Resveratrol protects the integrity of alveolar epithelial barrier via SIRT1/PTEN/p‐Akt pathway in methamphetamine‐induced chronic lung injury

Abstract Objectives SIRT1 is an antioxidative factor, but its mechanism in methamphetamine (MA)‐induced lung injury remains unclear. The purpose of this study is to determine whether MA can disrupt the integrity of alveolar epithelial barrier, whether SIRT1 is involved in MA‐induced chronic lung injury and whether Resveratrol (Res) can protect the integrity of alveolar epithelial cells by regulating ROS to activate SIRT1/PTEN/p‐Akt pathway. Materials and methods The rats were randomly divided into control group and MA group. Extracted lungs were detected by Western blot, HE staining and immunohistochemistry. The alveolar epithelial cells were treated with MA or/and Res, following by Western blot, LDH leakage assay and flow cytometry. MOE is used for bio‐informatics prediction. Results Chronic exposure to MA can cause slower growth ratio of weight, increased RVI and induced lung injury including the reduced number of alveolar sacs and the thickened alveolar walls. MA‐induced apoptosis was associated with SIRT1‐related oxidative stress. Res suppressed ROS levels, activated SIRT1, negatively regulated PTEN, phosphorylated Akt, reduced LDH leakage, increased the expression of ZO‐1 and E‐cadherin and inhibited the apoptosis of alveolar epithelial cells to attenuate MA‐induced higher permeability of alveolar epithelium. Conclusions MA disrupted the integrity of alveolar epithelial barrier. Res inhibited oxidative stress and reversed MA‐induced higher permeability and apoptosis of alveolar epithelium by the activation of SIRT1/PTEN/p‐Akt pathway.

Sirtuin1 (SIRT1) is an NAD + -dependent deacetylase, which regulates some cellular processes, such as apoptosis, mitochondrial biogenesis, lifespan extension, cell growth and inflammation. [7][8][9][10][11] can deacetylate histones, non-histones and some other crucial transcription factors to modulate the ROS production. 10 Phosphatase and tensin homolog gene (PTEN) is a cancer suppressor with bispecific phosphatase activity and is a multifunctional molecule expressed in various cells. 12 PTEN function can be negatively regulated by ROS. 13 PTEN degraded PIP3 and resulted in a decrease in Akt phosphorylation at Ser 473. PTEN is a suppressor of Akt-mediated signalling in the pulmonary epithelium. 14 Nonphosphorylated Akt is lack of biological activity. Once it is phosphorylated, p-Akt is able to regulate cell growth, apoptosis, adhesion, migration, infiltration and metabolism. 15 Epithelial cells establish closed contacts with their neighbours through intercellular junction complexes (ie tight junctions (TJs) and adherent junctions (AJs)). The TJs of alveolar epithelial cells create a firm intercellular sealing, control the transportation of water and molecules between adjacent cells, and are obligatory to the maintenance of the integrity of the alveolar barrier. 16 ZO-1 is one of the important components of TJs. The reduction in ZO-1 expression has been proven to play a key role in the injury and increased permeability of a variety of epithelium. 17 Cadherins are the AJs mediating cell-cell adhesion. Epithelial cadherin (E-cadherin) mediates the adhesion between the adjacent cells. 18  Resveratrol (3,4',5-trihydroxystilbene; Res) is a natural polyphenol in grapes, berries, peanuts and other plants. 19 The functions of Res were reported in cancer, cardiovascular diseases, ischaemic injury and acute poisoning. [20][21][22] The biological effects of Res may be explained by its antioxidant properties and the activation of SIRT1. SIRT1 is postulated to be a key in the pathophysiology of Res. 19,20 Based on the above, the current study is aimed to investigated whether MA can disrupt the integrity of alveolar epithelial barrier, whether SIRT1 is involved in MA-induced chronic lung injury and whether Res can inhibit oxidative stress and protect the integrity of alveolar epithelial cells by activating SIRT1/PTEN/p-Akt pathway.

| Establishment of animal models
Twenty male Wistar rats (200 ± 10 g) were purchased from Animal Resource Center, China Medical University (Certificate number: Liaoning SCXK 2015-0001) and were randomly divided into control group and MA-treated group. MA was from Criminal Investigation Police University of China. At the first week, the rats in the MA group were intraperitoneally injected with 10 mg/kg MA twice/day. Next, the dosage was increased by 1 mg/kg per week, until the daily dosage was increased to 15 mg/kg at the 6th week. The rats in the control group were injected with an equal volume of 0.9% physiological saline solution. 23 All rats were raised in a room with controlled temperature (18-22°C) and humidity (50%-70%) and were fed with solid food and water in an alternating 12 hours light and 12 hours dark cycle. All experimental procedures involving animals were followed by the guidelines of

| Cell culture and treatment
The alveolar epithelial cells A549 (Dingguo Changsheng) were cultured in RPMI 1640 medium (HyClone) supplemented with 10% foetal bovine serum (Clark) and 1% penicillin/streptomycin in 25 cm 2 culture flask at 37°C in 5% CO 2 . A549 cells were treated by MA with the dosage of 0.1, 0.5, 1 and 5 mmol/L for 6, 12 and 24 hours. 24  The concentration of DMSO in the medium never exceeded 0.1% to avoid its toxicity towards the A549 cells. To evaluate the effects of NAC, the cells were divided into control group, 5 mmol/L MA group, 5 mmol/L NAC group, and 5 mmol/L MA plus 5 mmol/L NAC group. 25 To evaluate the effects of Res, the cells were divided into control group, 5 mmol/L MA group, 20 μmol/L Res group and three MA plus Res (1, 5, 20 μmol/L) groups. 26

| HE staining
The inferior lobes of the right lung were fixed with 4% paraformaldehyde buffer overnight, dehydrated, paraffin embedded, sliced into 4 µm sections and stained with haematoxylin and eosin (Hongqiao).
The slides were observed under a light microscopy (Olympus BX 51).
Lung injury was calculated by the thickness of alveolar septum and the number of alveolar sacs (three visual fields selected randomly were analysed in each section; magnification, ×200 and ×400). 24

| Immunohistochemical assay
The sections were antigen-retrieved. The slices were, respectively, incubated with the primary antibody of anti-SIRT1, anti-E-cadherin or anti-ZO-1 at 4°C overnight. The slides were incubated with goat anti-rabbit biotinylated secondary antibody (MXB, Fuzhou) for 10 minutes at room temperature and then reacted with streptavidin-peroxidase (MXB, Fuzhou) conjugate for 10 minutes at room temperature.
Subsequently, the slices were treated with diaminobenzidine (DAB; Zhongshan Jinqiao) and counterstained with haematoxylin. The slices were dehydrated, mounted and observed under a light microscope.

| Western blotting analysis
The lungs or alveolar epithelial cells from different groups were homogenized with ice-cold RIPA and centrifuged at 12 000 g for 15 minutes. Protein concentrations were determined using a BCA Kit (Beyotime). Protein samples were loaded by a SDS-polyacrylamide gel electrophoresis. After blocked with 5% fat-free milk for 2 hours, the PVDF membranes (GE) were incubated with primary antibodies, respectively, at 4°C overnight (Table 1). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (Proteintech) were incubated for 2 hours at room temperature.
Immunoreactive bands were visualized by DNR Bio-Imaging systems, and densitometric analysis was determined by ImageJ software.

| Bio-informatics prediction
The structures of SIRT1 and PTEN are from RCSB PDB data bank. moe software (CCG) is used to dock model structure of SIRT1 with PTEN. The binding sites between them are analysed by moe software. Apoptosis was determined by staining cells with FITC-Annexin V/PI (Vazyme). Cells (1 × 10 6 cells/well) were collected after the treatments, washed twice with cold PBS and resuspended with 100 μL binding buffer with FITC-Annexin V (5 μL) and PI staining solution (5 μL). The cells were darkly incubated for 10 minutes at room temperature. The percentage of apoptotic cells was measured by flow cytometry.

| LDH assay for the damage of alveolar epithelium
LDH leakage from A549 cells into the culture medium was assessed using an LDH Cytotoxicity Assay Kit (Beyotime). The cells The LDH leakage rate was calculated by comparing with the LDH activity control group.

| Statistical analysis
All the data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed with IBM spss Statistics 22.0 and graphpad prIsm 6.0 (GraphPad). Differences between two groups were assessed by Student's t test, and differences in multiple groups were assessed by one-way ANOVA. The values of P < .05 and P < .01 were considered to indicate a statistically significant difference.

| Pulmonary injury induced by chronic exposure to MA
HE staining was used to show pathological changes in rat lungs in different groups. In the control group, the alveolar structure was intact and clear, and there was no inflammatory cell infiltration, bleeding or thickening of the alveolar walls ( Figure 1A). But in the MA group, rat lungs showed the marked infiltration of inflammatory cells into the alveolar cavity, more compact parenchyma, reduction in the number of alveolar sacs and the thickened alveolar walls ( Figure 1B,C). The percentage of weight gain in the MA group was significantly lower than that in the control group from the 4th week to 6th week ( Figure 1D). Right ventricular index (RVI) 0.18 ± 0.037 from the control group was obviously increased to 0.32 ± 0.008 in the MA group ( ** P < .01, Figure 1E).

| MA disrupted the integrity of alveolar epithelial barrier
To determine whether MA can increase the permeability of alveolar epithelium, it is necessary that TJ protein ZO-1 and AJ protein E-cadherin should be detected. Western blot analysis showed that E-cadherin and ZO-1 in lungs were dramatically decreased in the MA group, compared with the control group (

| MA-induced oxidative stress and apoptosis of alveolar epithelial cells
SIRT1 can modulate the ROS levels to protect against oxidative stress-mediated injury. Western blot analysis showed that SIRT1 expression in the control group was higher than that in the MA group ( Figure 3A,B), which was consistent with the tendency of SIRT1 expression by IHC ( Figure 3E). Oxidative enzyme superoxide dismutase (SOD) reflects the degree of oxidative stress injury. In our study, it was found that SOD2 in lungs was highly expressed in the MA groups ( Figure 3C). Antioxidative enzyme glutamylcysteine synthetase (GCS) can scavenge oxygen free radicals, so its expression was reduced by MA ( Figure 3D). Excessive produc-  Figure 4A). It was found that the expression of SIRT1 was significantly reversed from MA by NAC ( Figure 4B).
Meanwhile, the levels of caspase 3, cleaved-caspase 3 and Bax were markedly downregulated and Bcl-2 was obviously upregulated in the 5M + 5NAC group, compared with the 5M group ( Figure 4E-H). These results were confirmed that SIRT1 expression and apoptosis of alveolar epithelial cells were modulated by MA-induced oxidative stress.

| Res reversed MA-induced ROS production and apoptosis
In order to furtherly determine MA-induced apoptosis by SIRT1related oxidative stress, in the present study, the alveolar epithelial

| Res alleviated the disruption of alveolar epithelial integrity through SIRT1/PTEN/p-Akt pathway
According to the possible interaction between SIRT1 and PTEN, Res, a SIRT1 activator, was used to detect the effect of SIRT1 on PTEN. It was found that SIRT1 was dose dependently increased by pre-treated Res and that the increase in SIRT1 expression was particularly significant by 20 μmol/L Res ( Figure 7A  The chronic accumulation of MA resulted in the pulmonary complications attributed to its high uptake in lungs. 5,28 Lung toxicity includes alveolar oedema, exudation, thickening of the alveolar septa and inflammatory cell infiltration. 29 In our study, it was found of more compact lung parenchyma, reduction in the number of alveolar sacs, the thickened alveolar walls and infiltration of inflammatory cells, which suggested that the damages of alveolar epithelial

F I G U R E 3 (Continued)
integrity and an increase in the permeability of alveolar epithelial cells are involved in MA-induced lung injury.
Pulmonary epithelium as a barrier limits the access of various toxic to the respiratory system to maintain the pulmonary homoeostasis. 30 Therefore, alveolar epithelial cells are also considered to play an important role in the defence and anti-infection to reduce the surface tension of the alveoli, promote the gas exchanges and improve the restoration of lungs. 13 TJs and AJs, such as ZO-1 and E-cadherin, are located on the apical surface of the sidewalls of adjacent alveolar epithelial cells. 17 ZO-1 and E-cadherin provide the linkage between the cell membranes, and they are essential for the integrity of alveolar epithelial barrier. 17 The results from our study were that the epithelial barrier markers ZO-1 and E-cadherin were localized at the alveolar walls and that MA dramatically decreased the expressions of E-cadherin and

ZO-1 in lungs and in alveolar epithelial cells, which reflected that MA
induced the lack of the function of alveolar epithelial barrier.
Sirtuins are a unique of histone III deacetylases with seven subtypes SIRT1-7. 31 SIRT1 is a NAM adenine dinucleotide (NAD + )dependent histone deacetylase involved in multiple cellular functions, for example, oxidative stress. [32][33][34][35] Oxidative stress is mediated by ROS. 3 Some studies have revealed that there is a crosstalk between SIRT1 and oxidative stress. 36,37 It was founded that MA inhibited the expression of SIRT1 by inducing oxidative stress with elevated ROS levels, increased SOD and reduced GCS. MA-induced lung toxicity is linked to oxidative injury by directly activating NAPDH oxidase and generating of ROS that cause cellular damages. 10,38 Previous studies reported that MA increased ROS production in lungs, 24  In order to furtherly determine that MA-induced apoptosis is associated with SIRT1-related oxidative stress, the alveolar epithelial cells were pre-treated with Res, a SIRT1 activator, previous to MA. Flow cytometry analysis showed that the production of ROS was partially reversed by 20 μmol/L Res from MA. It was also found that in Res significantly decreased Bcl-2, caspase 3 and cleaved-caspase 3, and in- PTEN can dephosphorylate PIP3 into PIP2 and PIP2 to PIP to switch off the pathway of Akt phosphorylation, and then downregulated the levels of p-Akt. 41 Therefore, Res exerted the protective effects on the integrity of alveolar epithelial barrier via SIRT1/PTEN/p-Akt pathway.
In summary, chronic exposure to MA can disrupt the integrity of alveolar epithelial barrier. Resveratrol can inhibit oxidative stress and reverse MA-induced higher permeability and apoptosis of alveolar epithelium via SIRT1/PTEN/p-Akt pathway.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no competing interests. The data that support the findings of this study are available from the corresponding author upon reasonable request.