右美托咪定对大鼠骨折模型所致的焦虑情绪及海马内神经炎症的调节作用

Regulatory effect of dexmedetomidine on anxiety and hippocampal neuro in flammation in a rat fracture model

目的研究右美托咪定对大鼠骨折导致的焦虑情绪及海马内神经炎症的调节作用。方法采用自由饮水模式将40只成年雄性Wistar大鼠随机平均分为对照组、右美托咪定组、手术组(对大鼠进行右股骨截骨术)、手术+右美托咪定组(骨折后的大鼠腹膜内注射12μg/kg右美托咪定)。建立大鼠骨折模型。在模型建立成功后6 h、24 h、7 d通过惊吓反应测试大鼠的焦虑情绪,酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测大鼠血清及海马组织中炎症因子[肿瘤坏死因子α(tumor necrosis factor-α,TNF-α),白细胞介素1β(interleukin-1β,IL-1β),白细胞介素6(interleukin-6,IL-6)]的表达水平,TUNEL染色定量分析海马组织中的凋亡细胞,Western blot检测海马组织中TNF-α,IL-1β,IL-6,活性氧(reactive oxygen species,ROS)、神经型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)、烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NOX2)、4-羟基壬烯酸(4 hydroxynonenal,4-HNE)、Bax(BCL2-associated X,Bax)和Bcl-2(B-cell lymphoma-2,Bcl-2)的表达。结果随着声刺激的强度从亚阈值(74 dB)增加到接近阈值(90 dB)再到超阈值(110 dB)水平,各组声学惊吓反应显著增加;在74、90、100 dB阈值,右美托咪定组声学惊吓反应低于对照组,手术组声学惊吓反应高于对照组、右美托咪定组,手术+右美托咪定组声学惊吓反应高于右美托咪定组,低于手术组(P<0.05)。右美托咪定组血清TNF-α、IL-6和IL-1β水平低于对照组,手术组血清TNF-α、IL-6和IL-1β水平显著高于对照组、右美托咪定组,手术+右美托咪定组血清TNF-α、IL-6和IL-1β水平高于右美托咪定组,低于手术组(P<0.05)。右美托咪定组海马体组织TNF-α、IL-6和IL-1β表达水平明显降低于对照组,手术组海马体组织TNF-α、IL-6和IL-1β表达水平显著高于对照组、右美托咪定组,手术+右美托咪定组海马体组织TNF-α、IL-6和IL-1β表达水平高于右美托咪定组,低于手术组(P<0.05)。TUNEL染色显示右美托咪定组凋亡细胞数量显著减少,手术组凋亡细胞数量明显增多。右美托咪定组Bax表达水平显著低于对照组,Bcl-2表达水平高于对照组,手术组Bax表达水平显著高于对照组、右美托咪定组,Bcl-2表达水平低于对照组、右美托咪定组,手术+右美托咪定组Bax表达水平高于右美托咪定组,低于手术组,Bcl-2表达水平低于右美托咪定组,高于手术组(P<0.05)。右美托咪定组海马组织ROS含量低于对照组,手术组海马组织ROS含量高于对照组和右美托咪定组,手术+右美托咪定组海马组织ROS含量高于右美托咪定组,低于手术组,差异有统计学意义(P<0.05)。右美托咪定组海马组织NOX2和4-HNE蛋白表达低于对照组,nNOS蛋白表达高于对照组,手术组海马组织NOX2和4-HNE蛋白表达高于对照组和右美托咪定组,nNOS蛋白表达低于对照组和右美托咪定组,手术+右美托咪定组海马组织NOX2和4-HNE蛋白表达高于右美托咪定组,低于手术组,nNOS蛋白表达低于右美托咪定组,高于手术组,差异有统计学意义(P<0.05)。结论右美托咪定可明显改善骨折大鼠的焦虑状态,减轻神经炎症和神经元凋亡。

Objective To establish a rat fracture model to study the regulatory effects of dexmedetomidine on anxiety and hippocampal neuroinflammation caused by fracture in rats.Methods A total of 40 adult male Wistar rats were randomly divided into control group,dexmedetomidine group,operation group(right femur osteotomy),and operation+dexmedetomidine group(12μg/kg intraperitoneal injection of dexmedetomidine after fracture)using free drinking water mode.The rat fracture model was established.On 6 h,24 h,and 7 d after the successful establishment of the model,the anxiety of the rats was tested by scare-response,and the expression levels of inflammatory factors[tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)]in the serum and hippocampal tissue of the rats were detected by enzyme-linked immunosorbent assay(ELISA).Apoptotic cells in hippocampal tissues were quantitatively analyzed by TUNEL staining,and the expressions of TNF-α,IL-1β,IL-6,reactive oxygen species(ROS),neuronal nitric oxide synthase(nNOS),nicotinamide adenine dinucleotide phosphate(NOX2),4-hydroxynonenal(4-HNE),BCL2-Associated X(Bax)and B-cell lymphoma-2(Bcl-2)in hippocampal tissues were detected by Western blot.Results As the intensity of acoustic stimulation increased from sub-threshold(74 dB)to near threshold(90 dB)to over threshold(110 dB),the acoustic startle response(ASR)increased significantly.At the threshold of 74,90 and 100 dB,the ASR of dexmedetomidine group was lower than that of control group,but higher in operation group than in control group and dexmedetomidine group;The ASR was higher in operation+dexmedetomidine group than in dexmedetomidine group but lower than that of operation group(P<0.05).The serum levels of TNF-α,IL-6 and IL-1βin dexmedetomidine group were lower than those in control group,but significantly higher in operation group than in control group and dexmedetomidine group;the serum levels of TNF-α,IL-6 and IL-1βin operation+dexmedetomidine group were higher than those in dexmedetomidine group,but lower than that in operation group(P<0.05).The expression levels of TNF-α,IL-6 and IL-1βin the hippocampus of dexmedetomidine group were significantly lower than those of control group,but significantly higher in operation group than in control group and dexmedetomidine group.The expression levels of TNF-α,IL-6 and IL-1βin the hippocampus of operation+dexmedetomidine group were higher than those of operation group,but lower than those of operation group(P<0.05).TUNEL staining showed that the number of apoptotic cells in dexmedetomidine group decreased significantly,and the number in operation group increased significantly.The expression level of Bax in dexmedetomidine group was significantly lower than that in the control group,while the expression level of Bcl-2 was higher than that in the control group;the expression level of Bax in operation group was significantly higher than that in the control group and dexmedetomidine group,while the expression level of Bcl-2 was significantly lower than that in the control group and dexmedetomidine group.The expression level of Bax in the operation group+dexmedetomidine group was higher than that in the dexmedetomidine group,but lower than that in the operation group.The expression level of Bcl-2 was lower than that of dexmedetomidine group but higher than that of operation group(P<0.05).The hippocampal ROS level was lower in dexmedetomidine group than in control group,lower in operation group than in control group and dexmedetomidine group,and higher in operation+dexmedetomidine group than in operation group,but lower than that of operation group,with statistical significance(P<0.05).The expression of NOX2 and 4-HNE protein in hippocampus of dexmedetomidine group was lower than that of control group,while the expression of nNOS protein was higher than that of control group.The expression of NOX2 and 4-HNE protein in hippocampus of operation group was higher than that of control group and dexmedetomidine group,while the expression of nNOS protein was lower than that of control group and dexmedetomidine group.The expression of NOX2 and 4-HNE protein in hippocampus of operation+dexmedetomidine group was higher than that of dexmedetomidine group,but lower than that of operation group,while the expression of nNOS protein was lower than that of dexmedetomidine group but higher than that of operation group,with statistical significance(P<0.05).Conclusion Dexmedetomidine can significantly improve the anxiety state,reduce neuroinflammation and neuronal apoptosis in rats with fracture.