Contents lists available at ScienceDirect Experimental and Toxicologic Pathology journal homepage: www.elsevier.com/locate/etp Full Length Article Buffalo casein derived peptide can alleviates H 2 O 2 induced cellular damage and necrosis in fibroblast cells Savita Devi, Naveen Kumar, Suman Kapila ⁎ , Sanusi Bello Mada, Srinu Reddi, Rishika Vij, Rajeev Kapila Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India ARTICLE INFO Keywords: Fibroblasts Oxidative stress Antioxidative Reactive oxygen species Hydrogen peroxide ABSTRACT Oxidative stress is one of a critical pathogenic factor in the progression of aging and chronic diseases such as cancer, myocardial inflammation and diabetes. In the present scenario, peptides with short half life and more biological specificities are gaining much attention as prodrugs. Thus, the present investigation carried out to screen potential antioxidative peptide, VLPVPQK to cope with the cellular oxidative damage. Our results showed that treatment of rat fibroblast cells with 0.2 mM H 2 O 2 for 6 h significantly declined different oxidative stress biomarkers such as SOD, CAT, GSH, and promoted LDH activity. In addition, ROS and TNF-α levels were also increased upon H 2 O 2 exposure for 6 h and thereby, it induced cell death. Amazingly, pretreatment of the peptide (VLPVPQK) significantly elevated cell survivability, by reversing all H 2 O 2 induced alterations in fibroblast cells. Therefore, our results indicated that, the peptide (VLPVPQK) acted as a potential cytoprotective agent, who restored redox balance and cell homeostasis in cultured fibroblast cells, even after H 2 O 2 exposure, suggesting that the peptide can be valuable as an effective remedy in treatment of oxidative stress related diseases and skin inflammation related disorders. 1. Introduction Fibroblasts are the main cell type present in the dermis of skin responsible for the synthesis of extracellular matrix, thus play a crucial role in the protection against the development of patho- physiological conditions of the skin (Gillitzer and Goebeler, 2001). Human body consists of a well developed antioxidative defense system against metabolic oxidative stress (Simic, 1988). Oxidative stress can be defined as elevated ROS production above the critical point in impaired antioxidative defense of cell, which leads to the development of oxidative stress and inflammation related diseases, such as cancer, atherosclerosis, hypertension and arthritis (Frenkel, 1992; Halliwell and Gutteridge, 1984; Sliwa-joźwik et al., 2002; Crujeiras et al., 2008; Laveti et al., 2013). Hydrogen peroxide (H 2 O 2 ) has been demonstrated as an intra and extracellular stimulant of oxidative stress, due to its stable and diffusible nature through the membranes of many cell types (Yuan et al., 2003). Therefore, H 2 O 2 -induced oxidative stress model served as a potential tool for screening antioxidative compounds (Feng et al., 2013). SOD, catalase and GSH are the major regulators of antioxidative defense, which neutralizes the excessive ROS, produced in the metabolic process. Several studies have reported that SOD, catalase and GSH were markedly decreased in fibroblast and other different cells upon H 2 O 2 treatment (Troyano et al., 2003; Dash et al., 2007; Feng et al., 2013). LDH is a soluble protein which is involved in the cellular anaerobic biochemical process and is released out of the cell, thus, it can be used as a cell injury marker. Moreover, Dash and colleagues, demonstrated the use of LDH as cell damage marker in fibroblast cell (Dash et al., 2007). ROS and the consequential cellular redox imbalance work as strong stimulants in apoptotic signal transduction (Kannan and Jain, 2000). Caspases are the major candidates, responsible in cellular programmed cell death (Troyano et al., 2003). Apart from nutritional resource, milk proteins also possess several health benefits including antioxidative, antihypertensive, antimicrobial, anti-inflammatory, and osteogenic effects which can be attributed to the encrypted bioactive peptides (Kumar et al., 2010; Shanmugam et al., 2015; Reddi et al., 2016a,b,c). Recently, four novel peptides from buffalo milk casein were http://dx.doi.org/10.1016/j.etp.2017.04.009 Received 2 December 2016; Received in revised form 15 March 2017; Accepted 16 April 2017 ⁎ Corresponding author. E-mail addresses: savitajohn4@gmail.com (S. Devi), nshiwal@gmail.com (N. Kumar), suman_ndri@yahoo.com (S. Kapila), sbmada@abu.edu.ng (S.B. Mada), bioreddy.srinu89@gmail.com (S. Reddi), rishikavij@gmail.com (R. Vij), rkapila69@rediff.com (R. Kapila). Abbreviations: VLPVPQK, Valine Leucine-Proline-Valine-Proline-Glutamine-Lysine; SOD, superoxide dismutase; LDH, lactate dehydrogenase; ROS, reactive oxygen species; DMEM, Dulbecco’s modified minimal essential medium; FBS, Fetal bovine serum; DCFH-DA, 2’,7’-dichlorodihydrofluorescein–diacetate; PI, propidium iodide; MTT, 3–(4, 5–dimethylthiazo- l–2–yl)–2, 5–diphenyl tetrazolium bromide; DMSO, dimethyl sulfoxide; DAPI, 4', 6-diamidino-2-phenylindole; ABTS, 2, 2 azinobis (ethylbenzthiazoline-6-sulfonic acid); AAPH, 2, 2’- azobis (2-amidino-propane) dihydrochloride; FITC, fluorescein isothiocyanate; NAC, N-acetyl-L-cysteine Experimental and Toxicologic Pathology xxx (xxxx) xxx–xxx 0940-2993/ © 2017 Elsevier GmbH. All rights reserved. Please cite this article as: Devi, S., Experimental and Toxicologic Pathology (2017), http://dx.doi.org/10.1016/j.etp.2017.04.009