1. 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 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.
2. 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 two-week 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 beta-actin 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.
3. 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.
4. 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 Liu et al. (2017) 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 N-terminal 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].
Ethical Considerations
Compliance with ethical guidelines
This study obtained its ethical approval from the Research Ethics Committee of Tabriz University of Medical Sciences (Code: IR.TBZMED.VCR.REC.1397.389).
Funding
This study was extracted from the PhD. dissertation of first author approved by Faculty of Physical Education and Sports Sciences, University of Tabriz, Tabriz, and received financial support from the Deputy for Research and Postgraduate Studies of the University of Tabriz.
Authors' contributions
Conceptualization and investigation: Ali Zarghami Khameneh; Writing, editing & review: Afshar Jafari; Data analysis: Saeed Nikookheslat; Investigation: Pouran Karimi
Conflicts of interest
The authors declare no conflict of interest
Acknowledgements
The authors would like to thank and appreciate the efforts of all colleagues, the head of the Animal Laboratory of the Neuroscience Research Center of Tabriz University of Medical Sciences, and Sara Cellular Research Center in Tabriz, Iran.


References

1. Preedy VR. Caffeine: Chemistry, analysis, function and effects. London: Royal Society of Chemistry; 2015. https://books.google.com/books/about/Caffeine.html?id=1GsoDwAAQBAJ
2. Monteiro J, Alves MG, Oliveira PF, Silva BM. Pharmacological potential of methylxanthines: Retrospective analysis and future expectations. Critical Reviews in Food Science and Nutrition. 2019; 59(16):2597-2625. [DOI:10.1080/10408398.2018.1461607] [PMID]
3. Lu PZ, Lai CY, Chan WH. Caffeine induces cell death via activation of apoptotic signal and inactivation of survival signal in human osteoblasts. International Journal of Molecular Sciences. 2008; 9(5):698-718. [DOI:10.3390/ijms9050698] [PMID] [PMCID]
4. Bode AM, Dong Z. The enigmatic effects of caffeine in cell cycle and cancer. Cancer Letters. 2007; 247(1):26-39. [DOI:10.1016/j.canlet.2006.03.032] [PMID] [PMCID]
5. Nakaso K, Ito S, Nakashima K. Caffeine activates the PI3K/Akt pathway and prevents apoptotic cell death in a Parkinson’s disease model of SH-SY5Y cells. Neuroscience Letters. 2008; 432(2):146-50. [DOI:10.1016/j.neulet.2007.12.034] [PMID]
6. Wang L, Lu L. Pathway-specific effect of caffeine on protection against UV irradiation-induced apoptosis in corneal epithelial cells. Investigative Ophthalmology & Visual Science. 2007; 48(2):652-60. [DOI:10.1167/iovs.06-1007] [PMID] [PMCID]
7. Liu H, Zhou Y, Tang. Caffeine induces sustained apoptosis of human gastric cancer cells by activating the caspase-9/caspase-3 signalling pathway. Molecular Medicine Reports. 2017; 16(3):2445-54. [DOI:10.3892/mmr.2017.6894] [PMID] [PMCID]
8. Diamantis A, Magiorkinis E, Sakorafas GH, Androutsos G. A brief history of apoptosis: From ancient to modern times. Onkologie. 2008; 31(12):702-6. [DOI:10.1159/000165071] [PMID]
9. Favaloro B, Allocati N, Graziano V, Di Ilio C, De Laurenzi V. Role of apoptosis in disease. Aging (Albany NY). 2012; 4(5):330-49. [DOI:10.18632/aging.100459] [PMID] [PMCID]
10. Harvey PA, Leinwand LA. The cell biology of disease: Cellular mechanisms of cardiomyopathy. Journal of Cell Biology. 2011; 194(3):355-65. [DOI:10.1083/jcb.201101100] [PMID] [PMCID]
11. Xiong FY, Tang ST, Su H, Tang HQ, Jiang P, Zhou Q, et al. Melatonin ameliorates myocardial apoptosis by suppressing endoplasmic reticulum stress in rats with long term diabetic cardiomyopathy. Molecular Medicine Reports. 2018; 17(1):374-81. [DOI:10.3892/mmr.2017.7841] [PMID]
12. Corsetti G, Pasini E, Assanelli D, Bianchi R. Effects of acute caffeine administration on NOS and Bax/Bcl2 expression in the myocardium of rat. Pharmacological Research. 2008; 57(1):19-25. [DOI:10.1016/j.phrs.2007.07.007] [PMID]
13. Woo SM, Min KJ, Seo BR, Nam JO, Choi KS, Yoo YH, et al. Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression. Cell Death & Disease. 2014; 5(11):e1514. [DOI:10.1038/cddis.2014.472] [PMID] [PMCID]
14. Sasidharan SR, Joseph JA, Anandakumar S, Venkatesan V, Ariyattu Madhavan CN, Agarwal A. An experimental approach for selecting appropriate rodent diets for research studies on metabolic disorders. BioMed Research International. 2013; 2013:752870. [DOI:10.1155/2013/752870] [PMID] [PMCID]
15. Jafari M, Rabbani A. Studies on the mechanism of caffeine action in alveolar macrophages: Caffeine elevates cyclic adenosine monophosphate level and prostaglandin synthesis. Metabolism. 2004; 53(6):687-92. [DOI:10.1016/j.metabol.2003.08.004] [PMID]
16. Saiki S, Sasazawa Y, Imamichi Y, Kawajiri S, Fujimaki T, Tanida I, et al. Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition. Autophagy. 2011; 7(2):176-87. [DOI:10.4161/auto.7.2.14074] [PMID] [PMCID]
17. Farhadi H, Siahkohian M, Lotfali B, Pouran K. [Effects of aerobic training and hypoxia on expression angiogenic factors in cardiac male Wistar rats (Persian)]. Journal of Sport in Biomotor Sciences. 2016; 2(16):70-9. http://journals.hsu.ac.ir/sbs/article-1-708-en.html
18. Huang PC, Wang GJ, Fan MJ, Asokan Shibu M, Liu YT, Padma Viswanadha V, et al. Cellular apoptosis and cardiac dysfunction in STZ-induced diabetic rats attenuated by anthocyanins via activation of IGFI-R/PI3K/Akt survival signaling. Environmental Toxicology. 2017; 32(12):2471-80. [DOI:10.1002/tox.22460] [PMID]
19. Wang G, Bhoopalan V, Wang D, Wang L, Xu X. The effect of caffeine on cisplatin-induced apoptosis of lung cancer cells. Experimental Hematology & Oncology. 2015; 4:5. [DOI:10.1186/2162-3619-4-5] [PMID] [PMCID]
20. He Z, Ma W-Y, Hashimoto T, Bode AM, Yang CS, Dong Z. Induction of apoptosis by caffeine is mediated by the p53, Bax, and caspase 3 pathways. Cancer Research. 2003; 63(15):4396-401. [PMID]
21. Rahimi MR, Khabiri P, Faraji H. Effects of caffeine ingestion on resistance exercise-induced apoptosis in athletes: A randomized, double-blind, placebo-controlled, crossover study. Progress in Nutrition. 2018; 20(4):563-9. [Doi:10.23751/pn.v20i4.6442]
22. Zarghami-Khameneh A, Jafari A. [The effect of different doses of caffeine and a single bout of resistant-exhaustive exercise on muscle damage indices in male volleyball players (Persian)]. Feyz. 2014; 18(3):220-8. http://feyz.kaums.ac.ir/article-1-2304-en.html
23. Sinha RA, Farah BL, Singh BK, Siddique MM, Li Y, Wu Y, et al. Caffeine stimulates hepatic lipid metabolism by the autophagy-lysosomal pathway in mice. Hepatology. 2014; 59(4):1366-80. [DOI:10.1002/hep.26667] [PMID]