Compound 19 inhibitor

Mechanistic target of rapamycin-mediated autophagy is involved in the alleviation of lipopolysaccharide-induced acute lung injury in rats

Acute lung injury (ALI) is a complex clinical syndrome with high morbidity and mortality rates. Autophagy is an adaptive process that plays a complex role in ALI. The aim of this study was to investigate the effects of au- tophagy on lipopolysaccharide (LPS)-induced lung injury by establishing a rat ALI model and to further explore the possible mechanisms involved. Rats were pretreated with the autophagy inhibitor 3-methyladenine (3-MA) or the autophagy activator rapamycin before they were challenged with the intratracheal instillation of LPS (5 mg/kg). The level of autophagy in the lung tissue was detected. Lung injury and vascular permeability were assessed. The role of the mechanistic target of rapamycin (mTOR)-mediated Unc-51-like kinase 1 (ULK1) and the class III PI3 kinase VPS34 in autophagy regulation was examined. LPS challenge induced autophagy and ra- pamycin pretreatment enhanced autophagy activity in LPS-induced ALI rats. LPS caused severe lung injury and high pulmonary vascular permeability, which could be alleviated by enhancing autophagy. In addition, the inhibition of mTOR upregulated the expression of ULK1 and VPS34 and thus increased LPS-induced autophagy. Autophagy plays a protective role in LPS-induced ALI, and enhancing autophagy via the inhibition of mTOR alleviates lung injury and pulmonary barrier function. Moreover, mTOR negatively mediates ULK1 and VPS34 to regulate LPS-induced autophagy in rats.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are a complex clinical syndrome character- ized by acute inflammatory responses in the lung airspace and par- enchyma, thus resulting in diffuse alveolar injury, increased vascular and epithelial permeability, pulmonary edema, and gas exchange abnormalities [1–4]. Despite the better understanding of ARDS/ALI pa-
thogenesis and intensive care medicine, the morbidity and mortality rates of ARDS/ALI remain high [4,5], with a morbidity rate of ap- proximately 50–80 per 100,000 person-years and a current overall mortality rate of approximately 30–40% [6,7]. Lipopolysaccharide
(LPS) is the primary component of the outer membrane of gram-nega- tive bacteria, and LPS-induced lung injury is a widely acceptable method to model the consequences of bacterial sepsis [8].Autophagy is an evolutionarily conserved cellular process in which long-lived proteins, damaged organelles and misfolded proteins are delivered to lysosomes for degradation, clearance and recycling [9]. Under normal physiological conditions, autophagy occurs to maintain cellular homeostasis at a basal level [10]. Under stress or injury situa- tions (e.g., nutrient deprivation, starvation, and trauma), autophagy primarily attempts to serve as an adaptive and defensive mechanism for cell survival [9]. Mechanistic target of rapamycin (mTOR) is a central regulator of many major cellular processes, including cell growth, proliferation and autophagy initiation [11,12]. mTOR negatively reg- ulates autophagy machinery. Under nutrient deprivation conditions, mTOR is inhibited, resulting in Unc-51-like kinase 1 (ULK1) and class III phosphatidylinositol 3-kinase (PI3KC3/VPS34) kinase activation [13–15]. Immediately thereafter, PI3KC3/VPS34 phosphorylates phosphatidylinositol to produce phosphatidylinositol-3-phosphate
(PI3P/PtdIns3P), which promotes autophagosome formation [16,17]. Autophagy activity has been reported to be involved in various pulmonary diseases, including ALI [18–20]. The pathogenesis of ALI is associated with multiple stresses, including sepsis, hyperoxia, trauma,
inflammatory damage and other damaging insults [19], most of which can lead to the induction of autophagy. However, the role and reg- ulation of autophagy in ALI have not been extensively studied. Au- tophagy may contribute to cell survival or lead to cell damage in some ALI models, and clarifying its role may contribute to the development of novel treatment protocols for ALI. Therefore, we investigated the effects of autophagy on LPS-induced lung injury by establishing a rat ALI model and further analyzed the function of the mTOR pathway in mediating LPS-induced autophagy.

2.Materials and methods
Male Sprague-Dawley rats weighing 200–220 g were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. [Animal certificate number: SCXK(Jing)-2016-0006]. Rats were housed on a 12-hour light/dark cycle in a temperature-and humidity-controlled room and maintained on a standard diet and water ad libitum. Animal pro- tocols were approved by the Ethics Committee of the Academy of Military Medical Science (Beijing, China). Experimental studies were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All surgery was carried out under sodium pentobarbital anesthesia.

2.2.Chemicals and reagents
LPS (Escherichia coli 055:B5) and 3-methyladenine (3-MA) (M9281) were purchased from Sigma-Aldrich. Rapamycin (Rap) was purchased from Selleck (S1039). The following antibodies were purchased from Cell Signaling Technology: anti-mTOR (#2972), anti-phospho(p)- mTOR (#2971), anti-ULK1 (#8054), anti-p-ULK1 Ser757 (#6888),anti-PIK3C3 (#4263), anti-sequestosome 1/p62 protein (#5114), anti- GAPDH (#5174), and anti-rabbit IgG (#7074). The anti-microtubule- associated protein 1-light chain 3 B (LC3B, ab192890) and anti-zonula occludens-1 (ZO-1, ab59720) antibodies were purchased from Abcam. The Alexa Fluor 647-conjugated secondary antibody was purchased from Beyotime Biotechnology (A0468).

2.3.Experimental protocols
After a 1-week acclimatization period, forty rats were randomly divided into four groups of ten per group: sham group, LPS group, 3-MA group and Rap group. Rats in the 3-MA group were pretreated with 3- MA (15 mg/kg, intraperitoneally), and rats in the Rap group were pretreated with rapamycin (1 mg/kg, intraperitoneally) 4 h prior to LPS addition. Rats in the sham and LPS groups received the same dose of normal saline.

2.4.Construction of the ALI model
Rats were anesthetized with an intraperitoneal injection of pento- barbital sodium (30 mg/kg) solution (1 mL/kg of 3% pentobarbital so- dium solution). Skin preparation was performed at the throat of the rats. After regular disinfection, the throat was incised longitudinally with the rat head in the higher position. Skin and subcutaneous tissue were separated layer by layer until the trachea was exposed. Then, the rats for the ALI model (LPS, 3-MA and Rap groups) received a single intratracheal instillation of 5 mg/kg LPS (2.5 mg/mL, diluted with phosphate-buffered saline (PBS), 0.2 mL/100 g body weight) to induce ALI. The control rats from the sham group were intratracheally ad- ministered an equal volume of sterile PBS via the same method. Finally, the incision was sutured.

2.5.Collection of bronchoalveolar lavage fluid and lung tissues
Eight hours after LPS administration, ten rats in each group were sacrificed by exsanguination, and the thoracic cavity was surgically exposed. The hilum of the right lung was ligated, and the left lung was flushed with ice-cold PBS three times. The bronchoalveolar lavage fluid (BALF) was collected and the bilateral lungs were dissected, and the left lungs were snap frozen in liquid nitrogen and stored at −80 °C.

2.6.Analysis of BALF
The BALF was centrifuged at 1500 rpm for 10 min at 4 °C, and the supernatant was stored at −80 °C for protein and cytokines measure- ment. The precipitated cells were resuspended in PBS for total cell counts using a hemocytometer, and the ratio of neutrophils was counted via optical microscopy after Wright-Giemsa staining. The total protein concentration in the BALF was measured with a BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin- 6 (IL-6) in the BALF were determined by enzyme-linked im- munosorbent assay (ELISA) kits (R&D Systems Inc., Minneapolis, MN) according to the manufacturer’s instructions.

2.7.Lactate dehydrogenase (LDH) activity assay
The activity of LDH in BALF was measured using a Lactate dehy- drogenase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol. The absor- bance at 440 nm was determined with an automatic multiwell spec- trophotometer.

2.8.Histological examination
The middle lobe of the right lung was excised, fixed in 4% paraf- ormaldehyde for 24 h at 4 °C, embedded in paraffin, and sectioned (4 mm thick). Hematoxylin and eosin (H&E) staining was performed according to a standard protocol. Pathological changes in lung tissue were observed under a light microscope. The lung injury scores were based on four categories: interstitial inflammation, neutrophil infiltra- tion, edema, and congestion. Those indexes were graded as follows
[21]: no injury = score of 0; injury in 25% of the field = score of 1; injury in 50% of the field = score of 2; injury in 75% of the field = score of 3; and injury throughout the field = score of 4. Each sample was analyzed in ten microscopic fields, and the severity of lung injury was evaluated by the average score.

2.9.Lung wet/dry weight ratio
The inferior lobe of the right lung was excised, and the wet weight was recorded before it was placed in an incubator at 60 °C for 72 h to obtain the dry weight. The lung wet/dry weight ratio was calculated for each group.

2.10.Transmission electron microscopy (TEM)
Lung pieces were fixed with 2.5% glutaraldehyde at 4 °C overnight and further fixed in 1% buffered osmium tetroxide for 30 min at 37 °C. All fixed samples were then dehydrated in graded alcohols, embedded in EMbed, and sectioned with an ultramicrotome. Thin sections were stained with uranyl acetate and lead citrate. Three sections from each block were randomly selected for observation. The number of autop- hagic structures was counted under TEM from 30 randomly selected fields.

Frozen sections (4 μm thick) from lung tissues were fixed in 4% paraformaldehyde and blocked with 5% goat serum in PBS containing 0.25% Triton X-100. The sections were then incubated with the rabbit anti-LC3B antibody (1200) overnight at 4 °C. After washing with PBS, the sections were incubated with the Alexa Fluor 647-conjugated sec- ondary antibody at room temperature for 2 h. 4′,6-Diamidino-2-phe-
nylindole (DAPI) was used to visualize nuclei. Images were captured using a fluorescence microscope (Nikon Ti-A1, Tokyo, Japan).

2.12.Western blot analysis
Lung tissues were homogenized in lysis buffer containing protease and phosphatase inhibitors. The homogenate was incubated on ice for 45 min and then centrifuged at 4 °C (12,000 rpm for 5 min). The su- pernatant protein concentration was determined using a BCA protein assay kit. Equal amounts of protein from each group were separated by SDS-polyacrylamide gel electrophoresis and transferred to poly- vinylidene difluoride (PVDF) membranes. Subsequently, the mem- branes were blocked and incubated with primary antibodies against LC- 3, p62, p-mTOR, mTOR, p-ULK1, ULK1, VPS34, ZO-1 and GAPDH overnight at 4 °C. After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody. Protein bands were detected using a chemiluminescence kit (Millipore, MA, USA) via the Odyssey system (Germany). The optical density of the bands was analyzed using ImageJ software.

2.13.Statistical analysis
Data were analyzed using SPSS version 17.0 statistical software (SPSS, Chicago, IL, USA). All data are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for multiple comparisons among groups. Student’s t-test was used to test the differences between two groups. A P-value < 0.05 was considered statistically significant. 3.Results 3.1.Induction of autophagy in the LPS-induced ALI rat model To test the effects of endotoxin on autophagy, we employed an LPS- induced lung injury rat model and applied the autophagy inhibitor 3- MA and the autophagy inducer rapamycin to regulate autophagy. The conversion of LC3B-I to LC3B-II is widely used as a marker to determine autophagosome formation [22]. The degradation of p62 can serve as a marker to detect autophagic flux [23]. As shown in Fig. 1A and B, compared with the sham group, LPS stimulation increased the LC3B-II protein level but decreased that of p62, indicating autophagy induction. However, these effects were significantly inhibited by 3-MA. In con- trast, pretreatment with rapamycin enhanced the effects. In the im- munofluorescence assay, punctate LC3B staining was gradually in- creased after LPS challenge compared with that of the sham group. 3- MA reduced the protein expression of LC3B-II, while rapamycin ele- vated it (Fig. 1C). In addition, TEM revealed an increased number of autophagic vacuoles in the LPS group (Fig. 1D). These data demon- strated that the intratracheal instillation of LPS induced autophagy and that rapamycin pretreatment enhanced autophagy activity in the LPS- induced ALI rat model. 3.2.Autophagy alleviated LPS-induced lung injury To investigate the effects of autophagy on LPS-induced lung injury in rats, we evaluated the lung histological damage scores and measured LDH activity, cell counts and inflammatory factors concentrations in the BALF. As shown in Fig. 2A and B, lungs from the LPS group showed more severe lung pathological damage and higher ALI scores than the sham group. These effects were further increased by the autophagy inhibitor 3-MA but were attenuated by the autophagy inducer rapa- mycin. Similar trends were observed in the LDH activity, which is an indicator of acute damage in lung cells. As shown in Fig. 2C, the LDH activity was greatly increased after LPS challenge, but decreased after rapamycin treatment. In addition, LPS caused a significant increase of the total cells (Fig. 2D) and neutrophils (Fig. 2E) in BALF, whereas the rapamycin treatment significantly decreased these counts. ELISA results revealed that the concentrations of TNF-α and IL-1β were higher in the LPS group than in the sham group. 3-MA pretreatment induced a marked increase while rapamycin pretreatment induced a significant decrease compared with the LPS group (Fig. 2F). Together, these results show that LPS caused severe lung injury and inflammation responses that were alleviated by enhancing autophagy. 3.3.Autophagy improved pulmonary barrier function after LPS stimulation To detect the effects of autophagy on pulmonary barrier function, we evaluated pulmonary edema, total protein concentrations in the BALF, and zonula occludens-1 (ZO-1) expression in lung tissues. The wet/dry ratio is an index of pulmonary edema. Rats subjected to LPS displayed significantly elevated wet/dry ratios compared to the sham group. The ratios were markedly higher after 3-MA injection and lower after rapamycin treatment (Fig. 3A). The total protein concentration in the BALF is used to evaluate pulmonary capillary permeability. Com- pared with the value in the sham group, the capillary permeability in the LPS group was much higher. Increased capillary permeability re- sults in pulmonary edema. The inhibition of autophagy by 3-MA further promoted higher capillary permeability after LPS stimulation, whereas the rapamycin treatment reversed the effect of LPS on capillary per- meability (Fig. 3B). Western blot analysis also showed that LPS chal- lenge significantly decreased the expression of the endothelial junction protein ZO-1. The inhibition of autophagy by 3-MA further decreased the expression of ZO-1, while the enhancement of autophagy by rapa- mycin notably increased it (Fig. 3C and D). Collectively, our data de- monstrated that enhancing autophagy improved pulmonary barrier function in the LPS-induced ALI rat model. 3.4.mTOR negatively mediates ULK1 and VPS34 to regulate LPS-induced autophagy To explore the possible mechanisms involved in the regulation of autophagy induced by LPS, we examined the activity of the mTOR signaling pathway in the lung. Autophagy is activated when mTOR is inhibited. Rapamycin is the most commonly used autophagy inducer, which acts by inhibiting mTOR [24]. mTOR suppresses autophagy in- duction in part by phosphorylation and inhibition of ULK1, a VPS34 complex [25]. 3-MA blocks autophagy by inhibiting the class III PI3K VPS34 [17]. As shown in Fig. 4A and B, the western blot analysis re- vealed that the expression of p-mTOR protein was downregulated in lung tissues after LPS challenge. Meanwhile, the p-ULK1 and VPS34 protein levels were increased in the LPS group compared to the sham group. 3-MA did not change the expression of p-mTOR or p-ULK1 but significantly inhibited VPS34 activity. Moreover, the inhibition of mTOR upon rapamycin pretreatment enhanced the kinase activity of ULK1 and VPS34. Taken together, these data indicate that mTOR ne- gatively mediated the activity of ULK1 and VPS34 to regulate LPS-in- duced autophagy. 4.Discussion The key pathophysiological feature of ALI is the disruption of en- dothelial-epithelial barriers [4]. Animal models are an indispensable method of exploring ALI pathogenesis and discovering new treatment approaches. Sepsis is the primary risk factor for ALI/ARDS [26], and (caption on next page) Fig. 1. Effects of LPS on autophagy in rats. (A) Representative western blot image of LC3B, p62 and GAPDH in lung tissues from different (sham, LPS, 3-MA and Rap) groups. (B) Quantitative data for the indicated proteins; LC3B-II normalized to LC3B-I expression and p62 normalized to GAPDH expression. (C) Representative LC3B immunofluorescence images from the lung tissues of the sham, LPS, 3-MA and Rap groups. Scale bar = 40 μm. (D) Representative transmission electron microscopy (TEM) images of autophagic ultrastructures in the lung tissues of the sham and LPS groups. Autophagosomes and autolysosomes are indicated by thick arrows and double thin arrows, respectively. N, nucleus. Scale bar = 1 μm. (E) Quantitative analysis of the number of autophagic vacuoles in different groups. Values are expressed as the mean ± SD.*P < 0.05, **P < 0.01 vs the sham group. #P < 0.05, ##P < 0.01 vs the LPS group bacteria or endogenous infections, such as LPS, are usually used to in- duce animal sepsis model. Furthermore, LPS-induced injury is a very useful experimental in vivo model that closely resembles ALI/ARDS in humans [8]. The intravenous administration of LPS induces the pro- duction of inflammatory mediators, thus resulting in a disruption in the alveolar-epithelial barrier. As a consequence, the alveolar-capillary permeability increases, which leads to the accumulation of protein-rich edema fluid within the pulmonary alveolus and interstitium [26,27].In different ALI animal models, autophagy may present a state of enhancement or inhibition. For instance, several previous studies found that LPS challenge enhanced the autophagy level in mice [28–31]. Another previous study observed that autophagy was activated in traumatic brain injury-induced ALI [32]. Moreover, Angara et al. re- ported that hyperoxia induced autophagy in lung epithelial cells Fig. 2. Effects of autophagy on LPS-induced lung injury in rats. (A) Representative lung histological image stained with hematoxylin and eosin. Scale bar = 200 μm. (B) Quantitative data for lung injury scores. (C) Relative LDH activity in BALF. (D) Total cell count in BALF. (E) Neutrophils count in BALF. (F) Concentrations of TNF-α and IL-1β in the BALF. Values are expressed as the mean ± SD. **P < 0.01 vs the sham group. ##P < 0.01 vs the LPS group. Fig. 3. Effects of autophagy on pulmonary barrier function in LPS-induced ALI rats. (A) Pulmonary edema was evaluated by the wet/dry weight ratio of lung tissues. (B) Pulmonary vascular permeability was determined by the total protein con- centration in the BALF. (C) Representative western blot image of ZO-1 in the lung tissues from each group. (D) The relative expressions of ZO-1. Values are expressed as the mean ± SD. **P < 0.01 vs the sham group. ##P < 0.01 vs the LPS group neonatal mice lungs [33]. These results were consistent with our finding that the autophagy level was elevated by the intratracheal in- stillation of LPS in rats, which was reflected by the increased expression of LC3B-II, decreased expression of p62 and the corresponding increase in the number of autophagic vacuoles. The autophagy-related (Atg) protein LC3B (homolog of yeast Atg8) plays an important role in autophagosome formation. Cytoplasmic LC3 is conjugated to phos- phatidylethanolamine to produce LC3-PE (or LC3-II), which is subse- quently incorporated into the double-membrane structure of the au- tophagosome [34]. Thus, the conversion of LC3-I (unconjugated cytosolic form) to LC3-II (autophagosomal membrane-associated PE- conjugated form) can be used as a marker to determine autophagosome Fig. 4. Role of the mTOR pathway in mediating autophagy in LPS-induced ALI rats. (A) Representative western blot image of mTOR, ULK1, VPS34 and GAPDH in the lung tissues from each group. (B) Quantitative data for the indicated proteins: p- mTOR normalized to mTOR expression, p-ULK1 normalized to ULK1 expression and VPS34 normalized to GAPDH expression. Values are expressed as the mean ± SD. *P < 0.05, **P < 0.01 vs the sham group. #P < 0.05, ##P < 0.01 vs the LPS group formation. However, increased LC3B-II or autophagosomes could be due to a high autophagic flux or a downstream blockage of autophagy [14,34,35]. The turnover of p62 is an alternative method widely used to detect autophagic flux [36]. As an autophagic substrate, p62 binds to specific cargo proteins and carries them to autophagosomes; then, the cargo and p62 itself are degraded by autolysosomes. Thus, the down- regulation of p62 suggests an occurrence of autophagic flux.The autophagy level was upregulated in this rat model of LPS-in- duced ALI. However, the cells in the lung that exhibit increased au- tophagy have not been identified. The level of autophagy maybe de- creases in certain cells or increases in others. For example, autophagy is functionally active in regulatory T cells that respond to immune and inflammatory signals [37]. Importantly, enhancing autophagy in T cells upon mTOR blockade with rapamycin might benefit in systemic lupus erythematosus [38,39]. In addition to the maintenance of regulatory T cells, autophagy is also required for the survival of memory B cells [40,41]. However, other studies showed that the baseline level of au- tophagy was reduced in human pulmonary endothelial cells after LPS stimulation [42,43]. Hence, further cell experiments are required to clarify the alteration of autophagy in different cells of ALI. Accumulating research has suggested that autophagy plays a com-plex role in ALI, where it can have protective or deleterious effects upon various stimuli and in different models [14,20]. For example, Hu et al. observed that the induction of autophagy increased alveolar macro- phage apoptosis in intestinal ischemia/reperfusion (IR)-induced lung injury and subsequently disrupted pulmonary homeostasis and con- tributed to the development of ALI [44]. Slavin et al. also reported that inhibiting autophagy by 3-MA could reverse LPS-induced endothelial cell barrier dysfunction [28]. In contrast, several recent studies have demonstrated the beneficial effects of autophagy upregulation in LPS- induced lung injury and showed that treatment with autophagy in- hibitor aggravated LPS-induced vascular leakage and inflammatory cytokine release in the lung [29,31,42,45]. Our current results are consistent with these later studies, demonstrating that autophagy plays a protective role in LPS-induced ALI rats.In the present study, LPS exposure was found to induce pathological damage of lung tissue and lead to the recruitment of neutrophil into lungs and production of the pro-inflammatory cytokines. However, these effects were alleviated by enhancing autophagy with rapamycin. These findings suggest that autophagy helps alleviate LPS-induced lung injury. Autophagy maintains cellular homeostasis through the clear- ance of aggregated proteins, damaged organelles and invading patho- gens [18,20]. In addition, autophagy is upregulated as an adaptive mechanism for cell survival under conditions of nutrient starvation or energy depletion [9]. The specific characters of autophagy reasonably elucidate its protective functions in LPS-induced lung injury. We also found that LPS exposure induced high pulmonary vascular permeability and lung edema, and this effect was significantly improved by enhan- cing autophagy. Moreover, rapamycin pretreatment reversed the de- creased level of ZO-1 induced by LPS. These findings suggest that au- tophagy contributes to maintaining the integrity of the endothelial barrier. Pulmonary edema results from increased alveolar-capillary permeability and the accumulation of a large number of proteins in fluid [46]. ZO-1 is one of the key members of tight junction proteins that regulates the barrier formation of endothelial cells [47,48]. To date, the exact mechanism of LPS-induced autophagy in ALI rats has not been fully clarified. Autophagy is regulated by a complicated signaling network [49,50]. Emerging evidence has shown that mTOR signaling plays a critical role in autophagy. mTOR is a well-known upstream regulator of autophagy in mammals that regulates multiple aspects of the autophagy process, such as initiation, autophagosome formation, and autolysosome degradation by modulating the activity of the ULK1 complex and the VPS34 complex [13,25,51,52]. ULK1 (also known as Atg1) interacts with ATG13, ATG101 and focal adhesion ki- nase family interacting protein of 200 kD (FIP200), which composes the ULK1 protein kinase complex [13,53,54]. Beclin-1 (Atg6 in yeast),Atg14L, VPS34 and VPS15 form the PI3KC3/VPS34 complex [55]. The ULK1 complex and the VPS34 complex are two major regulators re- quired for the initiation of autophagy [13,18]. In our study, we found that LPS inhibited mTOR expression and then recovered the activity of ULK1 and VPS34. Moreover, the inhibition of mTOR by rapamycin further activated ULK1 and VPS34, resulting in upregulated autophagy levels. These results suggest that mTOR negatively regulates the activity of autophagy-related ULK1 and the PIK3C3 complex in response to LPS exposure. Further comprehensive studies are required to explore addi- tional molecular mechanisms. 5.Conclusions In this rat model of LPS-induced ALI, the autophagy level was in- creased, and enhancing autophagy by rapamycin alleviated lung his- topathological injury, reduced inflammatory cytokine release and im- proved pulmonary barrier function. The mechanism by which autophagy protects against LPS-induced ALI may be Compound 19 inhibitor associated with downregulation of the mTOR signaling pathway. These results suggest that the regulation of autophagy could be a novel therapeutic target for ALI.