Swimming training reduced inflammation and apoptotic changes in pulmonary tissue in type 1 diabetic mice

Abstract

Background

Despite the vulnerability of pulmonary tissue to diabetic conditions, there are few reports related to the detrimental effects of hyperglycemia and therapeutic modalities on lung parenchyma. Here, the apoptotic changes were monitored in the diabetic pulmonary tissue of mice (DM1) subjected to a four‒week swimming plan.

Methods

The mice were randomly allocated into Control; Control + Swimming (S); Diabetic group (D); and Diabetic + Swimming (D + S) groups (each in 8 mice). In the D and D + S groups, mice received intraperitoneally 50 mg/kg of streptozotocin (STZ). After 14 days, swimming exercise was done for four weeks. The expression of il-1β, bcl-2, bax, and caspase-3 was investigated using real-time PCR analysis. A histological examination was performed using H&E staining.

Results

DM1 significantly upregulated il-1β, bax, and caspase-3, and down-regulated bcl-2 compared to the non-diabetic mice (p < 0.05). We noted that swimming exercises reversed the expression pattern of all genes in the diabetic mice and closed to basal levels (p < 0.05). Data indicated that swimming exercise could diminish emphysematous changes, and interstitial pneumonitis induced by STZ. Along with these changes, swimming exercise had protective effects to reduce the thickness of the inter-alveolar septum and mean alveolar area in diabetic mice.

Conclusion

These data demonstrated that swimming exercises could decrease DM1-related pathologies in mouse lungs by regulating apoptosis and inflammatory response.

Introduction

Diabetes mellitus (DM) is initiated due to abnormal insulin function and can affect a lot of people worldwide [1, 2]. During the diabetic changes, pancreatic β cells lose the ability to produce and secret insulin meanwhile the affinity of other cell types for insulin is reduced [3, 4]. To date, both type 1 (DM1) and type 2 (DM2) have been described in the clinical setting with several complications. In DM1, pancreatic β cell failure leads to abnormal insulin production, while DM2 is recognized with the progressive resistance of the body’s cells to insulin [5, 6]. Of note, several clinical studies have reported the detrimental properties of diabetic conditions on pulmonary tissue [7, 8].

Systemic inflammatory response, vasculitis, oxidative stress, and deposition of collagen fibers are the main pathological outcomes within the pulmonary tract [7, 9, 10]. For example, it was suggested that serum levels of cytokines such as CRP, TGF-β1, and TNF-α were increased in bronchial alveolar lavage (BAL) fluid after the onset of diabetic conditions [11]. Along with these changes, the activation of pro-apoptotic factors like Caspase-3, Bax, and BCL-2 can intensify pathological injuries within the pulmonary tract in rats with DM1 [11, 12]. It is suggested that the local elevation of TGF-β1, TNF-α, ICAM-1, and VCAM-1 can lead to prominent interstitial bronchopneumonitis following DM1 and 2 [12, 13].

With a variety of medicinal compounds used for the alleviation of DM-related injuries, physical activity especially exercise is a noteworthy means to prevent and/or reduce the severity of different pathologies like DM [14]. Data have confirmed the interventional role of exercise in the suppression of apoptotic changes following several pathological conditions [15–19]. For instance, Moradi and collaborators proved the synergistic therapeutic effect of swimming exercises and Crocin, a phyto-carotenoid compound, in the reduction of myocyte apoptosis in obese rats [20]. Programmed swimming protocols can regulate the function of hepatic and cardiac tissues by the reduction of plasma inflammatory factors in diabetic patients [21–24]. Because of the lack of comprehensive insight regarding putative impact of programmed exercise on diabetic lungs, we, here, explored the protective impacts of swimming training on apoptotic response in DM1 mice.

Materials and methods

Animal and ethical issues

Adult male BALB/c mice (32 mice), weighing 25–30 g, were accommodated in the Animal House of Drug Applied Research Center (affiliated with Tabriz University of Medical Sciences) under recommended standard conditions. All protocols and manipulations used in this study were under the supervision of the Local Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1399.005). After seven days, male mice were randomly allocated to different experimental groups as follows; Control (C); Swimming (S); Diabetic (D), and Diabetic + Swimming (D + S) groups.

Induction of DM1

To induce DM1, 8-hour fasted mice received a single dose of 50 mg/Kbw STZ (dissolved in 0.1 mM Na₃C₆H₅O₇) via the intraperitoneal route. Three days after induction, the glucose contents were determined using a glucometer strip. Mice with glucose higher than normal concentrations (X ≥ 250 mg/dl) were selected and subjected to the experimental procedure [25, 26].

Swimming exercise

Using a water-filled swimming tank (100 cm × 150 cm) and a temperature of 24–26°C, a programmed exercise was done in the respective groups. On the 1st day of the swimming exercise, mice were put in a tank for 10 minutes and this period was increased to 60 minutes until day 6. Two weeks after the induction of DM1, the swimming program was applied for the duration of 4 weeks [1h/day for 3 days in each week]. Mice were subjected to swimming exercise 9:00 a.m. to 12:00 p.m. After completion of the swimming exercise (on week 6), the mice from different groups were sacrificed as recommended previously (Figure. 1) [16]. To see the detrimental effects of diabetic condition in lungs, we allowed the animal to experience these conditions.

Fig. 1.

Brief description of the experimental procedure in this study

Histological examination

Right lungs were placed in 10%formalin solution for 24 hours. Using the ascending concentration of ethanol from 70 to 100%, tissues were dehydrated and paraffin-embedded. For histological examination, 4-µm thick slides were stained with H & E solution. The abnormal tissue remodeling was monitored in each group [27]. The thickness of the inter-alveolar septum and mean alveolar area were measured using appropriate software (ImageJ; ver. 1. 4; NIH) in 20 random high-power fields.

Monitoring apoptosis and inflammation in diabetic lungs

bcl-2, bax, caspase-3, and il-1β transcripts were followed in left lung tissue using conventional real-time PCR analysis [28]. After the extraction of RNAs from different samples (RNA extraction kit; Cat no: YT9065), cDNAs were synthesized (Cat no: Cat No.YT4500) [29]. Primers were designed using Oligo7 software. According to standard protocols, SYBR Green qPCR Master Mix and Rotor-Gene Q system PCR reaction were done. The raw CT data were calculated using the 2^-∆∆Ct method and compared to the expression of gapdh [30].

Statistical analysis

Using the Tukey test with One-Way ANOVA (GraphPad Prism software; ver. 5), statistical analysis was performed. P values < 0.05 were considered statistically significant. Experiments were set at least three times otherwise mentioned.

Results

Swimming exercise decreased inflammation in DM1 mice

We monitored the expression of il-1β in diabetic lungs before and after swimming exercise (Fig. 1). It was suggested that the expression of IL-1β was increased in D and D + S groups compared to the non-diabetic lungs (p < 0.001). The transcription rate was reduced in diabetic mice after swimming exercise compared to the diabetic mice (D group) (p < 0.05; Fig. 2).

Fig. 2.

The levels of IL-1β mRNA expression in the lung tissues of control animals (C group), Swimming (S group), diabetic animals (D group), diabetic + Swimming (D + S group) (for each group, n = 8). Bars represent the mean ± SEM. Statistical differences between control and diabetic groups: +++; p < 0. 001. Statistical differences between D group and D + S group:*; p < 0. 05

Swimming exercise decreased apoptosis in DM1 mice

We also monitored the activity of bcl-2, bax, and caspase-3 genes in diabetic lungs before and after swimming exercise (Fig. 3). Data indicated that the induction of DM1 can significantly up-regulate pro-apoptotic genes bax, and caspase-3, and increase the BAX/BCL-2 ratio (Fig. 2). By contrast, transcription of an anti-apoptotic gene, bcl-2, was reduced in diabetic lungs related to non-diabetic samples (Fig. 3). As expected, the expression of pro- and anti-apoptotic genes was not significantly altered in the S group related to non-diabetic samples (Fig. 3). Data confirmed that a 4-week swimming exercise can reduce the activity of bax, and caspase-3 genes in the D group compared to the diabetic mice without programmed exercise (p < 0.05). Similar to these changes, the BAX: BCL-2 ratios were diminished in D + S group in comparison with the D group (Fig. 3). However, we found non-significant differences between the lung samples belonging to mice from C and S groups (Fig. 3). It was notified that swimming exercise had the potential to increase the expression of BCL-2 related to diabetic lungs (p < 0.001; Fig. 3). Again, the changes caused statistically significant results in the D + S group related to the non-diabetic samples. These data demonstrated that the application of swimming exercise in DM1 mice can return, in part, the expression of apoptosis-related genes reaching near-to-normal levels.

Fig. 3.

Measuring the transcription of Bax (A), Bcl-2 (B), Caspase-3 (C), and Bax/Bcl-2 ratio (D) mRNA in the lung tissues of control animals (C group), Swimming (S group), diabetic animals (D group), diabetic + Swimming (D + S group) (for each group, n = 8). Bars represent the mean ± SEM. Statistical differences between control and diabetic groups: +++; p < 0. 001. Statistical differences between the D group and DM group:*; p < 0.05,**; p < 0.01 and ***; p < 0.001

Table 1.

List of primers used in this study

Gene	Sequence	ACCESSION Number	Tm
Bcl-2	5’-CCTGTGGATGACTGAGTACCTG-3’ 5’-AGCCAGGAGAAATCAAACAGAGG-3’	NM_009741.5	61
IL-1β	5’-TGGACCTTCCAGGATGAGGACA-3’ 5’-GTTCATCTCGGAGCCTGTAGTG-3’	NM_008361.4	63
Bax	F: 5’-TGGCAGCTGACATGTTTTCTGAC-3’ R: 5’-TCACCCAACCACCCTGGTCTT-3’	NM_007527.4	63
Casp3	F: 5’-GGACAGCAGTTACAAAATGGATTA-3’ R: 5’-CGGCAGGCCTGAATGATGAAG-3’	NM_009810.3	60
GAPDH	F: 5’-AACTTTGGCATTGTGGAAGG-3’ R: 5’-ACACATTGGGGGTAGGAACA-3’	NM_001289726.2	60

Swimming exercise blunt pathological changes in DM1 lungs

Histological analysis showed that the onset of DM1 caused slight to moderate interstitial pneumonia indicated by thickened alveolar septa (Fig. 4A-B). Diabetic conditions directed the immune cells into the lung niche compared to the control mice. Several emphysematous foci were indicated in diabetic samples along with the increase in mean alveolar thickness (Fig. 3A-B). According to our data, 4-week swimming exercise can blunt these effects. We found no prominent changes in the mouse lung samples belonging to the S group related to non-diabetic lungs. These data indicated that 4-week swimming exercise could reduce pathological changes in pulmonary tissues in DM1 mice.

Fig. 4.

Bright-field images from lung tissue sections stained with H&E solution. The diabetic condition led to the generation of interstitial pneumonitis (yellow arrows) and emphysematous foci (red asterisks). The analysis revealed that the mean intra-alveolar septum thickness and alveolar area increased under the diabetic condition. These conditions were reversed in mice subjected to swimming training. Bars represent the mean ± SEM. Statistical differences between control and diabetic groups: +++p < 0.001. Statistical differences between the D group and the DM group: ***p < 0.001

Discussion

DM is a complex disease that leads to systemic hyperglycemia due to insulin deficiency or resistance [31]. Some data pointed to the fact that the lungs are susceptible to metabolic disorders like DM1 and 2 [13, 32, 33]. The apoptotic changes after the inflammatory response are the most complication of hyperglycemic conditions in lung parenchyma [16, 34, 35]. Local accumulation of cytokines like TNF-α, IL-1β, IL-6, and NF-can lead to progressive apoptosis in pulmonary tissue in diabetic rats [36, 37].

To date, the therapeutic effects of programmed exercise have not been evaluated on the pulmonary tissue under DM. Here, the anti-apoptotic effects of swimming exercise were examined in the lungs of diabetic rats. Current data indicated that the promotion of DM1 can alter the activity of several genes involved in apoptosis and pro-inflammatory responses. The induction of il-1β, caspase-3, and bax and down-regulation of bcl-2 indicated enhanced inflammatory status and apoptotic response in DM1 pulmonary tissues. It was suggested that a 4-week swimming exercise in DM1 mice reverted the expression pattern of the above-mentioned factors and closed to basal levels. Consistent with current data, Kanter et al. previously declared that low-intensity physical activity can reduce the generation of singlet oxygen, free radicals, and apoptotic changes in the hearts of diabetic rats after 4 consecutive weeks [38]. They also indicated that exercise promotes enzymes associated with antioxidant system. Likewise, the intracellular content of malondialdehyde and vacuoles were reduced in diabetic rats subjected to low-intensive exercise. It seems that the combination of swimming exercise with phytocompounds can contribute to better regenerative outcomes during diabetic conditions [39]. In support of this notion, Kermanshah and co-workers indicated the reduction of apoptotic hepatocytes in male diabetic rats subjected to regular exercise and resveratrol administration [39]. Similarly, the simultaneous application of continuous training and Crocin led to the suppression of apoptotic changes in diabetic rat hepatocytes after consumption of a high-fat-diet regime [40]. Besides the protective properties of exercise on the activity of pro-apoptotic factors, anti-apoptotic genes are induced under diabetic conditions subjected to continuous and programmed exercise [41]. In an experiment, it was indicated that the number of apoptotic cardiomyocytes is reduced in diabetic rats via the induction of the pro-apoptotic gene Bcl-2 and stimulation of the IGFI-R/PI3K/Akt axis [42]. In line with the previous findings, we noted that programmed exercise could reduce detrimental properties of DM1 on mouse lungs by reduction of pathological remodeling. The intensity of hyperemia, interstitial pneumonitis, atelectasis, and emphysema is diminished in DM1 lungs after 4-week swimming exercise. This experiment faces some imitations that need further attention. Monitoring protein levels of apoptosis-related factors in association with the DM signaling pathway should be assessed in varied animal model types. In conclusion, the use of swimming exercises can reduce apoptosis in the lungs of DM1 mice, leading to the reduction of DM1-related pathological changes.

Authors Contribution

N.A., A. R., F.M.B., R.R., A.G.N., J.R., and A.D. performed all analyses and prepared draft. M. A. supervised the study.

Funding

This work was supported by a grant from Molecular Medicine Research Center of Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1399.005).

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the Local Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1399.005).

Consent for publication

None declared.