The protective effect of hydrogen sulfide on systemic sclerosis associated skin and lung fibrosis in mice model
- Zhi Wang†1,
- Xiaoya Yin†1,
- Luyan Gao1,
- Sheng Feng1,
- Kai Song1,
- Lingyun Li1,
- Ying Lu1 and
- Huaying Shen1Email author
© The Author(s) 2016
Received: 11 April 2016
Accepted: 6 July 2016
Published: 15 July 2016
Systemic sclerosis (SSc) caused fibrosis can be fatal and it still lack of effective treatment. Hydrogen sulfide (H2S) appears to be an attractive therapeutic candidates. This study aimed to investigate the protective effect of H2S on SSc-associated skin and lung fibrosis.
We developed a model of SSc by subcutaneous injecting BLM to female C3H mice. The mice received daily subcutaneous injections of NaHS (56 and 112 μg/kg), an H2S donor. On days 7, 28, and 42, the mice were killed and blood samples were collected to measure the plasma H2S concentration, the skin and lung tissues was harvested for microscopic examination, immunohistochemistry and quantify biological parameters (hydroxyproline content, RT-qPCR and Western blot).
In model group, the dermis of skin tissues at different time points gradually thickened, collagen deposition increased. The lung tissues presented pathological changes such as obvious inflammatory cell infiltration, increased collagen deposition and the plasma H2S concentrations points significantly decreased. Administration of NaHS markedly decreased the biomarkers of fibrosis such as α-smooth muscle actin, collagen-I, collagen-III, fibronectin, transforming growth factor-β1, Smad2/3 phosphorylation and inflammation including the marker protein of monocyte/macrophage and monocyte chemoattractant protein-1 in the lung. Compared to the low dose group, the expression in the high dose group have decreased trend, but the difference was not significant.
We demonstrate the beneficial effects of H2S on SSc-associated skin and lung fibrosis. H2S may be a potential therapy against this intractable disease.
Systemic sclerosis (SSc) is a severe connective tissue disease of unknown etiology. SSc is characterized by multivisceral fibrosis resulting from inflammation, vascular injury and excessive collagen deposition. Skin sclerosis can cause discomfort and organ involvements—such as pulmonary fibrosis—can be fatal. However, the mechanisms of fibrosis in SSc have not been fully elucidated and effective drugs are still scarce (Walker et al. 2012). Immunosuppressants, used to treat SSc, can also cause important adverse effects, such as hepatic and renal functional lesion, bone marrow depressions and infecting etc. Therefore, new therapy options need to be found.
Hydrogen sulfide (H2S) is a newly recognized endogenous gasotransmitter analogic to nitric oxide and carbon monoxide (Wang 2012). As a new emerging gaseous signalling molecule, for a long time, its study was limited to its toxicity. In the 1990s, it was discovered that endogenous H2S participated in anti-inflammation, anti-oxidation, and the regulation of cell proliferation and apoptosis (Gao et al. 2012; Moody and Calvert 2011; Vandiver and Snyder 2012). Past studies have confirmed that exogenous H2S attenuates aortic-coarctation-induced cardiac hypertrophy and fibrosis (Huang et al. 2012), inhibit renal interstitial fibrosis caused by obstructive nephropathy (Song et al. 2014), relieve peritoneal mesothelial cell injury caused by high-glucose peritoneal dialysis fluids (Lu et al. 2015) and carbon tetrachloride-induced cirrhosis (Tan et al. 2011). The potential mechanisms of H2S anti-fibrotic may be account for inhibiting the activation and migration of inflammatory cells likewise myofibroblasts, subsequently decrease the production of pro-inflammatory cytokines and extracellular matrix accumulation (Li et al. 2008; Tan et al. 2011). Therefore, we hypothesized that H2S may against organ fibrosis caused by immune reaction. Importantly, the present of H2S in a variety of mammalian tissues and organs support it may be without overt adverse effect.
We conduct this study to investigate the effect of H2S on SSc-associated skin and lung fibrosis in mice model established by subcutaneous injections with BLM, and also examined the underlying anti-fibrotic mechanisms.
Establishment of animal models
After adaptive feeding for 1 week, 75 clean-grade female C3H mice with a body weight of 18–22 g (SLAC, Shanghai) were randomly divided into 5 groups: (1) control group: local subcutaneous injection of PBS 0.1 ml + intraperitoneal injection of PBS 1 ml; (2) model group: local subcutaneous injection of 1 mg/ml bleomycin (BLM, Nippon Kayaku Co. Ltd.) 0.1 ml + intraperitoneal injection of PBS 1 ml; (3) low dose treatment group: local subcutaneous injection of 1 mg/ml BLM 0.1 ml + intraperitoneal injection of 56 μg/kg/d NaHS (Sigma, USA); (4) high dose treatment group: local subcutaneous injection of 1 mg/ml BLM 0.1 ml + intraperitoneal injection of 112 μg/kg/d NaHS; (5) H2S group: local subcutaneous injection of PBS 0.1 ml + intraperitoneal injection of 112 μg/kg/d NaHS. BLM (0.1 ml) dissolved in PBS at a concentration of 1 mg/ml, was subcutaneously each day for 4 consecutive weeks. Intraperitoneal injection of 56 μg/kg/d or 112 μg/kg/d NaHS began at the same time with BLM injection and lasted for 6 weeks. Five mice in each group were randomly sacrificed on days 7, 28, and 42. Serum samples of mice were collected to measure the H2S concentrations. Skin and lung tissues of mice were collected for pathological staining usinghaematoxylin-eosin (HE) stain and Masson stain to observe the degrees of inflammation and fibrosis in lung tissues. The alkaline hydrolysis method was performed to determine the concentrations of hydroxyproline (HYP) in lung tissues (Jiangcheng Bioengineering Institute, Nanjing). PCR and western blot were performed to detect the expression of fibrosis indicators such as α-smooth muscle actin (α-SMA), collagen-I (Col-I), collagen-III (Col-III), fibronectin (FN), transforming growth factor-β1 (TGF-β1) and Smad2/3 phosphorylation (p-Smad2/3). Immunohistochemistry was performed to detect the expression of inflammatory cytokines including the marker protein (ED-1) of monocyte/macrophage and monocyte chemoattractant protein-1 (MCP-1). The α-SMA, FN and p-Smad2/3 antibodies antibodies were purchased from Santa Cruz; all other antibodies were purchased from Abcam.
Skin tissues and left lung tissues from the injection locations in the neck and back of mice were fixed in 4 % paraformaldehyde, embedded in paraffin, and cut into 5 μm sections. HE and Masson stains were performed to observe the changes of gross morphology, inflammation, and collagen deposition.
Measurement of the plasma H2S levels
The NaHS standard was used for a twofold serial dilution to obtain concentrations from 1.56 to 100 μM. The DNA-Az (H2S probe) was dissolved in anhydrous ethanol to obtain the final concentration of 2.2 mM. One black 96-well plate was obtained, and 10 μl of 2.2 mM DNS-Az stock solution was then added toeach well followed by 100 μl fresh plasma or the NaHS standard immediately to obtaina DNS-Az concentration of 200 μM. The absorbance value of each well was measured in a microplate reader. The excitation wavelength was 360 nm, and the emission wavelength was 528 nm. The NaHS results were used to plot a standard curve. The absorbance values of plasma wells were compared with the standard curve to obtain the plasma H2S levels.
Measurement of HYP concentration
Lung tissues of mice at a wet weight of 30–100 mg were collected. The HYP levels in lung tissues were detected using alkaline hydrolysis colorimetry. Lung tissues were hydrolysed using an alkaline solution. Chloramine T, perchloric acid, and 4-(dimethylamino) benzaldehyde were sequentially added. After mixing thoroughly, samples were incubated in a 60 °C water bath for 15 min. The samples were cooled down and centrifuged, and the supernatant was collected. The absorbance values at 550 nm wavelength and 1 cm optic path were measured. Calibration was performed using a blank tube and a standard tube. The results arepresented as μg/mg.
Primers for polymerase chain reaction
94 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min (30)
94 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min (30)
94 °C, 30 s; 52 °C, 30 s; 72 °C, 1 min (30)
94 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min (30)
The frozen right lung tissues of mice were added to the tissue lysis solution. After homogenization and centrifugation, the supernatant was collected. Protein concentrations were determined using the BCA method, and the protein samples were stored in a −80 °C freezer for future usage. A 10 % polyacrylamide gel was prepared for electrophoresis. After the samples were transferred to a membrane and blocked, the primary antibody was added (1:1000 dilution) and incubated at 4 °C overnight. The secondary antibody was then added (1:5000 dilution) and incubated at room temperature for 1 h. The results were developed, and fibrosis indicators such as α-SMA, FN, TGF-β1 and p-Smad2/3 were detected.
The streptavidin–biotin peroxidase complex (ABC) method was used for immunohistochemistry. The dilution of the primary antibodies was 1:100 for ED1and 1:100 for MCP-1. Five fields were randomly selected under a microscope (400×). After the positive regions were localized, images were taken using a colour camera. The results were input into a colour image analysis system. After the positive regions were precisely segmented, the quantities of ED1 and MCP-1 positive cells were measured, and the mean values were calculated.
Measurement data are presented as mean ± SD. Comparison among multiple groups was performed using one-way analysis of variance. A threshold α = 0.05 was used as the detection level. Data were analysed using the SPSS17.0 software.
Pathological changes in skin tissues
Pathological changes in lung tissues
Changes in the plasma H2S concentrations
H2S reduces the expression level of HYP in the lung tissues of mice induced by BLM on the 42th day
H2S inhibits the expression of α-SMA, Col-I, Col-III and FN in lung tissues on the 42th day
H2S reduces the number of ED-1-positive cells and MCP-1-positive cells in lung tissues on the 7th day
H2S inhibits the expression of TGF-β1 and p-Smad2/3 in lung tissues on the 28th day and the 42th day
The present study demonstrated that inflammatory cell infiltration in skin and lung tissues increases and the synthesis of fibrogenic cytokines increases in a BLM-induced SSc mice model. Previous studies have suggested that exogenous H2S exerted a protective effect of pulmonary fibrosis by attenuating lipid peroxidation injury (Fang et al. 2009). And this study confirmed that exogenous H2S could relieve SSc through an anti-inflammation and anti-fibrosis mechanism, which provided a novel therapeutic approach to SSc.
In the present study, the mice model of SSc was established by subcutaneous injection of BLM. Consistent with previous studies (Yamamoto and Nishioka 2002; Yamamoto et al. 1999), we could observe the destruction of the structure of skin and lung tissues, such as disordered structure, increased inflammatory cell infiltration, collagen deposition, significant skin and pulmonary fibrosis in mice from the model group at different time points. Furthermore, studies confirmed that the skin and lung tissues of SSc mice exhibited increased inflammatory cell filtration and upregulation of TGF-β1 expression at the early stage (Bienkowski and Gotkin 1995; Bonniaud et al. 2005). This study confirmed that BLM could induce inflammatory cell filtration and increase of the expression of fibrogenic factors in skin and lung tissues, these findings were consistent with previous studies indicated that the SSc mice model induced by BLM in this study was successfully established.
The relation of H2S and SSc associated fibrosis is not well defined. The results of this study suggested that the plasma H2S levels are decreased in the model group after subcutaneous injection of BLM. Which was consistent with the conclusion of previous studies showing that “organ fibrosis might be associated with H2S deficiency” (El-Seweidy et al. 2011; Tan et al. 2011). Therefore, we investigate the role of H2S on SSc-associated skin and lung fibrosis.
Our study indicate that H2S exhibited an anti-inflammatory property by reducing macrophage recruitment in lung tissues. The inflammatory cytokines such as ED-1 and MCP-1 were downregulated by NaHS in the 7-day group. Substantial data have suggested that H2S is protective against inflammation in multiple fibrosis-related diseases (Babaei-Karamshahlou et al. 2012; Huang et al. 2012; Lu et al. 2015; Song et al. 2014; Whiteman and Winyard 2011). Our team previous study indicated that administration at a relatively low dose (56 μg/kg/d) exogenous H2S could inhibit renal interstitial mononuclear cell infiltration and TNF-α expression in a unilateral ureteral obstructive (UUO) model, suggesting that H2S could exert its anti-inflammation function through the inhibition of the activation of macrophages (Song et al. 2014). Therefore, we speculated that the anti-pulmonary fibrosis function of H2S was associated with its anti-inflammation function. Inflammation plays an important role at the initial stage of systemic sclerosis, and inflammatory cell infiltration is the upstream event and one of the significant pathological features of organ fibrosis. The above study indicated that H2S inhibits the progression of skin and lung fibrosis through its anti-inflammation function.
In addition to the factor of inflammatory reaction, cytokines particular TGF-β1 also play an important role in the occurrence and development of fibrosis diseases (Flanders and Burmester 2003; Pittet et al. 2001). Substantial data have demonstrated that TGF-β1 promotes the transdifferentiation of fibroblasts into myofibroblasts (Scotton and Chambers 2007). Our results showed that NaHS reduced the expression level of HYP and suppressed mRNA and protein expressions of a-SMA, FN, Col-1and Col-3 induced by BLM in the lung tissues. Importantly, NaHS also attenuated the protein expressions of TGF-β1 at 28 and 42 days. These data suggested that H2S inhibited the fibroblast differentiation and extracellular matrix production partially by blocking the expression of TGF-β1. It is well known that TGF-β1 to be working main through its signal protein p-Smad2/3. Our study showed that after NaHS administration, the protein expression level of p-Smad2/3 in the treatment group was significantly downregulated on day 42. These results consistent with the previous study (Bei et al. 2013). This finding suggests that the anti-fibrotic effect of H2S may be connected with its inhibition of TGF-β/Smads pathway.
The above study confirmed that exogenous H2S had a protective function on SSc-related organ fibrosis. And importantly the present of H2S in a variety of mammalian tissues and organs support it may be without overt adverse effect. However, currently acquirable H2S donors like NaHS used in present study are not suitable for therapy because they release H2S uncontrolled. Tries are being made to obtain more appropriate H2S precursors for therapeutic aim. GYY4137 and SG1002 are two administrated H2S-releasing compounds that have been proven to be beneficial in various diseases such as hepatocellular carcinoma, diabetes, and chronic heart failure (Kondo et al. 2013; Lu et al. 2014; Wei et al. 2014). Such compounds may become useful for treatment of SSc-related organ fibrosis.
In conclusions, this study confirmed that H2S could improve SSc-related organ fibrosis. The mechanism was achieved through the inhibition of the inflammatory reaction and the reduction of TGF-β1 expression, thus reducing the accumulation of extracellular matrix. This study provides new insight into the treatment of SSc-related organ fibrosis in the clinic. A detailed understanding of the molecular mechanism and the related cell signalling pathways still awaits further study.
ZW participated in conception, design, acquisition, analysis and interpretation of data and drafted the manuscript. XYY participated in acquisition of data and samples, analysis and interpretation of data and drafted the manuscript. LYG and SFparticipated in design, statistical analysis and interpretation of data. KS, LYL and YL participated in analysis and interpretation of data and were also involved in drafting the manuscript and revising it. HYS participated in conception, design, analysis and interpretation of data and was also involved in drafting the manuscript and revising it. All authors read and approved the final manuscript.
This work was supported by grants from the National Nature Science Foundation of China (81302584, 81400762 and 81200495).
The authors declare that they have no competing interests.
The authors can confirm that they have read and understood the contents of the section on compliance with ethical standards of the journal. The study complies with current animal rights and ethical consideration.
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