Department of Surgery, Baylor Scott and White Medical Center, Baylor Scott and White Research Institute, Temple, TX, USA.
Texas A&M University Health Science Center College of Medicine, Temple, TX, USA.
Sci Rep. 2022 Mar 30;12(1):5415. doi: 10.1038/s41598-022-09394-4.
The main objective of this study was to determine the cellular and molecular effects of doxycycline on the blood-brain barrier (BBB) and protection against secondary injuries following traumatic brain injury (TBI). Microvascular hyperpermeability and cerebral edema resulting from BBB dysfunction after TBI leads to elevation of intracranial pressure, secondary brain ischemia, herniation, and brain death. There are currently no effective therapies to modulate the underlying pathophysiology responsible for TBI-induced BBB dysfunction and hyperpermeability. The loss of BBB integrity by the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) is critical to TBI-induced BBB hyperpermeability, and doxycycline possesses anti-MMP-9 effect. In this study, the effect of doxycycline on BBB hyperpermeability was studied utilizing molecular modeling (using Glide) in silico, cell culture-based models in vitro, and a mouse model of TBI in vivo. Brain microvascular endothelial cell assays of tight junction protein immunofluorescence and barrier permeability were performed. Adult C57BL/6 mice were subjected to sham versus TBI with or without doxycycline treatment and immediate intravital microscopic analysis for evaluating BBB integrity. Postmortem mouse brain tissue was collected to measure MMP-9 enzyme activity. It was found that doxycycline binding to the MMP-9 active sites have binding affinity of -7.07 kcal/mol. Doxycycline treated cell monolayers were protected from microvascular hyperpermeability and retained tight junction integrity (p < 0.05). Doxycycline treatment decreased BBB hyperpermeability following TBI in mice by 25% (p < 0.05). MMP-9 enzyme activity in brain tissue decreased with doxycycline treatment following TBI (p < 0.05). Doxycycline preserves BBB tight junction integrity following TBI via inhibiting MMP-9 activity. When established in human subjects, doxycycline, may provide readily accessible medical treatment after TBI to attenuate secondary injury.
本研究的主要目的是确定强力霉素对血脑屏障（BBB）的细胞和分子作用，以及在创伤性脑损伤（TBI）后防止继发损伤。TBI 后 BBB 功能障碍导致的微血管通透性增加和脑水肿会导致颅内压升高、继发性脑缺血、脑疝和脑死亡。目前，尚无有效的治疗方法来调节导致 TBI 诱导的 BBB 功能障碍和通透性增加的潜在病理生理学。蛋白水解酶基质金属蛋白酶-9（MMP-9）对 BBB 完整性的破坏对 TBI 诱导的 BBB 通透性增加至关重要，强力霉素具有抗 MMP-9 作用。在这项研究中，使用分子建模（使用 Glide）进行了体内实验，体外细胞培养模型以及体内 TBI 小鼠模型来研究强力霉素对 BBB 通透性的影响。进行了脑微血管内皮细胞紧密连接蛋白免疫荧光和屏障通透性的测定。对成年 C57BL/6 小鼠进行假手术对照和 TBI 对照，并用强力霉素治疗，并立即进行活体显微镜分析以评估 BBB 完整性。死后收集小鼠脑组织以测量 MMP-9 酶活性。结果发现，强力霉素与 MMP-9 活性部位的结合具有-7.07 kcal/mol 的结合亲和力。强力霉素处理的细胞单层可防止微血管通透性增加并保持紧密连接完整性（p <0.05）。强力霉素治疗可使 TBI 后小鼠的 BBB 通透性降低 25％（p <0.05）。TBI 后强力霉素治疗可降低脑组织中的 MMP-9 酶活性（p <0.05）。强力霉素通过抑制 MMP-9 活性来保持 TBI 后 BBB 的紧密连接完整性。如果在人类中得到证实，强力霉素可能为 TBI 后提供易于获得的治疗方法，以减轻继发性损伤。
