Korean J Physiol Pharmacol.  2022 Mar;26(2):95-111. 10.4196/kjpp.2022.26.2.95.

Metformin alleviates chronic obstructive pulmonary disease and cigarette smoke extract-induced glucocorticoid resistance by activating the nuclear factor E2-related factor 2/heme oxygenase-1 signaling pathway

• Tao F
• Zhou Y
• Wang M
• Wang C
• Zhu W
• Han Z
• Sun N
• Wang D

• KMID: 2526729
• DOI: http://doi.org/10.4196/kjpp.2022.26.2.95

Abstract

Chronic obstructive pulmonary disease (COPD) is an important healthcare problem worldwide. Often, glucocorticoid (GC) resistance develops during COPD treatment. As a classic hypoglycemic drug, metformin (MET) can be used as a treatment strategy for COPD due to its anti-inflammatory and antioxidant effects, but its specific mechanism of action is not known. We aimed to clarify the role of MET on COPD and cigarette smoke extract (CSE)-induced GC resistance. Through establishment of a COPD model in rats, we found that MET could improve lung function, reduce pathological injury, as well as reduce the level of inflammation and oxidative stress in COPD, and upregulate expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), multidrug resistance protein 1 (MRP1), and histone deacetylase 2 (HDAC2). By establishing a model of GC resistance in human bronchial epithelial cells stimulated by CSE, we found that MET reduced secretion of interleukin-8, and could upregulate expression of Nrf2, HO-1, MRP1, and HDAC2. MET could also increase the inhibition of MRP1 efflux by MK571 significantly, and increase expression of HDAC2 mRNA and protein. In conclusion, MET may upregulate MRP1 expression by activating the Nrf2/HO-1 signaling pathway, and then regulate expression of HDAC2 protein to reduce GC resistance.

Keyword

Chronic obstructive pulmonary disease; Glucocorticoid resistance; Metformin; Nuclear factor E2-related factor 2

Figure

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  Fig. 1 Chemical structure of MET. MET, metformin.

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  Fig. 2 Histopathological analysis of the lung in rats. (A) Pathological changes in the lung tissues (Scale bars: 100 μm). The magnification is indicated with arrows. (B) Average score of airway inflammation. (C) Alveolar damage index (DI). Data are expressed as means ± SD. MET, metformin; NAC, N-Acetylcysteine. *p < 0.01 vs. control group; #p < 0.05 or ##p < 0.01 vs. model group (n = 3).

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  Fig. 3 Effects of MET on inflammatory factors and oxidative stress parameters in lung tissue of COPD rats. (A) Effect of MET on the level of IL-8 in lung tissues of COPD rats. (B) Effect of MET on the level of TNF-α in lung tissues of COPD rats. (C) Effect of MET on the activity of GSH-Px in lung tissues of COPD rats. (D) Effect of MET on the activity of SOD in lung tissues of COPD rats. (E) Effect of MET on the level of MDA in lung tissues of COPD rats. Data are expressed as means ± SD. MET, metformin; NAC, N-Acetylcysteine; COPD, chronic obstructive pulmonary disease; IL, interleukin; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; TNF, tumor necrosis factor. *p < 0.01 vs. control group; #p < 0.05 or ##p < 0.01 vs. model group (n = 5).

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  Fig. 4 Immunohistochemical analysis of lung tissues in rats. (A) The immunohistochemistry results of MRP1, Nrf2, and HO-1 proteins in each group (scale bars: 50 μm). The magnification is indicated with arrows. (B) The IOD of MRP1, Nrf2, and HO-1 proteins in each group were analyzed. Data are expressed as means ± SD. Nrf2, nuclear factor E2-related factor 2; HO-1, heme oxygenase-1; MRP1, multidrug resistance protein 1; IOD, integral optical density; MET, metformin; NAC, N-Acetylcysteine. *p < 0.01 vs. control group; #p < 0.01 vs. model group (n = 5).

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  Fig. 5 Effects of MET on protein expression of Nrf2, HO-1, MRP1, and HDAC2 in lung tissues of COPD rats. Data are expressed as means ± SD. Nrf2, nuclear factor E2-related factor 2; HO-1, heme oxygenase-1; MRP1, multidrug resistance protein 1; MET, metformin; NAC, N-Acetylcysteine; HDAC2, histone deacetylase 2; COPD, chronic obstructive pulmonary disease. *p < 0.01 vs. control group; #p < 0.01 vs. model group (n = 3).

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  Fig. 6 Survival of 16HBE in the presence of different concentrations of MET for 12, 24, and 48 h. Data are expressed as means ± SD. MET, metformin; 16HBE, human bronchial epithelial. *p < 0.05 or **p < 0.01 vs. control group (n = 3).

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  Fig. 7 Effect of MET on IL-8 inhibition rates in each group. Data are expressed as means ± SD (n = 3). MET, metformin; IL, interleukin; CSE, cigarette smoke extract; DEX, dexamethasone.

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  Fig. 8 Effects of MET on the expression of Nrf2, HO-1, MRP1, HDAC2 proteins in cells with glucocorticoid resistance. Data are expressed as means ± SD. Nrf2, nuclear factor E2-related factor 2; HO-1, heme oxygenase-1; MRP1, multidrug resistance protein 1; MET, metformin; HDAC2, histone deacetylase 2; CSE, cigarette smoke extract; DEX, dexamethasone. *p < 0.01 vs. control group; #p < 0.05 or Δp < 0.01 vs. CSE group; Δp < 0.01 vs. DEX group or MET group (n = 3).

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  Fig. 9 Effect of SnPP on IL-8 inhibition rates. Data are expressed as means ± SD (n = 3). SnPP, tin protoporphyrin; IL, interleukin; CSE, cigarette smoke extract; DEX, dexamethasone; MET, metformin.

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  Fig. 10 Effect of MK571 on IL-8 inhibition rates. Data are expressed as means ± SD (n = 3). IL, interleukin; CSE, cigarette smoke extract; DEX, dexamethasone; MET, metformin.

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  Fig. 11 Effects of MET and MK571 on efflux function of MRP1. (A) Effects of MET and MK571 on 5-CF level. (B) Mean fluorescence intensity (MFI) in each group. Data are expressed as means ± SD. MRP1, multidrug resistance protein 1; MET, metformin; 5-CF, 5-carboxyfluorescein; CSE, cigarette smoke extract; DEX, dexamethasone; 5-CFDA, 5-carboxyfluorescein diacetate. *p < 0.01 vs. control group; **p < 0.01 vs. 5-CFDA group; #p < 0.01 vs. CSE group; Δ p < 0.01 vs. DEX+MET group (n = 5).

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  Fig. 12 MK571 significantly reduced the expression of HDAC2 protein and mRNA induced by MET. (A) Effect of 20 μM MK571 on the expression of HDAC2 protein. (B) Quantitative analysis results of protein expression in each group. Data are expressed as means ± SD. *p < 0.01 vs. control group; #p < 0.01 vs. CSE group; Δp < 0.01 vs. DEX+MET group (n = 3). (C) Effect of MK571 on mRNA expression of HDAC2. Data are expressed as means ± SD. *p < 0.01 vs. control group; #p < 0.01 vs. CSE group; Δp < 0.01 vs. DEX+MET group (n = 5). (D) Effects of different concentrations of MK571 on the expression of HDAC2 protein. (E) Quantitative analysis results of protein expression in each group. Data are expressed as means ± SD. MET, metformin; HDAC2, histone deacetylase 2; CSE, cigarette smoke extract; DEX, dexamethasone. *p < 0.05 or #p < 0.01 vs. control group (n = 3).

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  Fig. 13 The potential mechanism of MET alleviating COPD and CSE-induced GC resistance through activating Nrf2/HO-1 signaling pathway, which can promote the function and expression of MRP1 to increase the expression of HDAC2. MET, metformin; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; GC, glucocorticoid; Nrf2, nuclear factor E2-related factor 2; HO-1, heme oxygenase-1; MRP1, multidrug resistance protein 1; HDAC2, histone deacetylase 2; 4-HNE, 4-hydroxynonenal; ROS, reactive oxygen species; SOD, superoxide dismutase.

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