Liraglutide Inhibits Pyroptosis In Focal Cerebral Infarction Mice And Its Related Mechanisms

Cerebral infarction,also known as ischemic stroke,is a major chronic non-communicable disease that seriously endangers the health of people in the world,and is the second leading cause of death in the world,causing a heavy economic burden to patients and society.There are many risk factors for cerebral infarction,including age,diabetes,hypertension,hyperlipidemia,hyperhomocysteinemia,atrial fibrillation,patent foramen ovale,etc.After the occurrence of cerebral infarction,it is divided into the central infarct area and the peripheral ischemic penumbra area.If an excessive inflammatory reaction occurs in the ischemic penumbra area,a cascade of cerebral infarction aggravation can occur.For example,inflammatory stimulation can promote brain cells.Programmed cell death-pyroptosis occurs,thereby aggravating the severity of the acute phase of cerebral infarction.In recent years,great progress has been made in the treatment of acute cerebral infarction:appropriate extension of intravenous thrombolysis in the posterior circulation,thrombolytic therapy for stroke after awakening,application of new thrombolytic drugs,and mechanical thrombectomy after thrombolysis.treatment,etc.However,due to the strict time window and hemorrhagic transformation,neurotoxicity and complications,there are still not many patients who receive the above treatment and benefit within the specified time,and many patients are left with severe disability.still very limited.For this reason,it is necessary to seek other drug therapy in acute cerebral infarction and explore its mechanism for the treatment of cerebral infarction.Liraglutide is a novel long-acting glucagon-like peptide-1（GLP-1）analog approved for the drug treatment of type 2 diabetes.Liraglutide binds to GLP-1 receptors distributed in the central nervous system through the blood-cerebrospinal fluid barrier（BBB）.Previous studies have found that liraglutide has pharmacological effects such as anti-inflammatory,anti-oxidation,affecting autophagy,promoting angiogenesis,and promoting nerve cell plasticity.Dementia has a good protective effect.In previous studies,we found that liraglutide can play a role in brain protection by promoting angiogenesis.Previous studies have found that liraglutide plays a protective role in hepatocytes by inhibiting pyroptosis in a non-alcoholic liver cirrhosis research model.However,there are few studies on whether liraglutide can play a role in brain protection by inhibiting pyroptosis.report.Therefore,in this study,we first conducted a retrospective study on the patients with acute cerebral infarction admitted to our department,to explore the relationship between hypersensitive-c-reactive-protein（hs-CRP）and the severity of acute cerebral infarction.At the same time,the animal model of distal middle cerebral artery occlusion（d MCAO）through liraglutide intervention and the establishment of an oxygen-glucose deprivation model by culturing microglia in vitro to further explore the treatment of liraglutide Whether it can promote the improvement of neurological function and reduce cerebral ischemia injury by inhibiting the classical pathway of pyroptosis,NLRP3/Caspase-1/IL-1β.The research is divided into the following four parts,the content of each part is summarized below.Part One Correlation between serum hs-CRP and the severity of acute cerebral infarctionObjective:To investigate the correlation between hs-CRP and the risk of acute cerebral infarction.Methods:The clinical data of 102 patients with acute cerebral infarction and 101 patients with transient ischemic attack were reviewed.The levels of hs-CRP,white blood cells and neutrophils between the two groups were analyzed,the correlation between hs-CRP level and NIHSS score in acute cerebral infarction was analyzed;the subgroup analysis of acute cerebral infarction was performed to evaluate the correlation of hs-CRP level in NIHSS score of patients with large artery stenosis and non-large artery stenosis.The SPSS 25.0was used for Statistical analysis.Results:1.The results of the general data comparison of patients with acute cerebral infarction and patients with transient ischemic attack show that there is no difference in gender,hypertension,diabetes,and coronary heart disease between patients with acute cerebral infarction and transient ischemic attack.There was no statistical significance（P>0.05）;however,the age of onset of acute cerebral infarction（60.71±9.82）was greater than that of transient ischemic attack（56.57±11.29）（P<0.05）,which was statistically significant.2.The levels of high-sensitivity C-reactive protein（hs-CRP）,white blood cells and neutrophils in patients with acute cerebral infarction and patients with transient ischemic attack showed that:102 patients with acute cerebral infarction and the level of hs-CRP were 4.42±7.12,the hs-CRP level of 101patients with transient ischemic attack was 2.43±4.55,which was statistically significant（P<0.05）;there was no significant difference in white blood cells and neutrophils between the two groups.significance（P>0.05）.3.The results of the study on the correlation between the level of hs-CRP and NIHSS in patients with acute cerebral infarction showed that the level of hs-CRP in patients with acute cerebral infarction was positively correlated with the NIHSS score of patients.The CRP level was most closely related to the NIHSS score,R=0.71 P=0.000.Summary:There is a non-infectious inflammatory response in patients with acute cerebral infarction,and there is positive correlation between the level of hs-CRP and the NIHSS score,especially the level of hs-CRP in the patients with large vessel stenosis was moderately correlated with the NIHSS score.Part Two Effects of Liraglutide on neuroprotoration in focal cerebral infarction miceObjective:To evaluate the effect of Liraglutide on post-stroke neurological recovery,we measured the neurobehavioral function of mice with focal cerebral ischemia.Methods:Male C57BL/6 mice were used as research objects to establish d MCAO model.Male C57BL/6 mice were randomly divided into the following groups:d MCAO group:animals only received d MCAO;Liraglutide-treated group:Three days before d MCAO,immediately after d MCAO,and consecutive three days after d MCAO,Liraglutide（246.7μg/kg/day）was intraperitoneally injected.Rota-Rod test,m NSS neurological function score,body weight and blood glucose measurement were performed on each group before and day 1,2,3 after d MCAO.Cerebral infarct volume was determined by TTC staining at day 3 after d MCAO.Gait analysis was performed on each group before and day 1,2,3 after d MCAO.Cerebral blood flow（CBF）was monitored by laser speckle imager before surgery and 1,2,3 days after surgery.Results:1.The results of the blood and weight measurement indicated that there was no significant difference at different time in blood glucose levels or body weight between the d MCAO+Lg group and d MCAO group.2.TTC staining was used to measure the infarct volume in the different groups at day 3 and day 7 after d MCAO to analyze neurons.Compared with the d MCAO group,the d MCAO+Lg group exhibited a significant reduction in the infarct volume（P<0.05）.3.Rota-Rod test results showed that compared with the d MCAO group,there was significant neurological improvement at day 1,2 and 3 in the d MCAO+Lg group（P<0.05）.4.The m NSS results showed that compared with the d MCAO group,the score was higher at day 1,2 and 3 in d MCAO+Lg group（P<0.05）.5.The gait analysis results showed that compared to that of the untreated d MCAO animals,the instantaneous running speed of the mice in the d MCAO+Lg group increased continuously over three days,with the increase being particularly pronounced on day 3.The mice in d MCAO+Lg group had a shorter stride length,smaller average print area and improved LF-Gait angles on day3（P<0.05）.6.The results of laser speckle showed that in the d MCAO+Lg group there was higher CBF of the ipsilateral hemisphere at day 3 after surgery compared with the d MCAO group（P<0.05）.However,no significant difference was observed in the CBF of ipsilateral hemisphere at day 3 after surgery compared with the d MCAO+Lg group（P<0.05）.Summary:Liraglutide significantly reduced the infarct volume in mice with focal cerebral infarction and improved the neurological function of mice with cerebral infarction,suggesting that liraglutide has a cerebral protective effect.Part Three Effects of Liraglutide on pyroptosis in focal cerebral infarction miceObjective:To evaluate whether liraglutide inhibits pyroptosis in animal models of cerebral infarction by measuring pyroptosis-related factors in mice with focal cerebral infarction after liraglutide intervention.Methods:Male C57BL/6 mice were used as subjects to establish a d MCAO model.The male C57BL/6 mice were randomly divided into the following groups:Sham group:d MCAO group:animals only received d MCAO;Liraglutide-treated group:Three days before d MCAO,immediately after d MCAO,and consecutive three days after d MCAO,Liraglutide（246.7μg/kg/day）was intraperitoneally injected.The pyroptosis-related factors were detected by RT-PCR,Western Blot and Immunofluorescence techniques at 1day after operation;Results:1.The m RNA expression of pyroptosis-related genes on day 1 after surgery revealed that the m RNA expression of NLRP3,IL-1β,Caspase1 and Gsdmd was upregulated in d MCAO mice compared with sham-operated mice（P=0.0002,0.0008,0.0182,and 0.0108）and that administration of Lg induced a downward trend in the m RNA levels of these molecules（P=0.0019,0.0006,0.0284 and 0.0346）.2.Western Blot results showed that the protein levels of NLRP3,Pro IL-1β,Cleaved IL-1β,Caspase-1,Cleaved Caspase1（p10）and Gsdmd were all reduced by Lg treatment（P=0.0006,0.0228,0.0045,0.0016,0.0044,and0.0346,respectively;indicated by the arrowheads）.However,we did not observe a significant difference in the gene or protein expression of Asc following Lg treatment.3.Immunohistochemical analysis of the brain tissues showed that the numbers of cells positive for NLRP3（red）,IL-1β（red）,and Caspase1（red）were markedly reduced in the Lg-treated d MCAO group compared to the untreated d MCAO group（P=0.0110 for NLRP3;P=0.0041 for IL-1β;P=0.0414 for Caspase1;）.Summary:Liraglutide significantly down-regulated the expression of pyroptosis-related factors in a mouse model of focal cerebral infarction,suggesting that liraglutide inhibits pyroptosis after cerebral infarction.Part Four Effects of Liraglutide on pyroptosis in focal cerebral infarction miceObjective:The oxygen glucose deprivation model was constructed in vitro to detect the mechanism of the of Lg,to evaluate whether Lg anti-pyroptosis after ischemic stroke via NLRP3/Caspase-1/IL-1βsignaling pathway.Methods:The Bv2 cells were used as the subjects to establish an OGD model.Experiment 1:BV2 cells were treated with OGD and were divided into the following groups according to different concentrations of liraglutide:Lg 1n M group,10 n M group,100 n M group,200 n M group,and 1000 n M group.CCK-8 was used to detect cell viability and determine the best time for OGD to carry out the next experimental study.Experiment 2:The BV2 cells were divided into the following groups:Sham group;OGD group:cells received OGD for 6 h;OGD+Lg group:cells received OGD for 6 h and given 200 n M Lg;the effect of Lg on apoptosis-related factors was measured by Western Blot.The secretion function of IL-1βand IL-18 was detected by Elisa.Experiment 3:The BV2 cells were divided into the following groups:Sham group;OGD group:cells received OGD for 6 h;OGD+Lg group:cells received OGD for 6 h and given 200 n M Lg;OGD+Mcc950 group:cells received OGD for 6 h and given10n M Mcc950;by Western Blot measurement to evaluate whether Liraglutide exerts an anti-pyroptotic effect through the NLRP3/Caspase-1/IL-1βsignaling pathway.Results:1.In vitro,Liraglutide（1 n M,10 n M,100 n M,200 n M,1000 n M）have protective effects on OGD-treated BV₂ cells（P<0.05）.We selected Lg 200 n M dose for subsequent in vitro experiments.2.In vitro,OGD treatment for 6 h led to approximately 50%cell death,so this time period was selected for the following experiments.3.In vitro,the enzyme-linked immunosorbent assay（ELISA）was used to detected the secretion of IL-1βand IL-18 in OGD-treated BV2 cells.The results showed that compared with the Sham group,OGD-treated BV2 cells significantly increased IL-1βand IL-18,and there was a statistically significant difference between the two groups（P<0.0001）,compared with the OGD group,Lg significantly inhibited the secretion of IL-1βand IL-18,with significant statistical significance between the two groups（P<0.01）4.In vitro,the protein expression of NLRP3,Pro IL-1β,Cleaved IL-1β,Pro Caspase-1,and Cleaved Caspase-1（p10）was upregulated in microglial cells exposed to OGD,and Lg treatment significantly reduced the expression levels of these proteins（P<0.0001;）.We employed Mcc950,a potent and specific small-molecule inhibitor of NLRP3,to further investigate the molecular mechanism underlying the effect of Lg.Compared with the OGD,treatment with Mcc950 decreased the protein levels of NLRP3,Pro Caspase1,Cleaved Caspase1（p10）,IL-1β,and Cleaved IL-1βin BV2 cells,indicating that Mcc950 effectively suppressed the expression of the key molecules involved in pyroptosis.Summary:In vivo,Liraglutide inhibited pyroptosis,and it exerts an anti-pyroptotic effect by inhibiting the NLRP3/Caspase-1/IL-1βpathway,and NLRP3 may be a key target of liraglutide in the anti-pyroptotic effect.Conclusions:1.There is a non-infectious inflammatory response in patients with acute cerebral infarction,and the level of inflammation may be related to the severity of cerebral infarction.2.Liraglutide promotes the recovery of neurological function in mice in the animal d MCAO model,reduces the volume of cerebral infarction,and has a protective effect on the brain.3.In vivo and in vitro experiments,liraglutide inhibited pyroptosis after cerebral infarction.4.Liraglutide may play an anti-pyroptotic effect by inhibiting the NLRP3/Caspase-1/IL-1βpathway,and NLRP3 may be a key target of liraglutide in the anti-pyroptotic effect.