ANA-12靶向抑制BDNF/TrkB信号缓解奥沙利铂诱导化疗大鼠的痛觉行为

doi:10.11958/20220518

天津医药 ›› 2023, Vol. 51 ›› Issue (1): 35-40.doi: 10.11958/20220518

ANA-12靶向抑制BDNF/TrkB信号缓解奥沙利铂诱导化疗大鼠的痛觉行为

赵佳佳¹(), 万文军², 杨荷雨¹, 谢敏¹, 刘玲¹^,^∆()

1 湖北科技学院医学部药学院（邮编437100）
2 湖北科技学院附属浠水医院

收稿日期:2022-04-15 修回日期:2022-07-20 出版日期:2023-01-15 发布日期:2023-01-17
通讯作者: 刘玲 E-mail:1396904895@qq.com;liuling0306@163.com
作者简介:赵佳佳（1997），女，硕士在读，主要从事痛觉传导机制研究。E-mail：1396904895@qq.com
基金资助:
国家自然科学基金青年基金资助项目(81901149);国家自然科学基金青年基金资助项目(32100823);湖北省教育厅科学研究计划项目(Q20212804)

ANA-12 relieves oxaliplatin-induced chemotherapy pain in rats by targetly inhibiting BDNF/TrkB signal

ZHAO Jiajia¹(), WAN Wenjun², YANG Heyu¹, XIE Min¹, LIU Ling¹^,^∆()

1 School of Pharmacy, Xianning Medical College, Hubei University of Science and Technology, Xianning 437100, China
2 Xishui Hospital Affiliated to Hubei University of Science and Technology

Received:2022-04-15 Revised:2022-07-20 Published:2023-01-15 Online:2023-01-17
Contact: LIU Ling E-mail:1396904895@qq.com;liuling0306@163.com

赵佳佳, 万文军, 杨荷雨, 谢敏, 刘玲. ANA-12靶向抑制BDNF/TrkB信号缓解奥沙利铂诱导化疗大鼠的痛觉行为[J]. 天津医药, 2023, 51(1): 35-40.

ZHAO Jiajia, WAN Wenjun, YANG Heyu, XIE Min, LIU Ling. ANA-12 relieves oxaliplatin-induced chemotherapy pain in rats by targetly inhibiting BDNF/TrkB signal[J]. Tianjin Medical Journal, 2023, 51(1): 35-40.

摘要/Abstract

摘要：

目的探讨ANA-12靶向抑制脑源性神经营养因子（BDNF）/酪氨酸激酶受体B（TrkB）信号缓解奥沙利铂（OXA）化疗痛的作用及机制。方法将18只雄性SD大鼠按照随机数字表法分为3组：对照组、OXA组以及OXA+ANA-12组，每组6只。OXA组和OXA+ANA-12组腹腔注射OXA（4 mg/kg，连续5 d）构建化疗痛模型，模型构建成功后OXA+ANA-12组进行ANA-12鞘内给药（20 g/L）。给药完毕后，对各组大鼠进行痛觉行为学检测，记录大鼠自发性缩足反射次数和机械痛觉阈值的变化;采用HE染色、免疫印记和免疫荧光检测脊髓炎性细胞浸润情况及白细胞介素（IL）-1β、肿瘤坏死因子（TNF）-α、离子钙结合衔接分子1（Iba1）、BDNF、TrkB和核转录因子κB（NF-κB）表达水平变化。结果行为学分析显示，与对照组相比，OXA连续注射可显著增加自发缩足次数以及机械刺激敏感性;与OXA组相比，鞘内注射ANA-12显著降低自发缩足次数，增加机械性痛觉阈值。形态学及蛋白表达分析显示，与对照组相比，OXA诱导脊髓炎症，激活BDNF/TrkB信号，上调IL-1β、TNF-α、Iba1和NF-κB的表达;与OXA组相比，ANA-12显著抑制BDNF/TrkB信号，下调IL-1β、TNF-α、Iba1和NF-κB的表达水平。结论 ANA-12鞘内给药通过阻断BDNF/TrkB信号来抑制脊髓炎症，缓解化疗痛。

关键词: 化学疗法，肿瘤，局部灌注, 脊髓炎, 小神经胶质细胞, 脑源性神经营养因子, 受体，trkB, 奥沙利铂, ANA-12

Abstract:

Objective To investigate the effect and mechanism of ANA-12 on relieving oxaliplatin (OXA) induced neuropathic pain by targeting brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) signaling in rats. Methods Eighteen male SD rats were randomly divided into 3 groups according to table of random number: the control group, the OXA treated group and the OXA + ANA-12 group (OXA+ANA-12), with 6 rats in each group. The OXA group and the OXA + ANA-12 group received an intraperitoneal injection of OXA (4 mg/kg for 5 days) to construct a chemotherapeutic pain model. After the model was successfully established, ANA-12 (20 g/L) was intrathecally administered in the OXA + ANA-12 group. After administration, pain behavior tests of rats in each group were performed, and changes in number of spontaneous flinches and mechanical pain threshold were recorded. The infiltration of spinal inflammatory cells and changes in expression level of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, ionized calcium binding adapter molecule 1 (Iba1), BDNF, TrkB and nuclear factor kappa B (NF-κB) were detected by HE staining, immunofluorescence and Western blot assay. Results Compared with the control group, behavioral analysis showed that continuous injection of OXA significantly induced pain hyperalgesia and increased number of spontaneous flinches. Compared with the OXA group, intrathecal injection of ANA-12 significantly decreased the number of spontaneous flinches and increased mechanical pain threshold of rats. Morphological and protein expression analysis showed that OXA administration induced spinal inflammation, up-regulated BDNF/TrkB signaling and increased the expression levels of IL-1β, TNF-α, Iba1 and NF-κB compared with those of the control group. Compared with the OXA group, ANA-12 treatment significantly inhibited BDNF/TrkB signaling and down-regulated the expression levels of IL-1β, TNF-α, Iba1 and NF-κB. Conclusion Intrathecal administration of ANA-12 inhibits spinal inflammation and relieves chemotherapy pain by blocking BDNF/TrkB signal.

Key words: chemotherapy, cancer, regional perfusion, myelitis, microglia, brain-derived neurotrophic factor, receptor, trkB, oxaliplatin, ANA-12

中图分类号:

R730.53

图/表 13

表1 3组大鼠行为学检测结果比较（n=3，$\bar{x}±s$）

Tab.1 Comparison of behavioral test results in rats between the three groups

组别	PWT（g）	缩足次数（次）
对照组	15.10±0.26	5.33±0.58
OXA组	6.00±0.26^a	25.00±1.00^a
OXA+ANA-12组	10.20±0.46^ab	6.67±1.15^b
F	533.400^＊＊	407.625^＊＊

表2 各组大鼠脊髓炎症评分和IL-1β荧光强度比较（n=3，$\bar{x}±s$）

Tab.2 Comparison of inflammatory score and fluorescence intensity of IL-1β in spinal cord of rats between the three groups

组别	炎症评分（分）	IL-1β
对照组	2.28±0.10	1.00±0.00
OXA组	2.94±0.07^a	1.64±0.11^a
OXA+ANA-12组	2.35±0.06^b	0.77±0.07^ab
F	72.300^＊＊	109.509^＊＊

图1 各组大鼠脊髓背角炎性细胞浸润情况（HE染色，标尺= 20 μm）

Fig. 1 Inflammatory cell infiltration of spinal dorsal horn in each group (HE staining, scale bar=20 μm)

图2 各组大鼠脊髓背角IL-1β的表达（免疫荧光染色，标尺=20 μm）

Fig. 2 Expression of IL-1β in spinal dorsal horn of rats in each group (Immunofluorescent staining, scale bar=20 μm)

图3 Western blot检测各组大鼠脊髓组织促炎细胞因子IL-1β和TNF-α表达水平

Fig.3 Expression levels of pro-inflammatory cytokines IL-1β and TNF-α in spinal cord of rats in each group detected by Western blot assay

表3 各组大鼠脊髓组织中IL-1β和TNF-α表达水平比较

Tab.3 Comparison of expression levels of IL-1β and TNF-α in spinal cord of rats between the three groups （n=3，$\bar{x}±s$）

组别	IL-1β	TNF-α
对照组	1.00±0.00	1.00±0.00
OXA组	2.37±0.06^a	2.04±0.14^a
OXA+ANA-12组	1.57±0.09^ab	1.58±0.16^ab
F	355.028^＊＊	54.769^＊＊

图4 各组大鼠脊髓背角小胶质细胞标志物Iba1的表达（免疫荧光染色，标尺=20 μm）

Fig.4 Expressions of Iba1 in spinal dorsal horn of rats in each group (Immunofluorescent staining, scale bar=20 μm)

表4 各组大鼠脊髓组织中Iba1表达水平比较

Tab.4 Comparison of expression levels of Iba1 in spinal cord of rats between the three groups

组别	荧光强度	蛋白表达水平
对照组	1.00±0.00	1.00±0.00
OXA组	1.71±0.04^a	1.40±0.05^a
OXA+ANA-12组	1.11±0.09^b	0.88±0.06^ab
F	144.794^＊＊	99.441^＊＊

图5 Western blot检测各组大鼠脊髓组织小胶质细胞标记物Iba1表达水平

Fig. 5 Expression levels of microglial marker Iba1 in spinal cord of rats in each group detected by Western blot assay

图6 各组大鼠脊髓背角BDNF的表达（免疫荧光染色，标尺=20 μm）

Fig.6 Expression of BDNF in spinal dorsal horn of rats in each group (Immunofluorescent staining, scale bar=20 μm)

图7 Western blot检测各组大鼠脊髓组织中BDNF、磷酸化TrkB（Y705）和总的TrkB表达水平

Fig.7 Expression levels of BDNF, phosphorylated TrkB (Y705) and total TrkB in spinal cord of rats in each group detected by Western blot assay

表5 各组大鼠脊髓组织中BDNF、磷酸化TrkB（Y705）和总的TrkB表达水平的作用比较

Tab.5 Comparison of expression levels of BDNF, phosphorylated TrkB (Y705) and total TrkB in spinal cord between three groups of rats

组别	荧光强度	BDNF	pTrkB/TrkB
对照组	1.00±0.00	1.00±0.00	1.00±0.00
OXA组	1.60±0.12^a	1.42±0.04^a	1.70±0.03^a
OXA+ANA-12组	0.70±0.02^ab	1.10±0.06^ab	0.88±0.05^ab
F	122.231^＊＊	99.464^＊＊	466.694^＊＊

图8 Western blot检测各组大鼠脊髓组织中NF-κB表达水平

Fig.8 Expression levels of NF-κB in spinal cord of rats detected by Western blot assay

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ANA-12靶向抑制BDNF/TrkB信号缓解奥沙利铂诱导化疗大鼠的痛觉行为

ANA-12 relieves oxaliplatin-induced chemotherapy pain in rats by targetly inhibiting BDNF/TrkB signal

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