Department of Neurosurgery, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
Department of Neurosurgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
J Neuroinflammation. 2017 Sep 13;14(1):186. doi: 10.1186/s12974-017-0959-6.
The NLRP3 inflammasome is a multiprotein complex that regulates the innate immune inflammatory response by activating caspase-1 and subsequent IL-1β and IL-18. Fluoxetine has been shown to have the anti-inflammatory properties in many disease models. However, the effects and mechanisms of these effects of fluoxetine in early brain injury after subarachnoid hemorrhage (SAH) have not been defined.
The SAH model was induced by an endovascular perforation in adult male Sprague-Dawley (SD) rats weighing 300-320 g. N-Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (AC-YVAD-CMK) was injected intraperitoneally (5 mg/kg) 1 h after SAH. Fluoxetine was administered via intravenous route 6 h after SAH. 3-Methyladenine (3-MA) was intracerebroventricularly injected 20 min before SAH. SAH grade, neurological function, brain water content, propidium iodide (PI) staining, western blot, double immunostaining, and transmission electron microscopy were performed.
Expression of caspase-1 increased and peaked at 24 h after SAH. Caspase activation was along with the increased necrotic cells, which occurred mainly in neurons. Necrotic cell death of microglia and astrocyte were also found. Administration of AC-YVAD-CMK, a caspase-1 inhibitor, reduced the expression of IL-1β and IL-18 and the number of PI-positive cells, attenuated brain edema, and improved neurological function, which was also observed in fluoxetine-treated rats. Furthermore, fluoxetine treatment significantly decreased the expression of NLRP3 and cleaved caspase-1 and upregulated the expression of beclin-1, a marker for autophagy. Finally, the effects of fluoxetine in NLRP3 inflammasome activation were reversed by additional 3-MA administration.
Together, our present study indicated that NLRP3 inflammasome and caspase-1 activation play a deleterious role in early brain injury and fluoxetine mitigates NLRP3 inflammasome and caspase-1 activation through autophagy activation after SAH, providing a potential therapeutic agent for SAH treatment.
NLRP3 炎性小体是一种多蛋白复合物，通过激活半胱天冬酶-1 以及随后的白细胞介素-1β和白细胞介素-18 来调节先天免疫炎症反应。氟西汀已在许多疾病模型中显示出抗炎特性。然而，氟西汀在蛛网膜下腔出血 (SAH) 后早期脑损伤中的这些作用及其机制尚未确定。
通过成年雄性 Sprague-Dawley (SD) 大鼠血管内穿孔诱导 SAH 模型，体重 300-320g。SAH 后 1 小时经腹腔内注射 N-乙酰基-Tyr-Val-Ala-Asp-氯甲基酮 (AC-YVAD-CMK，5mg/kg)。SAH 后 6 小时通过静脉途径给予氟西汀。SAH 前 20 分钟经侧脑室注射 3-甲基腺嘌呤 (3-MA)。进行 SAH 分级、神经功能评分、脑水含量测定、碘化丙啶 (PI) 染色、Western blot、双免疫荧光染色和透射电镜检查。
SAH 后 24 小时 caspase-1 的表达增加并达到峰值。半胱天冬酶激活伴随着坏死细胞的增加，主要发生在神经元中。也发现小胶质细胞和星形胶质细胞的坏死性细胞死亡。给予 caspase-1 抑制剂 AC-YVAD-CMK 可减少 IL-1β和 IL-18 的表达以及 PI 阳性细胞的数量，减轻脑水肿并改善神经功能，这在氟西汀治疗的大鼠中也观察到。此外，氟西汀治疗还显著降低 NLRP3 和裂解的半胱天冬酶-1 的表达，并上调自噬标志物 beclin-1 的表达。最后，通过额外给予 3-MA 给药逆转了氟西汀对 NLRP3 炎性小体激活的作用。
综上所述，本研究表明，NLRP3 炎性小体和半胱天冬酶-1 的激活在早期脑损伤中起有害作用，氟西汀通过激活自噬减轻 SAH 后 NLRP3 炎性小体和半胱天冬酶-1 的激活，为 SAH 的治疗提供了一种潜在的治疗药物。
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