赵峰,樊少卿,程晓燕,李小娜,李长生,马浩杰
ZHAO Feng,FAN Shaoqing,CHENG Xiaoyan,LI Xiaona,LI Changsheng,MA Haojie

• 1:河南省中医院麻醉科
• 2:河南省肿瘤医院麻醉与围术期医学科

摘要(Abstract)：

目的 探讨山奈酚(kaempferol, KAE)对脊神经结扎(spinal nerve ligation, SNL)大鼠神经性疼痛的作用机制。方法 采用脂多糖(lipopolysaccharide, LPS)诱导大鼠星形胶质细胞建立炎症模型，用山奈酚单独或与miR-340-5p抑制剂共同处理细胞，RT-qPCR检测miR-340-5p表达，ELISA检测炎症因子白细胞介素(interleukin, IL)-1β和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平；荧光素酶报告基因实验验证AKT3与miR-340-5p的靶向关系；Western blotting检测自噬标志蛋白的表达。此外，建立SNL大鼠模型，山奈酚口服给药后，检测miR-340-5p表达，炎症因子以及自噬标志蛋白水平，并测定大鼠机械缩爪阈值和缩爪潜伏期。结果 在LPS诱导的星形胶质细胞中，山奈酚上调miR-340-5p表达，抑制IL-1β和TNF-α分泌，转染miR-340-5p抑制剂则逆转山奈酚对miR-340-5p表达的促进作用以及对炎症因子分泌的抑制作用。AKT3被确定是miR-340-5p的靶基因。山奈酚上调miR-340-5p表达，进而阻断AKT3/mTOR信号通路，增强星形胶质细胞自噬。山奈酚给药的SNL大鼠表现出miR-340-5p表达上调，炎症因子分泌减少，自噬相关蛋白表达增加，机械缩爪阈值增加，缩爪潜伏期延长。结论 山奈酚通过上调miR-340-5p靶向抑制AKT3/mTOR信号通路，抑制星形胶质细胞炎症反应，增强自噬，减轻SNL大鼠神经性疼痛。
Objective To explore the effects and mechanism of kaempferol(KAE)on neuropathic pain in spinal nerve ligation(SNL)rats.Methods Rat astrocytes were induced by lipopolysaccharide(LPS)to establish inflammation model, and cells were treated with kaempferol alone or together with miR-340-5p inhibitor.The expression of miR-340-5p was measured by RT-qPCR.The levels of inflammatory cytokines interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by ELISA.Moreover, luciferase reporter gene assay verified the binding between AKT3 and miR-340-5p.The expression of autophagy marker proteins was detected by Western blotting analysis.Furthermore, SNL rat model was constructed and kaempferol was administered orally in rats.We detected miR-340-5p expression, inflammatory cytokine and autophagy marker protein levels, the paw withdrawal threshold and the paw withdrawal latency of rats.Results Kaempferol upregulated the expression of miR-340-5p, and inhibited the secretion of IL-1β and TNF-α in LPS-induced astrocytes.Transfection of miR-340-5p inhibitor reversed the promoting effect of kaempferol on the expression of miR-340-5p and the inhibiting effect on the secretion of inflammatory factors.AKT3 was identified as the target gene of miR-340-5p.Kaempferol increased the expression of miR-340-5p, blocked the AKT3/mTOR signaling pathway, and enhanced autophagy in astrocytes.SNL rats orally administered kaempferol showed the upregulated expression of miR-340-5p, decreased secretion of inflammatory factors, increased expression of autophagy-related proteins, increased mechanical paw withdrawal threshold and prolonged paw withdrawal latency.Conclusion Kaempferol reduces inflammation, enhances autophagy in astrocytes, and relieves neuropathic pain in SNL rats via inhibiting AKT3/mTOR signaling pathway by upregulating miR-340-5p.

关键词(KeyWords)： 神经性疼痛;山奈酚;miR-340-5p;自噬;AKT3/mTOR信号通路
neuropathic pain;kaempferol;miR-340-5p;autophagy;AKT3/mTOR signaling pathway

Abstract:

Keywords:

基金项目(Foundation): 河南省医学科技攻关计划项目(201702242);; 河南省科技攻关国际联合项目(182102410015)

作者(Author): 赵峰,樊少卿,程晓燕,李小娜,李长生,马浩杰
ZHAO Feng,FAN Shaoqing,CHENG Xiaoyan,LI Xiaona,LI Changsheng,MA Haojie

DOI: 10.14066/j.cnki.cn21-1349/r.2021.0423

参考文献(References)：

• [1] FINNERUP N B,SINDRUP S H,JENSEN T S.Recent advances in pharmacological treatment of neuropathic pain[J].F1000 Med Rep,2010,2:52.
• [2] LIN M K,YU Y L,CHEN K C,et al.Kaempferol from Semen cuscutae attenuates the immune function of dendritic cells[J].Immunobiology,2011,216(10):1103-1109.
• [3] DEVI K P,MALAR D S,NABAVI S F,et al.Kaempferol and inflammation:from chemistry to medicine[J].Pharmacol Res,2015,99:1-10.
• [4] CHEN M J,JIN H W,CHEN Z S,et al.The effect of kaempferol on COX-2 and Inos expression and generated production in LPS-induced RAW264.7 cells[J].Journal of Shenyang Pharmaceutical University(沈阳药科大学学报),2013,30(12):982-985,990.
• [5] QIN Y,CUI W,YANG X,et al.Kaempferol inhibits the growth and metastasis of cholangiocarcinoma in vitro and in vivo[J].Acta Biochim Biophys Sin,2016,48(3):238-245.
• [6] ALKHALIDY H,MOORE W,ZHANG Y,et al.Small molecule kaempferol promotes insulin sensitivity and preserved pancreatic β-cell mass in middle-aged obese diabetic mice[J].J Diabetes Res,2015,2015:532984.
• [7] ABO-SALEM O M.Kaempferol attenuates the develop-ment of diabetic neuropathic pain in mice:possible anti-inflammatory and anti-oxidant mechanisms[J].Open Access Maced J Med Sci,2014,2(3):424-430.
• [8] SINGH H,ARORA R,ARORA S,et al.Ameliorative potential of Alstonia scholaris(Linn.)R.Br.against chronic constriction injury-induced neuropathic pain in rats[J].BMC Complement Altern Med,2017,17(1):63.
• [9] SOMMER C,LEINDERS M,üEYLER N.Inflammation in the pathophysiology of neuropathic pain[J].Pain,2018,159(3):595-602.
• [10] ZHANG Y,MOU J,CAO L,et al.MicroRNA-142-3p relieves neuropathic pain by targeting high mobility group box 1[J].Int J Mol Med,2018,41(1):501-510.
• [11] MOALEM G,TRACEY D J.Immune and inflammatory mechanisms in neuropathic pain[J].Brain Res Rev,2006,51(2):240-264.
• [12] ANDERSEN H H,DUROUX M,GAZERANI P.MicroRNAs as modulators and biomarkers of inflammatory and neuropathic pain conditions[J].Neurobiol Dis,2014,71:159-168.
• [13] LI L,LI J H,GAO Y,et al.Research progress in the protective effect of wine on central nervous system injury[J].Journal of Shenyang Pharmaceutical University(沈阳药科大学学报),2021,38(3):314-320,327.
• [14] QIAN Z,CHANG J,JIANG F,et al.Excess administration of miR-340-5p ameliorates spinal cord injury-induced neuroinflammation and apoptosis by modulating the P38-MAPK signaling pathway[J].Brain Behav Immun,2020,87:531-542.
• [15] GAO L,PU X,HUANG Y,et al.MicroRNA-340-5p relieved chronic constriction injury-induced neuropathic pain by targeting Rap1A in rat model[J].Genes Genomics,2019,41(6):713-721.
• [16] CHEN H,HU Y,XIE K,et al.Effect of autophagy on allodynia,hyperalgesia and astrocyte activation in a rat model of neuropathic pain[J].Int J Mol Med,2018,42(4):2009-2019.
• [17] GUO J S,JING P B,WANG J A,et al.Increased autophagic activity in dorsal root ganglion attenuates neuropathic pain following peripheral nerve injury[J].Neurosci Lett,2015,599:158-163.
• [18] CORUM D G,TSICHLIS P N,MUISE-HELMERICKS R C.AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1[J].Faseb J,2014,28(1):395-407.
• [19] CAI L,LIU X,GUO Q,et al.MiR-15a attenuates peripheral nerve injury-induced neuropathic pain by targeting AKT3 to regulate autophagy[J].Genes Genomics,2020,42(1):77-85.
• [20] SHI J,JIANG K,LI Z.MiR-145 ameliorates neuropathic pain via inhibiting inflammatory responses and mTOR signaling pathway by targeting Akt3 in a rat model[J].Neurosci Res,2018,134:10-17.
• [21] DING Y,ZHANG J,WANG R.Inhibition of tissue transglutaminase attenuates lipopolysaccharide-induced inflammation in glial cells through AKT/mTOR signal pathway[J].Biomed Pharmacother,2017,89:1310-1319.