(percentage of turning to the left)and forelimb placement test(rate of successful placement of the left forelimb).The dry-wet specific gravity method was used to detect the water content of the brain tissues.HE staining was used to observe pathological changes in the brain tissues.The levels of serum interleukin 18(IL-18)and interleukin 1β(IL-1β)were detected by ELISA.Western blotting was used to detect the protein expression levels of NOD-like recep-tor protein 3(NLRP3),cysteine aspartate protein hydrolase 1(Caspase-1),and gasdermin D(GSDMD)in the brain tis-sues.We detected the mRNA expression of NLRP3,Caspase-1,and GSDMD in brain tissues by real-time fluorescent quantitative PCR.Double immunofluorescence staining(GSDMD-positive microglia number)and TUNEL staining(TU-NEL-positive cell rate)were used to observe pyroptosis in the brain tissues.Results The model group showed visible in-flammatory cell infiltration,nerve cell swelling and loss of cell nuclei,and disorderly cell arrangement.Compared with the model group,inflammatory cell infiltration and cell swelling decreased,cell arrangement was relatively neat,and cell con-solidation and lysis were reduced in the melatonin group and the inhibitor group,and the changes were more obvious in the melatonin group.Compared with the sham operation group,the percentage of turning to the left and the rate of successful placement of the left forelimb decreased in the melatonin group,the inhibitor group,and the model group(all P<0.05);compared with the model group and the inhibitor group,the percentage of turning to the left and the rate of successful place-ment of the left forelimb increased in the melatonin group(all P<0.05).The mNSS,brain tissue water content,serum IL-18,serum IL-1β levels,the number of brain tissue GSDMD-positive microglia,brain tissue TUNEL-positive cell rate,as well as the mRNA and protein relative expression levels of NLRP3,Caspase-1,and GSDMD in the brain tissues of rats in the sham operation group,melatonin group,inhibitor group,and model group sequentially increased,with statistically sig-nificant difference between every two groups(all P<0.05).Conclusion Melatonin attenuates the SBI condition in ICH rats,and the mechanism may be related to the reduction of microglial pyroptosis and attenuation of neuronal inflammatory response by inhibition of NLRP3/Caspase-1/GSDMD pathway.
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