Development of sandwich ELISA and lateral flow assay for the detection of Bungarus multicinctus venom.

Snakebite envenoming adversely affects human health and life worldwide. Presently, no suitable diagnostic tools for snakebite envenoming are available in China. Therefore, we sought to develop reliable diagnostic tests for snakebite management. We conducted affinity purification experiments to prepare species-specific antivenom antibody (SSAb). In brief, affinity chromatography with an antibody purification column (Protein A) was conducted to purify immunoglobulin G from Bungarus multicinctus (BM) venom hyperimmunized rabbit serum. The cross-reactive antibodies were removed from commercial BM antivenin by immune adsorption on the affinity chromatography columns of the other three venoms, Bungarus Fasciatus (FS), Naja atra (NA), and O. hannah (OH), generating SSAb. The results of western blot analysis and enzyme-linked immunosorbent assay (ELISA) showed the high specificity of the prepared SSAb. The obtained antibodies were then applied to ELISA and lateral flow assay (LFA) to detect BM venom. The resulting ELISA and LFA could specifically and rapidly detect BM venom in various samples with the limits of quantification as 0.1 and 1 ng/ml, respectively. This method could effectively detect snake venom in experimentally envenomed rats (simulating human envenomation), which could distinguish positive and negative samples within 10-15 min. This method also showed promise in serving as a highly useful tool for a rapid clinical distinguishing of BM bites and rational use of antivenom in emergency centers. The study also revealed cross-reactivity between BM and heterogenous venoms, suggesting that they shared common epitopes, which is of great significance for developing detection methods for venoms of the snakes belonging to the same family.

Effect of Several Naja atra Antivenom Injection Methods on the Rabbit Model of Naja naja atra Bite Poisoning.

Snakebite is a global public health concern, which often occurs in tropical and subtropical underdeveloped areas, but it is often neglected. In the southern China, Naja naja atra (Chinese cobra) is a common venomous snake that causes swelling and necrosis of local tissues, even amputation and death. Currently, the main therapy is the administration of Naja atra antivenom, which greatly reduces mortality. However, the antivenom is not particularly effective in the improvement of local tissue necrosis. Clinically, antivenom is mainly administered intravenously. We speculated that the method of injection influences the efficacy of antivenom. In this study, the rabbit model was used to explore the effects of different antivenom injection methods on systemic and local poisoning symptoms. If topical injection of antivenom contributes to ameliorate tissue necrosis, then we need to reconsider the use of Naja atra antivenom.

Effects of Mitochondrial ATP-Sensitive Potassium Channel in Rats with Acute Myocardial Infarction and Its Association with the AKT/mTOR Pathway.

BACKGROUND: Myocardial infarction is associated with the autophagy and apoptosis of cardiomyocytes, and the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway plays a crucial role in this mechanism. METHODS: Acute myocardial infarction rat models were assessed 0.5, 2, 4, and 6 hours after the induction of the myocardial infarction using hematoxylin and eosin staining, triphenyl tetrazolium chloride staining, myocardial enzyme measurements, and levels of autophagic activity. Additionally, diazoxide, 5-hydroxydecanoate, and LY294002 were intraperitoneally administered to rat models at peak myocardial injury to assess their effects on cardiac injury. The expression levels of autophagy-related and apoptosis-related proteins, as well as p-AKT and p-mTOR, were measured. Electron microscopy was used to assess the ultrastructure and the number of autophagosomes in the cardiac tissue. RESULTS: We demonstrated that the degree of myocardial injury and the level of autophagy were significantly elevated in the experimental cohort compared with the control cohort. In addition, the myocardial infarct size was significantly smaller in diazoxide-treated acute myocardial infarction rats compared with untreated rats. Diazoxide also decreased the levels of myocardial injury markers, autophagy, and apoptosis, while it also induced the levels of AKT and mTOR phosphorylation, decreased the number of autophagosomes, and improved the myocardial ultrastructure of the acute myocardial infarction rats. 5-Hydroxydecanoate treatment resulted in an opposite effect to those observed upon diazoxide treatment. LY294002 was also able to reverse diazoxide treatment effects. CONCLUSION: Peak levels of myocardial tissue injury and autophagy were observed 2 hours post-acute myocardial infarction induction in rats. Diazoxide treatment inhibited myocardial autophagy and apoptosis while protecting cardiac tissue from ischemic injury, which is likely to have proceeded through activation of the AKT/mTOR pathway.

Effects of ferroptosis in myocardial ischemia/reperfusion model of rat and its association with Sestrin 1.

BACKGROUND: Ferroptosis is a type of iron-dependent programmed cell death. The inhibition of ferroptosis has been reported to alleviate myocardial ischemia/reperfusion injury (IRI). However, it is unknown whether this protective effect occurs in the ischemia or reperfusion phase. Sestrin 1 (Sesn1) possesses remarkable cytoprotective functions to diverse cellular stresses. However, whether Sesn1 is involved in the regulatory process of ferroptosis during myocardial IRI is unknown. OBJECTIVES: This study aimed to simulate an acute myocardial infarction (AMI) that occurs in rats within 6 h, verify the occurrence and effects of ferroptosis in the phases of ischemia and reperfusion, and further explore the relationship between ferroptosis, IRI and Sesn1. MATERIAL AND METHODS: The hearts of Sprague Dawley (SD) rats undergoing ischemia for varying lengths of time or having undergone ischemia followed by varying lengths of reperfusion were examined. The occurrence of ferroptosis was verified by detecting changes in ferroptosis biomarkers. In addition, ferrstatin-1 (Fer-1) was administered to demonstrate the effect of ferroptosis in myocardial IRI and to detect changes in Sesn1. RESULTS: The results showed that the myocardial damage was more severe with more prolonged myocardial ischemia. There were no significant changes in ferroptosis biomarkers in cardiac tissues during the ischemia phase, the levels of iron and malondialdehyde (MDA) were elevated, and the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) were decreased after myocardial IRI. Compared to the ischemia/reperfusion (I/R) group, the treatment with Fer-1 before reperfusion can attenuate myocardial IRI, reverse the decrease in GPX4 and FTH1 expression, and decrease the rise in iron content and MDA. In addition, we found that the expression of Sesn1 was reduced in hearts that suffered IRI; however, the treatment with Fer-1 can reverse this situation. CONCLUSIONS: Ferroptosis occurred during the myocardial reperfusion phase but not ischemia. The inhibition of ferroptosis exerted beneficial effects on myocardial IRI, providing a theoretical basis for targeted therapy in patients with AMI. Sestrin 1, regulated by ferroptosis, may play an important role in myocardial IRI.

Changes in Serum Enzymes and Related Mechanisms of Respiratory Dysfunction in Patients after Venomous Snake Bite and Analysis of Anti-Venomous Snake Serum Treatment.

To explore the changes in serum enzymes in patients with a snake bite, the treatment of respiratory dysfunction, and the clinical effect of anti-snake serum treatment. Fifty snake bite patients admitted to the emergency medicine department were selected and rolled into a light group (n=27), heavy group (n=15), and critical group (n=8). Anti-venomous snake serum was injected intravenously. Patients with severe respiratory dysfunction were treated with mechanical ventilation. The white blood cell (WBC), C-reactive protein (CRP), interleukin-6 (IL-6), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) counts of the heavy group and the critical group were higher versus light group (P<0.05). The WBC, CRP, IL-6, ALT, AST, BUN, and Cr of the critical group were higher versus the heavy group (P<0.05). The prothrombin time (PT), activated partial thrombin time (APTT), and thrombin time (TT) of the heavy group and critical group were longer versus the light group (P<0.05). The PT, APTT, and TT of the critical group were longer than the heavy group (P<0.05). The fibrinogen (FIB) of the light group was higher in contrast to that in the other two groups (P<0.05), while the critical group was the lowest (P<0.05). In summary, the severity of snakebites in patients can be evaluated according to the indexes of WBC, IL-6, coagulation function, and liver and kidney function.