**1. Introduction**

The global burden of snake envenoming is estimated at around 400,000 cases/year, with 20,000 deaths/year. Due to poor notifications, 1,800,000 envenomations/year and 94,000 deaths/year may occur [1]. It is estimated that, on the African continent, more than 60% of these accidents happen in sub-Saharan Africa, of which 95% occur in rural regions, leading to about 12,000 to 32,000 deaths and more than 9000 amputations related to post-envenoming complications [1,2].

**Citation:** Megale, Â.A.A.; Magnoli, F.C.; Guidolin, F.R.; Godoi, K.S.; Portaro, F.C.V.; Dias-da-Silva, W. *Bitis arietans* Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages. *Toxins* **2021**, *13*, 906. https://doi.org/ 10.3390/toxins13120906

Received: 29 October 2021 Accepted: 10 December 2021 Published: 16 December 2021

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Except in some islands, at high altitudes, and in regions with long-lasting snow seasons, venomous snake species are distributed worldwide, living naturally in intimate contact with human populations [3]. In the different global regions, the number of venomous snake species is large and varied. In the Middle East and North Africa, 17 snake species are found, and in sub-Saharan Africa, encompassing the Central, East, South, and West regions of the African continent, 26 species are found. Among these snakes, the *Bitis* genus includes six species, which, due to the number and severity of envenomation incidents, are important: *B. arietans*, *B. somalica*, *B. parviocula*, *B. gabonica*, *B. rhinoceros,* and *B. nasicornis* [4–6]. *B. arietans* is found in North Africa and in all sub-Saharan regions and is considered one of the most relevant snakes of medical importance in the African continent [4,7–9].

Local and systemic symptoms are observed in human victims of *Bitis arietans* bite. Intense pain, blistering, edema, ecchymosis, hemorrhage, draining lymph node hypertrophy, and necrosis are the common local symptoms. The systemic symptoms are fever, neutrophilic leukocytosis, thrombocytopenia, hemolysis, and bleeding. These systemic symptoms result in anemia, lower resistance to infections, diffuse hemorrhages, myocardial injury, coagulopathy, blood hypotension, and sometimes permanent injury of the affected body regions. All of these can cause death [9–13].

The *Bitis arietans* venom (BaV) is a complex mixture of proteins (±90–95%), peptides, carbohydrates, nucleic-acid-derived segments, metallic cations, biogenic amines, lipids, and free amino acids. Among the proteins, there are substantial quantities of enzymes, such as snake venom serine proteases (SVSPs), snake venom metalloproteases (SVMPs), and phospholipase-A2. Aside from some non-enzymatic proteins such as disintegrins, type-C lectins, cystatins, and type-Kunitz protease inhibitors are also found [14–17]. More recently, our group showed the presence of some new and already known proline-rich peptides in BaV, also known as bradykinin-potentiating peptides (BPPs) [18].

Among the most common components of BaV are proteases, specially SVSPs and SVMPs, which represent 58% of the venom composition [15–17]. These two protease classes, already studied in other snake venoms, such as *Bothrops atrox* [19–21] and *Bothrops jararaca* [22–24], have been identified as important toxins for the local and systemic effects observed in envenomation.

Due to the importance of these molecules, our group has been working on the purification and characterization of SVSPs and SVMPs present in the BaV. Thus, initially, a serine protease with fibrinogenolytic and kinin-releasing activities, named Kn-Ba, was purified and characterized in vitro. Kn-Ba is also recognized by antibodies (Abs) present in the horse-serum anti-*Bitis* spp. venoms [25].

Based on the importance of the inflammatory process in snake envenomation [11,23,26–29], the in vivo inflammation induced by BaV was studied in mice peritoneal cavities. BaV induced local tissue vessel dilatation followed by plasma infiltration; erythrocytes and neutrophils accumulation with simultaneous production of the eicosanoids LTB4, LTC4, TXB2, and PGE2; as well as the local and systemic production of IL-6 and MCP-1. In general, this study demonstrated that the BaV contains toxins that trigger the inflammatory process, which is partially dependent on lipid mediators [30].

In this context, aiming to better understand the inflammatory mechanisms involved in *Bitis arietans* envenomation, the present study focused on the identification of inflammatory mediators induced by BaV and Kn-Ba in human macrophages differentiated from THP-1 cells (THP-1 macrophages).

#### **2. Results**
