Protective Effect And Mechanisms Of Melatonin/ATF6/CHOP Pathway In Secondary Brain Injury Of Intracerebral Hemorrhage

BACKGROUNDIntracerebral hemorrhage(ICH)is generally referred as spontaneous cerebral hemorrhage in the clinical practice.It is a cerebral vascular rupture process caused by various reasons,leading to intracerebral tissue or intraventricular hemorrhage.Trauma.and tumor-induced cerebral hemorrhage are generally not included in the scope of ICH,and are not the objectives of this study.ICH is the most common type of hemorrhagic stroke and has the highest rate of death and disability.The average incidence of ICH is 2.5/10,000 worldwide,45%of whom die within 1 month after acute onset,and 35%of patients survived with severe permanent disability,which means the overall mortality and morbidity rate of ICH is over 80%.ICH has become a serious challenge for the public health all over the world.In developing countries such as China,the incidence and prevalence rate are gradually increasing,thus posing a serious threat to the welfare of the publics and endangering social stability.Previous studies of ICH believed that the mechanical damage of the hematoma and the resulted brain tissue penumbra around the hematoma were the underlying mechanisms of brain damage after ICH.Therefore,basic and clinical studies focused on how to remove the hematoma and reduce the penumbra size.This view,however,after a long period of practice,has been proved unconvincing.It was found that the treatment based on this perspective did not significantly improve the prognosis of patients with ICH.In recent years,some scholars have studied the pathophysiological mechanisms of ICH,and proposed that brain damage after ICH can be separated into primary brain injury(PBI)and secondary brain injury(SBI).The former is the direct damage caused by hematoma,the latter is a collection of pathological processes and injuries induced by hematoma,including dysregulation of the innate immune system,iron toxicity,glutamate toxicity,free radical damage,inflammatory response,autophagy,neuronal apoptosis and white matter damage.By preventing or inhibiting SBI,the researchers demonstrated significant improvements in brain damage and neurological deficits in vitro and in vivo ICH models.Thus,the latest brain hemorrhage research focuses on how to improve SBI.The endoplasmic reticulum(ER)is an organelle with a unique topological membrane network system that provides a venue for protein modification,folding and assembly.Various insults that interfere with ER function can cause endoplasmic reticulum stress(ER stress).Inflammation,oxidative stress,mitochondrial calcium overload,toxic glutamate release and other pathophysiological processes can greatly activate ER stress response.Resultantly,overloaded proteins can further trigger unfolded protein response(UPR).UPR is mainly mediated by three types of ER stress proteins:inositol-requiring enzyme 1(Ire1),RNA activated protein kinase-like ER kinase(PERK)and activating transcription factor 6(ATF6).Among these proteins,ATF6 has been reported to play a key role in UPR,and ATF6 activates the downstream target C/EBP homologous protein(CHOP).CHOP is a transcription factor and is involved in ER stress-mediated apoptosis.Some scholars have reported that ATF6 inhibition provides neuroprotection in a model of cerebral ischemia,but the role of ATF6 has not been explored in ICH.Melatonin is an amine hormone secreted mainly by the pineal gland,which regulates the sleep-wake cycle.Recent studies have shown that in addition to sleep regulation,melatonin also has effects such as anti-oxidation,immune regulation,inhibition of inflammation and anti-apoptosis.Related studies of ischemic stroke have also confirmed that melatonin can reduce neuronal apoptosis and play a protective role in the brain.In summary,we hypothesized that inhibition of ATF6/CHOP-mediated ER stress by melatonin can alleviate neuronal apoptosis in brain injury secondary to ICH,and thereby exerting a neuroprotective effect.In this study,a rat model of autologous blood injection was used to establish a model of experimental ICH.The behavioral evaluation and molecular biological techniques were used to explore the protective effect and mechanisms of melatonin/ATF6/CHOP pathway.METHODSPART IArterial blood were taken through the femoral artery of adult male SD rats,and the experimental ICH model was established using stereotactic injection of autologous arterial blood into the right basal ganglia.Low dose(100mg/Kg)or high dose(150mg/Kg)of melatonin was injected intraperitoneally 1h after model establishment.Behavioral evaluation(24h/72h)and general observation(24h)were assessed to reveal whether melatonin exerts neuroprotective effects and whether the effects were dose-dependent.After confirming the dose-dependence and optimal dose of melatonin treatment,optimal dose was used to assess the mid-and long-term(1wk-3wk)neurological function in rats.PART ⅡSD rats were randomly divided into 7 groups:Sham group,ICH 3h(3 hours after ICH)group,ICH 6h group,ICH 12h group,ICH 24h group,ICH 48 group and ICH 72h group,respectively.Changes in ATF6 and CHOP on WB analysis at corresponding time points after ICH were studied.The sham operation group was treated exactly the same as the ICH group,except that there was no autologous blood injection.According to the expression changes of ATF6 and CHOP,the highest or lowest time point was selected and immunofluorescence was performed in order to explore the co-expression of ATF6 and CHOP and neurons.Based on these results,the research cross-section and the best time point to apply drug intervention for the next part of the study were determined.PART ⅢAs the continuation and deepening of part Ⅱ,this section focused on mechanism studies.SD rats were randomly distributed to Sham group,ICH+solvent control group(ICH+vehicle),ICH+melatonin optimal dose group(ICH+melatonin(150 mg/kg)),ICH+interfering RNA control group(ICH+scramble siRNA),ICH+CHOP interfering RNA group(ICH+siRNA CHOP),ICH+ATF6 interfering RNA group(ICH+siRNA ATF6).By selectively silencing CHOP or ATF6 expression by interfering RNA(siRNA)technology,their role in the molecular mechanisms could be verified.The RNA expression of ATF6/CHOP and downstream proteins was quantified by reverse transcription-polymerase chain reaction(RT-PCR)at the transcriptional level,and the expression of ATF6/CHOP and the downstream proteins was quantitatively studied by WB.The anti-apoptotic effect of melatonin and the effect of selective silencing of CHOP were verified at the cellular level by immunofluorescence staining and TUNEL staining of Cleaved caspase-3(activated caspase-3).RESULTSPART ⅠNeurobehavioral results showed severe neurological impairment after ICH,and both groups treated with melatonin showed improved short-term(24h/72h)and mid-and long-term(lw-3w)neurological severity scores and corner test scores,as well as improvement on foot fault test and the rotarod test.Moreover,the improvement in the high-dose group was more significant.Evans blue(EB)extravasation and brain water content were consistent with the former results.After ICH modeling,Evans blue significantly exudated through the blood-brain barrier,and the water content of the right cerebral hemisphere(treated side)increased significantly,suggesting that the blood-brain barrier was damaged and cerebral edema was obvious.The EB extravasation and the brain water content was remarkably reduced in the melatonin-treated groups,and the reduction in the high-dose group was more significant.Together,these results indicated that melatonin can effectively protect the integrity of blood-brain barrier and alleviate ipsilateral cerebral edema after ICH.Meanwhile,this protective effect is dose-dependent,and the high dose(150 mg/kg)is more favorable.PART ⅡThe results of WB showed that the expression of ATF6 and CHOP started to increase at 3h after ICH,and reached to a peak at 24h,and then decreased at 48h.Therefore,the 24h was selected as the observation time point,and the co-expression of ATF6 and the neuron marker(NeuN)was compared between the Sham group and the ICH 24h group.Immunofluorescence results showed that ATF was widely expressed in brain tissue and abundantly expressed in neurons,and the expression intensity of ATF6 appeared to increase at 24 hours after ICH,which was consistent with the results of WB.PART ⅢThe expression of MMP-9,ATF6 and CHOP increased significantly at 24h afterICH,suggesting pronounced blood-brain barrier destruction and ER stress response,respectively.After treatment with melatonin,the expression of the above three proteins was suppressed,indicating that melatonin exerted its neuroprotective effects through mitigating blood-brain barrier destruction and inhibiting ER stress.Compared with the scramble siRNA group,the expression of CHOP mRNA was significantly decreased in rats pretreated with CHOP siRNA,which proved that the inhibition of CHOP was effective and the ER stress was inhibited.At the same time,the expression of ATF did not change significantly,suggesting that ATF was not a downstream target of CHOP.It is worth noting that pretreatment of CHOP siRNA also down-regulated the expression of MMP-9,suggesting that activation of CHOP is a contributing factor to the destruction of the blood-brain barrier.The Bcl-2/Bax ratio was increased after treatment with melatonin,while the activation of Cleaved caspase-3 was decreased,indicating that melatonin has anti-apoptotic effect The CHOP siRNA pretreatment group showed similar changes compared with the scramble siRNA group,that is,the Bcl-2/Bax ratio was up-regulated and the Cleaved caspase-3 activation was inhibited.These results indicated that activation of CHOP not only promotes ER stress and blood-brain barrier disruption,but also increases apoptosis.Furthermore,pretreatment with ATF6 siRNA synchronously down-regulated the expression of ATF6,CHOP and cleaved caspase-3,whereas the Bcl-2/Bax ratio was increased,which demonstrated that inhibition of ATF6 can also effectively inhibit ER stress and apoptosis,and ATF6 is upstream of CHOP.RT-PCR analysis showed consistent results.While melatonin administration and ATF6 siRNA pretreatment reduced the mRNA concentration of ATF6,CHOP and caspase-3,CHOP siRNA pretreatment only reduced the mRNA level of CHOP and caspase-3,but not ATF6.The results of Cleaved caspase-3 and TUNEL fluorescence staining further confirmed that melatonin treatment or CHOP siRNA can significantly reduce the activation of caspase-3 and neuronal apoptosis.CONCLUSIONS1.After ICH,the neurological function of the rats was significantly impaired,the blood-brain barrier was destroyed,and brain edema occurred in the ipsilateral brain tissue.The administration of melatonin significantly improved the above-mentioned situation on a general level,and the improvement was dose-dependent.2.The expression levels of ATF6 and CHOP were significantly up-regulated 3h after ICH,then gradually increased,and reached the peak at 24 h.ATF6 is abundantly expressed in the brain and is co-expressed with neurons in large amounts.3.At 24h after ICH,the ATF/CHOP signaling pathway was significantly activated,accompanied by enhanced ER stress.Melatonin prominently inhibited the activation of this pathway,alleviated ER stress,and reduced apoptosis in ipsilateral cortex.Increased MMP-9 expression and impaired blood-brain barrier was also observed following ICH,which can be potently reversed by melatonin treatment.Selective silencing of CHOP or AFT6 gene expression can also downscale ER stress,reduce apoptosis and protect blood-brain barrier,suggesting that ATF6/CHOP activation underlies ER stress associated injuries,apoptosis and blood-brain barrier disruption after ICH.The protective effect of melatonin in SBI is most likely achieved by inhibiting the activation of ATF6/CHOP.