Cui Xu, Xu Xin, Ju Zhihai, Wang Guyan, Xi Chunhua, Li Junfa
Department of Anesthesiology, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
Department of Neurobiology and Center of Stroke, School of Basic Medical Science, Beijing Institute for Brain Disorders, Capital Medical University, Beijing, China.
J Cell Biochem. 2021 Apr 9. doi: 10.1002/jcb.29929.
Herkinorin is a novel opioid receptor agonist. Activation of opioid receptors, a member of G protein coupled receptors (GPCRs), may play an important role in Herkinorin neuroprotection. GPCRs may modulate NOD-like receptor protein 3 (NLRP3)-mediated inflammatory responses in the mechanisms of inflammation-associated disease and pathological processes. In this study, we investigated the effects of Herkinorin on NLRP3 and the underlying receptor and molecular mechanisms in oxygen-glucose deprivation/reperfusion (OGD/R)-treated rat cortex neurons. First, Western blot analysis showed that Herkinorin can inhibit the activation of NLRP3 and Caspase-1, decrease the expression of interleukin (IL)-1β, and decrease the secretion of IL-6 and tumour necrosis factor α detected by enzyme-linked immunosorbent assay in OGD/R-treated neurons. Then we found that Herkinorin downregulated NLRP3 levels by inhibiting the activation of nuclear factor kappa B (NF-κB) pathway, reducing the phosphorylation level of p65 and IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Instead, both the mu opioid receptor (MOR) inhibitor, β-funaltrexamine, and MOR knockdown reversed the effects of Herkinorin on NLRP3 (p < .05 or .01, n = 3 per group). Further, we found that the level of β-arrestin2 decreased in the cell membrane and increased in the cytoplasm after Herkinorin pretreatment in OGD/R-treated neurons. In co-immunoprecipitation experiments, Herkinorin increased the binding of IκBα with β-arrestin2, decreased the ubiquitination level of IκBα, and β-arrestin2 knockdown reversed the effects of Herkinorin on IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Our data demonstrated that Herkinorin negatively regulated NLRP3 inflammasome to alleviate neuronal ischemic injury through inhibiting NF-κB pathway mediated primarily by MOR activation. Inhibition of the NF-κB pathway by Herkinorin may be achieved by decreasing the ubiquitination level of IκBα, in which β-arrestin2 may play an important role.
赫基诺林是一种新型阿片受体激动剂。阿片受体作为G蛋白偶联受体（GPCRs）家族的一员，其激活可能在赫基诺林的神经保护作用中发挥重要作用。在炎症相关疾病和病理过程中，GPCRs可能在调节NOD样受体蛋白3（NLRP3）介导的炎症反应中发挥作用。在本研究中，我们研究了赫基诺林对氧糖剥夺/再灌注（OGD/R）处理的大鼠皮层神经元中NLRP3的影响及其潜在的受体和分子机制。首先，蛋白质免疫印迹分析表明，赫基诺林可以抑制OGD/R处理的神经元中NLRP3和半胱天冬酶-1的激活，降低白细胞介素（IL）-1β的表达，并通过酶联免疫吸附测定法检测到降低IL-6和肿瘤坏死因子α的分泌。然后我们发现，赫基诺林通过抑制核因子κB（NF-κB）通路的激活来下调OGD/R处理的神经元中NLRP3水平，降低p65和IκBα的磷酸化水平（p < 0.05或0.01，每组n = 3）。相反，μ阿片受体（MOR）抑制剂β-氟纳曲胺和MOR基因敲低均逆转了赫基诺林对NLRP3的作用（p < 0.05或0.01，每组n = 3）。此外，我们发现，在OGD/R处理的神经元中，赫基诺林预处理后，β-抑制蛋白2在细胞膜中的水平降低，在细胞质中的水平升高。在免疫共沉淀实验中，赫基诺林增加了IκBα与β-抑制蛋白2的结合，降低了IκBα的泛素化水平，β-抑制蛋白2基因敲低逆转了赫基诺林对OGD/R处理的神经元中IκBα的作用（p < 0.05或0.01，每组n = 3）。我们的数据表明，赫基诺林通过抑制主要由MOR激活介导的NF-κB通路，对NLRP3炎性小体进行负调控，从而减轻神经元缺血性损伤。赫基诺林对NF-κB通路的抑制作用可能是通过降低IκBα的泛素化水平来实现的，其中β-抑制蛋白2可能发挥重要作用。
