SQSTM1

Accepted Manuscript Autophagy induction by celastrol augments protection against bleomycin-induced experimental pulmonary fibrosis in rats: Role of adaptor protein p62/ SQSTM1 Thomas Divya, Anandasadagopan Sureshkumar, Ganapasam Sudhandiran PII:

S1094-5539(17)30034-2

DOI:

10.1016/j.pupt.2017.04.003

Reference:

YPUPT 1612

To appear in:

Pulmonary Pharmacology & Therapeutics

Received Date: 26 January 2017 Revised Date:

27 March 2017

Accepted Date: 3 April 2017

Please cite this article as: Divya T, Sureshkumar A, Sudhandiran G, Autophagy induction by celastrol augments protection against bleomycin-induced experimental pulmonary fibrosis in rats: Role of adaptor protein p62/ SQSTM1, Pulmonary Pharmacology & Therapeutics (2017), doi: 10.1016/ j.pupt.2017.04.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Autophagy induction by celastrol augments protection against bleomycin-induced experimental pulmonary fibrosis in rats: Role of adaptor protein p62/ SQSTM1

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Thomas Divya1, Anandasadagopan Sureshkumar2 and Ganapasam Sudhandiran1*

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Department of Biochemistry and Biotechnology, Central Leather Research Institute, Chennai-600 020, India

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Cell Biology Laboratory, Department of Biochemistry, University of Madras, Guindy Campus, Chennai – 600 025, India

*Corresponding author G.Sudhandiran, Ph.D Assistant Professor Department of Biochemistry University of Madras Guindy Campus, Chennai – 600 025, INDIA Mobile: +91 9790816696 E-mail: [email protected] [email protected]

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ACCEPTED MANUSCRIPT Abstract

Pulmonary fibrosis (PF) is a chronic pulmonary disease of unknown cause with high mortality. Autophagy is an important homeostatic process that decides the fate of cells under stress conditions. This study is aimed to investigate whether impaired autophagic activity leads to fibrosis and pharmacological induction of autophagy provides protection against

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bleomycin (BLM)-induced PF. A single dose of BLM (3 U/kg body weight) was administered intratracheally to induce fibrosis in rats. Celastrol, a triterpenoid (5 mg/kg/body weight, intraperitoneally) was given in every 81 hrs for a period of 28 days. Western blot and Confocal microscopic analysis of rat lung tissue samples revealed that celastrol induces

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autophagy in BLM-induced rats. Transmission electron microscopic analysis supports the above findings. Celastrol increased the expressions of Beclin 1 and Vps 34, promoted the upregulation of Atg5-Atg12-16 formation and enhanced the lipidation of LC3-I to LC3-II

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suggesting induction of autophagy by celastrol provide protection against lung fibrosis. Further, we revealed that celastrol activates autophagy by inhibiting PI3-K/Akt mediated mTOR expression. In addition, we show evidences that lack of autophagy leads to p62, an autophagy adaptor protein accumulation that is degraded by celastrol. This study helps to describe the importance of autophagic cell death as a possible therapeutic target against lung

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fibrosis, and celastrol as a potential candidate for the treatment options for PF.

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Key words: Autophagy, Pulmonary fibrosis, p62, Celastrol, bleomycin, mTOR

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ACCEPTED MANUSCRIPT Abbreviations

AEC: Alveolar epithelial cell; Atg: Autophagy-related; BLM: Bleomycin; DMSO: Dimethyl sulfoxide; FITC: Fluorescein isothiocyanate;

IgG: Immunoglobulin G; IL: interleukin;

Keap1: Kelch-Like ECH-Associated Protein 1; LC3: Light chain 3; mTOR: mammalian target of rapamycin; MT: Masson's trichrome; Nrf2: Nuclear factor erythroid 2- related

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factor; PF: pulmonary fibrosis; PI3K: phosphoinositide 3-kinase; SQSTM1: Sequestosome 1; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TRITC:

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Tetramethylrhodamine isothiocyanate; Vps34: Vacuolar protein sorting 34

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ACCEPTED MANUSCRIPT 1. Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic disease of the lung, characterized by extracellular matrix (ECM) degradation and collagen accumulation which destroys lung architecture [1]. Though the aetiology of this disease is yet to be identified,

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oxidative stress is considered as one of the major cause for its progression [2]. Bleomycin (BLM) induced pulmonary fibrosis (PF) is a well established model of experimentally induced PF in rodents [3]. BLM is shown to induce similar fibrotic changes such as epithelial damage, fibroblast proliferation, increased deposition of collagen and alveolar epithelial cell

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(AEC) apoptosis in the lungs of animals [4,5].

Autophagy is a defence mechanism of a cell that degrades damaged cytoplasmic

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constituents to maintain tissue homeostasis. Autophagy occurs through the formation of double-membrane vesicles (autophagosomes) that engulfs the cytoplasmic components to be digested. The autophagosomes further fuse with lysosomes to form autolysosomes and the damaged organelles and cytoplasmic components are degraded within autolysosomes by lysosomal hydrolases [6]. Autophagy is therefore essential for a normal cell to eliminate damaged organelles, the major sources of toxic reactive oxygen species (ROS) and is crucial

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to maintain the pool of functioning organelles [7]. However, autophagy does not have a protective role always. Cell type and particular stimulus determine whether the autophagic cell death is protective or destructive to the cellular environment [8,9]. Deficient or impaired autophagy is reported in the progression of various diseases including neurodegeneration,

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cardiovascular disorder etc. Recently, emerging reports suggest the involvement of autophagy in lung diseases also [10,11]. Under oxidative stress condition, cells initially activate

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autophagy as to exterminate damaged organelles to promote cell survival. However, when oxidative stress persists for long, lysosomal membranes are degraded by intracellular ROS that impairs the autophagic activity which ultimately leads to apoptotic cell death [12]. In addition, there are evidences that deficient autophagy results in cellular senescence, fibroblast proliferation and differentiation of fibroblast into myofibroblast which are the vital processes in the pathogenesis and progression of lung fibrosis [13]. These reports suggest a role of deficient autophagy in the pathogenesis of PF. However its precise role in the progression of PF is yet to be defined. Currently there are no proven therapies that prevent or reverse PF, highlighting the need to identify new molecular targets. In this context, we have reported that celastrol

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(C29H38O4), a triterpenoid derived from the Chinese herbal medicine Tripterygium wilfordii (Thunder God Vine) possesses anti-fibrotic effect by reducing collagen accumulation and inflammation through the enhancement of nuclear factor erythroid 2- related factor (Nrf2) mediated anti-oxidant enzymes in BLM induced rat model [14]. Interestingly, recent reports reveal a cross-talk between Nrf2 signalling and autophagy and have reported the functional

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importance of p62/sequestosome 1 (SQSTM1), an autophagy adaptor protein that intimately links these two pathways [15,16]. During autophagy, p62 and associated cargo are degraded in the lysosome. However, deficient autophagy leads to accumulation of p62 and associated cargo in the cytoplasm and thereby over expression of p62 is an indication for insufficient

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autophagy [17]. Cople et al have reported that p62 directly binds with Kelch-Like ECHAssociated Protein 1 (Keap1) and over expression of p62 dissociates Keap 1 from binding with Nrf2 and thereby decreases the half-life of Keap1 [18]. Other studies also suggest the

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functional role of p62 which links autophagy and Nrf2 [19,20]. These reports prompted us to analyze the fate of autophagic cell death and level of p62 sequestosome in BLM-induced experimental PF. To the best of our knowledge, the involvement of p62 in autophagic cell death in the context of PF has not been documented in detail. In this study, we show evidence that celastrol induces autophagic cell death through the degradation of p62/SQSTM1 and

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provide protection against the fibrotic responses induced by BLM. 2. Materials and Methods

2.1. Chemicals and reagents

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Primary antibodies for Beclin1 (Rabbit mAb), Vps 34 (Rabbit polyclonal IgG), Atg 5 (Rabbit mAb), Atg 7 (Rabbit mAb), Atg 12 (Rabbit mAb), Atg 16L (Rabbit mAb), LC3I/II

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(Rabbit mAb), and Atg 3 (Rabbit polyclonal IgG) were procured from Cell Signalling Technology, USA. Primary antibodies for PI3-K (mouse monoclonal IgG), Akt (mouse monoclonal IgG), mTOR (Rabbit polyclonal IgG), p62 (Rabbit polyclonal IgG) and β-actin (mouse monoclonal IgG) were procured from Santa Cruz Biotechnology, USA. Alexafluor 488, Tetramethylrhodamine isothiocyanate (TRITC) and Fluorescein isothiocyanate (FITC) conjugated secondary antibodies were purchased from Jacksons Laboratories, UK. Hoechest 333244 stain was purchased from InvitrogenTM, USA. Bleomycin sulfate was purchased from Sigma-Aldrich, USA. Celastrol (purity ≥ 98%) was procured from Cayman Chemicals, USA. High analytical grade chemical are used for this study unless otherwise mentioned.

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ACCEPTED MANUSCRIPT 2.2. Animals

Adult male Wistar albino rats (200-240g) were purchased from Central Animal House, King Institute of preventive medicine and research centre, Chennai, India. The rats were kept in polypropylene cages under conditions of temperature 22-24°C and humidity 4550 %, 12-h light–dark cycles and provided with standard rat feed (Hindustan Unilever Ltd,

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Mumbai, India) and water ad libitum. All the procedures involving animal experiments were approved by Institutional animal ethical committee (IAEC) and performed in accordance with the guidelines of IAEC. Approval No: IAEC No.12/01/2014.

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2.3. Induction of PF in rats

Intratracheal administration of BLM was given in rats as standardized earlier in our

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laboratory [21,22]. Rats were randomly selected as six rats in each group. Prior to BLM administration, rats were anesthetized via intraperitoneal injection of ketamine and xylazine at concentration of 0.5 mg/kg b.w and 1 mg/kg b.w respectively. Rats were administered with a single dose of BLM (3U/kg b.w) through a tracheal cannula that directly inserted into the

2.4. Experimental setup

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trachea. BLM was administered on day 1 of total experimental period of 28 days.

The experimental design of the study was as follows. Group I: Normal control rats; Group II: Rats induced with BLM (3U/Kg b.w); Group III: Rats administered with celastrol (5mg/Kg b.w), twice a week in BLM-induced rats; Group IV: Rats treated with celastrol

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alone at a dosage of 5 mg/kg b.w [14]. After the experimental period of 28 days, animals

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were sacrificed, lungs were collected and relative lung weights were calculated.

2.5. Histopathological analysis of collagen accumulation Lung tissue collagen content was analysed by Masson's trichrome staining and

picrosirius red staining. Lung tissues were fixed in 3.6% buffered formaldehyde and embedded in paraffin. Tissue sections of 4 µm thickness were used for the analysis. For Masson’s trichrome staining, after rehydration of the sections with alcohol series, the tissue sections were stained with Biebrich Scarlet and then placed in phosphomolybdic/ phosphotungstic acid solution. The sections were then stained with aniline blue and observed under a light microscope (Motic digital BA210, Hongkong). For picro-sirius red staining, the tissue sections after de-paraffinization and rehydration, were stained with Sirius red (Direct

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red 80) dissolved in picric acid solution and observed under light microscope (Motic digital BA210, Hong-kong). The images were processed with NIH Image J software to quantify the percentages of collagen deposition areas.

2.6. Electron microscopic analysis of lung tissue sample

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For ultra structural studies of lung tissues using transmission electron microscopy (TEM), the samples were first fixed in 2.5% gluteraldehyde for 6-8 h at 4°C followed by 1% osmium tetra oxide solution in phosphate buffer (0.1 M) for 2 h at 4°C. The sections were dried in acetone after dehydrated in ascending series of ethyl alcohol. The tissues were then

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placed in a resin mixture of 1,2-epoxy propane and Epon 812. The resin embedded tissues were then treated with dodecyl succinic anhydride (DDSA) and methyl nadicanhydride

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(MNA). Thin hardened tissues were trimmed and cut with an ultra-microtome (Leica Ultra R). The thin sections of 40-60 nm thickness were mounted on copper grids and stained with saturated solution of uranyl acetate and lead citrate. The sections were observed under a transmission electron microscope (Morgagni 268D). For scanning electron microscopy (SEM), small pieces of tissues were initially fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer for 6-8 h at 4°C followed by 1% osmium tetroxide solution in phosphate buffer (0.1

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M) for 2 h at 4°C. After fixation, the blackened specimens were treated with ascending series of ethanol (30%, 50%, 70%, 90% and 100%) for dehydration. The specimens were allowed to dry completely and dried specimens were mounted on copper grid. The specimens were

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examined in a Scanning electron microscope (Hitachi, Europe). 2.7. Immunofluorescence analysis

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For immunofluorescence analysis, lung tissue sections of 4 µm thickness were first de-paraffinized in xylene and rehydrated in descending series of ethanol solutions. The sections were treated with blocking agent (3% bovine serum albumin) for 2 h. The sections were then immunostained with primary antibodies for Atg5 (1:500) and LC3 II (1:500) for overnight in refrigerated condition. Washed the slides thrice with tris-buffered saline (TBS) and then incubated with Alexafluor 488 conjugated secondary antibody for 2 h in dark. The sections were then counter stained with Hoechest 333244 and images were captured with a fluorescence microscope (Nicon Eclipse 80i).

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Lung tissue sections of 4µm thickness were used for confocal microscopic analysis. Paraffin embedded lung tissue sections were de-waxed and re-hydrated and incubated overnight with primary antibodies for Beclin 1 (1:500) and p62 (1:500) at 4°C. The sections were then incubated with secondary antibody (TRITC for Beclin 1 and FITC for p62) and

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kept in dark for 2 hrs. After washed with TBS, the sections treated with Beclin 1 and TRITC, were counterstained with Hoechst 333244 and the sections treated with p62 and FITC, were

microscope (Leica, TCS-SP2-XL).

2.9. Western blotting analysis of lung tissue samples

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counterstained with propidium iodide (PI) in dark. The slides were then imaged in a confocal

Lung tissue homogenate was prepared in buffer containing NaCl (0.135M), Tris HCl

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(0.02M), ethylenediaminetetraacetic acid (EDTA, 2nM) and phenylmethylsulfonyl fluoride (PMSF, 1mM). The protein content of the sample was evaluated by CB-XTM assay kit. 40 µg of denatured protein samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences, USA). The membrane was blocked with blocking agent (5% non-fat dried milk in TBS/Tween) for 1.5 h at room temperature. The

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membrane was then incubated with primary antibodies [Beclin 1 (1:2000); Vps 34 (1:1000); LC3 I/II (1:2000), Atg5 (1:1000); Atg12 (1:1000); Atg16L1 (1:1000); Atg7 (1:2000); Atg3 (1:2000); PI3-K (1:1000); Akt (1:1000); mTOR, (1:1000); p62 (1:1000); β-actin (1:1000)] for overnight at 4°C. After washing the membrane thoroughly in TBS/Tween, the membranes

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were incubated with horseradish peroxidise (HRP) conjugated secondary antibody for 1 h at room temperature. The membranes were developed by Enhanced chemiluminescence (ECL)

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and detected the protein antibody complex formed.

2.10. Statistical analysis

All the data was analyzed using SPSS/10.0 software. The results were expressed as

mean ± standard deviation for six rats in each group from three independent experiments. One-way analysis of variance (ANOVA) and least significant difference (LSD) were used to determine statistical significant difference. p < 0.05 was considered as statistically significant.

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3.1. Celastrol alters morphological changes, reduce collagen deposition in BLM-induced PF BLM-induced PF is a well studied model of fibrosis and rat lungs exhibits severe

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fibrotic responses upon administration with BLM [23,24]. An evident pulmonary edema (fluid accumulation in the lungs) was observed in BLM- induced rats. Along with this, a higher value of absolute and relative lung weight was observed in BLM-injured rats (Figure 1A & B). Of interest, celastrol significantly modulated BLM-induced pulmonary edema.

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Similarly, celastrol significantly attenuated the absolute and relative lung weight in BLMinduced group of rats. The surface topography of lung describes the extend of alveolar damage and therefore scanning electron microscope (SEM) make it a valuable tool for

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morphologic analysis of normal as well as diseased lungs, as large surface areas can easily be examined at low or high magnification. Morphological examination by SEM showed a rounded polyhedral morphology with a smooth inner surface and clear alveolar space joining neighbouring alveoli in the lung tissue of control rats (Figure 2A). However, lung tissue of BLM-induced group of rats displayed an imprecise architecture with deformed alveolar

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spaces. Celastrol altered these morphological changes induced by BLM to some extent and celastrol alone treated group of rats exhibited morphology same as to control rats. Extracellular matrix (ECM) degradation and collagen accumulation is the major culprit in the progression of PF. Masson's trichrome (MT) and Sirius red are presented as the methods for

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collagen determination since these staining methods can distinguish collagen from other fibres. Figure 2B & 2C represents the accumulation of collagen in lung tissue of control and experimental group of rats, as evaluated by MT and picrosirius red staining. Celastrol

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significantly attenuated the BLM-induced collagen deposition in rat lungs which shows its anti-fibrotic effect. The quantification of MT and Picrosirius red staining is shown in Figure 2D.

3.2. Celastrol induces autophagy in BLM-induced PF 3.2.1. Initiation of autophagosome formation by celasteol To analyze the protective role of autophagy in the progression of PF, we have elucidated the autophagy pathway in detail in BLM-induced fibrosis model. Beclin 1-Vps 34, a class III phosphatidylinositol 3-kinase complex is very essential for the formation of the

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autophagosome membrane from the endoplasmic reticulum (ER) [25]. Beclin 1, a major protein which regulates autophagosome formation was analyzed with confocal microscopy (Figure 3A). An increased expression of Beclin 1 was evident in lung tissue of celastrol treated group of rats induced with BLM. Surprisingly, celastrol alone treated group of rats also exhibited an increased expression of Beclin 1. Figure 3B represents Western blotting

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analysis of Beclin 1 and Vps 34 in lung tissue of control and experimental group of rats. BLM-injured lung tissues revealed a decreased expression of these proteins which was increased upon treatment with celastrol. As expected, celastrol alone treated group of rats also showed increased expressions of both Beclin 1 and Vps 34 implying the role of celastrol

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in the formation of autophagosome.

3.2.2. Celastrol enhances the expression of proteins involved in the elongation of

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autophagosome

The expansion of autophagosomes depending on the formation and activation of two ubiquitin-like complexes [26,27]. First step is the conjugation of Atg12 with Atg5 in the presence of Atg7. Atg 16L is then recruited to Atg5-Atg 12 complex. The resulting complex helps in the elongation of the autophagosme [26]. In order to demonstrate the role of celastrol

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in the elongation of autophagosome, we have analysed the expression of Atgs involved in the formation of multimeric complex. Western blotting analysis of expression of Atg 5, Atg 7, Atg 12 and Atg 16L proteins are shown in Figure 4A. Two fold increases in the expression level of these proteins was evident in lung tissue of celastrol treated group of rats when

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compared with BLM-injured rat lungs. Figure 4B shows the immunofluorescence analysis of Atg 5 in lung tissue sections of control and experimental groups. In BLM-induced rat lungs, decreased expression of Atg5 was evident, whereas treatment with celastrol, an increased

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expression of Atg5 was observed which coincides with Western blotting analysis. 3.2.3. Role of celastrol in the final stage elongation and maturation of autophagosome The complete formation of autophagosome requires microtubule-associated protein-1

light chain 3 (LC3), a ubiquitin like protein and Atg 8 [28]. LC3 modification is one of the essential steps in the final stage of elongation of autophagosome. The conversion of LC3 to lipidated form ie., LC3-II is considered as an autophagosomal marker, and is used to study autophagy in experimental models. In order to elucidate the protective autophagy induction by celastrol, we have analyzed the expression level of LC3I/II and Atg 3 in lung tissue of control and experimental group of rats. Figure 5A represent the protein expression of LC3

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I/II and Atg 3 by Western blotting analysis. A significant increase in the LC3 II basal level in lung tissue of celastrol treated group of rats was observed. Similarly, we found an abundant expression of Atg3 in lung tissue of celastrol treated group of rats. Immnofluorescence analysis of LC3II was performed and depicted in Figure 5B. Celastrol significantly enhanced the expression of this autophagic marker in BLM-injured rat lungs which shows that celastrol

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enhances the modification of LC3 to LC3II. Electron microscopic analysis of autophagoseme is shown in Figure 6A. In contrast to the lungs of BLM injured rats, which shows rare autophagosme, numerous autophagosome vacuoles were observed in celastrol treated rat lungs (labelled with arrows) which supports the Western blot and immunofluorescence

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results. Quantification of autophagic vacuoles is depicted in Figure 6B. Together, these data point out that celastrol is an inducer of autophagy against BLM-induced experimental PF.

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3.3. Celastrol inhibits the expression of mTOR complex proteins and reduces BLMinduced fibrosis

mTOR signalling is known as one of the key regulators of autophagy and therefore, we have analyzed whether the expression of mTOR affect the progression of fibrosis in BLM model by Western blotting analysis. As shown in Figure 7, the lung tissue of BLM-induced

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rats exhibited enhanced expression of mTOR along with phosphatidyl inositol 3-kinase (PI3K)-Akt. However celastrol treatment significantly reduced the expression of these kinase complexes.

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3.4. Celastrol regulates p62 accumulation during BLM-induced PF The chaperone molecule p62 that carries cytoplasmic cargo to the autophagosome for degradation is greatly associates with autophagic cell death. The expression level of p62 is

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inversely correlates with autophagy [29]. The results of previous experiments show a defective autophagy upon the administration of BLM to rat lungs. Next, we assessed the expression level of p62 in rat lung tissues. The purpose was to determine whether defective autophagy causes accumulation of p62 in BLM-injured rat lungs and whether celastrol could modulate the accumulation of p62 sequestosome. Figure 8A represents the confocal microscopic analysis of p62 and shows an increased accumulation of p62 in BLM injured rat lungs. Treatment with celastrol resulted in significant decrease in the expression of this protein. Western blotting analysis of p62 is depicted in Figure 8B which shows higher level of p62 in BLM-injured rat lungs. A significant decrease in the expression of p62 was observed in lung tissue of celastrol treated group which indicates that autophagy deficiency

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leads to intracellular accumulation of p62 and celastrol regulates the over expression of p62 in BLM-induced PF. 4. Discussion Celastrol, a quinine methide triterpenoid is reported to possesses several effects such

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as anti-inflammatory, anti-cancer, anti-rheumatoid and anti-oxidant properties and it is being used for the treatment of autoimmune and neurodegenerative diseases [30-33]. It regulates the production of inflammatory mediators and cytokines including interleukins, tumour necrosis factor-α (TNF-α) and have reported as a potent inhibitor of NF-kappa β [34,35]. We

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have documented that celastrol induces Nrf2 dissociation from Keap1 by direct interaction with thiol group of cysteine residue in Keap 1 [14] in accordance with other reports [36]. Recent reports reveal that the therapeutic potential of celastrol may be based on its effects in

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regulating autophagy. Celastrol triggers both apoptosis and autophagy that represses the proliferation of osteosarcoma cells by regulating ROS/JNK signalling pathway in both in vivo and in vitro is reported [37]. Guo et al have reported that celastrol enhances autophagic cell death by targeting androgen receptors in prostate cancer [38]. This triterpenoid also prevents neurodegeneration through the activation of autophagic cell death in affected neuronal cells

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[39]. Therefore, we speculated that, if celastrol can induce protective autophagy against BLM-induced PF, it could be a better therapeutic option for the development of anti-fibrotic drug. Interestingly, other investigators have reported the functional role of p62, an autophagy adaptor protein in turnover and physical sequestration of Keap 1, which leads to Nrf2

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activation [40]. Therefore, the main question raised by this study is whether celastrol can induce autophagy and regulate the expression of p62 to provide protection against BLM-

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induced lung injury and fibrosis in experimental animals which is yet to be documented. Autophagic signalling pathway has been reported in the pathogenesis of pulmonary

diseases recently only[41-43]. It has been reported that, there is deficient expression of autophagic proteins in lung tissue samples from patients with IPF when compared with chronic obstructive pulmonary disease (COPD) patients and healthy controls [44]. Moreover, Patel et al have reported that autophagy plays a crucial role in TGF-β1 mediated fibrogenesis in IPF [45] in accordance with our findings. Autophagy begins with the recruitment of cytoplasmic elements into a cytoplasmic vesicle i.e a double-membrane autophagosome. The formation of autophagosome is a

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sequential process that includes isolation of membrane from ER, membrane nucleation, elongation and maturation [46]. In order to elucidate the functional involvement of celastrol in the induction of autophagy, we have analyzed the role of celastrol in each step in the formation and maturation of autophagosome. During autophagy, as the initial step in the formation of autophagosome membrane, Beclin 1-Vps34 complex plays a major role in

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isolation membrane nucleation from ER. Vps34 is associated with Beclin 1 to form the class III PI3K core complex which facilitate processes for the initial nucleation of autophagosome [47]. An increased expression of Beclin 1 and Vps 34 in celastrol treated group of rats when compared to BLM-injured rat lungs suggesting an induction of autophagosome formation by

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celastrol.

The subsequent step of the expansion of the isolation membrane is basically depends

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on a ubiquitin complex that recruits additional membranes. Atg5-Atg12-Atg16L is very much essential for the formation of pre-autophagosomes [48]. It is yet to known how Atg5-Atg12 complex recruits additional membranes, but it was reported that Atg7 activates the carboxyterminal glycine residue of Atg12 in an energy dependant process [49,50]. The Atg5-Atg12 complex subsequently associates with Atg16L1 to form Atg5-Atg12-Atg16 protein complex [51,52]. In lung tissue of BLM-induced rats, a decreased expression of these Atg proteins

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were evident whereas in treatment with celastrol significantly enhances the expressions which further confirm the protective effect of celastrol through the induction of autophagy which is deffective in BLM-induced rat lungs. Yet another ubiquitin-like protein complex system is necessary for the further elongation and matruration of autophagosome [53]. The

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cysteine protease Atg4 cleaves LC3 and Atgs 7 and 3 triggers the association of LC 3 with Phosphatidylethanolamines (PE). This lipidation of LC 3 to LC3-II is very much crucial and

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therefore the formation of LC3II is considered as a marker for autophagy-induction [54]. In this study, we have analyzed the expression level of LC3I/II and Atg 3 in lung tissue control and experimental group of rats by Western blotting. Celastrol significantly increases the expression of LC3II and Atg 3 in BLM-induced rat lungs. At the ultrastructural level, we have analyzed the formation of autophagosome by TEM. The formation of autophagosome vacuoles is evidently visible in sections of celastrol treated group of rats. However, TEM analysis has its own limitation that, there are chances for losing the distinguished physical characteristics of tissues while processing and therefore it might be less sensitive for taking up late-stage autophagosomes. In this point of view, we have analyzed the expressions of LC3II by immunofluorescence also and found increased expression of these proteins in

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celastrol treated group of rats. The observed role of celastrol in the induction of autophagy is quite interesting and it is evidently confirm that celastrol is a potent inducer of autophagy against BLM-induced PF. It is essential to mention that the expressions of autophagy proteins described in this work are confined to day 28 of BLM administration. Arguably, to give a clear demonstration, it is vital to describe the role of celastrol inducing autophagy in

validated through molecular docking (Data not shown).

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midphase (10-20 days) of fibrosis and interactions of celastrol with autophagy proteins as

Deficient autophagy is shown to be associated with increased cytoplasmic accumulation of p62 as it cannot be cleaved properly. Subsequently, the accumulated p62

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enhances the production of ROS inside cells which damages cellular organelles [55]. Cells possess several defence mechanisms in order to diminish the toxic effect of ROS. Nrf2-

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Keap1 signalling is one such defence mechanism of cells against oxidative stress [56]. Ni et al have demonstrated that impaired autophagy due to Atg 5 deficiency leads to p62 accumulation and prolonged Nrf2 activation [57]. These reports shed light into a possible link between Nrf2 and autophagy signalling. In this study, we assessed the expression of p62 in confocal microscopy and Western blotting and found higher level of p62 in BLM-induced tissue than control. Moreover, we have evidences that TGF-β trans-differentiated A549 cells

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treated with 3-methyladenine, a chemical inhibitor of autophagy show increased expression of Nrf2 and p62 (data not shown). Here, we proposed that celastrol, activates autophagy and degrades the accumulation of adaptor protein p62 and renders protection against PF.

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Interestingly, PI3K/Akt/mTOR complex is a negative regulator of autophagic cell death. There are evidences that the formation of autophagosomes is closely associated with the inhibition of mTOR protein [58]. Xia et al have reported an aberrant expression of

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PI3K/Akt proteins in IPF when compared with normal samples [59]. In this study, since it was observed that celastrol potentially induces autophagy, we tempted to study the level of PI3K-Akt mediated mTOR expression in rat lung tissues and our findings suggest that celastrol induces autophagy through the inhibition of these kinase complex. Yet another important observation of our study is that an increased expression of autophagic proteins is observed after BLM injury in rat lungs when compared to control rats. Alveolar epithelial cell injury modulates inflammatory cell infiltration into the lungs promoting the release of growth factors and cytokines that in turn disseminate epithelial apoptosis and lung inflammation [60]. In this condition, autophagy functions in the removal

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of dead cells to regulate the inflammation and apoptosis to modulate tissue damage [61]. This might be the reason for increased expression of autophagic proteins after BLM injury in rat lungs. However, the over production of free radicals and profibrotic cytokines further decreases autophagy, the survival mechanism that leads to worsening of the disease. We suggest that induction of autophagy could attenuate the progression of PF and p62 play a

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critical role in determining the fate of cell in autophagic cell death (Figure 9). Further works are in progress to demonstrate the mechanism of BLM induced fibrosis and the role of celastrol as a better drug regimen for the treatment modalities of PF.

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Conflict of interest The authors confirm that there are no conflicts of interest.

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Acknowledgment

DT would like to thank Council of Scientific and Industrial Research (CSIR), India, for the financial support as Senior Research Fellowship (09/115(0776)/2015-EMR-1). The authors thank Dean of research, Sri Ramachandra University, Chennai for the help in fluorescence imaging. The authors thank Prof. T. C Nag and Mr. Sandeep Arya, Sophisticated Analytical Instrument Facility (SAIF) - All India Institute of Medical Sciences

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(AIIMS), New Delhi, India, for their guidance and assistance in TEM analysis. The help of Dr.P.Palani, Assistant Professor, CAS Botany, University of Madras for SEM analysis is acknowledged. The authors would like to thank Dr.M.Susruthan, MD, Sri Ramachandra

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University, Chennai for his assistance in interpreting collagen specific staining.

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Figure 1: Effect of celastrol on BLM-induced pulmonary edema. Pulmonary edema was assessed in lung tissue of control and experimental group of rats. BLM (3U/kg B.W) was administered intratracheally to the rats. In BLM + Cel group, rats were i.p. injected with celastrol (5mg/kg B.W) twice a week. Lungs were collected after the experimental period of

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28 days. Absolute and relative lung weights are represented as graphs. Hypothesis testing method included one-way analysis of variance (ANOVA) and least significant difference (LSD) test. ‘p’ value <0.05 was considered to indicate statistical significance. Values are given as mean ± S.D for groups of 6 rats each.

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non significant, aBLM-induced vs normal

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control; bBLM+Cel vs BLM-induced, cCel alone vs control.

Figure 2: Effect of celastrol on BLM-induced fibrotic changes. A) Morphological changes

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were analyzed by SEM (a) Control rats shows normal lung morphology without any pathological deformities (b) BLM-induced group displays deformed architecture with collapsed alveolar spaces (c) Pathological alterations were reduced in celastrol treated group (d) Celastrol alone treated group of rats shows lung histology similar to that of control. Collagen accumulation was evaluated by B) Masson’s trichrome staining and C) Picrosirius

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red staining respectively. (a) Control lung section shows scarcely deposited collagen. (b) BLM-induced group shows increased collagen deposition. (c) Section of celastrol treated group of rats shows comparatively less collagen deposition to BLM-induced group. (d) Celastrol alone group of rats shows normal lung histology. The image shown is representative

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of three independent experiments. D) Collagen deposition areas were quantified. Hypothesis testing method included one-way analysis of variance (ANOVA) and least significant difference (LSD) test. ‘p’ value <0.05 was considered to indicate statistical significance. b

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Values are given as mean ± S.D for groups of 6 rats each. aBLM-induced vs normal control; BLM+Cel vs BLM-induced, cCel alone vs control.

Figure 3: Celastrol initiates autophagosome formation in BLM-induced rat lungs A) Confocal microscopic analysis of Beclin 1 in control and experimental group of rats. Primary antibody specific for Beclin 1 and TRITC conjugated secondary antibody was used. Hoechest 333244 was used as a counter stain. The sections were imaged in a Confocal laser scanning microscopy. Control section shows basal expression of Beclin 1. BLM-induced lung section exhibit few positive expression of Beclin 1. Celastrol treated group show abundant expression of Beclin 1. Celstrol alone treated group also exhibit positive expression of Beclin

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1. B) Western blot analysis of Beclin 1 and Vps 34. Lane 1: Control, Lane 2: BLM-induced, Lane 3: BLM + Celastrol, Lane 4: Celastrol alone. Data expressing the respective protein levels were quantitated using Image J software. The data is expressed as relative intensity of proteins normalized to β-actin expression. Values are given statistically significant at p < 0.05, aBLM-induced vs control; bBLM + Cel vs BLM-induced; cCel alone vs control. The

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data presented here represent the experiments performed in triplicate.

Figure 4: Effect of celastrol in the elongation of autophagosome. A) Western blot analysis of Atg 5-Atg 12-Atg 16 complex. Lane 1: Control, Lane 2: BLM-induced, Lane 3: BLM +

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Celastrol, Lane 4: Celastrol alone. Data expressing the respective protein levels were quantitated using Image J software. The data is expressed as relative intensity of proteins normalized to β-actin expression. Values are given statistically significant at p < 0.05, aBLM-

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induced vs control; bBLM + Cel vs BLM-induced; cCel alone vs control. The data presented here represent the experiments performed in triplicate. B) Immunofluorescence analysis of Atg 5. Primary antibody specific for Atg 5 and Alexafluor 488 conjugated secondary antibody was used. Hoechst 333244 was used as a counter stain. The sections were imaged in a fluorescence microscopy. Control rat lung tissue sections show fewer positive expression of

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Atg 5. BLM-induced rat lung sections show decreased expression of Atg 5. Celastrol treated group exhibits two fold increase in the expression of Atg 5. Celastrol alone treated group also exhibits increased expression of Atg 5. The image shown is a representative of three

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independent experiments. Sclae bar - 100µm.

Figure 5: Effect of celastrol in the final stage elongation of autophagosome. A) Western

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blot analysis of LC3/II and Atg 3. Lane 1: Control, Lane 2: BLM-induced, Lane 3: BLM + Celastrol, Lane 4: Celastrol alone. Data expressing the respective protein levels were quantitated using Image J software. The data is expressed as relative intensity of proteins normalized to β-actin expression. Values are given statistically significant at p < 0.05, aBLMinduced vs control; bBLM + Cel vs BLM-induced; cCel alone vs control. The data presented here represent the experiments performed in triplicate. B) Immunofluorescence analysis of LC3II. Primary antibody specific for LC3II and Alexafluor 488 conjugated secondary antibody was used. Hoechst 333244 was used as a counter stain. The sections were imaged in a fluorescence microscopy. Control section show basal level of LC3 II. BLM injured lung section exhibit decreased epression of LC3II when compared to control. Lung section of

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celastrol treated group exhibit abundant expression of LC3II as compared to BLM group. Celastrol alone treated group also exhibit positive expression of LC3II. Sclae bar - 100µm.

Figure 6: A) Autophgosome detection by TEM analysis. Representative electron microscopy images from lung tissue of (a) control, (b) BLM-induced, (c) BLM + Cel and (d)

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Cel alone treated group of rats. Yellow arrow in the figure indicates autophagosomes. Lung tissue section of control group of rats shows clear nuclei, mitochondria and cytoplasm. Tissue section of BLM-induced rat lungs shows more accumulation of collagen fibers in lungs. Celastrol treated rat lungs exhibits clear nucleus and more number of autophagosomes around

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the nucleus. Celastrol alone treated rat lungs exhibits intact cellular organelles. The image shown is a representative of three independent experiments. B) Quantification of autophagic vacuoles. Values are given statistically significant at p < 0.05. aBLM-induced vs control; BLM + Cel vs BLM-induced; cCel alone vs control.

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Figure 7: Effect of celastrol on the expression PI3K/Akt/mTOR kinase complex. Western blot analysis of PI3K-Akt mediated mTOR. Lane 1: Control, Lane 2: BLM-induced, Lane 3: BLM + Celastrol, Lane 4: Celastrol alone. Data expressing the respective protein levels were

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quantitated using Image J software. The data is expressed as relative intensity of proteins normalized to β-actin expression. Values are given statistically significant at p < 0.05. The data presented here represent the experiments performed in triplicate.

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Figure 8: Effect of celastrol on the expression of p62. A) Confocal microscopic analysis of p62 in control and experimental group of rats. Primary antibody specific for p62 and FITC

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conjugated secondary antibody was used. PI was used as a counter stain. The sections were imaged in a Confocal laser scanning microscopy. Control section shows negligible expression of p62. BLM injured lung section exhibit abundant expression of p62. Celastrol treated group of lung section exhibited reduced expression of p62 as compared to BLM group. The image shown is a representative of three independent experiments. B) Western blot analysis of p62. Lane 1: Control, Lane 2: BLM-induced, Lane 3: BLM + Celastrol, Lane 4: Celastrol alone. Data expressing the respective protein levels were quantitated using Image J software. The data is expressed as relative intensity of proteins normalized to β-actin expression. Values are given statistically significant at p < 0.05, NSNon-significant; aBLM-induced vs control; bBLM

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+ Cel vs BLM-induced; cCel alone vs control. The data presented here represent the experiments performed in triplicate. Figure 9: protective action of celastrol against bleomycin-induced pulmonary fibrosis. In this study celastrol was shown to be associated with initiation, elongation and maturation phase of autophagosome formation. Celastrol activates autophagy through the inhibition of

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PI3K/Akt/mTOR expression and the degradation of p62. Celastrol is therefore, regulate

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collagen accumulation and provide protection against BLM induced pulmonary fibrosis

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Table 1: An overview of Autophagy induction of Celastrol

Corresponding role Beclin 1 interacts with cofactors and regulates lipid kinase Vps-34 protein to form the class III PI3K core complex which facilitate initial nucleation of autophagosome. Bleomycin administration in rats results in decreased expression of Beclin 1 and Vps-34 indicates deficient autophagy leads to fibrosis in rat lungs.

Elongaion phase

Atg12 conjugates with the lysine residue of Atg5. This conjugation requires the activity of Atg7. Atg-15 complex in turns non-covalently associates with Atg16. This ubiquitin-like protein conjugation system is very much essential for the elongation of autophagosome membrane. Increased expressions of Atg5-Atg12-Atg16 complex along with Atg7 in celastrol treated rat lungs indicate the ability of celastrol in inducing autophagy which is decreased upon bleomycin administration.

Lipidation of LC3

During the maturation of autophagosome, LC3-I covalently links to phosphatidylethanolamine (PE) and is incorporated into autophagosome which is essential for recruiting cargo. Atg3 catalyzes the lipidation of LC3. This lipidation process converts cytosolic LC3-I into the active, autophagosome membrane-bound form, LC3-II. Celastrol enhanced the expressions of LC3 I/II and Atg3 which further confirm the autophagy induction by celastrol in bleomuycin-induced rat lungs.

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mTOR is a negative regulator of autophagy. PI3K/Akt pathway is an upstream modulator of mTOR. Suppression of mTOR activity leads to the activation of autophagy activating kinase 1 complex. Inhibition of PI3k/Akt/mTOR by celastrol may represent a mechanism by which celastrol induces autophagy in bleomycin-induced rat lungs.

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Negative regulator of autophagy

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Molecule Autophagy induction

Autophagy adapter protein p62 recognizes damaged cellular organelles and toxic cellular waste which in turn degraded by autophagy. Accumulation of p62 in bleomycin induced rat lungs indicates deficient autophagy upon bleomycin administration which represents one of the mechanisms for the disease progression. Decreased expression of p62 in celastrol treated rat lungs suggest that celastrol degrades p62 and provides protection against bleomycin-induced pulmonary fibrosis.

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Highlights

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Celastrol alters morphological changes, modulates edema and collagen accumulation

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in bleomycin-induced pulmonary fibrosis in rats Celastrol enhances the formation, elongation and maturation of autophagosome and induces autophagy in rat lungs administered with bleomycin

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Celastrol inhibits PI3k/Akt mediated mTOR expression in bleomycin-induced pulmonary fibrosis Celastrol decreases the expression of autophagy adaptor protein p62 during pulmonary fibrosis

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