| Objective:Network pharmacology was used to screen the interaction target of Saposhnikoviae Radix（SR）on pyroptosis and brain damage caused by arsenic exposure;The protective effect of SR on neuronal cells exposed to sodium arsenic（Na As O2）and the underlying mechanism were studied in vitro and in vivo.Methods:1.The network pharmacology technique was used to screen the action targets of SR on arsenic-induced pyroptosis of neuronsThe active ingredients and possible actin targets of SR were identified by searching the traditional Chinese medicine pharmacology and analysis platform（TCMSP）database.Through the disease database OMIM,Gene Cards collected genes associated with arsenic nerve damage and pyroptosis genes.Lead compounds and potential targets are screened through the Venny of SR and neuronal pyroptosis targets.The core targets are imported into the String database to analyze the protein interactions between targets.The R language and bioinformatics are used for ontology analysis to identify targets and visualization involvement in regulated,involving biological processes,cellular components,molecular functions,and signaling pathways.Autodock,Chem 3D,and Pymol software were used to perform molecular docking between the active ingredients and their corresponding target proteins to evaluate the interaction between the active ingredients and targets.2.Protective mechanism of SR on arsenic-induced neuronal pyroptosis was detected by in vivo experimentsAfter one week of adaptive feeding,thirty-six Kunming mice were randomly divided into six groups:the Control group,Na As O2 group,low-dose SR group,medium-dose SR group,high-dose SR group,and positive drug DMPS group.The mice were given twice a day in each group,group SR low-dose（3 g/kg）,SR medium-dose（6 g/kg）,SR high-dose（12 g/kg）,and positive drug DMPS（5 mg/kg）were given 4hours in advance,and the Control group and Na As O2 group were given the same volume of saline;After for 4 hours,the mice were administered with the model by Na As O2,in which Control group was given saline,and the other groups were given Na As O2（10 mg/kg）respectively and the experimental period was 30 days.After the end of the last day of intragastric administration,the O maze method was used to detect the activity time in the open arm area of Kunming mice in the experiment;The time of escape latency and the times of crossing the platform were measured by water maze;The morphological structure changes of brain tissue of mice were observed by HE staining;Detection of pyroptosis-related genes and proteins（NLRP3,Caspase-1,Cleaved-Caspase-1,GSDMD,GSDMD-N,IL-1β,IL-18）and TNF-αchanges of expression in brain tissue of mice in each group by RT-q PCR and Western blot.3.Protective mechanism of SR on arsenic-induced neuronal pyroptosis was detected by in vitro experimentsHT22 cells were used to be the research objects that were divided into different concentrations of Na As O2（0,6.25,12.5,25,50,100μM）group,SR（0,50,100,200,400,800μg/ml）group,After treatment of the cells for 24 hours,the changes of cell viability were detected by CCK8 method,and the safety concentration of SR and the concentration of Na As O2 injury model were obtained.After that,they were divided into the Control group,Na As O2 group,and Na As O2+SR group.The CCK-8 method and LDH release assay were used to evaluate the effects of SR and Na As O2 on the viability and rupture of membranes in HT22 cells,and to evaluate the efficacy of SR;Observe the effects of SR and Na As O2 on the number and morphology of HT22 cells by light microscope;The influence of SR and Na As O2 on the expression of pyroptosis-related genes and proteins（NLRP3,Caspase-1,Cleaved-Caspase-1,GSDMD,GSDMD-N,IL-1β,IL-18）and TNF-αwere detected by RT-q PCR and Western blot;The formation of pyroptotic bodies in HT22 cells treated with SR and Na As O2 was observed by scanning electron microscopy;Observation of GSDMD,IL-1βexpression,and TNF-α/Capase-1co-location by laser scanning confocal microscope Fluorescence in HT22 cells treated with SR and Na As O2;HT22 cells were pretreated with SR and TNF-αinhibitor C87,then treated with Na As O2 for 24 h,and the cell viability was detected by CCK8;The morphological changes of cells were observed by electron microscope;The pyroptosis-related genes and proteins（NLRP3,Caspase-1,Cleaved-Caspase-1,GSDMD,GSDMD-N,IL-β,IL-18）and TNF-αexpression changes were detected by RT-q PCR and Western blot;The formation of pyroptotic bodies was observed by scanning electron microscope in HT22 cells.Results:1.The 18 compounds in SR protected against Na As O2-induced HT22 pyroptosis and brain injury of mice by regulating 9 core targets.Input"Fangfeng"in the TCMSP database to screen 18 compounds and participate in regulating 78 targets;By searching the disease-related database,985 arsenic-nerve injury related targets and 185 pyroptosis-related targets were obtained,and 62 arsenic-neuronal pyroptosis-related targets were obtained by intersection;Nine core targets were obtained by making Venny of SR target and arsenic-neuronal pyroptosis target;The interaction network between target points included 9 nodes and 31 edges;The average degree of nodes is 6.89,and the average clustering coefficient is 0.898;The main compounds were wogonin,beta-sitosterol,ledebouriellol,and 5-O-Methylvisamminol;Molecular docking results showed that the active component-target binding was good and wogonin was the most involved in regulating the target;The GO results showed that the target involves 14 biological processes,such as the positive regulation of neuronal death process,the response to LPS,positive regulation of IL-1βproduction,inflammatory response,and cells to DNA damage stimulation,and the target involves 8 cell components,such as the death induction signal complex,membrane raft,and cytoplasm,at the same time;The target plays 14 molecular functions,such as binding transcription factor,binding death receptor,and tumor necrosis factor receptor;The enrichment analysis of KEGG pathways showed that the active components of the drug were involved in 19 signal pathways,including the cancer pathway,tumor necrosis factor signal pathway,neurotrophin signal pathway,and Alzheimer’s disease pathway and so on.2.SR inhibited Na As O2-induced pyroptosis and TNF-αexpression in mice brain tissueThe results of the O maze showed that compared with the Control group,the activity time of the open arm was significantly reduced in the Na As O2group（P<0.05）,while compared with the Na As O2group,the activity time of the open arm was significantly increased in the SR low-dose group,SR medium-dose group,SR high-dose group and positive drug DMPS group（P<0.05）;The results of water maze showed that compared with the control group,the escape latency time of Na As O2group was significantly longer and the number of times of crossing the platform was significantly reduced（P<0.05）,while compared with the Na As O2group,the escape latency time of SR low-dose group,SR medium-dose group,SR high-dose group and positive drug DMPS group were significantly shorter and the number of times of crossing the platform were significantly increased（P<0.05）;The HE results showed that compared with the control group,the cells in the cerebral cortex,hippocampus and cerebellum of the Na As O2group were disorderly arranged and the nuclei were shrunk,while compared with the Na As O2group,the brain tissue damage of the SR low-dose group,SR medium-dose group,SR high-dose group and positive drug DMPS group were reduced;The results of RT-q PCR and Western blot showed that compared with the Control group,the expression of pyroptosis-related genes and proteins（NLRP3,Caspase-1,Cleaved-Caspase-1,GSDMD,GSDMD-N,IL-1β,IL-18）and TNF-αwere significantly upregulated in the Na As O2 group of mice brain tissue（P<0.05）,while compared with the Na As O2 group,the SR low-dose group,SR medium-dose group,SR high-dose group and positive drug DMPS group pyroptosis-related genes and proteins（NLRP3,Caspase-1,Cleaved-Caspase-1,GSDMD,GSDMD-N,IL-1β,IL-18）and TNF-αexpression were reversed（P<0.05）.3.SR inhibited Na As O2-induced pyroptosis and expression of the TNF-αin HT22cellsThe results of CCK8 showed that SR reduces HT22 cell viability at 800μg/ml concentration,Na As O2 inhibited the viability of HT22 cells in a dose-dependent manner and suitable modeling concentrations were screened at 12.5μΜ（P<0.05）,SR（100,200,400μg/ml）reversed the cell viability decrease of HT22 cells induced by Na As O2（P<0.05）;SR significantly inhibited the release of lactic acid dehydrogenase caused by Na As O2（P<0.05）;Under electron microscopy,it was observed that the number of HT22 cells decreased,the number of dead cells increased,and cells became round or oval in the Na As O2 group;The RT-q PCR results showed that,when compared to the Na As O2 group,the Na As O2+SR group had no significant changes in pyroptosis-related genes NLRP3 and Caspase-1,while the expression of GSDMD,IL-1β,IL-18and TNF-αwas significantly decreased;Western blot results showed that the expression of pyroptosis-associated proteins（NLRP3,Caspase-1,Cleaved-Caspase-1,GSDMD,GSDMD-N,IL-1β,IL-18）and TNF-αin the Na As O2+SR group was significantly downregulated compared with the Na As O2group（P<0.05）;Scanning electron microscopy observed that compared with the Na As O2 group,the formation of pyroptotic bodies was reduced in the Na As O2+SR group;Compared with the Na As O2group,the fluorescence intensity of GSDMD,IL-1βand TNF-α/Capase-1 were significantly reduced in the Na As O2+SR group,and then TNF-α/Capase-1 co-expression was inhibited;After C87 was used to block the TNF-α,compared with the Na As O2+SR group,the cells viability were significantly increased in the Na As O2+SR+C87 group（P<0.05）,the number of cells was higher,and the synapses were more obvious;The m RNA expression levels of Caspase-1,GSDMD,IL-1βand IL-18 were not significantly changed,while NLRP3 and TNF-αwere more significantly down-regulated.At the protein level,except IL-1β,the expressions of other pyroptosis-related proteins and TNF-αwere reversed more significantly,and fewer pyroptotic bodies formed.Conclusions:1.SR alleviates the Na As O2-induced decrease in neuronal cells viability and increase in cytotoxicity,reduces neuronal pyroptosis,and protects the nervous system;2.SR antagonizes Na As O2-induced neuronal pyroptosis injury by inhibiting the TNF-α/Caspase-1 inflammatory axis. |
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