Due to its significant mortality and morbidity, scorpion sting is a public health concern worldwide, particularly in tropical and subtropical regions. Heterometrus bengalensis (HB) significantly impacts health; however, HB’s venom composition is the least characterized. The present study provides a comprehensive insight into the HB venom (HBV) profile by in vitro enzyme assay and tandem mass spectrometry analysis of SDS-PAGE-separated venom proteins and peptides. HBV showed calcium-dependent PLA2 and nucleotidase (ATPase, ADPase, and AMPase) activities, indicating myotoxicity and ATP-depletion-induced shock. Proteomic analysis of HBV identified 25 toxins belonging to 8 protein toxin families from searching the MS data against the Scorpionidae family (taxid 6888) of toxin entries, which collectively contribute to the pharmacological effects of HBV. The lethal dose of HBV in Swiss albino mice (SAM) was determined to be 20 mg/kg (i.v). In vivo, HBV-envenomed SAM revealed severe physiological disturbances, elevated liver enzymes, plasma glucose levels, histopathological evidence of organ damage, and myotoxicity. The serum proinflammatory cytokines (IL-1β, IL-6, and TNFα) in HBV-injected SAM were significantly increased (1–2 fold) compared to the controls. The current findings underscore the complexity of HBV, highlighting the urgent need for improved therapeutic strategies, including the development of species-specific and targeted antivenom.
