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ORIGINAL ARTICLE
Year : 2023 | Volume
: 13
| Issue : 5 | Page : 205-213
Modulatory effect of D-pinitol on bleomycin-induced pulmonary fibrosis in rats
Yu-Ling Duan, Zhi-Hua Wang, Yan-Xia Huo, Yang Zhang, Xiao-Ran Wu, Cui-Ke Gong, Lin-Lin Bai
Department of Respiration Medicine, Xingtai People’s Hospital, Xingtai, Hebei Province, China
Correspondence Address:
Lin-Lin Bai
Department of Respiration Medicine, Xingtai People’s Hospital, Xingtai, Hebei Province
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2221-1691.377407
Objective: To assess the effect of D-pinitol on pulmonary fibrosis induced by bleomycin.
Methods: Sprague-Dawley rats received intratracheal bleomycin (6 IU/kg) to induce pulmonary fibrosis, followed by administration of either D-pinitol (5, 10, or 20 mg/kg) or vehicle or methylprednisolone (10 mg/kg) over 28 days after bleomycin administration. Lung function, biochemical parameters, serum biochemistry, mRNA expressions, and histological features were observed.
Results: D-pinitol at 10 and 20 mg/kg significantly (P<0.05) attenuated bleomycin-induced bronchoalveolar lavage fluid, decreased myeloperoxidase, nitric oxide, malondialdehyde levels, and increased glutathione and superoxide dismutase level. D-pinitol also improved lung function (enhanced pause, frequency of breathing, expired volume, and tidal volume). Besides, D-pinitol significantly (P<0.05) upregulated Nrf2 and downregulated mRNA expressions of TGF-β, collagen-1, and Smad-3. Furthermore, considerably less inflammation (peribronchial, perivascular, and total), Ashcroft, and interstitial fibrosis scores were observed in the D-pinitol group.
Conclusions: D-pinitol exerts its effect against bleomycin-induced pulmonary fibrosis via antioxidative and anti-fibrotic pathways.
Keywords: Antioxidant; Bleomycin; Collagen-1; D-pinitol; Pulmonary fibrosis; Smad-3; TGF-β
Significance:
Bleomycin is a chemotherapeutic agent, and its long-term administration causes idiopathic pulmonary fibrosis (IPF). The current work evaluated the effect of D-pinitol, a cyclic polyol, against bleomycin-induced IPF in experimental rats. It could exert its effect via antioxidant and anti-fibrotic pathways, which will deliver valuable information to researchers as a possible alternative for IPF treatment.
Idiopathic pulmonary fibrosis (IPF) is a parenchymal respiratory illness characterized by fibrosis and inflammation in the lung. It is an aggressive, chronic disease associated with an unceasing decrease in lung function, high lethality, and respiratory failure[1]. It has been estimated that around 0.09-4.51 per 100000 population suffered from IPF worldwide, with the region-wise incidence being 0.57-4.51 in Asia-Pacific, 2.40-2.98 in North America, and 2.40-2.98 in Europe[1]. The increased prevalence is associated with the increased economic burden with an estimated annual cost of $20000 (United States Dollar in 2016) for a patient with IPF in North America, which is 2-3 times the national expenditure on healthcare[2].
Interstitial pneumonia is characterized by myofibroblasts and fibroblast clusters, extracellular matrix and collagen accumulation, and heterogenous fibrosis, which result in a distorted lung architecture and possible honeycombing of lungs[3]. The specific pathogenesis of IPF is not yet completely evaluated, current hypotheses point toward an abnormal repairing process in response to various environmental and host factors[4]. Various other factors, including genetics, are important in developing and maintaining IPF. They lead to modification in the epithelium, making it more susceptible to injuries due to environmental factors[5]. The damage to the epithelium disrupts the capillary barrier of alveoli, thereby causing the proteins (such as fibrin) to leak into its spaces, abnormal vascular remodeling, and activation of the coagulation cascade[1],[5].
Furthermore, a rapid increase in free radicals occurs, which further causes the upregulation of transforming growth factor-β (TGF-β)[6].
The treatment of IPF mainly comprises immunosuppressant like azathioprine and prednisolone with the addition of N-acetylcysteine[7]. However, this therapy was documented to be linked with a higher risk of hospitalization and death[7]. As a result of these findings, new therapeutics were developed using nintedanib and pirfenidone, which have been demonstrated to ameliorate elevated forced vital capacity and halt further development of IPF in both mild and severe cases[8]. However, several adverse effects are associated with using both nintedanib and pirfenidone, including gastrointestinal adverse effects, photosensitivity rash, and increased liver enzyme levels, some of which led to therapy discontinuation[9]. In several preclinical studies, alternative medicines such as traditional Chinese medicine, Korean herbal medicine, and ayurvedic medicine are quite effective against IPF[10]. A clinical trial on IPF patients using traditional Chinese medicine showed promising results in reduced acute exacerbations, relieved IPF symptoms, and delayed pulmonary deterioration[11]. Because these medications have few to no noticeable side effects, they are good candidates as therapeutic agents for IPF[11].
Cyclitol, a cyclic polyol, is a group of naturally occurring substances in many plants, and their byproducts are largely employed as traditional medicines to combat numerous diseases such as diabetes, cancer, etc[12]. One of the cyclic polyols, Inositol, has been reported to alleviate lipopolysaccharide-induced IPF and acute respiratory distress syndrome by regulating the signaling of hypoxia-inducible factor 1-alpha[13]. Sutherlandia frutescens is a tropical plant native to regions in southern Africa and has been reported for a wide range of health benefits. D-pinitol, a cyclic polyol is found abundantly in the leaves of Sutherlandia frutescens, a tropical plant in southern Africa and the Asian region. It is used as a traditional medicine due to its anti-inflammatory, antioxidant, and immunomodulatory activity[14]. D-pinitol has previously demonstrated anti-fibrotic activity against renal tubulointerstitial fibrosis by decreasing oxidative stress via activation of nuclear factor E2-related factor 2 (Nrf2)[15]. Furthermore, D-pinitol has been shown to have a potential antitumor agent against human lung cancer cells[16]. Therefore, the current study was conducted to evaluate the potential of this cyclic polyol on IPF.
2. Materials and methods2.1. Animals
Male rats of strain Sprague-Dawley with weights ranging from 180 to 220 g were obtained from the Xingtai People’s Hospital animal house. They were maintained at (24 ± 1) °C under a relative humidity of 45%-55% with a 12:12 h dark/light cycle throughout the experimental period. Rats were provided with standard pellet chow and filtered water.
2.2. Ethical statement
The Institutional Animal Ethics Committee of Xingtai People’s Hospital approved the protocol of the experiment [approval no. LL2022 (BD15)] and all the experiments were performed as per the National Institute of Health Guide for Care and Use of Laboratory Animals.
2.3. IPF model establishment
Induction of IPF was performed according to the method reported before[17]. Bleomycin (BLM) hydrochloride (Biochem Pharmaceutical Industries Ltd., India) at a dose of 6 IU/kg (in 0.9% NaCl) was administered to overnight fasted rats. Rats were randomly assigned to the following groups (n=16):
BLM control group received distilled water (10 mg/kg); MP10 group received methylprednisolone (MP, 10 mg/kg); P5 group received D-pinitol 5 mg/kg (purity 95%, Sigma Chemical Co., St Louis, MO, USA); P10 group received D-pinitol 10 mg/kg; P20 group, received D-pinitol 20 mg/kg.
The following groups did not receive BLM:
Normal group received distilled water (10 mg/kg); sham control group received intratracheal administration of saline followed by treatment of distilled water (10 mg/kg); per se group received D-pinitol (20 mg/kg).
All treatments lasted for 28 d. The doses of D-pinitol (5 or 10 or 20 mg/kg)[18] and methylprednisolone (10 mg/kg)[6] were selected based on previous reports. D-pinitol and methylprednisolone were prepared daily as a fresh solution in distilled water.
2.4. Lung function and peripheral blood oxygen measurements
The whole-body flow-through plethysmography (EMKA Technologies, France) was used to evaluate the respiratory dynamics, whereas the peripheral pulse Ox sensor (ChoiceMMed, V1.0CF3, MD300CF3, China) was used to determine in-vivo peripheral blood oxygen content[17].
2.5. Serum and bronchoalveolar lavage fluid (BALF) biochemical analysis
At the end of the experiment (on the 28th day), rats were anesthetized with ether for blood withdrawal by using the retro-orbital puncture method to collect the blood specimen, followed by separation of serum, which was further used for the analysis of lactate dehydrogenase and alkaline phosphatase using reagent assay kits procured from Accurex Biomedical Pvt. Ltd., Mumbai, India. Simultaneously, BALF was also collected to assess the total cell counts and levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), hydroxyproline, myeloperoxidase (MPO), and total protein as per the methods reported previously[19],[20].
2.6. Lung biochemical analysis
Rats were sacrificed by the cervical dislocation method, and lungs specimens were collected to determine the SOD, GSH, MDA, NO, and total protein levels.
2.7. Reverse transcriptase (RT)-PCR
Another specimen of lungs was maintained at –70 °C to determine collagen-1, Nrf2, Smad-3, and TGF-β mRNA expressions using total RNA Extraction kit and One-step reverse transcription polymerase chain reaction kit (MP Biomedicals India Private Limited, India) as per previously described method[21],[22]. The gene expression (Supplementary Table 1) [Additional file 1] was assessed semi-quantitatively by generating densitometry data for band intensities in different sets of experiments and analyzing the gel images using the software Image J program (Version 1.33, USA). The band intensities were compared with constitutively expressed β-actin.
2.8. Histological analysis
The remaining specimen of lung tissue was subjected to histopathological analysis using picro-sirius red and hematoxylin and eosin stains as described previously[17],[23]. Sections were examined under a light microscope (Olympus DP71, DP-BSW Ver.03.03, Olympus Medical Systems India Private Limited, India) to obtain a general impression of the histopathology features of the specimen and infiltration of cells in epithelium and sub-epithelium. The intensity of histological aberrations in the nasal and spleen tissue was graded as Grade 0 (not present or very slight); Grade 1 (mild); Grade 2 (moderate); and Grade 3 (severe) as described in the literature[6].
2.9. Statistical analysis
Data analysis was conducted using GraphPad Prism 5.0 software (GraphPad, San Diego, CA) and reported as mean ± standard error mean (SEM) and a value of P<0.05 was considered statistically significant. During one way ANOVA, a post hoc analysis was conducted using Tukey’s multiple ranges (for parametric tests) and the Kruskal-Wallis test (non-parametric tests).
3. Results3.1. Effect on body weight and lung index
There was no significant difference in body weight or lung index between the per se group and the sham or the normal groups (P>0.05). BLM significantly (P<0.05) reduced body weight and increased relative lung weight compared with the sham and normal groups. D-pinitol at 10 and 20 mg/kg treatment significantly (P<0.05) ameliorated alteration in body weight and relative lung weight compared with the BLM control group. D-pinitol at 5 mg/kg did not demonstrate any significant change [Table 1].
Table 1: Effect of D-pinitol on body weight, relative lung weight, pulse oximetry, serum ALP, and LDH (n=6).3.2. Effect on percent oxygen saturation
There was no significant difference in the percentage of oxygen saturation between the per se group and the sham or the normal groups (P>0.05). BLM significantly (P<0.05) decreased the percentage of oxygen saturation compared to the sham and normal groups. Treatment with D-pinitol at 10 and 20 mg/kg significantly increased the percentage of oxygen saturation (P<0.05). D-pinitol at 5 mg/kg did not demonstrate any significant change [Table 1].
3.3. Effect on levels of serum alkaline phosphatase and lactate dehydrogenase
There was no significant difference in alkaline phosphatase or lactate dehydrogenase between the per se group and the sham or the normal groups (P>0.05). Serum alkaline phosphatase and lactate dehydrogenase were significantly (P<0.05) increased in the BLM control group. D-pinitol at 10 and 20 mg/kg significantly reduced serum alkaline phosphatase and lactate dehydrogenase (P<0.05). D-pinitol at 5 mg/kg did not demonstrate any significant change [Table 1].
3.4. Effect on lung function
There was no significant difference in lung function between the per se group and the sham or the normal groups (P>0.05). Significantly higher peak expiratory flow, frequency of breathing, enhanced pause, and significantly lower peak inspiratory flow, expired volume, and tidal volume were seen in the BLM control group compared with the sham and normal groups (P<0.05). D-pinitol at 10 and 20 mg/kg significantly decreased peak expiratory flow, breathing frequency, enhanced pause, and significantly increased peak inspiratory flow, expired volume, and tidal volume (P<0.05). D-pinitol at 5 mg/kg did not demonstrate any significant change in lung function [Table 2].
3.5. Effect on BALF differential cell count
There was no significant difference in cell count between the per se group and the sham or the normal groups (P>0.05). The BLM control group showed a significant (P<0.05) increase in cell counts (total lymphocytes, neutrophils, monocytes, and eosinophils) in the BALF compared with the sham and normal groups. D-pinitol at 10 and 20 mg/kg reduced cell counts in BALF significantly (P<0.05). D-pinitol at 5 mg/kg did not demonstrate any significant change in differential cell count [Table 3].
3.6. Effect on hydroxyproline and MPO levels in the BALF and lung
There was no significant difference in hydroxyproline or MPO between the per se group and the sham or the normal groups (P>0.05). Significantly higher hydroxyproline and MPO levels were observed in the BALF and lung of the BLM control group (P<0.05). D-pinitol at 10 and 20 mg/kg significantly decreased levels of hydroxyproline and MPO in the BALF and lung (P<0.05). D-pinitol at 5 mg/kg did not demonstrate any significant change in levels of hydroxyproline and MPO in the BALF and lung [Figure 1]A and [Figure 1]B.
Figure 1: Effect of D-pinitol on hydroxyproline (A), MPO (B), SOD (C), GSH (D), MDA (E), and NO (F) in BALF and lung (n=6). One way ANOVA (post-hoc test: Tukey’s multiple ranges) was used for data analysis. #P<0.05 compared with normal (received DW at 10 mg/kg); &P<0.05 compared with sham (received saline followed by DW); *P<0.05 compared with the BLM control group (received BLM at 6 IU/kg and DW). DW: distilled water; BLM: bleomycin; MP: methylprednisolone; P: D-pinitol; GSH: glutathione; SOD: superoxide dismutase; NO: nitric oxide; MDA: malondialdehyde; MPO: myeloperoxidase.3.7. Effect on oxido-nitrosative stress in the BALF and lung
There was no significant difference in antioxidant indices between the per se group and the sham or the normal groups (P>0.05). The BLM control group had significantly (P<0.05) higher NO and MDA levels and significantly (P<0.05) lower SOD and GSH levels in the BALF and lung compared with the sham and normal groups. D-pinitol at 10 and 20 mg/kg significantly decreased MDA and NO, and significantly increased GSH and SOD (P<0.05). D-pinitol at 5 mg/kg did not demonstrate any significant change in these levels in BALF and lung [Figure 1]C,[Figure 1]D,[Figure 1]E,[Figure 1]F.
3.8. Effect on Nrf2, collagen-1, Smad-3, and TGF-β mRNA expression in the lung
There was no significant difference between the per se group and the sham or the normal groups (P>0.05). Significantly (P<0.05) downregulated Nrf2 mRNA and upregulated collagen-1, Smad-3, and TGF-β mRNA expressions were seen in the lungs of the BLM control group compared with sham and normal groups. D-pinitol at 10 and 20 mg/kg significantly upregulated the Nrf2 mRNA and downregulated collagen-1, Smad-3, and TGF-β mRNA expression (P<0.05). D-pinitol at 5 mg/kg did not show any significant change [Figure 2].
Figure 2: Effect of D-pinitol on Nrf2, collagen-1, TGF-β, and Smad-3 mRNA expression in the lung (n=6). A: gel image; B: Nrf2; C: collagen-1; D: TGF-β; E: Smad-3. One way ANOVA (post-hoc test: Tukey’s multiple ranges) was used for data analysis. #P<0.05 compared with normal; &P<0.05 compared with sham; *P<0.05 compared with the BLM control group.3.9. Effect on lung histological aberrations
[Figure 3]A, [Figure 3]B, [Figure 3]G&[Figure 3]H depict normal lung architecture in the sham and normal groups without any evidence of inflammatory infiltration and interstitial fibrosis. BLM caused inflammatory changes reflected by distorted morphology of lung including thickened intra-alveolar septa, alveolar injury, inflammatory infiltration, and fibrotic changes [Figure 3]C&[Figure 3]I. D-pinitol at 10 and 20 mg/kg effectively reduced inflammation infiltration, lung injury and peribronchial and interstitial fibrosis [Figure 3]E-[Figure 3]F & [Figure 3]K-[Figure 3]L. The BLM control group showed significantly (P<0.05) higher inflammation (peribronchial, perivascular, and total) [Figure 4]A, Ashcroft, and interstitial fibrosis scores [Figure 4]B & [Figure 4]C than the sham and normal groups. D-pinitol at 10 and 20 mg/kg significantly (P<0.05) reduced inflammation, Ashcroft score, and interstitial fibrosis. D-pinitol at 5 mg/kg did not significantly change inflammation, Ashcroft score, and interstitial fibrosis [Figure 4].
Figure 3: Effect of D-pinitol on airway histology of rats in the lung using hematoxylin and eosin staining (A-F) depicting peribronchial inflammation (black arrow) and perivascular inflammation (red arrow) and picro-sirius red staining (G-L) depicting interstitial fibrosis (black arrow) (n=3) at 40×.
Figure 4: Effect of D-pinitol on inflammation score (A), Ashcroft score (B), and interstitial fibrosis score (C) (n=3). Non-parametric one way ANOVA (post-hoc test: Kruskal-Wallis test) was used for data analysis. #P<0.05 compared with normal; &P<0.05 compared with sham; *P<0.05 compared with the BLM control group. 4. Discussion The finding of our study suggests a potential effect of D-pinitol against pulmonary fibrosis. It is also noted that the effect of D-pinitol at 20 mg/kg on lung function, biochemical parameters, serum biochemistry, mRNA expressions, and histological features was almost comparable to the effect of methylprednisolone, which is utilized clinically for treating pulmonary fibrosis.
IPF is mainly characterized by dysregulated healing of wounds in response to micro-injuries leading to fibrogenesis[1]. Extracellular matrix accumulation in the lung is characteristic of the disease[4]. This accumulation leads to the remodeling of the lung texture and the thickening of alveolar walls, ultimately decreasing gas exchange[4].
Intratracheal administration of BLM causes breaks in single or double DNA strands, leading to apoptosis and free radical formation[24]. These cause the death of epithelial cells, fibroblast accumulation, and increased inflammatory cell infiltration, which ultimately causes fibrosis development[24] and decreased gaseous exchange; thus, it is crucial to evaluate the oxygenated level of hemoglobin[25]. Percentage of oxygen saturation is a vital marker for checking blood oxygen content, which has been reported to decrease with the administration of BLM[17] Furthermore, as a result of inflammation, lung edema occurs, leading to an increase in relative lung weight[26]. The body weight has been also reported to decrease with the administration of BLM[27]. Consistent with these studies, our study shows that BLM administration resulted in decreased body weight, percent oxygen saturation, and increased relative lung weight and cell infiltration in BALF in rats. D-pinitol treatment markedly reduced relative lung weight and cell infiltration in BALF and increased body weight and percent oxygen saturation. This result shows that D-pinitol could ameliorate BLM-induced lung inflammation and injury.
The early phase of IPF is associated with increased free radicals[24]. These free radicals upregulate TGF-β1 expression, an essential cytokine in cellular function[6],[28]. This causes an increase in fibroblasts, thereby contributing to the pathogenesis of pulmonary fibrosis[29]. A homolog, Smad-3, is responsible for inducing pulmonary fibrosis by forming a transcriptional activation complex[6]. This complex binds to TGF-β and other elements to repress peroxisome proliferator-activated receptor gamma transcription, which regulates inflammation[6],[30]. The inflammatory activity increases due to the decreased transcription of peroxisome proliferator-activated receptor gamma[6]. This complex promotes the production of collagen-1, which deposits in the interstitium leading to the decreased tensile strength of the interstitium[6],[10]. In the current investigation, the elevated collagen-1, Smad-3, and TGF-β mRNA expressions were associated with BLM administration. The decrease in these mRNA expressions caused by D-pinitol further indicates its protective effect against pulmonary fibrosis. Furthermore, D-pinitol also upregulated the mRNA expression of Nrf2, which has been evidenced in controlling inflammation and fibrosis via the suppression of various proinflammatory molecules and regulating oxidative stress via regulating detoxifying enzymes in the body[31].
During the induction and maintenance of IPF, oxidative stress plays an important role[24]. GSH and SOD are important antioxidants for scavenging the body’s free radicals[32],[33]. A study in IPF patients noted a highly decreased level of GSH[32]. A decreased level of SOD has also been noted in fibrotic areas of interstitial pneumonia lungs[33],[34],[35]. Furthermore, it was noted that IPF patients had increased levels of MPO, MDA, and NO levels, which are markers of oxidative stress[33]. Consistent with this, the current study also showed increased MPO, MDA, and NO levels and decreased SOD and GSH levels in the lung and BALF of rats treated with BLM. However, these levels were considerably normalized by the administration of D-pinitol, which further points to its effect on pulmonary fibrosis via its antioxidant effect.
Cyclic polyol demonstrated a wide array of activities[12] and previously reported amelioration of IPF by reducing sphingosine kinase 1 signaling, enhancing TGF-β/Smad, and inhibiting inflammation[36],[37],[38]. A study on the effect of quercetin in an ex-vivo setting in IPF patients concluded that patients could benefit from quercetin as it decreased inflammatory markers and increased Nrf2 activity[32]. A pilot study on stable IPF patients found that quercetin in combination with dasatinib may improve physical function in these patients[39]. In the current study, D-pinitol demonstrated potent antioxidant and anti-fibrotic activities. It is also reported that D-pinitol has potential against nephrotoxicity, renal tubulointerstitial fibrosis, and diabetic cardiomyopathy[15],[40]. D-pinitol has been reported to be a potential treatment in type-2 diabetes mellitus patients as it prevented resistance to insulin and acted as an anti-hyperglycemic agent[41].
This study also has several limitations. Firstly, the anti-inflammatory effects of D-pinitol might play a vital role in anti-fibrotic effects against BLM-induced pulmonary fibrosis. BLM-induced pulmonary fibrosis is associated with the sequence of biphasic events, the preliminary acute inflammatory response occurred within an onset of 8-9 days lasting up to 14-16 days[17]. A previous study also evaluated the anti-inflammatory potential of glycosides against BLM-induced pulmonary fibrosis, where inflammatory markers (TGF-alpha and interleukins) were assessed on day 14[17]. An additional study is warranted to evaluate the anti-inflammatory potential of D-pinitol during BLM-induced pulmonary fibrosis. Another limitation of this study is only using a BLM model. Although the BLM-induced pulmonary fibrosis model has been widely used to evaluate potential therapies, a single animal model does not recapitulate the spectrum of human pulmonary fibrosis. For clinical implication, more different animal models including silica-induced pulmonary fibrosis model should be considered.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Funding
The authors received no extramural funding for this study.
Authors’ contributions
YLD: concept, design, literature search, data analysis, manuscript preparation, and manuscript review. ZHW: concept, data acquisition, manuscript editing and manuscript review. YXH: literature search, data analysis, manuscript editing and manuscript review. YZ: concept, design, data analysis, manuscript editing and manuscript review. XRW: concept, literature search, data acquisition, manuscript preparation, and manuscript review. CKG: concept, data acquisition, manuscript preparation, and manuscript review. LLB: concept, design, literature search, data acquisition, data analysis, manuscript preparation, manuscript editing and manuscript review.
References
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