Stachys pilifera Benth. Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats through the Antioxidant Pathways

Methods In this experimental study, 35 male Wistar rats (120–180 g) were divided into five groups (n = 7) as follows: intratracheal instillation of bleomycin (BLM, 7.5 IU/kg) was administered to group II. The third and fourth groups received BLM plus Stachys pilifera hydroalcoholic extract (SPHE) (300 mg/kg/day, gavage). Vitamin E (500 mg/kg/day, gavage) was given to group V in addition to BLM. After 14 days, the animals were euthanized to assess biochemical parameters and lung histopathology. Malondialdehyde (MDA), nitric oxide (NO), total thiol (TSH), and glutathione (GSH) levels were measured. In addition, hydroxyproline (HYP) levels along with histological changes in lung tissue were also assessed. Results MDA, NO, and HYP elevations induced by BLM toxicity were significantly inhibited by SPHE (300 and 600 mg/kg), and Vit E. SPHE also significantly increased GSH and TSH levels in comparison to the BLM group.HPLC analyses showed the presence of thymol (55.47 ng/mL) and carvacrol (109.91 ng/mL) in SPHE as potential bioactive phenolic compounds. Conclusion The results suggest that SPHE alleviates the development of BLM-induced pulmonary fibrosis by inhibiting the proliferation of fibroblasts mediated by antioxidant pathways. Other mechanisms underlying this Effect of SPHE need to be clarified through further research.


Introduction
Idiopathic pulmonary fbrosis (IPF) is a chronic and progressive lung disease characterized by proliferation and diferentiation of fbroblasts and accumulation of extracellular matrix, primarily collagen, leading to irreversible damage to lung architecture [1,2].In some acute lung injury survivors, excessive alveolar injury stimulated the proliferation and repair system.As a result, many pulmonary interstitial cells and fbroblasts flled the damaged alveolar interstitium with neovascularization, causing excessive alveolar repair and pulmonary fbrosis [3].Due to the limited possibility of initial diagnosis and intervention, the average life of patients with IPF is 3 to 5 years after diagnosis.Te incidence of IPF is estimated to range from 6.8 to 16.3 per 100,000 [4].Although the cause of IPF is unknown, environmental pollution, viral infections, genetic factors, infammation, and oxidative stress are involved [5].In addition, some drugs, such as bleomycin (BLM), methotrexate, and nitrofurantoin, induce pulmonary fbrosis [6].Lungs are more exposed to exogenous oxidants than other organs due to their unique position and function.Antioxidants in epithelial lining fuid counteract these oxidants.However, this defense system can be disrupted by the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in disease states.In order to reduce the toxicity caused by ROS, enzymatic antioxidants such as GPX, SOD, and catalase and molecular antioxidant systems such as Vit C and Vit E are activated [7].Other antioxidants include thiol-containing compounds such as glutathione (GSH) and thioredoxin [8].Numerous studies have proven the role of oxidative stress in the pathogenesis of IPF [5,9,10].Increased levels of oxidative stress markers have been shown in patients with pulmonary fbrosis [5].
Bleomycin (BLM) is a chemotherapeutic antibiotic used to treat a variety of malignancies.Since one of the most crucial side efects of BLM during the chemotherapy process is pulmonary fbrosis, it is a classic combination for modeling pulmonary fbrosis in laboratory animals [6,11].Intratracheal BLM injection causes interstitial and alveolar infammation, macrophage activation, and increased infammatory factors.It also stimulates ROS production, including superoxide and hydroxyl radicals [12].Tere is currently no efective treatment for most types of IPF [1].For example, using methylprednisolone efectively improves BLM-induced IPF, but it has disadvantages such as inhibiting the immune system.Terefore, new therapeutic compounds with minimal side efects for preventing and treating PF have been considered [10].
Stachys pilifera is a perennial and fragrant plant belonging to the Lamiaceae family [13].In traditional medicine, some Stachys species have been utilized as antinephritis, antidiarrhea, and wound disinfectants [14,15].Moreover, the antibacterial, antioxidant, and cytotoxic efects of some Stachys species have been confrmed [16].Diferent species of this genus contain phytochemicals such as favonoids, iridoids, diterpenoids, and phenolic acids [17].Farjam et al. used the DPPH test to prove the antioxidant properties of S. pilifera [18].In our previous studies, the efects of hepatic and renal protection of the plant were shown by reducing oxidative stress [19,20].Hence, the main objective of this study was to evaluate the protective efect of S. pilifera hydroalcoholic extract (SPHE) on bleomycin-induced pulmonary fbrosis in rats.

Plant Materials.
Te aerial parts of Stachys pilifera were collected from the Kakan region in Kohgiluyeh and Boyer-Ahmad Province in spring 2018.Dr. Azizollah Jafari confrmed the collected samples from the Department of Botany, Center for Research in Natural Resource and Animal Husbandry, Yasuj University, Yasuj, Iran, where a voucher specimen (herbarium No. 1897) was deposited.Te plant materials were transferred to the laboratory and dried in a dark place.

Extract Preparation.
Te air-dried powdered plant (100 g) was macerated in 70% methanol (500 mL) and kept at room temperature for 48 hours (three times 500 mL each).Having fltrated the extract by Whatman No. 1, the solvent was evaporated by rotary apparatus to yield a dark green gummy residue.Te extract was stored in the refrigerator at 4 °C for further experiments.Briefy, a portion of the extract was dissolved in methanol/water using sonication and stored in a cold place [21,22].Te fltrated extract was defatted using n-hexane through the liquid-liquid extraction (LLE) method.Te remained fraction defatted was stored in the refrigerator for technical HPLC analysis.

Quantitative Reverse-phase HPLC Analysis of a Defatted
Stachys pilifera Portion.In order to calculate the amount of thymol and carvacrol as simple phenolics in SPHE, a highperformance liquid chromatography (HPLC; Model KNAUER ® ) apparatus was applied equipped with a qua- ternary K-1001 controller pumps and a K-2550 UV absorbance detector.A binary mobile phase consisting of 45% water and 55% MeCN was gone through a stainless steel analytical column (Zorbax SB-C 18 ; 250 mm × 3.9 mm i.d., 5 μm).Moreover, the fow rate and the UV-Vis light detector were set at 1.1 mL/min and 220 nm, respectively.In order to have a calibration curve, about 2 mg of standard thymol and carvacrol (each 1 mg) was dissolved in methanol (HPLC grade) to reach the fnal concentration of 2000 μg/mL.Te calibration curves for standards were prepared using concentrations of 0.5 to 2000 μg/mL [23].

Laboratory Animals.
Fifty male Wistar rats 120-180 g were purchased from Razi Institute in Shiraz.Rats were kept in an animal laboratory at a controlled temperature (22 ± 2 °C) with a 12-hour light/dark-light cycle and access to adequate food and water.All ethical issues approved by the Committee for Supervision of Laboratory Animals were followed in this work.Te code of IR.YUMS.REC.1396.124was enclosed by the ethics committee of Yasuj University of Medical Sciences.

BLM-Induced Lung
Fibrosis.Animals were sedated with intraperitoneal injections of ketamine (50 mg/kg) and xylazine (5 mg/kg).A single intratracheal instillation of BLM (7.5 mg/kg, Nippon Kayaku, Japan) was administered to anesthetized rats by placing them on a biased panel and suspending them by their upper canine teeth, followed by a single intratracheal instillation of Next (7.5 mg/kg, Nippon Kayaku, Japan).Te placebo group received the same volume of intratracheal normal saline as the BLM group.Te animals were euthanized two weeks following PLF induction [24].Group I received a single intratracheal instillation of normal saline (control group) Group II I received a single intratracheal instillation of BLM solution (7.5 IU/kg in 0.9% NaCl) (BLM-treated group) Group III was given a single intratracheal instillation of BLM solution (7.5 IU/kg in 0.9% NaCl) and a daily SPHE (300 mg/kg, gavage) for two weeks (BLM/ PPX 0.5) Group IV received a single intratracheal instillation of BLM solution (7.5 IU/kg in 0.9% NaCl) and a daily SPHE (600 mg/kg, gavage) for two weeks (BLM/PPX 1) Group V was given a single intratracheal instillation of BLM solution (7.5 IU/kg in 0.9% NaCl) and a daily Vit E (500 mg/kg, gavage) for two weeks (BLM/Vit E) Te number of animals and the dose of drugs were selected according to previous studies [20,24,25].
On the 14 days, the rats were anesthetized with diethyl ether, and blood samples were taken from their cardiac.After that, serum was isolated to assess the indicators of oxidative stress.After sacrifcing rats, lung tissue was removed, and a portion was kept in 10% formalin for histological evaluation.After homogenizing and centrifuging were used to quantity oxidative stress and hydroxyproline markers.

Evaluation of Oxidative Stress Markers
2.8.1.Determination of MDA.Serum and tissue levels of MDA were determined based on reaction with TBA.In this method, 1 mL of MDA reagent (mixture of TCA and TBA in 0.25% HCl) was mixed with 500 μL of sample in a microtube.Te microtubes were placed in Ben Marie for 95 minutes at 95 °C and then in ice for 5 minutes.After centrifugation, the adsorption of the supernatant was read at 535 nm [22,26].

Determination of Nitric
Oxide.Since nitric oxide (NO) is an unstable compound rapidly converted to nitrate and nitrite, its direct measurement is a bit difcult, so the amount of nitrite as a stable NO metabolite was measured by the grease method.In summary, 10 μL of serum or tissue homogenate with 100 μL grease reagent (1% sulfanilamide, 0.1% naphthyl ethylene amide, and hydrochloride in phosphoric acid (5.2%)) was transferred to a 96-well plate and placed in the dark for 15 minutes at room temperature.Te absorbance of the samples was then read at 541 nm [27].

Determination of Glutathione Concentration. Te
Tietze method was used to determine glutathione.In this method, an enzyme cycle in which glutathione reductase (GR) plays a signifcant role is used to quantify the glutathione present [28].

Determination of Total Tiol.
Te amount of total thiol (TSH) was determined based on the reaction of DTNB with thiol groups and the formation of TNB (2-nitro-5thiobenzoic acid) and disulfde compounds.Briefy, 10 μL of DTNB, 150 μL of tris-EDTA, and 790 μL of methanol were added to microtubes containing 25 μL of the sample.After incubation at room temperature, the samples were centrifuged, then the adsorption of the supernatant was read at 412 nm, and the amount of TSH was calculated using a molar absorption coefcient of 13,600 M −1 •cm −l [29].

Determination of Ferric Reducing Ability of Plasma.
Benzie and strain method was used to measure ferric reducing antioxidant power (FRAP) activity.In this method, the ability of plasma to reduce ferric ions is measured.Following the reduction of ferric (Fe 3+ ) ions and their conversion to ferrous (Fe 2+ ) at an acidic pH in the presence of tripyridyl-S-triazine, the Fe-TPTZ complex is formed in blue.Te resulting color intensity was measured by spectrophotometry at 593 nm [30,31].

Determination of Hydroxyproline in Lung Tissue.
Te Wesner method was used to measure hydroxyproline (HYP) levels in lung tissues [32].
2.9.Lung Histology.Lung tissue samples were kept in 10% formalin bufer.Tese samples were dehydrated with alcohol and immersed in parafn.Tissues were placed in parafn, and 5 μm sections were prepared.After staining with hematoxylin-eosin, lung tissue was examined by light microscopy.

Statistical Analysis.
Data were analyzed by SPSS software for normality and expressed as Mean ± SEM.Statistical calculations were performed for signifcant diferences between the experimental groups using analysis of variance (ANOVA) and Tukey post hoc test.P < 0.05 was considered signifcant.

Lung Tissue Oxidative Stress Markers.
Our fndings showed that the levels of MDA and NO in the BLM group increased signifcantly compared to the control group (p).However, using SPHE at doses of 300 and 600 mg/kg caused a substantial decrease in these indices.BLM administration signifcantly reduced TSH and GSH levels compared to the control group.However, in the groups receiving SPHE + BLM, these markers increased signifcantly compared to the BLM-treated group.Vitamin E treatment also had a similar efect to SPHE (Figure 2).Evidence-Based Complementary and Alternative Medicine

Serum Oxidative Stress Markers.
As shown in Figure 3, the MDA, FRAP, and NO levels in BLM rats were signifcantly increased compared to the control group.Tese markers were signifcantly reduced in the groups receiving SPHE + BLM and Vit E. Meanwhile, TSH levels in BLM + SPHE groups showed a signifcant increase compared to the BLM group.

Hydroxyproline Assay.
It was observed that BLM increased the level of hydroxyproline, which was signifcant compared to the control group (p).However, SPHE signifcantly reduced hydroxyproline when compared to the BLM group alone (Figure 4).

Histological Changes.
In the control group, the cellular structures of pulmonary bronchioles and alveoli were normal.In the bleomycin group, tissue necrosis, severe hyperplasia, and alveolar deterioration were observed compared with the control group.In the SPHE + BLM group, ductal hyperplasia and moderate alveolar destruction occurred, but no tissue necrosis, infammation, or pulmonary infltration was observed (Figure 5).

Discussion
BLM is employed as a chemotherapeutic drug for treating various cancers.It can damage DNA on both the single- Evidence-Based Complementary and Alternative Medicine strand and double-strand levels, prevent DNA synthesis, stop cancer cells from proliferating, and trigger apoptosis pulmonary fbrosis (PF) is one of the most important of its side efects.Tis compound damages lung tissue by increasing extracellular matrix remodeling and redox imbalance [34].Tese destructive changes provide a model for assessing pulmonary fbrosis in laboratory animals.Tus, it is possible to comprehend their potential therapeutic benefts and novel therapeutic approaches by investigating how various substances, including novel drugs and herbal medicines, afect this model.Reference [35].
Medicinal plants and their bioactive phenolic and favonoid compounds have gained more attention due to their benefcial properties, such as antioxidant and antiinfammatory potencies [36].
Te present study investigated the possible protective efects of SPHE against the harmful efects of BLM on pulmonary tissue.In a manner similar to IPF, BLM destroys alveolar epithelial cells and provokes a pronounced infammatory response.Reference [37].It is assumed that lung damage can be seen in this model on 7 to 14 days, which begins with oxidative and infammatory events and ends with fbrotic damage [6].
Based on our results, it was determined that BLM caused signifcant hyperplasia and alveolar damage in the animals.BLM administration dramatically elevated the concentrations of hydroxyproline (HYP), MDA, and NO, and reduced TSH and GSH levels.Oral treatment with SPHE alleviated BLM-induced pulmonary damage by restoring lung tissue architecture and normalizing the indicated oxidative stress markers.
Te infammatory mediators' cascade was activated following infammatory cell infltration in the early stages of pulmonary fbrosis.In the later stages, fbroblasts activate  Evidence-Based Complementary and Alternative Medicine and stimulate collagen secretion [34].As a result of excessive collagen deposition and remodeling processes, the lung tissue architecture is destroyed, and fbrosis results.It has been reported that approximately 13.14% of collagen fbers are hydroxyproline, so its amount in the tissue is a hallmark of the collagen level and the PF severity [32].
In accordance with Bahri et al. [35] and Huang et al. [34] studies, intratracheal administration of BLM markedly increased the levels of f HYP in lung tissue compared to the saline-treated group.Treatment with SPHE signifcantly reduced the elevation of HYP levels in the lung tissue due to BLM intoxication.
Tis activity could be attributed to phenolic and favonoid ingredients in SPHE that produce anti-infammatory and antioxidant efects [15].In this line, our previous studies have reported anti-infammatory activities and antioxidant properties of SPHE [38].Histological examination of the lung tissues exhibited that SPHE could suppress the accumulation of collagen, duct hyperplasia, alveolar degeneration, and penetration of infammatory cells due to BLM lung toxicity.One of the characteristics of cancer treatment and pulmonary fbrosis is weight loss.Reference [37].Intratracheal delivery of BLM during the study decreased animal weights and increased body lung index, which is consistent with earlier fndings.Reference [25].Tese changes may be induced by side efects of BLM on appetite and protein catabolism, consistent with the previous studies [20].
Te critical role of oxidative stress during the PF process has been demonstrated.Increased ROS and oxidant/antioxidant imbalances have been shown to play a critical role in lung tissue damage in IPF patients [39,40].Te activation of transcription factors, cytokines, and apoptosis are some ways ROS contribute to worsening IPF [1].In addition, ROS interferes with the extracellular matrix degradation process [34].Tus, targeting oxidative stress processes can be considered a therapeutic approach to managing PF [4].BLM stimulates the overproduction of ROS in animal models of PF [1].In the presence of a compromised antioxidant system, free radicals attack saturated fatty acids in the cell membrane, which leads to lipid peroxidation and the formation of MDA [41].In this context, it has been reported that various respiratory conditions increase MDA levels in the body fuids of patients.Reference [42].According to our results, MDA serum and pulmonary levels increased in the group treated with BLM.It was consistent with previous studies, which showed that MDA levels in BLM-treated animals were signifcantly higher than in saline-treated animals.Reference [40], A signifcant reduction in MDA levels was observed after SPHE treatment.
Tere is increasing evidence that NO plays an essential role in the pathogenesis of numerous lung diseases.An isoform of NO called iNOS is responsible for producing cytokines in lung diseases.Tis isoform is involved in the pathogenesis of PF by producing ROS such as peroxynitrite and nitrogen dioxide.A signifcant increase in NO levels was observed in the BLM-treated group compared with the control group, which was consistent with the results reported by Kseibati et al. [11].In the SPHE receiving groups, NO was signifcantly reduced.Tere was good agreement between this fnding about SPHE and previous studies of the plant [14,16,20].SPHE-treated groups had lower FRAP levels than BLMtreated groups, which is in line with our previous fndings.As antioxidants, the thiol groups of proteins and glutathione play a signifcant role in inhibiting ROS and RNS.Oxidative stress reduces -SH reserves by oxidizing the -SH groups into disulfde or sulfonic acid.References [43,44].Furthermore, the relationship between a decrease in GSH and an increase in ROS in the BLM-induced pulmonary fbrosis animal model has been confrmed [1].In agreement with the study of Gamed et al., the levels of GSH and TSH in the BLM group were signifcantly lower than in the control group.Reference [40].Te Results showed that GSH and TSH levels rose signifcantly in SPHE-treated groups.
However, Vit E as an antioxidant behaved similarly to SPEH in the evaluated parameters, but it did not signifcantly afect GSH levels.Polyphenols such as resveratrol, mangiferin, quercetin, and dihydroquercetin mitigate lung damage after BLM induction in animal models [31].It is also reported that quercetin and gallic acid restore lung SOD and GSH levels while decreasing the infammation factors, NO, and IL-6 levels in BLM-induced pulmonary fbrosis in rats [32].Carvacrol and thymol are two of the essential components of SPHE.Several studies have demonstrated the antioxidant efects, free radical scavenging properties, antiinfammatory properties, and benefcial properties of the compounds in various animal disease models [45][46][47].Salimoglu Sen et al. found that preadministration of carvacrol or pomegranate extract reduced MDA levels, total oxidant status, and oxidative stress index in methotrexateinduced lung injury rats.Reference [24].Hence, polyphenol ingredients of SPHE such as carvacrol and thymol may be partially responsible for the protective efect on BLMinduced fbrosis.
Future research could address two signifcant limitations of the study.First, SPHE is also examined for its efects on apoptosis and infammation in BLM-induced lung fbrosis.Te second step is to prepare and evaluate the extract's diferent fractions or principal components.

Conclusion
Te evidence from this study suggests SPHE on BLMinduced fbrosis.Histological examination of lung tissue confrmed the biochemical results.Tus, it can be postulated that a defatted SPHE involving thymol and carvacrol as the signifcant constituents may efectively reduce BLM damage due to its antioxidant efects.More research is required to determine the precise mechanisms of SPHE in managing pulmonary fbrosis.

Data Availability
Te data used to support the fndings of this study are included within the article.Te raw materials of data are available from the corresponding author upon reasonable request.

2
Evidence-Based Complementary and Alternative Medicine 2.7.Study Design.Out of ffty male Wister rats, 35 were randomly divided into the following 5 groups (n � 7):