β-榄香烯对骨癌痛大鼠吗啡耐受的影响及作用机制研究

Effect ofβ-elemene on morphine tolerance in rats with bone cancer pain and its molecular mechanism

目的研究β-榄香烯对骨癌痛大鼠吗啡耐受的影响及作用机制。方法雄性Wistar大鼠随机分为对照组、吗啡组及β-榄香烯低、高剂量(0.7、2.8 mg/kg)组和艾芬地尔(5 mg/kg)组,每组15只;对照组予以生理盐水胫骨注射并ip二甲基亚砜,其余各组构建骨癌痛-慢性吗啡耐受模型后,分别ip相应药物,通过检测机械撤退阈值和热退潜伏期对各组大鼠进行疼痛行为学评估;采用qRT-PCR检测各组大鼠脊髓组织肿瘤坏死因子(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、μ阿片受体(μopioid receptor,MOPR)、N-甲基-D-门冬氨酸受体亚单位2B(N-methyl-D-asparate receptor subunit 2B,NR2B)和环磷酸腺苷(cyclic adenosine monophosphate,cAMP)的mRNA表达;采用Western blotting检测各组大鼠脊髓组织NR2B、cAMP和MOPR的蛋白表达。分别采用不同质量浓度(5、10、20、25µg/mL)的β-榄香烯处理人神经母细胞瘤SH-SY5Y细胞,采用CCK-8法检测SH-SY5Y细胞活性,采用试剂盒测定细胞内cAMP含量,确定β-榄香烯作用于SH-SY5Y细胞的适宜质量浓度;设置对照组、吗啡组、β-榄香烯(5µg/mL)组和艾芬地尔(10µmol/L)组,对照组予以常规培养,其余各组采用10µmol/L吗啡作用SH-SY5Y细胞48 h,以构建吗啡耐受细胞模型,而后β-榄香烯组和艾芬地尔组分别给予相应药物,采用qRT-PCR检测TNF-α、IL-1β、IL-6、NR2B、cAMP和MOPR的mRNA表达,采用Western blotting检测NR2B、cAMP和MOPR的蛋白表达。结果给药第1天,相较于对照组,其余各组大鼠机械撤退阈值和热退潜伏期明显升高(P<0.001),随给药时间延长,吗啡组机械撤退阈值和热退潜伏期逐渐下降,各给药组机械撤退阈值和热退潜伏期明显高于吗啡组(P<0.05、0.01、0.001)。β-榄香烯显著抑制SH-SY5Y细胞活性(P<0.001),且呈剂量和时间相关性;β-榄香烯对SH-SY5Y细胞内cAMP含量无明显影响。吗啡耐受可促进大鼠脊髓组织和SH-SY5Y细胞中TNF-α、IL-1β、IL-6、NR2B及cAMP表达(P<0.001),抑制MOPR表达(P<0.001);给予艾芬地尔或β-榄香烯后,则呈现相反趋势(P<0.01、0.001),且β-榄香烯的作用呈剂量相关性。结论β-榄香烯具有一定的镇痛作用,可通过调控MOPR/NR2B有效缓解骨癌痛大鼠吗啡耐受。

Objective To study the effect and mechanism ofβ-elemene on morphine tolerance in rats with bone cancer pain.Methods Male Wistar rats were randomly divided into control group,morphine group,β-elemene low-and high-dose(0.7,2.8 mg/kg)groups and ifenprodil(5 mg/kg)group,with 15 rats in each group.Control group was given normal saline tibia injection and ip dimethyl sulfoxide.After bone cancer pain-chronic morphine tolerance model was established in other groups,rats were ip corresponding drugs respectively,mechanical withdrawal threshold and thermal withdrawal latency were detected to evaluate pain behavior;qRT-PCR was used to detect tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,μopioid receptor(MOPR),N-methyl-D-aspartate receptor subunit 2B(NR2B)and cyclic adenosine monophosphate(cAMP)mRNA expressions in spinal cord tissues of rats in each group;Western blotting was used to detect the protein expressions of NR2B,cAMP and MOPR in spinal cord tissues of rats in each group.Human neuroblastoma SH-SY5Y cells were treated withβ-elemene(5,10,20,25µg/mL),activity of SH-SY5Y cells was detected by CCK-8 method,and kit was used to detect intracellular cAMP content of SH-SY5Y cells,to determine the appropriate concentration ofβ-elemene acting on SH-SY5Y cells;Control group,morphine group,β-elemene(5µg/mL)group and ifenprodil(10µmol/mL)group were set,control group was routinely cultured,and other groups were treated with 10µmol/L morphine for 48 h to build a morphine-resistant cell model,and thenβ-elemene group and ifenprodil group were given with corresponding drugs,mRNA expressions of TNF-α,IL-1β,IL-6,NR2B,cAMP and MOPR were detected by qRT-PCR,protein expressions of NR2B,cAMP and MOPR were detected by Western blotting.Results On 1st day of administration,compared with control group,mechanical withdrawal threshold and thermal withdrawal latency of rats in other groups were significantly increased(P<0.001).With the extension of administration time,mechanical withdrawal threshold and thermal withdrawal latency decreased gradually in morphine group.Mechanical withdrawal threshold and thermal withdrawal latency of rats in each administration group were significantly higher than those of morphine group(P<0.05,0.01,0.001).β-Elemene significantly inhibited the activity of SH-SY5Y cells(P<0.001)in a dose-and time-dependent manner;β-elemene had no significant effect on intracellular cAMP content of SH-SY5Y cells.Morphine tolerance promoted the expressions of TNF-α,IL-1β,IL-6,NR2B and cAMP in spinal cord tissue of rats and SH-SY5Y cells(P<0.001),and inhibited the expression of MOPR(P<0.001).The opposite trend(P<0.01,0.001)was observed after the addition of ifenprodil orβ-elemene,and the effect ofβ-elemene was dose-related.Conclusionβ-Elemene has a certain analgesic effect,and can effectively relieve morphine tolerance in rats with bone cancer pain by regulating MOPR/NR2B.