Anti-Lung Cancer Mechanism Of VPA Combined With CoQ₁₀ By Improving Mitochondrial Function Of T Cells In Mice

Objective:The aim of this study was to investigate the effect of improving T-cell mitochondrial function on benzo[a]pyrene（Bap）-induced lung carcinogenesis.To clarify the ameliorative effect of CM（the combination of valproic acid and coenzyme Q10）,medications that improves T-cell mitochondrial function,on Bap-induced lung cancer in mice,and to explore its mechanism of the effect.Contents and methods:1.Screen medications that improve mitochondrial function of T cellsAccording to the literature and the results of previous pre-experiments,the interventions used in this part concluding Bombyx Batryticatus（BB,5 g/kg）,Raw Pinellia（RP,5 g/kg）,and VPA and Co Q10 in combination（CM,150 mg/kg VPA+100mg/kg）.Balb/C mice were randomly divided into control group（gavaged with olive oil）,model group（gavaged with benzene）and intervention group（gavaged with benzene and medications）which were gavaged for 5 days per week for 4 weeks.By measurement of peripheral blood T-cell mitochondrial function,pathological examination of spleen and thymus,evaluation of immune function and antioxidant function,the effect of different medications on immune function of mice was clarified and medications that can effectively improve T-cell mitochondrial function were selected.2.Effect of CM on the incidence of Bap-induced lung cancerIn vitro experiments,primary mouse T cells were used to observe the effect of Bap on mitochondrial function of T cells,and whether medication interventions could effectively prevent Bap induced mitochondrial damage of T cells.The co-culture technique was used to observe T cells function of killing tumor cells.In vivo,male Kunming mice were randomly grouped into control,Bap,Bap+VPA,Bap+Co Q10,and Bap+CM group.Bap was gavaged once a week for 8 weeks,and the medication groups were administered daily for 22 weeks from the beginning of the experiment.At the end of the experiment,the effects of the different medications on the incidence of lung cancer in mice were monitored,and the effects of the different medications on the antioxidant capacity,immune factor levels,lung immune microenvironment,mitochondrial function of peripheral,thymus and spleen T cells in mice were determined,to clarify the effects of VPA,Co Q10 and their combined medications on the mitochondrial function of Bap-induced T cells and lung cancer in mice.3.Investigate mechanism of medications on prevention Bap-induced lung cancerThe protein expression level of T cells in the control group,Bap group and intervention group were measured by proteomics technology.The significantly changed proteins were selected and enriched by bioinformatics analysis.The signaling pathways that may related with Bap-induced tumors were obtained by comparing the control group with the Bap group,and the possible signaling pathways for CM prevention of lung cancer in mice were obtained by comparing the Bap group with the CM group.The differential proteins obtained were carefully compared and analyzed to obtain the key proteins.The expression of the screened proteins was verified by immunohistochemistry to determine their role in the process of lung cancer in mice.Mice T cells with low expression of key protein（KP）and overexpressed of KP T cells were obtained by using lentivirus.The effect of KP was determined by observing its effect on mitochondrial function.Cells were divided into control groups,Bap group,Bap+KP^OE group to determine the effect of KP on Bap-induced mitochondrial functional damage.Control group,KP^KD group,CM+KP^KD,was set up to determine the regulatory effect of CM on KP and its effect on mitochondrial function.Results:1.CM can significantly improve the mitochondrial function of T cellsThe levels of malondialdehyde（MDA）,superoxide dismutase（SOD）,and glutathione peroxidase（GSH）were increased in plasma,thymus and spleen tissues after benzene intervention.Benzene induced the oxidative stress response in mice,accompanied with structural damage to spleen and thymus tissues,imbalance of T-cell ratio and impairment of mitochondrial function of immune cells,which caused a significant increase in ROS levels and a significant decrease in ATP levels,leading to the appearance of immune damage.After BB,RP,and CM interventions,MDA levels were significantly reduced,SOD and GSH levels were significantly increased,tissue structural damage was reduced,and T cell ratios were restored compared to the benzene exposed group,meanwhile all three medications prevented benzene-induced increase in T cell ROS expression levels and increased peripheral blood mononuclear cell（PBMC）adenosine triphosphate（ATP）levels,and protected T-cell mitochondrial function.Among them,CM had the most significant effect on the improvement of T-cell mitochondrial function2.CM decrease the incidence of Bap-induced lung cancerIn vitro experiments revealed that Bap exposure led to a dose-dependent decrease in T cell mitochondrial function,mainly manifested by a significant increase in T cell ROS expression levels and a significant decrease in ATP levels after Bap exposure,as well as a significant decrease in T cell activity.co-cultured T cells with tumor cells revealed that Bap significantly reduced the tumor-killing ability of T cells,mainly manifested by promoting tumor cell migration and reducing tumor cell apoptosis.In the presence of Bap exposure,CM intervention prevented Bap-induced T cell mitochondrial damage and improved T cell survival,while significantly increasing the tumor-killing function of T cells.In vivo experiments showed a significant increase in lung carcinogenesis in mice with Bap（71.4%）compared to the control group（16.7%）,leading to a significant increase in the proportion of CD4⁺/CD8⁺T cells in the lung.Meanwhile,Bap exposure increased MDA expression levels and decreased SOD,GSH expression levels,leading to increased ROS levels and decreased ATP levels in PBMC,spleen and thymus T cells,accompanied by an imbalance of T cell ratio in PBMC and thymus.Compared with the Bap group,the use of VPA（47.6%）,Co Q10（45.5%）,and CM（40.0%）significantly reduced the incidence of lung cancer in mice,with the most significant reduction in the CM group.CM also reduced the proportion of CD4+/CD8+T cells in lung tissue,increased the levels of antioxidants SOD,GSH,reduced the levels of oxidized MDA,and decreased the levels of PBMC,ROS expression in thymic T cells,and increased ATP expression level.Among them,CM had the most significant effect on the improvement of mitochondrial function of thymic T cells.3.Bap affects T cell oxidative phosphorylation signaling pathwayBap induced 281 proteins upregulated and 327 proteins downregulated in thymic T cells of lung cancer mice compared to controls.The enrichment of all differential proteins by KEGG revealed that Bap induced changes in the expression levels of proteins related to apoptosis,cell cycle,antigen processing and other signaling pathways in lung cancer mice.And the results of thymocyte apoptosis level showed that the percentage of thymocyte apoptosis was significantly higher in the Bap-induced group of mice compared with the control group,and the level of thymocyte apoptosis was alleviated after CM intervention.GO enrichment analysis of upregulated proteins revealed that mitochondrial functional execution-related processes,such as mitochondrial protein complexes,TCR signaling vesicles,immune receptor differentiation,antigen expression and NADP metabolic processes,were significantly upregulated after Bap intervention.Meanwhile,downregulated protein GO enrichment results showed that mitochondrial protein complexes,NAD metabolic processes,NADH metabolism and other mitochondrial biological metabolic processes were altered,and KEGG signaling pathway analysis showed that the T cell oxidative phosphorylation（OXPHOS）signaling pathway was significantly downregulated after Bap exposure,and in addition,differential protein analysis combined with Bap-induced and CM intervention groups showed that the mitochondrial protein complex-related protein ATP5MF was significantly downregulated.Complex-related proteins ATP5MF and SDHD may play a key role in Bap-induced lung carcinogenesis and CM prevention of lung carcinogenesis.Subsequently,immunohistochemistry was used to validate the mitochondrial protein complex subunits SDHD and ATP5MF,and Bap exposure increased ATP5MF expression levels and decreased SDHD expression levels compared with the control group,and CM decreased ATP5MF and increased SDHD expression levels compared with the Bap group.The results showed that ATP5MF was consistent with the expression trend determined by proteomics.4.Changes in ATP5MF expression levels affect mitochondrial function in T cellsTo further elucidate the effect of ATP5MF on T cell mitochondrial function,overexpression of ATP5MF lentiviral vector was used to increase the expression level of ATP5MF in T cells.The results showed that overexpression of ATP5MF significantly improved the Bap-induced increase in mt ROS level and decrease in ATP content level in T cells in the Bap-induced T cell injury model.Meanwhile,overexpression of ATP5MF significantly increased the expression of mitochondrial biogenesis-related protein PGC-1αand TFAM compared with the Ba P group,and the above results indicated that overexpression of ATP5MF significantly ameliorated the Bap-induced decrease in T cell mitochondrial function.The results showed that the reduced expression level of ATP5MF in T cells led to an increase in ROS expression and a significant decrease in PGC-1αand TFAM expression in T cells.And compared with the low ATP5MF expression group,the use of CM increased the cellular ATP5MF expression level,decreased the T cell ROS expression level,increased the T cell ATP expression level,and increased the PGC-1αand TFAM protein expression levels.Conclusion:1.CM significantly ameliorated benzene-induced immune damage and T-cell mitochondrial function impairment in mice.2.CM prevented Bap-induced mitochondrial damage and improved T-cell tumor killing function in vitro.3.CM reduced the incidence of Bap-induced lung cancer in mice.4.Bap-induced lung cancer in mice is associated with impaired T-cell oxidative phosphorylation process.5.Bap down-regulates ATP5MF leading to mitochondrial functional impairment,and CM up-regulates ATP5MF reversing Bap-induced mitochondrial functional impairment.