IP3R attenuates oxidative stress and inflammation damage in smoking‐induced COPD by promoting autophagy

Abstract Tobacco smoking is one of the most important risk factors for chronic obstructive pulmonary disease (COPD). However, the most critical genes and proteins remain poorly understood. Therefore, we aimed to investigate these hub genes and proteins in tobacco smoke‐induced COPD, together with the potential mechanism(s). Differentially expressed genes (DEGs) were analysed between smokers and patients with COPD. mRNA expression and protein expression of IP3R were confirmed in patients with COPD and extracted smoke solution (ESS)‐treated human bronchial epithelial (HBE) cells. Moreover, expression of oxidative stress, inflammatory cytokines and/or autophagy‐related protein was tested when IP3R was silenced or overexpressed in ESS‐treated and/or 3‐MA‐treated cells. A total of 30 DEGs were obtained between patients with COPD and smoker samples. IP3R was identified as one of the key targets in tobacco smoke‐induced COPD. In addition, IP3R was significantly decreased in patients with COPD and ESS‐treated cells. Loss of IP3R statistically increased expression of oxidative stress and inflammatory cytokines in ESS‐treated HBE cells, and overexpression of IP3R reversed the above functions. Furthermore, the autophagy‐related proteins (Atg5, LC3 and Beclin1) were statistically decreased, and p62 was increased by silencing of IP3R cells, while overexpression of IP3R showed contrary results. Additionally, we detected that administration of 3‐MA significantly reversed the protective effects of IP3R overexpression on ESS‐induced oxidative stress and inflammatory injury. Our results suggest that IP3R might exert a protective role against ESS‐induced oxidative stress and inflammation damage in HBE cells. These protective effects might be associated with promoting autophagy.


| INTRODUC TI ON
Chronic obstructive pulmonary disease (COPD), a general term for a series of airflow limitation diseases, has been predicted to become the third leading cause of death in 2030 by the World Health Organization (WHO). In recent years, COPD seriously threatened the health of humans and reduced the quality of normal life. The symptoms of patients with COPD always include chronic cough, sputum production and dyspnoea, which are mainly associated with the inflammatory response in the airway and alveolar epithelium. 1 Symptoms could be induced by a variety of reasons, among them, tobacco smoking, especially in elderly smokers, is one of the main risk factors for COPD. 2 More than 4000 chemicals are contained in tobacco smoke, of which at least 20 are carcinogenic substances. 3 The pathogenic mechanisms of tobacco smoke mainly include inflammation, oxidative damage and cell senescence. 4 In addition, previous studies have reported that changes in gene expression and epigenetic modification are related to tobacco smoke-induced inflammation; 1,5 meanwhile, a large number of protein-coding genes are associated with COPD exacerbation. 6,7 However, the gene and protein whose alteration is most critical in tobacco smoke-induced COPD remain poorly understood. Therefore, first, this study used bioinformatics methods to explore the biomarkers of COPD induced by tobacco smoke based on the gene chip databases. We found inositol 1,4,5-trisphosphate receptor was one of the common different expression genes of patients with COPD and normal smokers compared with the non-smoker control, separately. IP 3 R can regulate the release of intracellular Ca 2+ through binding the second messenger IP3 from the endoplasmic reticulum (ER). 8 In the molecule, IP 3 R is essentially regulated by IP3 and Ca 2+ , 9 and intracellular signals such as the redox state, 10 ATP 11 and cAMP 12 can modulate its regulation. Moreover, IP 3 R was found to play an important role in both normal physiological processes and disease. The central function of IP 3 R is its delivery of Ca 2+ to mitochondria or lysosomes, by which it participates in regulatory processes such as oxidative phosphorylation and cell apoptosis. [13][14][15] Dysregulation or mutation of IP 3 R is associated with neurodegenerative disorders. 16,17 In addition, IP 3 R signalling pathway might be a target for cancer therapeutics by inducing autophagy. 18 The latest studies demonstrated that IP 3 R-associated calcium signalling plays an essential role in the oxidative stress process in human endothelial cells. 19 However, whether IP 3 R contributes to the smoke-induced respiratory inflammatory response and oxidative stress remains not obvious.
Airway inflammation, which might be related to increased oxidative stress, is widespread in patients with tobacco smoke-induced COPD. 20,21 Numerous oxidants can be directly measured in tobacco smoke, and tobacco smoking can deplete antioxidants. 22,23 Moreover, oxidative stress remains high due to the presence of active inflammatory cells in tobacco smoke-induced COPD. The inflammatory response was also shown to be further amplified in this process by activation of the NF-κB pathway. 24 Multiple cell and proinflammatory cytokines participate in the inflammatory response. 25 IL-4, IL-5, IL-9 and IL-13 mediate inflammation, whereas TNFα and IL-1β further amplify the inflammatory response. 21,26 In tobacco smokeinduced COPD patients, an imbalance between oxidative and antioxidative processes caused by the inflammation response eventually leads to disease progression. 27 Autophagy is a widespread basic activity in eukaryotic cells 28 that plays an essential role in the inflammatory response to stress in the airway and lung. 29,30 Furthermore, there is increasing evidence that the autophagy process plays critical roles in the progression of COPD-emphysema. [31][32][33][34][35][36] It has been demonstrated that exposure to tobacco and/or e-cig/nicotine vapour promotes oxidative stress reaction and inflammatory response that could lead to autophagy-flux impairment, further impeding the important cellular homeostatic processes involved in the removal of misfolded proteins and bacterial/viral pathogens, ultimately affecting cell survival. 33,34,37 In this process, Ca 2+ signalling is one of the basic mechanistic targets for modulating autophagic flux. 38 Previous studies proposed that IP 3 R could mediate Ca 2+ signalling as an essential target in starvationinduced autophagy. 39 Cytosolic Ca 2+ buffering could impede autophagy through the mTOR-related pathway. 40 However, whether cell autophagy-related protein mediated by IP 3 R participates in the regulation of oxidative stress and inflammation induced by tobacco smoke requires further investigation.
In the present study, we performed a bioinformatics analysis to identify the potential molecular targets of tobacco smoke-induced COPD. A subsequent study was performed to verify the differential expression of one of these selected targets (IP 3 R). Finally, we aimed to explore the role of IP 3 R in the process of oxidative stress and inflammation in a tobacco smoke-induced human bronchial epithelial (HBE) cell injury model and determine whether IP 3 R regulation is associated with autophagy.

| Data resources
Gene expression data used in this study were obtained from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI-GEO) database. We obtained the gene expression data sets GSE54837 and GSE37768. Microarray data from these two data sets were collected with the GPL570 platform ([HG-U133_Plus_2]).
The GSE54837 data set comprised data from 136 COPD patient, 84 smoker control and six non-smoker control samples. The GSE37768 data set comprised data from 18 COPD patient, 11 smoker control and nine non-smoker control samples. The batch effects in the expression data were adjusted using distance-weighted discrimination methods. 41

| Data pre-processing of differentially expressed genes
Differentially expressed genes (DEGs) between COPD/non-smoker controls and smoker/non-smoker controls were identified via GE2O online tools. The DEGs between these pairs of groups were aggregated. A P-value <.05 and a |logFC| > 2 were used as the cut-off criteria for DEG screening. Then, raw data were analysed with the Venn online website to determine common DEGs between the COPD/ non-smoker control and smoker/non-smoker pairs of groups.

Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses
DAVID is an online bioinformatics tool that is widely used to identify gene and protein functions. 42,43 In the present study, gene ontology

| HBE cell cultures and transfection
The HBE cell line was used in the present study. HBE cells were thawed and cultured in six-well cell culture plates. RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin was used to culture the cells in an incubator set at 37°C and 5% CO 2 .
To explore the function of IP 3 R in tobacco smoke-induced oxidative stress and inflammation, we purchased lentiviruses expressing si-RNA against IP 3 R, which were transduced into HBE cells according to the manufacturer's instructions. In addition, HBE cells transduced with lentiviruses expressing control si-RNA were used as a control group. Subsequently, IP 3 R was overexpressed in the experimental group with the same method, and HBE cells transfected with lentiviruses containing a scrambled sequence were used as the control group.

| Pre-treatment with aqueous cigarette smoke extract
Tobacco smoke was extracted based on previous reports. 44,45 Three cigarettes from different companies (Liqun, Nanjing, China; Hongtashan, Yunnan, China; and Jiaozi, Sichuan, China) were burned, and the smoke mixtures were extracted through a vacuum pump and dissolved in 10 mL of PBS. The pH of the extracted smoke solution (ESS) was adjusted to 7.4, and the ESS was filtered through a 0.22μm filter. HBE cells in the experimental group were cultured in medium containing 5% ESS. HBE cells cultured in medium containing a 5% extract of normal air prepared in a manner similar to that used to prepare the experimental extract were used as a control group.

| IP3R double immunofluorescence of HBE cells
Human bronchial epithelial cells were pre-treated for 1 hour with 5% ESS, and the medium was then replaced. After pre-treatment for 24 hours, the HBE cells and medium were collected. HBE cells after ESS exposure and control group were fixed with formaldehyde, and treated with 0.1% Triton X-100 to permeabilize the cell membrane.
The cells were then blocked with non-immunized animal serum and incubated with antibodies against IP3R (1:500; Cell Signaling Technology) at 4°C and incubated with a fluorescently labelled secondary antibody (1:200; Cell Signaling Technology) for 2 hours at room temperature. DAPI was applied to stain the nuclei, and results were imaged using a fluorescence microscope.

| Determination of CAT, MDA, SOD and GSH-Px levels
Human bronchial epithelial cells were pre-treated for 1 hour with 5% ESS, and the medium was then replaced. After pre-treatment for

| Measurement of ROS formation
Human bronchial epithelial cells were collected 24 hours after pretreatment for 1 hour with 5% ESS. Digested cells were harvested after centrifugation, and precipitated cells were resuspended in binding buffer after washing twice. The final concentration of HBE cells was 1 × 10 5 /mL. As shown in previous research, 46 HBE cells were incubated with 10 μmol/L DCFH-DA for 30 min in an incubator.
Finally, ROS formation was quantified via flow cytometry (Becton Dickinson) and is shown as the fluorescence intensity.

| Quantification of HBE cell viability
Human bronchial epithelial cell viability was quantified using the MTS method. HBE cells were evenly cultured in a 96-well assay plate and pre-treated for 1 hour with 5% ESS, following which the cell medium was recycled with fresh medium, and the incubation was continued for 24 hours. Subsequently, the cells were cultured in the incubator for 2 hours after the addition of 20 μL of MTS assay reagent to each well. Absorbance values at 490 nm were determined using a microplate reader (BioTek Epoch).

| Real-time quantitative PCR (RT-qPCR) analysis
Human bronchial epithelial cells were collected after pre-treatment with ESS as previously described. Gene expression levels of TNFα, IL-1β, IL-4, IL-6 and IP 3 R were determined via RT-qPCR, and the primer sequences are provided in Table 1. β-Actin was used as a housekeeping gene. Total RNA was isolated with TRIzol reagent, and cDNA was synthesized with a PrimeScript RT kit according to the manufacturer's instructions. RT-qPCR was performed with a SYBR Premix TaqTM Kit using a 7500 real-time PCR system (Applied Biosystem). After pre-denaturation for 30 seconds at 90 o C, RT-qPCR was performed as 40 cycles of denaturation for 5 seconds at 95°C and annealing for 34 seconds at 60°C. Relative target genes were quantified with the comparative CT (2 − ΔΔC t ) method and then compared with target gene expression in the control group. The gene expression of each target in each sample was measured in three independent experiments.

| Western blot
After pre-treatment with ESS for 1 hour, HBE cells were collected 24 hour later. Total protein was extracted using an Invent protein extraction kit (containing 1% PMSF), and the protein concentration was quantified using BCA kits. Equal quantities of protein (30-60 μg) were separated by 8%-12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked for 2 hours, followed by incubation with primary antibodies at 4°C overnight.
The membranes were washed three times and incubated with HRP-conjugated anti-rabbit antibodies (1:5000) for 1 hour at room temperature. An enhanced ECL substrate was used to measure the optical densities of the bands. β-Actin was used as the loading con-

| Statistical analysis
Statistical analyses in this study were performed with SPSS 17.0, and GraphPad Prism software was used to prepare figures.

TA B L E 1 Primer sequences used for RT-qPCR in this study
Student's t test was used to analyse the variance between experimental groups and the control group. One-way analysis of variance followed by the Student-Newman-Keuls (SNK) test was used for comparisons between multiple means. Sample data from independent experiments are shown as the mean ± standard deviation (SD).
Differences for which *P < .05 or **P < .01 were considered statistically significant. Subsequently, we used the Venn diagram website to determine the DEGs common to smokers and COPD patients, and finally obtained 30 common DEGs (Table 2 and Figure 1E).

| GO and KEGG pathway enrichment analyses of DEGs in smokers and COPD patients
The results of GO analysis and KEGG pathway related to the selected DEGs in tobacco smoke-induced COPD are shown in Table 3 and Figure S1.

| IP 3 R was decreased in COPD and ESS-treated HBE cells
To verify the differential expression of the IP 3 R gene, we analysed its mRNA levels in tobacco smoke-induced COPD lung samples and non-smoker control samples using a GEO data set (GES103174, 11739563_a_at). Figure 2A shows that the IP 3 R mRNA was significantly decreased in tobacco smoke-induced COPD patients compared with the controls (P < .05). In addition, we performed F I G U R E 1 Determination of DEGs among COPD patients and smoker samples. A and B, Volcano plots of DEGs between COPD patient and non-smoker control samples; C and D, Volcano plots of DEGs between smoker control and non-smoker control samples; E, DEGs common to COPD patients and smoker samples via a Venn diagram RT-qPCR analysis to further determine the IP 3 R mRNA level in peripheral blood samples from patients with COPD (who had smoked for more than 15 years) and volunteer non-smokers, which showed that the transcription of IP 3 R was also significantly reduced in the patients with COPD (P < .05, Figure 2B). Subsequently, we analysed both the mRNA expression and protein expression of IP 3 R in ESStreated HBE cells. The results in Figure 2C,D indicated that both the mRNA expression (P <.01) and protein expression (P <.05) of IP 3 R were markedly decreased after ESS exposure. In addition, microphotographs showed that the number of HBE cells was decreased and that the area of intercellular adhesion was thinner in the ESS-treated group compared with the control ( Figure 2E). Moreover, we found that ESS significantly decreased cell viability but increased ROS formation compared with the control group (both P <.01; Figure 2F-H).
These results showed that ESS exposure could cause HBE cell injury and decrease the transcription of IP 3 R.

| Silencing of IP 3 R enhanced oxidative stress and inflammation damage in ESS-treated HBE cells
To assess the effect of IP 3 R in ESS-treated HBE cells, the IP 3 R was silenced by lentiviral transfection. As shown in Figure 3A- with the control group (P < .05 or P < .01). Moreover, we detected that ESS exposure significantly elevated MDA but decreased levels of CAT, SOD and GSH-Px (P < .05 or P < .01), while si-IP 3 R further enhanced these functions. However, there were no significant differences in SOD and GSH-Px ( Figure 3D-G). Furthermore, as shown in

| Overexpression of IP 3 R decreased oxidative stress and inflammatory damage in ESS-treated HBE cells
Next, we overexpressed the IP 3 R gene by lentiviral transfection in si-IP 3 R HBE cells. The mRNA and protein expression levels of IP 3 R are shown in Figure 4A,B, respectively. The results demonstrated that both the mRNA and protein levels of IP 3 R were statistically increased in the overexpression (IP 3 R + ) group compared with the control group (P < .01). The fluorescence intensity was significantly lower in the IP3R+ group than that in the control group after treatment with ESS, which indicated that ROS formation was decreased with the overexpression of IP 3 R ( Figure 4C). As shown in Figure 4D, the MDA level was obviously decreased in the IP 3 R + group compared with the control group (P < .01). Furthermore, the CAT, SOD and GSH-Px activities were specifically increased in the IP 3 R + group compared with the control group (P < .05 or P < .01; Figure 4E-G).
Additionally, the relative gene expression levels of TNFα, IL-1β, IL-4 and IL-6 were all obviously decreased in the IP 3 R + group compared with the control group (P < .05 or P < .01; Figure 4H-K). These data implied that overexpression of IP 3 R could decrease oxidative stress and inflammatory damage in ESS-treated HBE cells.

| Aberrant expression of IP 3 R changed autophagy level in ESS-treated HBE cells
To further explore the role of IP3R in autophagy, we analysed the protein expression of Atg5, LC3, P62 and Beclin1 in ESS-treated HBE cells. Relative expression of the Atg5 and LC3 was markedly decreased in the si-IP 3 R group but increased in the IP 3 R + group compared with the control group (P < .05 or P < .01; Figure 5A,B).
In contrast, the relative protein expression of p62 was significantly increased in the si-IP 3 R group and decreased in the IP 3 R + group compared with the control group (P < .05 or P < .01; Figure 5C). Experiments were performed at least three times, and data are shown as the mean ± SD. *P < .05, **P < .01 compared with the control group; #P < .05, ##P < .01 compared with the single ESS-treated HBE cells Moreover, we found that the protein expression of Beclin1 was decreased in the si-IP 3 R group and increased in the IP 3 R + group; however, no significant differences were detected ( Figure 5D).

| Protective effects of IP 3 R on ESS-treated HBE cells were through regulation of autophagy
To further determine whether IP 3 R had protective effects in ESStreated HBE cells by regulating autophagy, the cells were pre-

| D ISCUSS I ON
Numerous studies have reported the possible related mechanisms of COPD. The pathogenic mechanisms of tobacco smoke were previously reported to be closely associated with the inflammatory response, oxidative damage and autophagy in the airway. 4,47,48 However, the most critical gene and protein in tobacco smokeinduced COPD remain poorly understood. Therefore, this study aimed to investigate variations in this hub gene and protein in tobacco smoke-induced COPD and further explore the role of this key target.
In recent years, bioinformatics analysis has been widely used to determine increasingly reliable and crucial biomarkers of diseases. [49][50][51] We analysed gene expression data from GEO data sets, to  Autophagy is a widespread activity in the inflammatory response of COPD, 29,30 and autophagy was shown to be induced by the relatively excessive accumulation of ROS. 61 In the present study, we observed variations in a series of autophagy-related proteins in ESStreated si-IP 3 R HBE cells and IP 3 R + HBE cells compared with control cells. However, the regulatory mechanism of IP 3 R on autophagy is still not obvious. A previous research has confirmed that IP 3 binds to IP 3 R on the ER and induces the release of intracellular Ca 2+ stores.
The high level of intracellular Ca 2+ activates calmodulin, thereby blocking autophagy. 62 In addition, it has reported that endoplasmic reticulum stress (ERS)-induced IP 3 R can lead to the occurrence of autophagy through different signal transduction pathways, but the mechanism of ERS that can cause autophagy through IP 3 R remains not obvious. 63 Therefore, we suspected that the effects of IP 3 R on ESS-treated HBE cells may be also due to the involvement of autophagy. To further verify our speculation, we investigated autophagyrelated proteins. An increase in Beclin1, LC3 and Atg5 indicates active cell autophagy. 64 Furthermore, p62, an autophagy adaptor protein, could mediate Keap1 inactivation and induce the accumulation of Nrf2, finally causing inflammation. 65 In the present study, as shown by variations in autophagy-related protein expression, the autophagy level was decreased in si-IP 3 R HBE cells but increased in IP 3 R + HBE cells compared with control cells after treatment with ESS. To further investigate whether the protective function of IP 3 R in HBE cells was achieved through autophagy, autophagy was inhibited in ESS-treated IP 3 R + HBE cells with an inhibitor. As shown in our data, the protective function of IP 3 R was decreased in the inhibitor group. Based on these results, we suspected that IP 3 R might play a protective role in ESS-induced inflammation and oxidative stress in HBE cells and that the function of IP 3 R might be achieved associated with the autophagy-related proteins. However, a more detailed upstream mechanism and the molecular targets associated with the regulation of IP 3 R remain not obvious. In addition, functional in vivo animal model, such as knockout animal experiments, should be performed to verify the antioxidant and anti-inflammatory properties of IP 3 R, as well as the regulatory mechanism of IP 3 R on autophagy.
In summary, the study demonstrated that IP 3 R had protective effects in ESS-treated HBE cells and suggests IP 3 R as a potential target to prevent tobacco smoke-induced COPD.

ACK N OWLED G EM ENT
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.