The Role Of NLRP3InFlammasome In Mitochondrial Toxicity Caused Bysmokeless Tobacco Water Extract

Part one Mitochondrial toxicity study ofsmokeless tobacco water extractObjective: To study the mitochondrial damage effect of smokeless tobaccowater extract(STWE). Methods: we chose the human hepatoma carcinoma cellHepG2and the human embryo lung fibroblast cell MRC-5as the target cells. Thecells were routinely cultured with proper medium which contained10%fetal bovineserum and1%Penicillin-streptomycin (10000unit/ml-10000μg/ml) in37℃,5%carbon dioxide incubator. When they reached more than90%density, a series ofexperiment could begin.(1) Detection of half inhibitory concentration of STWE onHepG2cells and MRC-5cells. Both the two cells were separately plated into the96-well plates and stimulated with different dosages of STWE24hours. The cellproliferation inhibiton rate was dertermined by MTT method and the half inhitoryconcentration was caculated by using the SPSS16.0. The maximum concentration ofSTWE was10mg/ml and50%diluted to different concentrations, set a row ofblank wells. The measuring wavelength was490nm.(2) Based on the MTT results,we selected the625.00μg/ml,312.50μg/ml and156.25μg/ml STWE as theeffective concentrations, set the blank control. HepG2and MRC-5cells wererespectively seeded into the6-well plates and exposured to the three doses STWE24hours, the blank control were treated with medium contained1%fetal bovine serum24hours. Observation of cell morphology. The cell morphological changes wereobserved by using the transmission electron microscope.(3) Assesment ofmitochondrial toxicity.①Measurment of mitochondrial membrane electric potential.The mitochondrial membrane electric potential changes were determined with themitochondrial membrane electric potential assay kit with JC-1by using the Backmanflow cytometry.②Measurment of ROS levels. The levels of ROS were determinedwith the GEMNED reactive oxygen assay kit by using the Backman flow cytometry.③Measurment of mitochondrial mtDNA encoded genes. The total RNA wasextracted and synthesized to cDNA. RT-PCR was applied to determin the expressionof12S rRNA, NDⅠ, COXⅡ genes.(4) Mesurement of cells apoptosis rate. The cellsapoptosis rates were determined with the FITC Annexin V Apoptosis Detection Kit Ⅰ by using the Backman flow cytometry.(5) Measurement of cell cycle associatedgenes. The total RNA was extracted and synthesized to cDNA. RT-PCR was appliedto determin the expression of P53, CDK2and MDM2genes. Resluts:(1) MTTresults suggested that along with the increasing of the STWE concentraion, theproliferation rates of HepG2and MRC-5were decreased. The half inhibitoryconcentrations of HepG2and MRC-5were respectively447.00μg/ml and441.00μg/ml. So we selected the625.00μg/ml,312.50μg/ml and156.25μg/ml as thestimulation concentrations.(2) The transmission electron microscope results showedthat compared with the blannk control, cells of the STWE stimulated groupsdiaplayed the following morphology changes: microvilli decreased or evendisappeared, the cells became swelling and some had cavity and even losed themembrane integrity, nucleus became shrinking.(3)The mitochondrial toxicityexperiment results.①The flow cytometry results showed that the mitochondrialmembrane potentials of HepG2and MRC-5decreased in a STWE dosage dependentway.②The flow cytometry results showed that the ROS levels of HepG2andMRC-5increased along with the increasing dosage of STWE.③Compared with theblank control,12S rRNA, NDⅠ and COXⅡ expression of HepG2and MRC-5increased along with the increasing dosage of STWE.(4) The flow cytometry resultsshowed that the apoptosis rates of HepG2and MRC-5cells increased along with theincreasing dosage of STWE.(5) RT–PCR results of HepG2showed that comparedwith the blank group, STWE could induce the upregulation of CDK2, MDM2expression in a dosage independent way, while we couldn’t detect P53geneepression in HepG2cells. RT–PCR results of MRC-5showed that STWE couldinduce the upregulation of CDK2, MDM2and downregulation P53gene expressionin a dosage independent way. Conclusin: The STWE could damage themitochondrion of the HepG2and MRC-5and along with the increasing dosage ofsmokeless tobacco water extract, the mitochondrial menmbrane potential decreasedwhile the cell apoptosis rate and ROS production rate both increased. Part two The role of NLRP3inflammasome inmitochondrial toxicity caused by smokeless tobaccowater extractObjective: To investigate the activation and expression of NLRP3inflammasome and its downstream functional effectors in HepG2cells, MRC-5cellsand C57BL/6mice after exposoured to different dosages of STWE. To study therelationship between NLRP3inflammasome and mitochondrial toxicity. Methods:1. In vitro studies. Based on the part one results, we chose the312.50μg/ml and156.25μg/ml STWE as the stimulated dosages. The experiments carried out asbelow: HepG2and MRC-5cells were respectively seeded into6-well plates andexposured to156.25μg/ml STWE24hours,0.10μg/ml LPS4hours,0.10μg/mlLPS priming4hours and156.25μg/ml STWE24hours,0.10μg/ml LPS priming4hours and312.50μg/ml STWE24hours, and the blank control was cultured withmedium contained1%fetal bovine serum24hours.(1) The total RNA of cells indifferent experiment groups were extracted and determined by using the RT-PCR toassay the expression of NLRP3, ASC and Caspase-1.(2) The total proteins wereextracted and determined by using the western blot to assay the expression ofNLRP3, ASC and Caspase-1.(3) The cell supernatants of the four groups werecollected respectively and the concentrations of IL-1β were assayed by the IL-1βimmunoassay kit.2. In vivo studies. Healthy C57BL/6mice were randomlydivided into blank control group (5males and5females,intragastric administeredwith physiological saline),5mg/kg STWE group (5males and5females, po)、10mg/kg STWE group (5males and5females, po) and20mg/kg STWE group (5males and5females, po). After administration for consecutively15days, all themice were sacrificed.(1) The lung tissue pathology changes were analyzed byobserving pathological section.(2) The protein NLRP3, ASC, Caspase-1, IL-1β andIL-18were measured by the immunohistochemical. Results:1. In vitro studies.(1)The RT-PCR results showed that compared with the blank control, LPS priming andthen STWE stimulated groups significantly increased the gene expression of NLRP3, ASC and Caspase-1(P<0.05).(2) The western blot results showed that comparedwith the blank control, LPS priming and then STWE stimulated groups significantlyincreased the expression of NLRP3, ASC and Caspase-1protein.(3) The ELISAresults showed that compared with the blank control, LPS priming and STWEstimulated groups significantly increased the expression levels of IL-1β (P<0.05).2.In vivo study.(1) The pathology results of the10mg/kg and20mg/kg STWEstimulated groups showed the macrophages infiltration and local inflammatory reaction.while the blank control and the5mg/kg STWE stimulated groups did not have theseinflammation phenomenon.(2) Immunohistochemistry results suggested that alongwith the increase concentrations of STWE, the expression of NLRP3, ASC,Caspase-1, IL-1β and IL-18displayed an upregulation trend. Conclusion: TheSTWE could provoke the NLRP3inflammasome and induce the expression ofNLRP3, ASC, Caspase-1. And in vivo results suggested high dosage of STWEexpousure could induce the secreation of IL-1β and IL-18. Part3Breeding, reproducing and genetypeidentifying NLRP3gene konckout mice, ASC genekonckout mice and Caspase-1gene konckout miceObjective: Breeding, reproducing and identifying gene konck-out mice ofNLRP3, ASC and Caspase-1respectively. Methods: The NLRP3gene knock-outheterozygote mice (NLRP3+/-), ASC gene knock-out heterozygote mice (ASC+/-) andCaspase-1gene knock-out heterozygote mice (Caspase-1+/-) we got were respectivelybred and copulated in specific pathogen-free housing. When the baby mice werebirthed, they would be one of the following three genetypes: wild genetype,heterozygote genotype and homozygote genotype. So we extracted the genomic DNAfrom the21-days offsprings mice ears and then sent those DNA to PCR and T7endonuclease to identifying the genotype. As soon as we got the homozygote mice, letthe mice mate with the heterosexual heterozygote mice for acquiring homozygotebaby mice according to Mendel law. Results: the three gene knock-out types ofmice were bred and reproduced successfully and also we got some heterozygotegenotype mice and many homozygote genotype mice. Conclusion: Appropriatemethods for breeding, reproducing and identifying are the effective way for acquiringNLRP3-/-, ASC-/-and Caspase-1-/-mice from heterozygote mice.