Neuroprotective Effect Of Hydrogen-rich Saline In Focal Cerebral Infarction Mice And Its Anti-inflammatory Mechanism

Ischemic stroke has become the leading cause of death in China.Due to its high morbidity,mortality and disability,it has brought heavy economic burden and life stress to the population.Currently,thrombolytic therapy and intervention treatments in the early stage have significant effects,but the application is limited due to strict time windows.Therefore,there is an urgent need to find effective drugs to improve the neurological function recovery and prognosis of stroke patients.The inflammatory response following cerebral ischemia is one of the key mechanisms leading to brain damage,in which inflammasomes play a crucial role and are integral components of the innate immune system.The NOD-like receptor family Pyrin Domain Containing 3(NLRP3)is currently known as the most characteristic inflammasome.The activation of NLRP3 can trigger the assembly of the apoptosis-associated speck-like protein(ASC),which contains the CARD domain,and the caspase precursor Pro-Caspase-1into a cytoplasmic protein complex,the NLRP3 inflammasome.Subsequently,the activation of pro-Caspase-1 through self-proteolysis leads to the cleavage of Pro-interleukin 1β(IL-1β),promoting the inflammatory response.Some studies have found that the increased production of ROS is a key factor in the activation of NLRP3 inflammasome.In addition,research has found that the ROS scavengers N-acetylcysteine(NAC)can block the activation of NLRP3 inflammasome.Currently,NLRP3 is the most widely studied inflammasome in central nervous system disease.Therefore,a proper understanding of NLRP3 inflammasome is essential for finding new anti-inflammatory drugs.In recent years,research on the mechanism of hydrogen has made rapid progress.Hydrogen is a new medium with strong anti-inflammatory,antioxidant,and anti-apoptotic effects.Hydrogen has been found to have protective effects in multiple organs such as the brain,heart,and gastrointestinal tract.Hydrogen-rich saline(HS)is currently widely used and has a neuroprotective effect on ischemic injury.The experimental results showed that intraperitoneal injection of hydrogen-rich saline could protect mice from experimental acute pancreatitis by inhibiting NLRP3 inflammasome.However,there are few studies on whether HS can play a neuroprotective role in cerebral infarction through ROS-NLRP3 inflammasome signaling pathway.Therefore,in this study,we observed whether hydrogen-rich saline could exert its neuroprotective effect on focal cerebral infarction in mice by inhibiting the activation of the ROS-NLRP3 inflammasome signaling pathway.Firstly,we collected outpatient normal population and acute cerebral infarction inpatients in our department to explore the levels of NLRP3 inflammasome expression in acute cerebral infarction patients.Subsequently,we established an mouse model of distal middle cerebral artery occlusion(d MCAO)and lipopolysaccharide(LPS)induced BV2 microglia cells in vitro.We discussed whether hydrogen-rich saline can relieve brain injury by inhibiting the ROS-NLRP3 inflammasome signaling pathway and promote neural function recovery.This experiment is divided into four parts,and each part is summarized as follows.Part 1 Expression level and clinical significance of NLRP3 inflammasome in Patients with cerebral infarctionObjective: To analyze the m RNA level of NLRP3 inflammasome in patients with different degrees ofacute cerebral infarction compared to the normal control group.Methods: A total of 62 patients with cerebral infarction admitted to our department from January 2021 to December 2021 were selected as the case group according to the inclusion and exclusion criteria,while 60 healthy individuals were selected as the control group during the same period.All patients in the case group underwent head MRI+MRA+DWI examination to confirm the diagnosis of acute cerebral infarction and their disease severity was evaluated based on the National Institutes of Health Stroke Scale(NIHSS)score.The patients were then divided into three groups according to their NIHSS scores,with 21 cases in the mild group(NIHSS score 0-4),20 cases in the moderate group(NIHSS score 5-15),and 21 cases in the severe group(NIHSS score ≥16).Fasting blood samples were collected from both groups,and RT-q PCR was used to determine the m RNA level of NLRP3 inflammasome.SPSS21.0 was used for statistical analysis.Results:Compared with the control group,the m RNA level of NLRP3 inflammasome in the case group was significantlyincreased(P < 0.05).The statistical results of NLRP3 m RNA level in patients with different degrees of cerebral infarction showed that as the severity of cerebral infarction increased,the m RNA level of NLRP3 gradually increased.The m RNA level was lowest in the mild group,highest in the severe group(P < 0.05).What’s more,the m RNA level of NLRP3 was positively correlated with NIHSS score(r = 0.78,P < 0.001).Conclusion: The NLRP3 m RNA level increased in patients with cerebral infarction,and the level gradually increased with the severity of the disease,which was closely related to the severity of the disease.Part 2 Neuroprotective effects of hydrogen-rich saline on focal cerebral ischemia in miceObjective: to investigate the protective effect of hydrogen-rich saline on neurological function in mice with focal cerebral ischemia.Methods: Male C57BL/6 mice(21-25g)were used to establish the d MCAOmodel using electrocoagulation.In the study,mice were divided into two groups: d MCAO groupand HS group(d MCAO+10 ml/kg/day HS).Animals received continuous intraperitoneal injection of hydrogen-rich saline for 3 days before and after surgery,while the d MCAO groups received the same amount of physiological saline.Modified neurological severity score(m NSS),Rotarod test and weight monitoring were used to evaluate neurological function in each experimental group at day 1 and 3 after stroke.The brain infarct volume was measured using TTC staining at day 3 after stroke,and the cerebral blood flow of the infarct hemisphere was monitored by laser speckle imaging on day 1 and 3 after stroke.Results:1.The m NSS showed that there was no significant difference in neurological function between the groups before cerebral ischemia.However,the neurological function in the d MCAO group significantly decreased after stroke,while the HS group showed improved neurological function.Compared with the d MCAO group,a significant difference was observed in the HS group at day 3(P < 0.05).2.Rotarod test results showed that,compared with the d MCAO group,the HS group displayed a significant improvement of neurological function on day 3(P < 0.05).However,there was no statistical significance between the two groups at day 1(P > 0.05).3.Weight monitoring results showed that the weight of mice lost after operation in the three groups.Compared with the d MCAO group,the weight of mice in HS group increased at each time point,but there was no statistical significance(P > 0.05).4.TTC staining results showed that there was no infarct in the Sham group.Compared with the d MCAO group,the infarct volume was significantly decreased in the HS group(P < 0.01).5.Cerebral blood flow: The blood flow in the right cerebral hemisphere of mice immediately decreased(P< 0.01).Compared with the d MCAO group,the cerebral perfusion in the right cerebral hemisphere of mice in HS group recovered faster,with statistical significance on day 3(P < 0.05).Conclusion: Intraperitoneal injection of HS in mice can improve behavioral scores,reduce infarct volume,promote cerebral blood flow recovery and improve neurological function.Part 3 Effect of Hydrogen-rich Saline on ROS-NLRP3 Signaling Pathway in d MCAO MiceObjective: To determine the levels of the ROS-NLRP3 signaling pathway and the inhibitory effects of hydrogen-rich saline in mice with focal cerebral artery occlusion.Methods: The animal model is similar to the second part.The animals were randomly divided into three groups: Sham group,d MCAO group,and HS group(d MCAO + 10ml/kg/day HS).One day after surgery,RT-q PCR,Western blot,and laser confocal microscopy techniques were used to detect the m RNA and protein levels of NLRP3,Caspase-1,and IL-1β in each group and the number of positive cells in brain tissue slices.The DCFH-DA fluorescent probe was used to detect the expression level of ROS in brain tissue.Results:1.Changes in the localization of NLRP3 and Caspase-1: Laser confocal microscopy was used to determine the localization of NLRP3 and Caspase-1expression in the cortex.In normal brain tissue(Sham group),NLRP3 and Caspase-1 were mainly expressed in the nucleus,but after d MCAO,they were mainly expressed in the cytoplasm.2.Hydrogen-rich saline inhibited the expression of ROS,NLRP3,caspase-1,and IL-1β in brain tissue.1)Following d MCAO,the ROS expression was notably increased(P <0.001),while after HS treatment,itwas decreased(P < 0.001).2)Western blot results showed that the protein level of NLRP3,Caspase-1(P20),and Cleaved IL-1β were significantly increased(P < 0.01)in the d MCAO group compared to the Sham group.The expression of NLRP3,Caspase-1(P20),and Cleaved IL-1β was significantly reduced in the HS group(P < 0.05).3)PCR results showed that compared to the Sham group,the m RNA level of NLRP3,Caspase-1,and IL-1β were significantly increased(P < 0.001)in the d MCAO group,while were further reduced after HS treatment(P <0.01).4)Laser confocal microscopy results showed that the number of positive cells of NLRP3,Caspase-1,and IL-1β in the d MCAO group was significantly increased(P < 0.001)compared to the Sham group.The number in the HS group was significantly reduced(P < 0.01)compared to the d MCAO group.Conclusion: The expressions of ROS,NLRP3,Caspase-1,and IL-1β in brain tissue were significantly increased after d MCAO,while the levels of these factors downregulated in the HS group,indicating that HS has anti-inflammatory and neuroprotective effects.Part 4 The anti-inflammatory mechanism of hydrogen-rich saline in neuroprotectionObjective: To construct an vitro inflammation model of LPS-induced BV2 microglial cells,and investigate the anti-inflammatory mechanism of hydrogen-rich saline.Methods: BV2 cells were divided into the following groups: 1)Sham group;2)LPS group: cells were treated with 1μg/m L LPS for 6h;3)LPS+H group: cells were treated with 1μg/m L LPS and the culture medium was replaced with hydrogen-rich medium(concentration of 0.8mmol/L);4)LPS+NAC group: cells were treated with 1μg/m L LPS and 10 n M NAC was added;5)LPS+H+NAC group: cells were treated with 1μg/m L LPS and10 n M NAC,and the culture medium was replaced with hydrogen-rich medium.Cell viability was detected by CCK-8 assay,and the protein level of NLRP3,Caspase-1,and IL-1β were detected by Western Blot.The level of ROS was detected by DCFH-DA fluorescence probe and immunofluorescence imaging.Results:1.The cell viability with LPS-treated cells decreased by about 50% at 6h(P<0.05),which was selected as the subsequent experimental time point.Furthermore,the cell viability was significantly increased in the HS group compared with the LPS group(P < 0.001).2.The level of ROS was significantly increased after LPS treatment compared with the Sham group(P < 0.001).Both HS and NAC treatment could reduce the level of ROS,and the difference was statistically significant(P < 0.001).Furthermore,compared with the NAC group,the combination of NAC and HS could further significantly reduce ROS expression(P < 0.001).The level of ROS observed by laser confocal microscopy was consistent with the above results(no statistical analysis was performed).3.The protein level of NLRP3,Caspase-1,and IL-1β were up-regulated in BV2 cells after LPS treatment in vitro(P < 0.001).The expression of NLRP3,Caspase-1,and IL-1β was down-regulatedin the HS group compared with the LPS group(P < 0.05).NAC treatment could also reduce the expression of NLRP3,Caspase-1,and IL-1βcompared with the LPS group(P< 0.05).The combination of NAC and HS could further significantly reduce the protein level of the three factors compared with the NAC alone(P < 0.05).Conclusion: Our results suggest that HS suppresses the activation of the ROS-NLRP3 inflammasome signaling pathway and exerts neuroprotective effects after ischemic stroke.