The Effects of Resveratrol on Silica-Induced Lung Oxidative Stress and Inflammation in Rat

This study was approved by the Research Ethics Committee of Hamadan University (No. IR. UMSHA.REC.1399.444).

This study was financially supported by 10.13039/501100004697 Hamadan University of Medical Sciences , Hamadan, Iran (project number: 9906183906).

As shown in Fig. 2 , the silica group revealed a significant elevation in LDH activity as compared to control group (p < 0.001). In contrast, treatment with Res induced a significant reduction in LDH activity in comparison with silica group (p < 0.001). Fig. 2 The effect of daily oral Res (20 mg/kg) on BALFs LDH activity: silica-induced lung injure was induced intratechally instillation (50 mg/kg in 0.1 ml saline/rat). Res treatment began 24 h after silica instillation and continued for 90 days. Results are presented as the mean ± standard error of 5 rats in each group. ∗∗p˂0.01 significantly different Vs Silis group; ∗∗∗p˂0.001 significantly different Vs Silis group (one-way ANOVA followed by Bonferroni post hoc test). Fig. 2 The effect of daily oral Res (20 mg/kg) on BALFs LDH activity: silica-induced lung injure was induced intratechally instillation (50 mg/kg in 0.1 ml saline/rat). Res treatment began 24 h after silica instillation and continued for 90 days. Results are presented as the mean ± standard error of 5 rats in each group. ∗∗p˂0.01 significantly different Vs Silis group; ∗∗∗p˂0.001 significantly different Vs Silis group (one-way ANOVA followed by Bonferroni post hoc test). Based on Table 1 , Table 2 , intratracheal silica installation significantly increased the serum and lung MDA levels as compared with the control group (p < 0.001 and p < 0.001, respectively), While daily oral Res administration reversed these changes in serum and lung, as compared with silica group (p < 0.001 and p < 0.001, respectively). The serum level of MDA in the Silica + Res groups was higher in comparison with the MDA level of lung tissues. Table 1 The effects of daily oral RES treatment on serum TNF-α, oxidative, and anti-oxidative markers Table 1 Parameters Control Silica Res Silica + Res (20 mg) MDA (μM/L) 1.81 ± 0.33∗∗∗ 5.99 ± 0.49 2.66 ± 0.55∗∗ 4.12 ± 0.43∗∗ SOD (U/ml) 1.95 ± 0.92∗∗∗ 0.95 ± 0.12 1.89 ± 0.07∗∗ 1.49 ± 0.19∗∗∗ CAT (U/ml) 174.8 ± 13.26∗∗∗ 107.2 ± 9.63 156.8 ± 6.53∗∗∗ 160.6 ± 9.86∗∗∗ TNF-α (pg/mL) 10.92 ± 1.21∗∗∗ 19.63 ± 2.41 11.67 ± 2.02∗∗ 15.26 ± 1.5∗∗ Silicosis was induced intratechally instillation of silica (50 mg/kg in 0.1 ml saline/rat). Res treatment began 24 h after silica instillation and continued for 90 days. Results are presented as the mean ± standard error of five rats in each group. ∗∗p˂0.01 significantly different Vs Silis group; ∗∗∗p˂0.001 significantly different Vs Silis group (one-way ANOVA followed by Bonferroni post hoc test); MDA, malondialdehyde; CAT, catalase; SOD, superoxide dismutase; TNF-a, tumor necrosis factor. Table 2 The effects of daily oral RES treatment on lung TNF-α, oxidative, and anti-oxidative markers Table 2 Parameters Control Silica Res Silica + Res (20 mg) MDA (μM/g tissue) 1.27 ± 0.27∗∗∗ 3.14 ± 0.48 1.64 ± 0.36∗∗∗ 2.09 ± 0.15∗∗ SOD (U/mg tissue) 1.58 ± 0.32∗ 0.79 ± 0.22 1.49 ± 0.28 1.39 ± 0.11 CAT (U/mg tissue) 422.22 ± 61∗∗ 283.2 ± 17.91 553.14 ± 52.44 318.84 ± 22.5 TNF-α (pg/mg tissue) 296.8 ± 43.05∗∗∗ 561.8 ± 33.48 274.8 ± 45.83∗∗∗ 397.76 ± 54.13∗∗ Silicosis was induced intratechally instillation of silica (50 mg/kg in 0.1 ml saline/rat). Res treatment began 24 h after silica instillation and continued for 90 days. Results are presented as the mean ± standard error of five rats in each group. ∗∗p˂0.01 significantly different Vs Silis group; ∗∗∗p˂0.001 significantly different Vs Silis group (one-way ANOVA followed by Bonferroni post hoc test); MDA, malondialdehyde; CAT, catalase; SOD, superoxide dismutase; TNF-a, tumor necrosis factor. The effects of daily oral RES treatment on serum TNF-α, oxidative, and anti-oxidative markers Silicosis was induced intratechally instillation of silica (50 mg/kg in 0.1 ml saline/rat). Res treatment began 24 h after silica instillation and continued for 90 days. Results are presented as the mean ± standard error of five rats in each group. ∗∗p˂0.01 significantly different Vs Silis group; ∗∗∗p˂0.001 significantly different Vs Silis group (one-way ANOVA followed by Bonferroni post hoc test); MDA, malondialdehyde; CAT, catalase; SOD, superoxide dismutase; TNF-a, tumor necrosis factor. The effects of daily oral RES treatment on lung TNF-α, oxidative, and anti-oxidative markers Silicosis was induced intratechally instillation of silica (50 mg/kg in 0.1 ml saline/rat). Res treatment began 24 h after silica instillation and continued for 90 days. Results are presented as the mean ± standard error of five rats in each group. ∗∗p˂0.01 significantly different Vs Silis group; ∗∗∗p˂0.001 significantly different Vs Silis group (one-way ANOVA followed by Bonferroni post hoc test); MDA, malondialdehyde; CAT, catalase; SOD, superoxide dismutase; TNF-a, tumor necrosis factor. The activities of antioxidant enzymes, including SOD and CAT, were measured in the serum and lung tissue. As shown in Table 1 , Table 2 , intratracheal instillation of silica significantly decreased the activities of SOD, and CAT in serum and lung (p < 0.01 and p < 0.01, respectively) compared to the control group. However, orally Res administration could improve the alterations of SOD activities in serum and lung tissues compared to the silica group (p < 0.001and p < 0.05), although the activities of CAT increased in Silica + Res groups, the changes were not statically significant compared to the silica group. In the current study, silica instillation was associated with a significant increase in serum and lung level of TNF-α compared with control group (p < 0.001 and p < 0.001, respectively). Based on results, daily RES treatment meaningfully decreased serum and lung TNF-α levels compared with silica group (p < 0.01 and p < 0.001, respectively), and details are indicated in Table 1 , Table 2 . Histopathological assessments of the Masson’s trichrome staining of the pulmonary tissue are shown in Figs. 3 A–D and 4 . Lungs of rats in control and Res group showed normal lung structure without any signs of lesion ( Fig. 3 A–B) (P < 0.001). In silica group, silica-induced lung toxicity caused marked increase in collagen deposition ( Fig. 3 C). However, treatment with Res significantly ameliorated these histopathological changes ( Figs. 3 D and 4 ) (P < 0.01). Fig. 3 Photomicrograph of Masson’s trichrome staining of lung tissue, control groups (A), resveratrol treatment (Res 20 mg/kg) (B), silica (IT instillation) after 90 days (C), silica (IT instillation) along with 20 mg/kg of Res (D). Fig. 3 Fig. 4 Deposition of collagen is evident (magnification 200 × ), control groups (A), resveratrol treatment (Res 20 mg/kg) (B), silica (IT instillation) after 90 days (C), silica (IT instillation) along with 20 mg/kg of Res (D); ∗∗∗p˂0.001 (compared to control group); ∗∗p˂0.01 (compared to silica group). Fig. 4 Photomicrograph of Masson’s trichrome staining of lung tissue, control groups (A), resveratrol treatment (Res 20 mg/kg) (B), silica (IT instillation) after 90 days (C), silica (IT instillation) along with 20 mg/kg of Res (D). Deposition of collagen is evident (magnification 200 × ), control groups (A), resveratrol treatment (Res 20 mg/kg) (B), silica (IT instillation) after 90 days (C), silica (IT instillation) along with 20 mg/kg of Res (D); ∗∗∗p˂0.001 (compared to control group); ∗∗p˂0.01 (compared to silica group).

Crystalline silica (1–2 μm) was purchased from Nanozarat (Tehran, Iran). The surface characteristic was determined by Oxford scanning electron microscope ( Fig. 1 ). Res were purchased from (FIRAT ECZANEST, Turkey, and Code: 166354). The other chemical substances used in this research were of high available grade of purity. Fig. 1 SEM images of crystalline silica particles used in the study. White bar represents 1–2 μm. Fig. 1 SEM images of crystalline silica particles used in the study. White bar represents 1–2 μm. Male Wistar rats (150–180 g), aged 4 weeks, were purchased from the Animal Care Center, Hamadan University of Medical Sciences. All animal treatment protocol was approved by the guidelines established by the Review Board of Hamadan University of Medical Sciences with the code number of IR. UMSHA.REC.1399.444. The rats were kept in air-conditioned room at 21–23°C, humidity (50 ± 10%), and a regular 12 h light/12 h dark cycle. The animals were permitted free water and food during investigational procedure. The ethics in experiments were cautiously monitored. The silica-induced lung toxicity was performed according to Hemmati et al. study [ 3 ]. The crystalline silica particles were weighted, suspended in normal saline (0.9% w/v) and autoclaved (in order to sterilize), and mixed with 20,000 IU penicillin. Silica suspension was used as intratracheally instillation (IT) (50 mg/rat, 0.1 mL/rat), and ketamine and xylazine were used for anesthetizing . In order to study the effect of Res on silica-induced lung injure in rats, a total of 20 male Wistar albino rats were randomly classified into four groups (n = 5): control group received 1 ml of saline; Res group received orally 20 mg/kg of Res (based on previous studies [ 3 , 18 ]. Silica group received a single dose of 1 ml silica suspension IT; silica and Res group received co-administration of single IT silica and oral Res (20 mg/kg/day). Res (100 mg/60 tablet) purchased from (FIRAT ECZANEST), and Turkey were suspended in deionized water and administered orally by gavage to rats for 90 days (20 mg/kg/day). Finally, animals were deeply anesthetized by ketamine and xylazine injections. Blood samples were collected from left ventricle. Serum was separated, alliquted, and stored at -80˚C for subsequent analyses. In order to collect Bronchoalveolar lavage fluid (BALF), the lungs were lavaged three times with 6 ml of sterile saline, and the recovery ratio was 70%–80%. BALF was centrifuged (1500 g, 10 min at 4 C), and supernatant was stored at -80. The lung protein content measurement was conducted by the Bradford technique, and bovine serum albumin was used as standard.

Our results demonstrated that silica caused oxidative stress and inflammation in serum and lung tissue of rat model. We indicated the evidence that Res has protection effects on these harmful effects of silica by an increase in antioxidants and a decrease in oxidative stress and inflammatory biomarkers in rats. Moreover, Res ameliorated histopathological changes induced by silica. There were some limitations in this study that should be considered in future studies. Since lung injuries induced by silica are a chronic respiratory disease, it is necessary to administrate silica in the inhalation route. It is required to investigate molecular mechanisms involved in the anti-inflammatory and antioxidant effects of Res against silica-induced lung impairment. Furthermore, we recommended next studies on other animals closely associated with humans and eventually in a clinical setting. Undoubtedly, such studies can help to provide a novel vista in the nature-based therapeutic strategy for silicosis.

The current study evaluated the effects of Res on silica-induced lung injury. Successful silica-induced lung injure induction in the rat model was approved by detecting morphological and pathological changes in lung tissue. Also, we measured oxidative, anti-oxidative, and inflammatory markers in serum and lung. The molecular mechanism of silica-induced lung toxicity is still the topic of various studies. It is described chiefly by huge pulmonary fibrosis due to proliferation and progressive increase in connective tissue, which is substituted for normal functional parenchyma [ 1 ]. As a result, it has a detrimental effect on patients' quality of life. The pulmonary fibrosis mechanisms induced by silica are distinct from other pulmonary fibrosis types. According to Heppleston and Styles' hypothesis, silica acid groups (in crystalline silica particles) can interact with the membrane of macrophages, impair the cell membrane, and ultimately discharge silica crystals and digesting enzymes [ 21 ]. Silica crystals are insoluble particles that can activate alveolar macrophages, epithelial, and fibroblast cells. These lead to the proliferation and migration of macrophages, the production of inflammatory markers and collagen fibers, and ultimately lung injury [ 3 ]. These events are considered the major causes of oxidative and inflammatory mediators releasing, including TNF-α, ROS, and RNS, which can recall neutrophils and lymphocytes [ 12 ]. Oxidative stress and inflammation are regarded as indicators of silicosis [ 19 ]. ROS generated at the surface of silica particles and phagocytic cells are involved in silica particles' digestion [ 5 ]. Accumulating evidence demonstrated that oxidative stress has a vital role in initiating and developing fibrotic lung disease [ 3 ]. The plasma levels of MDA can be an important biomarker of silicosis severity [ 22 ]. The high level of oxidant radicals can disturb the balance between free radical production and the biological antioxidant systems [ 10 ]. SOD is a pivotal enzyme involved in cell redox potential, and CAT involves in hydrogen peroxide decomposition [ 23 ]. These enzymes are regarded as the first line of antioxidant defense [ 24 ]. Our results confirmed that silica increased lipid peroxidation as evidenced by increased serum and lung levels of MDA, concomitant with suppression of CAT and SOD levels in serum and lung. In line with our results, Abdelaziz et al. reported that silica increased pulmonary oxidative markers such as MDA and decreased anti-oxidative markers such as Glutathione (GSH) content and SOD activity in lung tissue [ 19 ]. In another study, Orman et al. indicated an increase in plasma MDA levels and a decrease in erythrocyte GSH levels in construction workers exposed to cement dust [ 25 ]. In a rat model, Peng et al. showed that silica exposure-induced p-AKT/NF-ƙβ expression increased collagen content and inflammatory cytokines (i.e., TNF-α and TGF-β) in the lung, as well as increased serum MDA level [ 5 ]. Another study was conducted on cement plant workers, and their results indicated that plasma MDA levels in workers exposure silica dust were significantly high than control group [ 25 ]. In a similar study, Fahmy, F. C., et al. reported that occupational exposure to silica dust could increased induce oxidative stress in workers exposure silica dust [ 26 ]. Anlar et al. and Azari et al. indicated a significant increase in MDA plasma levels of workers in the field of ceramics and glass sandblasters exposed to crystalline silica, than workers unexposed, respectively [ 27 , 28 ]. Intracellular ROS has a fundamental role in the activation of NF-κB, which has a significant function in inflammatory responses. NF-κB is a major mediator of TNF-α, a pleiotropic cytokine [ 29 ]. Previous research has reported that in the silicosis model, TNF-α released from activated macrophages is involved in inflammation and lung fibrosis [ 30 ]. TNF-α can activate neutrophils, eosinophils, superoxide, and lysosomal enzymes. In addition, this inflammatory cytokine can promote the proliferation of fibroblasts and collagen synthesis [ 31 ]. The current study indicated high levels of serum and lung TNF-α, following silica intratracheal instillation compared with the control group. In line with our observation, Peng et al. confirmed that TNF-α generated at the initial phase of inflammation in silica group, as well as significantly increased and persisted 14 days after silica instillation [ 5 ]. Some plants or herbal combinations can act as strong antioxidants [ 32 ]. Our previous studies proved the protective effects of Res against diazinon-induced oxidative stress in the liver and kidney [ 33 ]. In this study, Res treatment improved antioxidant status (including SOD and CAT) and diminished oxidative stress markers, such as MDA. Meanwhile, Res mitigated inflammation via a decrease in serum and lung levels of TNF-α. Also, the results of this study indicated that Res administration decreased BALF LDH activity and moderated histopathological changes following silica exposure. Many preclinical studies documented lung-protective effects of Res due to its anti-inflammatory, antioxidant, and antifibrotic properties [ 34 ]. Research has proven the anti-inflammatory effects of Res in mice model of COPD caused by smoking [ 35 ]. In addition, in COPD patients, Res can suppress inflammation in macrophages of bronchoalveolar lavage fluid and bronchial smooth muscle cells [ 36 , 37 ]. In this regard, epidemiological and pathological results indicated that silica exposure has a close association with COPD, independent of silicosis. Alveolar epithelial type II is important for sustaining lung hemostasis [ 38 ]. In vitro studies have shown that Res can suppress inflammation in type II alveolar epithelial cell line via decreasing expression of IL-1B and IL-6 and increase in IL-10 and surfactant proteins expressions. These effects were mediated by the inhibition of NF-κB pathway [ 39 ], an increase in SIRT1, and a decrease in FoxO3a. SIRT1 enhances PGC-1 α expression, which can attenuate ROS production via detoxifying enzymes induction. Also, PGC-1α activates mitochondrial biogenesis and has a significant role in the regulation of antioxidant enzymes, such as SOD, CAT, and Plasma glutathione peroxidase (GSH-Px) [ 40 ]. Indeed, Res enhances mitochondrial oxidative capacity through AMPK-SIRT1-PGC-1α axis. Preclinical researches have shown that inhaled formulation of Res increased the mitochondrial function of the lung and preserved lung structure [ 41 ]. Consistent with our study, Hamza et al. demonstrated that Res with free radical scavenging activity and antioxidant properties improved lung histopathological changes and nicotine-induced oxidative stress status [ 42 ]. The study of Kashef DH et al., in silica model, proved a decrease in the level of nuclear elytroid 2-related factor 2 (Nrf-2) and HO-1 in BALF. Nrf-2 is a significant molecule in the activation of antioxidant enzymes transcription [ 43 ]. Moreover, this transcription factor is involved in inflammation protein degradation; thus, Nrf-2 is critical in the regulation of oxidative stress [ 44 ]. In vitro studies have shown that Res can protect human alveolar epithelial cells against oxidative stress induced by cigarette smoke extract (CSE), and Res increases GSH level through Nrf2 activation [ 45 ]. Res improved antioxidant capacity and attenuated apoptotic processes through Nrf2/HO signaling activation in human bronchial epithelial cells exposed to CSE [ 46 ]. In addition, Res increased SIRT1 expression and augmented antioxidant enzymes, including SOD and CAT, in lung injury induced by organophosphate pesticide [ 47 ]. These effects showed that Res can decrease lung injury and may potentially decrease anti-inflammatory drugs requirement with disadvantages and complications [ 47 ]. Several clinical trials indicated anti-inflammatory effect of Res including reduce in IL 6 and TNF-α [ 48 , 49 ]. Also, human studies indicated that Res treatment diminished oxidative stress markers (such as protein carbonyl content and O2-level of PBMCs) and increased plasma antioxidant capacity [ 50 ]. Res had a beneficial effect in prolonged job-related exposure to electromagnetic fields [ 51 ]. These evidence suggested that Res supplementation may be candidate agents in inflammatory lung diseases, particularly silica-induced lung injury.

Silicosis is a dangerous chronic occupational disease caused by prolonged inhalation exposure to crystalline silica (silicon dioxide), a major component of sand or rock [ 1 ]. There are various forms of crystalline silica, including silicon dioxide and alpha quartz, as the most abundant form of silica [ 2 ]. Annually, millions of workers involved in industrialization processes (e.g., sandblasting, drilling, pulverizing, cutting, grinding tools, jackhammering, drilling, mining, construction, and stone cutting) are exposed to free crystalline silica [ 3 ]. In these industries, the inhalation of crystalline silica particles smaller than 10 μm can lead to silicosis or cancer [ 4 ]. The lung as a vital organ is seriously affected by crystalline silica particles. The pathological process of silicosis is characterized by lung tissue inflammation and fibrosis [ 5 ]. A silicosis patient suffers from breathing difficulty and symptoms such as chronic obstructive pulmonary disease (COPD) [ 3 ]. Silicosis happens in two forms: (i) accelerated silicosis due to exposure to low silica concentration over a long period and (ii) acute silicosis caused by short-term exposure to a very large amount of silica [ 6 ]. Identifying the molecular mechanism of cell injury induced by silica is the main purpose of in vivo and in vitro studies [ 7 , 8 ]. After the first exposure to crystalline silica, alveolar macrophages begin to inflammatory responses. The consistency exposure stimulates oxidative stress in many macrophages by reactive oxygen species (ROS) and hydrogen peroxide production [ 9 ]. These oxidants can damage lung cells and increase the expression of tumor necrosis factor (TNF)-α and other inflammatory cytokines [ 10 ]. Previous studies have described that oxidative stress is involved in pulmonary fibrosis [ 11 ]. However, the actual molecular mechanisms are still unclear. There is no effective treatment to control lung fibrosis [ 12 , 13 ]. This issue is important, particularly in workers exposed to silica crystals. Resveratrol (Res) (5-[( Z )-2-(4hydroxyphenyl) ethenyl] benzene-1, 3-diol), as a small polyphenol compound, is abundant in different plant species, including berries, nuts, pistachios, and grapes [ 14 ]. This natural product has several protective effects, including anti-oxidative and anti-inflammatory properties, capillary protective action, anti-mutagenic effect, heart and neuroprotective effects, and anti-allergic activities [ 15 , 16 ]. Hemmati et al. (2008) indicated that grape seed extract could reduce the fibrogenic effect of silica in male albino Wistars [ 3 ]. In another study, Liu et al. (2017) reported that supplementation with grape seed extracts improves the quality of life of lung fibrosis patients [ 17 ]. The present study aimed to investigate the possible protective effects of Res against silica-induced lung injure in a rat model.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Lactate Dehydrogenase Activity (LDH) activity was evaluated using commercially available Pars Azmun kits (KSOD88, Iran). The procedures were conducted as supplied manufacturer's instructions; the change in absorbance was recorded at λ340 nm. Lung and serum malondialdehyde (MDA) level (as end production of lipid peroxidation) were determined by thiobarbituric acid method (TBA) based on Esfahani et al. studies [ 18 ]. Serum and lung activities of superoxide dismutase (SOD) and catalase (CAT) were evaluated by Kiazist kits (KSOD96 and KCAT96, respectively). The serum and lung level of TNF-α were measured by Carmona Pars Gene (Iran, Cat. No: KPG-RTNF96). The left lung tissues were excised, washed with iced-cold saline, and prepared for lung homogenization according to previous studies [ 19 ]. Also, the left lung was rinsed with iced cold saline, following then fixed in 4% paraformaldehyde for the assessment of morphological changes, in order to evaluate pulmonary fibrosis size, and deposition of extracellular matrix (ECM) deposition was used for Masson’s trichrome staining. In order to evaluate the collagen deposition, the sections were inspected and photographed by use of a light microscope with digital camera (Nikon E800, Japan). Histopathological scoring of pulmonary fibrosis was reported according to Ashcroft score [ 20 ]. The SPSS-20 software ((SPSS, Inc., Chicago, IL, USA) was used to perform data analyses. The normal distribution of data was assessed by Kolmogrov–Smirnov test. Multiple group comparisons were accomplished by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. The data are presented as mean ± SEM. In all cases, P < 0.05 was considered significant.