Thymosin β4 Suppresses LPS-Induced Murine Lung Fibrosis by Attenuating Oxidative Injury and Alleviating Inflammation

Inflammation plays a critical role in the progression of pulmonary fibrosis. Thymosin β4 (Tβ4) has antioxidant, anti-inflammatory, and antifibrotic effects. Although the potent protective role of Tβ4 in bleomycin-induced pulmonary fibrosis has been validated, the underlying mechanism is not clear; moreover, the influence of Tβ4 on lipopolysaccharide (LPS)-induced lung injury/fibrosis has not been reported. Expression of Tβ4 in fibrotic lung tissues was assessed by real-time quantitative reverse-transcription PCR (rt-PCR), immunohistochemistry (IHC), and western blotting. The effects of intraperitoneal adeno-associated virus-Tβ4 (AAV-Tβ4) on LPS-induced lung injury and fibrosis were observed through the evaluation of collagen deposition and α-smooth muscle actin (SMA) expression. In vitro tests with HPAEpiC and HLF-1 cells were performed to confirm the effects of Tβ4. In this study, we evaluated the role of Tβ4 in pulmonary fibrosis and explored the possible underlying mechanisms. Tβ4 was markedly upregulated in human or mouse fibrotic lung tissues. AAV-Tβ4 markedly alleviated LPS-induced oxidative damage, lung injury, inflammation, and fibrosis in mice. Our in vitro experiments also showed that LPS inhibited mitophagy and promoted inflammation via oxidative stress in HPAEpiC, and Tβ4 significantly attenuated LPS-induced mitophagy inhibition, inflammasome activation, and transforming growth factor-β (TGF)-β1-induced epithelial–mesenchymal transition (EMT) in HPAEpiC. Moreover, Tβ4 suppressed the proliferation and attenuated the TGF-β1-induced activation of HLF-1 cells. In conclusion, Tβ4 alleviates LPS-induced lung injury, inflammation, and subsequent fibrosis in mice, suggesting that Tβ4 has a protective role in the pathogenesis of pulmonary fibrosis. Tβ4 is involved in attenuating oxidative injury, promoting mitophagy, and alleviating inflammation and fibrosis. Modulation of Tβ4 might be a novel strategy for treating pulmonary fibrosis.


INTRODUCTION
Pulmonary fibrosis (PF) is a chronic, progressive, irreversible, and fatal lung disease, marked by progressive dyspnea and, ultimately, respiratory failure [1]. Although 1 it is a rare disease, its poor prognosis, with a median survival of 2-5 years, has made it a considerable challenge for clinicians [2]. Cigarette smoking, exposure to organic and inorganic dust, and genetic factors have been shown to play important roles in disease pathogenesis [3].
Oxidant/antioxidant imbalance and the excessive production of proinflammatory and profibrotic cytokines are involved in the pathogenesis of PF. These stimulations then lead to alveolar epithelial injury, followed by proliferation of type II alveolar epithelial cells and myofibroblasts, and deposition of extracellular matrix (ECM) proteins, and finally, parenchymal remodeling [4]. Antioxidant, anti-inflammatory, and antifibrotic therapies are often used in the treatment of PF. However, none of these treatments have been proven available, and lung transplantation is now the only option for a small number of PF patients [5,6].
Thymosin β4 consists of 43 amino acids and belongs to a highly conserved β-thymosin family [7]. It spreads in nearly all cells and is released to body fluids, including tears, saliva, blood, and plasma with important regulatory roles in cell functions [8,9]. Tβ4 has been reported to participate in wound healing, inflammation, fibrosis, and tissue regeneration, and recent studies have shown that Tβ4 prevents inflammation and fibrosis in the eye, skin, heart, liver, and bleomycin-induced pulmonary fibrosis [8,10,11]. Tβ4 has shown a long-term protective effect in scleroderma patients with pulmonary fibrosis [12]. However, the underlying mechanism by which Tβ4 regulates these fibrotic processes is not fully understood.
Autophagy is a conserved process by which cytoplasmic components, including damaged proteins and organelles, are degraded by lysosomes [13]. An increasing amount of evidence have shown that autophagy limits NLRP3 inflammasome activation by targeting ROS-producing mitochondria; the process by which mitochondria are degraded by autophagy is called mitophagy [14,15]. Recent studies have shown that Tβ4 limits inflammation by contributing to autophagosome formation and membrane remodeling during autophagy [16], and Tβ4 could also prevent oxidative stress by upregulating the antioxidant enzyme Cu/Zn superoxide dismutase (SOD) [17]. However, no studies have examined whether mitophagy regulates inflammation via Tβ4 during PF.
In the present study, we constructed a recombinant adeno-associated virus (rAAV) to achieve persistent expression of Tβ4 in LPS-induced PF models so as to explore the possible role of Tβ4 in regulating mitophagy and inflammation in vitro.

Histological Sampling
We collected surgical resected paraffin-embedded human fibrotic lung tissues specimens (10 cases) and pathologically normal para-tumor lung tissue specimens (10 cases) from the Department of Pathology, the First Affiliated Hospital of Xi'an Jiaotong University, with the approval of the Institutional Review Board. Immunoreactions were performed on selected lung sections.

Preparation of Recombinant AAV
Self-complementary recombinant adeno-associated virus were constructed by applying an AAV Helper-Free System (Cell Biolabs, San Diego, CA, USA). The coding DNA of human Tβ4 (GenBank NM_021109.3) was inserted into pscAAV-MCS to yield the pscAAV-Tβ4 plasmid. Recombinant AAV containing Tβ4 (AAV-Tβ4) was generated via co-transfection of pscAAV-Tβ4, pHelper, and pAAVRC5 into AAV-293 cells using polyethylenimine (PEI). Recombinant AAV carrying LacZ (AAV-LacZ) was constructed as a control virus. Next, 72 h after the transfection, the cells were collected for viral particle isolation, purification, and quantitative analysis.
TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was employed to determine the recombinant AAV (rAAV) titters and the abundance of the rAAV in the lung. The primers against the cytomegalovirus promoter region were as follows: 5′-AGA CTT GGA AAT CCC CGT GAGT-3′ (forward) and 5′-CGT ATT AGT CAT CGC TAT TAC CAT GGT-3′ (reverse). The sequence of the probe was 5′-6FAM-AAC CGC TAT CCA CGC CCA TTG ATG -TAMRA-3′. The collected data were analyzed by the standard curve method.

Animals
Specific pathogen-free, 6-week-old male ICR mice, weighing 25-30 g were obtained from the Experimental Animal Center, School of Medicine, Xi'an Jiaotong University. The mice were housed under pathogen-free conditions under a 12/12 h light/dark cycle at constant temperature (22 ± 2 °C) and humidity, with free access to water and standard laboratory chow. All the mice were acclimatized to the abovementioned conditions for 1 week before initiating experiments. All efforts were undertaken to minimize the suffering of the mice.
To test the transduction efficiency of repeated intraperitoneal (i.p.) rAAV injection, 24 mice were divided into three groups: PBS, AAV-LacZ, and AAV-Tβ4. Mice in PBS group were injected with PBS, mice in AAV-LacZ group were injected with AAV-LacZ [4 × 10 10 viral genome (vg)], and mice in AAV-Tβ4 group were injected with AAV-Tβ4 (4 × 10 10 vg) on day 0. Two mice from each group were randomly euthanized on day 14 and day 28. The remaining mice were injected again with AAV-LacZ and AAV-Tβ4 on day 28 and were sacrificed on day 42. The lungs of the euthanized mice were harvested for further examination.

AAV-Mediated Tβ4 Expression upon LPS-Induced Lung Injury and Fibrosis
To verify the expression of Tβ4 in mouse lung after LPS treatment, 35 mice were divided into normal saline (NS, n = 5) and LPS (n = 30) groups. A septic lung injury model was established by i.p. injection of 5 mg/kg LPS for 5 consecutive days [18]. Five mice from the LPS group were euthanized on days 7, 14, 21, 28, 35, and 42, while all the mice in the NS group were euthanized on day 7. Mouse lungs were collected for HE and picrosirius red staining, western blotting, and other analyses.
To investigate the effects of Tβ4 on acute lung injury and fibrosis, 40 mice were divided into four equal groups: NS, NS + LPS, LPS + AAV-LacZ, and LPS + AAV-Tβ4. Mice in the AAV groups were i.p. injected with AAV (AAV-LacZ or AAV-Tβ4, 4 × 10 10 vg) for the first time, while mice in the other two groups were injected with an equal volume of NS. Two days later (day 0), the mice were i.p. instilled with NS or LPS. Five mice in each group were sacrificed on day 7. The remaining mice received the second i.p. administration of AAV or NS on day 26 (4 weeks after the first adenovirus administration) and were sacrificed on day 42, and then the lungs and serum were harvested for subsequent experiments. The mice were weighed during LPS modeling, and their lung coefficient was calculated (lung coefficient = lung wet weight/body weight × 100).

Bronchoalveolar Lavage (BAL)
BAL was carried out on day 7 following LPS injection. After the mice had been sacrificed, their lungs and trachea were extracted immediately, and a 20G intravenous catheter was inserted into their trachea. Next, 1 mL PBS was instilled into the lungs and withdrawn three times via the catheter. More than 85% of the fluid was recovered as bronchoalveolar lavage fluid (BALF), which was then centrifuged at 1000 rpm for 10 min at 4 °C. The supernatants were collected and stored at −80 ℃, and the precipitate was washed with red blood cell lysis buffer and resuspended in 500 µL PBS for further tests.

Measurement of Malondialdehyde (MDA) and Myeloperoxidase (MPO)
MDA content and MPO activity in mouse lung tissue were detected using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer's protocols.

Measurement of Hydroxyproline Content
Pulmonary hydroxyproline content was detected using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer's protocols.

Measurement of JC-1 Staining and CellROX Evaluation
JC-1 staining (Thermo Fisher Scientific, Waltham, MA, USA), which shows red fluorescence in normal mitochondrial potential and green fluorescence in damaged mitochondrial potential was used. HPAEpiC cells were seeded in 6-well plates and treated with 400 µM H 2 O 2 for up to 1 h. JC-1 was added in each well at 10 mg/ mL and incubated for 10 min at 37 °C in the dark. The cells were harvested and detected in flow cytometer.
CellROX deep red oxidative stress reagent (Thermo Fisher Scientific, Waltham, MA, USA) which is nonfluorescent while in a reduced state and upon oxidation exhibits strong fluorogenic signal was used. HPAEpiC cells were seeded in 6-well plates and treated 100 µM H 2 O 2 for up to 2 h. CellROX deep red was added to each well at 10 ug/mL and incubated for 15 min. The cells were harvested and detected in flow cytometer.

Cell Culture, Proliferation Assay, and Reagent Treatment
The HPAEpiC were cultured in DMEM, while HIF-1 cells were cultured in F-12 K medium supplemented with 10% fetal bovine plasma and 2 mM L-glutamine at 37 °C in 95% air and 5% CO 2 -humidified atmosphere.
The cells were inoculated to 96-well plates at 500 cells/well and allowed to adhere for 24 h. The cells were then treated with Tβ4 at different concentrations (0, 75, and 150 nM) and incubated for another 72 h. Cell viability was assessed using CCK-8 (Dojindo, Kyushu, Japan) assay at 24, 48, and 72 h, in accordance with the manufacturer's protocols.

Immunohistochemistry
Immunoreactions were performed on selected lung sections. Antigens were detected by the primary antibodies: anti-thymosin β4 (ab167650, Abcam, Cambridge, UK) and anti-α-SMA (#56,856, Cell Signaling Technology, Danvers, MA, USA), followed by appropriate secondary antibodies. The slides were then observed under a Nikon Eclipse microscope (Tokyo, Japan) coupled to a digital camera.

Statistical Analysis
The results are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS software 13.0 (SPSS, Inc., Chicago, IL, USA). The Shapiro-Wilk test and Levene statistic were used to evaluate the normality and homogeneity of the variance, respectively. On that basis, t tests or Mann-Whitney U tests were used to evaluate differences between two groups. Correlations between two quantitative groups were analyzed using Pearson or Spearman correlation tests. The χ2 test was used for comparisons between two groups. The two-sided P values < 0.05 were considered statistically significant.

Tβ4 Expression Is Elevated in Human and Mouse Fibrotic Lung Tissues
Immunohistochemical staining showed a marked increase in Tβ4 expression in fibrotic human lung tissues, resulting in a significant increase in the average IOD compared with that in normal tissues (Fig. 1A). In LPS-treated mice, qRT-PCR and western blot showed markedly elevated expression of Tβ4 from day 14 after LPS treatment and thereafter (Fig. 1B, C). The expression of Tβ4 was also confirmed by immunohistochemistry (Fig. 1D).

Intraperitoneal Administration of Adeno-associated Virus Efficiently Transduces Mouse Lung Tissue
To verify the transduction efficiency of recombinant adeno-associated viruses in mouse lung, we used qRT-PCR to determine the abundance of vector DNA in mouse lung. As shown in Fig. 2A, qRT-PCR revealed the presence of vector DNA in mouse lung after the administration of recombinant adeno-associated viruses.
Moreover, western blot showed that the expression levels of Tβ4 after the second injection of recombinant adeno-associated viruses were comparable to those observed after the first injection, which indicated that the Fig. 1 Tβ4 expression is elevated in human and mouse fibrotic lung tissues. Immunohistochemistry showed that type II alveolar epithelial cells were stained with anti-Tβ4 antibody in normal human lung tissue and Tβ4 expression was drastically elevated in fibrotic human lung tissues (A). The expression of Tβ4 in mouse lung tissues was upregulated at both the mRNA (B) and protein (C) levels. Immunohistochemical staining showed that the expression levels of Tβ4 in normal and fibrotic mouse lung tissues were similar to those in the normal and fibrotic human lung tissues (D). Scare bars = 100 µm. realization of prolonged ectopic expression by repeated injection of recombinant adeno-associated viruses was feasible (Fig. 2B).

Tβ4 Protects Mice from LPS-Induced Lung Injury and Inflammation
Body weight continuously decreased, while lung coefficient markedly increased after LPS treatment, and AAV-Tβ4 significantly attenuated these changes (Fig. 3A,  B). We found lower tissue MDA content in AAV-Tβ4 group than in NS + LPS or LPS + AAV-LacZ groups, both on day 7 and on day 42 (Table 1). Histological examination showed lung injury and inflammation (interstitial edema, inflammatory cells infiltration, and hyaline membrane formation), and these changes were alleviated by AAV-Tβ4 (Fig. 3C). Moreover, LPS increased in BALF protein content (Fig. 3D), and total cell number (Fig. 3E) is significantly reduced by AAV-Tβ4.
Tissues MPO activity, an indicator of oxidative injury as well as neutrophil infiltration, was elevated by LPS treatment, but its increase was attenuated by AAV-Tβ4 (Table 1). To further explore the antiinflammatory function of Tβ4 in LPS-treated mice, the BALF level of inflammatory mediators, such as TNF-α, IL-1β, and IL-6 in fibrotic mouse lungs, was tested; the results showed that AAV-Tβ4 significantly mitigated the increase ( Table 2).

Tβ4 Attenuates LPS-Induced Lung Fibrosis in Mice
Days after LPS treatment, pulmonary hydroxyproline content was markedly increased (Fig. 4A). HE and picrosirius red staining showed that many spindleshaped fibrotic cells clumped together, and collagen fibers accumulated (Fig. 4C, D), with increased fibrosis score in LPS-treated mice (Fig. 4B). All these fibrotic changes were significantly alleviated by AAV-Tβ4, while the usage of AAV-LacZ showed no significant effect (Fig. 4A-D).
The expression of α-SMA was significantly lower in LPS + AAV-Tβ4 group than that in NS + LPS and LPS + AAV-LacZ group (Fig. 4E), and this result was also verified by western blot (Fig. 4F) and immunohistochemistry (Fig. 4G).

LPS Promotes Inflammatory Responses and Inhibited Mitophagy in HPAEpiC
We next investigated the effect of LPS in HPAEpiC. We first confirmed that the ROS donor, H 2 O 2 , decreased mitochondrial membrane potential (MMP) and promoted ROS accumulation and inflammatory responses in a dose-dependent manner. Moreover, the antioxidant NAC decreased the LPS/H 2 O 2 -induced    (Fig. 5A-F). Recently, mitophagy has been shown to alleviate inflammation via inhibiting the NLRP3 inflammasome. Thus, we examined whether ROS induce inflammatory responses through mitophagy. The usage of oligomycin, a mitophagy inhibitor, promoted LPS/H 2 O 2 -induced IL-1β secretion; moreover, the usage of FCCP, a drug dissipating MMP and inducing mitophagy by activating PINK1, rescued HPAEpiC from LPS/H 2 O 2 -induced inflammatory responses (Fig. 5G, H). Because ROS-induced inflammatory responses in HPAEpiC were modulated by mitophagic inhibitor and inducer, we further examined whether ROS regulated mitophagy in HPAEpiC.
As the initiator of mitophagy, PINK1 phosphorylates ubiquitin to activate Parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins. We found that incubation with H 2 O 2 led to decreased expression of PINK1 in a dose-dependent manner. Mitophagy inhibition leads to an increase in Tom40 protein level. The usage of H 2 O 2 promoted Tom40 accumulation in a dose-dependent manner (Fig. 5I).

Tβ4 Attenuates LPS-Induced Mitophagy Inhibition, Inflammasome Activation, and TGF-β1-Induced EMT in HPAEpiC
We firstly tested whether Tβ4 affects mitophagy and inflammatory responses in HPAEpiC; we found that Tβ4 alleviated the LPS/H 2 O 2 -induced decreased expression of PINK1 and accumulation of Tom40 (Fig. 6A). We also revealed that Tβ4 successfully suppressed the LPS/ H 2 O 2 -induced NLRP3 activation and IL-1β secretion in HPAEpiC (Fig. 6B, C). qRT-PCR showed that although Tβ4 did not affect the basal expression levels of vimentin and α-SMA, it markedly opposed the TGF-β1-induced upregulation of vimentin and α-SMA in HPAEpiC (Fig. 6D, E).

Tβ4 Suppresses the Proliferation and Attenuates the TGF-β1-Induced Activation of HLF-1 Cells
The CCK-8 assay showed that Tβ4 significantly inhibited the growth of HLF-1 cells (Fig. 7A). qRT-PCR revealed that Tβ4 did not affect basal expression of α-SMA and vimentin but markedly attenuated the TGF-β1-induced elevation of α-SMA and vimentin in HLF-1 cells (Fig. 7B, C).

DISCUSSION
Pulmonary fibrosis is a heterogeneous disease with significant global morbidity and mortality. The pathogenesis of PF is still poorly understood. Recent studies have shown that PF mainly results from inflammation and consequent fibroblast proliferation, which leads to abnormal deposition of extracellular collagen [3]. In the present study, we found the increased expression of Tβ4 in human and mouse fibrotic lung tissues. The role of Tβ4 in alleviating hepatic, renal, and cardiac fibrosis has been confirmed in   ◂ recent studies [9,19,20]. The increased production of local Tβ4 in mice is an adaptive response to lung injury, but the increased expression of endogenous Tβ4 might not be sufficient to alleviate lung injury and fibrosis. Our data revealed protective effect of Tβ4 in pulmonary fibrosis, and AAVmediated dramatic overexpression of Tβ4 in mouse lung successfully alleviated LPS-induced lung injury and fibrosis in mice. Our results also indicate that the protective role of Tβ4 may involve suppressing oxidant damage and inflammasome activity and, consequently, alleviating fibrosis. The lung is susceptible to high oxygen tension and exogenous oxidants, and pollutants can increase oxidant production in the lung [21]. Previous studies have revealed that ROS play a role in the pathogenesis of lung inflammation; the generation of mitochondrial ROS is crucial for NLRP3 inflammasome activation, leading to the release of IL-1β [22]. Here, our in vitro data demonstrated that ROS promoted inflammation in alveolar epithelial cells; alveolar epithelial injury leads to the impairment of air exchange function and, more importantly, the secretion of IL-1β [23]. We also found that LPS treatment induced ROS generation in HPAEpiC, thereby leading to activation of NLRP3 inflammasome, but this effect was alleviated by NAC, an antioxidant.
Chronic inflammation participates in the pathogenesis of many human diseases, including PF. These diseases are characterized by excessive ROS production; dysfunctional mitochondria have been implicated in these disorders, acting as both a source and a target of ROS [24]. Mitophagy is a special type of autophagy that degrades damaged mitochondrial. In the present study, mitophagy decreased in HPAEpiC, and this phenomenon was alleviated by NAC. Moreover, we found that FCCP, a mitophagy inducer, alleviated LPS/H 2 O 2 -induced IL-1β secretion, whereas oligomycin, a mitophagy inhibitor, promoted LPS/H 2 O 2 -induced IL-1β secretion in HPAEpiC. Defective mitophagy leads to the accumulation of damaged ROS-generating mitochondria and activation of NLRP3 inflammasome; our data revealed for the first time that ROS promote inflammation via mitophagy inhibition in HPAEpiC.
The antioxidant effect of Tβ4 has been reported in previous studies [11,17]; in the present study, we observed that Tβ4 significantly attenuated both the LPSinduced elevation of mouse pulmonary MPO activity, MDA content and proinflammatory cytokines in vivo, and the LPS/H 2 O 2 induced mitophagy inhibition and inflammasome activation in vitro. Inflammation is likely involved in the initial pathogenesis of PF; dysfunction of alveolar epithelial cells and subsequent inflammation trigger the fibrogenic process, resulting in the deposition of matrix and remodeling of the lung [25]. Our data demonstrated that Tβ4 alleviated LPS-induced lung inflammation and fibrosis in mice and suppressed the fibrogenic process in HPAEpiC and HLF-1 cells.
In conclusion, the present study demonstrates that Tβ4 alleviates LPS-induced lung injury, inflammation, and subsequent fibrosis in mice, indicating the protective role of Tβ4 in the pathogenesis of PF. In addition, this study also indicates that the protective effect of Tβ4 may involve the attenuation of oxidative injury, promotion of mitophagy, and subsequently, alleviation of inflammation and fibrosis. Modulation of Tβ4 may be a novel strategy for treating PF.

DECLARATIONS Ethics Approval and Consent to Participate
The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of Xi'an Jiaotong University. This study followed the national guidelines and protocols of the National Institutes of Health and was approved by the Local Ethics Committee for the Care and Use of Laboratory Animals of Xi'an Jiaotong University.