二氧化硫对大鼠肢体缺血再灌注致急性肺损伤中肺泡巨噬细胞凋亡的影响

Effects of sulfur dioxide on alveolar macrophage apoptosis in acute lung injury induced by limb ischemia/reperfusion in rats

目的:探讨二氧化硫(sulfur dioxide,SO2)在肢体缺血再灌注(ischemia/reperfusion,I/R)致急性肺损伤(acute lung injury,ALI)保护作用中对肺泡巨噬细胞(alveolar macrophage,AM)凋亡的影响,为控制炎症反应寻找新的靶点。方法:分离培养AM,应用肢体缺血再灌注致ALI大鼠血清制备细胞模型,给予外源性SO2,然后检测线粒体膜电位以及线粒体通透性转换孔(mitochondrial permeability transition pore,m PTP)开放情况,AM凋亡情况及凋亡相关Bcl-2、Caspase-3分子蛋白表达情况。结果:与对照组相比,I/R组红、绿荧光的比值下降,吸光度显著降低,AM凋亡率增加到43.81%±2.40%,Caspase-3蛋白表达升高,Bcl-2蛋白表达下降;而与I/R组比较,I/R+SO2组红、绿荧光的比值升高,吸光度增高,AM凋亡率减少37.01%±1.93%,Caspase-3蛋白表达降低,Bcl-2蛋白表达升高。结论:外源性SO2可通过抑制线粒体途径改善巨噬细胞的凋亡。

Objective: To investigate the effect of sulfur dioxide (SO2) on the apoptosis of alveolar macrophage (AM) in lung protection of limb ischemia/reperfusion (I/R) induced acute lung injury (ALI), and to find a new target for the control of inflammatory response. Methods: Twenty pathogen-free, adult male Sprague-Dawley (SD) rats (180-230 g) were used in this study. Five rats were to be used for limb ischemia/reperfusion, then plasma was extracted as ischemia/reperfusion serum stimulation. Fifteen rats were to be used for extracting AM by bronchoalveolar lavage. The AM was isolated and cultured, then the cell count was adjusted to 1×10^6/mL, and randomly divided into the following 4 groups (n =6): control group, I/R group, SO2 group, and I/R+SO2 group. The I/R group was given ischemia/reperfusion serum (500 μg/L) to stimulate 6 h;the SO2 group was given an SO2 donor, Na2SO3/NaHSO3 [(0.54 mmol/kg)/(0.18 mmol/kg)];and the I/R+SO2 group was given the same ischemia/reperfusion serum and Na2SO3/NaHSO3 at the same time. The level of mitochondrial membrane potential, the state of mitochondrial permeability transition pore (mPTP), the rate of AM apoptosis, the expression of Bcl-2 and Caspase-3 proteins were detected by flow cytometry, microplate reader and Western blotting. Results: Compared with the control group, in the I/R group, the ratio of red to green fluorescence and the absorbance decreased significantly, the percentage of apoptotic cells increased obviously, the apoptotic rate was 43.81%±2.40%, Caspase-3 protein expression increased, Bcl-2 protein expression decreased. While compared with the I/R group, in the I/R+SO2 group, the ratio of red to green fluorescence and the absorbance increased significantly;the apoptotic rate decreased to 37.01%± 1.93%, Caspase-3 protein expression decreased, Bcl-2 protein expression increased. Conclusion: Exo- genous SO2 has the effect of accelerating AM apoptosis by stimulating mPTP to open and mitochondrial membrane potential to decrease;besides, exogenous SO2 could stimulate AM to secrete more anti-inflammatory cytokines and less inflammatory cytokines. In conclusion, exogenous SO2 can reduce macrophage apoptosis by inhibiting mitochondrial pathways.