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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (2): 239-244. doi: 10.19723/j.issn.1671-167X.2019.02.007

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

Yan-rui ZHAO,Yang LIU,Dong WANG,Wen-rui LV,Jun-lin ZHOU()   

  1. Department of Orthopedics, Beijing Chao-yang Hospital, Capital Medical University, Beijing 100020, China
  • Received:2017-04-06 Online:2019-04-18 Published:2019-04-26
  • Contact: Jun-lin ZHOU E-mail:doctorzyr@163.com
  • Supported by:
    the National Natural Science Foundation of China(81070050);Beijing Natural Science Foundation(7152061)

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Abstract:

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.

Key words: Sulfur dioxide, Reperfusion injury, Acute lung injury, Macrophages, alveolar

CLC Number: 

  • R364.12

Table 1

Effect of exogenous SO2 on mitochondrial membrane potential and mPTP opening of AM (n=8)"

Group Ratio (Red/Green) Absorbance
Control 4.94±0.26 0.45±0.03
SO2 5.00±0.29 0.44±0.02
I/R 1.75±0.24* 0.24±0.02*
I/R+SO2 2.12±0.15# 0.27±0.03#

Figure 1

Detection of AM apoptosis by Annexin V-FITC and PI double-label flow cytometry A, representative flow cytometric dot plots. X-axis, Annexin-V staining; Y-axis, PI staining. Q1, detection error within the permitted range; Q2, late apoptotic cells and necrotic cells; Q3, early apoptotic cells; Q4, normal living cells (The apoptotic cells are Q2+Q3). B, quantitation of apoptotic cells population as shown in figure A. All the results are expressed as x ? ±s (n=8). *P<0.01 vs. control group; #P<0.05 vs. I/R group. "

Figure 2

The expression of Bcl-2 and Caspase-3 proteins of AM in different groups A, representative bands of Western blots; B, bar graphs show the relative expression of Bcl-2 and Caspase-3 proteins of AM in different groups. All the results are expressed as x ? ±s (n=3). *P<0.01 vs. control group; #P<0.05 vs. I/R group. "

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