Volume 28, Issue 4 (7-2021)                   RJMS 2021, 28(4): 1-12 | Back to browse issues page

Ethics code: IR.TBZMED.VCR.REC.1397.389
Clinical trials code: IR.TBZMED.VCR.REC.1397.389

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Jafari A, Zarghami khameneh A, Nikookheslat S, Karimi P. The effect of caffeine supplementation combined with high-intensity interval training on the levels of the cardiac tissue apoptosis-related proteins in diabetic rats. RJMS 2021; 28 (4) :1-12
URL: http://rjms.iums.ac.ir/article-1-6330-en.html
University of Tabriz, Tabriz, Iran , ali.zarghami64@gmail.com
Abstract:   (1815 Views)
Background & Aims: The prevalence of type 2 diabetes (T2DM) is a major public health problem that is approaching epidemic proportions globally (1). T2DM is associated with severe arteriosclerosis, hypertension and etc. All of which further contribute to the development of cardiovascular disease that the development of diabetic cardiomyopathy (DCM) involves mitochondrial dysfunction (2). In this regard, Fang-Yuan et al (2018) showed that cardiac dysfunction elong with anti-apoptotic proteins (Bcl-2) downregulation, while levels of pro-apoptotic proteins  (Bax, Cas-3) were upregulated in rats with long-term diabetic cardiomyopathy (3). However, a number of evidence suggests that apoptotic processes may be affected by some pharmacological and oral interventions. In this regard, several epidemiological and experimental studies have reported that caffeine may have ability to suppress cell proliferation and induce apoptosis via regulation some oncogenic signalling pathways, including the phosphatase and tensin homolog (PTEN), PI3K/Akt, p53 and mammalian target of rapamycin (mTOR) pathways (5, 6).  For example, Hanyang Liu et al (2017) have found that caffeine treatment significantly up-regulates mRNA expression levels of PTEN and p53 proteins, increased the activation of caspase-9 and -3, and increased the expression levels of Cyt-c in on human gastric cancer cells (6). On the other hand, Rahimi et al (2018) have reported that ingestion of caffeine reduced in the expression of Bax and increased Bcl-2 serum protein levels immediately after resistance training (8). Whereas, Moradi et al (2019) showed 8-weeks high-intensity  interval  training  (HIIT) at the  intensity of 80-85% of the maximum speed could significantly increase Bcl-2 and decrease Bax on diabetic rats (12).
In this regard, this study aimed to investigate the effects of 8-weeks of caffeine administration alone and in combination with HIIT intervention on the levels of some proteins in the mitochondrial pathway of apoptosis i.e. Bax, Bcl-2, Caspase-3 in the myocardium of diabetic rats as a sensitive somatic tissue to cell death.
Methods: The present study is a type of animal experiments clinical intervention that was conducted in the form of a two-factor post-test control group designs. Fifty white male Wistar rats (two-month-old with a mean body weight of 225-300 g) were acclimated in a specific laboratory setting and then, at the end of the period (acclimatization), the subjects were randomly divided into five groups (n=10 per group) including: Healthy control (C), Diabetic control (D), Diabetic with trainining (D+T), Diabetic with caffeine (D+CA), Diabetic with training and caffeine (D+T+CA). Type 2 diabetes was induced two weeks after the animals became acclimatization with the method used in a study by Sasidharan et al (2013), so that the studied animals became diabetic after two weeks of high-fat diet (45% fat) and intraperitoneal injections of single dosage of 35 mg.kg-1 body weight of STZ. After one week of the diabetic procedure (14), blood glucose samples were collected and levels more than 300 mg.dl were used as type 2 diabetic animals (12). In addition, hydrated caffeine (saline solution) was intraperitoneal injection in D+CA and   D+T+CA groups during a period of 8 weeks, 5 days a week, using insulin syringe (70 mg.kg-1 b.w per day, approximately equivalent to 14 mg of caffeine per 200 grams of rats b.w) (16). Caffeine was administered to rats during their waking hours and early in the activity period between 18-19 pm (60 min before training). The animals in D+T and D+T+Ca groups, for 8 weeks and 5 d wk-1 in the form of high-intensity interval training (6 to 13 bouts of 2-min-1 at a 0 degree slope with intensity of 85-90% of the maximum running speed followed by a 1-min-1 rest between bouts) run on animal treadmill during hours 19-20 pm (15). Finally, all rats were deeply anesthesia with ketamine HCl (90 mg.kg-1) and xylazine (10 mg.kg-1) intraperitoneal injection following the 48-hours after the last training session and after 12-14 hours fasting. We used an immunoblotting assay to measure the levels of the some proteins involved in apoptosis pathways (Bax, Cas-3, Bcl-2), following the Santa Cruz manufacturer’s instructions (17). Data were analyzed by of one-way ANOVA followed by Tukey post hoc test in significance level of P≤0.05.
Results: The results showed that levels of pro-apoptotic protein level (Bax and Cas-3), in diabetic control (D) was (∆=94% & ∆=618%), in diabetic with caffeine (D+CA) was (∆=106% & ∆=951%) and in diabetic with training and caffeine (D+T+CA) was (∆=221% & ∆=1071%) more than healthy control group (C) respectively (F=60.37; P=0.001 & F=2102.37; P=0.001). On the one hand, the level of this proteins in the D+T group was about (Bax=  ∆=-81% & Cas-3= ∆=-423%) lower than that in the D group (P=0.038). Moreover, the Cas-3 protein expression in D+CA group was about 333% significantly higher than the D group (P=0.001) (Fig.1&3). Furthermore, Bcl-2 protein level in D group was significantly lower than healthy C group (∆=-37%) (P=0.001). However, the levels of this protein in D+CA and D+T+CA were about (∆=-64%) and (∆=-70%) lower than those of the C group, respectively (P=0.001). Also, Bcl-2 protein in the D+T group was insignificantly lower than (∆=-20%) the D group (P=0.32) (Fig.2).
Conclusion: In line with the results of the present study, Fang-Yuan et al (2018) showed induction of diabetes caused significant decrease of Bcl-2 gene expression and an increase in Bax and p53 gene expression in heart tissue of diabetic rats (3). It has been well established that diabetic-mediated apoptotic cell death effect on mitochondrial outer membrane permeabilization (MOMP) leading to the irreversible release of intermembrane space proteins (e.g. Bid, Bim, Bad and Bax), thereby activating the caspase cascade (18).
However, caffeine administration for two months leads to an significant increased in apoptotic markers by exacerbation of pro-apoptotic proteins expression of Bax and Caspase-3 and reduction in anti-apoptotic Bcl-2 protein. These findings were supported by Hanyang Liu et al (2017), indicated that caffeine treatment increased the activation of caspase-9 and -3 (cleaved caspase-9 and -3), and increased the expression levels of Cyt-c and Bax proteins (6). Researchers suggested the caffeine triggered intrinsic apoptotic pathways via; inactivation of PI3K-Akt-mTOR cell survival signaling pathways and activation of cell death such as p21-activated protein kinase 2 (PAK2) and N-terminal and C-Janus kinases (JNK), thereby increased levels of Bax and cleaved caspase 3 and phosphorylating of Bcl-2 protein tha cause initiation of apoptotic events (9-11). In contrast of this paper, Rahimi et al (2020) reported that the caffeine intake decresed Bax protein and significantly increaesd Bcl-2 levels after acute resistance exercise protocol (8). Though, the protective effects of exercise training on apoptosis have been well established. For example, Moradi et al (2019) showed that HIIT training (80-85% maximum speed) could significantly increase Bcl-2 and decrease Bax and p53 in muscle tissue of the diabetic rats (12). Nonetheless, contrary to the results of these studies, some studies also Shirpour  Bonab  et al (2017) conclusion that the Bax expression was higher in HIIT intervention after 8-weeks  in aged female (27). While, the intervention of 8-weeks HIIT alone in the present study reduced the expression of Bax and Caspase-3 proteins and increased the Bcl-2 in myocardial tissue.
In other words, HIIT training ameliorates myocardial apoptosis. while, the combination of two independent variables in present study (e.g caffeine with HIIT) even induced deterioration the apoptotic cell death process in the myocardial tissue of diabetic rats. Perhaps the combination of caffeine with strenuous or prolonged exercise training  can result increased tissue damage during contractile activity along with an increase in ROS levels, calcium release from sarcoplasmic reticulum and increases in concentrations of inflammatory cytokines production have been shown to modulation apoptotic processes.
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Type of Study: Research | Subject: Exercise Physiology

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