Contributed equally
Interstitial lung disease is the most common complication of systemic sclerosis (SSc) and is associated with a high rate of mortality. Due to the complex pathogenesis of SSc, the therapies currently available remain limited. In the present study, the effect of asiatic acid (AA) on SSc-associated pulmonary fibrosis (PF) and its association with the transforming growth factor-β1 (TGF-β1)/Smad2/3 signaling pathway were evaluated. A hypochlorous acid (HOCl)-induced model of SSc was used to evaluate the therapeutic effect of AA on PF in SSc, where AA was administered to SSc mice by gavage. PF was alleviated in the AA-treated SSc mice groups when examined under light microscopy. In addition, there was a decrease in histopathological progression and collagen in the lungs. AA significantly reduced expression of type I collagen in the lungs of mice with SSc. It also significantly suppressed α-smooth muscle actin expression, which attenuated the conversion of fibroblasts into muscle fibroblasts. These AA-associated antifibrosis and anti-immune effects were mediated through the significant downregulation of advanced oxidation protein product, E-selectin, and anti-DNA topoisomerase-1 autoantibody levels in the serum. Furthermore, the expression levels of TGF-β1 and the phosphorylated-Smad2/3/Smad2/3 ratios in AA-treated SSc mice were similar to the control. The presence of pulmonary inflammation and fibrosis was confirmed in the HOCl-induced SSc mice and the results demonstrated that selective inhibition of reactive oxygen species prevented PF. By focusing on the classical TGF-β1/Smad2/3 signaling pathway, a mechanism of action of AA was identified to be associated with the inhibition of Smad2/3 activation through negative regulation of Smad2/3 phosphorylation.
Systemic sclerosis (SSc), also known as scleroderma, is a multisystem connective tissue disease, and its pathogenesis is associated with several factors, including inflammation, autoimmune antibodies, extensive fibrosis and microvascular changes (
Asiatic acid (AA) is a triterpenoid extracted from
Overall, it is essential to characterize the pathogenesis of SSc and associated interstitial pneumonia, as well as identify a fibrosis-inhibiting drug that is therapeutically effective with few side effects. In the present study, whether AA ameliorates PF, and modulates myofibroblast differentiation, immune dysfunction and oxidative stress was determined. In addition, it was determined whether any of these effects are mediated through the classical TGF-β1/Smad2/3 signaling pathway.
A total of 40, six-week-old female-specific pathogen-free BALB/c mice with a mean weight of 25 g were housed at the Experimental Animal Center at Wenzhou Medical University (Zhejiang, China) at 20–24°C with 40–60% humidity and with a regular light-dark cycle. The animals were allowed free access to water and standard mouse chow. The present study was approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. Efforts were made to minimize animal suffering and the number of animals used in experiments.
Sodium hypochlorite (NaClO), potassium dihydrogen phosphate (KH2PO4) solution, sodium carboxymethylcellulose (CMC-Na) and AA were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). AA was dissolved in dimethyl sulfoxide, and then diluted with a 0.5% CMC-Na solution to create separate solutions with concentrations of 0.1 and 0.4 mg/ml. Advanced oxidation protein products (AOPP; 10572-09m), E-selectin (E-sel; 10316-09m) and anti-DNA topoisomerase I autoantibody (TOP1-Ab; 10821-09m) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Shanghai Boyun Biochemical Institute (Shanghai, China;
HOCl was synthesized by adding 166 µl NaClO solution (2.6% active chlorine) to 11.1 ml KH2PO4 solution (100 mM; pH 7.2) as previously described (
The mice were randomly divided into control, model, treatment with a low dose of AA of 2 mg/kg/day (LAA), and treatment with a high dose of AA of 8 mg/kg/day (HAA) groups (n=10 mice/group). AA was dissolved in a 0.5% CMC-Na solution to create separate solutions with concentrations of 0.1 and 0.4 mg/ml. Mice in the model group received 300 µl HOCl subcutaneously and 0.5% CMC-Na solution by gavage every day for 6 weeks, while mice in the control group received 300 µl sterilized PBS subcutaneously and 0.5% CMC-Na solution orally every day for 6 weeks. Mice in the LAA and HAA groups received 0.5 ml AA solution (2 and 8 mg/kg/day, respectively) by gavage, as well as received HOCl injections as aforementioned for the model group.
Two weeks after treatment was halted, the mice were sacrificed, and their lungs and serum harvested. Lower right lung lobe samples were washed with cold PBS, fixed in 4% buffered paraformaldehyde at 4°C for 24 h, and then embedded in paraffin for histological and immunohistochemical studies. The remaining lung tissue was snap-frozen in liquid nitrogen and stored at −80°C until processed for protein extraction. Expression of AOPP, E-sel, and TOP1-Ab was measured in the collected mouse serum.
The lower right lobes of the collected lungs were fixed in 4% paraformaldehyde at 4°C overnight, dehydrated in a graded ethanol series, embedded in paraffin, and then sectioned into 5-µm thick slices. Histopathological changes were assessed using hematoxylin and eosin (H&E) staining. Tissue sections were deparaffinized in xylene and hydrated gradually through graded alcohol series and processed with PBS buffer solution. Hematoxylin was applied for 3 min in room temperature, and then rinsed in H2O for 5 min. Eosin was used for staining for 1 min in room temperature, and then H2O for 1 min. The sections were dehydrated with an ascending alcohol series (75, 85, 95 and 100%) for 3 min each, and then xylene was used twice for 3 min each. Another set of sections was stained to visualize interstitial collagen using Masson's trichrome method (
Expression levels of AOPP, E-sel and TOP1-Ab in serum were measured using the relevant ELISA kits according to the manufacturer's protocol. A total of 50 µl of each standard and sample were added into the appropriate well. 10 µl Biotinylated antibodies to AOPP and E-sel, and antigen to TOP1 were coated to samples separately. 50 µl chain enzyme avidin-HRP was added and incubated at 37°C for 60 min, followed by washing four times with washing buffer (provided in the kit) for 30 sec. Developer (100 µl) was added and kept in the dark at 37°C for 10 min. The stop solution (50 µl) was added to each well to terminate the reaction. The optical density of each sample was read at 450 nm. The results were calculated using the linear regression equation based on the standard curve.
The lungs were homogenized in RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) (RIPA: PMSF 100:1) and then centrifuged at 15,000 × g for 30 min at 4°C. The resulting supernatants were collected and determined using the BCA protein assay. Subsequently, 20 µg of protein/lane was separated using 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. The membranes were then blocked at room temperature for 1 h with 5% milk and incubated overnight at 4°C with primary antibodies against TGF-β1 (1:500), Smad2/3, and p-Smad2/3 (1:500) and GAPDH (1:500). Subsequently, the membranes were incubated with the relevant HRP conjugated secondary antibody (goat anti-rabbit IgG for GAPDH, 1:10,000; goat anti-mouse IgG for the other primary antibodies, 1:10,000) at room temperature for 1 h and then developed using Clarity Western ECL Substrate (cat. no. 1705060; Bio-Rad Laboratories, Hercules, CA, USA). Images were collected and analyzed using the Image Lab program 5.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Data are expressed as the mean ± standard deviation. All calculations were performed using SPSS version 19.0 (IBM Corp., Armonk, NY, USA). Differences between groups were identified by one-way analysis of variance (ANOVA) with post hoc contrasts by Student-Newman-Keuls test. P<0.05 was considered statistically significant.
In the model group, the lungs were pale and stiff, and were harder compared with lungs from the control group following treatment for 6 weeks with HOCl (
The therapeutic antifibrotic effects of AA on lung pathology was assessed in a HOCl-induced SSc mouse model. Immunohistochemical staining was used to evaluate α-SMA and Col-I expression in lungs prior to and following AA treatment (
To evaluate the effects of AA on oxidative stress, damage to the epithelium and vessels, and immune response, AOPP, E-sel and TOPI-Ab expression was evaluated in serum from the mice (
To identify the potential mechanism underlying the effect on AA treatment of mice with SSc and SSc-associated ILD, TGF-β1/Smad2/3 signaling way was examined by measuring TGF-β1 expression (
SSc is a multisystem disease with a variable clinical course (
It was previously demonstrated that ROS serve an essential role in SSc-associated ILD pathogenesis (
TGF-β is the most potent profibrogenic cytokine and its expression is increased in almost all fibrotic diseases (
AA reduces the occupancy of Smad2/3 elements in response to TGF-β. Previous studies have reported that AA treatment inhibits TGF-β1 and Smad2/3 expression in cardiac hypertrophy, and liver and renal interstitial fibrosis (
In conclusion, the results of the present study confirmed the presence of pulmonary inflammation and fibrosis in a murine model of HOCl-induced SSc, and demonstrated that selective inhibition of ROS reduces PF in this model. By focusing on the classical TGF-β1/Smad2/3 signaling pathway, it was observed that AA significantly inhibited the phosphorylation and, thus, the activation of Smad2/3. This phenotype was even more significant when high concentrations of AA were used. This pathway may be one of the most important ways in which AA affects SSc-associated ILD. A drug similar to AA that regulates the TGF-β1/Smad2/3 signaling pathway may be a novel therapeutic for treatment of SSc.
The authors would like to thank Dr Kate Huang (The First Affiliated Hospital of Wenzhou Medical University) and Dr Jianbo Wu (The First Affiliated Hospital of Wenzhou Medical University) in the Department of Pathology for expert technical assistance and the Dr Yicheng He (Wenzhou Medical University) and Dr Zhenni Zhou (Wenzhou Medical University) for help with the mice experiments.
The present study was supported by a grant from the Wenzhou Science and Technology Bureau Project, Zhejiang Province, China (grant no. Y20140250).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
LW and YT conceived and designed the experiments; XX and CD performed the animal experiments; YT, HY performed the pathological examination; XH analyzed the data; AC contributed materials and western blot analysis; XX, CD and LW wrote the paper.
The present study was approved by the institutional Animal Care and Use committee of Wenzhou Medical University (wydw2016-0067).
Not applicable.
The authors have declared that they have no competing interests.
Histological evaluation of mouse lungs. Representative pathological findings from lungs from each cohort stained with H&E and Massome's trichrome at ×100 (top line) and ×400 (bottom line) magnification. Scale bar, 100 µm. H&E, hematoxylin and eosin; AA, asiatic acid; LAA, low dose of AA (2 mg/kg/day); HAA, high dose of AA (8 mg/kg/day).
Effect of AA on α-SMA and Col-I expression in SSc and normal lungs. The effect of AA on (A) α-SMA and (B) Col-I expression in each mouse group. (C) Immunohistochemical staining was used to evaluate α-SMA and Col-I expression in mouse lungs. Images are presented at magnification, ×100. **P<0.01, and ***P<0.001, compared with the model group. ###P<0.001, compared with the LAA group. Image-ProPlus software was used to calculate the relative expression levels. Scale bar, 100 µm. AA, asiatic acid; LAA, low dose of AA (2 mg/kg/day); HAA, high dose of AA (8 mg/kg/day); α-SMA, α-smooth muscle actin; Col-I, type I collagen; OD, optical density.
Effect of AA on AOPP, E-Sel, TOP1-Ab, TGF-β1 and p-Smad2/3 expression levels. Effects of AA on (A) AOPP, (B) E-Sel and (C) TOP1-Ab levels in mouse serum. (D) TGF-β1 and (E) p-Smad2/3/Smad2/3 levels were measured by western blotting. **P<0.01, and ***P<0.001, compared with the model group. #P<0.05, ##P<0.01, and ###P<0.001, compared with the LAA group. AA, asiatic acid; LAA, low dose of AA (2 mg/kg/day); HAA, high dose of AA (8 mg/kg/day); TGF-β1, tumor growth factor-β1; p-, phosphorylated; AOPP, advanced oxidation protein products; E-sel, E-selectin; TOP1-Ab, anti-DNA topoisomerase I autoantibody.