右美托咪定对甲醛致疼痛小鼠炎症介质释放的干预研究

Interventional study of dexmedetomidine on inflammatory release in mice with formaldehyde-induced pain

目的探讨右美托咪定对甲醛致疼痛小鼠炎症介质P物质(SP)、降钙素基因相关肽(CGRP)及血清中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-6的影响。方法将40只雄性C57小鼠分成对照组、低剂量组、中剂量组和高剂量组,每组10只。低、中、高剂量组小鼠分别给予腹腔内注射10、20、30μg/kg右美托咪定,对照组小鼠给予腹腔注射10 mL/kg等渗NaCl溶液。注射后30 min,在异氟烷麻醉下于各组小鼠右足底皮下注射2%甲醛20μL,记录各组小鼠的舔舐时间。采用免疫荧光染色观察小鼠脊髓和神经节SP和CGRP表达水平,并行酶联免疫吸附测定(ELISA)检测脊髓和神经节上清中SP、CGRP以及血清中TNF-α、IL-1β及IL-6表达水平。结果足底注射甲醛后,对照组小鼠的舔舐时间为(12.9±5.0)s,而低、中、高剂量组小鼠的舔舐时间分别为(8.4±3.1)s、(7.9±2.7)s、(7.8±2.5)s。4组小鼠舔舐时间比较,差异有统计学意义(F=19.472,P=0.018),且对照组小鼠舔舐时间较低、中、高剂量组小鼠均显著延长(P均<0.05)。免疫荧光染色结果显示,对照组小鼠脊髓背角和结状神经节处SP表达量较高,可见亮绿色荧光纤维,低、中剂量组SP表达水平较对照组显著降低,高剂量组小鼠SP表达最少;且各组小鼠脊髓背角和结状神经节处CGRP表达均较低,未见明显红色阳性纤维和神经元胞体。ELISA结果显示,对照组、低剂量组、中剂量组和高剂量组小鼠脊髓背角处SP [(43±9)、(33±7)、(31±7)、(20±5)ng/L]、结状神经节处SP [(30±7)、(20±5)、(22±5)、(13±3)ng/L]以及血清中TNF-α[(381±60)、(340±54)、(330±48)、(289±41)ng/L]、IL-1β[(68±19)、(55±16)、(52±16)、(41±13)ng/L]和IL-6 [(55±15)、(53±15)、(45±11)、(39±10)ng/L]表达水平比较,差异均有统计学意义(F=6.527、5.269、35.612、33.843、37.175,P=0.031、0.022、0.036、0.048、0.029)。进一步两两比较发现,低、中、高剂量组小鼠脊髓背角处SP、结状神经节处SP、血清中TNF-α和IL-1β以及中、高剂量组小鼠血清中IL-6表达水平均较对照组显著降低,且高剂量组较低、中剂量组小鼠脊髓背角处SP、结状神经节处SP以及血清中TNF-α、IL-1β和IL-6表达水平更低(P均<0.05)。结论脊髓背角及结状神经节处的神经肽SP可能参与了疼痛相关的炎症介质释放,而右美托咪定可抑制其表达,同时减轻血清中的炎症因子水平。

Objective To investigate the effect of dexmedetomidine on inflammatory mediators of substance P(SP),calcitonin gene related peptide(CGRP),serum tumor necrosis factor-alpha(TNF-α),interleukin-1beta(IL-1β),and IL-6 in mice with formaldehyde-induced pain.Methods Forty male C57 mice were divided into a control group,a low dose group,a medium dose group and a high dose group,10 in each group.Mice in the low,medium and high dose groups were given intraperitoneal injection of 10,20,and 30μg/kg dexmedetomidine respectively,while mice in the control group were given intraperitoneal injection of 10 mL/kg isosmotic NaCl solution.Thirty minutes later,20μL of 2%formaldehyde was subcutaneously injected into the right foot of mice under isoflurane anesthesia,and the licking time was recorded in each group.The expression levels of SP and CGRP in the spinal cord and ganglion were observed by immunofluorescence staining.The expression levels of SP,CGRP in the spinal cord and ganglion supernatant and TNF-α,IL-1β,IL-6 in serum were determined by enzyme-linked immunosorbent assay(ELISA).Results After the plantar injection of formaldehyde,the licking time was(12.9±5.0)s in the control group,(8.4±3.1)s in the low dose group,(7.9±2.7)s in the medium dose group,and(7.8±2.5)s in the high dose group.It was significantly different among these four groups(F=19.472,P=0.018),and mice had significantly prolonged licking time in the control group than in the other three groups(all P<0.05).Immunofluorescence staining showed that the SP expression level at the dorsal horn of spinal cord and nodular ganglia was higher in the control group than in the other three groups,with bright green fluorescent fibers,and that it was minimal in the high dose group.In the meantime,the CGRP expression level at the dorsal horn of spinal cord and nodular ganglia was low in each group,and no red positive fibers and neuronal cell bodies were obviously found.ELISA showed that the expression levels of SP at the dorsal horn of spinal cord[(43±9),(33±7),(31±7),(20±5)ng/L],SP at nodular ganglia[(30±7),(20±5),(22±5),(13±3)ng/L],TNF-α[(381±60),(340±54),(330±48),(289±41)ng/L],IL-1β[(68±19),(55±16),(52±16),(41±13)ng/L],and IL-6[(55±15),(53±15),(45±11),(39±10)ng/L]were statistically different among the control group,low dose group,medium dose group and high dose group(F=6.527,5.269,35.612,33.843,37.175;P=0.031,0.022,0.036,0.048,0.029).In further pairwise comparison,the expression levels of SP at the dorsal horn of spinal cord and nodular ganglia,TNF-αand IL-1βin the low,medium and high dose groups,and IL-6 in the medium and high dose groups were significantly lower than those in the control group,and those were lowest in the high dose group(all P<0.05).Conclusion The neuropeptide SP at the dorsal horn of spinal cord and nodular ganglia may be involved in the release of pain-related inflammatory mediators,while dexmedetomidine inhibits their expressions and reduces levels of inflammatory factors in serum.