Abstract

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal lung disease characterized by progressive dyspnea, decreased lung function, and death (1). The etiology of IPF is not clear, but several associations have been reported: smoking; exposed to wood and metal dust; chronic viral infections; exposure to certain medications; and genetic factors (2, 3). The occurrence and development of IPF are caused by abnormal damage and activation of alveolar epithelial cells, leading to the secretion of profibrotic, coagulation, and inflammatory cytokines, controlling the proliferation, activation, and differentiation of fibroblasts into myofibroblasts (4, 5). Currently, there is a lack of effective therapy for IPF, as the only approved treatments for IPF (Nintedanib and Pirfenidone) only slow the progression of the disease (6). Hence, there is an urgent need to find new therapies for IPF patients.

Schisandrin B (Sch B) is an active monomer of Schisandrin. Pharmacological studies have shown that Sch B plays an important role in liver protection and neuroprotection by removing free radicals and achieving antioxidant effects (7, 8). In term of cardiac actions, Sch B can inhibit cell apoptosis, downregulate inflammatory cytokines to prevent ischemia-reperfusion injury and doxorubicin induced cardiac toxicity (9, 10). In addition, Sch B plays a certain role in tumor treatment by inducing cell cycle arrest (11). Pharmacological studies have suggested that Sch B has anti-fibrosis effect (12, 13). Sch B can alleviate bleomycin-induced (BLM-Induced) pulmonary fibrosis in mice by inhibiting inflammatory response and oxidative stress (14). In addition, Sch B is associated with autophagy in liver injury mechanism, and autophagy regulation is closely related to the pathogenesis of pulmonary fibrosis. Autophagy regulation involves transforming growth factor-β (TGF-β) and mTOR. TGF- β an important pro-fibrosis factors in the pathogenesis of pulmonary fibrosis. It can activate mTOR to inhibit autophagy in BLM-Induced animals, leading to pulmonary fibrosis (15-17).

Currently, there are rarely reports about the autophagy regulation of Sch B in the process of inhibiting pulmonary fibrosis, so the study aims to investigate the autophagy regulation mechanism of Sch B in BLM-Induced pulmonary fibrosis. We hypothesized that autophagy is involved in the inhibitory effect of Sch B on pulmonary fibrosis. The results may provide a theoretical basis for the clinical development and application of Sch B in the treatment of pulmonary fibrosis.

Methods

BLM-induced pulmonary fibrosis model

Adult male C57BL/6 mice with weight from 18 to 22 g were provided by the animal center of Wuhan University, and all experimental protocols were approved by the institutional animal care and use committee. Mice were randomly divided into saline control group, BLM-induced group, Sch B group, Sch B + bleomycin group (Sch B treated group) and Sch B + bleomycin + chloroquine group, with 6 mice in each group. Then, we used bleomycin to induce the early inflammation model and the pulmonary fibrosis model (18). On day 0, C57BL/6 mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (5 ml/kg) and intratracheal injection of 2mg/kg BLM (Kayaku Co., Ltd., Tokyo, Japan). The saline control group was given normal saline according to the same procedure. mice were gavaged with normal saline or Sch B once per day from day 1 to day 7 after bleomycin administration at a dose of 3 mg/kg body weight. In the early inflammation model induced by bleomycin, mice were sacrificed on the day 8 to collect alveolar lavage fluid, serum and lung tissue, and analysis the levels of inflammatory factors and biomarkers of oxidative stress were analyzed. In the bleomycin induced pulmonary fibrosis model, on the day 14, the forced vital capacity (FVC) of mice was recorded by animal lung function tester, and the lung tissue was taken for the determination of hydroxyproline and histopathology (Figure 1A).

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Figure 1.

Sch B attenuates BLM-Induced pulmonary fibrosis in mice. A. Informative figure on the different groups of mice and a timeline in treatment and different measurements. B. The representative photographs of the effects of Sch B against BLM-induced pathological changes (Masson staining. Bars, 100 μm). C. The mean optical density (MOD) of positive areas was quantified by Image Pro Plus 6.0 to evaluate the levels of collagen expression. D. Degree of lung fibrosis was quantified by Ashcroft score system. E. The forced vital capacity of the mice. F. The Lung Coefficient of the mice. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.

Results

Sch B inhibits myofibroblast activation and collagen fibers deposition

The expression of α-SMA and collagen I in the alveolar and interstitium of pulmonary were observed by immunohistochemistry on the 14th day. In the saline control group (normal lung tissue), the immunostaining was exceedingly weakly positive. In the BLM-Induced group (fibrotic lung tissues), high expression of α-SMA and collagen I were observed in the lung interstitium by the large positive immunolocalization area of lung slices (Figures 2A and ​andC).C). In the Sch B treated group, the expression of α-SMA (Figures 2A and ​andB)B) and collagen I (Figures 2C and ​andD)D) were significantly reduced compared with BLM-Induced group.

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Figure 2.

Sch B attenuates myofibroblast activation and collagen fibers deposition. A. Representative images of immunohistochemistry for α-SMA in BLM-induced pulmonary fibrosis in mice. B. The mean optical density (MOD) of positive areas was quantified by Image Pro Plus 6.0 to evaluate the levels of a-SMA expression. C. Representative images of immunohistochemistry for collagen I in BLM-induced pulmonary fibrosis in mice. D. The mean optical density (MOD) of positive areas was quantified by Image Pro Plus 6.0 to evaluate the levels of type I collagen expression. E. The type I collagen content and type III collagen content was measured by using polarized light. F. The mean optical density (MOD) of positive areas was quantified by Image Pro Plus 4.5 to evaluate the levels of type I collagen content and type III collagen content expression. G. COL1A1 mRNA expression. H. COL3A1 mRNA expression. I. CTGF mRNA expression. J. FN mRNA expression. K. Immunofluorescence of VIMENTIN and a-SMA. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.

The type I and type III collagen fibers were clearly stained with Sirius scarlet staining. We observed that type I collagen fibers were closely arranged and showed strong birefringence by polarized light. It appeared bright red or orange yellow. The type III collagen fibers showed weak birefringence and appeared green (Figure 2E). We observed the type I collagen deposition in Sch B treated group was significantly lower than that in the BLM-Induced group (Figure 2F). Fibrosis related genes were also detected by RT-PCR, and the results showed that BLM could significantly induce mRNA expression of genes such as COL1A1, COL3A1, CTGF, and FN, while Sch B could inhibit the increase of mRNA expression (Figures 2 G-J). In addition, we conducted immunofluorescence staining of VIMENTIN and α-SMA to mark fibroblasts, Sch B could inhibit BLM-Induced increase of fibroblasts (Figure 2K).

Sch B decreases inflammatory response and oxidative stress in early stage

Inflammatory response and oxidative stress in early stage play an important role in the process of pulmonary fibrosis, so we have established bleomycin-induced early inflammation model (Figure 1A). In the early inflammation model induced by bleomycin, inflammatory cytokine IL-6 and TNF-α in BALF and serum of BLM-Induced group was significantly higher than that in the saline control group. In the BALF of BLM-Induced group, the levels of inflammatory cytokines were decreased after Sch B intervention (Figures 3A and ​andC).C). In the serum of different groups, we found the consistent results (Figures 3B and ​andD).D). In addition, we also observed a decrease in SOD level and an increase in MDA level in lung tissue homogenate in BLM-Induced group. In contrast, we observed the opposite changes after Sch B intervention (Figures 3E and ​andF).F). In addition, we conducted immunofluorescence staining of CD45 to represent infiltration of inflammatory cells, Sch B could reduce immune cell infiltration caused by BLM (Figure 3G).

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Figure 3.

Sch B inhibits inflammatory response and oxidative stress in early stage. A. Effects of Sch B on inflammatory mediators of IL-6 in BALF. B. Effects of Sch B on inflammatory mediators of IL-6 in serum. C. Effects of Sch B on inflammatory mediators of TNF-α in BALF. D. Effects of Sch B on inflammatory mediators of TNF-α in serum. E. Effects of Sch B on oxidative stress markers of SOD in lung tissues of BLM-treated mice. F. Effects of Sch B on oxidative stress markers of MDA in lung tissues of BLM-treated mice. G. Immunofluorescence of CD45. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.

Sch B inhibits pulmonary fibrosis by promoting autophagy

The autophagic flux is directly related to the pathogenesis of pulmonary fibrosis. We detect the autophagic marker proteins Beclin-1 and LC3 by immunofluorescence staining. In the BLM-Induced group, autophagy is activated to a certain degree, representing the basis of the autophagic flux in fibrosis process. However, we observed that the levels of Beclin-1 and LC3 increased significantly after Sch B administration, which could be blocked to the basis state again by chloroquine (Figure 4A). Western blot analysis showed the sham expression of LC3 and Beclin-1 in different groups. Moreover, western blot analysis showed Sch B could decrease the expression of α-SMA and collagen I to attenuate BLM-Induced pulmonary fibrosis. Furthermore, chloroquine inhibited the autophagic flux increased by Sch B, the expression of fibrosis protein increased again. Therefore, Sch B could inhibit pulmonary fibrosis by promoting autophagy (Figures 4B-F).

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Figure 4.

Sch B inhibits pulmonary fibrosis by promoting autophagy. A. Effects of Sch B on the expression of Beclin-1 and LC3 in lung tissues of BLM-treated mice by confocal microscope analysis. B. Western blot analyses expression of a-SMA, collagen I, LC3 and Beclin-1 in different groups. C. Quantification of a-SMA expression is achieved using densitometric values. D. Quantification of collagen I LC3-II expression is achieved using densitometric values. E. Quantification of Beclin-1 expression is achieved using densitometric values. F. Quantification of LC3-II expression is achieved using densitometric values. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.

Sch B promotes autophagy by enhancing the dephosphorylation of AKT-mTOR

To clarify the mechanism of Sch B regulating autophagy, we evaluated the AKT-mTOR pathway by western blot analysis. The results showed that p-AKT could be induced to dephosphorylate by bleomycin to a certain degree. However, the phosphorylation of AKT and mTOR were decreased significantly by Sch B, which could be rescue by 3-Methyladenine. The above results show that Sch B promotes autophagy by promoting the dephosphorylation of AKT-mTOR pathway (Figures 5A-C).

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Figure 5.

Sch B promotes autophagy by promoting the dephosphorylation of AKT-mTOR pathway. A. Western blot analyses expression of AKT-mTOR pathway in different groups. B. Quantification of p-AKT expression is achieved using densitometric values. C. Quantification of p-mTOR expression is achieved using densitometric values. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

IPF is a chronic progressive interstitial lung disease, which is characterized by aggressive and poor prognosis. IPF has a complex etiology and high mortality (20). So far, lack of effective drugs for IPF is a major difficulty at present. Sch B has a variety of pharmacological activities, it can play a pharmacological role through the regulation of a variety of signal pathways (21). Sch B isolated from Schisandra chinensis is a natural non-enzymatic antioxidant with low toxicity, low cost, and broad application prospects. It has been proven to have multiple pharmacokinetic properties, including antioxidant, anti-inflammatory, cardioprotective, and neuroprotective properties (11). Studies have shown that lignans from Sch can improve clinical symptoms, enhance quality of life, increase exercise tolerance, prolong survival, and partially improve lung function in patients with IPF (11, 22). A study in vitro has shown that Sch B can inhibit epithelial mesenchymal transition, which is closely related to airway remodeling in fibrotic lung disease (23, 24).

In our study, we found that Sch B could improve the lung function of BLM-Induced pulmonary fibrosis in mice. In addition, Sch B could attenuate BLM-Induced pulmonary fibrosis in mice by inhibiting the proliferation and differentiation of myofibroblasts and the deposition of collagen fibers. Results from inflammation models indicate Sch B could reduce lung inflammation and oxidative stress in mice with BLM-induced pulmonary fibrosis in early stage. Moreover, analysis on Belin-1 and LC3 level indicate Sch B could promote dephosphorylation of AKT-mTOR pathway, therefore promoting autophagy to inhibit pulmonary fibrosis. These results should provide a theoretical basis for exploring potential therapeutic drugs for IPF.

Sch B attenuates BLM-induced pulmonary fibrosis via inhibiting myofibroblast activation and lung interstitial ECM deposition. Myofibroblast can inhibit fibroblast differentiation and reduce pulmonary fibrosis (11, 14). α-SMA is a recognized biomarker of myofibroblast activation, and type I collagen is closely associated with ECM deposition in lung mesenchymal tissue (25). Zhou et al found that aucubin could reduce BLM-induced pulmonary fibrosis by decreasing α-SMA expression (26). Our study showed similar effect for Sch B, as the expression of α-SMA and type I collagen decreased after Sch B intervention in the lung tissue of BLM-induced pulmonary fibrosis in mice. These findings support that Sch B attenuates BLM-induced pulmonary fibrosis via inhibiting myofibroblast activation and lung interstitial ECM deposition. As animal models of BLM-induced fibrosis can simulate the clinical pathogenesis of IPF, our findings also suggest a clinical potential of Sch B (21).

Sch B could inhibit inflammatory responses and could inhibit pulmonary fibrosis. The key component of BL-induced pulmonary fibrosis is inflammatory responses, as BLM-induced lung injury lead to inflammatory responses due to the production of excess free radicals (27, 28). The results from the BLM-induced early inflammation model indicated Sch B could significantly decrease inflammatory responses, therefore have the potential for alleviating pulmonary fibrosis. This finding is comparable to previous research. Liu et al. found that AF-1 was able to inhibit TGF-β induced proliferation by reducing the production and release of inflammatory cells and inflammatory factors (29). Moreover, Chitra et al. found that berberine attenuated BLM-induced fibro-proliferation and ECM deposition by improving antioxidant status and blocking the expression and release of inflammatory mediators (30). Collectively, similar results support our findings in the anti-fibrosis effect of Sch B.

Sch B may inhibit pulmonary fibrosis by upregulating autophagy genes. Autophagy is a metabolic process of eukaryotic self-catabolic process (31). Impairment of autophagy is closely related to the promotion of pulmonary fibrosis, and previous research showed that the regulation of autophagy response played an important role in inhibiting ECM accumulation. Taken together, autophagy is considered a key regulator in the pathogenesis of pulmonary fibrosis (32, 33). Recent research has documented that activating autophagy can significantly reduce BLM-induced pulmonary fibrosis and local inflammation in fibrotic tissues. Consequently, insufficient autophagy can lead to cell senescence and myofibroblast (34). Our results indicate BLM can significantly down-regulate autophagy. The decrease in the expression of autophagy protein LC3-II in lung tissues was observed (30), and Sch B increased the expression levels of Beclin-1 and LC3-II in mice with BLM-induced pulmonary fibrosis. This finding strongly suggests that the level of autophagy was up-regulated after the intervention of Sch B. These results suggest that Sch B could attenuate pulmonary fibrosis in mice with BLM-induced pulmonary fibrosis by activating autophagy.

In addition, our study clarified the mechanism of Sch B regulating autophagy. mTOR signaling pathway is an important molecular signaling pathway for the regulation of autophagy. Several studies have reported the relationship between autophagy and the mTOR signaling pathway (35, 36). Recent studies have also shown that TGF-β inhibits autophagy by activating mTOR. Administration of rapamycin (an mTOR inhibitor) in BLM-induced animals increased the expression of autophagy proteins LC3 and Beclin-1, followed by decreased α-SMA and fibronectin, resulting in attenuated pulmonary fibrosis. In addition, the silencing of the autophagy proteins Beclin-1 and LC3 resulted in enhanced levels of α-SMA and fibuconin in response to TGF-β (15). Our western blot analysis focused on the effect of Sch B on autophagy status and examined the effects of Sch B on AKT-mTOR. The results showed that Sch B could induce significant dephosphorylation in p-AKT, which could be rescue by 3-Methyladenine. Our results prove the potential of Sch B in promoting autophagy through the AKT-mTOR pathway, and it is a possible mechanism for attenuating BLM-induced pulmonary fibrosis injury.

Collectively, this study provides evidence for the potential of Sch B as a drug for the treatment of pulmonary fibrosis. However, Sch B has many other pharmacological activities beside its effect in the autophagy mechanism. Whether other mechanisms involve in the inhibition of pulmonary fibrosis remains unclear. In addition, the formation of pulmonary fibrosis involves many pathogenic mechanisms, and mTOR signaling pathway is only one of the important molecular mechanisms of IPF. The mechanism of Sch B in BLM-induced pulmonary fibrosis needs further clarification with clinical studies. In conclusion, our study demonstrated that Sch B exerts anti-fibrotic effects in BLM-induced pulmonary fibrosis. The effect of Sch B may be accomplished by activating autophagy through AKT-mTOR signaling pathway. Therefore, Sch B may be a potential agent for the treatment of IPF.

References