Interferon-gamma modified mesenchymal stem cells (MSCs) induced apoptosis in lung tumor cells through caspase-3 activation. The percentage of activated-caspase-3-positive tumor cells in IFN-γ modified cultured MSCs was significantly higher than in control cultured MSCs. The results of this study provide a new strategy for tumor therapy that utilizes IFN-γ modified MSCs (40).

Anti-inflammatory effect of IFN-γ via down-regulation of TGF-β1 and pro-collagen I and III gene expression in a mouse model of lung fibrosis was demonstrated (41). Interactions of IFN-γ and IL-13, on the mouse model of airway inflammation, showed a negative correlation between increased IFN-γ (number of Th1 cells) with airway hyperreactivity. Also, IFN-γ is able to modulate the effects of IL-13-induced airway hyperreactivity and goblet cell hyperplasia. Intranasal administration of IFN-γ inhibits IL-13-induced goblet cell hyperplasia and airway eosinophilia. Co-administration of IFN-γ and IL-13 showed synergic effects on increased IL-6 level as well as numbers of natural killer (NK) cells and CD11c-positive cells in the airways of mice (42).

Interferon-gamma has been shown to be effective in the treatment of patients with idiopathic pulmonary fibrosis (IPF) (43). Decreased IFN-γ levels in leukocyte cultures from SM exposed patients were also reported (44). In a clinical study, 36 SM exposed patients with SM bronchiolitis were divided into case and control groups. The case group was treated with a combination of 200 mg IFN-γ and 7.5 mg prednisolone for 6 months. Pulmonary function tests (PFT) including FEV₁ and forced vital capacity (FVC) were not significantly different at the baseline between the two groups. However, FEV₁ and FVC were significantly increased in the case group during the subsequent months (45). In another similar study, the effect of interferon IFN-γ on respiratory symptoms, quality-of-life, and oxidative parameters in SM exposed patients was investigated. IFN-γ (100 μg) was administered every other day for 6 months. Severity and frequency of respiratory symptoms, quality-of-life, serum levels of different cytokines, and oxidative stress parameters were assessed at the baseline and at the end of the study. The results showed that IFN-γ therapy not only elevated FEV₁, but also reduced the severity of cough, dyspnea, and frequency of sputum occurrence. IFN-γ therapy also is associated with improvements in quality-of-life. Serum levels of IL-4, IL-6, IL-10, calcitonin gene-related peptide (CGRP), MMP-9, tumor necrosis factor-alpha (TNFα), TGFβ, and malondialdehyde (MDA) as oxidative stress marker were also decreased while the level of IL-2, IFN-γ, and glutathione (GSH) were increased at the end of study (46).

The above studies suggest the therapeutic potential of IFN-γ in SM-induced lung injury by reduction of inflammatory mediators and oxidative stress as well as respiratory symptoms but increased PFT values in SM exposed patients with lung disorders.

The administration of theophylline (10 or 20 μg/ml) 2 h before human TGF-β1 stimulation suppressed TGF-β-induced type I collagen (COL1) mRNA expression in lung fibroblasts and also inhibited fibroblast proliferation (48). It was reported that theophylline (1–50 mg/kg–1, i.p.) significantly inhibited the inflammation at the early (4 h) and late (48 h) phases of inflammatory reaction induced by carrageenin (1%) in a murine model of pleurisy (49). Theophylline reduced airway inflammation and pathologic changes of lung tissue induced by cigarette smoke in a COPD model of rats (50). Theophylline (3 and 30 mg/kg) reduced the influx of inflammatory cells into the BALF of Guinea pigs exposed to LPS (30 μg/mL). Furthermore, theophylline improved histological changes induced by LPS, including accumulation of inflammatory cells in the lung parenchyma, swelling of the alveolar walls, and goblet cell hyperplasia in the airways (51).

Treatment of children with moderate to severe cystic fibrosis (CF) by theophylline for 10 days showed protective effects on arterial oxygen desaturation during sleep, increased wakefulness and decreased sleep efficiency (52). Histone deacetylase complex (HDAC) is the main enzyme responsible for regulating inflammatory gene expression activity induced by oxidative stress. Theophylline can restore reduction of this enzyme. Moreover, theophylline may be able to reverse steroid resistance in COPD and other inflammatory lung diseases (53). Treatment of asthmatic smokers with theophylline (400 mg) in combination with inhaled beclometasone (200 mg) per day after four weeks significantly improved peak expiratory flow (PEF) and FVC but a borderline increase in pre-bronchodilator FEV₁ (54).

In a clinical study, SM-exposed patients were treated with oral slow releasing (SR) theophylline (250 mg), omeprazole (20mg), NAC (600 mg), salmetrol, and fluxitide (2 puffs) twice a day, during 8 weeks of therapy. The results showed that the low dose of theophylline and other mentioned drugs was partially able to decelerate the reductions in PFT values of these patients (55).

The decline of the reductions in PFT values in SM-exposed patients and its effect on CF and improvement of PFT values in asthmatic smokers by theophylline suggest its therapeutic effect on SM-induced lung disorders.

It was reported that doxycycline (2 mg/kg, p.o.) reduced lung pathology (neutrophil migration, septal thickening, and alveolar destruction), intra-alveolar inflammatory cells (mainly neutrophils), bacterial number, and MMP-2 and MMP-9 lipopolysaccharide-induced lung injury in C57Bl/6 mice (58). The effects of doxycycline (20 mg/kg or 60 mg/kg, orally), on virulent influenza A/Aichi/2/68 (H3N2) virus-induced lung injury in mice showed a significant decrease in inflammation and protein leakage in the lungs. Treatment with doxycycline also reduced the levels of MMP-2 and MMP-9 activity, T1-α (type I alveolar epithelium protein), and thrombomodulin (endothelial protein) compared to the non-treated group. These results demonstrated that doxycycline treatment is able to reduce lung damage but it did not affect virus titers and body weights (59).

Administration of doxycycline (2 mg/kg) in the drinking water in mice challenged with intratracheal LPS, significantly decreased the number of neutrophils and shed syndecan-1 in BALF. In addition, doxycycline had no significant effect on total BALF protein and the whole lung caspase-3 activity (60). The protective effect of doxycycline on acute lung injury induced by cardiopulmonary bypass (CPB) in 30 healthy mongrel dogs showed that administration of doxycycline (30 mg/kg) in feeding food significantly decreased WBC count, alveolar-arterial oxygen difference, respiratory index, total protein, and myeloperoxidase (MPO) activity in the BALF compared to the control group. Also, doxycycline (60 mg/kg) significantly decreased the concentration of MMP-9 compared to the control group (61).

Administration of doxycycline (2 mg/kg) in drinking water prior to intratracheally administration of bleomycin which induced pulmonary fibrosis, significantly decreased the number of neutrophils, although the total number of cells in the BALF remained unchanged. Dxycycline also attenuated gelatinase activities and reduced production of gelatinase B in BALF (62). Pretreatment of SM exposed guinea pigs with doxycycline resulted in the reduction of gelatinases activity (MMP-2 and MMP-9), decreased lung inflammation (cellularity and protein levels in BAL), and decreased lung pathological changes (epithelial lesions) (63). These results indicated that doxycycline has potent therapeutic effects on LPS and bleomycin-induced pulmonary fibrosis and inflammation in animal models with a similar action to SM-induced lung injury.

In a clinical study, pharyngeal Chlamydia trachomatis patients were treated with azithromycin (n = 78) and doxycycline (n = 64). Treatment failure in patients treated with azithromycin and doxycycline was 8.78 (10%) and 1.64 (2%), respectively. Treatment with doxycycline (100 mg) twice a day for 7 days was associated with less treatment failure of oropharyngeal chlamydia compared with azithromycin (1 g), (64). The effect of doxycycline on cystic fibrosis exacerbation in a randomized, double-blind, placebo-controlled study was studied and bio-specimens were collected at the start and the end of the study. Treatment with doxycycline (100 mg) orally twice daily in participants (n = 20) was given over an 8-day period during hospitalization, significantly reduced the levels of MMP-9 in sputum, which was also associated with the reduction in active MMP-9 levels (56.5%), and increased TIMP-1 in sputum. Doxycycline also improved forced expiratory volume in the first second (FEV1) and increased the time to next exacerbation compared to the placebo participants (n = 19). Furthermore, treatment with doxycycline reduced total hospital days compared with the placebo-treated group (65).

Using nuclear magnetic resonance (NMR)-based metabolomics to obtain serum metabolic profiles of doxycycline-treated (n = 60) and standard therapy of COPD patients (n = 40), doxycycline (100 mg) significantly increased the values of FEV1/forced vital capacity (FVC) and improved the COPD assessment test (CAT) scores after 3 months’ treatment. In addition, doxycycline significantly down-regulated serum levels of lactate and fatty acid, while, up-regulated the levels of formate, citrate, imidazole, and L-arginine compared to the pre-treatment level. The post doxycycline treatment significantly reduced serum level of the folate compared to COPD patients (66).

The results of experimental and clinical studies indicated that doxycycline could attenuate lung injury and pulmonary edema through anti-inflammatory effects such as degradation of the cell membrane, pulmonary neutrophil infiltration, and PFT test. These results suggest its therapeutic value in COPD and lung injury induced by SM.

Sulfur mustard analog exposure led to the formation of fibrin-rich in the airways which caused airway obstruction (68). The intra-tracheal administration of tPA (0.15–0.7 mg/kg, 5.5 and 6.5 h) in adult rats exposed to SM analog completely eliminated mortality at 48 h (0%), and improved morbidity (90–100%). Treatment with tPA also normalized plastic bronchitis and hypercarbia. It also improved respiratory distress, pulmonary gas exchange, and oxygen utilization while reduced airway fibrin casts (69).

Intra-tracheal tPA treatment reduced mortality at 48 h (0%) and significantly improved lung injury after lethal SM inhalation (100% death in controls). In addition, tPA improved respiratory distress and normalized hypoxemia, hypercarbia, and lactic acidosis induced by SM. Moreover, tPA was given via airway 6h after SM exposure prevented death from lethal SM inhalation, and normalized oxygenation and ventilation defects (70).

The effect of intravenous recombinant human tPA (rt-PA) was compared to urokinase in 45 patients with pulmonary embolism (PE). The results showed improvement in lung scan reperfusion in the two treatment groups after 24 h. The reduction in fibrinogen did not differ significantly between the two treated groups. These results also indicated that rt-PA acts more rapidly and is safer than urokinase in the treatment of acute PE (71).

Therefore, tPA reduced mortality and respiratory distress as well as normalized hypoxemia and hypercarbia induced by lethal SM inhalation which suggest the therapeutic effect of tPA on SM-induced lung disorders.

Pre and post-exposure treatment of rats with recombinant TFPI (rTFPI) significantly inhibited LPS-induced pulmonary vascular injury and coagulation abnormalities. Treatment with rTFPI also significantly inhibited increases of TNF-α, cytokine-induced neutrophil chemo-attractant, and myeloperoxidase in lung tissue. The administration of rTFPI significantly reduced the expression of TNF-α messenger RNA (mRNA) and inhibited TNF-α production in the lungs after stimulation by LPS. The results of this study suggested the effect of rTFPI on pulmonary vascular injury by inhibiting leukocyte activation in LPS-administered rats (74). Intravenous injection of rTFPI immediately before the introduction of tumor cells also reduced metastasis by 83% in mice (75).

Intra-tracheal administration of TFPI decreased fibrin-containing formation and limited severe hypoxemia in SM analog-induced lung injury in animals. TFPI limited thrombin activation in airways by reduction of prothrombin consumption and decreased thrombin- antithrombin complex (TAT) in BALF which led to a reduced airway obstruction and an improved gas exchange in rats (76). Therefore, the therapeutic effect of TFPI on LPS and SM analog-induced lung injury was documented.

The involvement of IL-8 in the pathogenesis of Bleomycin (BLM)-induced lung injury has been suggested and its level was elevated in a bronchial epithelial cell line (BEAS-2B cells). However, BLM-induced expression of IL-8 protein and mRNA in BEAS-2B cells was inhibited by NAC (78). Pre-incubation with NAC (5 × 10^–5M, 24 h) also significantly reduced peroxynitrite (ONOO–) and O^2–production in lung macrophages obtained from systemic sclerosis (SSc) patients (79).

Administration of NAC in lead-exposed animals, reduced or reversed lead-induced oxidative stress. Lead-exposed rats resulted in signs of anemia such as anisocytosis, poikilocytosis, and alterations in hemoglobin and hematocrit. It also causes an alteration in lipid peroxidation such as, increased MDA content and a decrease in GSH which are reversed by NAC (80). It has been reported that pretreated animals with NAC or liposomally-entrapped N-acetylcysteine (L- NAC) (25 mg/kg, iv), and challenged with lipopolysaccharide (LPS) (Escherichia coli, LPS 0111:B4) prevents increased lung weights, decreased lung angiotensin-converting enzyme (ACE) (the injury marker for pulmonary endothelial cells), and significantly reduced the LPS-induced increase in plasma TNF-α levels. In addition, NAC significantly decreased myeloperoxidase (MPO) and chloramine concentrations in the lungs and the extent of lipid peroxidation in liver tissues (81).

The enhancement of endothelial survival via GSH against SM injury of the endothelium was also reported. In this study, exposure to 500 μM SM resulted in increased activation of the nuclear transcription factor (NF_kB) binding to its consensus sequence 5 h after exposure. Pretreatment with NAC suppressed SM-induced NF_kB activation which is an important transcription factor for a number of cytokine genes (e.g., TNF) and is activated following stress in endothelial cells (82). GSH-induced enhancement of cell viability at 2.5 and 5 μM was shown after SM exposure. Pretreatment with hioninesulfoximine (BSO) an inhibitor of GSH synthesis, alone did not show toxicity effect but potentiated the toxicity of SM (82). Similarly, SM exposure led to a dose and time-dependent decrease in GSH content in human skin fibroblast cell line (HF2FF). NAC increased intracellular GSH level and protected the cells against SM-induced reactive oxygen species formation and lactate dehydrogenase leakage. In contrast, BSO pretreatment reduced cellular GSH and enhanced the cytotoxic effects of SM on HF2FF cells (83).

Treatment with intra-tracheal administration of NAC on pulmonary edema formation induced by phosgene exposure in rabbit lungs lowered pulmonary artery pressure, lung weight gain, the concentration of peptide leukotrienes LTC₄, D₄, and E₄, and lipid peroxidation which were increased following exposure to phosgene (84).

Animal exposure to SM leads to inflammatory cell accumulation in the airways and lung as well as structural and functional alterations in the respiratory tract (18, 85). The role of oxygen species and free radicals in the pathophysiology of inflammation-induced pulmonary lesions was described previously (86). However, treatment with NAC reduced the number of neutrophils in mice that were exposed to SM and developed lung injuries (87). Following animal exposure to SM vapor (100 μg/kg) for 10 min, arterial blood oxygen saturation levels (SaO₂), arterial blood pH and bicarbonate (HCO3-) were significantly decreased. Also, arterial blood carbon dioxide (PaCO₂) and shunt fraction were significantly increased. After treatment with inhaled doses of NAC (1 ml of 200 mg) arterial blood oxygen saturation, HCO3- level, and shunt fraction were significantly improved compared to those of the SM exposure. In addition, infiltration of neutrophils and concentrations of protein in BALF were significantly decreased compared to the SM group (88).

In clinical studies, treatment with NAC in patients with COPD, asthma, and acute bronchitis improved respiratory symptom, lung function, and quality of life. Treatment with NAC also scavenged reactive oxygen species and inhibited mediator release (89, 90). It was reported that the administration of NAC, 600 mg daily for 12 months in patients with COPD, reduced oxidative stress (91, 92) and also the oxidative burst of poly morphonuclear (PMN) cells and showed protective effects on peripheral granulocytes in COPD patients (93, 94).

Intravenous administration of NAC (190 mg/kg/day) in adult patients (n = 42) with respiratory distress syndrome (Pao2/Fio2 ≤ 200 mm Hg) significantly decreased the lung injury score between days 1 and 3 compared to the placebo group (95).

Treatment with NAC (40 mg/kg/day) intravenously for 3 days in 61 adult patients with mild-to-moderate acute lung injury (NAC, n = 32 and the placebo groups, n = 29 patients) improved ventilation compared to the placebo group. The oxygenation index (PaO₂/FIo₂) significantly was improved from day 0 to day 3 only in the NAC treated group and the lung injury score was improved in the NAC treated group during the first 10 days of treatment but, no change was observed in the placebo group (96).

Treatment of SM-induced bronchiolitis obliterans patients (n = 144) with NAC (1,200 mg daily), improved dyspnea, wake-up dyspnea, and cough after 4 months of treatment compared to the control group. Additionally, NAC reduced sputum from (76.9%) of cases before the study to (9.6%) of cases at the end of trial. The values of FEV1, FVC, and FEV1/FVC were also significantly improved in NAC treated patients compared to the placebo group (97).

In another clinical study, treatment with NAC (1,800 mg daily) in 144 bronchiolitis obliterans patients 18 years after SM exposure improved clinical signs and symptoms of patients including; dyspnea, cough, sputum, wake-up dyspnea, and also increased PFT values (98). It has been reported that administration of NAC (oral, IV, IP or IT) is effective in the management of SM induced acute lung injury due to inhibition of oxidative stress, inflammatory responses and apoptosis in a time-dependent manner. Furthermore, oral NAC alone at high doses for 4 months or in combination with clarithromycin (500 mg/day) at a dose 600 mg/day for 6 months improved clinical and paraclinical pulmonary parameters of patients with bronchiolitis obliterans induced by SM exposure (99). The efficacy of NAC in reducing SM toxicity in different studies (in vitro and in vivo) model of lung injury as well as the safe and efficacy of NAC in treating patients suffering the long-term and chronic pulmonary effects of SM exposure in veterans was reported (100).

Both experimental and clinical studies suggest the possible therapeutic effect of NAC on SM-induced lung disorders and other respiratory diseases via possible anti-inflammatory and anti-oxidant mechanisms.

The effects of vitamin C, vitamin E, and rINN, alone or in combination in placental villi culture after exposure to nicotine-induced endothelial dysfunction reduced placental cell proliferation until cell death (101). Vitamin E reduced phospholipidosis in cultured human skin fibroblasts chronically exposed to amiodarone and desethylamiodarone (DEA) and inhibited cumulative uptake of the drugs in a dose-dependent manner (102). Vitamin E pretreatment on human fibroblast cells significantly protected against ciprofloxacin (CPFX)-induced cytotoxicity. It also significantly increased the total GSH content and reduced the level of lipid peroxidation (103).

The effects of liposome-encapsuled vitamin E on a mouse model for airway inflammation induced by inhalation exposure to the alkylating nitrogen mustard melphalan showed that vitamin E (50 mg/kg) reduced inflammatory cell influx, and inhibited collagen formation in lung tissue (27). The administration of vitamin E (600 mg/kg), dexamethasone (5 mg/kg) or their combination in guinea pigs exposed to SM caused improvement in the pathological changes in the livers and kidneys (104).

The effect of vitamin E on tracheal responsiveness (TR) and lung inflammation in SM exposed animals also showed that TR to methacholine, total and differential WBC count in lung lavage, and serum levels of IL-4 were significantly decreased in treated animals with vitamin E compared to the untreated SM-exposed group (105). In addition, vitamin E treatment reduced pathological changes in guinea pigs exposed to SM. The antioxidant effect of tocopherol acetate (vitamin E) was also reported (106, 107) which supports the above results.

Tocopherol acetate (200 mg/kg, i.p.) increased GSH levels and decreased MDA level in systemic toxicity due to percutaneous administration of SM in animals. Furthermore, total antioxidant status was increased but red blood corpuscles and hemoglobin content were significantly decreased in the tocopherol acetate treated group (108).

Liposomes containing tocopherol (α, γ, δ) showed protective effects on the accumulation of RBC in the bronchi, alveolar space, arterioles and veins, and fibrin and collagen deposition in the alveolar space on 2-chloroethyl ethyl sulfide (CEES) (a mono-functional analog of sulfur mustard)-induced lung injury in a guinea pig. Moreover, tocopherol decreased lipid peroxidation level and hydroxyproline in the lungs (109). The treatment with liposome of tocopherol (α, γ, δ) significantly blocked the CEES-induced increase in the protein levels of the cell cycle protein (cyclin D1), and a cell differentiation marker (PCNA). Additionally, tocopherol protected the lungs against CEES-induced inflammation and alveolar infiltration of neutrophils and eosinophils (110).

The effect of vitamin E on CEES and alkylating nitrogen mustard-induced lung inflammation, as well as its effect on lung oxidative stress, lung inflammation, lung pathology, and tracheal responsiveness due to similar mechanisms in SM-exposed subjects indicated its therapeutic value in SM and other chemical-induced lung injuries.

Dietary intakes of vitamin E in 178 men and women aged (70–96 year) indicated that intake of vitamin E may influence lung function by increasing FVC, FEV1 and showed protective effects on wheeze in elderly in three European countries including Finland (n = 1,248), Italy (n = 1,386), and the Netherlands (111, 112). Treatment of 33 mild atopic asthmatics subjects for 16 weeks, with high doses of -α-tocopheryl acetate significantly decreased F2-isoprostanes, and reduced allergen-provoked concentrations of IL-3 and IL-4, and increased levels of IL-12 in BALF. In addition, natural source of vitamin E improved airway responsiveness to methacholine (113).

In a placebo-controlled randomized clinical trial the effects of natural vitamin E (500 mg) or matched placebo in 72 participants aged (18–60 years) for 6 weeks, had no significant impact on FEV1, asthma symptom scores, or serum immunoglobulin levels (114). Investigation of the influence of maternal antioxidant intake in pregnancy on the development of asthma and eczema in children showed that maternal vitamin E intake during pregnancy is negatively associated with wheeze in the absence of “cold” and childhood eczema in the children’s with 24 months of age (115).

The therapeutic effects of pharmaceutical drugs on SM-induced lung disorders in experimental and clinical studies were summarized in Tables 1, 2, respectively. The possible mechanisms of the therapeutic effects of pharmaceutical drugs on SM-induced lung disorders were also summarized in Figure 2.