OTA was purchased from Merck (CAS No.: 303-47-9, Merck Co Inc. Kenilworth, NJ, USA). Western blotting primary antibody diluent buffer (Cat no. C05-07001) was purchased from Bioss Biotechnology Co., Ltd (Beijing, China). Tris Buffered Saline with Tween 20 (TBST, Cat no. T1081), PBS phosphate buffer (Cat no. 1022), Bovine serum albumin (BSA) blocking solution (Cat no. A8020), and BCA protein assay kit (Cat no. PC0020) were purchased from Beijing Solarbio company (Beijing, China). Rabbit monoclonal $\beta$-actin antibody (cat no: ab8226, 1:1500 dilution), anti-p16 antibody (cat no: ab189034, 1:2000 dilution), anti-p21 (cat no: ab109199, 1:1000 dilution), Ki67 (cat no: ab15580, 1:1000 dilution) and $\alpha$-SMA (cat no: ab5694, 1:1500 dilution) were purchased from Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). Polyvinylidene fluoride (PVDF) membrane (0.22 $\mu$m pore size, Cat no. GVWP09050) was obtained from Millipore (Boston, MA, USA). Fetal bovine serum (Cat no.12484028) and Dulbecco’s Modification of Eagle’s Medium (DMEM) culture medium (Cat no. 11965092) were purchased from Thermo Fisher Company (Shanghai, China). Mouse IL-1$\beta$ ELISA kit (Cat no. SEKM-0002) and mouse IL-6 ELISA (Cat no. SEKM-0007) were purchased from Solarbio Biotechnology Co., Ltd (Beijing, China). Total Superoxide Dismutase (T-SOD, Cat no. BC0170), MDA (malonaldehyde, Cat no. BC0025), and GSH (glutathione, Cat no. BC1175) kits were purchased from from Solarbio Biotechnology Co., Ltd (Beijing, China).

OTA was purchased from Merck (CAS No.: 303-47-9) (Shanghai, Chian). Western blotting primary antibody diluent buffer was purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). 10$\times$ TBST, PBS phosphate buffer (0.01 M pH 7.2–7.4), Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS‒PAGE) electrophoresis solution, BSA blocking solution, BCA protein assay kit, 5X SDS‒PAGE protein loading buffer, hematoxylin and eosin dye were purchased from Beijing Solarbio company (Beijing, China). Rabbit monoclonal $\beta$-actin antibody (cat no: ab8226, 1:1500 dilution), anti-p16 antibody (cat no: ab189034, 1:2000 dilution), anti-p21 (cat no: ab109199, 1:1000 dilution), Ki67 (cat no: ab15580, 1:1000 dilution) and $\alpha$-SMA (cat no: ab5694, 1:1500 dilution) were purchased from Abcam (Cambridge, UK). HRP-conjugated goat anti-rabbit IgG was purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). PVDF membrane (0.22 $\mu$m pore size) was obtained from Millipore (MA, USA). A protein extraction kit was purchased from Jiangsu Kaiji Biotechnology Co., Ltd (Nanjing, China). Toluidine blue was purchased from Beijing Biotechnology Co., Ltd (Beijing, China). Fetal bovine serum and DMEM culture medium were purchased from Thermo Fisher Company (Shanghai, China). Mouse IL-1$\beta$ ELISA and mouse IL-6 ELISA kits were purchased from Solarbio Biotechnology Co., Ltd (Beijing, China). A T-SOD, MDA, and GSH kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd (Nanjing, China).

TC-1 and MLE-12 murine lung epithelial cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in an incubator with a 5% CO${}_2$ atmosphere.

Cell density was observed and monitored under a microscope. When the cell density was greater than 75%, the medium in the cell culture flask was discarded and washed three times with normal saline. Cells were digested using trypsin and digestion, serum-containing DMEM medium was added and the cell suspension was inoculated into cell culture plates.

All animal procedures were reviewed and approved by the affiliated Hospital of Guangdong Medical University (IACUC-2210-A061). Forty (40) male C57 mice (body weight 20–22 g, 8 weeks old) were divided into a control group and three OTA groups with 10 mice in each group. Mice were treated with OTA for 30 days by gavage. In the control group the mice were treated with 0.1 mL olive oil by gavage every day, and then 0.1 mL of NaHCO${}_3$ was given 2 h later. In the OTA groups the mice were treated with 0.1 mL of olive oil by gavage every day, and then treated with low-dose (1 mg/kg), medium dose (5 mg/kg), or high-dose OTA (10 mg/kg) 2 h later.

Cells were washed three times with PBS, cell lysis buffer was added, and the plate placed on ice for 30 min. Cells were subsequently collected and transferred into 1.5 mL microfuge tubes. The protein lysates were centrifuged at 12,000 rpm for 15 min, and the cleared supernatant was transferred into another microfuge tube. 30 $\mu$g of protein was subjected to SDS‒PAGE, protein samples were electro-transferred to PVDF membranes. PVDF membranes were subsequently blocked in 5% skim milk at room temperature for 1 h. Primary antibodies were added and incubated overnight at 4 °C with shaking. After washing the membrane three times with TBST, secondary antibody diluted in 2% skim milk was added and incubated at room temperature for 2 h. After washing the membrane three times with TBST (10 min/times), enhanced chemiluminescence (ECL) solution was added to the PVDF membrane and protein bands visualized using a Bio-Rad ChemiDoc${}^{\text{TM}}$ XRS system (ImageLab developed by Bio-rad company, Hercules, CA, USA).

Following blood collection, the mice were sacrificed using the cervical dislocation method. The mice were subsequently fixed on a wooden board, and the abdominal and thoracic cavity were opened from bottom to top using surgical scissors. After opening the thoracic cavity, the intact lungs were carefully extracted and weighed on an analytical balance. Following this, the lungs were divided into two pieces, one piece was placed in 4% polymethanol tissue fixative for immunohistochemistry and the second piece was washed with sterile normal saline, after which it was blotted dry with filter paper and then placed in a cryopreservation tube and stored at –80 °C for subsequent detection of biochemical indicators and protein content.

Following dissection of whole mouse lungs, lungs were weighed, and a mouse lung coefficient was calculated per the formula: mouse whole lung weight (g)/mouse body weight (g) $\times$ 100%.

The lung tissues of mice in each group were fixed in 4% paraformaldehyde solution. The fixed tissues were dehydrated, embedded, and sectioned on a rotary microtome. The tissue sections were stained with hematoxylin for 10–20 min, rinsed with tap water for 1–3 min, and de-stained with hydrochloric acid alcohol for 5–10 seconds, after which the tissue sections were rinsed with tap water for 1–3 min. Finally, the tissue sections were placed into warm water followed by placing into 85% alcohol for 3–5 min, staining with eosin for 3–5 min, and dehydrated with alcohol. Finally, the slices were deparaffinized with xylene, mounted with neutral gum, and then placed under a microscope to observe the tissues.

Tissue sections were subjected to Masson staining according to the manufacturer’s instructions. In brief, tissue sections were stained with Weigert’s iron hematoxylin staining solution for 5–10 min, and sections were then soaked in a 0.2% glacial acetic acid aqueous solution for 5–10 min. The sections were de-stained using 1% aqueous phosphotungstic acid (3–5 min). After this, the sections were stained with toluidine blue for 5 min. After staining, the sections were washed with 0.2% glacial acetic acid aqueous solution for 5–10 min. After the sections were soaked and washed, they were deparaffinized with a series of 95% ethanol, absolute ethanol, and xylene. Finally, the sections were sealed with neutral gum and then observed under a microscope.

Tissue sections were blocked using 5% BSA in a –37 °C incubator for 1 h. After washing three times with PBS, the blocking solution was discarded, and the primary antibody, diluted in 3% BSA, was added dropwise until the tissue was covered. The tissue sections was incubated at –4 °C overnight and subsequently the tissues were washed three times with PBS. After this, a solution containing HRP-conjugated secondary antibody was added dropwise to the tissues and they were incubated at RT for 1 h. After three washes with PBS, DAB (3,3’-Diaminobenzidine Tetrahydrochloride) was added to the tissue, and tap water was used to stop the reaction when the sections were sufficiently developed, and subsequently observed under a microscope.

Tissue sections were blocked with 5% BSA and incubated in a 37 °C incubator for 1 h. After the blocking solution was removed, primary antibody diluted in 5% BSA was added dropwise to cover the tissues, and the sections were incubated at 4 °C for overnight. After washing with PBS three times, fluorescently-labeled secondary antibody diluted in 3% BSA was added dropwise, and the tissue was covered and incubated in a 37 °C incubator for 2 h. After three washes with PBS, the tissue samples were mounted with glycerol and the tissues were examined by fluorescence microscopy.

Cells in logarithmic growth were seeded in 6-well cell culture plates, and OTA was added and incubated at different time points. After incubation, the medium was discarded, the cells were washed three times with PBS, and collected into centrifuge tubes. After the samples were centrifuged, the supernatant was discarded, and cells were sonicated. The supernatant of the cells was placed in a 96-well plate, and various reagents were added in sequence according to the manufacturer’s instructions. The samples were then tested for indicated oxidative stress indicators using commercial SOD (Cat no. BC0170, Solarbio Science & Technology Co., Ltd, Beijing China), MDA (Cat no. BC0025, Solarbio Science & Technology Co., Ltd, Beijing, China), and reactive oxygen species (ROS, Cat no. S0033S, Beyotime Biotechnology company, Shanghai, China) kits.

Lung cells in the logarithmic growth phase were plated in 96-well cell culture plates at a density of 1.5 $\times$ 10${}^4$ cells/well, and cultured for an additional 24 h. Following this, indicated concentrations of OTA were added and the cells further incubated for 24–48 h. After this the cells were washed, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added (final concentration: 0.5 $\mu$g/mL), and the plates incubated for 4 h. After the addition of DMSO (Dimethyl sulfoxide), absorbance was measured at 450 nm using a microtiter plate reader (ST-360, Thermo Fisher, Houston, TX, USA).

Cells in the logarithmic phase were seeded into 6-well cell culture plates, indicated concentrations of OTA were added, and the cells were incubated for 24 h. After this, the supernatant was discarded, cells were washed with PBS, 1 mL of PFA was added to each tube, and the cells were fixed for 15 min at RT (room temperature). Following this, the fixative was discarded, and the cell samples were washed with PBS. After adding 1 mL of staining working solution to each well, plates were covered in parafilm and incubated overnight in a 37 °C incubator. Five fields of view were randomly selected for each sample and ImageJ software (version: 1.8.0, developed by National Institutes of Health, LOCI, University of Wisconsin, Madison, WI, USA) was used to count senescent cells.

Freshly prepared lungs were homogenized with a tissue homogenizer. The homogenate was placed in a benchtop refrigerated centrifuge and centrifuged at 3000 rpm for 20 min. The supernatant was collected after centrifugation. TNF-$\alpha$ (PT513, Solarbio Science & Technology Co., Ltd, Beijing, China), IL-1$\beta$ (SEKM-0002, Solarbio Science & Technology Co., Ltd, Beijing, China) and IL-6 (SEKM-0007, Solarbio Science & Technology Co., Ltd, Beijing, China) assays were performed according to the instructions of the ELISA kit.

Data are expressed as the mean $\pm$ standard deviation. Statistical analysis was performed using SPSS 22.0 software (IBM Corp., Chicago, IL, USA). All data were analyzed by ANOVA and multiple comparisons using Fisher’s Least Significant Difference (LSD) test. p 0.05 is used to indicate a significant difference; p 0.01 indicates an highly significant difference.

Previous studies found that OTA causes cytotoxicity [18]. However, in the current study, we analyzed the effect of OTA on lung cell. MTT assays were used to determine the effect of different concentrations of OTA on the viability of cultured lung cells. The results indicated that the effect of OTA on the viability of lung cells was not apparent at low OTA concentrations. However, when OTA concentration was increased, cell viability was significantly decreased (Fig. 1A). Based on these MTT assay results, we selected three concentrations of OTA, 1, 5 and 10 $\mu$M, for use in the outlined experiments.

Fig. 1.

OTA induced Lung Cell Senescence. (A) Effect of OTA treatment on cell viability. (B) OTA treatment increased ratio of SA-$\beta$-Gal-positive ratio. (C) OTA treatment reduced the expression of Ki67. (D) OTA caused cell cycle arrest. Different lowercase letters indicate significant differences (p 0.05).

Senescence-associated $\beta$-galactosidase (SA-$\beta$-Gal) is one of the most important markers of senescent cells. As shown in Fig. 1B, with increasing OTA concentration, the percentage of Sa-$\beta$-gal-positive cells increased. Furthermore, the expression of Ki67 was also significantly downregulated (Fig. 1C). Moreover, cell cycle distribution showed that OTA resulted in significant cell cycle arrest (Fig. 1D).

P16 and p21 are cell cycle regulatory proteins, and the expression of these proteins is significantly upregulated in senescent cells. In the current work, the expression of p16, p21, and p53 was evaluated by western blotting. The results showed that OTA treatment increased the protein expression of each of these proteins compared to the control treated cells (Fig. 2).

Fig. 2.

Effect of OTA on the expression of senescence-related marker molecules. Effect of OTA on p16/p21/p53 protein expression in TC1 (A) and MLE-12 (B). The protein samples were subjected to SDS‒PAGE, Western-blot analyses were them performed. Different lowercase letters indicate significant differences (p 0.05).

We next evaluated the effect of OTA on oxidative stress. We found that the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the cells were significantly increased compared with those in the control group. In contrast, the levels of SOD, CAT and GSH were significantly downregulated. Compared with the control group, 1 $\mu$M OTA increased ROS/MDA levels to 12.8% and 21%; 5 $\mu$M OTA increased ROS/MDA levels to 26.8% and 37%; and 10 $\mu$M increased ROS/MDA levels by 62.5% and 74.5%, respectively (Fig. 3A). In addition, these various concentrations of OTA significantly decreased the levels of SOD, GSH and CAT. Furthermore, OTA significantly downregulated the expression of Nrf2 (Nuclear Factor erythroid 2-Related Factor 2), HO-1 (heme oxygenase 1) and MnSOD (manganese superoxide dismutase) (Fig. 3B).

Fig. 3.

Effect of OTA on oxidative stress. (A) Effects of OTA on oxidative stress (ROS, MDA, SOD, CAT and GSH) on TC-1 and MLE-2 cells. Cells in the logarithmic growth phase were seeded in 6-well cell culture plates, and OTA (1, 5 and 10 $\mu$M) was added, respectively. (B) Effects of ochratoxin A on Nrf2, HO-1 and MnSOD protein expression. The protein samples were subjected to SDS‒PAGE, after which the protein samples were transferred to PVDF membranes. After western-blot, Blots were exposed using the Bio-Rad ChemiDocTM XRS system. Different lowercase letters indicate significant differences (p 0.05).

We investigated the effect of OTA on the secretion of senescence-associated secretory phenotype (SASP) factors in cultured lung cells. SASP is one of the key phenotypic features of senescent cells, and includes proinflammatory factors, such as IL-6, IL-1$\beta$ and TNF-$\alpha$, CXCL1 chemokine, and MMP-1 metalloproteinase. Here, we analyzed the effect of OTA on inflammation in lung cells, and the results showed that the expression of each of these inflammation-related factors was significantly increased, indicating that OTA increased the inflammation of lung cells (Fig. 4).

Fig. 4.

Effects of OTA on lung cell inflammation. ELISA kit (TNF$\alpha$, IL-6) was used to detect the effect of OTA on lung cell inflammation according to the kit instructions (n = 5). Different lowercase letters indicate significant differences (p 0.05).

As shown in Fig. 5A, the average weight of mice in each of the OTA-treated groups was lower than that in the control group. Specifically, the average weight of mice in the 5 mg/kg and 10 mg/kg OTA groups was significantly reduced compared with the control group. Further, as indicated in Fig. 5B, the lung weight/body weight ratio in the OTA-treated groups was much higher than that in the control group.

Fig. 5.

Effects of OTA on body weight and lung coefficient. (A) Effect of OTA on average body weight (n = 10). (B) The ratio of lung weight/body weight was increased in the OTA group (n = 10). Different lowercase letters indicate significant differences (p 0.05).

OTA-induced changes in mouse lung tissues were observed by Hematoxylin and Eosin (H&E) staining. In the control group, the structure of the bronchial branches in the lungs at all levels was typical, the bronchial ciliated epithelial cells in the lungs were neatly arranged, and there was no apparent inflammatory infiltration. In the low-dose OTA treatment group, the structure of the bronchial branches in the lungs was typical, and mild alveolar inflammation was observed in the lungs. In the moderate and high dose OTA groups, moderate alveolar inflammation was observed in the lungs, with inflammatory infiltration of many macrophages and lymphocytes. As illustrated in the histograms, inflammatory cells in the lungs of mice in the high-dose OTA group covered 30% of the total lung area. This may potentially lead to functional necrosis of the lungs (Fig. 6).

Fig. 6.

Analysis of lung tissue by HE staining. The lung tissues of mice in each group were fixed in 4% paraformaldehyde solution. The sections were stained with hematoxylin. Different lowercase letters indicate significant differences (p 0.05).

We next analyzed the effects of OTA on lung tissue aging. Sa-$\beta$-gal staining showed that staining was significantly increased, indicating that the lungs displayed significant senescence (Fig. 7A). Further, the expression of p16 and p21 was also significantly increased compared to that in the control group (Fig. 7B). and the expression of the proliferation marker Ki67 was increased (Fig. 7C). These findings suggest that OTA induces lung aging.

Fig. 7.

Effect of OTA treatment on lung tissue aging. (A) Analysis of lung tissue aging by Sa-$\beta$-gal staining. (B) The effect of OTA on p16 and p21. (C) Effect of OTA on Ki67. The integrated optical density (IOD) values of each slide (tissue) was obtained with the software Image-Pro Plus 6.0. Different lowercase letters indicate significant differences (p 0.05).

Fibrosis is also an important manifestation of organ aging [19]. Masson staining showed that the bronchial branches of the lung in the control group were relatively normal with no alveolar wall or bronchial wall fibrosis evident, nor was obvious damage to the lung tissue structure observed. Moderate pulmonary fibrosis, alveolar wall, and bronchial wall fibers were observed in the OTA group. The degree of collagenization was also found to be increased, and collagen fibers were raised significantly (Fig. 8A). Further study showed that the expression of $\alpha$-SMA (alpha smooth muscle actin) was clearly elevated compared with that in the control group (Fig. 8B).

Fig. 8.

Effect of OTA on Lung Fibrosis. (A) Analysis of fibrosis in lung tissue by Masson staining. (B) Effect of OTA on $\alpha$-SMA expression. The protein samples were subjected to SDS‒PAGE and Western-blot. Blots were exposed using the Bio-Rad ChemiDocTM XRS system. Different lowercase letters indicate significant differences (p 0.05).

As shown in Fig. 9A, the MDA and ROS levels in the control group were much lower than those in the OTA-treated groups. Additionally, Fig. 9B illustrates that the SOD content in the control group was much higher than that in the OTA group. Similarly, the contents of GSH and CAT in the control group were much higher than those in the OTA group. These findings indicated that OTA exposure results in severe oxidative stress. We further analyzed the changes in Nrf2, HO-1 and MnSOD expression in mice lungs in vivo. As illustrated in Fig. 9C, compared with the control group, the expression of Nrf2 in the OTA group was obviously lower as were the expression levels of HO-1 and MnSOD in OTA-treated mouse lungs when compared to the control group.

Fig. 9.

Effect of OTA on oxidative stress. (A,B) Effects of OTA on oxidative stress-related protein expression. (C) Effects of OTA on the expression of Nrf2, HO-1 and MnSOD. The protein samples were subjected to SDS‒PAGE and Western-blot. Blots were exposed using the Bio-Rad ChemiDocTM XRS system. Different lowercase letters indicate significant differences (p 0.05).

We determined the effect of OTA on lung inflammation by measuring inflammatory markers such as IL-1$\beta$, IL-6 and TNF$\alpha$. As shown in Fig. 10A, when OTA-treated mice were compared with those in the control group, the levels of TNF$\alpha$, IL-1$\beta$ and IL6 in the OTA group were found to be clearly elevated. In addition, histochemical analysis showed that the inflammatory damage caused by OTA was evident (Fig. 10B).

Fig. 10.

OTA exposure induced Lung Inflammation. (A) Effects of OTA on IL-1$\beta$, IL-6 and TNF$\alpha$. Histochemical analysis of IL-1$\beta$ and IL-6 expression. (B) Effects of OTA on the TLR4/MyD88/NF-$\kappa$B signaling pathway protein expressions. The protein samples were subjected to SDS‒PAGE and Western blot. Blots were exposed using the Bio-Rad ChemiDocTM XRS system. Different lowercase letters indicate significant differences (p 0.05).

Additionally, TLR4, MyD88 and NF-$\kappa$B expressions were analyzed. Fig. 10C indicates that the expression of TLR4 in the OTA-treated group was higher than that in the control group, and, similarly, the expression levels of MyD88 and NF-$\kappa$B were much higher than the levels observed in the control group.