The Mechanism Of Apoptosis And Inflammatory Response Induced In Mouse Spleen Lymphocytes By Hypoxia

ObjectiveOxygen is necessary tosustain human and animal life activities.When the body is hypoxia,it causes irreversible damage to tissues and cells.Spleen is the largest peripheral immune organ of the body,and splenic lymphocytes are important adaptive immune response cells,which are essential for maintaining immune homeostasis.Hypoxia exposure can induce apoptosis in splenic lymphocytes and spleen,and inflammatory response of spleen.However,the mechanism is still unclear.Therefore,in this study,splenic lymphocytes and spleen of mice were taken as the research objects to explore the effect and mechanism of hypoxia exposure on splenic lymphocytes apoptosis.To further explore whether this mechanism plays a role in apoptosis of spleen induced by hypoxia,and to clarify the mechanism of inflammatory response of spleen under hypoxia exposure.MethodsLymphocytes were isolated from the spleen of C57BL/6J mice and cultured in hypoxia（1%O₂）and normoxia（21%O₂）environment,respectively.The survival rate of lymphocytes was observed by trypan blue staining.Scanning electron microscope（SEM）was used to observe the morphological changes of lymphocytes.The number,proliferation,apoptosis,reactive oxygen species（ROS）and mitochondrial membrane potential（MMP）of lymphocytes,in addition to T lymphocytes and B lymphocytes were detected by flow cytometry.RT-q PCR and Western Blot were used to detect the m RNA and protein expressions levels of apoptosis-related factors（Bak,Bcl-2,Fas,Fas L,Cytochrome C,Apaf-1,caspase-3,caspase-6,caspase-7,caspase-8,and caspase-9）.C57BL/6 mice were exposeed in plain normoxia（400m,PSC group）and plateau hypoxia（4,200m,HST group）for 30 days,respectively;to establish the plateau hypoxia animal model.Transcriptome sequencing was performed by RNA-seq technology.Bioinformatics analysis technology was used to identify key（Hub）genes（SOD2,MRPS16,NDUFS6,TUFM,and NDUFA3）related to mitochondrial damage and apoptosis under hypoxia exposure.RT-q PCR and Western Blot were used to detect the m RNA and protein expression of Hub genes.Tandem mass spectrometry（TMT）and liquid chromatography-tandem mass spectrometry（LC-MS/MS）were used for proteomics and non-targeted metabolomics sequencing,respectively.Combined analysis of the two omics was conducted to explore the key proteins,metabolites,and mechanisms of inflammatory response in spleen tissue under hypoxia exposure.RT-q PCR was used to verify the m RNA expression of key proteins（Alox15,Hpgds,and Ptgs2）.Results1.Hypoxia exposure for 12 h,24 h,and 48 h could reduce the number of T and B lymphocytes,inhibit the proliferation of lymphocytes,and promote lymphocytes apoptosis.SEM indicated that at the same time point,the characteristic morphological changes of apoptosis of lymphocytes in the hypoxia group appeared earlier.The m RNA and protein expression levels of Bak and caspase-3 were up-regulated,while the m RNA and protein expression levels of Bcl-2 were down-regulated in lymphocytes exposed to hypoxia at 12 h,24 h and 48 h.2.Hypoxia exposure at different times can reduce the survival rate of lymphocytes,increase the apoptosis rate of lymphocytes,and make the morphological changes of apoptotic characteristics of cells appear earlier.By grouping T and B lymphocytes,it was found that the apoptosis of lymphocytes was first dominated by T lymphocytes,and then dominated by B lymphocytes.ROS production increased and MMP decreased.RT-q PCR results showed that the m RNA expression levels of Bak,Fas,Fas L,Cytochrome C,Apaf-1,caspase-3,caspase-6,caspase-7,caspase-8,and caspase-9 were up-regulated in lymphocytes exposed to hypoxia.The expression of Bcl-2 m RNA was down-regulated.Western Blot indicated that the protein expression levels of Bak,Fas,Fas L,Cytochrome C,and caspase-3 were up-regulated,while Bcl-2 was down-regulated under hypoxia environment.3.Transcriptome analysis revealed that 4,173 genes were significantly changed in the spleen tissue of mice exposed to hypoxia,of which 454 genes were related to mitochondrial damage.The GO and KEGG enrichment results of 454 differential mitochondria-related genes（DE-MFRGs）represented that DE-MFRGs were significantly enriched in mitochondrial function and apoptosis-related pathways.Five Hub genes（SOD2,MRPS16,MRPL12,NDUFS6,TUFM,and NDUFA3）were identified by constructing a protein-protein interaction（PPI）network.The m RNA and protein expression levels of 5 Hub genes were down-regulated,which was consistent with the transcriptome results.The correlation analysis between the 5 Hub genes and differential apoptosis-related genes（DE-ARGs）showed a positive or negative correlation.The m RNA and protein expression levels of apoptosis-related genes Bak and caspase-3 were up-regulated,and the m RNA and protein expression levels of Bcl-2 were down-regulated in spleen tissue exposed to hypoxia.4.Proteomics results showed that 166 proteins were significantly up-regulated and 39 proteins were significantly down-regulated in the plateau hypoxia group compared with the plain normoxia group.Bioinformatics analysis of 166 up-regulated differentially expressed proteins（DEPs）revealed that mineral absorption,neuroactive ligand-receptor interaction,arachidonic acid（AA）metabolism,IL-17 signaling pathway,and NOD-like receptor signaling pathway were significantly enriched.Metabonomics results represented that 4,285 differential metabolites（DAMs）were significantly down-regulated and 4,026 DAMs were significantly up-regulated.Combined proteomics and metabolomics analysis showed that mineral absorption,neuroactive ligand-receptor interaction,and AA metabolism pathways were significantly enriched in KEGG rich sets.Comparing the expression levels of proteins and metabolites involved in the AA metabolic pathway,it was found that Alox15,Hpgds,Ptgs2,and Pld3 were significantly up-regulated.The downstream metabolites Pga2 and Pgd2 were significantly up-regulated.RT-q PCR and Western Blot results revealed that the m RNA and protein expression levels of Alox15 and Hpgds were significantly up-regulated.The m RNA expression level of Ptgs2 was not significantly different,but the protein expression level was significantly up-regulated.Conclusions1.Hypoxia exposure can inhibit lymphocytes proliferation,promote apoptosis,further accelerate the morphological changes of lymphocytes apoptosis characteristics,and induce the changes of apoptosis-related factors.It is suggested that the decrease of lymphocytes number induced by hypoxia exposure may be mainly caused by apoptosis.2.Hypoxia can induce lymphocytes apoptosis through Fas receptor-dependent apoptosis pathway and mitochondria-dependent apoptosis pathway,thereby affecting the number and activity of lymphocytes and mediating the functional damage of lymphocytes.3.Based on transcriptome analysis,hypoxia mediates mitochondrial dysfunction by down-regulating SOD2,MRPS16,NDUFS6,TUFM and NDUFA3,and then induces apoptosis injury of spleen.4.Through the combined analysis of proteomics and metabolomics,it was found that the related proteins and downstream metabolites of AA metabolism pathway in spleen were changed under hypoxia exposure.These results suggest that AA metabolism may be involved in hypoxia-induced spleen inflammation in mice.