基于p38 MAPK/MSK1/CREB转导通路研究痛泻要方对肝郁脾虚型D-IBS大鼠痛觉敏化的干预作用

Intervention Effect of Tongxie Yaofang on Pain Sensitization in D-IBS Rats with Liver Depression and Spleen Deficiency Based on p38 MAPK/MSK1/CREB Transduction Pathway

目的研究痛泻要方对腹泻型肠易激综合征(Diarrhea-predominant irritable bowel syndrome,D-IBS)大鼠的痛觉敏化的干预机制。方法将60只SPF级Wistar大鼠随机分为空白组、模型组、匹维溴铵组(0.02 g·kg^-1)和痛泻要方低、中、高(2.25、4.5、9 g·kg^-1)剂量组,采用番泻叶灌胃联合慢性束缚应急法连续14 d复制肝郁脾虚型D-IBS模型;模型复制结束后第2天,予以匹维溴铵和痛泻要方治疗21 d。采用酶联免疫吸附(ELISA)法检测大鼠脊髓背根神经节(DRG)中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-4(IL-4)的含量变化;采用实时荧光定量聚合酶链式反应(Real-time PCR)法检测脊髓DRG中p38丝裂原活化蛋白酶(p38 MAPK)、丝裂原和应激蛋白激酶1(MSK1)、环磷腺苷效应元件结合蛋白(CREB)mRNA的表达;蛋白免疫印迹(Western Blot)法检测脊髓DRG中p38 MAPK、MSK1、CREB蛋白含量;采用腹部回缩反射(AWR)评价内脏高敏状态。结果痛泻要方中、高剂量组均提高D-IBS模型大鼠腹部回缩反射压力阈值(P <0.01);升高脊髓DRG中IL-4的含量(P <0.01),降低IL-1β、IL-6含量(P <0.01,P <0.05);降低D-IBS模型大鼠脊髓DRG中MSK1 mRNA表达和p38 MAPK、MSK1、CREB蛋白含量(P <0.01)。痛泻要方高剂量组还可同时降低p38 MAPK、CREB mRNA表达(P <0.01)。结论痛泻要方可通过上调D-IBS大鼠脊髓DRG中IL-4和下调IL-6、IL-1β含量,抑制p38 MAPK、MSK1、CREB mRNA的表达和蛋白的含量,降低了内脏痛觉敏化而达到治疗腹泻型肠易激综合征。

Objective To investigate the effects of Tongxie Yaofang(TXYF) on pain sensitization of rats with diarrhea-predominant irritable bowel syndrome(D-IBS). Methods Sixty SPF-grade Wistar rats were randomly divided into 6 groups,including the control group,the model group,pinaverium group,TXYF high,medium and low dose groups. Model rats were induced by senna leaf(F)gavage combined with chronic restraint(S)emergency method for 14 days. From the next day after modelling,rats were given pinaverium and TXYF treatment for 21 days.The levels of interleukin-1 β(IL-1 β),interleukin-6(IL-6)and interleukin-4(IL-4)in the dorsal root ganglia of the rats’ spinal cords(DRG)were detected by ELISA. The mRNA expression levels of p38 MAPK,MSK1 and CREB in DRG were detected by qPCR. The protein contents of p38 MAPK, MSK1 and CREB in DRG were detected by Western Blot. The visceral sensitivity was measured by abdominal withdrawal reflex(AWR). Results Medium and high doses of TXYF significantly increased the thresholds of abdominal reflex pressure in D-IBS model rats(P < 0.01);increased the contents of IL-4 in DRG of spinal cord(P < 0.01),decreased the contents of IL-1βand IL-6(P < 0.01,P < 0.05),decreased the expression of MSK1 mRNA and the contents of p38 MAPK,MSK1 and CREB protein in DRG of spinal cord(P < 0.01). High dose of TXYF decreased p38 MAPK,CREB mRNA expression(P < 0.01). Conclusion TXYF may treat D-IBS through increasing IL-4 and decreasing IL-1β,IL-6 contents in DRG, inhibiting the expression of p38 MAPK, MSK1, CREB mRNA and protein contents, and reducing the pain sensitization.