Article Moringa oleifera Extract Extenuates Echis ocellatus Venom-Induced Toxicities, Histopathological Impairments and Inflammation via Enhancement of Nrf2 Expression in Rats Akindele O. Adeyi 1,*, Sodiq O. Adeyemi 1, Enoh-Obong P. Effiong 1, Babafemi S. Ajisebiola 2, Olubisi E. Adeyi 3 and Adewale S. James 3 1 Animal Physiology Unit, Department of Zoology, University of Ibadan, Ibadan P.M.B. 200284, Oyo State, Nigeria; [email protected] (S.O.A.); [email protected] (E.-O.P.E.) 2 Department of Zoology, Osun State University, Oshogbo P.M.B. 230212, Osun State, Nigeria; [email protected] 3 Department of Biochemistry, Federal University of Agriculture, Abeokuta P.M.B. 2240, Ogun State, Nigeria; [email protected] (O.E.A.); [email protected] (A.S.J.) * Correspondence: [email protected]; Tel.: +234-80-3069-2698 Abstract: Echis ocellatus snakebite causes more fatalities than all other African snake species combined. Moringa oleifera reportedly possesses an antivenom property. Therefore, we evaluated the effective- ness of M. oleifera ethanol extract (MOE) against E. ocellatus venom (EOV) toxicities. Thirty male rats were grouped as follows (n = 5): Group 1 (normal control received saline), groups 2 to 6 were administered intraperitoneally, 0.22 mg/kg (LD50) of EOV. Group 2 was left untreated while group 3 to 6 were treated post-envenoming with 0.2 mL of polyvalent antivenom, 200, 400, and 600 mg/kg of MOE respectively. MOE significantly (p < 0.05) normalized the altered haematological indices and Citation: Adeyi, A.O.; Adeyemi, blood electrolytes profiles. MOE attenuated venom-induced cellular dysfunctions, characterized by a S.O.; Effiong, E.-O.P.; Ajisebiola, B.S.; Adeyi, O.E.; James, A.S. Moringa significant increase in NRF2, and concomitant downregulation of increased antioxidant enzymes oleifera Extract Extenuates Echis (SOD and CAT) activities in the serum and heart of the treated rats. MOE normalized the elevated ocellatus Venom-Induced Toxicities, TNF-α and IL-1β in serum and heart tissues. Furthermore, the IgG titre value was significantly Histopathological Impairments and (p < 0.5) higher in the envenomed untreated group compared to the MOE-treated groups. Hemor- Inflammation via Enhancement of rhagic, hemolytic and coagulant activities of the venom were strongly inhibited by the MOE dose, Nrf2 Expression in Rats. dependently. Lesions noticed on tissues of vital organs of untreated rats were abolished by MOE. Pathophysiology 2021, 28, 98–115. Our findings substantiate the effectiveness of MOE as a potential remedy against EOV toxicities. https://doi.org/10.3390/ pathophysiology28010009 Keywords: Moringa oleifera; antivenom; Echis ocellatus; inflammation Received: 23 December 2020 Accepted: 26 February 2021 Published: 5 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral Snakebite-related pathology affects as many as 2.7 million people annually, most who with regard to jurisdictional claims in live in remote, poorly developed and marginalized tropical communities of the world [1]. published maps and institutional affil- Snakebites accounts for annual death ranging from 81,000 to 138,000, while over 400,000 iations. surviving victims suffer permanent psychological and physical disabilities [1,2]. Conse- quently, envenoming from snakebites is thus a priority among neglected tropical disease by the World Health Organization [3,4]. In Nigeria, the incidence of snake envenoming in remote communities and some parts of urban areas has been on the rise [5]. The up- surge in cases of snake bites might be due to the unique ecosystem, most especially in the Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. North-Eastern and central parts of Nigeria, where the vegetation favours breeding and This article is an open access article multiplication of snakes [5]. distributed under the terms and Echis ocellatus (Carpet viper) is regarded as the most medically significant species of conditions of the Creative Commons viper in West Africa. Its envenoming is poisonous and life-threatening, and accounts for Attribution (CC BY) license (https:// more death than all other African species combined [6,7]. The venom of E. ocellatus is highly creativecommons.org/licenses/by/ toxic, consists primarily of peptides and proteins majorly; snake venom metalloproteinases 4.0/). (SVMPs) possess hemorrhagic, nephrotoxic, cardiotoxic and anti-coagulant effects [8]. Pathophysiology 2021, 28, 98–115. https://doi.org/10.3390/pathophysiology28010009 https://www.mdpi.com/journal/pathophysiology Pathophysiology 2021, 28 99 Thus, envenoming by E. ocellatus is responsible for several clinical complications of severe systemic and local pathologies, such as swelling, blistering, necrosis and haemorrhages [9]. Furthermore, the venom of vipers is known to elicit specific immunologic responses in vitro and in vivo or activate inflammatory responses mediated by some cytokines [10,11]. Studies have also shown that toxins in snake venoms generate reactive oxygen species (ROS) associated with inflammatory responses. These free radicals ROS are directly in- volved in oxidative stress and play a significant role in venom-induced toxicities [12]. Induction of oxidative stress could, in turn, activate a transcription factor known as nu- clear factor erythroid 2-related factor 2 (NRF2)—a protein that is known to modulate the expression of antioxidant proteins to the cell protection against oxidative damage triggered by injury and inflammation [13]. Several in vivo and in vitro studies have re- ported various pathophysiological effects of viper envenoming with concerns focused on E. ocellatus [9,14,15]. Over the years, researchers have made several efforts to develop effective therapies for the treatment of snakebites, but to date, antivenom remains the only definitive treatment for snake envenoming. Antivenom has limitations associated with acute and delayed reactions such as anaphylactic and serum sickness reactions combined with its ineffectiveness against local effects after snake envenoming [16,17]. These effects develop very rapidly after snake envenomation, making neutralization by antivenom difficult, especially if treatment is delayed due to either late access to hospital or scarcity of antivenom [18]. All these and more, place a constraint on the use of antivenom in treating snake envenoming. Development of an effective therapy that can overcome drawbacks of current snakebite treatment is, therefore, a clinical priority. Natural means of combating snakebites relies on the use of plants with anti-ophidic effects, with little or no worry about limitations currently associated with antivenom [19]. In the phytotherapy of snakebites, effectiveness of Moringa oleifera (Lam) has been ascertained in neutralizing snake venoms of different species [19,20]. However, limited information exists on efficacy of M. oleifera on the treatment of complications arising from envenoming by E. ocellatus. Accordingly, this study evaluated the bioactivities of ethanol crude extract of M. oleifera leave against E. ocellatus venom-induced toxicities in vivo and in vitro. 2. Materials and Methods 2.1. Snake Venom and Antivenom E. ocellatus venom was procured from the Department of Veterinary Pharmacology and Toxicology, Ahmadu Bello University Zaria, Nigeria. A polyvalent antivenom (EchiTAb- Plus ICP) produced at Instituto Clodomiro Picado, University of Costa Rica, Costa Rica was used as standard control treatment for this study. 2.2. Ethical Consideration Animal experiments was approved by the University of Ibadan-Animal Care and Research Ethics Committee (UI-ACUREC), with Assigned number: UI-ACUREC: 18/0108 per the guidelines of the National Code for Health Research Ethics. All animal experiments complied with the National Research Council’s publication on guide for the care and use of laboratory animals [21]. 2.2.1. Experimental Animals Eighty male albino Wistar rats weighing 120–160 g used for this study were procured from the animal house of the Department of Zoology, University of Ibadan, Nigeria. Animals were housed in well-ventilated, pathogen-free cages at room temperature (27 ◦C) and allowed to acclimatize for 14 days under standard conditions (12 h light-dark cycles) before the study commenced. Rats were fed with standard pelleted feed and distilled water were provided ad libitum. Pathophysiology 2021, 28 100 2.2.2. Evaluation of Lethality Dose (LD50) of E. ocellatus Venom Lethal concentration dose (LD50) of the venom was done using a published method [22] with slight modifications. Exactly, 20 rats were randomly divided into four groups (n = 5) and injected intraperitoneally (i.p) with varying doses of E. ocellatus venom. Group 1 served as control and was injected with 0.2 mL of saline while Group 2 to 4 received 0.1 mL of 0.14, 0.28 and 0.56 mg/kg−1 of the venom dissolved in 1 mL of normal saline respectively. The LD50 of the venom was calculated with confidence limit at 50% probability using probity analysis of death occurring within 48 h after the venom administration. 2.3. Collection, Identification and Extraction Procedure of Plant Materials M. oleifera leaves were freshly harvested from Botanical Garden of the University of Ibadan, and identified in the Herbarium of Department of Botany, University of Ibadan (voucher number UIH-224601). Leaves were air-dried at room temperature for two weeks and blended into fine powder using a super-master electric blender (Model no: SMB-2977). Approximately 3 kg of powdered leaf was extracted in 4 L of absolute ethanol for 72 h using the cold maceration method [23]. The content was then filtered and concentrated using a rotary evaporator at 40 ◦C. 2.4. Anti-Ophidic Experiment Experimental Design, Envenoming and Treatment Procedures Thirty male albino rats were randomly selected into six groups (n = 5). Group 1 was in- jected with saline (normal control). Group 2 to 6 were injected intraperitoneally with 0.2 mL −1 of 0.22 mg/kg (LD50) of E. ocellatus in 1 mL of normal saline. Group 2 was not treated post-envenomation (venom control). Group 3 was treated with 0.2 mL of polyvalent an- tivenom (EchiTab ICP-Plus) intravenously while Groups 4, 5 and 6 were administered orally with (0.2 mL) of 200, 400, and 600 mg/kg−1 of M.