Histological and ultrastructural studies on the effect of Cassia alata methanolic leaf extracts against chemically induced lung adenocarcinoma in rats

ABSTRACT The present work aims to evaluate anticancer performance of Cassia alata methanolic leaf extracts (CMLE) in ethyl carbamate-stimulated lung adenocarcinoma (LAD) in differentiation to the function of Cisplatin (CIPL). Rats were divided into four groups: (1) control (CONT), (2) lung-adenocarcinoma (LAD) injected intra-peritoneally with 1g/kg ethyl carbamate once weekly for a month, (3) LAD+CMLE administered 500 mg/kg CMLE orally for the last two months of the experiment, and (4) LAD+CIPL treated group, injected 2.5 mg/kg Cisplatin intraperitoneally once weekly for the last two months of the experiment. Light and electron microscopic examinations revealed adenocarcinoma development in terminal bronchiole besides some histopathological changes in the LAD group such as atypical, exaggerated collagen fibers, increment of mucinous content, and increasing of PCNA positive immunoreactivity whereas electron microscopy investigation exposed that papillary adenocarcinoma originated from Clara cells in the LAD group. The LAD+CMLE treated group showed no tumor masses and nearly all with normal lung histology. It also recovered the normal ultrastructure of bronchiolar Clara cells. CMLE treatment offers a new alternative cure with less toxicity than Cisplatin for lung cancer therapy. Hence, CMLE would be employed as a novel supply of anti-cancer compounds combating lung cancer.


INTRODUCTION
According to Egypt's National Population-Based Registry Program (NCRPE), lung cancer, along with breast, brain, liver, and urinary bladder cancer, is one of the five most common types of cancer (Hamzawy et al., 2022).Adenocarcinoma is the most common type of lung cancer, with 6,538 people (4,851 men and 1,687 women) newly diagnosed in Egypt in 2020 (Sung et al., 2021).
Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are the two most common histological forms of lung cancer, accounting for around 85% and 15% of cases, respectively.Surgery, chemotherapy, radiotherapy, and immuno-therapy are now accessible (Wilcox et al., 2008;Ju et al., 2012, Travis, 2012).
Ethyl carbamate, often known as urethane, is a carbamic acid ester that has been detected as a byproduct of fermented beverages and foods.Because of unwitting fermentation through the storing or making of fermented beverages and foods, it is greatly spread (Dennis et al., 1989).Because of formation of vinyl carbamate, an active metabolite, Ethyl carbamate produced malignant lesions in various body locations.Continual exposure to ethyl carbamate which is found in fermented beverages and foods enlarges lung cancer risk through irreversible malfunction of mitochondria and increment of cellular mitotic activity (Hamzawy and Abo Youssef, 2015).
Cisplatin is a well-known platinum-based chemotherapeutic drug that is effective against a variety of cancers such as lung cancer, ovarian cancer, and breast cancer (Santarpia et al., 2017).Cisplatin destroys cells in two distinct ways.For starters, it can cause cytoplasmic mitochondrial apoptosis.Second, it enters the cell membrane for hydration, substituting one chloric ion with one water molecule, and then enters the nucleus to attach to a single nitrogen on a DNA nucleotide.Ethyl carbamate, often known as urethane, is a carbamic acid ester that has been detected as a byproduct of fermented beverages and foods.After that, one molecule of water replaces the second chloric ion, causing it to bind to a second nucleotide (Tanriverdi et al., 2007).Cisplatin binds with DNA, forming covalent adducts with purine DNA bases, and this reaction is the main cause of cisplatin's cytotoxic effect (Yousef et al., 2009).Cisplatin therapy causes a number of toxic side effects, in heart, kidney and liver (Jongh et al., 2003;Al-Majed, 2007).
Chemotherapy containing Platinum is still the recommended treatment for most progressive non-small cell lung-cancer (NSCLC) patients (Ibrahim et al., 2014).The creation of a more effective and safe alternative treatment strategy is necessary due to the excessive toxicity and chemoresistance linked to traditional chemotherapies, which lower the success rates of the present chemotherapeutic regimen (Swetha et al., 2022).Numerous studies suggest that natural polyphenols work through a variety of molecular mechanisms to combat different types of cancer in either a chemo-preventive or chemotherapeutic way (Zhou et al., 2019).
The family Fabaceae is considered the second largest widespread in the plant kingdom.It includes a big number of phytochemicals (saponins, alkaloids, flavonoids, phenolic acids, lectins, and carotenoids) with an assortment of health advantages, most notably anti-cancer activity (Usman et al., 2022).
C. alata leaf has been shown to contain phenolics and to have cytotoxic effect against cancer cells.Chrysoeriol, kaempferol, quercetin, palmitic acid, stearic acid, and other important chemical compounds are present, the bulk of these compounds have been found to have cytotoxic and anti-inflammatory effect against lung-cancer (Levy and Lewis, 2011;Olarte et al., 2013).
This study aimed to investigate the effect of C. alata extracts against chemically induced lung adenocarcinoma in rats.
Fresh C. alata leaves were collected during July 2021 at Cairo University's Agriculture Farm, dried at room temperature in the shade, and milled to a soft powder.Methanol was used to extract 2 kg of the powder and then concentrated under reduced pressure at 40 °C.The methanolic extract remnant (22.57percent) was dried using RE-2010 rotary evaporator (BIOBASE, China) at 40 °C.The collected components were kept until usage at -20 °C (Ali et al., 2017).
Adult albino rats (40 male rats, 150±20 g, 8-9 weeks old) were attained from Egypt's New Veterinary Office.Rats were housed in cages under condition of 12-hour light/dark cycles in a temperature-monitored setting.During the experiment, water and food were freely offered.The experiment was agreed to by the ethics committee of the Institutional Animal Care and Use Committee for Laboratory Animals at Zoology Department, Science Faculty, Helwan University (Approval number: HU-IACUC/Z/MI1101-23).
The rats were grouped into four main groups (10 rats for each) as follows: Group 1-Control (CONT) group: rats received intraperitoneal injections of phosphate buffered solution for 7 months.
Group 2-Lung adenocarcinoma (LAD) group: Animals served as the carcinogenic non-treated group.For induction of lung cancer, for a month, rats were intraperitoneally injected with 1g/kg/rat/week ethyl carbamate (Janker et al., 2018).Thirty adult male albino rats were given intra-peritoneal injections of ethyl carbamate (1g/kg) once weekly.Lung cancer developed after a 5-month latency period following the initial urethane injection.This group consists of ten rats that were left untreated for two months.
Group 3-LAD+CMLE treated group: Rats were orally administered with CMLE (500 mg/kg) daily (Onyegeme-Okerenta et al., 2017).Treatment began after the fifth month and continued until the experiment was completed.
Group 4-LAD+CIPL treated group: Lung cancer rats were injected intraperitoneally with Cisplatin (2.5 mg/kg/week) (Jafri et al., 2010).Treatment began after the fifth month and continued until the trial was completed.
At the end of the experiment, rats were sacrificed using isoflurane anesthesia at concentration of 6% and excised their lungs rapidly and cleansed in saline.Each lung was divided into two sections, one for histopathologic staining in neutral-buffered formalin at concentration of 10% and the other fixed in glutaraldehyde for transmission electron microscope of bronchiolar Clara cells.
The fixed lung specimens were processed for paraffin section preparation at thickness of 5 μm (Spencer and Bancroft, 2013).The slides were observed after staining by a light microscope of LEICA model and taken pictures by a Zeiss camera.
For general histopathological observation of lung tissue, we used hematoxylin and eosin stain (H&E) (Bancroft and Layton, 2019).It was also used to assess lung bronchiolar wall thickness, alveolar lumen diameter, and inflammatory score.We used a semi-quantified inflammatory score, with grades 0 denoting no inflammation, 1 implying minimal inflammatory cells in some microscope fields, 2 revealing mild inflammation with an influx of a ring of cells 1 cell layer deep, 3 indicating moderate inflammation with a ring of cells 2-4 cells deep, and 4 inferring severe inflammation with a ring of cells > 4 cells deep (Dong et al., 2012).
ImageJ analysis software (NIH, version, 1.46, LOCI, University of Wisconsin, USA) was used to measure and statistically analyze the bronchiolar wall thickness (µm) and alveolar lumen diameter (µm).The mean bronchiolar wall thickness, alveolar lumen diameter, and inflammatory score for each group were calculated using six random microscopic fields at (40×) magnification.Some lung paraffin sections from each group were dyed with MT stain and photographed at Arq. Bras.Med.Vet.Zootec., v.75, n.5, p.857-871, 2023 (40×) power (Silva et al., 2020).Masson's trichrome staining turns collagen fiber blue, cytoplasm, muscle, and erythrocytes pink, and nuclei dark brown to black (Zhou and Moore, 2017).ImageJ software was utilized to calculate the optical density of Masson's trichromestained collagen fiber.Some paraffin sections of lungs from each group were stained with Alcian blue-PAS stain (Bancroft and Gamble, 2013).The stained sections were photographed at high magnification power (40×).Acid mucin was dyed blue, whereas neutral mucin was dyed magenta.Image analysis software was used to measure mucin content in the lung across ten fields/each group.
In each group, lung sections were stained with anti-PCNA stain and photographed under high magnification (40×).
Streptavidin-biotin immunoperoxidase complex staining was utilized for immunohistochemical staining.The positive reaction for PCNA appears as a brown nuclear reaction (Ahmed et al., 2019).
For the evaluation of the ultra-structural characteristics of the bronchiolar Clara cells in the lung of various groups, transmission electron microscopy (TEM) processing was done (Graham and Orenstein, 2007).A portion of the lung was immediately fixed in cold glutaraldehyde (5%) for 24 hours, washed three times in 0.1 ml of phosphate-buffer (pH 7.2), and then post-fixed for 1.5 hours with 1 percent osmium tetroxide.The specimens were then dehydrated at various alcohol concentrations and implanted in epoxy resin following a second rinse in phosphate buffer.To evaluate the area that had been cut extremely thinly, semi-thin slices (1 micrometer thick) were made using an LKB ultratome, toluidine blue was added to them, and they were then examined under a light microscope.At the center, Regional Mycology and Biotechnology of Al-Azhar University in Cairo, Egypt, the ultra-thin slices were observed by using a transmission electron microscope (TEM) (JEOL1010 EX II, Japan).
Mean area-percentage of mucin, collagen fiber as well as PCNA positive reactions in ten nonoverlapping high-power fields (40×) of paraffinized sections of lungs per each group were measured and statistically evaluated using image analysis software (Imagej version,1.46).Version 25 of the Statistical Package for the Social Sciences (SPSS) was utilized to conduct a statistical analysis of all numerical data.The post-hoc Tukey's test was utilized to calculate whether there were any significant differences among groups, and one-way analysis of variance (ANOVA) was utilized to compare the means of multiple groups.If the p value was less than 0.05, the outcome was deemed statistically significant.

RESULTS
The CONT group had normal lung histoarchitecture in male albino rats.The murine lung was made up of a terminal bronchiole, an alveolar duct, an alveolar sac, and regular-sized alveolar spaces that were separated by a thin inter-alveolar septum and thick interstitial tissue (Fig. 1A).The LAD group had abnormal structure of lung tissue, with infiltrative papillary bronchiolar adenocarcinoma growing as a tumor lesion invading and obliterating the lumen of terminal bronchiole.The tumor mass was made of irregular papillae lined by hyperplastic columnar epithelial cells together with basal vesicular nuclei and some pleomorphic cells that differed in shape and size (Fig. 1B).The LAD+CMLE treated group had no tumor mass and a terminal bronchiole lined by some hyperchromatic low columnar cells (Fig. 1C).The LAD+CIPL-treated group had no tumor mass in the folded terminal bronchiole, and RBC extravasation was found in the air gaps (Fig. 1D).Cisplatin treatment left some lung lesions in the form of RBC extravasation after treatment of lung cancer bearing rats while CMLE treatment restored of normal lung histology when both treatments compared to control rats thus, CMLE treatment is preferable and safer than Cisplatin treatment.
The semi-quantitative evaluations of lung histoscoring for bronchiolar wall, alveolar lumen diameter, and inflammatory score among the study groups were shown in table (1) and figure (2).The mean bronchiolar wall thickness in the LAD group (96.20 6.37) was substantially higher than in the CONT group.When compared to the LAD group, CMLE treated and CIPL treated (34.05±2.38)groups significantly reduced the mean bronchiolar wall thickness (Fig. 2A).The alveolar lumen diameter in the LAD group (18.30±1.15)was substantially smaller than in the CONT group (39.14 3.41).The treatment with CMLE (35.60±3.35)and CIPL (40.24±5.14)increased the alveolar lumen diameter significantly as compared with the LAD group (Fig. 2B).Furthermore, inflammatory cell scoring in the LAD group (4.00 0.38) was substantially higher than in the CONT group (1.04±0.27).When compared to the LAD group, CMLE (1.78±0.33)and CIPL (2.42 0.19) treated groups significantly reduced inflammatory cell infiltration (Fig. 2C).Cisplatin treatment resulted in higher values of bronchiolar wall thickness, alveolar lumen diameter, and inflammatory score than CMLE treatment after treatment of lung cancer bearing rats when both treated groups compared to control group therefore, CMLE treatment is preferable in amelioration of pathological changes associated with lung cancer induction in male albino rats.The morphometric area percent of collage fibers, mucin content and PCNA positive immune reaction in the study groups were shown in Table 2 and Figure 3.The collagen fiber area % in the LAD group increased significantly as compared with the CONT group.When compared to the LAD group, rats in the CMLE and CIPL groups had a significant decrease in collagen fiber area percent.When compared to the CONT group, the LAD group showed a substantial increase in the area percent of total mucins.When compared to the LAD group, rats in the CMLE and CIPL groups had a significantly lower area percent of total mucins.When compared to the CONT group, the LAD group demonstrated a substantial increase in the area percent of PCNA positive reaction.When compared to the LAD group, rats in the CMLE and CIPL groups had a significantly lower area percent of PCNA.Cisplatin treatment resulted in higher values of collagen fibers content, AB-PAS positive reaction, and anti-PCNA positive immunoreaction than CMLE treatment when both treated groups compared to control group therefore, CMLE treatment is preferable in amelioration of lung fibrosis, high mucin production and higher cellular proliferation associated with lung cancer in male albino rats.
Blue collagen fibers were distributed normally within interstitial tissue and around the terminal bronchiole in the CONT group (Fig. 4A).The LAD group had an aberrant distribution of collagen fibers around and within the terminal bronchiole wall (Fig. 4B).The LAD+CMLEtreated group had moderate collagen fiber deposition around the terminal bronchiole and between alveoli (Fig. 4C).The LAD+CIPLtreated group had moderate collagen fiber deposition in the terminal bronchiole and between the alveoli (Fig. 4D).
The AB-PAS reaction for total neutral mucinous substance was moderately positive in the CONT group (Fig. 5A).The LAD group demonstrated a robust positive AB-PAS reaction for neutral mucin surrounding the terminal bronchiole and acidic mucin within the tumor mass and the epithelial lining of the terminal bronchiole (Fig. 5B).The LAD+CMLE group had a moderately positive AB-PAS reaction for total neutral mucin (Fig. 5C).The LAD+CIPL group had a moderate positive AB-PAS reaction of total neutral mucin content (Fig. 5D).In the entire section, the CONT group demonstrated an overall mild to moderate positive immunoreaction for staining of PCNA in the lung (Fig. 6A).The LAD group had overall strong positive PCNA immune reactivity in the lung (Fig. 6B), emphasizing cellular proliferation as a hallmark of cancer progression in the lung.In lung sections, the LAD+CMLE group had a net moderate positive PCNA immunoreaction (Fig. 6C).In the section, the LAD+CIPL treated group demonstrated a moderate positive PCNA total immunoreaction (Fig. 6D).
An ultrathin section of a lung terminal bronchiole from the CONT group revealed normal bronchiolar epithelium consisted of Clara cells and ciliated cells.Clara cells had a primary body and a projecting apex pointing toward the lumen.Clara cell's apex included several pale mitochondria and a few dense secretory granules, while the major body was composed of a notched central nucleus surrounded by scattered spherical or elongated mitochondria, many electron dense secretory granules, and smooth endoplasmic reticulum (Fig. 7A).
The LAD group had abnormally proliferating Clara cells in the terminal bronchiole epithelial lining, resulting in a tumor mass of tightly packed pleomorphic cells identified as Clara cells by dense secretory granules.The nuclei of these cells were pleomorphic and surrounded by vacuolated pale cytoplasm.Most cells did not rest on the basement membrane (Fig. 7B).The LAD group's high magnifications revealed some pleomorphic Clara cells creating a tumor mass with deformed cell borders, each cell contained pleomorphic nuclei surrounded by vacuolated cytoplasm, dilated smooth endoplasmic reticulum, and a few secretory granules (Fig. 7C).
The LAD+CMLE treatment group improved the ultrastructure of Clara cells, which are supported by a thick basement membrane.Clara cells had multiple electron rich secretory granules at the apex, whereas the main body consisted of a club-shaped nucleus surrounded by basal rough endoplasmic reticulum and apical elongated mitochondria (Fig. 7D).The LAD+CIPL-treated group restored the ultrastructure of Clara cells to lesser degrees.Clara cells protruded far into the lumen, enveloping neighboring ciliated cells.Clara cells had a lobed nucleus surrounded by apical dilated smooth endoplasmic reticulum, several electron dense secretory granules, and some Mitochondria in their main body (Fig. 7E).The apex of Clara cell contained many electron dense secretory granules (SG).The main body of Clara cell formed of club-shaped nucleus (N) that surrounded by basal rough endoplasmic reticulum (rER) and apical elongated mitochondria (M).The lower ciliated cells have cilia (red arrow) run toward airway lumen (L) while the upper cell appeared with detached cilia with remaining basal bodies (green arrow).(E) LAD+CIPL treated rats group showed more or less restoration of ultra-structure of ciliated cells (Ci) and Clara cells (Cl).Clara cells protruded much into lumen (L) covering adjacent ciliated cells.The main body Clara cell contained lobed nucleus (N) surrounded by apical dilated smooth endoplasmic reticulum (sER), many electrons dense secretory granules (SG) some Mitochondria (M).Bar = 500 nm excluding figure B = 2 µm. .Regarding TEM, CMLE treatment resulted in improved the ultrastructure of Clara cells than Cisplatin with some lesions when both treated groups compared to control group therefore, CMLE treatment is preferable in amelioration of ultra-structural features of Clara cells forming this type of lung cancer in male albino rats.
The above results showed that Cisplatin treatment left some lung lesions in the form of RBC extravasation after treatment of lung cancer bearing rats while CMLE treatment restored normal lung histology when both treatments compared to control rats.Moreover, CMLE treatment resulted in ameliorated values of bronchiolar wall thickness, alveolar lumen diameter, inflammatory score, collagen fibers content, AB-PAS positive reaction, and anti-PCNA positive immunoreaction than Cisplatin treatment when both treated groups compared to control group.Therefore, CMLE treatment allows a new alternative along with less toxicity than the Cisplatin drug in lung-cancer therapy.

DISCUSSION
The induced lung-cancer model employed in this study was the same as that used by Radwan et al.
(2021) who had used 4 intraperitoneally injections of ethyl carbamate 0.375 g/kg over a 12-week period with a three week gap between treatments.Janker et al. (2018) determined that three times of ethyl carbamate injection (1 mg/gm in one week) induced significantly more tumor nodules than one-time injection in mice, despite 0% death and morbidity and 100% tumor incidence rate.In the experimental paradigm, the length of time the animals were given to ethyl carbamate determines the induction of adenocarcinoma (Stakisaitis et al., 2014).
The ethyl carbamate's carcinogenic activity in our model could be attributed to the formation of vinyl carbamate, an active metabolite, which is a strong mutagen and stimulates the formation of electrophilic species that which interacts by DNA to generate 2-oxoethyl adducts as vinyl chloride (Ding et al., 2014).
Ethyl Carbamate injection caused progressive bronchiolar adenocarcinoma, which manifested as a tumor lesion infiltrating and partially obliterating the lumen of terminal bronchiole.The tumor mass was made up of hyperplastic columnar epithelial cells together with basal vesicular nuclei and pleomorphic cells with different cell sizes and shapes.According to the recent study, the bronchiolar adenocarcinoma cells' nuclei were pleomorphic, in addition to frequent mitosis, penetration of tumor cell into bronchiolar lumen, and wall push on the surrounding tissues, these pathologic features was in accordance with Stakisaitis et al. (2015) observations.
According to Gurukumar et al. (2010), the ethanolic leaf extract of C. alata displayed good anticancer effectiveness against generated cancer of prostate appearing in In vivo models utilizing N-methyl N-nitrosourea and Testosterone.
The existence of observable lesions in the LAD+CIPL treated group, for example RBC extravasation and inflammation in airway spaces, may be attributed to Cisplatin, which is not selective for cancer-cells and affects normal-cells as well (Sak, 2012).
Collagen fibers increased significantly in the LAD group compared toward the CONT group, confirming the findings of Anandakumar et al. (2015) who discovered enhanced collagen deposition in lung cancer-induced mice.The high collagen fiber content in the lung cancer group could be attributable to immunological and inflammatory processes that cause fibroblast activation and collagen deposition (Zidan, 2011).The current study suggests that the improvement in collagen fibers after CMLE treatment may be due to CMLE's anti-inflammatory characteristics (Patrick-Iwuanyanwu et al., 2011).

The
Alcian blue-PAS method is a straightforward method for identifying and characterizing muco-substances in tissue slices that appear to distinguish clearly between acid and neutral mucins (Ionilă et al., 2011).Chronic mucus hypersecretion has been displayed to be a significant predictor of lung cancer death (Lange et al., 1990).In this work, CMLE therapy restored normal mucin content in lung cancerbearing rats.
PCNA, proliferating cell nuclear antigen, is indeed a marker of cell proliferation (Naryzhny, 2008).PCNA seems to be a nuclear peptide with 36 kDa molecular weight that has been labeled as a DNA polymerase delta auxiliary protein (Nanji and Tahan, 1996).The terminal bronchiolar lining cell's nuclei as well as inflammatory peribronchiolar cells strongly responded positively to PCNA in the present study, significantly increasing the area% of PCNA in the LAD group as compared to the CONT group.This finding was supported by Wang et al. (2018) who demonstrated that expression of PCNA was raised in NSCLC tissues and cells and that overexpression of PCNA in lung-cancer cells led to proliferation of cells, clonal formation, along with tumor growth.In the current study, rats given CMLE had a moderate PCNA positive reaction in the lung.This discovery could be Arq.Bras.Med.Vet.Zootec., v.75, n.5, p.857-871, 2023 attributed to CMLE's anti-proliferative properties (Modarresi Chahardehi et al., 2021).
Cisplatin anticancer efficacy in the present work was consistent with Hamzawy et al. (2022) who revealed that carboplatin treatment of urethaneinduced lung cancer in BALB/C mice was substantially improved in all molecular, biochemical, and histological studies.
The lung cancer in this investigation is made up of neoplastic Clara cells.This conclusion was compatible with Reznik-Schuller (1976) who observed that long-term administration of nitrosamine derivatives to hamster rats, results in the production of bronchogenic carcinomas composed of neoplastic Clara cells.Tumor cells of the Clara cell type have a cluster of electrondense secretory granules, whereas tumor cells of the type II alveolar epithelial cell type have lamellar inclusion bodies inside the cytoplasm (Hou et al., 2020).
Clara cells of the LAD group had abundant secretory vesicles at the apex, pleomorphic nuclei, peri-nuclear rough endoplasmic reticulum, vacuolated cytoplasm, and dilated smooth endoplasmic reticulum with flocculent material.The altered ultrastructure of Clara cells forming tumor mass in this work was consistent with Van Winkle et al. (1999) who formed that following acute naphthalene injury of bronchiolar Clara cells In vivo, the early phases of Clara cell injury as smooth endoplasmic reticulum swelling, and bleb development preceded the rise in permeability of cell membrane.The following major events occur during acute injury of Clara cell in vivo: cytoplasmic apical blebbing, cytoplasmic vacuolization, SER swelling, and permeability of cell membrane which markedly by exfoliation of apical parts of injured Clara cells into airway lumen.Phimister et al. (2005) found that significant GSH depletion produces significant swelling, actin cytoskeleton disturbances, and plasma membrane blebbing in Clara cells, which explains the altered ultrastructure of Clara cells in ethyl carbamate-induced adenocarcinoma.These cellular alterations have been seen in cells exposed to a variety of toxins and may represent a general cell response to GSH depletion and/or oxidative stress that occurs during the early stages of cell death (Manygoats et al., 2002).Blundell (2006) confirmed our Clara cell findings by observing nuclear chromatin margination and clumping, mitochondrial swelling, endoplasmic reticulum dilation, Clara cells become enlarged, and numerous vacuoles form adjacent to the cellular membranes after toxic substances enter the lungs via inhaled air.Clara cells are thought to be vital in protecting the airways from the negative effects of a hazardous external environment (Reynolds and Malkinson, 2010).The extract's anti-proliferative action may be responsible for the positive impact of CMLE treatment on bronchiolar Clara cell ultrastructure found in this investigation (Modarresi Chahardehi et al., 2021).

Figure 1 .
Figure 1.Cross section of rat lung in the study groups (H&E stain).(A) CONT group showed normal histoarchitecture of lung including terminal bronchiole (TB), alveolar sac (AS), alveoli (A), thin inter-alveolar septum (black arrow) and thick interstitial tissue (red arrow).(B) LAD group showed bronchiolar papillary adenocarcinoma exhibited well-defined tumor mass (TM) formed of irregular papillae lined by hyperplastic columnar epithelial cells (black arrow) with basal vesicular nuclei in the epithelial lining of terminal bronchiole (TB) and some pleomorphic that different in their shape and size (red arrows), terminal bronchiole supported by hypertrophied discontinuous muscle fiber (MF) layer and surrounded by peri-bronchiolar inflammation (PBI).(C) LAD+CMLE treated group showed terminal bronchiole (TB) that lined with some hyper-chromatic low columnar cell (white arrows), many alveoli (A) separated by thin inter alveolar septum (black arrow) and by thick interstitial tissue (red arrow).(D) LAD+CIPL treated group showed folded terminal bronchiole (TB), extravasation (EV) of RBCs in bronchiolar lumen, alveolar sac (AS), alveoli (A), blood vessel (BV).Bar= 50 µm.

Figure 2 .
Figure 2. Semi-quantitative evaluations of lung histo-scoring of: (A) the bronchiolar wall thickness, (B) Alveolar lumen diameter and (C) Inflammatory score in H&E stained lung sections in different study groups.The data are expressed as the mean ± S.E.M. *: significance against CONT group, #: significance against LAD group.P < 0.05.

Figure 3 .
Figure 3. Quantitative analysis of mean area percent of Masson trichrome-stained collagen fibers, AB-PAS positive reaction and PCNA positive immunoreaction in the lung of different groups.Values are mean ± S.E.M. *: significance against CONT group, #: significance against LAD group.P < 0.05.

Figure 4 .
Figure 4. Photomicrograph of transverse section in lung of different groups stained with Masson trichrome.(A) CONT group showed normal distribution of minimal amount of blue collagen fibers between lung alveoli (A) , alveolar sac (AS) (arrow head) and around terminal bronchiole (TB) (arrow).(B) LAD group showed abnormal distribution of excessive amount of collagen fibers around and within wall of terminal bronchiole (TB) (black arrow), terminal bronchiole (TB) lined with tumor mass (TM) surrounded by disrupted layer of smooth muscle fibers (MF).(C) LAD+CMLE treated group showed moderate collagen fiber deposited around terminal bronchiole (TB) (black arrow) and in between alveoli (A) (arrowhead).(D) LAD+CIPL treated group showed moderate collagen fiber deposited around slightly folded terminal bronchiole (TB) (black arrow) and in between alveoli (A) (arrowhead).Thick congested blood vessels (BV) existed.Bar = 50µm.

Figure 6 .
Figure 6.Sections in lung showed different anti-PCNA immunohistochemical reactions in lung cells nuclei of different groups.(A) CONT group showed moderate PCNA positive immunoreaction.A moderate reaction (blue arrow) appeared in interstitial cells among alveolar sacs (AS) and alveoli while mild reaction (red arrow) appeared in in some cells of terminal bronchiole (TB).(B) LAD group showed strong PCNA positive immunoreaction.Differentially, the strong positive reaction (black arrow) appeared in some cells of tumor mass (TM) occupying terminal bronchiole (TB) lumen and in nuclei of peri-bronchiolar inflammatory cells (PBI) whilst moderate reaction in some cells of (TM) but weak PCNA positive reaction found in muscle layer of (TB).(C) LAD+CMLE treated group showed moderate PCNA positive immunoreaction.Differentially, strong reaction (black arrow) appeared in few interstitial cells among alveoli (A), moderate reaction (blue arrow) in bronchiolar epithelial cells of terminal bronchiole (TB) whilst weak reaction (red arrow) appeared in many interstitial cells.(D) LAD+CIPL treated group showed moderate PCNA positive immune reactivity.Differentially, moderate reaction (blue arrow) in in some interstitial cells whilst weak reaction (red arrow) appeared in wall of terminal bronchiole and in some interstitial cells.Bar = 50µm.

Table 1 .
Histology scoring of bronchiolar wall thickness, alveolar lumen diameter and inflammatory score in the study groups Scoring Groups *#Values are Mean ± S.E.M. *: significance against CONT group, #: significance against LAD group, P < 0.05.

Table 2
. Area percent of collagen fiber, AB-PAS positive reaction and PCNA Positive immunoreaction in the study groups Area Groups # Values are Mean ± S.E.M. *: significance against CONT group, #: significance against LAD group, P < 0.05.