Inflammatory and proapoptotic effects of inhaling gasoline fumes on the lung and ameliorative effects of fenugreek seeds

Impacts of inhaling gasoline fumes on the lungs of adult male rats and the alleviating role of fenugreek seeds were evaluated. Twenty-four rats were divided into four groups, unexposed control and fenugreek groups, gasoline exposed groups for 6 h/6 day/week for 10 weeks with and without supplementation of fenugreek seed powder in food (5% w/w). Rats exposed to gasoline fumes showed significant elevation in lung tumor necrosis factor-α, as an inflammatory marker, and the proapoptotic marker Bax with a reduction in the antiapoptotic marker Bcl2. Moreover, remarkable elevations in transforming growth factor-β1, collagen and hydroxyproline were observed as fibrotic markers. Lung oxidative stress markers (hydrogen peroxides, malondialdehyde, and protein carbonyl) increased significantly along with marked decrease in total antioxidant capacity, superoxide dismutase, and catalase levels. Additionally, marked decreases in white and red blood cell counts, hemoglobin content, platelet count, accompanied by elevated red cell distribution width percentage were observed, supporting the inflammatory status. Histopathological changes represented by hematoxylin&eosin, immunohistochemistry staining for Bax&Bcl2, and transmission electron microscopy supported the negative impacts of gasoline fumes compared to the control group. Fenugreek seeds supplementation with gasoline exposure showed pronounced alleviation of lung biochemical and histopathological changes compared to the gasoline-exposed group.

Exposure to gasoline fumes. The Egyptian commercial leaded gasoline RON 80 was purchased from a filling station at Mansoura City. Rats were exposed to gasoline fumes through a stainless-steel exposure chamber with dimensions of (1.5 m × 0.9 m × 2.1 m), provided with two upper holes (10 cm diameter) and two side holes (5 cm diameter) in other sides, except the lower side. Two calibrated 1000 ml beakers, each containing 500 ml of leaded gasoline, were placed at the bottom of the chamber. Beakers were allowed one hour before the exposure to ensure chamber saturation with gasoline fumes, as reported by Uboh et al. 26 . Gasoline exposure was continued for 10 successive weeks, 6 h daily, and 6 days/week. The average volume of freely vaporized gasoline daily during the time of exposure was approximately 16 cm 3 .
Experimental protocol. After the acclimation period, rats were classified into four groups (n = 6 per group): the control (CN) group, rats that received normal food and water, fenugreek (FN) group, rats supplemented with fenugreek seed powder mixed with normal food (5% w/w). Neither the CN nor FN groups were exposed to gasoline fumes or any source of pollution. In the gasoline (GS) group, rats were exposed to gasoline fumes as mentioned above, and in the (GS + FN) group, a fenugreek diet (5% w/w) was administrated during gasoline exposure.
This research was done in accordance with the ARRIVE guidelines and regulations for animal experiments.
Blood and tissue sampling. Twenty-four hours after the last day of exposure, rats were sacrificed after anesthesia by intraperitoneal (I.P) injection of a mixture of ketamine (0.08 ml/g) and xylazine (0.008 ml/g) where each rat received (0.001 ml/g) from this mixture. Blood samples were received on EDTA to estimate hematological parameters, including white blood cells (WBCs), red blood cells (RBCs) count, hemoglobin (Hb) content, hematocrit (HCT)%, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), red cell distribution width (RDW)% and total platelet (PLT) count, using a fully automatic hematological analyzer (Sysmex XE-2100, Japan) according to Dacie and Lewis 27 . Next, some of the lung tissues of the different investigated groups were immediately removed, weighed, homogenized in cooled distilled water, and centrifuged at 855×g for 15 min. The supernatants were preserved at − 80 °C for biochemical analysis.
Lung biochemical measurements. The following lung biochemical parameters were assessed according to protocols enclosed in enzyme-linked immunosorbent assay (ELISA) kits. The proinflammatory marker tumor necrosis factor-α (TNF-α) was estimated using the method provided by Alpco Diagnostics, USA, Catalog No. 45-TNFRT-E01. 1 Quantitative morphometric measurements of Bax and Bcl2. The percent of area appeared with Bax and Bcl2 reactions in lung sections stained by immunostaining reaction was illustrated in each group at X100 magnification by BLeica Quin 500^ image analyzer computer system (Leica image system Ltd.; Cambridge, England).
Ultrastructural examination. Transmission electron microscopy (TEM) was performed on lung specimens of different groups fixed in buffered 2.5% glutaraldehyde (pH 7.4) followed by buffered 1% osmium tetraoxide at 4℃. Samples were dehydrated in an ascending series of ethanol, cleared in acetone, and embedded in epoxy resin 31 . Ultrathin sections were cut with LKB Ultratome IV, mounted on grids, stained with uranyl acetate and lead citrate, and examined on a Joel 100CX1 transmission electron microscope (Mansoura University, Egypt).

Statistical analysis.
The obtained data were analyzed using the GraphPad Prism software program (v 5.04 GraphPad Software Inc., La Jolla, CA) using one-way ANOVA followed by Tukey's test, where statistically significant data were considered at p < 0.05. All results were recorded as the mean ± SD.
Ethical approval. This study was performed in line with the principles of the Declaration of Helsinki.
Approval was granted by the Ethics Committee of the Faculty of Science, Mansoura University (No. Sci-Z-P-2021-37).
Histopathological changes. H&E sections. Lung sections of different studied groups stained by H&E are represented in Fig. 3 and show control (A) and fenugreek (B) groups with normal lung structures of alveolar sacs and alveoli with thin and thick interalveolar septa. In addition, control lung group showed normal lung bronchioles and blood vessels. However, the gasoline-exposed group (C) exhibited thickening (hypertrophy) of the muscular layer of the bronchial wall, congested blood vessels, and marked proliferation of interstitial tissue showing hemorrhage and forming thickened interalveolar septa with a decreased number of alveoli that were collapsed. However, the lungs of rats in the gasoline + fenugreek group (D) showed diminished hypertrophy of bronchioles, collapsed alveoli with thin and thick alveolar septa, and decreased hemorrhage, indicating lung amelioration with fenugreek seed supplementation during gasoline exposure.
Immunohistochemical (IHC) staining. The BAX immune reaction was less detected in the alveolar tissue of both the unexposed control and fenugreek groups (Fig. 4A,B). However, immunohistochemical staining showed a marked increase in the content of Bax in the alveolar tissue, which appeared dark brown in color in the gas- Superscripts refer to significant changes (p < 0.05). a: All groups compared to CN group. b: GS + FN group compared to GS group. www.nature.com/scientificreports/ oline-exposed group, indicating increased cell apoptosis (Fig. 4C) compared to the control. Furthermore, the reaction of the apoptotic immune marker (Bax) appeared to be mild with the administration of fenugreek seeds during exposure to gasoline when compared to the gasoline group (Fig. 4D).
On the other hand, Bcl2 increased the immune reaction in normal alveolar cells of the control and fenugreek groups, where a dark brown color was deposited (Fig. 5A,B). However, no obvious color was observed in the gasoline-exposed group, indicating the cell tendency for apoptosis (Fig. 5C), but with fenugreek administration during gasoline exposure, the Bcl2 reaction was moderately observed in alveolar cells, indicating the role of fenugreek seeds in counteracting the proapoptotic effect of gasoline (Fig. 5D).
Quantitative morphometric measurements of Bax and Bcl2. Morphometric results for area percent of Bax protein expression in lung sections showed high percent area of brown color with exposure to gasoline fumes com-  www.nature.com/scientificreports/ pared to the control. This percent was decreased with fenugreek administration during gasoline exposure to values near the control group, as shown in Fig. 4E. However, Bcl2 protein expression exhibited low percent area with exposure to gasoline fumes, that was increased with fenugreek seed supplementation compared to gasolineexposed group (Fig. 5E).
Ultrastructural changes. At the ultrastructural level, TEM in the lungs of both the control and fenugreek groups showed normal alveoli with thin alveolar wall and microvilli. Alveoli composed of normal pneumocytes II with large nucleus occupied almost the cell body. Rough endoplasmic reticulum and mitochondria were observed. Moreover, some cells have several vacuoles, and electron-dense inclusions. Additionally, the lining wall is provided with numerous blood capillaries and some collagen fibers (Fig. 6A,B). However, the lungs of the gasolineexposed group showed several pathological changes represented by pyknosis of compact dense chromatin materials of type II pneumocytes and pneumocyte I. The cytoplasm contained rough endoplasmic reticulum and cytoplasmic vacuoles. Moreover, a considerable increase in the number of mast cells in inflamed alveolar tissue was also observed. In addition, large masses of collagen fibers were noticed in interstitial tissue of the alveolar wall, in addition to the appearance of damaged blood capillaries (Fig. 6C). On the other hand, supplementation www.nature.com/scientificreports/ of fenugreek seeds with exposure to gasoline fumes improved pathological signs, including the appearance of normal structures of pneumocyte II with nuclei and cytoplasmic granules. Microvilli and mitochondria were observed, In addition to a decrease in deposited collagen fibers and mast cells (Fig. 6D).

Discussion
Misuse or occupational exposure to leaded gasoline fumes can adversely affect various body organs and systems 24,32 . However, the respiratory tract seems to be more vulnerable to gasoline toxicity due to its large surface area, which makes gasoline fumes readily absorbed by the lung, causing lung toxicity 33 . The present study mimicked occupational exposure to leaded gasoline fumes for 6 h/day, 6 days/week for 10 weeks. The obtained results showed that rats exposed to gasoline fumes exhibited a significant increase in TNF-α, an inflammatory marker, compared to the control group. This inflammatory cytokine was reported to be released from mast cells recruited to the inflammatory site 34 . Several studies have supported this, as under normal physiological conditions, no mast cells can be detected in the lung, while in the case of inflammation, they can be observed 34,35 . This www.nature.com/scientificreports/ was broadly consistent with the present study through lung TEM sections, where mast cells were observed in the gasoline-exposed group compared to the control group. In this regard, WBCs count in circulating blood is one of the systemic inflammatory responses in the body 36 . Although the evidence of leukocytosis in the literature was inconsistent, the present study showed a significant decrease in blood WBCs count with exposure to gasoline fumes compared to the control. Jabbar and Ali 11 attributed this to chronic exposure to petroleum products that could reduce leucocyte number in a way affecting the immune process making the body more vulnerable to pathological pathways. Moreover, the present study dealt with leaded gasoline, where lead was reported to affect the immune response either in humans 11 or animals 9 . Amor-Carro et al. 15 explained that lead exposure could stimulate cascade events, including oxidative stress and inflammation, accompanied by reduced immune status. Moreover, the current study exhibited a significant increase in Bax expression and a decrease in Bcl2. This was in accordance with Wang et al. 6 , who found a strong relationship between the body inflammatory response and apoptotic reaction. Wang et al. 6 explained that increasing the degree of inflammation may lead to activation of pathological apoptosis signaling pathways, increased expression of Bax, as proapoptotic protein, and decreased expression of Bcl2, an antiapoptotic protein. Immunohistochemical investigations and morphometric analysis in the current study also supported this result, which may reflect the death of the alveolar epithelium in the gasolineexposed group compared to the control. Therefore, the inflammatory response is a highly regulated process through which the balance between cell survival and apoptosis occurs to drive and resolve inflammation. On the other hand, the obtained data showed a significant increase in TGF-β1, COL-1, and Hyp, fibrotic markers, in the lungs of gasoline-exposed rats compared to control rats. Shen et al. 37 attributed this to phagocytes that try to clear www.nature.com/scientificreports/ apoptotic cells and contribute to resolution by generating TGF-β1. However, the increased production of TGF-β1 stimulates the migration and proliferation of fibroblasts to deposit extracellular matrix, particularly COL-I as the most abundant collagen of the body, to initiate the repair process 38 . In addition, Hyp a major component of collagen, was significantly increased in the gasoline-exposed group, reflecting a case of pulmonary fibrosis 39 . Furthermore, Amor-Carro et al. 15 attributed cell death to the increased formation of ROS, which leads to oxidative damage of mitochondrial DNA. Su et al. 40 recommended that lipid peroxidation may serve as a common mediator of apoptosis in response to toxicants and pathological conditions. El-Sayed 41 added that highly oxidized lipids from gasoline exposure could attack nearby proteins forming more protein carbonyls that are accompanied by several inflammatory reactions. The present study was in accordance with these findings, where significantly increased levels of oxidative stress markers (H 2 O 2 , MDA and PC) in the lungs of rats exposed to gasoline fumes compared to the control group were observed. Elevated levels of H 2 O 2 reflect increased formation of ROS, which could attack the lipoprotein membrane of the lung, causing oxidation of both lipids and proteins, yielding MDA and PC, respectively. Moreover, increased production of H 2 O 2 could inhibit parenchymal cells of the lung to produce antioxidants, which may explain the decreased levels of TAC, SOD and CAT causing an imbalance in the lung oxidant/antioxidant levels 2 . In turn, this could result in loss of cell and tissue integrity, as seen in the current study, through histopathological changes in the lungs of the exposed group compared to the control group. This was in accordance with Roda et al. 42 , who attributed hemorrhage to the leakage of fluid into the extravascular space because of lipid and protein oxidation. The authors added that exposure to ROS harms type II pneumocytes, resulting in alveolar collapse, and interstitial inflammation, as seen in H&E and TEM lung sections in the present study.
On the other hand, blood is the most important body fluid that controls respiration 43 and primarily provides useful information on general health after exposure to extrinsic damage 11 . The present study also indicated a significant reduction in RBCs count, Hb content, HCT%, MCV, and MCH in gasoline-exposed rats compared to the control. These results agreed with the data of Teklu et al. 44 , who attributed this to the cytotoxic effects of the benzene constituent of gasoline, which depresses hematopoiesis in bone marrow, causing aplastic anemia. Moreover, naphthalene, as one of the gasoline constituents, is also known to affect the red cell membranes, leading to hemoglobin denaturation and hemolytic anemia 45 . Furthermore, Nathan et al. 46 reported that low Hb content accompanied by elevated levels of RDW% (measures the variation in RBC size) was previously mentioned to be related to pulmonary disorders caused by an underlying state of pulmonary inflammation accompanied by changes in erythropoiesis. This agreed with the present study supporting the obtained lung inflammatory status. Moreover, the decreased PLT count (thrombocytopenia) in gasoline-exposed rats may be related to the formation of lipid peroxides within platelet membranes, thus provoking platelet lysis and decreasing the platelet count. This may participate in pulmonary hemorrhage, as seen in intraalveolar and interstitial areas of the lungs of the gasoline-exposed group compared to the control (H&E sections), suggesting disrupted gas exchange and lung function 42 .
Nutritive natural plants have been reported to play an essential role in alleviating several health problems 16 . Fenugreek seeds possess several phytochemicals, including vitamins, flavonoids, alkaloids, terpenoids, carotenoids, coumarins, curcumins, lignin, and saponins 18 . Although there is no direct study between the addition of fenugreek seed powder to food and the lung response during gasoline exposure, several investigators have demonstrated the anti-inflammatory and antioxidant potential of fenugreek seeds in experimental animals 19,24,47 . Abdrabouh 24 attributed this to the high levels of phenolics and tannins, represented in gallic acid, in addition to quercetin and saponins, as well as lower values of radical scavenging activity (IC50%). Bafadam et al. 22 and Yusharyahya 47 reported that the interesting activities of fenugreek seeds are related to higher amounts of trigonelline and diosgenin steroids as well as the alkaloid and flavonoid contents. However, Tewari et al. 17 attributed the high antioxidant potential of fenugreek seeds to the reduction of ROS, which leads to a feedback regulation loop suppressing the levels of oxidative stress markers. This may explain the obtained significant decrease in oxidative stress markers (H 2 O 2 , MDA and PC) with fenugreek administration during gasoline exposure along with increased TAC, SOD and CAT as antioxidants. Indeed, the amelioration of oxidant/antioxidant balance may participate in the inflammatory status induced by gasoline inhalation. This was clear in the pronounced decrease in TNF-α and mast cells in TEM sections compared to the gasoline-exposed group. Bafadam et al. 22 explained that fenugreek seeds could inhibit the production of phorbol-12-myristat-13-acetate induced inflammatory cytokines such as TNF-α. However, Emtiazy et al. 48 attributed the anti-inflammatory effect of fenugreek seeds to flavonoids, especially quercetin, which inhibits the activation of mast cells. Another explanation was provided by Durga et al. 49 , where quercetin ligands interact with the protein constituent of cytokines, forming hydrogen bonds and then suppressing their over release. In turn, apoptotic and antiapoptotic proteins were significantly ameliorated compared to those in the gasoline-exposed group, as shown by biochemical and immunohistochemical analyses supported by image analysis. The percentage of Bax decreased and Bcl2 increased, reflecting the antioxidant, anti-inflammatory and antiapoptotic activities of fenugreek seeds. This may explain the consequent significant decrease in TGF-β1 along with COL-I and Hyp in the gasoline group administered fenugreek seeds compared to the gasoline group.
These observations were also accompanied by a significant increase in WBCs count, reflecting the potential role of fenugreek seeds in increasing the immune response. Moreover, fenugreek seeds are rich in amino acids (lysine and threonine), minerals (iron and copper), and vitamins (folate and ascorbic), which are all essential components of hemoglobin synthesis 50 . This may explain the observed alleviation of blood Hb and other blood indices with fenugreek administration compared to the gasoline group. As a result, histopathological changes in the lung showed pronounced improvement in most pulmonary architecture in the exposed group administered fenugreek seeds compared to the gasoline group. Yao et al. 51 attributed this to the high antioxidant activity of fenugreek seeds, which could restore oxidant/antioxidant balance. www.nature.com/scientificreports/ In conclusion, exposure to gasoline fumes is intimately related to lung inflammatory and proapoptotic disorders resulting from released ROS and helps release fibrotic factors that affect the lung architecture. However, supplementation with a fenugreek seed diet during gasoline exposure helped to alleviate all biochemical and histological alterations in alveolar tissue. Thus, fenugreek seed powder is a powerful plant that is recommended to add to food, especially for those exposed to gasoline fumes.

Data availability
All data generated or analyzed during this study are included in this published article.