The Mitochondria/Caspase Dependent Pathway Mechanism Of Apoptosis In Cultured Rats Spleen Lymphocyte Induced By Aluminum

Spleen lymphocytes were cultured in vitro to investigate the mitochondria/caspase-dependentpathway apoptosis induced by aluminum in rats. The spleen lymphocytes were isolated fromWistar rats and cultured in RPMI-1640medium. The lymphocytes were exposed to AlCl3·6H2O infinal concentration of0(control group,GC),0.30(low-dose group, GL),0.61(middle-dose group,GM), and1.21(high-dose group, GH) mmol·L-1, respectively. Lymphocytes morphology, apoptosisindex, DNA ladder, mitochondrial transmembrane protential (Δψm), CytC expression of cytoplasmand the activity of Caspase-3,9were detected using Acridine Orange/Ethidium Bromide (AO/EB)staining, flow cytometry, agarose gel electrophoresis, JC-1and Western blot. The activity ofCaspase-3,9were detected under microplate reader. And the mRNA expressions of Bcl-2, Bax,caspase-3,9were determined by real-time fluorescence quantitative PCR (RT-PCR).The resultsshowed as follows：1Lymphocytes in GC were green, round or elliptical under fluorescence microscopic, butlymphocytes were pyknotic in Al-treated groups. Early apoptotic cells were green pyknotic, lateapoptotic cells were orange-red pyknotic and necrotic cells were orange-red round. Apoptosisindexes of early and late lymphocyte in Al-treated groups were significantly higher than those ofGC, and there were dose-cffect relationships. It indicated aluminum could induce spleenlymphocyte apoptosis in rats.2Typical DNA ladder appeared in Al-treated groups while not in GC, which indicated thataluminum could induce DNA damage of spleen lymphocyte in rats.3The fluorescence red/green in Al-treated groups was significantly lower than that in GC. Itindicated aluminum induced lymphocyte mitochondrial damage by decreaseΔψm.4The CytC expressions of cytoplasm in Al-treated groups increased significantly. It indicatedaluminum induced cytotoxicity by causing CytC release from mitochondria.5Caspase-3,9activity and mRNA expressions in Al-treated groups were significantly higherthan those in GC. It indicated Al could induce cytotoxicity by inducing lymphocyte csapasecascade reaction.6The Bcl-2, Bax mRNA expressions in Al-treated groups all increased gradually with theincreased Al concentration. It indicated aluminum could regulated lymphocyte apoptosis by induce apoptosis gene expression.The above results showed that aluminum could induce spleen lymphocyte apoptosis throughmitochondria/caspase-dependent pathway in rats. The results could enrich aluminumimmunotoxicology and provide theoretical basis for preventing immunotoxicity of aluminum.