4-phenylbutyric acid alleviates bleomycin-induced pulmonary brosis in mouse via inhibition of endoplasmic reticulum stress

Background: 4-phenylbutyric acid (4-PBA) is a chemical chaperone that may aid the folding of proteins and alleviate endoplasmic reticulum (ER) stress by inhibiting the unfolded protein response (UPR). This study explores the effects of 4-PBA on idiopathic pulmonary brosis (IPF) using a murine model of bleomycin (BLM)-induced pulmonary brosis. Methods: Pulmonary brosis was induced in C57BL/6 mice by intratracheal injection of BLM. Sixty mice were randomly allocated into three groups: BLM group (n=20), BLM+4-PBA group (n=20), and control group (n=20). Lung tissues and lung function were analyzed to evaluate the degree of pulmonary brosis and the survival of the mice was noted. The expression levels of the ER stress markers activating transcription factor 6 (ATF6) and C/EBP Homologous Protein (CHOP) were analyzed in lung tissues from IPF patients and healthy controls as well as the mice. Results: Lung tissues from IPF patients expressed signicantly higher levels of ATF6 and CHOP compared to those from healthy controls. BLM induced signicant collagen deposition in the lungs of the mice, which was prevented by 4-PBA. 4-PBA also dramatically improved pulmonary function and increased the survival rate in the BLM+4-PBA group compared to that in the BLM group. Both the protein and mRNA expression levels of ATF6 and CHOP were signicantly reduced in mouse lung tissue after 2 weeks of 4-PBA treatment. Conclusions: This study demonstrated that 4-PBA treatment could alleviate BLM-induced pulmonary brosis in mice via the attenuation of ER stress.


Background
Idiopathic pulmonary brosis (IPF) is a lung disease of unknown etiology characterized by chronic, progressive, and diffuse parenchymal pathology. Despite decades of research, there is still no effective treatment available for IPF and the underlying pathogenesis is poorly understood. Accumulating evidence suggests that repetitive injury to alveolar epithelial cells activate the differentiation of pulmonary broblasts to myo broblasts, causing extracellular matrix deposition and lung remodeling [1,2].
Endoplasmic reticulum (ER) stress is emerging as a vital player in alveolar epithelial cell apoptosis and the proliferation of pro brotic cells in lung tissues during the course of IPF [3].
The ER is the largest organelle in cells and plays a critical role in many cellular processes, including protein folding and transport [4]. When the inner stability of the ER is disrupted by extracellular stimuli and changes in intracellular homeostasis, the unfolded protein response (UPR) may be activated due to the accumulation of misfolded and unfolded proteins in the ER lumen. If the UPR fails to restore ER homeostasis, ER stress occurs, disrupting the protein folding process in the ER [5].
Three major signaling pathways are involved in ER stress in mammalian cells: inositol-requiring enzyme 1 alpha (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6) pathways. Under normal conditions, IRE1, PERK, and ATF6 are bound by chaperone proteins such as immunoglobulin heavy-chain-binding protein (BiP; also known as GRP78) in an inactive state. Chaperone proteins assist in the normal folding of proteins. Owning to the higher a nity of BiP for misfolded proteins, BiP dissociates from the three ER sensors as misfolded proteins accumulate in the ER lumen, which initiates signaling cascades to protect and restore the function of the ER [6]. However, severe or prolonged ER stress instead activates the downstream cell death pathways through caspase-12, C/EBP homologous protein (CHOP), and c-Jun NH2-teiminal kinase (JNK) [7].
ER stress and UPR have been found to be involved in a number of human diseases such as cardiac brosis, diabetes, some neurological diseases, and also IPF [8][9][10]. The potential link between ER stress and pulmonary brosis was rst suspected after studying a family with familial interstitial pneumonia (FIP) and a mutation in surfactant protein C (SFTPC) [11]. Subsequently, Baek et al. [12] identi ed the high expression of UPR markers such as CHOP and ATF6 in the alveolar epithelium of patients with sporadic IPF and FIP, even when SFTPC mutations are absent. In addition, murine models have revealed enhanced lung brosis following BLM treatment if the alveolar epithelium was subjected to ER stress. The underlying mechanism was proposed to be, at least in part, increased apoptosis of alveolar epithelial cells [2]. Recently, Xu et al. [13] found that particulate matter with a diameter ≤2.5 mm (PM 2.5 ) could stimulate ER stress and thereby enhance BLM-induced pulmonary brosis in rats. Our previous studies [14,15] revealed that the ER stress markers, ATF6 and CHOP, were strongly associated with the development and progression of IPF.
The chemical chaperone 4-Phenylbutyric acid (4-PBA) can act as an ammonia scavenger, a weak histone deacetylase inhibitor, and also an ER stress inhibitor [16][17][18][19]. Recent research has demonstrated that 4-PBA can modulate UPR activation [20]. 4-PBA has been shown to have protective effects on various diseases such as myocardial hypertrophy, acute kidney injury, and cardiac brosis [9,18,19]. However, the use of 4-PBA in IPF has not been extensively investigated. The present study aimed to explore the effects of 4-PBA in a BLM-induced murine model of pulmonary brosis, which may lead to a better understanding of the pathogenesis of IPF and ultimately provide a novel treatment strategy for IPF.

Immunohistology of human lung tissues
Human lung tissues were kindly provided by Prof. Hui-ping Li (Shanghai Pulmonary Hospital, Tongji University). Lung tissues from IPF patients undergoing lung transplantation were collected. Unused healthy donor lungs served as the controls. Written informed consent was obtained from all study participants. The study was approved by the Ethics Committee of Shanghai Pulmonary Hospital Lung tissue was xed in formalin and embedded in para n. The expression levels of ATF6 and CHOP protein were visualized by immunohistochemistry. Positive expression was shown as a dark brown color.
The sections were counterstained with hematoxylin and eosin. The percentages of ATF6-and CHOPpositive cells were examined under a light microscope (20×, LEICA SCN400).

Animal model and 4-PBA treatment
Speci c pathogen-free grade wild-type male C57BL/6 mice (Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China) were housed for 1-week acclimation under a 12h light/ 12h dark cycle, controlled temperature (22-24°C) and humidity (50-60%) with free access to water and standard rodent chaw (normal diets with 4% of energy from lipid, XieTong Organism, China). After 1-week acclimation, mice were randomly divided into three groups: BLM group, BLM+4-PBA group, and control group, with 20 mice in each group. The study protocol was approved by the Institutional Animal Care and Use Committee at Soochow University (Approval No.: K18-028).
On day 0, all mice were injected intraperitoneally with sodium pentobarbital (150 mg/kg body weight solution in saline, Bio-Light Biotech Co. Ltd., Shanghai, China). Following anesthetization, mice in both the BLM group and BLM+4-PBA group received intratracheal injections of BLM (5.0 U/kg body weight solution in saline, Nippon Kayaku Co. Ltd., Tokyo, Japan) to induce lung brosis, while mice in the control group received equivalent volumes of vehicle (saline). In accordance with previous protocols [21], the mice were injected intraperitoneally with 4-PBA (500 mg/kg body weight solution in PBS) daily starting from day 15 for 2 weeks. Mice in the BLM and control groups were injected with equivalent volumes of saline. The mice were followed up for 4 weeks and their survival until day 28 was recorded. The work ow is shown in Figure 1.

Mouse lung function
The mice underwent tracheostomies and were intubated with an 18G intravenous catheter under sodium pentobarbital anesthesia (intraperitoneal injection). The mice were then placed in a Pulmonary Maneuvers System body plethysmograph (DSI's Buxco Electronics, Minnesota, USA) [22]. Lung function measurements were performed according to the manufacturer's instructions as previously described. For each animal, the average of three acceptable measurements was used. The parameters that were of main interest were forced vital volume (FVC), forced expiratory volume in 50 milliseconds (FEV50), and dynamic compliance (Cdyn).

Lung Collagen Measurements
Total collagen content in mice lung tissue (30-40 mg) were quanti ed using a hydroxylproline assay (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer's instructions. Hydroxyproline is a major component of the protein collagen. Using the alkaline hydrolysis method, hydroxyproline concentration is re ected as a colorimetric (550 nm) product that can be read with a spectrophotometer (Epoch2 microplate reader, BioTek, Vermont, USA), and then calculated using a standard formula according to the manufacturer's protocol.
Histopathology of mice lung tissue Mice were sacri ced using sodium pentobarbital anesthesia and cervical dislocation on day 21 or day 28 and the lung tissue was collected. Formalin-xed and para n-embedded mice lung tissue was sectioned and mounted on adhesion microscope slides for staining with hematoxylin and eosin or Masson's trichrome according to established protocols. The histopathological scoring for in ammation and brosis were performed by two experienced pathologists. The score standards were determined according to the Ashcroft score on lung histology [23].
Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted by using the EZgene TM Tissue RNA Miniprep Kit (BIOMIGA, San Diego, CA, USA) according to the manufacturer's instructions. The ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) was used for reverse transcription of RNA to cDNA. Quantitative real-time PCR was carried out using the ABI7500 system (Applied Biosystems). Primers for ATP6, CHOP, and β-actin (housekeeping gene) were obtained from Sangon Biotech (Shanghai, China) and their sequences are listed in Table 1. The PCR reaction was performed using the Thunderbird SYBR qPCR Mix Kit (TOYOBO, Osaka, Japan). The comparative cycle time (CT) method was used to determine the relative mRNA expression levels. The fold changes were calculated using the 2 −△△ CT method normalized to the housekeeping gene β-actin.

Western blot
Mice lung tissue was homogenized and the total protein extracted using the radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Shanghai, China). The lysate was centrifuged and the supernatant was collected. The bicinchoninic acid (BCA) Protein Assay Kit (Biotechwell, Shanghai, China) was used to determine the protein concentration. For the analysis of ATF6 and CHOP protein expression, 15-20 µg of lysates were separated on 10-12% SDS-PAGE Tris Bis gels and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were incubated with 5% w/v bovine serum albumin in tris-buffered saline with Tween 20 (TBST; Sigma-Aldrich) to block non-speci c binding for 2 h at room temperature. After three washes, the membranes were incubated with the following primary antibodies: anti-ATF6 Ab (1:1000, Abcam, Cambridge, USA), anti-CHOP Ab (1:1000, Cell Signaling), or antiβ-actin (1:2000, Arigo Biolaboratories, Shanghai, China) overnight at 4°C. The membranes were subsequently incubated with horseradish peroxidase-labeled secondary antibodies (1:2000, Huabio, Hangzhou, China) for 1 h at room temperature. The protein bands were visualized using the ChemiDoc™ XRS+ System (Bio-Rad, Hercules, CA, USA). Protein expression levels were determined based on the relative uorescence intensity of the protein band and normalized to the housekeeping protein β-actin.
Image analysis was carried out using Image Lab™ Software (Bio-Rad, Hercules, CA, USA).

Statistical analysis
All data were presented as the mean ± standard error (SEM). Comparison of continuous variables was carried out using the unpaired Student's t-test for two groups or one-way analysis of variance (ANOVA) with Bonferroni's post hoc analysis for multiple groups. Survival data for the mouse model was analyzed using Kaplan-Meier survival curves and log-rank test. p values ≤ 0.05 were considered statistically signi cant. All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA).  (Fig. 2).

4-PBA increased survival of mice with BLM-induced pulmonary brosis
Survival analysis showed that the BLM-treated mice who also received 4-PBA treatment from day 15 to day 28 showed a higher survival rate than the BLM-only group (Fig. 4). Hydroxyproline, which is a surrogate marker for collagen deposition, increased following BLM treatment (day21 median,  (Fig. 5). These results strongly suggested that 4-PBA had a positive effect on the survival of mice with BLM-induced pulmonary brosis and markedly reduced collagen deposition.

4-PBA improved lung function of mice with BLM-induced pulmonary brosis
To determine whether normal pulmonary function in mice was impaired by BLM and improved by 4-PBA, the mice were placed in a plethysmograph and the lung function parameters FVC, FEV50, and Cydn were measured.  (Fig. 6A, B, and C).

4-PBA decreased the level of ER stress and prevented the expression of ATF6 and CHOP
To evaluate the potential effect of 4-PBA in ER stress, the mRNA and protein expression levels for the ER stress markers ATF6 and CHOP in mice lung tissue were analyzed. Mice from the BLM group expressed higher levels of both ATF6 and CHOP mRNA and protein in their lungs than the control mice. 4-PBA reduced ATF6 and CHOP mRNA and protein expression levels after 1 week of treatment (Fig. 7A, B, and C). It should be noted that both decreases were more signi cant after 2 weeks of 4-PBA treatment (Fig.  7D, E, and F). Baek et al. [12] identi ed an upregulation in the expression of UPR markers such as GRP78 as well as CHOP and ATF6 in lung tissues from both BLM-treated mice and IPF patients. The present study has demonstrated that the expression levels of ATF6 and CHOP were increased both in IPF patients and in a BLM-induced mouse model of pulmonary brosis, which is consistent with the results in the published literature. Moreover, 4-PBA effectively inhibited the expression of ER stress markers and thereby alleviated brosis progression in mice.

4-PBA has been shown to exert protective effects on brotic processes [27]
. Rishikof et al. [28] found that 4-PBA could reduce type I collagen deposition in human lung broblasts. In the transforming growth factor (TGF)-β1-induced mice and human broblasts, 4-PBA was shown to reduce the expression of UPR markers such as GRP78, X-box binding protein 1, and ATF6a, along with the levels of myo broblast markers such as a-SMA and type I collagen, which suggested that 4-PBA was able to attenuate ER stress and inhibit the myo broblastic differentiation of broblasts in IPF [12]. In RLE-6TN cells, 4-PBA was found to suppress TGF-β1-induced epithelial-mesenchymal transition via the inhibition of ER stress, which in turn blocked the progression of IPF [29]. In the present study, we observed that 4-PBA reduced collagen deposition in BLM-induced pulmonary brosis. We also found that 4-PBA improved the lung function and survival rate. Moreover, our data showed that the expression of CHOP and ATF6 mRNA and protein decreased with 4-PBA treatment, which suggested that 4-PBA alleviated BLM-induced pulmonary brosis via attenuation of the ATF6 pathway. However, ER stress is a ubiquitous protection mechanism. Further study is required to determine the optimal time and course of 4-PBA treatment for IPF.

Conclusion
Taken together, the present study demonstrated that 4-PBA protected mice from BLM-induced pulmonary brosis via the inhibition of ER stress. This may provide new insights for the development of future therapies for IPF. Consent for publication: All authors approved the nal manuscript.
Availability of data and material All data generated or analyzed during this study are included in this published article and its supplementary information les.