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Total extract of Yupingfeng attenuates bleomycin-induced pulmonary fibrosis in rats.

ABSTRACT

Yupingfeng is a Chinese herbal compound used efficaciously to treat respiratory tract diseases. Total glucosides of Yupingfeng have been proven effective in anti-inflammation and immunoregulation. Nevertheless, the role of total extract of Yupingfeng (YTE) in pulmonary fibrosis (PF), a severe lung disease with no substantial therapies, remains unknown. Present study was conducted to elucidate the anti-fibrotic activity of YTE. The rat PF model was induced by intratracheal administration of bleomycin (BLM, 5 mg/kg), and YTE (12 mg/kg/d) was gavaged from the second day. At 14 and 28 days, the lungs were harvested and stained with H&E and Masson's trichrome. The content of hydroxyproline (HYP) and type 1 collagen (Col-1) were detected, while the protein expression of high-mobility group box 1 (HMGB1), transforming growth factor-beta 1 (TGF-[beta]1), Col-I and [alpha]-smooth muscle actin ([alpha]-SMA) were analyzed by immunohistochemistry or Western blot. As observed, YTE treatment attenuated the alveolitis and fibrosis induced by BLM, reduced the loss of body weight and increase of lung coefficient. Meanwhile, YTE strongly decreased the levels of HYP and Col-I, and reduced the over-expression of HMGB1, TGF-[beta]1, Col-1 and [alpha]-SMA. In conclusion, YTE could ameliorate BLM-induced lung fibrosis by alleviating HMGB1 activity and TGF-[beta]1 activation, suggesting therapeutic potential for PF.

Keywords:

Total extract of Yupingfeng

Astragalus membranaceus

Pulmonary fibrosis

High-mobility group box 1

Transforming growth factor-beta 1

Introduction

Pulmonary fibrosis (PF) is one of the interstitial lung diseases characterized by abnormal increase of fibroblast population and excessive accumulation of extracellular matrix (ECM), leading to serious respiratory impairment (Todd et al. 2012). Despite the clinical performance of PF consists of varying degrees of interstitial fibrosis and parenchymal inflammation, additional diagnostically relevant findings remain largely elusive. Moreover, no substantial therapies have been developed to reverse established PF or even halt the chronic progression to respiratory failure. Eventually, the widely accepted therapeutic schedule that curing PF with a combined therapy of corticosteroids, and an immunosuppressive agent plus high-dose oral N-acetylcysteine is no longer appropriate (Behr 2013). Thus, extensive efforts are urgent need to develop novel strategies to prevent or cure PF.

Transforming growth factor-beta 1 (TGF-[beta]1) is one member of TGF-[beta] superfamily, activation of which is considered as a hallmark in PF. It can be targeted to ECM and regulate a wide variety of cellular functions involved in PF (Koli et al. 2008; Kang et al. 2007). Moreover, TGF-[beta]1 is a master inducer of epithelial-mesenchymal transition (EMT) in alveolar epithelial cells and transdifferentiation of quiescent fibroblasts into myofibroblasts (MFb), which contribute to ECM deposition (Todd et al. 2012; Sureshbabu et al. 2011). Strategies that utilize proteins or small chemicals to inhibit TGF-[beta]1 signaling or block the associated signal transduction have therapeutic potential in the clinical treatment for PF. Recent studies have demonstrated that high-mobility group box 1 (HMGB1) acts as an inflammatory mediator in the acute exacerbation of PF and it can directly stimulate fibroblast proliferation while anti-HMGB1 antibody protects mice from bleomycin (BLM)-induced PF (Ebina et al. 2011; Hamada et al. 2008). The receptor for advanced glycation end-products (RAGE) is one of the receptors of HMGB1 known to be involved in PF (He et al. 2007). It has been confirmed that RAGE deficiency can lead to reduced TGF-[beta]1 level, while HMGB1-induced EMT can be partly mediated by TGF-[beta]1 (He et al. 2007; Lynch et al. 2010). Besides, damaged alveolar epithelial cells induce HMGB1 release and accelerate alveolar epithelial repair via activation of TGF-[beta]1 (Pittet et al. 2013). The evidence reveals that HMGB1 signal may stimulate TGF-[beta]1 activation in PF. Thus, further investigation of their potential interplay in PF is essential and meaningful.

As is known to all, traditional Chinese medicines (TCM) provide a vast source for the discovery of new drugs, so researches of the medicines such as paeoniflorin and thymoquinone may provide us novel strategies for blocking fibrotic development (Zeng et al. 2013; Ghazwani et al. 2014). The glucosides from some medicinal herbs also exert anti-inflammatory and immunoregulatory activities (Gao et al. 2009; Xu et al. 2013, 2014). Yupingfeng powder, a traditional Chinese herbal formula, has been confirmed efficacious in preventing respiratory tract diseases such as viral infections and chronic bronchitis (Liu et al. 2013; Song et al. 2013). Total extract of Yupingfeng (YTE) is a mixture extracted from Yupingfeng. Our previous data show that total glucosides from Yupingfeng have anti-inflammatory and immunoregulatory activities (Gao et al. 2009). Further studies elucidate that some ingredient pertain to YTE is effective to antagonize HMGB1 activity, collagen production, and attenuate myocardial fibrosis and hepatic fibrosis by disrupting TGF-[beta]1 signaling (Huang et al. 2012; Li et al. 2013; Chen et al. 2011; Liu et al. 2009). However, it has not yet any studies characterizing the therapeutic potential of YTE for PF. In this study, we focused on evaluating the potential activity of YTE on BLM-induced PF in rats and explored its effect on HMGB1 activity and TGF-[beta]1 activation.

Materials and methods

Animals

Healthy adult female Sprague-Dawley rats weighing 180-220 g were provided by the Experimental Animal Center of Anhui Medical University, Hefei, Anhui, China. In compliance with the relevant guidelines, all of the animals received humane care and had free access to food and water during the study. All of the procedures were approved by the University Animal Care and Use Committee.

Preparation of YTE and its content determination

Yupingfeng composes of Astragali Radix (Huangqi; the root of Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao), Atractylodis Macrocephalae Rhizoma (Baizhu; the rhizomes of Atractylodes macrocephala Koidz.) and Saposhnikoviae Radix (Fangfeng; the roots of Saposhnikovia divaricata (Turcz.) Schischk.) in a dry weight ratio of 3:1:1. These herbs were obtained from the First Affiliated Hospital of Anhui Medical University, and identified by Professor Hua-Sheng Peng (Anhui University of Traditional Chinese Medicine), Hefei, China. Firstly, these herbs were mixed and soaked in 10 l of 80% ethanol for 1 h. Secondly, the mixture was heated for 2 h starting from the boiling time, filtered while hot, and then recovered the ethanol solution. Subsequently, the residue was refluxed two times with 10 l of distilled water, 1 h each time starting from the boiling time and filtered while hot. Finally, the three solutions were combined, evaporated and stored at 4[degrees]C for one night. The next day, the frozen crude extract containing the glucosides was purified in a macroporous resin column, successively eluted with distilled water, 40% ethanol and 70% ethanol which last solution was collected and evaporated under vacuum at 90[degrees]C to give the pale, dull yellow, powdery extract (yield 0.45%).

To determine the purity of YTE, astragaloside IV, the quantitative indicator and main effective components of Astragali Radix that described in China Pharmacopoeia 2010, was used as the standard. Meanwhile, 5% vanillin-glacial acetic acid and perchlorate were used as the developer, colorimetric method was applied. Firstly, YTE and astragaloside IV were precisely weighed and diluted with methanol in different test tubes, and then dried the solvent at 80[degrees]C, and successively added 0.2 ml of 5% vanillin-glacial acetic acid, 0.8 ml perchlorate and heated in water bath at 70[degrees]C for 20 min. Subsequently, all the tubes were took out and put into ice water for cooling, and then quickly added 5 ml glacial acetic acid. Then the reagent blank was used as a control and Shimadzu UV-1700 ultraviolet spectrophotometer was used to scan the maximum absorption wavelength of astragaloside IV. In this study, the maximum absorption wavelength was 578 nm, so we chose it for measuring the absorbance. Finally, the obtained purity of YTE was 63% (w/w).

HPLC-ELSD analysis of YTE

Subsequently, YTE and the extracts from Astragalus membranaceus were analyzed by HPLC-ELSD with gradient elution. The extraction method of Astragalus membranaceus was the same as that of YTE. Shimadzu LC-10AT (Japan) high performance liquid meter and LP-ODS (4.6 mm x 250 mm, 5 [micro]m, Shimadzu Company) were used in this analysis. The mobile phase contained acetonitrile (A) and 0.2% formic acid (B) with constant volume flow (1 ml/min) and column temperature (40[degrees]C). The drift tube temperature was 110[degrees]C and the nitrogen volume flow rate was 3.10 1/min. The chromatograms showed that astragaloside II and IV, the main glycosides as well as active ingredients of Astragalus membranaceus, were included in YTE (Fig. 1).

Drug solution

In the study, each vial of BLM [A.sub.5] hydrochloride (120528, Laiboten Pharmaceutical Co., Ltd, China) was dissolved in 0.9% sodium chloride injection with a volume of 1.6 ml just before using. Sufficient YTE and prednisone (Pred) (1210246, Xinhua Pharmaceutical Co., Ltd, China) were all diluted with 0.5% CMC-Na solution just before gavage once per day.

Antibodies and reagents

Anti-HMGB1 antibody (ab79823), anti-TGF-[beta]1 antibody (ab64715) and anti-type I collagen antibody (ab34710) were Abeam products, while anti-a-smooth muscle actin ([alpha]-SMA) antibody (bs10196R) was purchased from Bioss and anti-[beta]-actin antibody (E1A7018) was from EnoGene. Peroxidase conjugated affnipure goat anti-mouse IgG (ZB-2305) and peroxidase conjugated affnipure goat anti-rabbit IgG (ZB-2301) were from ZSGB-BIO, China. In addition, the Masson's trichrome kit (MST-8003/8004, Maixin-Bio, China), hydroxyproline (HYP) Assay Kit (A030-2, Nanjing Jiancheng Bioengineering Institute, China) and type I collagen (Col-I) ELISA Kit (41582, Shanghai yuanye Bio-Technology Co., Ltd, China) were also used in this study.

Experimental design

All the experimental rats were randomly assigned to four groups: normal saline (NS) group, only instilled with saline; BLM group, only instilled with BLM; Pred group, instilled with BLM and treated with Pred (5 mg/kg); YTE group, instilled with BLM and treated with YTE (12 mg/kg). The PF model was induced by intratracheal instillation of BLM. Subsequently, Pred (5 mg/kg/d) and YTE (12 mg/kg/d) (dissolved in 0.5% CMC-Na) and equal volume of 0.5% CMC-Na were respectively given to the rats in Pred group, YTE group, NS group and BLM group by gavage administration 1 day after BLM instillation for 28 days. The dosage of YTE was based on the conversion from adult dosage to the rat dosage. On days 14 and 28, the rats were weighed and anesthetized with 10% chloral hydrate (2.2 ml/kg) intraperitoneally, and then the lungs were harvested, weighed, stained with H&E and Masson's trichrome. Meanwhile, the content of HYP in lung tissues and Col-1 in lung homogenates were measured. Besides, the expression of HMGB1, TGF-[beta]1, Col-I and [alpha]-SMA were observed by immunohistochemical (IHC) or Western blot analysis.

Histological analysis

The left lung tissues were fixed in 10% formalin for 48 h, dehydrated in a graded ethanol series and subsequently embedded in paraffin. Sequential 5 [micro]m lung sections were placed on slides and stained with routine H&E and Masson's trichrome, respectively, for morphological analysis and locating collagen expression by using the standard protocols. Average scores of the six random visual fields of the sections from each group were evaluated under x200 magnification in a blinded fashion for the pathologic grades of alveolitis and fibrosis. The method was reference to Szapiel et al. (1979): 0. no lung abnormality; 1, presence of inflammation and fibrosis involving less than 20% of the lung parenchyma; 2, lesions involving 20-50% of the lung; and 3, lesions involving more than 50% of the lung. The slides were investigated under a light microscope (Olympus Opticals, Japan) with the same magnification times (x200).

Effect of YTE on rat body weight and lung coefficient

On days 0, 7, 14 and 28, all rats were weighed to observe the changes of rat body weight in each group. On days 14 and 28, the rats were sacrificed and the lungs were harvested and weighed. Lung coefficient was determined by the equation: lung coefficient = lung weight (g)/body weight (kg) x 100%.

Effect of YTE on the levels of HYP in lung tissues and Col-I in lung homogenates

The obtained lung tissues saved in 80 [degrees]C or liquid nitrogen. Then the deputy lobes of each group were took out to detect the content of HYP ([micro]g/mg) in concordance with the instruction manual of the kit while the lung homogenates were to measure Col-I content (ng/l) by ELISA. The absorbance of HYP and Col-I in each samples were measured using an automated microplate reader at a wavelength of 550 nm and 450 nm.

Immunohistochemical analysis

Briefly, the formaldehyde-fixed and paraffin-embedded tissue sections were dewaxed in xylene, hydrated through graded ethanol, and rinsed with tap water and distilled water. Then the endogenous peroxide activity was blocked using 0.3% [H.sub.2][O.sub.2] in methanol for 30 min. Antigen retrieval was performed in citrate buffer for 20 min. Nonspecific protein staining was blocked with 1.5% normal goat serum in TBS with 0.5% bovine serum albumin. Subsequently, the sections were incubated with primary anti-HMGB1, anti-TGF-[beta]1, anti-Col-I, and anti-[alpha]-SMA antibodies at 1:550, 1:50, 1:500, and 1:500 dilution at 37[degrees]C for 30 min, and then stayed overnight at 4[degrees]C. On the next day, the slides were incubated in goat anti-mouse or anti-rabbit secondary antibodies for 1 h corresponding to the primary antibodies. Visualization was performed with diaminobenzidine followed by washing with water, then counterstained with hematoxylin, dehydrated and transparented, sections were cover-slipped with oil of cypress and dried. The images were photographed with the same microscope and magnification times (x400). Six random views in each group were selected to detect the integral optical density (IOD) value which was performed by Image-Pro Plus 6.0.

Western blot analysis

The right lung lobes weighing 80-100 mg were homogenized in ice-cold radio-immunoprecipitation lysis buffer (P0013C, Beyotime Institute of Biotechnology, China) and proteinase inhibitor PMSF (Amresco 0754, Biosharp, USA) cocktail. All the lytic process was performed as the instruction manual described. After centrifugation (12,000 r/min, 10 min at 4[degrees]C), the supernatant was collected. Before using, the loading buffer was mixed into the supernatant in a ratio of 1:4 in EP tubes and put them into boiling water for 10 min. Proteins in the supernatant were separated by SDS-PAGE on a 12% gel and then transferred to PVDF membranes (20130107054, Millipore, USA). The blotted membranes were blocked with 5% non-fat dry milk (w/v) (2013022311, Guangming, China) in TBS buffer (AR0031, BOSTER, China) and incubated at 4 [degrees]C overnight with anti-HMGB1 (1:10,000 diluted, 25 kDa), anti-TGF-[beta]1 (1:500 diluted, 45 kDa), anti-[alpha]-SMA (1:500 diluted, 42 kDa), and anti-[beta]-actin (1:2000 diluted, 43 kDa) antibodies at 4 [degrees]C overnight. On the next day, the blots were incubated with goat anti-mouse or anti-rabbit secondary antibodies for 1 h. Immunodetection was developed with enhanced chemiluminescence and exposed on an X-ray film. The densitometry was performed on protein bands using Image J analysis software (ChemiQ 4600, Bioshine, China). The IOD value was performed by Image-Pro Plus 6.0 and [beta]-actin was used as an internal reference for relative quantification.

Data analysis and statistics

All data were expressed as means [+ or -] SD (standard deviation). Difference among groups was performed using one-way ANOVA with a post hoc LSD test or Dunnett's T3 test. The scores of alveolitis and fibrosis were evaluated using the Mann- Whitney test. All analyses were performed by SPSS 13.0 software, and probability values of 0.05 or less were considered statistically significant.

Result

Treatment with YTE attenuated BLM-induced lung abnormalities

Gross features

The abnormalities of gross features in lungs after BLM treatment were first observed. In NS group, the lungs were pink, soft and smooth without abnormalities. However, parts of the lungs became hard and gray, while small nodules and hemorrhagic spots were observed on the lung surface on day 14 in BLM group. By day 28, the lungs became pale and the white nodules were evident. In addition, the hardness of the lungs was increased and part of the lobes became dark red. However, on day 14, the lungs in Pred group and YTE group were relatively soft and red. Besides, the hemorrhagic spots and white nodules on the lung surface were less than those of BLM group on both days.

Microscopic findings

In order to elucidate the pathologic changes in each group, the lung histopathology abnormalities were evaluated with H&E staining analysis (Fig. 2a). In NS group, the alveolar structure was complete without inflammatory cell infiltration or widened alveolar interval. After BLM administration, the alveolar structure was collapsed and part of alveolar space was filled with inflammatory cells first, and then the alveolar septa was marked thicken, the alveolar structure was seriously collapsed and filled with superabundant ECM on day 28. Impressively, we observed remarkable improvement in these pathological changes after the administration of YTE.

YTE reduced collagen deposition in lungs

As determined by Masson's trichrome staining, BLM administration led to obvious collagen deposition in lungs (the blue area), while it was few after saline treatment (Fig. 2b). As expected, YTE and Pred could largely reduce fibrillar collagen production induced by BLM, especially after treatment with YTE, revealing that YTE exerts its inhibition of collagen accumulation.

YTE decreased the pathologic scores of alveolitis and fibrosis

As stained by H&E and Masson's trichrome in lungs, the pathologic scores of alveolitis and fibrosis were evaluated (Table 1). The results indicated that there were no evident alveolitis and fibrosis in NS group. In contrast, BLM could markedly increase the scores of alveolitis and fibrosis (p < 0.01), and respectively reached a maximum on days 14 and 28. However, the scores were significantly decreased by YTE, suggesting that YTE has anti-fibrotic action.

YTE increased body weight and decreased lung coefficient

Afterwards, we analyzed the data of rats' body weight on days 0, 7, 14, and 28 (Fig. 3a). Briefly, the loss of rats' body weight in BLM group was significantly evident (p < 0.01). However, compared with BLM group, YTE administration reduced body weight loss from day 7, especially on days 14 and 28. Surprisingly, Pred displayed little effect on the weight loss induced by BLM. As shown in Fig. 3b, the lung coefficient was significantly increased after BLM administration when compared to NS group on days 14 and 28 (p < 0.01). In treatment groups, both YTE and Pred could significantly inhibit the increase of lung coefficient.

YTE reduced the levels of HYP in lung tissues and Col-1 in lung homogenates

Next, we measured the lung HYP content to quantify the extent of fibrosis, as HYP is a major constituent of collagens. Compared with the BLM group, the HYP level was significantly reduced after treatment with YTE (Fig. 3c). Furthermore, the Col-I level in lung homogenates were evaluated (Fig. 3d). The results indicated that BLM treatment significantly up-regulated Col-I level as early as day 14 and reached the maximum at day 28 compared with NS group. Impressively, the Col-I level was evidently reduced after Pred and YTE treatment, especially the reduced level of Col-I by YTE (p < 0.01). Collectively, these data further illustrate the reversed effect of YTE on collagen accumulation.

YTE inhibited the positive expression of HMGB1, TGF-[beta]1, Col-I and [alpha]-SMA in lungs

Because HMGB1 plays an important role in activating the profibrogenic master cytokine TGF-[beta]1 and reduction of HMGB1 is likely to be beneficial for attenuating PF (Pittet et al. 2013; Ebina et al. 2011; Hamada et al. 2008; He et al. 2007). We then examined the effect of YTE on the distribution of HMGB1. As illustrated by IHC, the positive expression of HMGB1 was markedly increased after treatment with BLM (Fig. 4a). As expected, the increased HMGB1 -positive expression was reversed after YTE and Pred treatment. It provides evidence that YTE inhibited the increase of HMGB1 expression. The suppressed expression of HMGB1 prompted us to explore whether YTE have therapeutic potential for PF by its direct action of disrupting TGF-[beta]1. As numerous studies have recognized TGF-[beta]1 as a profibrogenic master cytokine and the profibrogenic effect of HMGB1 may partly mediated by TGF-[beta]1 (Todd et al. 2012; Sureshbabu et al. 2011; Koli et al. 2008; Kang et al. 2007; Lynch et al. 2010). Therefore, the effect of YTE on TGF-[beta]1 expression was detected (Fig. 4b). Our results demonstrated that TGF-[beta]1 expression was markedly increased in BLM-treated groups and mainly expressed in alveolar epithelial cells and smooth muscle cells. However, very few expression of TGF-[beta]1 was observed in NS group. In the presence of YTE, the raised positive expression and IOD value of IGF-[beta]1 induced by BLM were markedly decreased. It indicates that anti-fibrotic effect of YTE may be partly due to the direct inhibition of TGF-[beta]1 signaling.

As we know, fibroblasts and MFb undergo autonomous proliferation and produce excessive matrix proteins, which resemble a wound-healing process during PF (Todd et al. 2012), we subsequently investigated the ability of YTE on the modulation of Col-1 and expression of [alpha]-SMA, the latter is the key marker of MFb. As shown in Fig. 4c and d, the rats receiving saline showed few positive staining of Col-I and [alpha]-SMA, while BLM administration resulted in markedly increased expression of them. From the representative images, most of Col-I-positive expression was located in fibroblasts or MFb, while that of [alpha]-SMA was located in MFb. In YTE-treated group, the increased expression of Col-l was largely reversed and the accumulation of [alpha]-SMA-positive MFb was dramatically reduced. Furthermore, the IOD value of their positive expression was both reduced by treatment with YTE when compared with that of BLM group (p < 0.01). These results indirectly reflect the inhibitory effect of YTE on MFb proliferation, may be due to which the collagens (such as Col-I) accumulation is decreased and then BLM-induced PF is attenuated in rats.

YTE down-regulated the expression of HMGB1, TGF-[beta]1, and [alpha]-SMA with Western blot analysis

To further clarify the role of YTE on BLM-mediated PF, we detected the changed protein expression of HMGB1, TGF-[beta]1 and [alpha]-SMA with Western blot analysis (Fig. 5). Similarly, our results revealed that YTE significantly inhibited the up-regulated expression of HMGB1, TGF-[beta]1 and [alpha]-SMA induced by BLM (p < 0.05). Therefore, these results further support that the anti-fibrotic effect of YTE may be via inhibiting HMGB1 release, TGF-[beta]1 expression as well as MFb proliferation.

Discussion

PF is a chronic lung disease characterized by excessive deposition of ECM and remodeling of lung architecture (Todd et al. 2012). In the early stage of PF, the lung pathological changes are mainly inflammatory cell infiltration, edema and congestion (Razzaque and Taguchi 2003). Then the changes are converted to abnormal proliferation of fibroblasts, production of ECM including collagens and destruction of lung structure, resulting in dysfunction of lung. Despite considerable studies of PF, current therapeutic trials have not provided solid and unequivocal efficacy. Recently, pirfenidone has been demonstrated preferable activity for treating PF, but the general agreement has not been reached yet (Papiris et al. 2012). Thus it naturally draws great attention of extensive experimental researches and clinical observations to further understand and beat PF. To elucidate the etiopathogenesis of human PF, the BLM-induced animal model remains the best available experimental tool for studying its pathogenesis and testing of novel pharmaceutical compounds. In this study, we found that YTE had an inhibitory effect on BLM-induced PF and the protective role may be partly achieved by alleviating HMGB1 release and TGF-[beta]1 signaling.

Here, we observed that treatment of rats with BLM caused typical PF such as excessive ECM accumulation and destructed lung structure. The pathological scores of alveolitis and fibrosis revealed that the PF model was in accordance with the pathological progression of PF. In addition, BLM-induced lung injury and fibrosis were obvious biochemically characterized by the up-regulated expression of HMGB1 and TGF-[beta]1, excessive ECM accumulation including Col-I and markedly raised HYP level in lung tissues. Furthermore, administration of BLM resulted in a significant up-regulation of [alpha]-SMA expression, loss of body weight and increase of lung coefficient.

However, compared with Pred treatment, the histological analysis evidently demonstrated that YTE exerted a great attenuation of BLM-induced alveolar spaces collapse, alveolar wall thickening, inflammatory cell infiltrate, and ECM deposition. Moreover, YTE evidently decreased collagen production as well as the pathological scores of alveolitis and fibrosis, suggesting that YTE treatment not only suppressed BLM-induced acute inflammatory injury but also attenuated the fibrotic changes such as collagen accumulation and lung structure destruction. HYP is the main constituent of collagens, so it may serve as a marker of fibrosis severity and success of anti-fibrotic therapy. Meanwhile, Col-I is one of the collagens, and its production can partly reflect the collagen deposition. Our results revealed that YTE treatment could markedly decrease the levels of HYP and Col-I as well as the positive expression of Col-l in lungs, suggesting that YTE possessed an inhibitory effect on collagen deposition. Furthermore, YTE treatment obviously decreased the body weight loss and lung coefficient.

It is well known that various cytokines associated with inflammation and fibrosis could be drug development targets. HMGB1 has been demonstrated as an important inflammatory mediator and profibrotic factor in the pathogenesis of PF, and HMGB1 signal may be involved in the up-regulation of TGF-[beta]1 activity (Ebina et al. 2011; He et al. 2007; Hamada et al. 2008; Pittet et al. 2013; Lynch et al. 2010). Therefore, HMGB1 may be a therapeutic target for PF. In our study, YTE treatment could partly inhibit BLM-induced elevation of HMGB1 expression. It provides evidence that the inhibitory effects of YTE on alveolitis and fibrosis might be partly due to suppressing HMGB1 expression. Furthermore, TGF-[beta] has been confirmed as a pleiotropic growth factor in PF by stimulating ECM production and epithelial cells injury, while its over-expression aggravates PF both in animal models and patients (Todd et al. 2012; Sureshbabu et al. 2011). However, TGF-[beta]1 has many essential roles including immune regulation, cancer surveillance and wound healing. In addition, the inactivation of TGF-[beta] signaling may increase inflammation as a side effect, even while inhibiting fibrosis (Hahm et al. 2000). In this study, we clearly found that YTE administration not only reduced BLM-induced TGF-[beta]1 elevation, but also decreased HMGB1 release. Considering the central role of activated MFb in fibroblastic foci formation, collagen accumulation and fibrogenesis in lung (Todd et al. 2012), we detected the expression of [alpha]-SMA, a key marker of MFb, which certainly reflect the abnormal proliferation of MFb. Here, we found that YTE could strongly inhibit [alpha]-SMA expression, revealing that YTE inhibits the proliferation of MFb and this effect may be partly due to the inhibition of YTE on TGF-[beta]1 activation. Compared with Pred treatment, YTE had more evident effects in reducing the expression of HMGB1, [alpha]-SMA, TGF-[beta]1 at day 28. Our encouraging results may reflect that the decreased lung inflammation and fibrosis might be partly due to inhibiting BLM-evoked HMGB1 release and TGF-[beta]1 signaling.

We here demonstrate that YTE ameliorates BLM-induced PF. We find that YTE treatment not only diminishes the expression of HMGB1 and TGF-[beta]1, but also reverses the collagen production and reduces the degree of PF induced by BLM. Our study provides new insights into the possible mechanism that YTE may be via repressing the increased HMGB1 release and subsequently blocking TGF-[beta]1 production, and thus leading to a remarkable decrease of ECM deposition and improvement of PF (Fig. 6). However, present data is not enough to illuminate the effect of YTE or Yupingfeng on PF before their possible application in clinical. Other compositions other than astragalosides in YTE may contribute synergistically for the anti-fibrotic effect. More detailed investigations will be performed to further explore the exact mechanism of YTE for PF treatment in vivo and in vitro. Moreover, whether the anti-fibrotic effect of YTE is superior to the single composition such as astragaloside IV in vivo and in vitro, it remains to be further investigated.

Conclusion

In a word, the anti-fibrotic effect of YTE provides evidence that YTE may be an attractive pharmacological tool for diminishing PF and offers a strategy that inhibiting HMGB1 activation may block the activation and release of TGF-[beta]1 from its latent form in a direct or indirect way. In addition, an investigation is going on whether the minor drug components of Yupingfeng contribute to the overall attenuation of PF and to which extent. Certainly, further elucidation of the interplay between HMGB1 and TGF-[beta]1 may provide new mind for PF treatment.

Conflict of interest

There was no conflict of interest.

ARTICLE INFO

Article history: Received 14 April 2014

Revised 6 September 2014

Accepted 26 October 2014

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (NSFC 81274172, 81473267, 30801535); the Open Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (SKLNMKF201206) and Traditional Chinese medicine research project of the Health Department of Anhui Province (2012zy53).

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Liucheng Li (a, b), Delin Li (b,c), Liang Xu (b,c), Ping Zhao (b,c), Ziyu Deng (a,d), Xiaoting Mo (a,b), Ping Li (e), Lianwen Qi (e), Jun Li (a) *, Jian Gao (b), **

(a) School of Pharmacy (Anhui Key Laboratory of Bioactivity of Natural Products), Anhui Medical University, Hefei 230032, China

(b) Pharmaceutical Preparation Section (Third-Grade Pharmaceutical Chemistry Laboratory of State Administration of Traditional Chinese Medicine (TCM-2009-202)), The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China

(c) School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038, China

(d) The Second Affiliated Hospital of Anhui Medical University, Hefei 230012, China

(e) State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China

* Corresponding author. Tel.: +86 551 65161001; fax: +86 551 65161001.

** Corresponding author. Tel.: +86 551 62922423; fax: +86 551 62922442.

E-mail addresses: lijun@ahmu.edu.cn (J. Li), gaojianayfy@163.com (J. Gao).

http ://dx.doi.org/10.1016/j.phymed.2014.10.011

Table 1
Effects of YTE on the pathologic scores of alveolitis and fibrosis.
Data are means [+ or -] SD (n = 6).

Croups       Alveolitis

             14 days                    28 days

NS group     0.17 [+ or -] 0.41         0.17 [+ or -] 0.41
BLM group    2.50 [+ or -] 0.55 **      2.00 [+ or -] 0.63 **
Pred group   1.67 [+ or -] 0.82 **, #   1.00 [+ or -] 0.63 *, #
YTE group    1.50 [+ or -] 0.84 **, #   1.17 [+ or -] 0.75 *, #

Croups       Fibrosis

             14 days                 28 days

NS group     0.00 [+ or -] 0.00      0.00 [+ or -] 0.00
BLM group    2.50 [+ or -] 0.55 **   2.83 [+ or -] 0.41 **
Pred group   1.50 [+ or -] 0.55 *    1.67 [+ or -] 0.52 **, #
YTE group    1.17 [+ or -] 0.75      1.50 [+ or -] 0.55 *, #

* p < 0.05.

** p < 0.01, vs. NS group.

# p < 0.05, vs. BLM group.
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Author:Li, Liucheng; Li, Delin; Xu, Liang; Zhao, Ping; Denga, Ziyu; Mo, Xiaoting; Li, Ping; Qi, Lianwen; Li
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jan 15, 2015
Words:6040
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