Indian Echis carinatus bite causes sustained tissue destruction at the bite site. Neutrophils, the major leukocytes in the early defence process, accumulate at the bite site. Here we show that E. carinatus venom induces neutrophil extracellular trap (NET) formation. The NETs block the blood vessels and entrap the venom toxins at the injection site, promoting tissue destruction. The stability of NETs is attributed to the lack of NETs-degrading DNase activity in E. carinatus venom. In a mouse tail model, mice co-injected with venom and DNase 1, and neutropenic mice injected with the venom, do not develop NETs, venom accumulation and tissue destruction at the injected site. Strikingly, venom-induced mice tail tissue destruction is also prevented by the subsequent injection of DNase 1. Thus, our study suggests that DNase 1 treatment may have a therapeutic potential for preventing the tissue destruction caused by snake venom.

TNF is a pleotropic cytokine known to be involved in the progression of several pro-inflammatory disorders. Many therapeutic agents have been designed to counteract the effect of TNF in rheumatoid arthritis as well as a number of cancers. In the present study we have synthesized and evaluated the anti-cancer activity of novel biscoumarins in vitro and in vivo. Among new compounds, BIHC was found to be the most cytotoxic agent against the HepG2 cell line while exhibiting less toxicity toward normal hepatocytes. Furthermore, BIHC inhibited the proliferation of various hepatocellular carcinoma (HCC) cells in a dose- and time-dependent manner. Subsequently, using in silico target prediction, BIHC was predicted as a TNF blocker. Experimental validation was able to confirm this hypothesis, where BIHC could significantly inhibit the recombinant mouse TNF-α binding to its antibody with an IC₅₀ of 16.5 μm. Furthermore, in silico docking suggested a binding mode of BIHC similar to a ligand known to disrupt the native, trimeric structure of TNF, and also validated with molecular dynamics simulations. Moreover, we have demonstrated the down-regulation of p65 phosphorylation and other NF-κB-regulated gene products upon BIHC treatment, and on the phenotypic level the compound shows inhibition of CXCL12-induced invasion of HepG2 cells. Also, we demonstrate that BIHC inhibits infiltration of macrophages to the peritoneal cavity and suppresses the activity of TNF-α in vivo in mice primed with thioglycollate broth and lipopolysaccharide. We comprehensively validated the TNF-α inhibitory efficacy of BIHC in an inflammatory bowel disease mice model.

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.