mTOR‐autophagy promotes pulmonary senescence through IMP1 in chronic toxicity of methamphetamine

Abstract It is growingly concerned about methamphetamine (MA)‐induced lung toxicity. IMP1 is identified as a key molecule for cell life processes, but the role of IMP1 in MA‐induced senescence remains unclear. The purpose of this study was to investigate whether chronic exposure to MA can cause autophagy and senescence of the lungs, whether there are interactions between Mammalian target of rapamycin (mTOR) and IMP1 and whether IMP1 is involved in pulmonary senescence promoted by mTOR‐autophagy. The rats were randomly divided into control group and MA group, following by H&E staining, immunohistochemistry staining and Western blot. The alveolar epithelial cells were proceeded by ß‐galactosidase staining, cell cycle detection, transfection and co‐immunoprecipitation. Long‐term exposure to MA led to the thickening of alveolar septum and more compact lungs. MA promoted the conversion of LC3‐I to LC3‐II and inhibited the activation of mTOR to induce autophagy. Bioinformatics and co‐immunoprecipitation results presented the interactions between IMP1 and mTOR. MA induced cell senescence by decreasing IMP1, up‐regulating p21 and p53, arresting cell cycle and increasing SA‐β‐gal. Overexpression of IMP1 reduced p21 and SA‐β‐gal to inhibit the senescence of alveolar epithelial cells. These results demonstrated that mTOR‐autophagy promotes pulmonary senescence through IMP1 in chronic toxicity of methamphetamine.

so that autophagy will occur after mTOR is inactivated. 8 Our previous studies found that MA-induced inactivation of mTOR promoted autophagy-mediated alveolar epithelial injury. 9 Senescence is the complex process of deterioration that occurs over the lifetime of an organism, resulting in progressive functional decline and eventual death. 10 Cellular senescence is a phenomenon that the cells lose the ability of self-proliferation. 11 Studies have shown that drug abuse, such as MA commonly used in HIV patients, can cause cell senescence and contributes to age-related diseases in nervous system, 2,12 but there are few studies on the effect of MAinduced senescence in the lungs. Although there are studies about the relationship between autophagy and senescence, their regulatory mechanism has been controversial. On the one hand, it is believed that autophagy is inhibited in ageing organisms, leading to the accumulation of harmful substances and damaged organelles. [13][14][15] On the other hand, it is reported that autophagy is positively regulating senescence, that is, the senescence process is accompanied by autophagy. 16,17 However, it has been unknown about the relationship between autophagy and senescence in MA-induced lung toxicity.
Insulin-like growth factor 2 messenger RNA-binding protein 1 (IGF2BP1, also known as IMP1) is a highly conserved RNA-binding protein and mainly expressed in embryonic stage but less expressed during adulthood and expressed in malignant transformation. [18][19][20] IMP1 can also stabilize c-Myc mRNA and prevent its binding with specific sequences to suppress its degradation to increase the protein level of c-Myc and promote cell proliferation accordingly. 21,22 Recovery of IMP1 expression in malignant cells not only promotes tumour progression, but also participates in the establishment and maintenance of tumour cell levels and increase the invasiveness of cancer. 19,23 However, IMP1 in breast cancer cells was significantly lower than that in colorectal cancer cells, and IMP1 had a positive role in inhibiting the growth and metastasis of breast cancer. 24 IMP1 is related to PI3K, mTOR and MAPKs pathways through protein-protein interactions. 21,23,25 It is generally acknowledged that mTOR acts as an autophagy inhibitor to regulate cellular autophagy. [26][27][28][29] Studies have shown that mTOR could catalyse the phosphorylation of IMP1 at Ser181 site and that IMP1 could bind to mRNA of mTOR. 30 Meanwhile, IMP1 deletion increases autophagy in Crohn's disease and ulcerative colitis. 31 Therefore, we make a hypothesis that there may be a protein-protein interaction between mTOR and IMP1. It is reported that IMP1 down-regulation can inhibit cell proliferation and induce cellular senescence. 32 Some studies have shown that the expression of p21, an indicator of senescence, is increased after IMP1 is knocked down in hepatocellular carcinoma, suggesting that IMP1 could regulate p21 expression to take a part in cellular senescence. 33,34 And it has also been reported that knocking down of IMP1 in hepatocellular carcinoma can result in G1 phase cell cycle arrest. 34 Based on the above, the current study is aimed to investigate whether chronic exposure to MA can cause pulmonary autophagy and senescence, whether there are interactions between mTOR and IMP1 and whether IMP1 is involved in pulmonary senescence promoted by mTOR-autophagy in chronic toxicity of MA.

| Animal models
Twenty male Wistar rats were obtained from the Animal Resource Center of China Medical University. They were divided into control group and MA group randomly. MA (China Criminal Police University, China) was injected intraperitoneally with the dosage of 10 mg/kg in the 1st week, and then, the daily dose was increased by 1 mg/kg per week, until the daily dose was increased to 15 mg/kg in the 6th week. 35 The control group was given intraperitoneal injection with the equivalent of 0.9% physiological saline. The rats were group housed in air-conditioned (the temperature is 18-22°C and the humidity is 50%-70%) rooms with a 12-hour light cycle and allowed free access to food and water. All the rats were weighted every day.
The percentage of weight gain in each group was calculated every week as the following Equation 1. After 6 weeks, the right ventricular index (RVI) was also calculated using Equation 2 to evaluate the remodelling of the right heart resulting from chronic pulmonary dysfunction. All experimental procedures involving animals were con-

| Transfection
The overexpression plasmids of IMP1 were designed and obtained from the GeneChem Group, Ltd. Cells were inoculated The percentage of weight growth = average of the weekly weight − average of the initial weight average of the initial weight × 100% The right ventricular index = right ventricular weight right ventricular weight + left ventricular weight × 100% and transfected with plasmids when 70% of the culture flask were covered. The protocol is referred to the Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific). The DNA and Lipo3000 were diluted with serum-free medium, respectively, and then mixed together at a 1:1 ratio. After incubation 10-15 minutes at room temperature, the mixture was added into normal cell medium. The cells were cultured at 37°C for 2-4 days.
Drugs were administrated at the time of highest transfection efficiency. Drug-treated cells were analysed by Western blot and senescence associate ß-galactosidase (SA-ß-gal) staining (Nikon MODEL ECLIPSE Ts2-FL).

| Cell cycle detection
The protocol is referred to the instruction of the cell cycle test-

| Western blot analysis
After sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred onto a PVDF membrane. The PVDF membrane was blocked for 2 hours and then incubated with corresponding primary antibodies (Table 1)

| H&E and Immunohistochemistry (IHC) staining
The lung was infused with 0.9% physiological saline and fixed slowly with the 4% paraformaldehyde solution through airway. The opening of the airway was upward. After the lower part of the lung was 3/4 inflated, the trachea was tighten, and then, the lung was immersed in the fixed solution for more than 24 hours for paraffin embedding.
The lung was sliced into 4-µm sections for morphological measurements. H&E staining was to evaluate the thickness of alveolar septum and the number of alveolar sacs to illustrate lung injury (three fields of view randomly selected were analysed in each section; magnification, ×200, ×400).

| Senescence-associated ß-galactosidase staining
The protocol was referred to the instruction of the senescence-asso-

| Bioinformatics prediction
The structures of IMP1 and mTOR are from RCSB PDB data bank.
MOE software is used to dock model structure of mTOR with IMP1.
The binding sites between them are analysed by MOE software (CloudScientific Technology Co., Ltd).

| Statistical analysis
All the data are expressed as mean ± SD. Statistical analysis was carried out by GraphPad Prism 5.0 (GraphPad Software, Inc). Nonparametric t test (Mann-Whitney test) and two-way analysis of variance (ANOVA) were used for statistical comparisons. ANOVA were followed by Bonferroni post-test. P < .05 was considered to have statistical significance.
F I G U R E 1 Chronic exposure to MA induced pulmonary injury in rats. A, The percentage of weight growth of rats in different groups. B, The right ventricular index of rats in different groups. C, MA induced chronic lung injury by H&E staining (Nikon, ECLIPSE TE2000-U, Japan, ×200 and ×400). The black arrows represent the alveolar septum. D, The alveolar septum thickness (μm) in different groups (magnification, ×400). E, The number of alveolar sacs in different groups (magnification, ×200). The quantification of chronic lung injury was analysed in three fields of view randomly selected in each section. Data are shown as mean ± SD, n = 7, *P < .05, ***P < .001, vs CON; CON, control group; MA, methamphetamine group

| Chronic exposure to MA-induced pulmonary injury in rats
For 6 weeks of administration with MA, the rat weight growth in MA group had been declined, especially from the 4th to 6th week ( Figure 1A). At the end of the 6th week, the RVI was significantly increased in MA group, compared with control group (Figure 1B), which indicated that it was successful to establish the chronic toxicity model of MA.
The results from H&E staining suggested that chronic exposure to MA can cause more compact parenchyma of lungs ( Figure 1C), including the thickened alveolar septum ( Figure 1D) and reduction of the number of alveolar sacs ( Figure 1E).

| MA-induced autophagy in lungs and alveolar epithelial cells
LC3 is a component of the autophagosome and represents the occurrence of autophagy. 37,38 Once autophagy is activated, LC3-I is converted to LC3-II. LC3-Ⅱ/LC3-Ⅰ ratio is widely used as a marker of autophagy activation. 39 In this study, it was found that compared with control group, LC3-Ⅱ expression was significantly increased (Figure 2A), and the ratio of LC3-Ⅱ/LC3-Ⅰ was up-regulated in MA group ( Figure 2B). The phosphorylation of mTOR was inhibited by MA, and the expression of p-mTOR was markedly reduced (Figure 2A,C), so that mTOR was inactivated to induce autophagy in lungs.
Western blot analysis showed that in alveolar epithelial cells, the protein levels of LC3-Ⅱ were notably increased at 5 mmol/L MA, compared with control groups ( Figure 2D). The ratio of LC3-Ⅱ/LC3-Ⅰ in 5 mmol/L MA group for 24 hours was much higher than other groups ( Figure 2E). Therefore, LC3-Ⅱ/LC3-Ⅰ was dose-dependently and time-dependently increased by MA in alveolar epithelial cells, which is consistent with that in lungs of rats. The protein expressions of mTOR and p-mTOR were remarkable decreased at 5 mmol/L MA for 24 hours, compared with control groups (Figure 2F-H). The results indicated that exposure to MA can suppressed mTOR activation dose-dependently and time-dependently.

| Protein-protein interactions exist between IMP1 and mTOR
IMP1 is involved in many signalling pathways and cellular processes. 19 Studies have shown that mTOR participates in the regulation of IGFs axis, that mTOR mRNA can bind to IMP1 by RNA immunoprecipitation and that mTOR protein can catalyse IMP1 phosphorylation at ser181 site. 30,40 Therefore, there may be a closer relationship between mTOR and IMP1.
MOE software was carried out to predict the molecular docking between IMP1 and mTOR. We obtained the protein structure of IMP1 and mTOR from Protein Data Bank (http://www.rcsb.org/) ( Figure 3A). In the most stable conformation, it was synthetically evaluated that there are seven pairs of hydrogen bonds between the two proteins ( Table 2). It was implied that there may be a link between IMP1 and mTOR. The results from co-immunoprecipitation were that IMP1 was also dragged down after mTOR was pulled down by agarose ( Figure 3B), which supported the bioinformatics prediction and indicated that there was protein-protein interactions between IMP1 and mTOR.

| MA caused the senescence in lungs
The relative protein expression of IMP1 in the rat lungs was shown by Western blot analysis ( Figure 4A). In MA group, the IMP1 expression was significantly declined compared with control group. The IHC results were also indicated that IMP1 was mainly located on the alveolar epithelium and that MA markedly reduced the expression of IMP1 in lungs ( Figure 4B). The senescence is regulated through p53-p21-pRb pathway, so p53 and p21 can be used as the markers of senescence. 41 The protein expressions of p21 and p53 in rat lungs in MA group were nearly twice as much as that in control group ( Figure 4C-E). IHC results showed that the expression of p21 in the MA group was much higher than that in control group ( Figure 4F), which indicated that MA could regulate the senescence-associated factors and lead to pulmonary senescence.

| MA-induced cellular senescence in human alveolar epithelial cells
Senescent cells can be identified by the expression of the above markers or SA-β-gal activity. 41 When cellular senescence occurs, the cell cycle is arrested with the increasing of G0/G1 phase and the decreasing of S phase, which can be induced by p53 via p21/CDKN1Adependent inhibition of cyclin A/E-CDK2. 42 The relative protein expression of IMP1 in the alveolar epithelial cells was shown by Western blot analysis ( Figure 5A)

| The role of IMP1 in MA-induced pulmonary senescence
It was reported that in hepatocellular carcinoma, knockout of IMP1 can lead to a significant increase of p21, suggesting that IMP1 may influence the expression of p21. 34 To investigate whether IMP1 can regulate p21, the IMP1 overexpression plasmids were transfected into the alveolar epithelial cells. After the incubation for 36 hours, 5 mmol/L MA was treated for 24 hours. Transfected efficiency was confirmed by Western blot analysis ( Figure 6A,B). The results showed that in comparison with control group, IMP1 was significantly increased in over-control group ( # P < .05, vs CON), which indicated that overexpression transfection is successful. Respectively compared with control group, negative control group or over-control group, MA markedly reduced the expression of IMP1 (*P < .05 vs CON, NCON and OCON, respectively). And the results of negative control group and negative MA group indicated that blank plasmid had no effect on the experimental results.

| D ISCUSS I ON
Although most studies of MA have been focused on the nervous system, about 78.3% deaths from methamphetamine toxicity are due to pulmonary diseases. 43,44 This study showed that long-term exposure to MA led to more compact lungs, such as the thickening of alveolar walls and reduced number of alveolar sacs. MA promoted the conversion of LC3-I to LC3-II, inhibited the activation of mTOR to induce autophagy. Bioinformatics and co-immunoprecipitation results presented the interplay between IMP1 and mTOR. MA down-regulated mTOR and caused the lower IMP1 by the interactions between IMP1 and mTOR, which were resulted in the occurrence of cellular senescence by the up-regulation of p21 and p53, the arrested cell cycle and the increased activity of SA-ß-gal, which represented.
Overexpression of IMP1 reduced the levels of p21 and decreased SA-ß-gal to inhibit the senescence of alveolar epithelial cells. These results demonstrated that mTOR-autophagy promotes pulmonary senescence through IMP1 in chronic toxicity of methamphetamine.
Autophagy is a highly conserved process for cell protection in various types of cellular stress (including nutritional deficiency, ROS, endoplasmic reticulum stress, bacterial infection). 40 Our previous study found that MA could induce endoplasmic reticulum stress and inflammatory lesions in rat lung tissue. 45 mTOR can inhibit autophagy, and phosphorylation is its active form, so that the down-regulation of mTOR or inhibition of phosphorylation means the occurrence of autophagy. 46,47 Autophagosomes are produced by autophagy. 37,38 And LC3 is one component of autophagosomes.
When autophagy is activated, LC3-I is converted into LC3-II. The

F I G U R E 3
Protein-protein interactions exist between IMP1 and mTOR. A, The results of bioinformatics prediction between IMP1 and mTOR. There are seven pairs of hydrogen bonds between IMP1 and mTOR, shown as amino acid sites on them. B, The results of co-immunoprecipitation between IMP1 and mTOR. Samples were made by capture mTOR protein with agarose beads, and IMP1 protein was detected by Western blot analysis TA B L E 2 The binding sites between IMP1 and mTOR by bioinformatics prediction mTOR IMP1
mTOR activation was also significantly suppressed after stimulation with MA. It implied that MA promoted pulmonary autophagy by accelerating the transformation from LC3-I to LC3-II and reducing the expression levels of p-mTOR in both dose-dependently and time-dependently way.
Autophagy disorder is a key cause of many diseases, including neurodegenerative diseases and some metabolic diseases. 48 Some researchers pointed out that since autophagy can be divided into general autophagy and selective autophagy, their regulatory mechanisms are different and they have different effects. 49 Selective autophagy inhibits senescence by GATA4, a key regulator of the senescence-associated secretory phenotype and senescence, while general autophagy induces senescence by TASCC (TOR-autophagy spatial coupling compartment). 49 But how does autophagy induce the senescence? IMP1 takes a part in many cell life processes, including cell proliferation and cell death. 19 Therefore, IMP1 is likely involved in cellular senescence.
We predicted the protein-protein binding of IMP1 and mTOR by molecular docking software and found that there were a great possibility of protein-protein interactions between IMP1 and mTOR, which were confirmed by co-immunoprecipitation. The interplay between mTOR and IMP1 may be the linkage between autophagy and senescence.
Furthermore, it was found that the expressions of p21 and p53 were significantly increased by MA. The results from cell cycle F I G U R E 4 MA caused the senescence in lungs. A, Western blot analysis of IMP1 in lungs. B, IHC results of IMP1 expression in lungs (magnification, ×200, ×400). The brown represented the expression of IMP1 located on alveolar epithelium with the black arrows. C, Western blot analysis of p21 and p53 in lungs. D, Relative expression of p21 in lungs. E, Relative expression of p53 in lungs. F, IHC results of p21 expression in lungs (magnification, ×200, ×400). The black arrows represented the expression of p21. Data were shown as means ± SD. **P < .01, ***P < .001 vs CON; CON, control group; MA, methamphetamine group In conclusion, it was discovered in this study that long-term exposure to MA can induce pulmonary autophagy and cellular senescence which may be mediated by the interplay between mTOR and IMP1. Therefore, mTOR-autophagy promotes pulmonary senescence through IMP1 in chronic toxicity of methamphetamine.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.