Thymosin β4 protect against LPS induced lung injury and inammation and subsequent brosis in mice

Background: Inammation plays a critical role in the progression of pulmonary brosis. Thymosin β4 (Tβ4) has antioxidant, anti-inammatory and antibrotic effects. Although the potent protective role of Tβ4 in bleomycin-induced pulmonary brosis has been validated, the mechanism is not clear, and its impact on lipopolysaccharide (LPS)-induced lung injury/brosis has not been reported. Method: Expression of Tβ4 in brotic lung tissues was assessed by real-time quantitative reverse transcriptase PCR (RQ-PCR), immunohistochemistry (IHC) and Western Blotting. The effects of intraperitoneal adeno-associated virus-Tβ4 (AAV-Tβ4) on LPS-induced lung injury and brosis 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 conrm the effects of Tβ4. Results: In this study, we evaluated the role of Tβ4 in pulmonary brosis and explored the possible underlying mechanisms. We found that Tβ4 was markedly upregulated in human or mouse brotic lung tissues. AAV-Tβ4 markedly alleviated LPS-induced oxidative damage, lung injury, inammation, and brosis in mice. Our in vitro experiments also showed that LPS inhibited mitophagy and promoted inammation via oxidative stress in HPAEpiC, and usage of Tβ4 signicantly attenuated LPS-induced mitophagy inhibition, inammasome activation and transforming growth factor-β (TGF)-β1 induced epithelial-mesenchymal transition (EMT) in HPAEpiC. Moreover, we found that Tβ4 suppressed the proliferation and attenuated the TGF-β1-induced activation of HLF-1 cells. Conclusions: In conclusion, Tβ4 lung and subsequent brosis suggesting pulmonary brosis


Introduction
Pulmonary brosis (PF) is a chronic, progressive irreversible and fatal lung disease marked by progressive dyspnea and, ultimately, respiratory failure [1]. Although it is a rare disease, its poor prognosis made it a considerable challenge for clinicians, with a median survival of 2-5 years [2]. Cigarette smoking, exposure to organic and inorganic dust, and genetic factors have been shown to play important roles in disease pathogenesis [3].
Oxidative/antioxidative imbalance and the excessive production of pro-in ammatory and pro-brotic cytokines are involved in the pathogenesis of PF. These stimulations then lead to alveolar epithelial injury, followed by proliferation of type alveolar epithelial cells and myo broblasts, and deposition of extracellular matrix (ECM) proteins, and then parenchymal remodeling [4]. Anti-oxidative, antiin ammatory and anti-brotic therapies are often used in the treatment of PF. However, none of these treatments has been proven available, and lung transplantation is now the only way to a small minority 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 exits in body uids, including tears, saliva, blood and plasma with important regulatory roles in cell functions [8,9]. Tβ4 has been reported participating in wound healing, in ammation, brosis and tissue regeneration, and recent studies show that Tβ4 prevents in ammation and brosis in the eye, skin, heart, liver and bleomycin-induced pulmonary brosis[8, 10,11]. Tβ4 has been shown a protective effect in the long run in the case of scleroderma patients with pulmonary brosis [12]. However, the underlaying mechanism of Tβ4 in regulating these brotic 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 in ammasome activating by targeting ROS-producing mitochondria, and the process by which mitochondria are degraded by autophagy is called mitophagy [14,15]. Some recent studies have shown that Tβ4 limits in ammation via contributing to autophagosome formation and membrane remodeling during autophagy [16], and Tβ4 could also prevent oxidative stress via upregulating anti-oxidative enzymes Cu/Zn superoxide dismutase (SOD) [17]. However, no studies have examined whether mitophagy regulates in ammation 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, we also explored the possible role of Tβ4 in regulating mitophagy and in ammation in vitro.

Histological sampling
We collected surgical resected para n-embedded human brotic lung tissues specimens (10 cases) and pathologically normal para-tumor lung tissue specimens (10 cases) from the Department of Pathology, the First A liated 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, SanDiego, CA, United States). 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. 72 hours after transfection, cells were collected for viral particle isolation, puri cation and quantitative analysis.
TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, United States) 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'-AGACTTGGAAATCCCCGTGAGT-3' (forward) and 5'-CGTATTAGTCATCGCTATTACCATGGT-3' (reverse). The sequence of the probe was 5'-6FAM-AACCGCTATCCACGCCCATTGATG-TAMRA-3'. The collected data were analyzed by the standard curve method.

Animals
Speci c pathogen free, 6-week-old male ICR mice, weighing 25-30g 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 hours light/dark cycle at constant temperature (22±2°C) and humidity, with free access to water and standard laboratory chow. All mice were acclimatized to the abovementioned conditions for one week before initiating experiments. All efforts were undertaken to minimize the suffering of the mice.
To test the transduction e ciency of repeated intraperitoneal (i.p.) rAAV injection, twenty-four mice were divided into 3 groups: PBS, AAV-LacZ and AAV-Tβ4. Mice in PBS group were injected with PBS, mice in AAV groups were injected were given AAV-LacZ [4x10 10 viral genome (vg)] or AAV-Tβ4 (4x10 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 sacri ced on day 42. The lungs of euthanized mice were harvested for further examination.

AAV-mediated Tβ4 expression upon LPS-induced lung injury and brosis
To verify the expression of Tβ4 in mouse lung after LPS treatment, thirty-ve mice were divided into normal saline (NS, n=5) and LPS (n=30) groups. Septic lung injury model was established by i.p. injection of 5mg/kg LPS for ve 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 experiments.
To investigate the effects of Tβ4 on acute lung injury and brosis, forty mice were equally assigned into four groups: NS, NS+LPS, LPS+AAV-LacZ and LPS+AAV-Tβ4. Mice in AAV groups were i.p. injected with AAV (AAV-LacZ or AAV-Tβ4, 4x10 10 vg) for the rst 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 sacri ced on day 7. The remaining mice received the second i.p. administration of AAV or NS on day 26 (four weeks after the rst adenovirus administration) and were sacri ced on day 42, when the lungs and serum were harvested for subsequent experiments. The mice were weighed during LPS modeling, and their lung coe cient was calculated (lung coe cient=lung wet weight/body weight×100).

Bronchoalveolar lavage (BAL)
BAL was carried out on day 7 following LPS injection. After the mice were sacri ced, their lungs and trachea were extracted immediately, and a 20G intravenous catheter was inserted into their trachea. 1mL PBS was instilled into the lungs and withdrawn three times via the catheter. More than 85% of the uid was recovered as bronchoalveolar lavage uid (BALF), which was then centrifuged at 1000rpm for 10minutes 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 500µL PBS for further tests.

Measurement of malondialdehyde (MDA) and myeloperoxidase (MPO)
MDA content and MPO activity in mouse lung tissue were detected with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols.

Measurement of hydroxyproline content
Pulmonary hydroxyproline content was detected with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols.

Measurement of IL-1β
IL-1β level was detected by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (eBioscience, San Diego, CA, USA) according to the manufacturer's protocols.

Cell culture, proliferation assay and reagents treatment
The HPAEpiC were cultured in DMEM, while HIF-1 cells were cultured in F-12K medium supplemented with 10% foetal bovine plasma and 2mM L-glutamine at 37°C in a 95% air, 5% CO 2 -humidi ed atmosphere.
Cells were trypsinized, and 500 cells were seeded onto 96-well plates and allowed to adhere for 24hours. Cells were then treated with Tβ4 at different concentrations (0, 75, and 150nM) and incubated for another 72hours. Cell viability was assessed using CCK-8 (Dojindo, Kyushu, Japan) assay at 24, 48, and 72hours according to the manufacturer's protocols.

Immunohistochemistry
Immunoreactions were performed on selected lung sections. Antigens were detected by the following primary antibody, followed by appropriate secondary antibodies: anti-Thymosin β4 (ab167650, Abcam, Cambridge, UK) and anti-α-SMA (#56856, Cell Signaling Technology, Danvers, MA, USA). The slides were then observed under a Nikon Eclipse microscope (Tokyo, Japan) coupled to a digital camera.

Statistical analysis
The results are expressed as the means ± 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, respectively, of the variance. According to the situation, t-tests or Mann-Whitney U tests were used to evaluate differences between two groups; correlations between two quantitative groups were analysed with Pearson or Spearman correlation tests. The χ2 test was used for comparisons between two groups. The reported P-values are two-sided, and P-values <0.05 were considered statistically signi cant.

Tβ4 expression was elevated in human and mouse brotic lung tissues
Immunohistochemical staining showed a marked increase in Tβ4 expression in brotic human lung tissues, resulted in a signi cant increase in the average IOD compared with that in normal tissues ( Figure  1A). In LPS-treated mice, qRT-PCR and western blot showed markedly elevated expression of Tβ4 from day 14 after LPS treatment and thereafter ( Figure 1B, C). The expression of Tβ4 was also con rmed by immunohistochemistry ( Figure 1D).

Intraperitoneal administration of adeno-associated virus e ciently transduced mouse lung tissue
To verify the transduction e ciency 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 Figure 2A, qRT-PCR revealed the presence of vector DNA in mouse lung after the administration of recombinant adenoassociated viruses. Moreover, western blot showed that expression levels of Tβ4 following the second injection of recombinant adeno-associated viruses were comparable to those observed following rst injection, which indicated that realizing prolonged ectopic expression by repeated injection of recombinant adeno-associated viruses was feasible ( Figure 2B).

Tβ4 protected mice form LPS-induced lung injury and in ammation
Body weight continuously decreased, while lung coe cient markedly increased after LPS treatment, AAV-Tβ4 signi cantly attenuated these changes ( Figure 3A, B). We found lower tissue MDA content in AAV-Tβ4 group than in NS+LPS or LPS+AAV-LacZ groups, both in day 7 and day 42 (Table 1). Histological examination showed lung injury and in ammation by interstitial edema, in ltration of in ammatory cells, and hyaline membrane formation, and all these changes were alleviated by AAV-Tβ4 ( Figure 3C). Moreover, LPS increases in BALF protein content ( Figure 3D), and total cell number ( Figure 3E) were signi cantly attenuated by AAV-Tβ4.
We found tissues MPO activity, an indicator of oxidative injury as well as neutrophil in ltration, was elevated by LPS treatment, and this increase was also attenuated by AAV-Tβ4 (Table 1). To further explore the anti-in ammatory function of Tβ4 in LPS-treated mice, the BALF level of in ammatory mediators, such as TNF-α, IL-1β and IL-6 in brotic mouse lungs were tested, and we found AAV-Tβ4 signi cantly mitigated the increase ( Table 2).

Tβ4 attenuated LPS-induced lung brosis in mice
42 days after LPS treatment, pulmonary hydroxyproline content was markedly increased ( Figure 4A). HE and picro-shaped red staining followed showed that lots of spindle-shaped brotic cells clumped together, and collagen bers accumulated ( Figure 4C, D), with increased brosis score in LPS-treated mice ( Figure 4B). All these above brotic changes were signi cantly alleviated by AAV-Tβ4, while usage of AAV-LacZ showed no signi cant effect (Figure4 A-D).
The expression of α-SMA was signi cantly lower in LPS+AAV-Tβ4 group than in NS+LPS and LPS+AAV-LacZ group (Figure 4E), and this result was also veri ed by western blot ( Figure 4F) and immunohistochemistry ( Figure 4G).

LPS promoted in ammatory responses and inhibited mitophagy in HPAEpiC
We next investigated the effect of LPS in HPAEpiC. We rst con rmed that the ROS donor H 2 O 2 led to Recently mitophagy has been shown to alleviate in ammation via inhibiting the NLRP3 in ammasome, we then tested whether ROS induce in ammatory responses through mitophagy. Usage of oligomycin, an ATP synthase inhibitor, promoted LPS-induced IL-1β secretion; moreover, usage of FCCP, a drug dissipates MMP and induces mitophagy by activating PINK1, rescued HPAEpiC from LPS-induced in ammatory responses ( Figure 5G, H). Because ROS-induced in ammatory responses in HPAEpiC was modulated by mitophagic inhibitor and inducer, we thus wondered whether ROS regulated mitophagy in HPAEpiC.
As the initiator of mitophagy, PINK1 phosphorylates ubiquitin to active 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. We found here that usage of H 2 O 2 promoted Tom40 accumulation in a dose-dependent manner ( Figure 5I).

Tβ4 attenuated LPS-induced mitophagy inhibition, in ammasome activation and TGF-β1 induced EMT in HPAEpiC
We rstly tested whether Tβ4 affects mitophagy and in ammatory responses in HPAEpiC, and found that Tβ4 alleviated LPS/H 2 O 2 -induced decreased expression of PINK1, and Tom40 accumulation ( Figure 6A), we also revealed that Tβ4 successfully suppressed LPS/H 2 O 2 -induced NLRP3 activation and IL-1β secretion in HPAEpiC ( Figure 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 ( Figure 6D, E).

Tβ4 suppressed the proliferation and attenuated the TGF-β1-induced activation of HLF-1 cells
The CCK-8 assay showed that Tβ4 signi cantly inhibited the growth of HLF-1 cells ( Figure 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 (Figure 7B, C).

Discussion
Pulmonary brosis is a heterogeneous disease with signi cant global morbidity and mortality. The mechanism of disease pathogenesis of PF is now poorly understood. Recent studies have shown that PF mainly results from in ammation and consequently broblast proliferation, which leads to abnormal deposition of extracellular collagen [3]. In the present study, we rstly found increased expression of Tβ4 in human and mouse brotic lung tissues. The role of Tβ4 in alleviating hepatic, renal and cardiac brosis has been con rmed by some recent researches [9,19,20]. The increased production of local Tβ4 in mice serves as an adaptive response to lung injury, and this increased expression of endogenous Tβ4 might not be su cient enough to alleviate lung injury and brosis. Our data revealed protective effect of Tβ4 in pulmonary brosis, AAV-mediated dramatic overexpression of in mouse lung successfully alleviated LPS-induced lung injury and brosis in mice. Our results also indicated that the protective role of Tβ4 may involve suppressing oxidant damage and in ammasome activity, and then alleviating brosis.
The lung is susceptible to high oxygen tension, 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 in ammation, the generation of mitochondrial ROS is crucial for NLRP3 in ammasome activation, leading to the release of IL-1β [22]. Here, our in vitro data demonstrated that ROS promotes in ammation 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 treatment of LPS induced ROS generation in HPAEpiC, leading to activation of NLRP3 in ammasome, and this effect was alleviated by NAC, an antioxidant.
Chronic in ammation participates in the pathogenesis of many human diseases, including PF. These diseases are characterized by excessive ROS production, and dysfunctional mitochondria have also been shown implicated in these disorders, act as both a source and a target of ROS [24]. Mitophagy is a special type of autophagy which degrades damaged mitochondrial. In the present study, we found mitophagy was 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, an mitophagy inhibitor, promoted LPS/H 2 O 2 induced IL-1β secretion in HPAEpiC. Defective mitophagy leads to accumulation of damaged ROS-generating mitochondria and then activation of NLRP3 in ammasome, our data revealed for the rst time that ROS promotes in ammation via mitophagy inhibition in HPAEpiC.
The anti-oxidative effect of Tβ4 has been conformed in many previous studies [11,17], in the present study, we observed that Tβ4 signi cantly attenuated LPS-induced elevation of mouse pulmonary MPO activity, MDA content and pro-in ammatory cytokines in vivo and LPS/ H 2 O 2 induced mitophagy inhibition and in ammasome activation in vitro. In ammation is thought participates in the initial period of pathogenesis of lung brosis, dysfunction of alveolar epithelial cells and subsequent in ammation trigger brogenic process, resulting in the deposition of matrix and remodeling of lung [25]. Our data demonstrated that Tβ4 alleviated LPS-induced lung in ammation and brosis in mice, and suppressed brogenic process in HPAEpiC and HLF-1 cells.
In conclusion, the present study demonstrates that Tβ4 alleviates LPS-induced lung injury, in ammation, and subsequent brosis in mice, suggesting the protective role of Tβ4 in disease pathogenesis of PF. In addition, this study also indicates that the protective effect of Tβ4 may involve attenuating oxidative injury, promoting mitophagy, and then alleviating in ammation and brosis. Modulating of Tβ4 may be novel strategies 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.
Consent to participate: Not applicable.
Consent for publication: Not applicable.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests.
Funding: This work was nancially supported by the National Natural Science Foundation of China (81800548). The sponsors had no role in the study design and in the collection, analysis, and interpretation of data.    Figure 1 Tβ4 expression is elevated in human and mouse brotic lung tissues Immunohistochemistry showed that type II alveolar epithelial cells were positively stained with anti-Tβ4 antibody in normal human lung tissue, and Tβ4 expression was drastically elevated in brotic human lung tissues (A). Expression of Tβ4 in mouse lung tissues was upregulated at both the mRNA (B) and protein (C) levels. Immunohischemical staining showed that the expression of Tβ4 in normal and brotic mouse lung tissues are similar to those in the normal and brotic human lung tissues (D).   AAV-Tβ4 alleviates LPS-induced lung brosis in mice Tβ4 signi cantly attenuated LPS-induced lung brogenesis in mice, as indicated by lower pulmonary hydroxyproline content (A), milder lung structure destruction (C), less picro-sirius red-positive collagen deposition (D) and lower brosis score (B) compared with those in NS+LPS and AAV-LacZ+LPS groups. AAV-Tβ4 signi cantly alleviated LPS-induced excess expression of α-SMA in mouse lung, as con rmed by rt-PCR (E), western blot (F) and immunohistochemistry (G), compared with those in NS+LPS and AAV-LacZ+LPS groups.  Tβ4 attenuates LPS-induced mitophagy inhibition, in ammasome activation and TGF-β1-induced EMT in HPAEpiC Tβ4 alleviated H2O2/LPS-induced decreased expression of PINK1 and accumulation of Tom40 (A). Tβ4 suppressed H2O2/LPS-induced NLRP3 in ammasome activation and IL-1β secretion (B, C). Tβ4 did not affect the basal expression of α-SMA, vimentin, but markedly attenuated the TGF-β1-induced upregulation of α-SMA and vimentin (D, E).