ABSTRACT

Aim

In this study, the effect of baicalein (BAI) on lung damage caused by formaldehyde (FA) is aimed to be examined via immunohistochemistry and gene expression techniques.

Materials and Methods

Within the scope of the study, 24 male Wistar-Albino rats were provided from experimental animal unit. Animals were divided into three groups as two experimental and a control group, using the simple randomization method. Control group received saline intraperitoneally for 14 days, the FA group received 10 mg/kg dose of FA intraperitoneally for 14 days and the FA+BAI group received 10 mg/kg dose of FA intraperitoneally and 200 mg/kg BAI daily for 14 days. At the end of the experimental process, lung tissue samples of rats were taken and analyzed in terms of gene expression and immunohistochemistry.

Results

FA group had high degree of histopathologic lung damage, immunochemically low endothelial  nitric oxide synthase (NOS)  and high inducible NOS expression. The FA+BAI group had similar findings with the FA group and did not display significant improvement on pathological findings (p<0.05). Superoxide dismutase and catalase expression levels were significantly increased in the FA+BAI group compared to the FA group (p<0.05). Compared with the control group, it was determined that Cytochrome-c expression increased in both FA group and FA+BAI group (p<0.05).

Conclusion

As a result, BAI treatment has no positive effects on FA-induced lung tissue damage. FA induces apoptosis in rat lungs via the intrinsic mitochondrial pathway and BAI has no positive effects on apoptosis at the expression level. However, our study reveals that BAI has an ameliorating effect on oxidative stress parameters at the expression level.

INTRODUCTION

Formaldehyde (FA) is an aldehyde that dissolves well in water, colorless, irritating in its pure form and has a pungent odor. FA is a commonly utilized chemical agent in a multitude of settings, including anatomy laboratories for the fixation of cadavers, histology and pathology laboratories for the fixation of tissues, disinfection processes within the medical field, and extensively within the food industry as a preservative (E240). However, due to its inherent properties, FA is classified as an irritating and poisonous chemical. It can exist in a gaseous state at room temperature in any environment, which poses a significant health hazard¹.

After an exposure, FA metabolizes to methanol and formic acid in the liver and erythrocytes. It excretes out of the body via urine and feces or with the respiratory tract by oxidizing to carbon dioxide. Numerous studies have shown that exposure to FA causes various symptoms such as sensory irritation, salivation, dyspnea, headache, insomnia, convulsions, behavioral disorders, and abnormal sperm production. The exposure of experimental animals to FA results in its rapid metabolic incorporation into DNA, RNA, and proteins^2-4. In experimental animal studies, FA induces a variety of toxic effects, possibly because of these macromolecular interactions. In studies conducted for many years, the toxic effect of FA on different tissues and potential protective agents has been investigated. Especially the respiratory tract and lungs are major damaged tissues due to the first affected area by inhalation^4-11. 

In the literature, numerous studies have shown that increased oxidative stress and apoptosis are involved in the damage-inducing mechanism of FA. It causes a decrease in lung epithelial cell viability and induction of apoptosis. Different mechanisms have been reported in the mechanism of apoptosis induced by FA, such as change in the Bax/Bcl-2 expression ratio, mitochondrial damage, emergence of toxic metabolites, and activation of the mitochondrial pathway of the apoptosis mechanism. Changes in the expression of Bcl-2 family proteins are generally thought to induce apoptosis by the intrinsic pathway. In addition, higher reactive oxygen species levels have been associated with cell death and pathological conditions caused by oxidative stress, in which excess ROS oxidizes DNA, lipids, proteins and cellular macromolecules, causing damage^10-14.

Apoptosis involves the activation, expression and regulation of certain genes¹⁵. Tumor suppressor gene p53, Bcl-2 family, and caspase family are among the genes associated with apoptosis. The mechanism of apoptosis includes receiving the apoptotic signal, interaction between molecules and caspase activation, which leads to a continuous reaction process¹⁶. Apoptosis, a dynamic process, activates caspases by releasing protein from mitochondria¹⁷. One of these proteins is cytochrome-c (Cyt-c), which is a component of the electron transport chain¹⁸. Cyt-c is localized between the inner and outer membranes of mitochondria. The release of Cyt-c from the outer membrane into the cytosol is the most important event triggering apoptosis¹⁹. The release of Cyt-c into the cytosol activates caspase. Caspases that play a role in apoptosis are divided into initiator caspase and effector caspase²⁰. Activated by Cyt-c, caspase-9 induces caspase-3. Thus, the caspase cascade begins, which will end with cell death. P53 is a tumor suppressor protein that controls the cell cycle, DNA replication and uncontrolled cell division. In healthy cells, p53 proteins are switched off. It is activated when cells are exposed to stress and uncontrolled division. However, when this protein is damaged or mutated, it cannot perform functions. This causes uncontrolled cell division and tumorigenesis²¹.

Nitric oxide (NO) is a signaling molecule synthesized from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial NOS (eNOS), neuronal NOS and inducible NOS (iNOS). It is synthesized by eNOS and is required for the normal functions of cells. In the caspase of tissue damage or stresses, extra NO is produced via iNOS. Due to increased NO production, an increase occurs in other free radicals such as peroxynitrite anion and hydroxyl, which are highly active. NO can also deplete antioxidants and inhibit their protective effects on organs against oxidative stress²².

Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and non-enzymatic markers such as reduced glutathione (GSH) contribute to the healthy functioning of physiological processes by providing defense against ROS species. SOD, glutathione peroxidase (Gpx), and CAT are the most important antioxidant enzymes in the investigation of tissue damage caused by oxidative stress, and changes in their activities are considered as oxidative stress markers. FA triggers both apoptosis and oxidative stress in cells by reacting with many molecules. SOD, Gpx and CAT are the most important antioxidant enzymes in the investigation of tissue damage caused by oxidative stress, and changes in their activities are considered as oxidant stress markers in studies conducted with FA^23-27.

Baicalein (BAI) is derived from the roots of the Scutellaria baicalensis plant. Studies have shown that BAI has anti-bacterial, anti-virus, anti-allergic, anti-oxidant, and anti-inflammatory properties. In recent studies, it has been found out that BAI has anti-cancer activities upon its effect on various biological processes such as cell proliferation, metastasis, apoptosis, and autophagy^28-30.

Our objective was to use this data to find out an answer to these two questions relevant to the topic. Initially, a link between the damage caused by FA and the production of eNOS, iNOS, apoptotic processes, and antioxidant responses in lung tissues were discovered. Furthermore, we investigated the influence of BAI on the lung damage caused by FA.

MATERIALS AND METHODS

Chemicals

BAI (BLDpharm BD6298, China), FA (Tekkim, Turkey).

Animals

A total of 24 male Wistar albino rats (weighing 20-24 g) were obtained from the Experimental Animals Unit of Trakya, Faculty of Medicine. Animals were maintained under standard laboratory conditions (22±1 ºC temperature, 55% humidity, 12-hour light/dark cycle) and fed with standard feed and tap water. Animals were brought to the laboratory environment 24 hours before the start of the experiment to ensure their adaptation to the experimental environment. The Trakya University Animal Experiments Local Ethics Committee granted approval for this study (decision no: 2019.03.01, date: 29.03.2019). 

Experimental Design

The study was planned to have three groups, including two experimental and a control group. Each group was formed with eight rats that were randomly selected. 

Control group: Intraperitoneal saline was given daily for 14 days.

FA group: Intraperitoneal FA was administered at a dose of 10 mg/kg diluted 1/10 with saline daily for 14 days. FA + BAI group: Intraperitoneal FA at a dose of 10 mg/kg diluted 1/10 with saline and BAI at a daily intraperitoneal dose of 200 mg/kg were given daily for 14 days. When subacute studies were examined in the literature to establish experimental FA toxicity, it was seen that intraperitoneal administration of FA was preferred. The doses of both FA and BAI were selected according to the literature^30-32.

At the end of the experiment, all rats were euthanized by removing lung tissues under 10 mg/kg xylazine hydrochloride (HCl) and 50 mg/kg ketamine HCl anesthesia. Half of the tissues were soaked into 10% formalin solution for pathological examination. The other parts were stored in liquid nitrogen at -80 °C until mRNA isolation after they were frozen quickly.

Determination of Gene Expression Levels

Quantitative reverse transcriptase (qRT)-PCR analyses were conducted on complementary DNA (cDNA) samples produced from total RNA extracted from lung tissue. Specially designed primers for the GSH, SOD, CAT, Cyt-c, P53, Caspase-3, Caspase-9, iNOS, and eNOS genes were utilized.

RNA Isolation

RNA isolation from lung tissues was performed using the Invitrogen by Thermo Fisher Scientific isolation kit. The isolation procedure was performed according to this kit’s method as described below. 1 mm diameter zirconium silicate r beads were placed in the samples for better homogenization. It was then kept in liquid nitrogen and passed through a tissue shredder. 1% mercaptoethanol and lysis buffer were added to the samples to accelerate the protein denaturation process in order to isolate the RNA easily. The same volume of 70% ethanol was added to the cell homogenates to remove water. It was centrifuged at 21380 G to remove the beads. 700 μL of the liquid from the centrifuge was taken into the columnar tubes included in the kit. The samples in these tubes were centrifuged at 12000 g for 15 seconds and then the filter sections of the tubes were transferred to the collection tubes. 700 µL of washing buffer 1 was added to the samples and centrifuged at 12000 g for 15 seconds, and the filter parts of the columnar tubes were taken into new tubes. 500 µL of washing buffer 2 was added to them and centrifuged at 12000 g for 15 seconds. 500 μL of washing buffer 2 was added again and centrifuged for 2 minutes, and then the tubes were taken into capped tubes and 50 μL of the RNase-free water composition was added to the tubes. The reason for adding 50 µL was that it was determined as the optimum volume in the studies. The mixture was centrifuged at 12000 g for 2 minutes, 30 seconds after incubation for 2 minutes. Filter parts were discarded and the lower parts were taken for measurement. 2 μL of the obtained RNA samples were taken, pipetted on the Nanodrop device and the purity and absorbance values ​​were determined by reading at 260-280 nm. The measured purity values ​​were measured in the range of 1.8-2.0, and c-DNA synthesis was performed from these samples.

Complementary Deoxyribonucleic Acid (cDNA) Synthesis

The cDNA synthesis was carried out from the isolated RNAs using the High-Capacity cDNA reverse transcription synthesis kit (Catalog no: 4368814) by following the appropriate protocol steps. The synthesized cDNAs were stored at -20 °C. Polymerase chain reaction (PCR) conditions step 1: 25 °C, 09:53 min; step 2: 37 °C, 120 min; step 3: The cDNA synthesis was performed by programming at 85 °C for 5 minutes. The cDNA synthesis protocol is shown in Table 1.

Real-Time Polymerase Chain Reaction (qRT-PCR) Expression

Active GSH, SOD, CAT, P53, Cyt-c, Caspase-3, Caspase-9, iNOS and eNOS gene expression levels were determined by qRT-PCR method. In gene expression studies, cDNAs obtained from RNA isolated as described in the “RNA isolation” section was used. In our study, the Quant Studio 6 Flex qRT-PCR system, which can read 384-well microplates, was used. qRT-PCR’ genes used and their sequences are listed in Table 2. Glyceraldehyde 3-phosphate dehydrogenase was used as calibration and correction factor and samples were analyzed. Expression levels of genes were determined using the SYBR Green method. qRT-PCR mix; cDNA contains SYBR Green and related genes. The contents of the qRT-PCR reaction mix consist of 6 µL of SYBR Green Master Mix, 2 µL of cDNA and 2 µL of RNase free water, and 0.5 µL of primer forward, 0.5 µL of reverse for each well of a 384-well plate. PCR program: 1 cycle of 2 min at 50 ºC and 10 min at 95 ºC, followed by 40 cycles of denaturation (95 ºC for 15 s) and annealing and extension (1 min at 60 ºC).

Histopathological and Immunohistochemical Evaluation

After the tissue samples were fixed in 10% formalin solution for 24 hours at room temperature, they were embedded in paraffin blocks and passed through an increasing rate of alcohol series (60%, 70%, 80%, 90.99.9%). Paraffin blocks were prepared after the tissues were exposed to 2 exchange xylene and paraffinization steps for transparency. After the deparaffinization process of the 4µ sections taken from the blocks, hematoxylin-eosin (H&E) staining was applied to one of them primarily for general tissue histological examination, and iNOS (Thermo Invitrogen PA3-030A) and eNOS (Thermo Invitrogen MA5-15559) antibodies were applied to the others. Ventana BenchMark XT Ultra IHC/ISH system platform was used for immunohistochemistry staining. Tissue slides were incubated at 37 °C overnight and 56 °C for 2 hours. Here, a 10-minute xylene series, 96%, 80%, 70% alcohol series and three times distilled water series were applied to the slides at 60^oC. “Citrate buffer 10 X pH 8.0” (code: 15-M820, Lot. 50930) was used for antigen retrieval (20 minutes at 95-100 °C and 20 minutes at room temperature/cooling). Phosphate-buffered saline was administered 10 minutes after endogenous peroxide blockade with 3% H₂O₂. Slides were incubated with iNOS and eNOS antibodies for 45 minutes at room temperature. Then, standard immunoperoxidase staining method steps were applied. While evaluating the stainings in the sections, the pathologist made an unbiased evaluation without knowing the group characteristics. The prepared preparations were evaluated under the Nikon Eclipse E600 model light microscope. Images were taken from the sections in the Visia imaging program and saved. All H&E sections were evaluated in 10 randomly selected high magnification fields (x400) of the microscope. Inflammatory cellular infiltration and foamy macrophages accumulation in the pulmonary interstitium, thickening, hemorrhage and epithelial cell shedding in the bronchiolar wall were observed. All rat’s sections were scored according to these findings from 0 to 4 on a predefined semi-quantitative scale. The degree of lung damage scored as 0: normal tissue; 1: minimal damage (<1-25% uptake); 2: moderate damage (25-75% involvement); and 3: severe necrosis (>75% involvement)³³. For the evaluation of eNOS and iNOS antibodies, the extension and density of positively stained cells were determined in each Caspase. The extent of staining was graded as 0 (0-5%), 1 (6-24%), 2 (25-49%), 3 (50-74%), and 4 (≥75%). Staining intensity was graded as 0 (negative), 1 (mild), 2 (moderate), and 3 (strong). The immunoreactivity score ranging from 0 to 300 was determined by multiplying the two obtained degrees³⁴.

Statistics Analysis

To define descriptive statistics, mean and standard deviation values were used. The conformity of the data to the normal distribution was determined by the one-sample Kolmogorov-Smirnov test. One-way ANOVA test was applied if the variables fit the normal distribution. The “Bonferoni post-hoc” test was performed for multiple comparisons between variables. The Kruskal-Wallis test was used for multiple comparisons between non-normally distributed variables. Pairwise comparisons between groups were evaluated with the Mann-Whitney U test. The significance limit for all statistics was considered as p<0.05. IBM SPSS Statistics 20.0 program was used for statistical analysis. GraphPad Prism 6 Windows Software was used to create the graphs.

RESULTS

Light Microscopy Findings

In control group, morphology of lung tissues of rats was similar to normal morphology. Microscopic findings of the FA group were quite remarkable since interstitial hemorrhage, inflammatory cells, macrophages and edema were seen in the FA group’s lungs. Terminal bronchioles with moderate or severe shedding of epithelial cells, thickening and bleeding were detected in all rats. Although the histopathologic findings of the FA+BAI group are less prominent than the FA group, there was statistically no significant difference between these two groups (p>0.05) (Figure 1).

Immunohistochemistry Findings

eNOS staining was moderate to severe in the control group and mild in the FA group. On the contrary, iNOS staining was not detected in the control group but it was moderate to severe in the FA group. The staining features in the BAI+FA group were similar with the FA group (p<0.05) (Figure 1).

In our study, while control group had normal range of findings, the FA group had higher degree of histopathologic lung damage, immunuchemically low eNOS and high iNOS expression. FA+BAI provided minimal reduction on lung damage and iNOS expression level in addition to the minimal increase on eNOS expression levels. However, these findings were not statistically significant (p<0.05).

Gene Expression Levels 

Compared to the control group, it is seen that GSH and CAT levels were lower in the FA group. SOD and CAT expression levels were significantly increased in the FA+BAI group compared to the FA group (p<0.05). The P53 expression level was lower in the FA group compared to the control group. When compared with the control group, it was observed that the expression level of sit-c, iNOS and eNOS increased in both FA and FA+BAI groups (p<0.05). The gene expression levels we obtained as a result of our study are given in Table 3.

DISCUSSION

FA is a colorless, flammable gas that is widely used in medical science and industrial sectors, and unfortunately it is one of the major air pollutants. Due to FA exposure in some professions such as pathologists, anatomists, and technicians, it is observed that there is an increase in the incidence of leukemias, brain tumors, liver, testes and lung cancers compared with the normal population. FA is well-known as mucosal irritant, serious respiratory system toxic agent and carcinogenic chemical, classified as Group 1 by International Agency for Research on Cancer^{35, 36}. FA is also associated with depressive symptoms mediated by systemic inflammation³⁷. In this study, we aimed to observe the connection between FA induced lung damage and eNOS, iNOS expression, antioxidant response and apoptotic process in lung tissues. On the other hand, we wanted to investigate the effects of baicaline on FA damage, which have never been studied before. 

FA, which is taken exogenously or formed by metabolic reactions, causes various toxic effects in tissues. FA exposure mostly appears with inhalation through exogenous and biosynthetic sources such as methanol metabolism, demethylation, histone/DNA/RNA demethylation in the body form FA as an intermediate product. Toxic effects of FA and its metabolites can be observed especially in parenchymal tissues. Previous studies confirmed the role of FA in biomolecular profile alterations and highlighted that the low occupational exposure on health care professionals could also result in measurable biological outcomes^{38, 39}.

The objective of this study was to ascertain the histopathologic effects of the toxic metabolites of FA metabolized in the liver on the lung parenchyma, as well as the apoptosis pathway, and to delineate the underlying causes of the observed damage, which included inflammation and oxidative stress. A rat model was established by direct (intraperitoneal) administration of FA to rats at doses previously determined in other studies. The results demonstrated that direct subacute FA exposure caused lung injury by inducing severe inflammation, apoptosis, and increased oxidative stress. These injury mechanisms resulted in histopathologic findings. 

The decrease in eNOS level due to FA exposure indicates that the cell loses its normal functions. The increase in iNOS can be seen as a sign of tissue damage development. The decrease in GSH and CAT levels also shows that the deterioration in oxidative balance is a factor that facilitates tissue damage and prevents the healing of the damage.

Although FA is caused cytotoxicity with cell death or apoptosis and genotoxicity via DNA and chromosomal damage, limited data suggested that oxidative stress caused by reactive oxygen species may contribute to damage. Excessive exposure to ROS is known to cause developmental toxicity through damage to cellular components such as DNA, lipids, and proteins⁴⁰. Both the induction and suppression of antioxidant enzymes by FA has been demonstrated in different tissues. While GPx, SOD, CAT and GSH protect cells against oxidative damage, malondialdehyde is an oxidative biomarker that their activity levels may use for indicating the level of oxidative damage⁴¹. Some studies have shown that FA induces the antioxidant defense mechanism in rodent testicular tissue and may impair its effects⁴². Lim et al.⁹ showed that FA induced an increase in lipid peroxidation formation, a marker of oxidative stress.

NO is generated through the action of iNOS in response to tissue injury or stress. The iNOS level increases but eNOS level decreases as an indicator of tissue damage. NO production causes an increase in free radicals. Another effect of NO is to reduce antioxidants such as SOD, Gpx, and CAT. As a result, tissue damage occurs due to oxidative stress. There were numerous studies about FA induced tissue damage, NO and oxidative stress mechanisms. First, the activator effect of FA on oxidative stress was evaluated⁴³. They were followed by studies showing the iNOS, eNOS, ROS, oxidative stress stimulating the effects of FA, degree of tissue damage and SOD, CAT, GPx levels in different tissues (brain, liver, kidney, lung, etc.). Mohammed et al.⁴⁴ evaluated genotoxic and heamatotoxic damage of FA. Zararsız et al.³¹ planned their study on lung tissue. Results of these studies are compatible with our data supporting the FA, NO and ROS relationship^{31, 43-45}.

Teng et al.⁴⁶ reported that even low concentrations of FA caused oxidative damage on isolated rat hepatocytes in their experimental study. Similarly, Sarsılmaz et al.⁴⁷ found that CAT activity decreased and SOD activity increased in liver tissue by administering FA to rats by inhalation. Zararsız et al.³¹ studied on lung tissue and they found out that CAT enzyme levels decreased and SOD enzyme activities increased in lung tissue of rats exposed to FA.

There is no study in the literature investigating the protective effects of BAI, an antioxidant, against FA toxicity at the expression level. However, there are studies evaluating the protective effects of BAI on different cell lines by in vitro studies at the expression level^{9, 43, 45}.

In our study, qRT-PCR results demonstrated that GSH and CAT expressions were downregulated as a result of damage to the lung tissue due to FA toxicity. This result shows that antioxidant defense is negatively affected. SOD and CAT expression levels were significantly increased in the FA+BAI group compared to the FA group. This situation was evaluated as a positive effect of BAI on providing antioxidant defense through SOD and CAT.

Two major pathways lead to apoptosis activation. First one is the extrinsic (death receptor-mediated) pathway that is initiated by tumor necrosis factor family members, as an activating complex for pro-caspase-8, and second one is the intrinsic (mitochondria-mediated) pathway with apoptosome, as an activating complex for procaspase-9. The intrinsic pathway is regulated by Bcl-2 family proteins. Several signaling molecules lead to the initiation of intracellular inflammatory mediator synthesis and/or apoptosis. Numerous studies have shown that apoptosis is one of the mechanisms of cell death during the FA induced lung injury^{12, 14, 44}. In our study, we investigated major members of the intrinsic pathway as Cyt-c, CASPASE-3, and CASPASE-9 via qRT-PCR.

The decrease in P53 expression level in the FA group compared to the control group was interpreted as the TP53 gene, which is a tumor suppressor gene. It was damaged due to oxidative stress and therefore the P53 expression level decreased. P53 expression level was found to be significantly lower in the FA+BAI group than in the control and FA groups. This was interpreted as BAI did not have an effect on the apoptotic process. As a result of oxidative stress due to FA toxicity, it was considered that the apoptotic process was triggered via the mitochondrial pathway. The increased expression of Cyt-c in the FA group compared to the control group may be an indicator of this. It is thought that raisedcellular stress may cause membrane damage due to DNA damage and lipid peroxidation, and as a result, Cyt-c release from mitochondria to the cytoplasm may have increased. Apoptotic effector or “executive” proteins in the apoptosome (Cyt-c, Apaf-1 and procaspase) and caspase cascade play roles in the mitochondrial-dependent apoptotic process. It is observed that the expression of Cyt-c increases to trigger cell death against the harmful effects of FA and triggers the formation of active caspase-9. Active caspase-9, as an initiator caspase, cleaves and activates effector caspase such as caspase-3 and caspase-7.  This situation increases the degree of cellular damage and DNA damage. There was no increase in caspase-3 expression levels in both the FA and FA+BAI groups. This suggested that the level of inactive caspase-3 in the cell was sufficient to trigger apoptosis. However, Cyt-c expression level was found to be high in the FA+BAI group. This suggests that BAI has no therapeutic effect on mitochondrial damage.

When compared to the control group, it was observed that the iNOS expression level increased in the FA group. In a different study with lung tissue, it was stated that iNOS expression increased due to FA toxicity. This result is compatible with our study⁴⁴. In our study, iNOS expression level was also higher than in the FA+BAI group when compared to the other two groups (p<0.05). NO reacts with the superoxide radical and forms peroxynitrite, which causes DNA damage. The increase in iNOS expression level in the FA+BAI group can be interpreted as the activation of the antioxidant system via NO. As a result of immunostaining, it was observed that iNOS immunoreactivity increased significantly in the FA group. This suggests that the response to cellular damage to this toxicity might be via NO. Similarly, iNOS immunoreactivity was found high in the FA+BAI group, but there was no significant difference compared to FA. It was determined that the eNOS expression level decreased significantly due to FA toxicity. Immunohistochemical staining also supports this result. It was considered that the decrease in eNOS immunoreactivity might be associated with epithelial tissue damage due to FA toxicity. A decrease in eNOS expression level and immunoreactivity intensity was observed in both FA and FA+BAI groups when compared to the control. This situation interpreted as BAI not efficient on eNOS.

At high concentrations of FA exposure, pulmonary effects such as cough, shortness of breath, wheezing at 10-20 ppm levels, edema and spasm in the larynx were observed. On microscopic examination, it is seen that even acute inhalation of low doses of FA causes inflammatory cell changes in the upper respiratory tract and lung parenchyma in humans and animals. Pulmonary inflammation, edema and pneumonia develop at doses of 50-100 ppm^37-38. Other important microscopic findings were an increase in thickness of the wall, hemorrhage and epithelial cell shedding of bronchioles. In literature, numerous studies showed FA induced damage and protective effect of various substances^{11, 32, 43-48}. In our study, easy identified microscopic lung damage was detected as a FA application.

Wogonin and BAI, extracted from S. baicalensis, exhibit similar pharmacological properties with respect to apoptotic, inflammatory, and oxidative effects. In vivo and in vitro studies have demonstrated that BAI protects organs such as the pancreas, liver, and kidney from inflammatory mediators and immune system damage^{49, 50}.

Study Limitations

That the parameters of the experimental study could not be examined biochemically and western blot analysis could not be performed in the tissue samples were the limitations of this study.

CONCLUSION

In this study, it was aimed to research the BAI, whose antioxidant and anti-inflammatory properties on different tissues have been proven, with experimental studies as a protective agent on lung tissue against FA damage. Bie et al.²⁸ investigated the anti-cancer effects of BAI against hepatocellular carcinoma and they found favorable results. In addition, Sowndhararajan et al.²⁹ highlighted the neuroprotective effects of BAI. However, we could not detect positive effects of BAI on FA induced lung damage. In rat lungs, FA induces apoptosis via the intrinsic mitochondrial pathway and BAI has no positive effects on apoptosis at the expression level. On the other hand, our study reveals that BAI has a curative effect on oxidative stress parameters at the expression level.