Silva Milena Daniela Souza, Lopes Jéssica Amaral, Paloschi Mauro Valentino, Boeno Charles Nunes, Rego Cristina Matiele Alves, de Oliveira Sousa Ortência, Santana Hallison Mota, Dos Reis Valdison Pereira, Serrath Suzanne Nery, da S Setúbal Sulamita, Lima Anderson Maciel, Soares Andreimar M, Zuliani Juliana P
Laboratório de Imunologia Celular Aplicada à Saúde, Fundação Oswaldo Cruz, FIOCRUZ Rondônia, Porto Velho, RO, Brazil.
Laboratório de Biotecnologia de Proteínas e Compostos Bioativos (LABIOPROT), Fundação Oswaldo Cruz, FIOCRUZ Rondônia, Porto Velho, RO, Brazil.
Int J Biol Macromol. 2022 Mar 31;202:597-607. doi: 10.1016/j.ijbiomac.2022.01.107. Epub 2022 Jan 21.
Bothropic venoms contains high amount of secreted phospholipases A (sPLAs) that play a significant role in leukocyte activation and inflammation. Monocytes and lymphocytes are highly functional immune system cells that mediate and provide efficient responses during the inflammation. NLRP3 inflammasome is a multiprotein complex found in immune system cells that is triggered by pathogen- and damage-associated molecular patterns, PAMPs and DAMPs, respectively. PLAs' effect on human peripheral blood mononuclear cells (PBMCs) is still incompletely understood. PBMCs were isolated by density gradient and incubated with RPMI (control), LPS, BthTX-I (PLA-Lys49) or BthTX-II (PLA-Asp49) isolated from Bothrops jararacussu venom, to evaluate viability, and the results showed that there was no cell death. RT-qPCR and immunoblot were used to assess the gene and protein expression of NLRP3 components. Results indicated that there was substantial amplification of ASC, Caspase-1, IL-6, and IL-1β in 1 h and NLRP3 in 2 h. Protein expression was measured, and the results revealed substantial expression of the NLRP3 inflammasome complex after 4 h. IL-1β and LDH was quantified in the supernatant of the cells. Taken together, the findings demonstrate that BthTX-I and BthTX-II activate the NLRP3 inflammasome complex in human PBMCs and contribute to the inflammatory response seen in envenoming.
具窍蝮蛇毒液含有大量分泌型磷脂酶A（sPLA），其在白细胞激活和炎症中起重要作用。单核细胞和淋巴细胞是高功能的免疫系统细胞，在炎症过程中起介导作用并提供有效反应。NLRP3炎性小体是一种存在于免疫系统细胞中的多蛋白复合物，分别由病原体相关分子模式（PAMPs）和损伤相关分子模式（DAMPs）触发。PLA对人外周血单个核细胞（PBMC）的影响仍未完全了解。通过密度梯度分离PBMC，并将其与从具窍蝮蛇毒液中分离出的RPMI（对照）、脂多糖（LPS）、BthTX-I（PLA-Lys49）或BthTX-II（PLA-Asp49）一起孵育，以评估细胞活力，结果显示没有细胞死亡。采用逆转录定量聚合酶链反应（RT-qPCR）和免疫印迹法评估NLRP3组分的基因和蛋白表达。结果表明，1小时内ASC、半胱天冬酶-1、白细胞介素-6和白细胞介素-1β大量扩增，2小时内NLRP3大量扩增。检测蛋白表达，结果显示4小时后NLRP3炎性小体复合物大量表达。对细胞上清液中的白细胞介素-1β和乳酸脱氢酶（LDH）进行定量。综上所述，这些发现表明BthTX-I和BthTX-II激活人PBMC中的NLRP3炎性小体复合物，并促成了蛇咬伤中毒时出现的炎症反应。
