Network Pharmacology And Experimental Studies For Exploring The Role And Mechanism Of Nrf2 Pathway In Salidroside Alleviating Methylmercury Induced Neurotoxicity

Background and objective:Methylmercury（Me Hg）is a heavy metal pollutant that widely exists in nature.Due to its strong biological enrichment,it is easy to enter the body through various ways and cause health threats.Me Hg is particularly harmful to the central nervous system,and the mechanism of its neurotoxicity has not been fully elucidated,which also leads to the current clinical treatment mainly based on chelating agents,which have great toxic and side effects.Therefore,finding a drug for the prevention and treatment of Me Hg with good efficacy and few side effects is an urgent public health problem to be solved.Nrf2 is a key molecule to maintain redox homeostasis and cope with oxidative stress,which is closely related to glutathione metabolism,cell viability,erythropoiesis and cellular detoxification.Many studies have shown that the mechanism of Me Hg toxicity is related to the activation of oxidative stress,in which Nrf2 plays an important role.Salidroside（SAL）is a natural adaptogen with multiple pharmacological activities.It is extracted from the rhizome of the plateau plant Rhodiola rosea L..It has natural activities such as anti-hypoxia and anti-oxidation,and plays an obvious protective role in a variety of degenerative diseases and heavy metal poisoning.It has been proposed that activating Nrf2 pathway by SAL has a protective effect on the occurrence and development of a variety of neurodegenerative diseases.Therefore,this study combined big data analysis with in vitro and in vivo experiments,used network pharmacology analysis,in vivo ICR mouse model and in vitro primary rat astrocytes model to investigate the protective effect of SAL against Me Hg toxicity,and to analyze the role and mechanism of Nrf2 in SAL alleviating Me Hg induced toxicity.The in vitro chelation experiment was carried out by UV-vis spectroscopy and ion trap mass spectrometry to explore whether SAL could chelate Me Hg,reduce its brain accumulation or promote its excretion.To provide scientific basis for the clinical prevention and treatment of Me Hg exposure.Methods:1.To explore the protective effect of SAL against Me Hg neurotoxicity in vitro and in vivo:Primary rat astrocytes were selected as the in vitro model.Astrocytes were exposed to different concentrations of Me Hg（1,2.5,5,and 10μM）for 6h,and MTT was used to evaluate the activity of the cell viability.SAL（5,10,20,and 40μM,24h）was used to pretreat the cells exposed to Me Hg（5μM,6h）.The optical microscope was used to track the morphological changes in the cells,the MTT assay was used to assess the cell viability,the LDH assay was used to test the cytotoxicity,the DCFH-DA fluorescent probe was used to measure the intracellular ROS level.ICR mice were treated with SAL（100mg/kg）by gavage for 7 days,and a single intraperitoneal injection of Me Hg（6mg/kg）was performed 24 hours after the last gavage.The effects of SAL on serum SOD activity and GSH level were detected,and the oxidative stress level was evaluated.The total mercury level in brain tissue of ICR mice was determined by DMA-80 direct mercury analyzer.2.To screen the key targets of SAL in antagonizing Me Hg neurotoxicity by network pharmacology:The targets of SAL and Me Hg were searched by CTD（Comparative Toxicogenomics Database）,Pharmmapper,Swiss targets prediction and SEA（Similarity ensemble approach）databases.The intersection of the targets was taken into account when screening the common targets using a Venn diagram.An enrichment study using GO（Gene Ontology）and KEGG（Kyoto Encyclopedia of Genes and Genomes）was performed on the common targets.Protein interaction analysis was performed by string analysis website and cytoscape.3.To explore the role of Nrf2 signaling pathway in SAL attenuating Me Hg neurotoxicity in vitro and in vivo:Primary rat astrocytes were selected as an in vitro model,and were divided into four groups:astrocytes control group,Me Hg（5μM,6h）exposure group,SAL（20μM,24h）+Me Hg（5μM,6h）exposure group,and SAL（20μM,24h）exposure group.Western blot was used to detect the protein expression of Nrf2,its downstream HO-1 and NQO1,and the expression of Nrf2 binding protein Keap1.Nrf2 inhibitor ATRA was used to inhibit the activation of Nrf2 signaling pathway proteins,and MTT assay was used to detect the cell viability.ICR mice were selected as the in vivo model,and were divided into control group,Me Hg single exposure group（6mg/kg）,SAL（100mg/kg,7d）+Me Hg（6mg/kg）single exposure group,and SAL group（100mg/kg,7d）.Twenty-four hours after Me Hg injection,the mice were anesthetized and sacrificed,and the brain tissues were analyzed.Western blot was used to detect the expression of Nrf2 and related proteins in the brain tissue of ICR mice.4.To explore the chelation of Me Hg by SAL in cell-free systemTotal mercury levels in brain tissues of ICR mice were measured by hyperthermic oxygen decomposition,catalytic adsorption,amalgam trapping and atomic absorption spectrometry.The mixture of SAL（1200,600,300,150μg/ml）and Me Hg（125μg/ml）was detected by ultraviolet spectrophotometer.The results of mixed SAL（1200,600,300,150μg/ml）and Me Hg（125μg/ml）mass spectrometry were detected by ion trap mass spectrometry.Results:1.Protective effect of SAL on Me Hg neurotoxicity:In vitro experiments showed that the cell viability of the Me Hg treatment group was decreased in a dose-dependent manner compared with the blank group.Pretreatment of Me Hg（5μM,6h）with SAL（20μM,24h）significantly inhibited the toxic characteristics of Me Hg.The cell viability was restored by about 10%,the LDH leakage rate was decreased by about 70%,In comparison to the Me Hg group,there were substantially more cells visible under the microscope,much better shape,and significantly lower ROS levels.In vivo experiments showed that SAL maintained the redox homeostasis in mice exposed to Me Hg,and restored the serum SOD activity and GSH level to normal levels.SAL may reduce the mercury concentration in brain tissue to a certain extent,although there was no statistical significance.Compared with 2,4 and 6mg/kg Me Hg,SAL pretreatment reduced the total mercury concentration in brain tissue by 15.67,77.39and 80.32μg/kg,respectively.2.Key targets of SAL in antagonizing Me Hg neurotoxicity:A total of 342 utility targets of SAL from Homo sapiens,918 utility targets of Me Hg,and 45common utility targets were obtained from each database.GO and KEGG enrichment analysis and protein interaction analysis were performed on 45 common targets.GO analysis mainly involved functions and components such as positive regulation of gene expression,positive regulation of cell proliferation,hypoxia response,response to poisons,oxidative stress response,glutathione metabolism,free radical scavenging,reactive oxygen reaction,redox homeostasis,protein binding,transcription factor binding,SOD activity,and antioxidant activity.KEGG signaling pathway was mainly involved in a variety of degenerative diseases,chemical cancer-reactive oxygen species,endoplasmic reticulum protein processing,MAPK signaling pathway,HIF-1 signaling pathway,m TOR signaling pathway,PI3K-Akt signaling pathway and other signaling pathways.AKT1,HIF1A,VEGFA,MAPK3,CAT,HSPA5,SOD1,MAPK1,PARP1,SOD2 and other related molecular targets were screened.3.Role of Nrf2 signaling pathway in SAL attenuating Me Hg neurotoxicity:In vitro experiments showed that SAL（20μM,24h）pretreatment with Me Hg（5μM,6h）significantly enhanced the protein levels of Nrf2 and its downstream HO-1 and NQO1 in astrocytes.The Nrf2 inhibitor ATRA significantly reduced the ability of SAL to alleviate the decrease in the cell viability of astrocytes induced by Me Hg.In vitro experiments showed that SAL（100mg/kg,7d）pretreatment significantly enhanced the expression of Nrf2 and its downstream HO-1 and NQO1 proteins in brain tissues of ICR mice exposed to a single intraperitoneal injection of Me Hg（6mg/kg）.4.Chelation of Me Hg by SAL:The mixture of SAL and Me Hg showed no new absorption peaks in UV spectra.The results of mass spectrometry showed that Me Hg lost one methyl group and combined with two hydroxyl groups of SAL to form a new substance C₁₅H₂₂Hg O₇ with a relative molecular mass of 514.92（M+H⁺）and a corresponding peak of 515.99 found on the mass spectrum.ITMS m/z calculated for C₁₅H₂₂Hg O₇ 514.92（M+H⁺）,found 515.99.Conclusion:1.SAL intervention could alleviate the toxic changes of cell viability,morphological changes,LDH leakage rate,ROS level,Serum SOD activity and GSH level caused by Me Hg.2.A total of 45 common targets of SAL and Me Hg were screened,and the functional components and signaling pathways enriched according to the common targets were mostly related to oxidative stress and redox homeostasis.3.SAL intervention activated the key factors of Nrf2 signaling pathway that may enable it to exert neuroprotective effects.It may be achieved by promoting Nrf2 nuclear translocation and inhibiting Keap1 protein expression.4.There is no new absorption peak was between SAL and Me Hg in the UV spectrum,indicating that there was no change in the conjugate bond between SAL and Me Hg.However,it was found that one molecule of SAL could combine with one molecule of Me Hg in the mass spectrum,suggesting that SAL has the potential to become a natural chelator of mercury.