Alluri Himakarnika, Grimsley Marcene, Anasooya Shaji Chinchusha, Varghese Kevin Paul, Zhang Shenyuan L, Peddaboina Chander, Robinson Bobby, Beeram Madhava R, Huang Jason H, Tharakan Binu
From the Departments of Surgery.
the Texas Bioscience Institute, Temple, Texas 76502.
J Biol Chem. 2016 Dec 30;291(53):26958-26969. doi: 10.1074/jbc.M116.735365. Epub 2016 Nov 8.
Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1β (IL-1β) that is up-regulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1β-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1β-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1β treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1β-induced calpain activity significantly (p < 0.05). IL-1β had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1β-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1β-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.
血脑屏障（BBB）破坏以及随之而来的微血管通透性增加和脑水肿是包括创伤性脑损伤（TBI）在内的几种脑部疾病的标志性特征。最近的研究表明，创伤后上调的促炎细胞因子白细胞介素-1β（IL-1β）也会促进血脑屏障功能障碍和通透性增加，但其潜在机制尚不清楚。本研究的目的是确定钙蛋白酶在介导血脑屏障功能障碍和通透性增加中的作用，并测试钙蛋白酶抑制对脑外伤后血脑屏障的影响。在这些研究中，暴露于钙蛋白酶抑制剂（钙蛋白酶抑制剂III和钙蛋白酶抑制蛋白）或转染了钙蛋白酶-1小干扰RNA的大鼠脑微血管内皮细胞单层显示出IL-1β诱导的单层通透性增加有所减轻。钙蛋白酶抑制可防止IL-1β诱导的紧密连接处闭合蛋白-1（ZO-1）丢失以及F-肌动蛋白细胞骨架组装的改变。IL-1β处理对ZO-1基因（tjp1）或蛋白表达没有影响。通过钙蛋白酶抑制剂III和钙蛋白酶抑制蛋白抑制钙蛋白酶可显著降低IL-1β诱导的钙蛋白酶活性（p < 0.05）。IL-1β对细胞内钙动员或内皮细胞活力没有可检测到的影响。此外，在使用伊文思蓝测定法和活体显微镜研究的TBI小鼠控制皮质撞击模型中，钙蛋白酶抑制可维持血脑屏障的完整性/通透性。这些研究表明，钙蛋白酶-1通过改变紧密连接完整性、促进ZO-1移位和细胞骨架组装紊乱，介导IL-1β诱导的血脑屏障完整性和通透性丧失。IL-1β介导的通透性改变既不是由于ZO-1表达的变化，也不是由于细胞活力的变化。钙蛋白酶抑制对TBI诱导的血脑屏障通透性增加具有有益作用。
