Effects Of Tricarboxylic Acid Cycle On Mitochondrial Function In Inflammatory And Anti-inflammatory Environments

Inflammation is a dual response of damage and anti-damage caused by external stimuli.Excessive or sustained inflammation will lead to tissue damage and may also lead to many diseases,such as diabetes,cardiovascular disease,tumor,etc.Mitochondria play an important role in cell energy metabolism,and the TCA circulation pathway is a key pathway for ATP production.Therefore,it is of great significance to study the correlation between inflammation and mitochondrial TCA cycle.Objective: To investigate the influence of inflammatory and anti-inflammatory environment on the expression of key enzymes in TCA cycle in mitochondria,combining the related changes of mitochondrial function.The correlation between TCA cycle and mitochondrial function was also analyzed and to provide theoretical basis for the treatment of related diseases in the future.Methods: Macrophages were stimulated with 1 ?g/m L lipopolysaccharide to induce inflammation,5 mmo L/L metformin was used for anti-inflammatory treatment,use the q RT-PCR and Western Blot technique to detect the change of key enzymes of TCA cycle in the modeling macrophages.Enzymes are CS,IDH2,MDH2 and OGDH.Western Blot was used to detect the changes of autophagy related protein LC3 b,and flow cytometry was used to detect the changes of mitochondrial membrane potential and mitochondrial reactive oxygen species,so as to infer the changes of mitochondrial function.Using six 8-week-old male C57BL/6J mice,intraperitoneal inject with LPS and Met,and taken lung tissues for HE staining to confirm the validity of the model.Western Blot was used to detect the change of key enzymes OGDH,CS,IDH2 and MDH2 in the mitochondrial TCA circulation pathway and the expression levels of autophagy related protein LC3 b in the mouse lung tissues.Results: Based on in vitro cell experiments,under LPS stimulation,q RT-PCR technique detected that m RNA expression levels of key enzymes CS,IDH2,MDH2 and OGDH in the TCA circulation pathway were all decreased,while detected by Western Blot technique,protein expression levels of key enzymes CS,OGDH and IDH2 in the TCA circulation were decreased.After the used Met to anti-inflammatory,m RNA expression levels of key enzymes CS,IDH2,MDH2 and OGDH in the TCA circulation pathway were all recovered detected by q RT-PCR technique,and protein expression levels of CS,OGDH and IDH2 were also recovered detected by Western Blot technique.The results of mice model experiments showed that after LPS injection,the expression levels of key enzymes OGDH,CS and MDH2 in TCA cycle were decreased detected by Western Blot technique.Protein expression of OGDH and MDH2 recovered after Met anti-inflammatory detected by Western Blot technique.That is,under the stimulation of LPS,the overall efficiency of mitochondrial TCA cycle was weakened,and the efficiency was recovered after anti-inflammatory.Based on in vitro cell experiments,flow cytometry technique showed that mitochondrial ROS increased and mitochondrial membrane potential decreased after LPS stimulation.Western Blot technique showed that autophagy related protein LC3 b increased in both cell model and mice model.After Met anti-inflammatory,mitochondrial ROS were decreased in the cell model,mitochondrial membrane potential was decreased,and autophagy related protein LC3 b was decreased in both the cell model and the mice model.That is,mitochondrial dysfunction under the stimulation after LPS,but was recovered after Met anti-inflammatory.Conclusion: Lipopolysaccharide induced macrophage inflammatory environment reduced the expression of four enzymes and affected mitochondrial function.The results of animal experiments were consistent with those of cell experiments.Mitochondrial function is closely related to the stability of the TCA cycle.