Effect of Chronic Caffeine Administration on Expression Ratio of Bax and Bcl-2 Proteins in Myocardial Tissue of Male Wistar Rats With Type 2 Diabetes

* Corresponding Author: Afshar Jafari, PhD. Address: Department of Sport Physiology, Faculty of Physical Eduction and Sport Sciences, University of Tabriz, Tabriz, Iran. Tel: +98 (914) 1168561 E-mail: af_jafari@sbu.ac.ir; afshar.jafari@gmail.com 1. Department of Sport Physiology, Faculty of Physical Eduction and Sport Sciences, University of Tabriz, Tabriz, Iran. 2. Department of Sport Biological Sciences, Faculty of Sport Sciences and Health, Shahid Beheshti University, Tehran, Iran. 3. Neurosciences Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Ali Zarghami Khameneh1 , *Afshar Jafari1,2 , Saeed Nikookheslat1 , Pouran Karimi3


Introduction
he prevalence of type 2 Diabetes (T2D) is a major public health problem that is becoming a global epidemic. T2D is mainly associated with atherosclerosis, hypertension, hyperlipidemia, obesity and inactivity. All of these risk factors for diabetes lead to the development T of cardiovascular disease and subsequently to diabetic cardiomyopathy, which is associated with impaired mitochondrial function. Meanwhile, cardiac dysfunction has been shown to cause apoptosis by increasing the activity of the cell death machine, which is measured by the negative regulation of anti-diabetic proteins (such as Bcl-2) and overexpression of pre-apoptotic proteins (such as Bax and Cas-3). However, some evidence suggests that apoptotic processes may be affected by some pharmacological interventions.
Several epidemiological and experimental studies have reported that caffeine may have the ability to suppress cell proliferation and induce apoptotis by regulating multiple signaling pathways including PTEN, PI3k / Akt, p53, and mTOR. For example, Liu et al. (2017) found that treatment with caffeine increased the activity of caspase-3 and caspase-9 proteins and increased the expression of cytochrome-c levels in cancer cells. Rahimi et al. (2018) reported that caffeine consumption decreases the expression of serum Bax level and increases the expression of Bcl-2 protein level following resistance training. Therefore, due to limited and contradictory findings, the present study aims to examine the effect of two months of caffeine administration on the expression of two proteins of the mitochondrial apoptotic pathway, Bax and Bcl-2, and their ratio (Bax/Bcl-2) in myocardial tissue of male Wistar rats following diabetes induction.

Materials and Methods
This is an experimental clinical study conducted on Male Wistar rats aged 10-12 weeks purchased based on a convenience sampling method and placed in separate cages under standard laboratory conditions. After a twoweek of adaptation, a high-fat diet (45% fat, 21% protein, and 34% carbohydrate) was given for two weeks to T2D and then 35 mg/kg-1 streptozotocin solution was administered intraperitoneally to ensure diabetic induction. Afterwards, rats were randomly assigned to three groups of 10 based on their blood glucose; Healthy control (C), diabetic control (D) and treated diabetic (D+CA). Caffeine was given to the experimental group in the form of pure dry powder for 5 days a week according to the body weight of rats (14 mg of caffeine per 200 g of body weight) and by intraperitoneal injection [4]. All rats were anesthetized and underwent operation painlessly by intraperitoneal injection of ketamine/xylazine solution 48 hours after the last supplementation session and after 12 to 14 hours of fasting. Then, a part of the apex of left ventricular (tip of the cone) in rats was carefully removed and after washing with normal saline, it was frozen in liquid nitrogen (-196°C) and kept in the freezer at -70°C. Then, Western blot method was used to evaluate the expression of Bax and Bcl-2 proteins. The band densities were measured by ImageJ software and the density of the target protein bands were normalized against a betaactin loading control. Finally, the results were presented as relative density (relative to the control group) [12]. Data were analyzed using Shapiro-Wilk test, one-way ANOVA, and Tukey's post hoc test in SPSS V. 22 software, considering a significance level of P<0.05.

Results
The results showed a significant increase in the expression of pre-apoptotic proteins (Bax) in group D by 94% and in group D+CA by 106% compared to group C (P =0.001). On the other hand, the expression of Bcl-2 protein was lower in group D+CA by about 64% than in group C (P=0.001). Thus, treatment by caffeine increased the Bax / Bcl-2 ratio.

Conclusion
Induction of T2D in rats caused a significant decrease in Bcl-2 expression (anti-apoptotic protein) and an increase in Bax expression (pre-apoptotic protein), resulting in a significant increase in apoptosis and an increase in Bax / Bcl-2 ratio. However, caffeine supplementation for two months increased the activity of the apoptotic cascade by increasing the Bax expression and decreasing the Bcl-2 expression. Consistent with these results, Hanyang  and Gan Wang et al. (2015) also concluded that the presence of caffeine increases apoptosis by increasing pre-apoptotic proteins and decreasing anti-apoptotic proteins in pathological conditions [13,14]. Researchers believe that the molecular mechanism by which caffeine stimulates the signaling of apoptotic pathways is the inactivation of the PI3K/Akt/mTOR cell survival signaling pathways and the activation of pathways involved in cell death such as p21-activated kinase (PAK2) and c-Jun Nterminal kinases (JNK) [15,16].
Apparently, the effects of caffeine on target cells are at least partly due to the condition of the target tissue and therapeutic amounts. Jaffari et al. (2004) and Sinha et al. (2014) stated that caffeine induces apoptotic and autophagic cell death pathways in equal amounts and above 50 μmol/L, respectively, as reported in the present study [17,18].