Federal Rural University of Pernambuco, Recife, PE, Brazil.
Federal University of Vale do São Francisco, Petrolina, PE, Brazil.
Phytomedicine. 2022 Jul 20;102:154186. doi: 10.1016/j.phymed.2022.154186. Epub 2022 May 18.
The osmotin from the medicinal plant Calotropis procera (CpOsm) has characteristics similar to adiponectin, a human protein with immunoregulatory actions.
This study aimed to investigate whether recombinant osmotin inclusion bodies from C. procera (IB/rCpOsm) produced in E. coli BL21(DE3) can prevent infection-induced inflammation. A virulent strain of Listeria monocytogenes was used as an infection model.
Cells of E. coli BL21(DE3) carrying the plasmid pET303-CpOsm were used to express the recombinant osmotin, which accumulated at reasonable levels as inclusion bodies (IB/rCpOsm). IB/rCpOsm were purified from induced cells and SDS-polyacrylamide gel electrophoresis followed by mass spectrometry analyses confirmed the identity of the major protein band (23 kDa apparent molecular mass) as CpOsm. Peritoneal macrophages (pMØ) from Swiss mice were cultured with IB/rCpOsm (1 or 10 µg/ml) in 96-well plates and then infected with L. monocytogenes. IB/rCpOsm (0.1, 1 or 10 mg/kg) was also administered intravenously to Swiss mice, which were then infected intraperitoneally with L. monocytogenes.
Pretreatment of the pMØ with IB/rCpOsm significantly increased cell viability after infection and reduced the intracellular bacterial load. The infiltration of neutrophils into the peritoneal cavity of mice pretreated with IB/rCpOsm at 10 mg/kg (but not 0.1 and 1 mg/kg) was reduced after infection. In these mice, the bacterial load was high in the peritoneal fluid and the liver, but histological damage was discrete. The treatments with IB/rCpOsm at 10 mg/kg significantly increased the expression of the anti-inflammatory cytokine IL-10.
This study shows that recombinant osmotin inclusion bodies from C. procera were bioactive and prompted anti-inflammatory actions at therapeutic dosages in the L. monocytogenes infection model.
来自药用植物牛角瓜（CpOsm）的 osmotin 具有类似于脂联素的特性，脂联素是一种具有免疫调节作用的人类蛋白。
本研究旨在探讨大肠杆菌 BL21(DE3) 中产生的重组 osmotin 包涵体（IB/rCpOsm）是否可以预防感染诱导的炎症。使用李斯特菌单核细胞增生李斯特菌作为感染模型。
携带质粒 pET303-CpOsm 的大肠杆菌 BL21(DE3) 细胞用于表达重组 osmotin，其以包涵体（IB/rCpOsm）的形式合理积累。从诱导细胞中纯化 IB/rCpOsm，并用 SDS-聚丙烯酰胺凝胶电泳和质谱分析证实主要蛋白带（23 kDa 表观分子量）为 CpOsm。在 96 孔板中用 IB/rCpOsm（1 或 10 μg/ml）培养瑞士小鼠的腹腔巨噬细胞（pMØ），然后用李斯特菌单核细胞增生李斯特菌感染。IB/rCpOsm（0.1、1 或 10 mg/kg）也通过静脉内给予瑞士小鼠，然后用李斯特菌单核细胞增生李斯特菌感染小鼠的腹腔。
IB/rCpOsm 预处理可显著提高感染后 pMØ 的细胞活力并降低细胞内细菌负荷。用 IB/rCpOsm 预处理（但不是 0.1 和 1 mg/kg）的小鼠腹腔内中性粒细胞浸润减少。在这些小鼠中，腹腔液和肝脏中的细菌负荷很高，但组织学损伤轻微。用 IB/rCpOsm 处理 10 mg/kg 可显著增加抗炎细胞因子 IL-10 的表达。
本研究表明，来自牛角瓜的重组 osmotin 包涵体具有生物活性，并在单核细胞增生李斯特菌感染模型中以治疗剂量引发抗炎作用。
