Rutin hydrate inhibits apoptosis in the brains of cadmium chloride-treated rats via preserving the mitochondrial integrity and inhibiting endoplasmic reticulum stress

ABSTRACT

Recent evidence has suggested that cadmium (Cd) ions-induced neurotoxicity is associated with increased oxidative stress and mitochondrial-dependent and endoplasmic reticulum (ER) stress-induced apoptosis. This study aimed to investigate if rutin hydrate (RH), a well-reported neuroprotective and an antioxidant flavonoid, can ameliorate cadmium chloride (CdCl2)-induced neurotoxicity by inhibiting the resultant ER stress. Rats were divided into 4 groups (n = 16/group) of control, control + RH (100 mg/kg), CdCl₂ (5 mg/kg), and CdCl₂ + RH. All treatments were administered orally for 30 days, on daily basis. Brain homogenates from CdCl₂-treated rats showed increased oxidative stress and induced activation of ER stress characterized by increasing mRNA and protein levels of GRP78, ATF-6, CHOP and Xbp-1 and protein levels of p-elF2α, p-JNK1/2 and cleaved caspase-12. Also, CdCl₂ significantly reduced Bcl-2, enhanced Bax translocation to the mitochondrial membrane, increased cytoplasmic levels of cytochrome-C and caspase-3, and reduced mitochondrial membrane potential (Δψm) (increased Vmax and reduced time to Vmax). In contrast, RH significantly enhanced levels GSH and activities of SOD, GSH-Px, decreased levels of MDA and inhibited mitochondrial permeability transition pore (mtPTP) in the brains of both control and CdCl₂-treated rats. Interestingly, in brain homogenates of CdCl₂-treated rats only, RH reduced all markers of ER stress, increased Bcl-2, reduced mitochondrial Bax translocation and improved mitochondrial coupling. It also reduced cytosolic levels of cytochrome-C, cleaved caspase-3, and cleaved caspase-12. Overall, these findings support the efficiency of RH to inhibit ER stress in brains CdCl2-treated rats which is added to its existing mechanisms of neuroprotection.

Abbreviations: ATF-6: activating transcription factor-6; Bax: Bcl-associated x; BBB: blood-brain barrier; Bcl-2: B-cell lymphoma 2; BiP: immunoglobulin heavy-chain-binding protein; [Ca²⁺]i: intracellular free Ca²⁺ concentration; Cd: cadmium; CdCl₂: cadmium chloride; CHOP: CCAAT/enhancer-binding protein-homologous protein; CMC: carboxymethyl cellulose; Δψm: mitochondrial membrane potential; elF2α: phospho-eukaryotic translation initiation factor 2-alpha; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERK1/2: extracellular signal-regulated kinases 1 and 2; GADD 153: growth arrest and DNA damage-inducible protein 153; GRP78, 78 kDa glucose-regulated protein; GSH: reduced glutathione; GSH: reduced glutathione; GSH-Px: glutathione peroxidase; GSSG: glutathione disulfide (oxidized glutathione); IRE-1: inositol-requiring enzyme-1; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; MDA: malondialdehyde; mTOR: Akt/mammalian target of rapamycin; mtPTP: mitochondrial permeability transition pore; ONOO⁻: peroxynitrite; PCR: polymerase chain reaction; PERK: protein kinase RNA-like ER kinase; p-JNK: phospho-JNK; qPCR: quantitative PCR; RCR: respiratory control ratio; RH: rutin hydrate; RHoGDI: Rho-GDP-dissociation inhibitor; ROS: reactive oxygen species; SOD: superoxide dismutase; UPR: unfolded protein response; VDAC: voltage-dependent anion channel; Vmax: maximal rate of pore opening; Xbp-1: X-box binding protein 1.

KEYWORDS:

• Cadmium chloride
• endoplasmic reticulum stress
• rutin hydrate
• brain
• rats

Acknowledgments

The authors wish to thank the staff of the animal house as well as the technical staff at the Department of Physiology and Biochemistry at the College of Medicine at King Khalid University for their help to the current study.

Authors’ Contribution

Dalia G. Mostafa, MD, Ph.D., is the principal investigator for the study provided and contributed by writing the proposal, measurements of biochemical parameters, running all RT_PCR, immunohistochemical analysis, and Western blot procedures preparation and data collection and assisted in writing the paper. Eman F. Khaleel MD, Ph.D. and Rehab M. BadiMD, Ph.D., assisted in writing the proposal, biochemical measurements, and qPCR, data collection and analysis, writing the paper. Ghada A. Abdel_Aleem, Ph.D. and Hanaa Maher Abdeen, Ph.D. assisted in immunohistochemical analysis, measurement of some biochemical parameters, data collection and analysis and writing the paper.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The authors declare that this is a self-funding project

Notes on contributors

Dalia G. Mostafa

Dalia G. Mostafa is a medical physiologist with a Master and MD in Medical Physiology, Faculty of Medicine, Assuit University, Assuit, Egypt. Now, I am an associate professor and teaching physiology at college of Medicine, King Khalid University, Abha, Saudi Arabia, to undergraduate medical, dental and pharmacy students. I have several publications in national and international (ISI) journals (Archives of Physiology and Biochemistry, Canadian Journal of Physiology and Pharmacology, Archives of Biological Sciences and Neurological Research) in diabetes mellitus, fatty liver and brain ischemia. I have also presented many posters at national conferences.

Eman F. Khaleel

Eman F. Khaleel is a physiologist with a diploma in pediatric, Master degree in physiology and MD in Physiology, Faculty of Medicine, Cairo University, Egypt. I have several international publications in diabetes, fatty liver diseases and CNS in experimental rats. I presented several posters in national conferences. I am an Associate professor, King Khalid University college of Medicine Saudi Arabia and Professor in Faculty of Medicine Cairo University Egypt. Also provides continuing lectures in Physiology for medical, dental, pharmacy and nursing students.

Rehab M. Badi

Rehab M. Badi is an assistant professor of physiology with a PhD in Human Physiology and holds a diploma in Molecular Medicine and Master’s degree in Health Professions Education. She has several publications in the beneficial effects of naturally occurring antioxidant, mainly Gum Arabic, in metabolic diseases and obesity. She has several poster presentations in international conferences. Originally, Rehab M. Badi is an assistant professor in the faculty of Medicine, University of Khartoum, Sudan. Currently she is an assistant professor in the Faculty of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia, lecturing on Human Physiology to undergraduate medical, dental and pharmacy students.

Ghada A. Abdel-Aleem

Ghada A. Abdel-Aleem is an associate professor, Department of Medical Biochemistry, Faculty of Medicine, Tanta University, Tanta, Egypt (Former at Department of Medical Biochemistry, College of Medicine, King Khalid University, Abha, Saudi Arabia). I have several international publications in diabetes, fatty liver diseases and CNS in experimental rats. I presented several posters in national conferences.

Hanaa M. Abdeen

Hanaa M. Abdeen M.B, B.Ch (Bachelor Degree) in Medicine and Surgery, Faculty of Medicine, Mansoura University, Mansoura, Egypt. M. Sc. and Ph. D degrees in Medical Biochemistry and Molecular Biology, Faculty of Medicine, Mansoura University. Assistant Professor at Medical Biochemistry and Molecular Biology dept., Faculty of Medicine, Mansoura University (Former Assistant Professor at Clinical Biochemistry dept., College of Medicine, KKU, Saudi Arabia).