Prevention of Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice by Bilobalide

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Bilobalide (BB) is a sesquiterpene isolated from Ginkgo biloba, and its role in IPF is poorly understood. Mice were intratracheally instilled with 2.5 mg/kg bleomycin (BLM) to induce IPF and then treated with 2.5, 5, and 10 mg/kg BB daily for 21 days. Treatment with BB ameliorated pathological injury and fibrosis of lung tissues in BLM-induced mice. BB suppressed BLM-induced inflammatory response in mice as demonstrated by reduced inflammatory cells counts (leukocytes, neutrophils, macrophages, and lymphocytes) and pro-inflammatory factors (CCL2 and TNF-α), as well as increased CXCL10 levels in BALF. The expression of BLM-induced hydroxyproline, LDH, and pro-fibrotic mediators including fibronectin, collagen I, α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-2, and MMP-9 in lung tissue was inhibited by BB treatment, and the tissue inhibitor of metalloproteinase-1 (TIMP-1) expression was increased. BB blocked the phosphorylation of JNK and NF-κB, and the nuclear translocation of NF-κB in the lung tissue of mice induced by BLM. Additionally, it abated the activation of NLRP3 inflammasome in lung tissue induced by BLM, which led to the downregulation of IL-18 and IL-1β in BALF. Our present study suggested that BB might ameliorate BLM-induced pulmonary fibrosis by inhibiting the early inflammatory response, which is probably via the inhibition of the JNK/NF-κB/NLRP3 signal pathway. Thus, BB might serve as a therapeutic potential agent for pulmonary inflammation and fibrosis.


Introduction
Idiopathic pulmonary fbrosis (IPF) is a common chronic progressive fatal lung disease characterized by alveolar epithelial cell damage, chronic infammation, and abnormal deposition of extracellular matrix (ECM), leading to progressive decline in lung function [1,2]. It was reported that there were 20 new cases per 100,000 people per year [3], and it usually has a short median survival of about 3-5 years from the time of diagnosis [4]. Although IPF afects millions of patients worldwide and causes scarring of the lungs, its etiology remains unclear [5]. In the past few years, several factors have been suggested to be involved in the pathogenesis of IPF, including infammation, epithelialmesenchymal transition (EMT), imbalance of ECM degradation and collagen deposition, and oxidative stress [6]. Oxidative stress and infammatory response may be two important factors that trigger the disease and allow its progression [7,8]. At present, treatment options for IPF are quite limited. Clinically, pirfenidone and nintedanib are the frst-line agents for the treatment of mild and/or moderate IPF, but their prognostic results are not satisfactory [9,10]. Tus, exploring efective antioxidant or anti-infammatory drugs is of great signifcance for the treatment of IPF.
Ginkgo biloba is an ancient Chinese tree that has been used to treat a variety of ailments. Studies have shown that it has benefcial efects on a variety of pathological conditions, including hepatoprotection, photoprotective efects, DNA repair mechanisms, antioxidant, and anti-infammatory activities [11]. Standardized ginkgo extract EGb761 is a well-known Ginkgo biloba extract that dates back over a century. It contains two main groups of bioactive components: 24% favonol glycosides and 6% terpene trilactones [12]. Bilobalide (BB) is one of the main components of terpene trilactone [13], approximately accounting for 2.9-3.2% of EGb76 [14]. Currently, BB has been used as a phytopharmaceutical or food supplement in numerous countries [15]. Considerable evidence has been suggested that BB has a wide range of therapeutic applications for neurological and vascular injuries [16]. BB could activate Akt/Nrf2 signal pathway to reduce oxidative stress, thereby alleviating cerebral ischemia injury [17]. Additionally, BB has been proven to have an anti-infammation efect. Goldie and Dolan [18] reported that BB could alleviate carrageenaninduced infammation and infammatory pain in rats by inhibiting infammatory factor release. BB also inhibited the expression of interleukin (IL)-17-induced infammatory factors and matrix metalloproteinases (MMPs) and alleviated infammatory damage in ATDC5 cells [19]. However, an extensive literature and the patent search revealed a lack of data on the efects of BB on IPF.
Based on the infammatory characteristics of IPF and the anti-infammation properties of BB, we speculated that BB could alleviate IPF by relieving infammatory response. Bleomycin (BLM) is an antibiotic used in the treatment of tumors. In clinical situations, the use of BLM was often associated with pulmonary toxicity [20]. BLM-induced IPF is the most commonly used animal model [21]. In our study, the efect and mechanism of BB on IPF were investigated in the BLM-induced mice model. Histopathological changes were determined by routine staining. Pathological indexes and markers of infammation and fbrosis were selected for analysis. In addition, the tentative mechanism by which BB alleviated IPF was discussed.

Material and Method
2.1. Animals. Male C57BL/6 mice were purchased from Hua Fukang Biological Technology Co., Ltd. (Beijing, China). Te animals were maintained under controlled temperature (22 ± 1°C) and humidity (50 ± 5%) laboratory condition, and all mice were acclimatized to the environment for 7 days. Ethical approval from the Animal Experimental Ethics Committee of the Afliated Hospital of Shandong University of Traditional Chinese Medicine was obtained for using mice (NO. 2021-44).
Mice were arbitrarily allocated into fve groups: control, BLM, BLM + 2.5 mg/kg BB, BLM + 5 mg/kg BB, and BLM + 10 mg/kg BB group (12 animals in each group). Te BLM group mice were challenged with BLM (2.5 mg/kg) (Dalian Meilun Biotechnology Co., Ltd., Dalian, China) by intratracheal injection to induce IPF. Mice in the shamoperated group were injected intratracheally with equal amounts of sterile saline. After 2 hours, mice in the BBtreated group were daily received diferent doses of BB (Aladdin regents Co. Ltd., Shanghai, China) by intraperitoneal injection for 21 days. BB was formulated in 20% PEG400 dissolved in saline. Control mice received the equivalent volume of the corresponding vehicle. Te mice in each group were anesthetized, and the bronchoalveolar lavage fuid (BALF) was collected. Ten, the mice were euthanized, and the lung tissues were obtained, photographed, and weighed.
Masson trichrome staining was used for the detection of IPF. Te 5 μm lung slides were stained with Regaud's hematoxylin solution for 6 min. Following rinsed thoroughly with water, sections were dipped in acid fuchsin solution (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 1 min, 1% phosphomolybdic acid solution (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 5 min, and aniline blue solution for 5 min. Ten, sections were immersed in 0.2% aqueous glacial acetic acid solution, dehydrated with gradient alcohol, clarifed with xylene, and closed with neutral resin. Finally, the tissue sections were observed under a microscope. Te severity of alveolitis and fbrosis was scored using the Szapiel scoring system [20].

BALF Cell
Count. BALF cells were resuspended in phosphate-bufered saline (PBS) solution, and leukocytes in BALF were quantifed by blood cell count. Subsequently, cell smears were fxed in methanol for 15 min and stained with Giemsa staining solution (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Diferential cell counts were analyzed under a light microscope. Group, Inc., Wuhan, China). Information for secondary antibodies was as follows: goat anti-rabbit horseradish peroxidase (HRP) and goat anti-mouse HRP (Beyotime Biotechnology, Shanghai, China).

Immunofuorescence (IF) and Immunohistochemistry
(IHC) Analysis. IF and IHC were carried out on parafnembedded samples after dewaxing and rehydration. For IF staining, the lung sections were incubated with primary antibody against NOD-like receptor family pyrin domain containing 3 (NLRP3) (ABclonal Biotechnology, Wuhan, China) at a dilution of 1 :100 at 4°C overnight and secondary antibody against Cy3-conjugated goat-anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) at a dilution of 1 : 200 at room temperature for an hour. 4′,6-diamidino-2-phenylindole (DAPI) (Aladdin regents Co. Ltd., Shanghai, China) was used for for nuclear labeling. Te sections were examined under a microscope.

Lactate Dehydrogenase (LDH) and Hydroxyproline
Content Assay. LDH and hydroxyproline levels in BALF or lung tissues were detected using an LDH assay kit or hydroxyproline assay kit (Jiancheng Bioengineering Institute, Nanjing, China).

Statistical
Analysis. Te data were analyzed using Graphpad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). All data are presented as mean ± standard deviation (SD). Signifcance was measured with a one-way analysis of variance (ANOVA) for multiple comparisons. P-value less than 0.05 was regarded as indicating statistical signifcance.

Bilobalide Attenuated BLM-Induced Pulmonary Fibrosis in Mice.
In BLM-induced IPF mice, there were signs of structural damage of the lung tissue and ameliorated by BB treatment (Figure 1(b)). Meanwhile, BB treatment obviously attenuated BLM-induced lung index increase (Figure 1(c)). Furthermore, H&E and Masson staining were performed to confrm the alleviation of BB for BLM-induced IPF. H&E staining showed the presence of interstitial septal thickening, infammatory infltration, fbrotic nodules, and alveolar structural changes after BLM administration (Figure 1(d)). Masson staining demonstrated the signifcant collagen fbrosis in the BLM group compared with the control group (Figure 1(f )). In addition, scores of alveolitis and lung fbrosis were signifcantly higher than that of the control group (Figures 1(e) and 1(g)). Remarkably, these phenomena were alleviated by BB treatment (Figures 1(d)-1(g)).
Hydroxyproline content is an important marker refecting the degree of collagen tissue metabolism and IPF [22]. Te high levels of hydroxyproline caused by BLM were significantly reversed by BB administration (Figure 1(h)). Overall, the above results suggested that BB could alleviate BLMinduced IPF.

Bilobalide Down-Regulated the Fibrosis Factor Expression in BLM-Induced
Mice. Subsequently, we measured the protein levels of fbronectin, α-SMA and collagen I, biomarkers of fbrosis, with Western blot. Figure 2(a) shows that those protein levels were enhanced in the BLM group. In contrast, these trends were reversed by BB (Figure 2(a)). At the same time, the gene expression of MMP-2 and MMP-9 was signifcantly decreased by BB administration and accompanied by increased TIMP-1 expression (Figures 2(b)-2(d)). Te expression of pro-fbrotic protein TGF-β1 followed the same trend as MMPs (Figure 2(e)).

Bilobalide Mediated Antipulmonary Fibrosis Response by
Inhibiting Infammation. In this model, the antiinfammation efect of BB was tested. Compared with untreated mice, leukocyte, neutrophil, lymphocyte, and macrophage counts in BALF were signifcantly increased in BLM-treated mice (Figures 3(a)-3(d)), whereas BB administration markedly decreased these cell counts    Evidence-Based Complementary and Alternative Medicine Furthermore, treatment with BB induced a dose-dependent increase in CXCL10 (Figure 3(e)).

Bilobalide Inhibited the Activation of NLRP3 Infammasome in BLM-Induced Mice.
To further explore the efect of BB on infammation, we also examined infammationrelated signal pathways. Data revealed that the protein levels of NLRP3, ASC, and cleaved-caspase-1 in mice exposed to BLM treatment exhibited an obvious elevation (Figure 4(a)). BB therapy remarkably attenuated these protein levels (Figure 4(a)). Indeed, comparable results of NLRP3 were obtained by immunofuorescence (Figure 4(b)). BB treatment signifcantly decreased the levels of IL-18, IL-1β, and LDH in BLM-induced IPF (Figures 4(c)-4(e)).

Bilobalide Suppressed BLM-Induced Infammation via JNK/NF-κB Pathway.
A previous study has suggested that JNK/NF-κB might involve in the activation of NLRP3 infammasome. [23]. As results show, BB signifcantly inhibited the BLM-induced increase in not only JNK phosphorylation ( Figure 5(a)) but also p65 phosphorylation ( Figure 5(b)). Meanwhile, the nuclear translocation of p65 was markedly reduced by BB administration (Figure 5(c)).

Discussion
IPF is a progressive disease with a poor prognosis and limited treatment options. Te initial stage of IPF pathogenesis is dominated by alveolar infammation and massive infammatory cell infltration, followed by massive fbroblast proliferation and collagen accumulation in the middle and/ or late stages, and fnally developed into irreversible interstitial lung fbrosis [24]. Research reported that BLMinduced IPF usually was accompanied by pulmonary oedema resulting in increased lung wet weight [25], which was consistent with our results, while BB could relieve this. BBinduced reduction in lung index showed potential for lung tissue protection. Wet lung weight is used as an indicator of pulmonary infammation and fbrosis [26]. Our HE and Masson trichrome staining results showed that BLM-treated mice exhibited signifcant lung histopathological damage as well as an increased degree of infammatory infltration and lung fbrosis, indicating successful modeling of IPF. In addition to histopathology, hydroxyproline measurement could also refect the status of IPF. Hydroxyproline is a unique component of the collagen fbrils in ECM, and one of the main features of IPF is the deposition of ECM [27]. In our results, the degree of alveolar infammation and fbrosis and hydroxyproline content was remarkably reduced after BB treatment suggesting a signifcant protective efect of BB on BLM-induced IPF mice. BLM-induced pulmonary fbrosis is thought to have an early partial infammatory phase, followed by a fbrotic phase [28]. Enhanced early infammatory events are critical for the development of pulmonary fbrosis in mice. Infammatory factors as well as chemokines have been suggested to be key factors in early infammatory events, during the early stage of pulmonary fbrosis [29]. TNF-α was involved in the pro-infammatory and pro-fbrotic activities of IPF, and its overexpression typically led to the development of IPF [30,31]. CCL2 belongs to the CC chemokine family and is formed under pathological conditions by pro-infammatory stimuli such as TNF-α [32]. Animal model studies have identifed a trend of increased CCL2 in IPF [33,34]. Our fnding showed that BB could reduce BLM-induced lung injury and infammation, which was supported by reduced cell counts (leukocyte, neutrophil, lymphocyte, and macrophage), the levels of CCL2 and TNF-α in BALF, and increased CXCL10 levels. A previous study has shown that the reduction in IPF was associated with a decrease in leukocyte, neutrophil, lymphocyte, and macrophage accumulation [35]. It was noteworthy that in our results, CXCL10 expression showed a gradual increase after BB treatment. CXCL10 is often recognized as an infammatory chemokine. However, Keane and colleagues [36] found that exogenous CXCL10 inhibits IPF, and its downregulation contributes to IPF lung development. It has antiangiogenic efects and inhibits fbroblast migration in response to the accumulation of fbroblasts in IPF. Tager et al. [37] likewise suggested that CXCL10 functions as an inhibitor of fbroblast chemotactic activity. Te changes in up-regulated or down-regulated expression of infammatory factors and cytokines elucidated the hypothesis of that BB treatment has a mitigating efect on BLMinduced IPF. Moreover, infammatory cell infltration and the release of infammatory factors are also accompanied by the deposition of ECM, which exacerbates the pathogenesis of IPF. TGF-β1 is an important and potent pro-fbrotic mediator in many fbrotic diseases including IPF [38]. It is involved in the development of IPF mainly by stimulating fbroblast activation and modulating EMT [39]. EMT, as an important part of the pathological process of IPF, is mainly manifested by increased expression of the mesenchymal marker α-SMA [40]. In addition, the activation of lung fbroblasts is the key rate-limiting step in IPF. Continuous pathogenic stimulation induces fbroblast activation and transforms into myofbroblasts, which secrete large amounts of ECM, leading to ECM deposition and thus accelerating the development of IPF [41]. Collagen I and fbronectin are fbroblast markers [42]. TGF-β1 has been proven to regulate the overexpression of fbrogenic proteins, such as Collagen I, fbronectin, and α-SMA, and thus exacerbate IPF [43]. Our results showed that BB treatment reduced the expression of Collagen I, fbronectin, α-SMA, and TGF-β1, indicating that BB might reduce these fbrogenic proteins by inhibiting TGF-β1 expression, thereby relieving IPF. Pan et al. [44] have reported that Ginkgo biloba extract EGb761 could decrease the expression of TGF-β1 and α-SMA to attenuate IPF. MMPs and TIMP-1 are key factors in ECM degradation and remodeling. MMPs promote the development of IPF, and its expression could be regulated by TIMP-1 [45]. Te inhibitory or promoting efect of Ginkgo biloba extract-EGb761 on MMPs and TIMP-1 has been shown [46], which is consistent with our results.
Activation of NLRP3 infammasome plays a crucial role in IPF [47]. Xiong et al. [48] reported that BLM induced a direct fbrogenic efect on IPF by up-regulating collagen expression and promoting infammatory factors release via JNK/NF-κB pathway. However, the relationship between BB and NLRP3 or JNK/NF-κB signaling pathways in IPF has not been clarifed. Te results of this study showed that BB signifcantly reduced BLM-induced activation of NLRP3 and NF-κB. In addition, BLM increased the phosphorylation of JNK, while BB treatment reversed this. Te results showed that BB was involved in the protective efect of lung fbrosis mice through NLRP3 and JNK/NF-κB signaling pathways. Tis result was supported by previous studies. Te report showed that high levels of JNK phosphorylation were closely associated with fbroblast to integrate fbroblast diferentiation in IPF [49]. By targeting downstream signaling molecules such as NF-κB, JNK exacerbated BLM-induced lung tissue fbrosis and infammation [50]. NF-κB is known to be upstream of NLRP3 [51], and the overexpression of NLRP3 exacerbates the release of infammatory factors IL-1β and IL-18. Te report has shown that Ginkgo biloba extract EGb761 had the potential to inhibit the activation of the NF-κB pathway [44]. In this study, we reported for the frst time that BB inhibited the activation of JNK/NF-κB and NLRP3 in  IPF, but we have only preliminarily explored the mechanisms. Endeavors to dissect the molecular mechanisms of JNK, NF-κB, and NLRP3 signaling will provide more valid and reliable evidence for the role of BB in protecting against BLM-induced pulmonary infammation and fbrosis.

Conclusions
In conclusion, we investigated the efects of BB on BLMinduced pulmonary fbrosis and elucidated the underlying mechanisms. Administered in the early infammatory phase, BB had a protective efect on pulmonary fbrosis in mice induced by BLM, which might be related to the inhibition of early infammatory response and amelioration of ECM deposition. Te underlying mechanism might be via the inhibition of the JNK/NF-κB/NLRP3 signal pathway. A summary of the proposed mechanism is shown in Figure 6. Overall, this study may provide some new insights into the mechanism of action of BB in the treatment of pulmonary infammation and fbrosis.

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

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this paper.  Evidence-Based Complementary and Alternative Medicine 9