Thymosin β4 protect against LPS induced lung injury and inflammation and subsequent fibrosis in mice

doi:10.21203/rs.3.rs-139980/v2

Download PDF

Research Article

Thymosin β4 protect against LPS induced lung injury and inflammation and subsequent fibrosis in mice

https://doi.org/10.21203/rs.3.rs-139980/v2

This work is licensed under a CC BY 4.0 License

Journal Publication

published 19 Aug, 2021

Read the published version in Inflammation →

You are reading this latest preprint version

Background: 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 mechanism is not clear, and its impact on lipopolysaccharide (LPS)-induced lung injury/fibrosis has not been reported.

Method: Expression of Tβ4 in fibrotic 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 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.

Results: In this study, we evaluated the role of Tβ4 in pulmonary fibrosis and explored the possible underlying mechanisms. We found that 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 usage of Tβ4 significantly attenuated LPS-induced mitophagy inhibition, inflammasome 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 alleviates LPS-induced lung injury, inflammation, and subsequent fibrosis in mice, suggesting a protective role of Tβ4 in disease pathogenesis of pulmonary fibrosis (PF). Tβ4 involves in attenuating oxidative injury, promoting mitophagy, and then alleviating inflammation and fibrosis. Modulating of Tβ4 might be a novel strategy for treating PF.

Pulmonology

Thymosin β4

adeno-associated virus

pulmonary fibrosis

oxidative stress

mitophagy

Pulmonary fibrosis (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-inflammatory and pro-fibrotic cytokines are involved in the pathogenesis of PF. These stimulations then lead to alveolar epithelial injury, followed by proliferation of typeⅡ alveolar epithelial cells and myofibroblasts, and deposition of extracellular matrix (ECM) proteins, and then parenchymal remodeling[4]. Anti-oxidative, anti-inflammatory and anti-fibrotic 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 fluids, including tears, saliva, blood and plasma with important regulatory roles in cell functions[8, 9]. Tβ4 has been reported participating in wound healing, inflammation, fibrosis and tissue regeneration, and recent studies show that Tβ4 prevents inflammation and fibrosis in the eye, skin, heart, liver and bleomycin-induced pulmonary fibrosis[8, 10, 11]. Tβ4 has been shown a protective effect in the long run in the case of scleroderma patients with pulmonary fibrosis[12]. However, the underlaying mechanism of Tβ4 in regulating 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 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 inflammation 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 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, we also explored the possible role of Tβ4 in regulating mitophagy and inflammation in vitro.

2.1 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.

2.2 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, purification 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.

2.3 Animals

Specific 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 efficiency 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 [4x10¹⁰viral genome (vg)] or AAV-Tβ4 (4x10¹⁰vg) 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 euthanized mice were harvested for further examination.

2.4 AAV-mediated Tβ4 expression upon LPS-induced lung injury and fibrosis

To verify the expression of Tβ4 in mouse lung after LPS treatment, thirty-five 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 five 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 fibrosis, 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, 4x10¹⁰ 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 (four weeks after the first adenovirus administration) and were sacrificed on day 42, when 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).

2.5 Bronchoalveolar lavage (BAL)

BAL was carried out on day 7 following LPS injection. After the mice were sacrificed, 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 fluid was recovered as bronchoalveolar lavage fluid (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.

2.6 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.

2.7 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.

2.8 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.

2.9 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% CO₂-humidified 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.

Cells were trypsinized, and 5×10⁵ cells were seeded onto plastic dishes and then treated with H₂O₂(0, 100, 200 and 400µM), LPS (1µg/mL), NAC (10mM), FCCP (10µM), Oligomycin (10µM) or TGF-β1 (5ng/mL).

2.10 Western Bloting

Protein extracts were prepared from cells and mouse lung tissues by RIPA Lysis Buffer supplemented with Complete EDTA-free protease inhibitor cocktail tablets (Roche Applied Science, Basel, Switzerland) and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein samples (50µg) were loaded onto SDS-PAGE gels and transferred onto PVDF membranes. After blocking in 5% evaporated milk at room temperature for 2hours, the membranes were then incubated with the indicated primary antibodies in 5% evaporated milk in TBS plus 0.1% Tween 20 overnight at 4°C. The following primary antibodies were used: anti-Thymosin β4 (ab167650, Abcam, Cambridge, UK), anti-α-SMA (#56856, Cell Signaling Technology, Danvers, MA, USA), IL-1β (#12703, Cell Signaling), PINK1 (#6946, Cell Signaling), anti-Tom40 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin as a loading control (no. 4970; Cell Signaling). and β-Actin as a loading control (#4970, Cell Signaling). Signals were developed using a chemiluminescent substrate and visualized through X-ray films.

2.11 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.

2.12 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 significant.

3.1 Tβ4 expression was elevated in human and mouse fibrotic lung tissues

Immunohistochemical staining showed a marked increase in Tβ4 expression in fibrotic human lung tissues, resulted in a significant 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 confirmed by immunohistochemistry (Figure 1D).

3.2 Intraperitoneal administration of adeno-associated virus efficiently transduced 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 Figure 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 expression levels of Tβ4 following the second injection of recombinant adeno-associated viruses were comparable to those observed following first injection, which indicated that realizing prolonged ectopic expression by repeated injection of recombinant adeno-associated viruses was feasible (Figure 2B).

3.3 Tβ4 protected mice form LPS-induced lung injury and inflammation

Body weight continuously decreased, while lung coefficient markedly increased after LPS treatment, AAV-Tβ4 significantly 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 inflammation by interstitial edema, infiltration of inflammatory 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 significantly attenuated by AAV-Tβ4.

We found tissues MPO activity, an indicator of oxidative injury as well as neutrophil infiltration, was elevated by LPS treatment, and this increase was also attenuated by AAV-Tβ4 (Table 1). To further explore the anti-inflammatory 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 were tested, and we found AAV-Tβ4 significantly mitigated the increase (Table 2).

3.4 Tβ4 attenuated LPS-induced lung fibrosis 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 fibrotic cells clumped together, and collagen fibers accumulated (Figure 4C, D), with increased fibrosis score in LPS-treated mice (Figure 4B). All these above fibrotic changes were significantly alleviated by AAV-Tβ4, while usage of AAV-LacZ showed no significant effect (Figure4 A-D).

The expression of α-SMA was significantly lower in LPS+AAV-Tβ4 group than in NS+LPS and LPS+AAV-LacZ group (Figure 4E), and this result was also verified by western blot (Figure 4F) and immunohistochemistry (Figure 4G).

3.5 LPS promoted inflammatory responses and inhibited mitophagy in HPAEpiC

We next investigated the effect of LPS in HPAEpiC. We first confirmed that the ROS donor H₂O₂ led to decreased mitochondria membrane potential (MMP) and promoted ROS accumulation and inflammatory responses in a dose-dependent manner. Moreover, the antioxidant NAC decreased the LPS/H₂O₂-induced inflammatory responses in HPAEpiC, suggesting that ROS plays a central role in LPS-induced inflammation in HPAEpiC (Figure 5A-F).

Recently mitophagy has been shown to alleviate inflammation via inhibiting the NLRP3 inflammasome, we then tested whether ROS induce inflammatory 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 inflammatory responses (Figure 5G, H). Because ROS-induced inflammatory 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 H₂O₂ 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 H₂O₂promoted Tom40 accumulation in a dose-dependent manner (Figure 5I).

3.6 Tβ4 attenuated 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, and found that Tβ4 alleviated LPS/H₂O₂-induced decreased expression of PINK1, and Tom40 accumulation (Figure 6A), we also revealed that Tβ4 successfully suppressed LPS/H₂O₂-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).

3.7 Tβ4 suppressed the proliferation and attenuated the TGF-β1-induced activation of HLF-1 cells

The CCK-8 assay showed that Tβ4 significantly 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).

Pulmonary fibrosis is a heterogeneous disease with significant global morbidity and mortality. The mechanism of disease pathogenesis of PF is now poorly understood. Recent studies have shown that PF mainly results from inflammation and consequently fibroblast proliferation, which leads to abnormal deposition of extracellular collagen[3]. In the present study, we firstly found 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 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 sufficient enough to alleviate lung injury and fibrosis. Our data revealed protective effect of Tβ4 in pulmonary fibrosis, AAV-mediated dramatic overexpression of in mouse lung successfully alleviated LPS-induced lung injury and fibrosis in mice. Our results also indicated that the protective role of Tβ4 may involve suppressing oxidant damage and inflammasome activity, and then alleviating fibrosis.

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 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 promotes 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 treatment of LPS induced ROS generation in HPAEpiC, leading to activation of NLRP3 inflammasome, and 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, 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/H₂O₂ induced IL-1β secretion，whereas oligomycin, an mitophagy inhibitor, promoted LPS/H₂O₂ induced IL-1β secretion in HPAEpiC. Defective mitophagy leads to accumulation of damaged ROS-generating mitochondria and then activation of NLRP3 inflammasome, our data revealed for the first time that ROS promotes inflammation 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 significantly attenuated LPS-induced elevation of mouse pulmonary MPO activity, MDA content and pro-inflammatory cytokines in vivo and LPS/ H₂O₂ induced mitophagy inhibition and inflammasome activation in vitro. Inflammation is thought participates in the initial period of pathogenesis of lung fibrosis, dysfunction of alveolar epithelial cells and subsequent inflammation trigger fibrogenic process, resulting in the deposition of matrix and remodeling of lung[25]. Our data demonstrated that Tβ4 alleviated LPS-induced lung inflammation and fibrosis in mice, and suppressed 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, 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 inflammation and fibrosis. Modulating of Tβ4 may be novel strategies for treating PF.

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 financially 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.

Authors' contributions: Zhen Tian contributed to the study conception and design. Experiments were performed by Naijuan Yao, Zhen Tian. Data analysis were performed by Zhen Tian, Fei Wang. The first draft of the manuscript was written by Zhen Tian and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgment: Zhen Tian wants to thank, in particular, for the love, support and patience from Jiajie Ma.

Sgalla, G., B. Iovene, M. Calvello, M. Ori, F. Varone, and L. Richeldi. 2018. Idiopathic pulmonary fibrosis: pathogenesis and management. Respiratory research 19: 32.
Wang, K., Q. Ju, J. Cao, W. Tang, and J. Zhang. 2017. Impact of serum SP-A and SP-D levels on comparison and prognosis of idiopathic pulmonary fibrosis: A systematic review and meta-analysis. Medicine 96: e7083.
Wolters, P.J., H.R. Collard, and K.D. Jones. 2014. Pathogenesis of idiopathic pulmonary fibrosis. Annual review of pathology 9: 157-179.
Fois, A.G., E. Sotgiu, V. Scano, S. Negri, S. Mellino, E. Zinellu, P. Pirina, G. Pintus, C. Carru, A.A. Mangoni, and A. Zinellu. 2020. Effects of Pirfenidone and Nintedanib on Markers of Systemic Oxidative Stress and Inflammation in Patients with Idiopathic Pulmonary Fibrosis: A Preliminary Report. Antioxidants 9 .
Oldham, J.M., S.F. Ma, F.J. Martinez, K.J. Anstrom, G. Raghu, D.A. Schwartz, E. Valenzi, L. Witt, C. Lee, R. Vij, Y. Huang, M.E. Strek, and I. Noth. 2015. TOLLIP, MUC5B, and the Response to N-Acetylcysteine among Individuals with Idiopathic Pulmonary Fibrosis. American journal of respiratory and critical care medicine 192: 1475-1482.
Laporta, H.R., P.M. Aguilar, C.M.T. Lázaro, and G.P. Ussetti. 2018. Lung Transplantation in Idiopathic Pulmonary Fibrosis. Medical sciences 6 .
Mannherz, H.G., and E. Hannappel. 2009. The beta-thymosins: intracellular and extracellular activities of a versatile actin binding protein family. Cell motility and the cytoskeleton 66: 839-851.
Jin, R., Y. Li, L. Li, D.H. Kim, C.D. Yang, H.S. Son, J.H. Choi, H.J. Yoon, and K.C. Yoon. 2020. Anti-inflammatory effects of glycine thymosin β4 eye drops in experimental dry eye. Biomedical reports 12: 319-325.
Han, T., Y. Liu, H. Liu, Z.Y. Zhu, Y. Li, S.X. Xiao, Z. Guo, and Z.G. Zhao. 2010. Serum thymosin beta4 levels in patients with hepatitis B virus-related liver failure. World journal of gastroenterology 16: 625-630.
Conte, E., E. Fagone, E. Gili, M. Fruciano, M. Iemmolo, M.P. Pistorio, D. Impellizzeri, M. Cordaro, S. Cuzzocrea, and C. Vancheri. 2016. Preventive and therapeutic effects of thymosin β4 N-terminal fragment Ac-SDKP in the bleomycin model of pulmonary fibrosis. Oncotarget 7: 33841-33854.
Shah, R., K. Reyes-Gordillo, Y. Cheng, R. Varatharajalu, J. Ibrahim, and M.R. Lakshman. 2018. Thymosin β4 Prevents Oxidative Stress, Inflammation, and Fibrosis in Ethanol- and LPS-Induced Liver Injury in Mice. Oxidative medicine and cellular longevity 2018: 9630175.
De Santis M, R. Inzitari, S.L. Bosello, G. Peluso, C. Fanali, F. Iavarone, G. Zizzo, M. Bocci, T. Cabras, I. Messana, L. Fuso, F. Varone, G. Pagliari, M. Castagnola, and G. Ferraccioli. 2011. β-Thymosins and interstitial lung disease: study of a scleroderma cohort with a one-year follow-up. Respiratory research 12: 22.
Levine, B., and G. Kroemer. 2008. Autophagy in the pathogenesis of disease. Cell 132: 27-42.
Lazarou, M., D.A. Sliter, L.A. Kane, S.A. Sarraf, C. Wang, J.L. Burman, D.P. Sideris, A.I. Fogel, and R.J. Youle. 2015. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524: 309-314.
Sorbara, M.T., and S.E. Girardin. 2011. Mitochondrial ROS fuel the inflammasome. Cell research 21: 558-560.
Renga, G., V. Oikonomou, S. Moretti, C. Stincardini, M.M. Bellet, M. Pariano, A. Bartoli, S. Brancorsini, P. Mosci, A. Finocchi, P. Rossi, C. Costantini, E. Garaci, A.L. Goldstein, and L. Romani. 2019. Thymosin β4 promotes autophagy and repair via HIF-1α stabilization in chronic granulomatous disease. Life science alliance 2 .
Kumar, S., and S. Gupta. 2011. Thymosin beta 4 prevents oxidative stress by targeting antioxidant and anti-apoptotic genes in cardiac fibroblasts. PloS one 6: e26912.
Suzuki, T., Y. Tada, S. Gladson, R. Nishimura, I. Shimomura, S. Karasawa, K. Tatsumi, and J. West. 2017. Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition. Respiratory research 18: 177.
Yuan, J., Y. Shen, X. Yang, Y. Xie, X. Lin, W. Zeng, Y. Zhao, M. Tian, and Y. Zha. 2017. Thymosin β4 alleviates renal fibrosis and tubular cell apoptosis through TGF-β pathway inhibition in UUO rat models. BMC nephrology 18: 314.
Peng, H., J. Xu, X.P. Yang, X. Dai, E.L. Peterson, O.A. Carretero, and N.E. Rhaleb. 2014. Thymosin-β4 prevents cardiac rupture and improves cardiac function in mice with myocardial infarction. American journal of physiology. Heart and circulatory physiology 307: H741-751.
Liu, M.H., A.H. Lin, H.F. Lee, H.K. Ko, T.S. Lee, and Y.R. Kou. 2014. Paeonol attenuates cigarette smoke-induced lung inflammation by inhibiting ROS-sensitive inflammatory signaling. Mediators of inflammation 2014: 651890.
Li, F., M. Xu, M. Wang, L. Wang, H. Wang, H. Zhang, Y. Chen, J. Gong, J.J. Zhang, I.M. Adcock, K.F. Chung, and X. Zhou. 2018. Roles of mitochondrial ROS and NLRP3 inflammasome in multiple ozone-induced lung inflammation and emphysema. Respiratory research 19: 230.
Downs, C.A., N.M. Johnson, G. Tsaprailis, and M.N. Helms. 2018. RAGE-induced changes in the proteome of alveolar epithelial cells. Journal of proteomics 177: 11-20.
Basit, F., van Oppen LM, L. Schöckel, H.M. Bossenbroek, van Emst-de Vries SE, J.C. Hermeling, S. Grefte, C. Kopitz, M. Heroult, W.P. Hgm, and W.J. Koopman. 2017. Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells. Cell death & disease 8: e2716.
O'Dwyer, D.N., S.L. Ashley, S.J. Gurczynski, M. Xia, C. Wilke, N.R. Falkowski, K.C. Norman, K.B. Arnold, G.B. Huffnagle, M.L. Salisbury, M.K. Han, K.R. Flaherty, E.S. White, F.J. Martinez, J.R. Erb-Downward, S. Murray, B.B. Moore, and R.P. Dickson. 2019. Lung Microbiota Contribute to Pulmonary Inflammation and Disease Progression in Pulmonary Fibrosis. American journal of respiratory and critical care medicine 199: 1127-1138.

Table 1 AAV-Tβ4 alleviates increases in MDA content and MPO activity in lung tissues

		NS	NS+LPS	LPS+AAV-LacZ	LPS+AAV- Tβ4
	Time	(n=5)	(n=5)	(n=5)	(n=5)
MDA	Day 7	0.64±0.68	1.42±0.12	1.46±0.32	0.95±0.17*
(nmol/mg prot.)	Day 42	0.78±0.35	1.71±0.45	1.69±0.29	1.16±0.29*
MPO	Day 7	0.45±0.43	0.89±0.23	0.99±0.34	0.53±0.17*
(U/g)	Day 42	0.62±0.23	1.07±0.26	1.02±0.19	0.79±0.17*

^*P<0.05 vs the NS+LPS or LPS+AAV-LacZ group

Table 2 AAV-Tβ4 alleviates LPS-induced upregulation of inflammatory factors in lung tissue

Concentration in BALF (pg/mL)	NS	NS+LPS	LPS+AAV-LacZ	LPS+AAV- Tβ4
	(n=5)	(n=5)	(n=5)	(n=5)
IL-1β	63.15±2.11	84.43±1.67^#	85.13±1.76^#	70.18±2.55*
IL-6	76.87±1.91	90.12±3.33^#	92.23±3.21^#	79.38±2.11*
TNF-α	6.76±1.89	15.56±7.23^#	16.15±7.15^#	9.05±1.97*

^#P<0.01 vs the NS group

^*P<0.01 vs the NS+LPS or LPS+AAV-LacZ group

Download PDF

Journal Publication

published 19 Aug, 2021

Read the published version in Inflammation →

Reviews received at journal
22 Apr, 2021
Editor assigned by journal
22 Apr, 2021
Reviewers invited by journal
22 Apr, 2021
First submitted to journal
21 Apr, 2021

You are reading this latest preprint version

Thymosin β4 protect against LPS induced lung injury and inflammation and subsequent fibrosis in mice

Status:

Journal Publication

Version 2

Abstract

Figures

1. Introduction

2. Materials And Methods

3. Results

4. Discussion

Declarations

References

Tables

Status:

Journal Publication

Version 2