Glutamine ameliorates Bungarus multicinctus venom-induced lung and heart injury through HSP70: NF-κB p65 and P53/PUMA signaling pathways involved.

Background: Bungarus multicinctus is one of the most dangerous venomous snakes prone to cardiopulmonary damage with extremely high mortality. In our previous work, we found that glutamine (Gln) and glutamine synthetase (GS) in pig serum were significantly reduced after Bungarus multicinctus bite. In the present study, to explore whether there is a link between the pathogenesis of cardiopulmonary injury and Gln metabolic changes induced by Bungarus multicinctus venom. We investigated the effect of Gln supplementation on the lung and heart function after snakebite. Methods: We supplemented different concentrations of Gln to mice that were envenomated by Bungarus multicinctus to observe the biological behavior, survival rate, hematological and pathological changes. Gln was supplemented immediately or one hour after the venom injection, and then changes in Gln metabolism were analyzed. Subsequently, to further explore the protective mechanism of glutamine on tissue damage, we measured the expression of heat-shock protein70 (HSP70), NF-κB P65, P53/PUMA by western blotting and real-time polymerase in the lung and heart. Results: Gln supplementation delayed the envenoming symptoms, reduced mortality, and alleviated the histopathological changes in the heart and lung of mice bitten by Bungarus multicinctus. Additionally, Gln increased the activity of glutamine synthetase (GS), glutamate dehydrogenase (GDH) and glutaminase (GLS) in serum. It also balanced the transporter SLC7A11 expression in heart and lung tissues. Bungarus multicinctus venom induced the NF-κB nuclear translocation in the lung, while the HO-1 expression was suppressed. At the same time, venom activated the P53/PUMA signaling pathway and the BAX expression in the heart. Gln treatment reversed the above phenomenon and increased HSP70 expression. Conclusion: Gln alleviated the glutamine metabolism disorder and cardiopulmonary damage caused by Bungarus multicinctus venom. It may protect lungs and heart against venom by promoting the expression of HSP70, inhibiting the activation of NF-κB and P53/PUMA, thereby delaying the process of snake venom and reducing mortality. The present results indicate that Gln could be a potential treatment for Bungarus multicinctus bite.

A Single Amino Acid Replacement Boosts the Analgesic Activity of α-Conotoxin AuIB through the Inhibition of the GABABR-Coupled N-Type Calcium Channel.

α-conotoxin AuIB is the only one of the 4/6 type α-conotoxins (α-CTxs) that inhibits the γ-aminobutyric acid receptor B (GABABR)-coupled N-type calcium channel (CaV2.2). To improve its inhibitory activity, a series of variants were synthesized and evaluated according to the structure-activity relationships of 4/7 type α-CTxs targeting GABABR-coupled CaV2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2-3-fold increase in the inhibition of GABABR-coupled CaV2.2 (IC50 is 0.74 nM); substitutions of position 9-12 with basic or hydrophobic amino acid and the addition of hydrophobic amino acid Leu or Ile at the second loop to mimic 4/7 type α-CTxs all failed to improve the inhibitory activity of AuIB against GABABR-coupled CaV2.2. Interestingly, the most potent form of AuIB[P7R] has disulfide bridges of "1-4, 2-3" (ribbon), which differs from the "1-3, 2-4" (globular) in the isoforms of wildtype AuIB. In addition, AuIB[P7R](globular) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study demonstrated that 4/6 type α-CTxs, with the disulfide bridge connectivity "1-4, 2-3," are also potent inhibitors for GABABR-coupled CaV2.2, exhibiting potent analgesic activity.

A new protein-coupled antigen of α-conotoxin MI displays high immunogenicity and can produce antiserum with high detoxification activity.

α-conotoxin (α-CTX) MI is a small peptide toxin with 14 amino acids and two disulfide bonds. It potently inhibits muscle-type nicotinic acetylcholine receptors (nAChRs), and poses a threat as a toxin to tropical fishermen. However, there are currently no effective drugs for the treatment of MI envenomation due to the toxin's low immunogenicity. In this report, we generated neutralizing antiserum and F(ab')2 to MI by synthesizing a new MI antigen through the coupling of alkynyl-modified MI and azide-modified bovine serum albumin (BSA), followed by immunization into mouse and horse. The new MI-BSA antigen generated high titers of mouse and horse antiserum (1:204,800 and 1:51,200, respectively), and both the antiserum as well as the horse F(ab')2 displayed highly potent neutralization and detoxification efficacy. 12.5 µL of mouse or horse antiserum preincubated with MI could completely neutralize a lethal dose of the MI (0.4 µg, 1.7 × LD50), while 6.25 µL (mouse) or 10.41 µL (horse) of the antiserum could exert complete detoxification of mice injected with 1.7 × LD50 of MI. Moreover, the mouse and horse antiserum exhibited medium cross-reactivity for highly toxic α-CTX GI. These results demonstrate that the integrity of MI's antigen epitope and carrier effect of BSA can improve MI's immunogenicity, and provides an effective detoxification treatment for highly toxic α-conotoxins as well as an effective method for the preparation of antiserum of small peptide toxins.