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弓状核内胱硫醚β-合成酶通过PKC的上调和GluN2B磷酸化参与慢性胰腺炎大鼠腹部痛觉过敏

郑航, 朱宏岩, 张晓宇, 王蒙, 肖颖, 徐广银^*, 蒋星红^*

苏州市疼痛基础研究和临床治疗重点实验室，苏州大学神经生物学和生理学系，神经科学研究所，苏州 215123

摘要

有报道显示初级感觉神经元中的硫化氢(H2S)参与内脏痛敏的形成，但其在中枢神经系统中的作用却鲜为人知。该研究旨在探讨下丘脑弓状核(arcuate nucleus, ARC)内H2S和其内源性合成酶是否参与慢性胰腺炎(chronic pancreatitis, CP)腹部痛觉过敏及其潜在机制。成年雄性Sprague-Dawley大鼠胰管内注射三硝基苯磺酸(trinitrobenzene sulfonic acid, TNBS)诱导产生CP模型，运用von Frey filament (VFF)评测大鼠腹部对机械刺激的反应频率，运用Western blot检测ARC内蛋白表达水平。TNBS注射4周后ARC内胱硫醚-β-合成酶(cystathionine β-synthetase, CBS)表达显著上调，而胱硫醚-γ-裂解酶(cystathionine-γ-lyase, CSE)的表达没有明显改变；CP显著提高了NMDA受体GluN2B亚单位的磷酸化水平，而总GluN2B不变；CP也显著上调了蛋白激酶Cγ (PKCγ)在ARC的表达。ARC内微量注射CBS的抑制剂O-(羧甲基)羟胺半盐酸盐(AOAA)能显著降低CP大鼠的腹部疼痛，也翻转了CP大鼠ARC中p-GluN2B和PKCγ的上调；ARC内微量注射GluN2B抑制剂或PKC特异性抑制剂白屈菜赤碱能显著减轻CP大鼠的腹部痛觉过敏，PKC特异性抑制剂能减少p-GluN2B表达。总之，以上数据表明，CP引起了ARC内CBS的上调，并可能通过PKCγ介导了GluN2B的活化，参与CP痛觉过敏。本研究在一定程度上揭示了CP痛觉过敏产生的中枢机制，并有助于发现CP疼痛治疗的新靶标。

关键词： 弓状核; 慢性胰腺炎; 腹部痛觉过敏; 硫化氢; NMDA受体; 蛋白激酶C

分类号：Q42

Upregulation of cystathionine β-synthetase in the arcuate nucleus produces pain hypersensitivity via PKC upregulation and GluN2B phosphorylation in rats with chronic pancreatitis

ZHENG Hang, ZHU Hong-Yan, ZHANG Xiao-Yu, WANG Meng, XIAO Ying, XU Guang-Yin^*, JIANG Xing-Hong^*

Key Laboratory of Pain Basic Research and Clinical Therapy, Department of Neurobiology &； Physiology, Medical College, Institute of Neuroscience, Soochow University, Suzhou 215123, China

Abstract

Hydrogen sulfide (H2S) contributes to visceral hyperalgesia in primary sensory neurons, but its role in central nervous system remains largely unknown. This study was to investigate the roles and underlying mechanisms of H2S and its endogenous synthesis enzymes in the arcuate nucleus (ARC) in rat pancreatic hyperalgesia. Chronic pancreatitis (CP) was induced in male adult Sprague-Dawley rats by intra-pancreatic ductal injection of trinitrobenzene sulfonic acid (TNBS). Abdominal hyperalgesia was assessed by referred somatic behaviors to mechanical stimulation of rat abdomen. Western blot analysis was performed to detect protein expression in the ARC. CP markedly upregulated cystathionine β-synthetase (CBS) expression but did not alter cystathionine-γ-lyase level in the ARC at 4 weeks after TNBS injection. Although the expression of total GluN2B was not altered, CP greatly enhanced the phosphorylation level of GluN2B in the ARC when compared with age- and sex-matched control rats. CP also significantly increased expression of protein kinase Cγ (PKCγ) in the ARC. Arcuate microinjection of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA, an inhibitor of CBS) significantly attenuated abdominal pain in CP rats in a dose-dependent manner and reversed the CP-induced upregulation of p-GluN2B and PKCγ in the ARC. Furthermore, the GluN2B inhibitor or specific PKC inhibitor chelerythrine significantly attenuated abdominal hyperalgesia in CP rats. The p-GluN2B expression was also suppressed by PKC inhibitor. Taken together, our results suggest that the upregulation of CBS in the ARC leads to an activation of GluN2B via PKCγ, which may play an important role in generation of pain hypersensitivity of CP.

Key words: Arcuate nucleus; chronic pancreatitis; abdominal pain; hydrogen sulfide; NMDA receptor; protein kinase C

收稿日期：2016-03-25　　录用日期：2016-06-29

通讯作者：徐广银,蒋星红　　E-mail: guangyinxu@suda.edu.cn, jiangxinhong@suda.edu.cn

引用本文：

郑航, 朱宏岩, 张晓宇, 王蒙, 肖颖, 徐广银, 蒋星红. 弓状核内胱硫醚β-合成酶通过PKC的上调和GluN2B磷酸化参与慢性胰腺炎大鼠腹部痛觉过敏[J]. 生理学报 2016; 68 (5): 575-584.

ZHENG Hang, ZHU Hong-Yan, ZHANG Xiao-Yu, WANG Meng, XIAO Ying, XU Guang-Yin, JIANG Xing-Hong. Upregulation of cystathionine β-synthetase in the arcuate nucleus produces pain hypersensitivity via PKC upregulation and GluN2B phosphorylation in rats with chronic pancreatitis. Acta Physiol Sin 2016; 68 (5): 575-584 (in Chinese with English abstract).