PTSD状态下HCN2-GLT-1在脊髓水平协同调控内脏高敏感性-中枢敏化的作用及机制研究

Effect and mechanism of the co-regulation of HCN2-GLT-1 on the spinal cord level of the visceral nociception and hyperalgesia following exposure to posttraumatic stress disorder-like stress in rats

目的研究创伤后应激障碍(PTSD)状态下,超极化激活环核苷酸门控阳离子通道2(HCN2)与谷氨酸转运体-1(GLT-1)在脊髓水平协同调控内脏高敏感性-中枢敏化的作用及机制。方法成年雌性SD大鼠随机分为3组,即正常对照组(n=14)、PTSD内脏高敏感模型组(n=15)、PTSD+头孢曲松(CTX)组[给予CTX预处理,n=15)。采用连续单一应激(SPS)联合足底电击刺激法建立PTSD内脏高敏感大鼠模型。建模7d后,采用结直肠扩张(CRD)-内脏运动反射(VMR)评估各组内脏敏感性的改变;采用CTX选择性诱发编码GLT-1的基因转录,并采用激光共聚焦及免疫荧光技术研究脊髓HCN2的表达及CTX对其的影响。结果与正常对照组比较,PTSD组脊髓HCN2表达明显上调(78.05±6.49 vs121.12±4.85,P<0.001);与PTSD组比较,PTSD+CTX组HCN2表达显著降低(121.12±4.85 vs 98.24±5.86,P=0.012);PTSD+CTX组HCN2表达比正常对照组明显升高(98.24±5.86 vs 78.05±6.49,P=0.024)。在20mmHg压力下行CRD时,PTSD组的AUCVMR明显高于PTSD+CTX组(0.2913±0.0229 vs 0.2175±0.0090,P=0.005);在40mmHg压力下行CRD时,PTSD组的AUCVMR明显高于正常对照组(0.6200±0.0278 vs 0.3786±0.0155,P<0.001),亦高于PTSD+CTX组(0.6200±0.0278 vs 0.5038±0.0336,P=0.006);在60mmHg压力下行CRD时,与正常对照组比较,PTSD组AUCVMR明显增高(0.7663±0.0262 vs 0.5271±0.0212,P<0.001),同时亦明显高于PTSD+CTX组(0.7663±0.0262 vs 0.6400±0.0245,P=0.001)。结论在PTSD内脏高敏感状态下,CTX可通过直接上调GLT-1表达并间接下调HCN2的表达协同发挥抗内脏伤害性刺激的作用,HCN2-GLT-1信号通路在脊髓水平协同参与了内脏高敏感性-中枢敏化调控作用,可能成为防止或抑制PTSD状态下内脏高敏感性-痛觉敏化新靶点之一。

Objective To explore the potential role of HCN2-GLT-1 [hyperpolarization-activated cyclic nucleotide-gated（HCN）-2 channel and astrocytic glutamate transporter 1（GLT-1）] in co-mediating visceral hyperalgesia at the spinal cord level following exposure to PTSD（posttraumatic stress disorder）-like stress in rats. Methods Adult female SD rats were randomly divided into normal control group（n=14）, PTSD group（n=15） and PTSD＋CTX group [pretreated with ceftriaxone（CTX）, n=15]. The animal model of PTSD was reproduced by using single-prolonged stress（SPS） combined with electric shock. The alteration of visceral sensitivity was evaluated by measuring the viscero-motor response（VMR） to graded colorectal distension（CRD） 7 days after PTSD. The transcription of the gene encoding the GLT-1 was selectively induced by CTX to up-regulate the GLT-1 expression. The expression of spinal HCN2 and the effect of CTX on it were evaluated by immunofluorescence. Results HCN2 expression increased significantly in the spinal cord within 7 days after the exposure to PTSD as compared with that of control group（78.05±6.49 vs 121.12±4.85, P0.001）. The expression of HCN2 significantly decreased when being treated with CTX（121.12±4.85 vs 98.24±5.86, P=0.012）. HCN2 expression significantly increased in PTSD＋CTX group compared with that of the control group（78.05±6.49 vs 98.24±5.86, P=0.024）. The value of AUCVMR in PTSD group（0.2913±0.0229） was obviously higher than that in PTSD＋CTX group（0.2175±0.0090） when the pressure of CRD was 20 mmHg（P=0.005）. When the pressure was changed into 40 mmHg and 60 mmHg, the values of AUCVMR in PTSD group（0.6200±0.0278, 0.7663±0.0262 respectively） were significantly higher than those in normal control group（0.3786±0.0155, 0.5271±0.0212, respectively, P0.001） and PTSD＋CTX group（0.5038±0.0336, 0.6400±0.0245, respectively, P=0.006, or P=0.001）. Conclusions CTX may exert an effect by directly up-regulating the expression of GLT-1 and indirectly down-regulating the expression of HCN2 I exposure to PTSD-like stress. The HCN2-GLT-1 pathway takes part in the regulation of visceral nociception and hyperalgesia in the spine cord, and it may play a role in the prevention or inhibition of the visceral nociception and hyperalgesia after exposure to PTSD-like stress.