The Scorpion Venom Heat-resistant Peptide Regulates Nrf-2 To Alleviate Neuronal Pyroptosis In The AD Model Induced By Endoplasmic Reticulum Stress Under PM_2.5 Exposure

Objective:In recent years,the effects of PM_2.5 exposure on the nervous system,especially neurodegenerative diseases,have received widespread attention.However,the regulatory mechanisms of PM_2.5 on Alzheimer’s disease（AD）have been less studied.In this experiment,we investigated pyroptosis and its regulatory mechanisms in a model of AD aggravated by PM_2.5 exposure,and further discussed the role of scorpion venom heat-resistant synthetic peptide（SVHRSP）on its repair and mechanism.Methods:1.PM_2.5 sample collection and processing.The PM_2.5 used in the experiment was collected in Langfang City,Hebei Province.The quartz filter paper containing PM_2.5 was cut into pieces,and PM_2.5 was removed from the quartz filter paper using double distilled water.The concentration of PM_2.5 was obtained by comparing the quality of the filter paper before and after elution.The PM_2.5 suspension is stored in a refrigerator at 4℃for subsequent experiments.2.Experimental grouping and model construction.In vitro experiments:In this experiment,Aβ_25-35 protein and PM_2.5 were used to treat rat adrenal pheochromocytoma cells（PC12）and mouse hippocampal neuronal cells（HT22）respectively or in combination to construct the AD model of PM_2.5 exposure,and the repair model was constructed with cells treated with scorpion venom heat-resistant peptide.Experimental groups:1)control group,Aβ_25-35 group,PM_2.5 group,Aβ_25-35+PM_2.5-treated 6h group,Aβ_25-35+PM_2.5-treated 12h group,Aβ_25-35+PM_2.5-treated 24h group,to discuss the effects of PM_2.5 exposure on endoplasmic reticulum stress and pyroptosis in AD model;2)control group,Aβ_25-35 group,PM_2.5 group,endoplasmic reticulum stress inhibitor 4-phenylbutyric acid（4-PBA）group,Aβ_25-35+PM_2.5 group,and Aβ_25-35+PM_2.5+4-PBA group,to discuss the regulation of cellular endoplasmic reticulum stress on pyroptosis under PM_2.5 exposure;3)control group,Aβ_25-35 group,PM_2.5group,Aβ_25-35+PM_2.5 group,to detect the changes of ROS level,MDA and SOD in cells under PM_2.5 exposure,as a reflection of the degree of oxidative stress in the organism;4)control group,Aβ_25-35 group,PM_2.5 group,ROS inhibitor N-acetylcysteine acid（NAC）group,Aβ_25-35+PM_2.5 group,Aβ_25-35+PM_2.5+NAC group,to discuss the effect of ROS on regulation of endoplasmic reticulum stress and pyroptosis;5)control group,Aβ_25-35group,PM_2.5 group,Aβ_25-35+PM_2.5 group,to detect the expression of Nrf-2 protein;6)control group,Aβ_25-35 group,PM_2.5 group,Nrf-2 activator（Bardoxolone）group,Aβ_25-35+PM_2.5 group,Aβ_25-35+PM_2.5+Bardoxolone group,to discuss the effect of Nrf-2 on ROS,endoplasmic reticulum stress and pyroptosis;7)control group,Aβ_25-35 group,PM_2.5group,scorpion venom heat-resistant peptide（SVHRSP）group,Aβ_25-35+PM_2.5 group,Aβ_25-35+PM_2.5+SVHRSP group,to discuss the restorative effect and regulatory mechanism of scorpion venom heat-resistant peptide.In vivo experiments:32 male Balb/c mice at 6-8 weeks were randomly divided into control group,Aβ_25-35group,Aβ_25-35+PM_2.5group,and Aβ_25-35+PM_2.5+SVHRSP group.All groups except the control group were injected with Aβ_25-35 using a brain stereotaxic locator to locate hippocampal tissue.mice in the Aβ_25-35+PM_2.5 group and the Aβ_25-35+PM_2.5+SVHRSP group were injected with PM_2.5 suspension by tracheal drip.The mice in the Aβ_25-35+PM_2.5+SVHRSP group were treated with intraperitoneal injection of scorpion venom heat-resistant peptide after the completion of tracheal drip.3.Relevant indicator detection.（1）Cell survival rate:The survival rate of PC12 cells and HT22 cells were detected after 24h treatment with different concentrations of Aβ_25-35,PM_2.5 and SVHRSP.（2）Endoplasmic reticulum stress-related indicators:Western blot to detect the expression levels of GRP78 and ATF-4 proteins;qRT-PCR to detect the levels of GRP78 and ATF-4genes;immunofluorescence to detect the fluorescence intensity changes of ATF-4 in HT22 and PC12 cells.（3）Pyroptosis-related indicators:Western blot to detect the expression levels of GSDMD,NLRP3 and IL-1βprotein;qRT-PCR to detect the expression levels of NLRP3 and GSDMD gene;immunofluorescence to detect the fluorescence intensity changes of GSDMD in HT22 and PC12 cells.（4）Oxidative stress-related indexes detection:malondialdehyde（MDA）assay kit and total superoxide dismutase（SOD）colorimetric assay kit to detect MDA and SOD levels;flow cytometry and fluorescence microscopy to detect ROS levels in cells;Western blot to detect Nrf-2protein expression levels.Results.1.In the CCK-8 experiment,after treating PC12 cells with different concentrations of PM_2.5 and Aβ_25-35protein for 24 h,cell survival was examined and the IC50 was found to be 56.05μM and 251.8μg/m L,respectively.Endoplasmic reticulum stress-related indicators GRP78 and ATF-4 and pyroptosis indicators NLRP3,IL-1β,and GSDMD were elevated and showed time-dependence,indicating that PM_2.5 exposure promotes endoplasmic reticulum stress and pyroptosis in AD cell models.2.After adding the endoplasmic reticulum inhibitor 4-PBA,GRP78,ATF-4,NLRP3,IL-1β,GSDMD gene and protein levels were reversed in the Aβ_25-35+PM_2.5+4-PBA group compared to the Aβ_25-35+PM_2.5group,suggesting that PM_2.5 may regulate pyroptosis in HT22 cells and PC12 cells under Aβ_25-35 protein exposure through endoplasmic reticulum stress.3.Cellular ROS levels were detected by flow cytometry and SOD and MDA levels were detected by the kit.It was found that the Aβ_25-35+PM_2.5 group had higher ROS and MDA levels and lower SOD levels compared to the Aβ_25-35 group,indicating that PM_2.5exposure leads to oxidative stress generation in neuronal cells.After adding the ROS inhibitor NAC,pyroptosis and endoplasmic reticulum stress-related indexes were inhibited in the Aβ_25-35+PM_2.5+NAC group compared to the Aβ_25-35 group,suggesting that PM_2.5 exposure may regulate endoplasmic reticulum stress and pyroptosis in neuronal cells in the AD model by promoting ROS production.4.Nrf-2 is often involved in cellular oxidative stress.Nrf-2 protein expression levels in PC12 cells and HT22 cells under PM_2.5 exposure were detected by Western blot,and PM_2.5 exposure was found to inhibit the expression of Nrf-2.The addition of Nrf-2activator Bardoxolone alleviated the elevated ROS,pyroptosis and endoplasmic reticulum stress induced by PM_2.5 and Aβ_25-35 protein co-exposure.5.The addition of scorpion venom heat-resistant peptide detected an increase in the expression level of Nrf-2,a decrease in the expression levels of GRP78,ATF-4,NLRP3,IL-1β,GSDMD protein,and a decrease in ROS content,indicating that scorpion venom heat-resistant peptide may regulate pyroptosis in the AD cell model by promoting the expression of Nrf-2.6.The hippocampal tissues of mice treated with scorpion venom heat-resistant peptide had intact HE stained cell morphology,tight arrangement and uniform nuclear staining.The endoplasmic reticulum stress and pyroptosis related indexes as well as ROS production were inhibited in the hippocampal tissues,and the expression level of Nrf-2was increased,indicating that the scorpionic heat-resistant peptide could repair the damage caused by PM_2.5exposure in AD mice,and it might be achieved by regulating the expression of Nrf-2.Conclusions.1.PM_2.5 may regulate pyroptosis in AD model by affecting Nrf-2 expression.2.Scorpion venom heat-resistant peptide repairs neuronal cell damage caused by PM_2.5exposure,and its repair effect may be achieved by regulating the expression of Nrf-2.