Anti-fibrosis effect for Hirsutella sinensis mycelium based on inhibition of mTOR p70S6K phosphorylation

Animals

Six–eight-wk-old C57BL/6J mice (male) weighing between 18 g and 22 g were purchased from the Animal Model Research Institute of Nanjing University (Nanjing, China) and fed for at least 1 wk before use. Procedures for animal care and use in the study were approved by the Ethics Committee of Animal Experiments of Nanjing University. The animals were kept under light–dark cycles of 12 h each at 21–23℃.

Preparation of the HSM

The HSM was originally obtained from Nanjing Zhongke Group (Nanjing, China), The HSM solution was prepared by dissolving HSM powder into saline at a concentration of 40 mg/ml, at 37℃ for 2 h, cooled to room temperature and centrifuged (Thermo Fisher Scientific, Waltham, MA, USA) at 500 g for 10 min at 4℃. Then, transport the supernatant to 5 mm filter (Millipore, Billerica, MA, USA) for sterilization and stored at –20℃ until use.

PF induced by bleomycin

Forty-five mice were randomly divided into four groups: six mice in normal control group (saline), six mice in HSM control groups (saline + 200 mg/kg HSM), 13 mice in bleomycin (BLM) group (4 mg/kg BLM + saline), 13 mice in BLM + HSM groups (4 mg/kg BLM + 200 mg/kg HSM). The HSM solution was prepared as described above. Lung fibrosis was induced by intratracheal instillation of BLM (Nippon Kayaku, Tokyo, Japan) at a single dose of 4 mg/kg on d 0 in 50 µl sterile saline. Control groups were treated with the same volume of sterile saline. The mice received HSM (200 mg/kg) or saline intra-gastrically daily for 21 d 2 h before BLM treatment. One animal died during the course, and the others were sacrificed at the end of 21 d after BLM or saline treatment. Bronchoalveolar lavage fluid (BALF) samples were obtained by washing the right lungs twice with 1 ml PBS, and stored on ice. Subsequently, the samples were centrifuged (500 g, 10 min, 4℃), the supernatants were stored at –80℃ for later analysis, the cells were re-suspended for flow cytometric analysis. The left lungs were removed for overnight (18 h) fixation with paraformaldehyde or storage at –80℃.

ELISA

TGF-β1 in the BALF of mice was measured using ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Flow cytometric analysis

Peritoneal macrophages were washed and fixed with 4% paraformaldehyde, followed by staining with PE-conjugated F4/80 and FITC-conjugated CD11c. Mouse spleen cells were isolated, passed through a 200-mesh sieve, centrifuged and re-suspended in red blood cell lysing buffer for 2 min. For intracellular IFN-γ, IL-4 or IL-17 staining, cells were incubated with 5 ng/ml PMA (Invitrogen, Carlsbad, CA, USA), 1 ng/ml ionomycin (Enzo Life Sciences, Solna, Sweden) and 10 ng/ml brefeldin A (Enzo Life Sciences) for 4 h. After washing, cells were stained with CD4-FITC (eBiosciences, San Diego, CA, USA). After permeabilization using the Cytofix/Cytoperm buffer (BD Biosciences, San Diego, CA, USA), cells were stained with IFN-γ–PE and IL-4–PE (eBiosciences). For intracellular Foxp3–PE staining, the cells were stained with CD4–FITC and CD25–APC (eBiosciences) after permeabilization. The fluorescence was measured by FACS Calibur (BD Biosciences), and data analyzed by FlowJo software (TreeStar, Ashland, OR, USA).

Quantitative real-time PCR

Total RNA was extracted by TRIzol reagent (Invitrogen). One µg RNA was reverse-transcribed into cDNA using a ReverTra Ace-a-RNeasy kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed on the Applied BioSystems Step-one plus PCR machine (Bio-Rad, Hercules, CA, USA) using SYBR Green PCR Master Mix (Invitrogen). Dissociation curve was analyzed for exclusion of amplification plot. The primer used in the PCR amplification were synthesized by Invitrogen and the sequences were as follows: mouse GAPDH,5’-AACGACCCCTTCATTGAC-3’(forward) and 5’-TCCACGACATACTCAGCAC-3’(reverse); mouse α-SMA:5’-CCCAGACATCAGGGAGTAATGG-3’(forward) and 5’-TCTATCGGATACTTCAGCGTCA-3’(reverse); mouse vimentin: 5’-CGTCCACACGCACCTACAG-3’(forward) and 5’-GGGGGATGAGGAATAGAGGCT-3’(reverse); mouse fibronectin: 5’-ATGTGGACCCCTCCTGATAGT-3’(forward) and 5’-GCCCAGTGATTTCAGCAAAGG-3’ (reverse); mouse mTOR: 5’-ACCGGCACACATTTGAAGAAG-3’(forward) and 5’-CTCGTTGAGGATCAGCAAGG-3’(reverse); human GAPDH: 5’-AGAAGGCTGGGGCTCATTTG-3’(forward) and 5’-AGGGGCCATCCACAGTCTTC-3’(reverse); human fibronectin: 5’-CGGTGGCTGTCAGTCAAAG-3’(forward) and 5’-AAACCTCGGCTTCCTCCATAA-3’(reverse); human vimentin: 5’-GACGCCATCAACACCGAGTT-3’(forward) and 5’-CTTTGTCGTTGGTTAGCTGGT-3’(reverse).

Histology and immunohistochemistry

Lung tissue sections were fixed with 4% paraformaldehyde and then were embedded in paraffin. Paraffin sections (3 µm) were dehydrated by a gradient series of ethanol, and processed for histology and immunohistochemistry staining. Masson’s trichrome staining was performed and observed by optical microscope. Immunostaining was carried out as follows: blocking with 5% BSA, incubated with primary Abs overnight at 4℃ and then incubation with secondary Abs for 1 h at room temperature (20–25℃). The bound secondary Abs were visualized with DAB substrate. Primary Abs used in the experiment included anti-α-SMA (1:200; Abcam, Cambridge, UK) and anti-p-P70S6K (1:200; Cell Signaling Technology, Danvers, MA, USA).

Cell viability analysis

A549 cell line was purchased from Cell Bank, China Academy of Sciences. A549 cells were cultured in DMEM complete medium [supplemented with 10% (v/v) heat-inactivated FBS, 1% penicillin streptomycin] at 37℃ under 5% CO₂. Cells were plated in 96-well plates at a density of 2 × 10³ cells/well, followed by treatment with HSM (100–800 µg/ml) and/or TGF-β1 (10 ng/ml) for 24 h. The cell viability was assessed by a Cell Counting Kit 8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol.

Western blotting

Western blotting was performed as described previously. The primary Ab including anti-mTOR, anti-p-mTOR, anti-p70S6K, anti-p-p70S6K, anti-GAPDH and anti-tubulin Abs were purchased from Bioworld Technology (St. Louis Park, MN, USA) and Cell Signaling Technology.

Detection of HYP in lungs

Briefly, 30–100 mg lung tissue samples were cut into pieces, and then hydrolyzed in 1 ml lysis buffer solution at 100℃ for 20 min. Impurities were absorbed by activated carbon. The pH of the supernatant was adjusted according to manufacturer's protocol. The absorbance of the colored products was measured at 550 nm. HYP was then estimated following the manufacturer's instructions. The kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Statistical analysis

All data in this experiment are presented as mean ± SEM. The statistical analysis in our research was completed by the GraphPad Prism 5 software and based on Turkey test or t-test using. P < 0.05 was considered statistically significant. Experiments were repeated at least three times, and each data point represents the mean of at least three samples.

Lung injury induced by BLM was attenuated by HSM

In the present study, we selected administration of BLM as a model of lung fibrosis to explore whether HSM has a protective effect on PF.

We performed Masson’s and immunohistochemical staining of lungs from each group. As observed by hematoxylin and eosin staining and Masson staining, BLM-treated mice histologically showed serious pulmonary injury and fibrosis than in the control group. HSM alleviates the injury and fibrosis and decreases the deposition of collagen (Figure 1b). α-SMA was mildly expressed in vascular and airway muscle cells in the control group (Figure 1b). By contrast, the expression of α-SMA was significantly increased in the BLM-treated group (Figure 1b), indicating that a significant number of myofibroblasts were trans-differentiated. OPEN IN VIEWER

Furthermore, the lung HYP content in the lung of BLM-treated mice was higher than that in the control group (Figure 1c). Treatment with HSM significantly reduced the lung HYP content in the lung.

TGF-β1 is regarded as a key factor of the pro-fibrogenic molecules. Thus, we assayed the TGF-β1 protein level in the BALF, which was found to be significantly higher in BLM-treated mice than that in the control group. The TGF-β1 levels were suppressed by HSM treatment (Figure 1d). To explore whether TGF-β1 is activated or not, we performed Western blot analysis, which showed that HSM decreased the level of p-Smad1/2 (Figure 1e), suggesting that HSM impeded the activation of TGF-β1 induced by BLM in the lung sections.

Furthermore, we examined the mRNA expression of several classic pro-fibrotic mediators (including α-SMA, fibronectin and vimentin) (Figure 1f). The expression of these pro-fibrotic cytokines was significantly increased compared with those in the control group and decreased by HSM treatment. Together, these results suggested that HSM improved BLM-induced lung fibrosis in wild type C57BL/6 J mice.

HSM suppresses the chemotaxis of alveolar macrophages and the expression of inflammatory mediators in lungs of BLM-treated mice

Alveolar macrophages are ideally placed to initiate a strong inflammatory response to the pathogenic material. Hence, we assessed the percentage of alveolar macrophages in each groups’ BALF by flow cytometric analysis. The results showed that CD11c⁺ F4/80⁺ alveolar macrophages significantly increased compared with those of the control group. However, the HSM treatment group obviously decreased the percentage of the macrophage population (Figure 2a, b), indicating that HSM hindered the chemotaxis of alveolar macrophages during PF. OPEN IN VIEWER

To further confirm the possibility that the anti-PF effect of HSM was associated with the inhibition of early inflammation in the lung, we examined the expression of inflammatory mediators, including IL-1β and IL-6 by Q-PCR (Figure 2c). The expression of these inflammatory cytokines in BLM group was significantly higher than those in control group and decreased by HSM treatment (Figure 2c).

HSM influenced the immune imbalance in PF mice induced by BLM treatment

Compared with controls, mice treated with BLM exhibited a higher proportion of Th2 (Figure 3b) and Treg (Figure 3c) cells, but lower Th1 (Figure 3a) and Th17 (Figure 3d) cells. HSM treatment reversed the imbalance, indicating that BLM can disrupt the balance of Th1/Th2 expression in the spleen and HSM may ameliorate the BLM-induced PF in mice by regulating the Th1/Th2 improving immunity. OPEN IN VIEWER

HSM alleviates mTOR overactivation in fibroblast foci of BLM-treated mice

Fibroblast foci have been recognized as an indicator of PF. Therefore, the mTOR activation in fibroblast foci during PF was investigated. As shown in Figure 4(a, b), HSM significantly alleviated mTOR overactivation both in mRNA and protein level. Then we performed immunohistochemical staining of p-p70S6K from each group lung sections (Figure 4c). p-p70S6K in the control group was found to be mildly expressed in vascular and airway muscle cells, whereas the protein expression remarkably improved in lungs from BLM-treated mice. HSM inhibited the phenotype, indicating that HSM alleviated overactivation of the mTOR signaling pathway. Western blotting also indicated that p-p70S6K was weakly expressed in the control lung tissue (Figure 4d). However, the protein was strongly increased in BLM-treated lung tissue, and HSM significantly suppressed the expression. We concluded that the mTOR signaling pathway was overactivated in the fibroblast foci in BLM-treated PF lung sections, and HSM alleviates PF may be associated with suppressing the overactivation in fibroblast foci. OPEN IN VIEWER

HSM suppresses mTOR activation stimulated by recombinant TGF-β1 in A549 cells during fibroblast–myofibroblast trans-differentiation

We first assessed the cellular toxicity of HSM in A549 cells through CCK-8 assay. As shown in Figure 5(a), up to 800 µg/ml 24-h treatment with HSM did not reduced cell viability obviously in resting A549 or TGF-β1-activated A549 cells. Resting A549 cells normally displayed a layer of cobblestone-like morphology but turned into spindle fibroblast-like appearance after stimulation by recombinant TGF-β1 (10 ng/ml) for 24 h. After treatment with HSM for 24 h, the spindle fibroblast-like phenotype was found to be reversed (Figure 5b). The expression of fibronectin and vimentin in TGF-β1-activated A549 cells was significantly increased compared with those in control group. Strikingly, the expression of these cytokines was reduced by HSM treatment (Figure 5c), indicating that TGF-β1-induced myofibroblast activation had been inhibited. OPEN IN VIEWER

To observe mTOR activation during the fibrosis process in vitro, we assessed the expression of p-mTOR and p-p70S6K protein in resting A549 or activatedA549 cells by Western blotting. After 24 h stimulation, p-p70S6K expression was elevated, while HSM treatment obviously reduced the expression of p-p70S6K, indicating that overactivated mTOR signaling stimulated by TGF-β1 was suppressed by HSM treatment (Figure 5d).