Zhao Y R, Liu Y, Wang D, Lv W R, Zhou J L
Department of Orthopedics, Beijing Chao-yang Hospital, Capital Medical University, Beijing 100020, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2019 Apr 18;51(2):239-244. doi: 10.19723/j.issn.1671-167X.2019.02.007.
To investigate the effect of sulfur dioxide (SO) 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.
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/mL, and randomly divided into the following 4 groups (n=6): control group, I/R group, SO group, and I/R+SO group. The I/R group was given ischemia/reperfusion serum (500 μg/L) to stimulate 6 h; the SO group was given an SO donor, NaSO/NaHSO [(0.54 mmol/kg) / (0.18 mmol/kg)]; and the I/R+SO group was given the same ischemia/reperfusion serum and NaSO/NaHSO 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.
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+SO 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.
Exogenous SO has the effect of accelerating AM apoptosis by stimulating mPTP to open and mitochondrial membrane potential to decrease; besides, exogenous SO could stimulate AM to secrete more anti-inflammatory cytokines and less inflammatory cytokines. In conclusion, exogenous SO can reduce macrophage apoptosis by inhibiting mitochondrial pathways.
探讨二氧化硫（SO）在肢体缺血/再灌注（I/R）诱导的急性肺损伤（ALI）肺保护中对肺泡巨噬细胞（AM）凋亡的影响，寻找控制炎症反应的新靶点。
本研究选用20只无特定病原体的成年雄性Sprague-Dawley（SD）大鼠（180-230g）。5只大鼠用于肢体缺血/再灌注，然后提取血浆作为缺血/再灌注血清刺激物。15只大鼠用于通过支气管肺泡灌洗提取AM。分离并培养AM，然后将细胞计数调整至1×10/mL，随机分为以下4组（n=6）：对照组、I/R组、SO组和I/R+SO组。I/R组给予缺血/再灌注血清（500μg/L）刺激6小时；SO组给予SO供体，亚硫酸钠/亚硫酸氢钠[（0.54mmol/kg）/（0.18mmol/kg）]；I/R+SO组同时给予相同的缺血/再灌注血清和亚硫酸钠/亚硫酸氢钠。通过流式细胞术、酶标仪和蛋白质印迹法检测线粒体膜电位水平、线粒体通透性转换孔（mPTP）状态、AM凋亡率、Bcl-2和Caspase-3蛋白表达。
与对照组相比，I/R组红绿荧光比值和吸光度显著降低，凋亡细胞百分比明显增加，凋亡率为43.81%±2.40%，Caspase-3蛋白表达增加，Bcl-2蛋白表达降低。而与I/R组相比，I/R+SO组红绿荧光比值和吸光度显著增加；凋亡率降至37.01%±1.93%，Caspase-3蛋白表达降低，Bcl-2蛋白表达增加。
外源性SO具有通过刺激mPTP开放和线粒体膜电位降低来加速AM凋亡的作用；此外，外源性SO可刺激AM分泌更多抗炎细胞因子和更少炎症细胞因子。总之，外源性SO可通过抑制线粒体途径减少巨噬细胞凋亡。
