Dexmedetomidine Protects Macrophage Injury Via Mitophagy

OBJECTIVE:Sepsis,a disease caused by a dysregulated host response to infection,is a common complication of burns,trauma and major surgery,and then immune disorders,immunosuppression and apoptosis are the main causes of death in patients with severe sepsis or septic shock.As an important innate immune-reactive cells,macrophages have played an important role in the development of sepsis.LPS was usually used to mimic inflammatory model.Mitochondria is the energy factory of all eukaryotic cells,during sepsis multiple organ dysfunction is supposedly associated with mitochondrial dysfunction.Mitophagy is a mitochondria quality control system,thus dampening inflammation,reducing cell death signals and preventing unwanted cell loss after cells encounter various stimulation.Dexmedetomidine?DEX?is a sedation drug,and increased evidences have shown that DEX have an anti-inflammation,anti-apoptosis and attenuate organ dysfunction effect,but the mechanism remains unclear.This study was to investigate the protective effect of dexmedetomidine on LPS treated macrophage and explore the mechanism of anti-inflammatory,anti-apoptotic and organ protection effects.METHOD:Mouse macrophage RAW264.7 was purchased from American ATCC company and cultured in DMEM with 10%FBS in a humidified incubator?5%CO₂?at 37°C and dislodged from the flask substrate with a cell scraper when the confluence was approximately 80%.The subcultivation ratio of 1:3 to 1:6 is recommended,and medium should be replaced about every 2 days.RAW264.7 cells?2×10⁵ cells/mL?were randomly seeded in dishes or plates.This experiment was divided into four parts.Part ?:The experiment was divided into seven groups,LPS group was treated with LPS,and Con group was given the same volume of PBS buffer.Cellular proteins were extracted at 4h,8h,12h,16h,20h and 24h,and the expressions of LC3and P62 were detected to determine the peak time.Then 3-MA and RAPA were administered at peak time respectively,and cell proliferation was measured by CCK-8 to observe the effect of autophagy on macrophages.Part ?:The experiment was divided into two groups,treatments was the same as part I,but the incubation time was choosed at the peak point of autophagy.Firstly,inflammatory cytokines and NO concentration in supernatant were detected to assess inflammation.Secondly,ATP,ROS,MMP and the expression of Tom20 were measured to evaluate mitochondrial function.Then Western Blotting was used to observe apoptosis in LPS-treated macrophage.Part ?:This part was divided into two small parts:1.Cell groups and treatments were the same as part II,incubation time was 12 hours.Western Blotting,confocal fluorescence and transmission electron microscopy were used to detected mitophagy or mitochondria morphology.2.Cells were divided into siCon group?siCon+LPS group?siPINK1 group and siPINK1+LPS group.siCon was transfected with vector,siPINK1 was transfected with shRNA targeting PINK1.Then the effects of PINK1 on inflammation,apoptosis and mitochondrial function were observed.Part ?:This part was divided into two small parts:1.Cells were divided into Con group,DEX group,LPS group and LPS+DEX group,LPS group was treated with LPS,DEX group was treated with dexmedetomidine?1?M?,DEX was added to the culture medium after LPS administration.Then the effects of DEX on inflammation,apoptosis,and autophagy was observed;2.The experiment was divided into siCon+LPS group,siCon+LPS+DEX group,siPINK1+LPS group and siPINK1+LPS+DEX group.Transfection was the same as part III.Then the role of PINK1 was observed in the protective effect of dexmedetomidine.RESULTS:Result ?:Cell viability was decreaed during LPS treatment?P<0.05?,while the expression of LC3-II was increased and P62 was decreased?P<0.05?,and the peak time of LC3-II expression was between 12h¹6h?P<0.05?.Cell viability was decreased when inhibit autophagy with 3-MA?P<0.05?and increased when promote autophagy with low concentration of RAPA?P<0.05?,while decreased when promote autophagy with high concentration of RAPA?P<0.05?.Result ?:The secretion of TNF-?,IL-1?and the concentration were increased?P<0.05?;intracellular ATP concentration and mitochondrial membrane potential were decreased?P<0.05?,and the expression of Caspase-9 and Cyt-c were increased?P<0.05?during LPS treatment.Result ?:LPS treatment of macrophages led to mitophagy,and the expression of PINK1 and Parkin was increased?P<0.05?.While inhibit the expression of PINK1by shRNA,the secretion of TNF-?,IL-1?and the concentration of ROS were increased?P<0.05?;intracellular ATP concentration and mitochondrial membrane potential were decreased?P<0.05?,and the expression of Caspase-9 and Cyt-c were increased?P<0.05?.Result ?:Cell viability and intracellular ATP concentration were increased?P<0.05?;ROS concentration,the secretion of IL-1?and TNF-?,and the expression of Cyt-c and Caspase-9 was decreased?P<0.05?during dexmedetomidine incubation.While inhibit the expression of PINK1 by shRNA,the secretion of TNF-?,IL-1?and the concentration of ROS were increased?P<0.05?;intracellular ATP concentration and mitochondrial membrane potential were decreased?P<0.05?,and the expression of Caspase-3 and Cyt-c were increased?P<0.05?.CONSLUSION:LPS treatment can cause macrophage inflammation,mitochondrial dysfunction and mitochondria-dependent apoptosis,while PINK1-mediated mitophagy plays a protective role in these reactions;Dexmedetomidine can reduce LPS-induced inflammation,mitochondrial dysfunction,and mitochondria-dependent apoptosis,and the protective effects are dependent on PINK1-mediated mitophagy.