Protective Effect of Eucalyptus globulus Extracts Against Bleomycin-Induced Pulmonary Fibrosis in Rats

Abstract

Pulmonary fibrosis (PF) is a fibrous interstitial pneumonia that causes damage to the lung tissue and thus alters all respiratory functions. In this study, we aim to investigate the therapeutic effects of fresh leaves of Eucalyptus globulus extracts on bleomycin (BLM)-induced (PF). Twenty-four rats were divided into four groups. The control group received no treatment, the BLM group received only intratracheally BLM (2 mg/kg), the essential water of Eucalyptus globulus (EWEG) group underwent administration of BLM followed by E. globulus hydrosol (2000 mg/kg), and the essential oil of Eucalyptus globulus (EOCG) group received BLM followed by E. globulus essential oil (10 mg/kg). Gas chromatography–mass spectrometry analysis showed that the main compounds of EOEG and EWEG are eucalyptol and spathulenol. Obtained results showed that BLM-induced PF caused a large accumulation of lymphocytes and monocytes in lung bronchoalveolar lavage fluid, a high fibrosis score, and an inflammatory index coupled to an oxidative stress state assessed by an increase in lipid peroxidation and depletion of the activities of antioxidant enzymes: superoxide dismutase and catalase. Otherwise, the treatment with EWEG and EOEG reversed the deleterious effects of reactive oxygen species and the inflammation raised by BLM. E. globulus extracts could improve BLM-induced PF, thus suggesting that the latter could serve as a potential therapeutic approach for PF.

INTRODUCTION

Pulmonary fibrosis (PF) is a chronic inflammatory disease that manifested in patients with severe impairment of respiratory functions and a feeling of difficulty in breathing (dyspnea).^1,2 Its pathogenetic mechanism remains complicated and is explained by a cascade of reactions, mainly inflammatory and oxidative stress. This disease is experimentally established by several agents, the most frequently used of which is bleomycin (BLM). This antitumor drug, isolated from cultures of a bacterium Streptomyces verticillus,³ is used in the treatment of various cancers and lymphomas.^4
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The currently available therapies for PF are based on corticosteroid therapy (prednisone) in combination with some immunosuppressants, namely, pirfenidone and tyrosine kinase inhibitors (nintedanib and imatinib).⁷ Without denying their therapeutic efficacy, these drugs have serious side effects and their cost remains expensive.⁸ Hence, the need to search for new therapeutic avenues and to develop new potential treatments.⁹ Among these avenues, a renewed interest in natural substances that have proven their effectiveness for the treatment of various pathologies and during several periods since antiquity (Chinese medicine, Indian Âyurveda, and herbal medicine).¹⁰

One of these substances is Eucalyptus globulus, a medicinal and aromatic plant belonging to the Myrtaceae family. These leaves are very rich in essential oils (EOs) whose chemotype is eucalyptol or (1,8-cineole) known for its anti-inflammatory and antioxidant properties.¹¹ Thus, its physicochemical characteristics make this compound a good remedy against respiratory infections, pancreatitis, colitis, and for pain relief.^11,12 Aromatherapy with volatile aromatic oils and hydrosol therapy developed by Bosson and Dietz¹³ may be useful in the continuing care and management of patients with chronic diseases such as cardiovascular disease, neurological disease, and chronic respiratory diseases.

Given the fact that E. globulus has high levels of bioactive compounds with beneficial health properties against inflammation and oxidative stress,¹⁴ our study aims to evaluate the chemical composition of aromatic extracts of E. globulus as well as their protective effects against BLM-induced PF on rat.

MATERIALS AND METHODS

Extraction of EOs from the leaves of E. globulus

The fresh leaves of E. globulus were collected in February 2020 from the forest of Tabarka, North West Tunisia. The extraction of EO from the leaves is carried out by hydrodistillation using a Clevenger-type device. Plant material (350 g) is brought into direct contact with distilled water (1000 mL) in a 2 L flask. The condensed vapor obtained corresponds to an organic phase (EO) that is separated from the aqueous phase (aromatic water or hydrosol) by decantation.¹⁵ The collected EO and essential water are placed in dark glass flasks and stored at 4°C until their analysis. The chemical composition of the different phytoconstituents of E. globulus extracts was determined by the gas chromatography method coupled with mass spectrometry.

Gas chromatography–mass spectrometry analysis

E. globulus extracts were analyzed with a Hewlett-Packard 6890N/5975B inert GC-MSD system (Agilent, USA) equipped with two caps. Columns were an HP-INNOWAX (30 m × 0.25 mm i.d., film thickness 0.25 μm) and an HP5MS (30 m × 0.25 mm i.d., film thickness 0.25 μm) (J&W Scientific, USA). The oven temperature was programmed isothermal at 50°C for 1 min, then rising from 50°C to 250°C at 28/min, and finally held isothermal at 250°C for 15 min; injector temperature, 250°C; ion source temperature, 230°C; carrier gas, He (high purity 99.99%; 1.2 mL/min); injection volume, 1 μL; and split ratio, 100:1. The electron impact ionization mode was used with an ionization voltage of 70 eV. Total ion chromatograms were obtained over the scan range of 30–800 a.m.u in the full-scan acquisition mode, and the compounds were identified using the NIST05 and Wiley 7 databases with a resemblance percentage above 85%.

Semiquantitative data were calculated from the gas chromatography peak areas without using correction factors and were expressed as a relative percentage (peak area %) of the total volatile constituents identified. Retention indices were determined for all the detected compounds based on the retention times (tr) of a homologous series of n-alkanes (C8–C30).¹⁶

Animals and treatment

The study involved 24 Wistar strain rats (Rattus norvegicus), supplied by a pet store of the Higher Institute of Biotechnology of Beja, used in accordance with the Local Ethics Committee of Tunis University for the use and care of animals in accordance with the recommendations of the International Council of Laboratory Animal Science. Animals were raised under standardized conditions and in polypropylene cages. The food and water were provided ad libitum and the temperature was maintained at 20°C ± 2°C with a light–dark cycle of 12–12 h. All experiments were performed according to the recommendations of the Ethics Committee of Tunis University (University of Jendouba) for the care and use of animals in conformity with the NIH guideline (National Research Council, 1985).

The rats are weighed and randomly divided into four groups according to the type of treatment administered. The rats were divided into four groups of six animals each. The first served as a normal control group (n = 6) that received any treatment, the second group served as a positive control (BLM, n = 6) that underwent only induction of fibrosis by BLM, the third group (essential water of Eucalyptus globulus [EWEG], n = 6) underwent administration of BLM followed by treatment with E. globulus hydrosol (2000 mg/kg body weight “bw”) for 21 days, and the fourth group (essential oil of Eucalyptus globulus [EOCG], n = 6) was instilled with BLM followed by E. globulus EO treatment (10 mg/kg bw) for 21 days.

Induction of fibrosis

Animals were anesthetized by intraperitoneal injection of sodium pentobarbital 100 μg/kg (Sandoz France laboratory). Each anesthetized rat was immediately suspended from the gallows. The induction of fibrosis was made by intratracheal injection of 2 mg/kg bw of a solution of BLM sulfate (Bleomycin^®) through a sterile polyvinyl chloride catheter.^17,18

Blood, bronchoalveolar lavage fluid, and organ sampling

At the end of the experimental period, the animals were euthanized by injection of a lethal dose of sodium pentobarbital (100 mg/kg bw). The blood was taken by cardiac puncture and put into a dry tube. The blood samples were centrifuged (Universal 320/320R) for 20 min and the serum was collected in an Eppendorf tube.

Then, we proceeded to remove the block (heart and lungs) and ligated the trachea at its bifurcation. The lobes of the right lung were used for the analysis of the metabolites of the bronchoalveolar lavage fluid (Balf). The Balf samples are obtained by intratracheal injections of saline solution (4–5 mL) via a catheter and the liquid aspirated by a soft suction injected between the two fractions. The lobes of the left lung were fixed by intratracheal injection of 10% formalin solution (6–8 mL) and immersed in it for 48 h before histological examination. Homogenates were centrifuged at 3500 rpm for 10 min at 4°C (Hettich^® Universal 320/320R centrifuge, UNIVERSAL 320), and the supernatant was retained for biochemical analyses.

Bronchoalveolar lavage fluid

The analysis was carried out in the Medical Analysis laboratory of Dr. Omar Ammous, Tabarka. The Balf was centrifuged at 3000 rpm for 5 min at 4°C (Labline Brushless Digital Centrifuge). The supernatant was removed and the cell cap was resuspended with 50 μL of saline solution. We took 10 μL of cell suspension that was pipetted, and the total number of cells stained with 0.4% Trypan blue was counted with a hemocytometer. Thirty microliters was pipetted, and cell smears were prepared and stained with May-Grünwald Giemsa staining (Organic-Diff kit; BioGnost) to distinguish different types of cells under a light microscope.¹⁹

Histological analysis of PF

Tissue samples were placed in formalin, dehydrated in a graded series of ethanol, embedded in paraffin, cut into 4-mm-thick serial sections, and stained with hematoxylin and eosin (H&E) to identify inflammatory cells and Masson's trichrome to identify collagen deposition. Histological grading of fibrous lesions was performed using a blind semiquantitative scoring system adopted by Ashcroft et al. ²⁰ for the extent and severity of inflammation and fibrosis in lung parenchyma.

The severity of inflammation was categorized as one of the following: Grade 0, absence of inflammation; Grade 1, minimal inflammation; Grade 2, minimal to moderate inflammation; Grade 3, moderate inflammation with thickening of the alveolar walls; Grade 4, moderate to severe inflammation; and Grade 5, severe inflammation with the presence of follicles. The severity of interstitial fibrosis was determined using the grading system, described by Ashcroft et al. and Hubner et al. ^20,21 The entire lung section was observed at × 100 magnification, and a score ranging between 0 (normal) and 8 (total fibrosis) was assigned.

The categories of PF were as follows: Grade 0, normal lung; Grade 1, minimal fibrous thickening of alveolar or bronchial walls; Grades 2–3, moderate thickening of walls without obvious damage to lung architecture; Grades 4–5, increased fibrosis with definite damage to lung architecture and the formation of fibrous bands or small fibrous masses; Grades 6–7, severe distortion of the structure and large fibrous areas, “honeycomb lung” was placed in this category; and Grade 8, total fibrotic obliteration of the field.

Protein determination

The protein concentration was determined by the method of Hartree, based on the ability of the protein–copper complex to reduce the Folin–Ciocalteu reagent inducing a blue coloration measured at 650 nm. After plotting the calibration curve (optical density [OD] as a function of the amount of protein in μg), the protein concentration of the samples is determined by reporting the ODs on the calibration curve produced using bovine serum albumin as a standard.²²

Determination of lipid peroxidation and antioxidant enzyme activity measurements

Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) using the double heating method.²³ Briefly, aliquots of lung tissue homogenates were mixed with a solution of BHT-trichloroacetic acid (TCA) containing 1% butylated hydroxytoluene (BHT) (w/w) dissolved in 20% (w/w) TCA and centrifuged at 1000 g for 5 min at 4°C. The supernatant was mixed with a solution containing 0.5 N HCl, 120 mM thiobarbituric acid buffer, and buffered in 26 mM Tris, and then heated at 80°C for 10 min. After cooling the samples, the OD is read with a spectrophotometer at 535 nm. The absorbance is directly proportional to the amount of MDA formed, thus giving an accurate assessment of membrane lipoperoxidation.

The colorimetric method has been used to measure superoxide dismutase (SOD) activity in lung tissue indirectly, using the epinephrine/adrenochrome system.²⁴ At basic pH, the superoxide anion induces the autoxidation of epinephrine to adrenochrome. In this reaction, SOD competes by inducing the formation of H₂O₂ from the superoxide anion, and catalase (CAT) catalyzes the formation of water from H₂O₂ to prevent the reformation of the superoxide anion. One unit of SOD is defined as the amount of extract that inhibits the rate of adrenochrome formation by 50%. The enzyme extract was added to 2 mL of reaction mixture containing 10 μL of bovine CAT (0.4 U/mL), 20 μL of epinephrine (5 mg/mL), and 62.5 mM of sodium carbonate/bicarbonate buffer (pH 10.2). Changes in absorbance were evaluated at 480 nm.

The activity of CAT was recorded by measuring the rate of disappearance of H₂O₂ by spectrophotometry at 240 nm, which will be degraded into H₂O and O₂.²⁵ The reaction mixture contained 33 mM H₂O₂ in 50 mM phosphate buffer (pH 7). Thus, the gradual decrease in OD corresponds to the decomposition of H₂O₂ by CAT. The OD reading time is fixed between 15 and 45 sec. The activity of CAT is expressed in μmoles of H₂O₂ per milligram of protein.

Determination of functional and metabolic parameters

The concentration of plasma glucose is determined by an enzymatic colorimetric test at 505 nm using a commercial assay kit (Biomaghreb, Tunisia).²⁶

The activities of aspartate aminotransferase (ASAT), as well as alkaline phosphatase or phosphatase alkaline and uric acid (U.Ac), were determined according to commercial kit methods supplied by Biomaghreb. These biomarkers were measured in frozen aliquots of serum by a biochemistry analyzer (Architect arc 8000i) in the biochemical analysis laboratory of Dr. AMMOUS, Tabarka, Tunisia.^27
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Statistical analysis

The experimental results are presented as mean ± standard deviation from the standard error of the mean. Data were analyzed using statistical software IBM SPSS 25.0. The difference is considered significant when the P value <.05.

RESULTS

Chemical composition of E. globulus extracts

Assessment of the content and chemical composition of E. globulus extracts was performed using gas chromatography-mass spectrometry (GC-MS) (Fig. 1). The main molecules detected in the EO and hydrosol are identified, and their molecular weight, the molecular formula, the retention time, and the surface percentages are indicated in Tables 1 and 2.

FIG. 1.

Gas chromatography–mass spectroscopy analysis of essential oil (A) and hydrosol (B) of Eucalyptus globulus.

Table 1.

Chemical Composition of Essential Oil of Eucalyptus globulus Leaves by Gas Chromatography–Mass Spectrometry Analysis

Peak	Compounds	Molecular weight	Molecular formula	Retention time	% Area
1	β-Cymene	134	C₁₀H₁₄	7.726	7.56
1	ο-Cymene	134	C₁₀H₁₄
2	(E)- β-Ocimene	136	C₁₀H₁₆	7.820	4.36
	Allo-ocimene	136	C₁₀H₁₆
	Sabinene	136	C₁₀H₁₆
3	Eucalyptol	154	C₁₀H₁₈O	7.883	34.47
4	Terpinen-4-ol	154	C₁₀H₁₈O	10.756	1.80
5	Crypton	138	C₉H₁₄O	10.939	7.88
5	3,5-Dimethylpyrazole	96	C₅H₈N₂
6	α-Terpineol	154	C₁₀H₁₈O	11.002	3.48
7	Benzenepropanal	134	C₉H₁₀O	11.914	1.68
	3-Phenylbutanal	148	C₁₀H₁₂O
	p-Acetylethylbenzene	148	C₁₀H₁₂O
8	Phellandral	152	C₁₀H₁₆O	12.559	1.08
8	Trans-3-Caren-2-ol	152	C₁₀H₁₆O
9	Carvacrol	150	C₁₀H₁₄O	12.973	1.23
	Thymol	150	C₁₀H₁₄O
10	Spathulenol	220	C₁₅H₂₄O	17.570	25.86
11	4, 4-Dimethyl-3-(3 methylbut-3-enylidene)-2-methylenebicyclo [4.1.0] heptanes	202	C₁₅H₂₂	17.649	1.54
12	Not identified			19.830	4.25
13	3-Carene	136	C₁₀H₁₆	20.129	4.82
13	Longipinocarvone	218	C₁₅H₂₂O
Total identified		100.01
Monoterpene hydrocarbons		(06)
Oxygenated monoterpenes		(10)
Sesquiterpene hydrocarbons		(01)
Oxygenated sesquiterpenes		(02)
Other aromatic compounds		(02)

The values in bold correspond to the majority molecules (with the highest percentages) in the extracts of Eucalyptus globelus.

Table 2.

Chemical Composition of Essential Water of Eucalyptus globulus Leaves by Gas Chromatography–Mass Spectrometry Analysis

Peak	Compounds	Molecular weight	Molecular formula	Retention time	% Area
1	ρ-Cymene	132	C₁₀H₁₂	7.731	17.10
1	β-Cymene	134	C₁₀H₁₄
2	β-Thujene	136	C₁₀H₁₆	7.826	7.02
	β-Phellandrene	136	C₁₀H₁₆
	Sabinene	136	C₁₀H₁₆
3	Terpinen-4-ol	154	C₁₀H₁₈O	10.761	6.38
4	Crypton	138	C₉H₁₄O	10.950	14.72
4	Furfural	96	C₅H₄O₂
5	α-Terpineol	154	C₁₀H₁₈O	11.007	5.41
	4-Carene	136	C₁₀H₁₆
	α-Terpinyl acetate	196	C₁₂H₂₀O₂
6	Propanal, 2-methyl-3-phenyl	148	C₁₀H₁₂O	11.925	7.22
6	Cuminaldehyde	148	C₁₀H₁₂O
7	Phellandral	152	C₁₀H₁₆O	12.570	7.40
7	2,5-Dimethylhex-5-en-3-yn-2-ol	124	C₈H₁₂O
8	Spathulenol	220	C₁₅H₂₄O	17.570	34.73
Total identified		99.98
Monoterpene hydrocarbons		(6)
Oxygenated monoterpenes		(7)
Sesquiterpene hydrocarbons		(0)
Oxygenated sesquiterpenes		(1)
Other aromatic compounds		(2)

The values in bold correspond to the majority molecules (with the highest percentages) in the extracts of Eucalyptus globelus.

The chemical analysis of the EO of fresh leaves of E. globulus by hydrodistillation revealed the presence of 21 constituents. The principal phytoconstituents identified are as follows: eucalyptol (34.47%), spathulenol (25.86%), crypton, 3,5-dimethylpyrazole (7.88%), and β-Cymene and o-Cymene (7.56%) (Fig. 1A and Table 1).

Concerning the hydrosol, the same analysis technique (gas chromatography–mass spectrometry [GC-MS]) allowed the identification of 16 compounds, the main ones of which are as follows: spathulenol (34.73%), p-Cymene and β-Cymene (17.10%), and crypton and furfural (14.72%) (Fig. 1B and Table 2).

Effect of aromatic compounds on BLM-induced PF

Mortality and morbidity

No rat succumbed to mortality during the experiment, or before and after the induction of fibrosis by BLM. The behavior of the rats was characterized by a return to normal in terms of food consumption and water intake. Besides, the gavage treatment showed no observable adverse side effects.

Body weight gain

All rats in the experimental groups were weighed every 7 days to follow the evolution of their body mass during treatment. The obtained results showed that the injection of BLM (2 mg/kg) induced fibrosis characterized by severe loss of body weight compared with the control group. In the treated groups, the administration of EOEG (10 mg/kg) and EWEG (2000 mg/kg) for 3 weeks decreased relatively this weight loss (Fig. 2).

FIG. 2.

Influence of the administration of Eucalyptus globulus extracts on body weight monitoring (n = 6, X ± S); P .05 versus BLM. BLM, bleomycin.

Histological analysis

The evolution of histological alterations in rat lungs after BLM instillation was determined by the H&E staining. The results under light microscope showed that the alveolar structure of rat in the BLM group was severely damaged, and the alveolar septum was significantly thickened with a large number of inflammatory cell infiltration and fibroblast proliferation compared with the control group (Fig. 3). The index of inflammation was significantly higher than the control group (Fig. 3E). On the contrary, the hydrosol treatment did not show any clear improvement over the BLM group, and its effects were observed locally in some areas of the lungs and not in others. Contrary, daily force-feeding with EOEG (10 mg/kg bw) showed an effectiveness of this extract for the protection of the lung parenchyma against the inflammation caused by BLM (P < .05).

FIG. 3.

Effect of Eucalyptus globulus on histological alterations in BLM-induced PF in the rat. The representative pictures, the paraffin sections were stained with hematoxylin and eosin: The BLM group is characterized by a significant peribronchial inflammation intra- and interalveolar (stars), a disruption of the alveolar architecture with alteration of epithelial coating coupled with the presence of plasma cells, lymphocytes, and macrophages (magnification, 100 × ; scale bar, 10 μm). (A) Control group; (B) BLM group; (C) EOEG (10 mg/kg); and (D) EWEG 2000 mg/kg; (E) Effect of E. globulus on the degree of inflammation of BLM-induced PF in rats (n = 6, X ± S); *P .05 versus control, ^# P .05 versus BLM and control groups. EOEG, essential oil of Eucalyptus globulus; EWEG, essential water of Eucalyptus globulus; PF, pulmonary fibrosis.

As shown in Figure 4, intratracheal instillation of BLM induced a significant increase of fibrosis score deduced from the high collagen accumulation in the perialveolar tissues observed by Masson's trichrome-stained sections. Compared with the control group, the alveolar structure of the BLM group was seriously damaged, and there was a marked increase of collagen fibers especially in the pulmonary interstitial septum (Fig. 4). Confirming the results of the H&E staining, the E. globulus extracts, especially EOEG treatment, showed their antifibrosis effects in reducing the accumulation of collagen in the various lung tissues.

FIG. 4.

Effect of Eucalyptus globulus on histological alterations in BLM-induced PF in the rat. The representative pictures, the paraffin sections were stained with Masson's trichrome: The BLM group is marked by significant accumulation of collagen, alveolar edema, or small fibrosing masses disrupting the structure of the extracellular matrix, especially at the level of the inflammatory infiltrate (stars) (magnification, 100 × ; scale bar, 10 μm). (A) Control group; (B) BLM group; (C) EOEG (10 mg/kg); (D) EWEG 2000 mg/kg; (E) effect of E. globulus on the degree of fibrosis of BLM-induced PF in rats (n = 6, X ± S); *P .05 versus control, ^# P .05 versus BLM and control groups.

Estimation of cell viability in Balf

The administration of BLM induced a significant increase in the markers of lung injury measured in the Balf, including total and individual lymphocyte cell count, in addition to a significant decrease in relative lung function (Fig. 5).

FIG. 5.

Counts of the total and the individual number of immune cells in broncho alveolar lavage fluid (n = 6, X ± S); *P .05 versus control, ^# P .05 versus BLM.

Our results showed that the total number of cells in the BLM control group was dramatically increased compared with that of the control group. In contrast, the EOEG-and EWEG-treated groups presented a significant decrease in total cells, monocytes, and lymphocytes compared with the BLM group, which suggested that eucalyptus extracts could effectively lower the total cell count of Balf.

Determination of functional and metabolic parameters

PF induced by BLM (2 mg/kg) caused impairment of biochemical parameters (Table 3). The results showed a significant increase (P < .05) in all the measured metabolic parameters.

Table 3.

Effects of Eucalyptus globulus Extracts on Biochemical Parameters After Bleomycin-Induced Pulmonary Fibrosis

Group	Control	Bleomycin	EWEG	EOEG
Glucose (g/L)	1.06 ± 0.03^b	1.29 ± 0.04^a	1.11 ± 0.05^b	1.23 ± 0.07^a
Uric acid (mg/L)	45.9 ± 2.57^c	58.3 ± 0.47^a	44.9 ± 3.32^c	50.1 ± 1.11^b
Aspartate aminotransferase (U/L)	25.9 ± 2.72^c	37.0 ± 1.77^a	25.3 ± 2.05^c	30.3 ± 3.15^b
Alkaline phosphatase (U/L)	70.1 ± 6.9^b	128 ± 4.78^a	71.9 ± 6.44^b	115 ± 5.78^a

For each parameter, different superscript letters indicate significant differences (P ≤ .05).

Results from EOEG treatment showed that glucose concentrations remained elevated (1.23 ± 0.07 g/L) compared with the control group (1.06 ± 0.03 g/L), while treatment with EWEG brought these concentrations back to baseline levels (1.11 ± 0.05 g/L).

In addition, the uric acid level slightly decreased in the EOEG-treated rats (50.1 ± 1.11 mg/L) besides the control group (45.9 ± 2.57 mg/L). Subacute treatment with EWEG significantly corrects the disturbance of this parameter (44.9 ± 3.32 mg/L).

On the contrary, the concentration of ASAT decreased slightly in the rats treated with EOEG by 30.3 ± 3.15 U/L compared with the control group (25.9 ± 2.72 U/L), while in the EWEG-treated rats, the level of this parameter decreased significantly (25.3 ± 2.05 U/L).

The induction of PF significantly increased the level of alkaline phosphatase compared with the control group (128 ± 4.78 U/L vs. 70.1 ± 6.9 U/L), which is a specific biomarker of inflammation. The results of treatment with EOEG showed a slight decrease in this parameter (115 ± 5.78 U/L), whereas treatment with EWEG brings these variations back to values close to normal (71.9 ± 6.44 U/L).

Effect of E. globulus extracts on BLM-induced lung lipoperoxidation

The BLM intratracheal instillation (2 mg/kg, body weight) causes an increase in the level of MDA in the pulmonary interstitium. However, the treatment with EWEG and EOEG significantly corrected lipid peroxidation (Fig. 6).

FIG. 6.

Effect of EOEG and EWEG extracts on the level of malondialdehyde, after BLM induction (2 mg/kg, body weight) (n = 6, X ± S); *P .05 versus control, ^# P .05 versus BLM.

Effect of E. globulus extracts on BLM-induced lung antioxidant enzyme activity depletion

The harmful effects of BLM are demonstrated in our results by the significant depletion of the activity of antioxidant enzymes (SOD and CAT). The EOEG and EWEG treatments significantly reversed all BLM-induced antioxidant enzyme depletion (Fig. 7).

FIG. 7.

Effect of the treatment with EOEG and EWEG on lipid peroxidation induced by BLM, the activity of antioxidant enzymes such as superoxide dismutase (A) and catalase (B) in the lungs (n = 6, X ± S); *P .05 versus control, ^# P .05 versus BLM.

DISCUSSION

The aim of the present study was to investigate the effects of E. globulus extracts in the treatment of PF induced by BLM as well as the implication of oxidative stress in such protection.

Our data were first started with a chemical composition analysis of E. globulus EO and hydrosol, done by GC-SM. It showed a high content of chemotypes, namely, eucalyptol (34.47%) and spathulenol (25.86%) that represent the major elements in EOs of E. globulus. Eucalyptol or 1,8-cineolem is a monoterpene oxide,¹² and spathulenol is an alcohol derived from all-aromadendrene.³⁰ On the contrary, spathulenol (34.73%) is the major compound in the phenolic profile of hydrosol. These results agree with those obtained by Sebei et al., who confirmed that despite the environmental, geographical, climatic, and genetic variations of the different Eucalyptus species in Tunisia, the major active compound of the leaves remains eucalyptol.³¹

Evaluation of fibrotic variations in lung tissue was performed using H&E and trichrome histological stains. The obtained results showed that BLM instillation raised a high inflammation and accumulations of collagen deposition especially in the extracellular matrix, proven by the increased inflammatory index and fibrosis score. Under the effect of treatment with EOEG, the degree of alveolar inflammation and the histological disorganization were significantly reduced. On the contrary, the treatment with hydrosol did not show any improvement, which could be explained by the absence of significant bioactive substances, more specifically eucalyptol.

Consistent with the pathological findings, induction of PF by BLM resulted in a significant elevation of inflammatory cells measured in Balf, and subsequently, an accumulation of lymphocytes and monocytes to high levels in the lungs. Treatments with EOEG and EWEG resulted in a significant decrease in the total and individual number of these immune cells, suggesting an involvement of eucalyptus extracts in the braking of inflammatory reactions and keeping the number of immune agents close to their normal value.

The adverse consequences of BLM in the context of increased inflammation are not limited to the lungs, the liver and kidneys too have been affected by this anticancer agent. This was confirmed by an abnormal increase in the concentrations of various elements of the biochemical balance such as glucose, uric acid, ASAT, and alkaline phosphatase. These imbalances of parameters and enzymes in the blood prove the existence of renal, especially hepatic, suffering, linked to inflammation.³² More importantly, the EWEG significantly corrected the increased rates of these parameters, and the percentage of protection is significantly higher than the EOEG-treated group.

Oxidative stress is one of the potential factors involved in the onset of PF. In this context, we referred to the enzymatic assay to confirm it, and thus to deduce the effectiveness of eucalyptus extracts against BLM-induced PF. As previously confirmed by Abidi et al., BLM caused the onset of a state of oxidative stress manifested by an increase in the level of MDA, a reflection of lipid peroxidation,¹⁸ which indirectly reflects the degree of damage to the cell membrane.³³ Treatment with EWEG and EOEG significantly corrected the increased rates of this stress parameter in the lung tissues.

It is well known that the oxidant–antioxidant system has been studied by analyzing the activity of enzymes, which are considered to be our body's first line of defense against reactive oxygen species. SOD is an enzyme that largely maintains the dynamic balance of free radical generation, and cleansing and catalyzing the dismutation of O₂ ⁻ into H₂O₂ and O₂.³⁴ On the contrary, CAT is a hemoprotein that catalyzes the reduction of hydrogen peroxides and protects tissues from hydroxyl radicals that are highly reactive.³⁵ In the same respect, BLM induced a significant decrease in the activity of antioxidant enzymes such as SOD and CAT in the lungs. Treatment with EWEG significantly corrected the depletion of these two markers. The protection offered in rats treated with EOEG is less demanding than those that received hydrosol.

In this round of oxidative stress, this study showed that the EO of eucalyptus could considerably reduce the content of MDA in the lung tissue of BLM group, and considerably increase the activity of SOD and CAT in lung tissues of the latter. These antioxidant effects, thus reducing lung tissue damage caused by lipid peroxidation, are probably due to the major components of E. globulus extracts (eucalyptol and spathulenol) known for their antioxidant, anti-inflammatory, antiproliferative, antimicrobial, and immunomodulatory effects.^11,36,37

In conclusion, our study revealed a beneficial and immediate action of E. globulus extracts just after the induction of PF by BLM. This remedy can be a promising source of bioactive compounds that may play an essential role in the treatment of PF while acting on the various hypothetical pathways involved in the development of this disease characterized by its physiopathology, which remains poorly understood. In fact, our results are encouraging and pave the way for other future therapeutic trials. Hence, the need for a more in-depth study on the mechanism of action of eucalyptol and spathulenol, which have interesting health benefits on many other chronic diseases.

Footnotes

ACKNOWLEDGMENTS

The authors are grateful to all the persons who helped to conduct this study especially the staff of the Sylvo-pastoral Resource Laboratory at the Sylvo-pastoral Institute of Tabarka, the laboratory of Dr. Omar AMMOUS (Tabarka), and Afifa ABDELLAOUI, technician in the Laboratory of Human and Experimental Pathology at the Pasteur Institute of Tunis, having helped enormously during all the analyses carried out.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

FUNDING INFORMATION

No funding was received for this article.

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