Protective Effect Of Perillaldehyde On Spinal Cord Ischemia-reperfusion Injury Via Regulation Of The Nrf2/HO-1 Signaling Pathway And Its Mechanisms

Objective:Spinal Cord ischemia-reperfusion injury（SCII）is a secondary nerve injury caused by primary spinal cord injury,which leads to motor and sensory dysfunction,paraplegia,paralysis or even death.Many clinical studies are dedicated to preventing spinal cord injury caused by spinal cord ischemia,yet current drugs such as Nimodipine,Mexiletine fail to have a good therapeutic effect.Some studies have found that microglia,the main immune cells in SCII site,are over-activated and induce neuronal death by phagocytosis or secretion of pro-inflammatory cytokines,thus aggravating the secondary nerve injury caused by SCII.Some drugs against SCII,such as dexmedetomidine and sevoflurane,also improve motor and sensory function of SCII mice by inhibiting the activity of microglia in SCII site.Besides,oxygen radical-mediated lipid peroxidation damage and inflammation in microglia are the two main mechanisms of SCII.Therefore,it is a potential method to inhibit oxidative stress and inflammatory response in microglia to improve motor and sensory function in SCII site.Perillaldehyde（PAH）is a natural monoterpene active extracted from Perilla frutescens that possesses a variety of pharmacological activities including anti-inflammation,anti-oxidation,vascular protection,and neuroprotection.Previous reports have suggested that PAH can ameliorate cerebral ischemia-reperfusion injury in rats.Mechanistically,PAH can exert a protective effect by activating the Nrf2/HO-1 signaling pathway in different disease models,which proves that Nrf2/HO-1 signaling pathway may be one of the potential targets for PAH to act.Meanwhile,the inhibitory effect of Nrf2/HO-1 signaling pathway activation on the activation of NLRP3 inflammasome has been reported in microglia of cerebral ischemia-reperfusion injury model.However,whether PAH ameliorates the oxidative stress and inflammatory responses in SCII progression via the Nrf2/HO-1 signaling pathway has not been confirmed.In view of the above investigation,our study intended to explore the functional role and molecular mechanism of PAH on inflammatory response and oxidative stress in SCII progression.This study will be divided into two sections,the initial segment aims to establish a rat model of SCII and explore whether PAH has a therapeutic effect on SCII.The subsequent part aimed to established oxygen and glucose deprivation/reoxygenation（OGD/R）-induced BV2 microglia model to investigate the effect of PAH on Nrf2 and NLRP3 activation,as well as to verify whether PAH could alleviate SCII symptoms via activating the Nrf2/HO-1 signaling pathway in microglia to suppress inflammatory response and oxidative stress.Methods:1 Part one:Protection effect of PAH on SCII mice model1.1、SCII rat model and drug treatmentMale Sprague-Dawley（SD）rats（180-200 g）were obtained from Liaoning Changsheng Biotech.All the procedures in animal experiments adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals（eighth edition,2011）and this protocol was approved by the First Hospital of China Medical University.All animals were free to drink and eat under 22±1°C,45%-55%humidity conditions with a 12-h light/dark cycle.Following 1 week for adaptation,rats were randomly divided into four groups（n=6 rats/group）:the sham group,the SCII group,the low-dose PAH group（PAH-L）,and the high-dose PAH group（PAH-H）.Based on a previous study,the PAH-L and PAH-H group rats were given intragastrical with 36 mg/kg and 72 mg/kg PAH for 7 consecutive days,while the other group rats obtained equal volume of vehicle.After the last administration,rats except the sham group were subjected to the SCII procedure according to a previous study.Under anesthetized with 100 mg/kg pentobarbital sodium,rats were fixed in a supine position and aorta was exposed from left renal artery to bifurcation of aorta,maintaining body temperature at 36.5±0.5°C.Five minutes before clamping aorta,rats were intravenously injected with heparin（130 U/kg）for anticoagulation.Two bulldog clamps were used to cross-clamp aorta from a point immediately inferior to the left renal artery to aortic bifurcation.Reduced blood flow was confirmed by the disappearance of distal femoral artery pulsation.After clamping for 30 min,blood flow was restored by removing bulldog clamps,and then the wound was sutured.Sham rats received the surgical procedure described above without SCII.Rats,except used for behavioral testing,were euthanized with 200 mg/kg pentobarbital sodium 24h after reperfusion.The lumbar spinal cord（L2-L5 segments）of rats was quickly removed for subsequent experiments.1.2、Neurological evaluationMotor function in each group of rats was assessed by the Basso,Beattie,and Bresnahan（BBB）grading scale,which ranges from 0-point for complete paraplegia to21-point for normal locomotion.The scores between 1 and 8 indicate rat limb movements that cannot stand or support weight,representing the early phase of recovery.The scores between 9 and 13 indicate rats that can stand and then step with different degrees of forelimb-hindlimb coordination,representing the intermediate phase of recovery.The scores between 14 and 20 indicate rats that are good walkers and gradual improvements in foot placement,toe clearance,tail position,and trunk stability,representing the late phase of recovery.BBB score was calculated at 1,2,3,and 7 d after reperfusion in a blinded manner.1.3、Hematoxylin-Eosin（HE）staining was used to evaluate the change of spinal cord tissueSpinal cord tissues obtained from each group of rats were fixed with 4%paraformaldehyde and embedded in paraffin.Tissues were then cut into 5μm-thick slices and deparaffinized.Slices were stained with Hematoxylin for 5 min and Eosin for 3 min,as well as dehydrated with ethanol and xylene.After mounting with neutral balsam,slices were observed under 40×light microscope.1.4、Nissl staining analysis of neuronal damage of spinal cord tissuesDeparaffinized spinal cord tissues of 5-μm thickness were stained with 0.5%cresyl violet for 10 min at room temperature and differentiated in 0.25%acetate ethanol for seconds.After dehydrating with ethanol and xylene,slices were observed under an Olympus BX53 light microscope at 40×magnification.1.5、The expression of NLRP3 inflammasome,cleaved caspase-1,and the Nrf2/HO-1/NF-κB pathway-related proteins in spinal cord tissues was determined by Western blotting.1.6、ELISA kits were employed to measure the levels of NSE,IL-1βand IL-18 in serum samples and the expression of IL-1βand IL-18 in the spinal cord tissues of each group of rats.1.7、Commercial kits were applied to examine the expression of oxidative stress indicators（SOD,mn-SOD,GSH-Px,MDA,and CAT）in spinal cord tissues.1.8、Double immunofluorescence was used to evaluate the expression of Nrf2 and microglia cell marker Iba-1 in spinal cord tissues.2 Part two:The regulatory effect of PAH on the oxidative stress and inflammation in microglia via activation of the Nrf2/HO-1 signaling pathway in vitro2.1、Cell model and drug treatmentFor OGD/R procedure,BV2 cells were switched to glucose-free DMEM and then incubated at 37°C in an anaerobic chamber containing 94%N₂,5%CO₂,and 1%O₂for 6 h.After OGD exposure,cells were returned to high glucose medium with normal air for 24-h reoxygenation.To investigate the effect of PAH on microglial activation,the 500μM PAH was used to pretreat the BV2 cells 30 min before OGD/R exposure.2.2、The toxic effect of PAH on BV2 cells was determined by MTT assayAt 90%confluency,BV2 cells were plated into a 96-well plate（4×10³ cells/well）and incubated at 37°C with 5%CO₂ in DMEM supplemented with different concentrations of PAH（0,0.01,0.1,1,10,50,100,500μM）for 30.5 h.Subsequently,cell viability was measured using the MTT assay kit according to the manufacturer’s protocols.2.3、si RNA was targeted to decrease the expression of Nrf2 in BV2 cellsA small interference sequences（si RNA）of Nrf2（si Nrf2）and its negative control（si NC）were obained from JTS scientific.BV2 cells were seeded in a 6-well plate and incubated overnight at 37°C with 5%CO₂.When achieved 70%confluency,cells were transfected with 100 pmol si Nrf2 or si NC using Lipofectamine 2000 following the manufacturer’s instructions.2.4、Western blotting was used to detect the expression of NLRP3 inflammasome,cleaved caspase-1,Nrf2,HO-1,p-NF-κB p65,and NF-κB p65 in BV2 cells.2.5、ELISA kits were employed to measure the levels of IL-1βand IL-18 in BV2cells.2.6、Commercial kits were performed to examine the expression of SOD,mn-SOD,GSH-Px,MDA,and CAT in BV2 cells.Results:1 Part one:Protection effect of PAH on SCII mice model1.1、PAH improves neurologic motor function and histopathologic injury of SCII rats Compared with sham group,the BBB score of SCII group was significantly lower at 1,2,3,and 7 d after reperfusion.However,low and high doses of PAH efficiently elevated the BBB scores with the prolonging of time.Next,the level of serum NSE in the SCII group was enhanced and high doses of PAH significantly decreased the serum NSE level.Results of HE staining revealed an obvious loss of motor neurons in the SCII group,whereas more intact motor neurons with a fine granular cytoplasm were observed in PAH-treated SCII group.The results of Nissl staining demonstrated that the number of Nissl bodies was markedly decreased after SCII,but treatment with PAH greatly alleviated neuron injury,exhibited by increased number of Nissl bodies.1.2、PAH inhibits NLRP3 inflammasome activation of SCII ratsCompared with sham group,the protein levels of NLRP3 and cleaved caspase-1 in spinal cord tissues of the SCII rats were significantly increased,while treatment with PAH downregulated both proteins expression.In addition,the levels of pro-inflammatory cytokines IL-18 and IL-1βwere significantly decreased after treatment with PAH.1.3、PAH suppresses oxidative stress of SCII ratsCompared with the sham group,SCII-induced rats underwent severe oxidative damage as revealed by the increase of MDA level,as well as the reduction of SOD,mn-SOD,GSH-Px and CAT contents,which were reversed after PAH treatment.Meanwhile,the results of Western blotting showed that the protein levels of Nrf2 and HO-1 were significantly lower than those in the sham group,while these protein levels were elevated after PAH treatment.immunofluorescence results confirmed that treatment with PAH significantly upregulated the expression of Nrf2.Besides,the structure merge of Nrf2 and microglia cell marker Iba-1 displayed that Nrf2expression was not directly colocalized but closely associated with Iba-1.Subsequently,we found that the expression of p-NF-κB p65 was remarkably upregulated in spinal cord tissues after SCII.PAH treatment efficiently inhibited phosphorylation of the spinal p-NF-κB p65.2 Part two:The regulatory effect of PAH on the oxidative stress and inflammation in microglia via activation of the Nrf2/HO-1 signaling pathway in vitro2.1、PAH activates the Nrf2 pathway in OGD/R-induced BV2 cellsThe results of MTT assay showed that BV2 cell viability did not change much when PAH concentration was in the range of 0-100μM,while it was notice ably reduced when PAH concentration was 500μM,suggesting that PAH concentration at 500μM was toxic to BV2 cells.Meanwhile,the protein level of Nrf2 was significantly decreased after transfected with si Nrf2 into BV2 cells.In addition,the protein levels of Nrf2 and HO-1 were markedly decreased under OGD/R stimulation,and increased after PAH treatment.However,this increase was greatly inhibited after Nrf2knockdown.The results of immunofluorescence confirmed that PAH activated the Nrf2 pathway in OGD/R-induced BV2 cells.Results of western blotting revealed the phosphorylation of NF-κB p65 in BV2 cells after OGD/R stimulation,but this effect was impeded by PAH administration.Furthermore,MDA content in OGD/R-induced BV2 cells was obviously increased,while SOD,mn-SOD,GSH-Px and CAT activities were decreased.PAH treatment significantly increased SOD,mn SOD,GSH-Px and CAT activities as well as decreased MDA content,but these effects were reversed after Nrf2 knockdown.2.2、PAH inhibits NLRP3 inflammasome activation by activating the Nrf2 pathway in vitroCompared with the control group,the expression of NLRP3 and cleaved caspase-1was increased obviously in OGD/R-induced BV2 cells,and PAH treatment downregulated their expression.However,expression levels of NLRP3 and cleaved caspase-1 were remarkably upregulated under PAH intervention after transfected with si Nrf2.Besides,the levels of IL-18 and IL-1βin OGD/R-induced BV2 cells were increased,but decreased after PAH intervention.After transfected with si Nrf2,the levels of IL-18 and IL-1βwere increased again.Conclusion:1、SCII model rats suffered severe neurological motor dysfunction and histopathological damage as evidenced by the loss of motor neurons and reduction of Nissl bodies.2、PAH administration significantly improved motor dysfunction and neuronal damage in SCII model rats by inhibiting oxidative stress and inflammatory response in spinal cord tissue.3、PAH treatment at the concentration of 500μM exerted significant toxic effects in microglia BV2.4、PAH suppressed the oxidative stress in OGD/R-induced BV cells via activation of the Nrf2/HO-1 signaling pathway.5、PAH inhibited NLRP3 inflammasome-induced inflammation in BV2 cells exposure to OGD/R by blocking the NF-κB signaling pathway.