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

doi:10.11958/20220518

Abstract

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

CLC Number:

R730.53

Figures/Tables 13

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^＊＊

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^＊＊

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

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

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

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^＊＊

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

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^＊＊

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

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

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

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^＊＊

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

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