SRT1720 Protects Against CSE-Induced Cellular Senescence via Accelerates of FOXO3-PINK1-mediated Mitophagy

Chronic obstructive pulmonary disease (COPD) is often associate with cigarette smoke extract (CSE)-introduced bronchial epithelial cell senescence, mitochondrial fragmentation. Sirtuin-1(Sirt1) has been reported to play a crucial role in mitochondrial homeostasis and confers a protective role against the onset and development of CSE introduced bronchial epithelial cell senescence in COPD although the precise mechanism(s) remain elusive. Here we hypothesized that SRT1720, a pharmacological SIRT1720 activator, exerts protect against COPD by activating PINK1 mediated mitophagy, en route to preserved mitochondrial homeostasis.


Abstract Background
Chronic obstructive pulmonary disease (COPD) is often associate with cigarette smoke extract (CSE)introduced bronchial epithelial cell senescence, mitochondrial fragmentation. Sirtuin-1(Sirt1) has been reported to play a crucial role in mitochondrial homeostasis and confers a protective role against the onset and development of CSE introduced bronchial epithelial cell senescence in COPD although the precise mechanism(s) remain elusive. Here we hypothesized that SRT1720, a pharmacological SIRT1720 activator, exerts protect against COPD by activating PINK1 mediated mitophagy, en route to preserved mitochondrial homeostasis. Methods COPD rats model was established by CS exposure. During 6 months of SRT1720 treatment, airway resistance, cellular senescence and mitochondrial injury, mitophagy in the lung tissues of model rats were examined by western blot(WB) and histochemical and immuno uorescence staining. Transmission electron microscopy was also carried to elucidate the effects of SRT1720.Human bronchial epithelial cells(HBEC) were used to clarify the underlying molecular mechanisms.

Result
During the introduction of CSE in cellular or rats, administration of SRT1720 improved airway resistance, cellular senescence and mitochondrial injury, accompanied with suppressed autophagy and mitophagy. Mitochondrial damage, cellular senescence and lung injury under contrast exposure were more severe in FOXO3 or Pink1 de cient cells and mice than in SRT1720 groups. Activation of Sirt1 by treating with SRT1720 induces autophagy enhanced. A Decrease in sirt1 expression caused by selisistat treatment promotes senescence.

Conclusions
Taken together, our data suggested that suppressed SIRT1/FOXO3/Pink1 signaling mediated mitophagy played a protective role in COPD by reducing mitochondrial reactive oxygen species (ROS).

Background
Chronic obstructive pulmonary disease (COPD) is one of the most common chronic and disabling diseases and a growing cause of morbidity and mortality [1]. It is currently the fourth leading cause of death worldwide, and the World Health Organization (WHO) predicts that it will become the third leading cause by 2030. In general, COPD is classi ed as chronic in ammatory disease associated cell metabolism disorder resulting from cigarette smoke, oxidative stress and in ammatory injury [2][3][4]. Accelerated cellular senescence resulting from cigarette smoke (CS) exposure with excessive reactive oxygen species (ROS) production has been implicated in the pathogenesis of COPD [5]. Nonetheless, the precise molecular mechanism(s) involved in the pathogenesis of COPD remains elusive.
Mitochondria, an organelle that maintains cell energy metabolism, is highly involved in cerebral ischemia/reperfusion injury [6]. The stability of its structure and function is considered an important therapeutic target. The process by which cells clear damaged or dysfunctional mitochondria and complete self-renewal to maintain mitochondrial quality control is called mitophagy [7,8]. The autophagy and renewal of mitochondria are the prerequisites for repetitive operation and continuous supply of energy to cells [9]. Moderate mitophagy can protect mitochondrial function and help maintain cell energy metabolism and survival, excessive or insu cient autophagy will paralyze mitochondria and lead to cell death [10].
Sirtuin deacylase enzymes are important modulators of mitochondrial energy metabolism. Sirtuin 1(SIRT1) is the mammalian orthologue of the yeast silent information regulator 2 (Sir2), which is function as NAD+-dependent deacetylases as well as ADP ribosyltransferases [11][12][13]. SIRT1 can modulates the activity of deacetylates histone and transcription factors such as p53 and forkhead box O3 (FoxO3) [14,15].Emerging evidences have shown sirt1, an anti-in ammatory and anti-aging protein, is reduced in lungs of patients with COPD [16]. Recently evidence has depicted the molecular mechanism of CSinduced cellular senescence via a SIRT1-FOXO3 axis protects against stress-induced premature senescence (SIPS) and various pathophysiological changes in COPD. [17] SRT1720, a pharmacological Sirt1 activator, was found a positive regulator for emphysema via upregulated the levels of foxo3, increased SIRT1 activity and inhibited AECII apoptosis [18][19][20]. Further studies also depicted a novel role for sirtuins in the pathogenesis and treatment of COPD. Moreover, the network of SIRT1-FOXO3-Pink1 was reported to govern mitophagy and mitochondrial balance between fusion and ssion [21]. However, the role of Sirt1 in the regulation in COPD has not been fully elucidated.
In this context, this work was designed to determined (a) whether SIRT1 imposes any effect on the regulatory role of autophagy in cigarette smoke extract (CSE)-induced cell senescence of primary HBEC, if any;(b) whether the foxo3a-pink1 signaling pathway is involved in mitophagy regulation triggered by SIRT1.

Mice
We purchased 45 male C57B1/6 wild type mice(8 weeks,200±25g) from Shanghai Laboratory Animal Co., Ltd.(shanghai, China).We maintained the mouse at 21℃-25℃ and 40%-60% relative humidity in a 12hour light/dark circle and provided them with water and food ad libitum in the Laboratory Animal Center of Zhejiang Industry University(Hangzhou, China).The mice were randomly sorted into 4 groups(15 mice/group):WT(group ),CS exposure(group ), WT +selisistat(group ) and CS exposure +selisistat(group ).All animal protocols were approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University.

CS exposure
A custom-designed cigarette smoking chamber stimuli were used for generating the mice model of COPD.
For studies involving 3 days of CS exposure, research grade cigarettes (Xiongshi; China Tobacco Zhejiang Industrial. Co., Ltd., China) were used to generate smoke. Brie y, cigarette smoke exposure were performed as follows mouse in group and group were placed in the cigarette smoking chamber(60cm×50cm×40cm). After an interval of 10 minutes, the smoke of 5 new cigarettes was delivered into the chamber. CS concentration was set at a value of approximately 300 mg/m 3 total particulate matter(TPM) by adjusting the ow rate of the diluted medical air, and the level of carbon monoxide in the chamber was 350 ppm [22,23], monitored by a real time aerosol monitor(MicroDust pro,Casella CEL, Bedford, UK). The mouse were exposed to smoke for 90 minutes daily for consecutive 24 weeks.

Administration of selisistat
Selisistat(30mg/kg body weight in 10%DMSO and 40%PEG300 and 5%Tween-80 and 45% saline,>95% pure by C-13 NMR and LCMS; synthesized by life chemicals) through oral gavage 1 hour prior to CS exposure daily. To study the therapeutic effect on emphysema, selisistat was orally administrated daily for 4 weeks after the development of CS induced emphysema.

Cell culture
Normal airways were obtained from 4st order bronchi from pneumonectomy and lobectomy specimens for primary lung cancer. Informed consent was obtained from all surgical participants as part of an approved ongoing research protocol by the ethics committee of Hangzhou medical college. HBEC were isolated with protease treatment and characterized as previously describe [24]. HBEC were serially passaged and used for experiments were performed with HBEC from non-COPD patients. HBEC was cultured in RPMI1640 with 10% fetal calf serum and penicillin-streptomycin(Gibo Life Technologies,15140-112).For in vitro study, HBEC were treated with CSE(1%) and SRT1720(4mM/L,>95% pure by C-13 NMR and LCMS; synthesized by life chemicals)for 48h.

Preparation of cigarette smoke extract(CSE)
Cigarette smoke extract (CSE) was prepared as previously described with minor modi cation [10]. Forty milliliters of cigarette smoke were drawn into the syringe and slowly bubbled into sterile serum-free cell culture media in 15-ml BD falcon tubes. One cigarette was used for the preparation of 10 ml of solution. CSE solution was ltered(0.22μm;Merck Millipore,SLGS033SS) to remove insoluble particles and was designated as a 100%CSE solution.

Measurement of lung mechanics
Lung mechanical properties, including lung compliance and RL, were determined as described previously [25]. Brie y, the mouse was weighed, deeply anesthetized by i.p. injection of pentobarbital (90 mg/kg BW) and pancuronium (0.5 mg/kg BW), and tracheostomized. The trachea was cannulated, and the cannula was connected to a computer-controlled small animal ventilator (FlexiVent; SCIREQ).

Lung morphometry
Mouse lungs (which had not been lavaged) were in ated with 1% low-melt agarose at a pressure of 25 cm H2O, then xed with 4% neutral buffered PFA [25,26]. Fixed lung was dehydrated, embedded in para n, and sectioned into 4-μm sections using a rotary microtome (MICROM International GmbH). H&E staining was performed on the lung midsagittal sections to determine Lm of airspace using MetaMorph software (Molecular Devices) [25]. Ten randomly selected ×100 elds per slide were photographed in a blinded manner, and the images were manually thresholded. The airway and vascular structures were eliminated from the analysis.
Senescence-associated-β-galactosidase (SA-β-gal) activity assay SA-β-gal activity was assessed using an in situ β-galactosidase staining kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. Lung tissues were xed in βgalactosidase stationary solution for 15 min, then washed 3 times for 10 min each in PBS. Sections were then incubated with 1 ml staining solution mixture (10 µl staining solution A, 10 µl staining solution B, 930 µl staining solution C and 50 µl X-gal solution) for 2 h at 37˚C. Following 3 washes with PBS, 5 elds of view from each of the 3 sections from each lung sample were examined using a light microscope (Olympus Corporation).

Transmission electron microscopy (TEM)
Murine lung tissues were xed in 2 % glutaraldehyde for at least 24 h. Tissues were then immersed in 2 % osmium tetroxide and 1 % aqueous uranyl acetate, each for 1 h. After washed with a series of ethanol solutions (50 %, 70 %, 90 % and 100 %), tissues were transferred to propylene oxide, incubated in a 1:1 mixture of propylene oxide and EMbed 812 (Electron Microscopy Sciences) for 1 hour and then placed in a 70 °C oven to polymerize. Sections (75-80nm) were cut using a Leica ultramicrotome equipped with a Diatome diamond knife and collected on 200-mesh copper grids. After poststained in 5% uranyl acetate for 10 min and in Reynold's lead citrate for 5 min, sections were observed using a 40-120 kV transmission electron microscope (FEI TECNAI G2 Spirit Biotwin, Hong Kong, China). For in vitro study, After treatment, HBEC were xed with 2% glutaraldehyde/ 0.1 M phosphate buffer (pH7.4) and in 1% osmium tetroxide/0.1 M phosphate buffer (pH7.4), and dehydrated with a graded series of ethanol. The operations subsequent were performed as described above.
Immuno uorescence straining Murine lung frozen sections were xed with 4% paraformaldehyde. After washing with PBS for several times and incubating with goat serum for 1 h, they were incubated overnight with LC3B antibody(1:100,Cell Signaling Technology, USA)at 4 ℃. HBEC expressing EGFP-LC3B grown on 6-well culture slides were xed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.03% Triton X-100(Wako,16024751) for 60 min. After blocking with 0.1%BSA(Sigma Aldrich,A2153) for 60 min, the primary and secondary antibodies were applied according to the manufacture's instructions. Confocal laser scanning microscopy analysis of mitochondria was performed by TOMM20 staining, assessed with a confocal microscope(Carl Zeiss LSM510,Tokyo,Japan).Mito Tracker Red CMX Ros (MTR; Molecular Probes-Life Technologies,M-7512) staining (200nM, 30 min at 37℃) was also performed to evaluate the integrity of the mitochondrial membrane potential.

Statistical analysis
All the experimental data are presented as the means ±SEM and analyzed by Prism version 6.0 (Graph-Pad Software, San Diego, USA). The t-test was performed to measure the differences between the two groups and one-way analysis of variance (ANOVA) followed by a Dunnett's test was performed to compare the differences among three or more groups. P-values<0.05 were considered statistical signi cance.

Effect of Sirt1 inhibitor on CSE induced changs in lung function
Expression of Sirt1 in mouse with selisistat treatment was con rmed using western blot analysis. Our result showed that Sirt1 protein expression was drastically decreased in selisistat treatment mouse lung exposed to CS compare with their WT littermates (Fig.1A). Lung morphometry to examine alveolar mean linear intercept(L m ).CS exposure for 6 months induced a modest airspace enlargement in WT mice, whereas SIRT1 de cient mice exhibit airspace enlargement (Fig.1B). To evaluate the effect of Sirt1 inhibitor on CSE induced changes in lung function, Lung mechanical properties was employed to evaluate lung compliance and R L at 3 cmH 2 O positive end-expiratory pressure were obtained by tting a model to each impedance spectrum. Our data showed that lung compliance was augmented in WT mice exposed to CS for 6 months, the effect of which was accentuated in selisistat treatment mice ( Fig.1C and D). Sirt1 de ciency decreased total lung resistance(R L ), although no signi cant change in R L in WT mice was observed after 6 months of CS exposure.

Effect of Sirt1 inhibitor on CSE-induced airspace enlargement, cellular senescence and mitochondrial morphology
To evaluate the effect of Sirt1 inhibitor on CSE induced lung injury, SA-β-gal activity assay and transmission electron microscopy were used to examine cellular senescence and mitochondrial morphology, respectively. Our nding revealed that CS exposure for 6 months induced a modest airspace enlargement in WT mice, which was further augmented in selisistat treated mice. As expected, CS exposure signi cantly the level of SA β gal activity in lungs of selisistat treated mice versus WT littermates. Transmission electron microscopy also revealed more swollen an damaged mitochondria in WT mice exposed to CS, the effect of which was augmented by Sirt1 inhibitor treated mice. The mitochodria were slightly changed in selisistat treated mice compared with WT mice. (Fig2)

Effect of Sirt1 inhibitor on CSE-induced autophagy regulation
To determine the effect of Sirt1 inhibitor on CES induced autophagy regulation, immuno uorescence and western blotting were used to examine LC3 puncta in frozen section of lungs and LC3B protein expression, respectively. Our data revealed that the number of LC3B puncta was signi cant decreased in WT mice exposed to CS, with a more pronounced response in Sirt1 inhibitor mice(Fig3A).Meanwhile western blot analysis revealed that the LC3 protein level were overtly downregulated in WT mice exposed to CS, the effect of which was accentuated by Sirt1 inhibitor(Fig3B).Sirt1 de ciency itself elicited a decrease in LC3 puncta and LC3 level.

Signaling mechanism(s) in Sirt1 inhibitor induced autophagy regulation in emphysema
To explore the possible mechanism(s) in Sirt1 inhibitor accentuation of CSE induces changes in autophagy, western blot analysis was employed to examine the level of Ac-Foxo3A and Pink1. our result revealed that the ratio of Ac-Foxo3a to Foxo3A was greatly increased following CSE exposure, the effect of which was exacerbated by more upregulated by Sirt1 inhibitor. Sirt1 de ciency also increased the ratio of Ac-Foxo3a to Foxo3a. PINK1 expression was reduced in WT mice exposed to CSE, the effect of which was unaffected by Sirt1 inhibitor.Sirt1 de ciency also decreased the level of PINK1. (Fig.4.) Alteration of levels of Sirt1 and LC3B by CSE in primary human bronchial epithelial cells HBEC were treated with 10% fetal calf serum and CSE (1%) in the presence or absence of SRT1720 for 48h,and the control cells were cultured with 10% fetal calf serum. Western blotting revealed that LC3 protein level began to decline and p62 expression began to rise at CSE(1%) and was further reduced at CSE(2.5%),consistent with the previous report [14].We next examined the time course of autophagy downregulation. HBEC were exposed to CS (2.5%) for 6-48 hour, while 10% fetal calf serum were used as control. A signi cant reduction of LC3 protein levels and increased p62 expression was observed from 24h,the effect of which was present throughout the entire examination period. The expression of Sirt1 began to decrease at 12h and remained consistent thereafter(Fig5).

Effect of Sirt1 overexpression on CSE induced ROS production, apoptosis, and mitophagy in HBEC
To determine the effect of Sirt1 overexpression on CSE induced the reduction of mitochondrial ROS production, DCFH-DA assays and MitoSOX Red staining were applied to evaluate the association between mitophagy and ROS production in HBEC. Our data showed that CSE treatment results in a rise of damaged mitochondria, total and mitochondrial ROS production, which was signi cantly reduced by SRT1720 (Fig6A,B). JC-1 staining revealed that mitochondrial membrane potential of CSE group was signi cantly decreased, compared with normal control group, the effect of which was largely counteracted by SRT1720 (Fig6C). Result from transmission electron microscopy showed that more obviously swollen and damaged mitochondria were observed in cells with CSE treatment, with were reserved by SRT1720 (Fig6D).

Effect of Sirt1 overexpression on CSE induced autophagy regulation
To examine the effect of Sirt1 overexpression on CSE induced autophagy regulation, confocal microscopy was performed in HBEC stably experssion EGFP-LC3. Colocalization of TOMM20-stained mitochondria and EGFP-LC3B dots was used to determine autophagosome formation,and western blotting determining LC3B and p62 protein level was used.Our results showed that although CSE induced EGFP-LC3B dot formation,colocalization with mitochondria was barely detected in the absence of SRT1720. SRT1720 treatment alone induced EGFG-LC3B dot formation accompanied by limited colocalization with TOMM20-stained mitochondria(yellow dots),CSE treatment markedly enhanced EGFP-LC3B dot formation concomitant colocalization with TOMM20-stained mitochondria in the presence of SRT1720 (Fig).For cellular exposed to CSE,the level of LC3 were with signi cantly difference after overexpressing of Sirt1. The upregulated expression of p62 in response to CSE challenge was reversed by SRT1720.

Effect of Sirt1 overexpression on the mechanism(s) involved in CSE induced autophagy regulation
To determine the effect of Sirt1 overexpression on the mechanism(s) involved in CSE induced autophagy regulation,western blotting analysis was applied to detect the expression of Ac-Foxo3A and Pink1. The results revealed that Sirt1 level was increased after SRT1720 treatment. In CSE condition ,Sirt1 and Pink1 were both reduced,while the ratio of Ac-Foxo3A to Foxo3a was increased.The effect of CSE treatment was obviously reversed by overexpressing Sirt1 and this effect was also noted in normal HBEC with SRT1720.

Discussion
Our study nding that CSE induced HBEC and COPD downregulates anti-aging sirt1 and suppressed the Sirt1-Foxo3A signaling cascade. It is the protein levels demonstrated decreased Pink1 in CSE group compared with control group. Concomitantly, CSE induced fragmented mitochondria accumulated and slightly mitophagy activation [27,28]. Activation of Sirt1 mediated Foxo3 deacetylation, resulting in an increase of Pink1 in HBEC treated with CSE, restoration of HBEC autophagy and protection against HBEC apoptosis [29,30]. SIRT1 inhibitor lead to suppressed Sirt1-Foxo3 signaling and Pink1 mediated mitophagy may participate in CSE induced downregulation of HBEC autophagy and increased HBEC apoptosis [31]. SRT1720 and selisistat experiments clarify that both Sirt1 and Foxo3 are indicate a likely role for mitophagy in the pathogenesis of CSE exposure [32][33][34]. Moreover, recent studies observed modestly upregulation of PINK1 in lung homogenates from COPD patients [35,36]. Therefore, the exact role of PINK1 in mitophagy has not been well done de ned because of the existing contradistinction.
By mitophagic eliminate of damaged mitochondria and damaged organelles within lysosome, autophagy maintains cellular homeostasis [37]. Improper mitophagic has been implicated in the pathogenesis to different types of pulmonary diseases, and recently papers nding insu cient autophagy associated with complications of pulmonary including acute lung injury and idiopathic pulmonary brosis [38]. The depressed autophagy was proved to have a detrimental role in lung function of CES induced COPD although contrary ndings have also been reported [39]. To elucidate the function role of mitophagy that using activation and inhibitor experiments in vitro and in vivo models. The CSE induced cellular damage was attenuated with Sirt1 overexpression, while the cellular damage was exacerbated in emphysema mice de cient in Sirt1, suggesting an insu cient role for mitophagy in COPD/emphysema [14,40].
In the line with the recent ndings CSE-induced emphysema lung dysfunction, the Sirt1-Foxo3-Pink1 pathway is involved in regulating the mitophagic elimination of CSE induced damaged mitochondria that modulates cellular senescence [41]. In this research, EM shows autophagy and mitophagy activation induced by Sirt1 result in attenuated mitochondrial numbers and enhanced degradation of mitochondrial debris (Fig 2).It has been reported that Pink1 levels in COPD smokers tend to be lower than those in control lung, and Pink1 overexpression increase mitophagy activity, indication that Pink1 is not only a pivotal predisposition to COPD development but also a crucial role in mitochondrial quality control in terms of regulation of senescence associated disorders [42,43]. In this work, we found suppressed Pink1 in CSE induced HBEC or de ciency of Sirt1 condition, in concordance with other papers. Some studies demonstrated an inhibited autophagic ux in COPD lungs. In our vivo experiment, autophagy and Pink1 mediated mitophagy both reduced 6 months after CSE exposure, which can be regard as advanced stages of COPD.
Sirt1, as one of the intracellular deacetylases, is an important role in many processes of mitochondrial function [13]. Intriguingly, Studies demonstrated that Sirt1 may act as a tumor suppressor in several types of cancers. In response to CSE stimulus, Sirt1 was de ciency is increased apoptotic and mitochondrial dysfunction in bronchial epithelial cell. Airspace were enlarged obviously and the amount of alveoli was signi cantly decrease in selisistat treat mice than control group mice, along with more depressed mitophagy. Sirt1 protects against emphysema through Foxo3-mediated maintain mitochondrial homeostasis and reduction of cellular senescence. Overexpression of Sirt1 inverted Sirt1-Foxo3a-Pink1 signaling mediated mitophagy and senescence of epithelial cell, indicating that Sirt1-Foxo3a-Pink1 signaling was a crucial pathway involved in overexpression Sirt1 promotion of alveoli survival under CSE exposure [11,44,45]. Recent advances demonstrated in ammatory reactions involved in Sirt1 induced regulation of mitophagic degradation [46]. It was reported that Sirt1 activated mitophagy by inducing TLR9(toll-like receptor 9) recognition and activation of IL1B/interleukin 1β,via deacetylation Foxo3 with the subsequent downregulation of NALP3 in ammasome [47,48].

Conclusion
In summary, We demonstrated that the CSE induced mitophagy and autophagy by Sirt1-Foxo3-Pink1 path in HBEC. Suppressed Sirt1-Foxo3-Pink1 signaling and reduced Pink1 expression levels in COPD lung that downregulation of mitophagy and acceleration of cell senescence. Although the precise mechanism through other positive regulators regulated epithelial cell mitophagy in the setting of CSE exposure is still unclear. Our current study further supported the notion that the effect of Sirt1-Foxo3 signaling and Pink1association mitophagy may explain the mechanism of insu cient mitophagy in COPD lung.

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