Rho激酶抑制剂对脓毒症大鼠肠损伤的作用及机制研究

Effect and mechanism of Rho kinase inhibitor on intestinal injury in septic rats

目的探讨Rho激酶抑制剂对脓毒症大鼠肠损伤的影响及其可能机制。方法将32只成年雄性SD大鼠按随机数字表法分为假手术组(Sham组)、Rho激酶抑制剂Y-27632对照组(Y+Sham组)、脓毒症模型组〔盲肠结扎穿孔术(CLP)组〕及Y-27632预处理组(Y+CLP组), 每组8只。采用CLP法制备大鼠脓毒症模型;Sham组和Y+Sham组只游离拨动盲肠、不进行结扎穿孔。Y+Sham组和Y+CLP组大鼠于术前15 min腹腔注射Y-27632溶液5 mg/kg进行预处理;Sham组及CLP组大鼠则腹腔注射等量磷酸盐缓冲液(PBS)。于术后24 h取大鼠心脏血, 采用酶联免疫吸附试验(ELISA)测定血清二胺氧化酶(DAO)含量。收集小肠组织, 苏木素-伊红(HE)染色后, 光镜下观察肠组织病理学变化, 并进行肠黏膜损伤评分(Chiu’’s评分);采用免疫组化法检测肠组织Rho相关卷曲螺旋激酶1(ROCK1)和核转录因子-κB(NF-κB)阳性表达;采用ELISA法检测肠组织肿瘤坏死因子-α(TNF-α)和白细胞介素-10(IL-10)的含量。结果 Sham组和Y+Sham组肠组织结构完整, 黏膜纤毛排列整齐;与Sham组相比, CLP组肠道黏膜纤毛排列紊乱, 大量炎症细胞浸润, Chiu’’s评分显著升高(分:3.83±0.27比0.12±0.11, P<0.05), 说明大鼠发生了脓毒症肠损伤;与CLP组相比, Y+CLP组大鼠肠上皮细胞坏死程度减轻, 可见少量炎症细胞浸润, Chiu’’s评分显著降低(分:2.85±0.21比3.83±0.27, P<0.05), 说明Y-27632预处理可以减轻脓毒症大鼠肠损伤。与Sham组相比, CLP组肠组织ROCK1和NF-κB阳性表达、血清DAO及肠组织TNF-α含量显著增加〔ROCK1表达(A值):0.19(0.18, 0.22)比0.10(0.09, 0.11), NF-κB表达(A值):0.40±0.02比0.15±0.01, DAO(ng/L):287.81±23.31比144.92±17.72, TNF-α(ng/L):101.08±5.62比74.81±5.56, 均P<0.05〕, 而肠组织IL-10含量显著降低(μg/L:55.16±5.20比95.95±7.53, P<0.05);与CLP组相比, Y+CLP组肠组织ROCK1和NF-κB的阳性表达、血清DAO及肠组织TNF-α的含量均显著降低〔ROCK1表达(A值):0.15(0.13, 0.18)比0.19(0.18, 0.22), NF-κB表达(A值):0.28±0.01比0.40±0.02, DAO(ng/L):243.34±19.76比287.81±23.31, TNF-α(ng/L):90.41±8.79比101.08±5.62, 均P<0.05〕, 而肠组织IL-10的含量则显著增加(μg/L:66.15±5.74比55.16±5.20, P<0.05), 说明Y-27632预处理对脓毒症肠损伤大鼠的保护作用可能与调节RhoA/ROCK1/NF-κB信号通路有关。结论 Rho激酶抑制剂可以减轻脓毒症大鼠肠损伤, 其机制可能与抑制肠道RhoA/ROCK1/NF-κB信号通路, 减轻肠道炎症反应有关。

Objective To explore the effect of Rho kinase inhibitor on intestinal injury in septic rats and its possible mechanism.Methods Thirty-two male Sprague-Dawley(SD)rats were randomly divided into sham operation group(Sham group),Rho kinase inhibitor Y-27632 control group(Y+Sham group),sepsis model group[cecal ligation and puncture(CLP)group]and Y-27632 pretreatment group(Y+CLP group),with 8 rats in each group.Rat sepsis model was reproduced by CLP.The rats in the Sham group and Y+Sham group were only separated and moved the cecum without ligation and perforation.The rats in the Y+Sham group and Y+CLP group were pretreated with intraperitoneal injection of Y-27632 solution 5 mg/kg 15 minutes before operation;the rats in the Sham group and CLP group were intraperitoneally injected with the same amount of phosphate buffered saline(PBS).Twenty-four hours after operation,the heart blood was collected and the serum diamine oxidase(DAO)content was determined by enzyme-linked immunosorbent assay(ELISA).Then the small intestine tissue was collected,the pathological changes of the intestinal tissue were observed under the light microscope after hematoxylin-eosin(HE)staining,and Chiu's score was performed.The positive expressions of Rho-related coiled-coil kinase 1(ROCK1)and nuclear factor-κB(NF-κB)in intestinal tissue were detected by immunohistochemistry.ELISA was used to detect the levels of tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)in intestinal tissue homogenate.Results The intestinal tissue structure of the Sham group and Y+Sham group was intact and the mucosa was arranged neatly.Compared with the Sham group,the intestinal mucosa of the CLP group was arranged disorderly,with a large number of inflammatory cells infiltration,and the Chiu's score was significantly increased(3.83±0.27 vs.0.12±0.11,P<0.05),indicating that those rats suffered from septic intestinal injury.Compared with the CLP group,the degree of necrosis of intestinal epithelial cells in the Y+CLP group was reduced,a small amount of inflammatory cells infiltration was seen,and the Chiu's score was significantly decreased(2.85±0.21 vs.3.83±0.27,P<0.05),indicating that Y-27632 pretreatment could alleviate intestinal injury in septic rats.Compared with the Sham group,the positive expressions of intestinal tissue ROCK1 and NF-κB,the contents of serum DAO and intestinal homogenate TNF-αin the CLP group were significantly increased[ROCK1 expression(A value):0.19(0.18,0.22)vs.0.10(0.09,0.11),NF-κB expression(A value):0.40±0.02 vs.0.15±0.01,DAO(ng/L):287.81±23.31 vs.144.92±17.72,TNF-α(ng/L):101.08±5.62 vs.74.81±5.56,all P<0.05],the level of intestinal homogenate IL-10 was significantly decreased(μg/L:55.16±5.20 vs.95.95±7.53,P<0.05).Compared with the CLP group,the positive expressions of intestinal tissue ROCK1,NF-κB,the contents of serum DAO and intestinal homogenate TNF-αin the Y+CLP group were significantly decreased[ROCK1 expression(A value):0.15(0.13,0.18)vs.0.19(0.18,0.22),NF-κB expression(A value):0.28±0.01 vs.0.40±0.02,DAO(ng/L):243.34±19.76 vs.287.81±23.31,TNF-α(ng/L):90.41±8.79 vs.101.08±5.62,all P<0.05],while the level of intestinal homogenate IL-10 was significantly increased(μg/L:66.15±5.74 vs.55.16±5.20,P<0.05),indicating that the protective effect of Y-27632 pretreatment on sepsis intestinal injury rats might be related to the regulation of RhoA/ROCK1/NF-κB signaling pathway.Conclusion Rho kinase inhibitors can reduce intestinal injury in septic rats,and the mechanism may be related to inhibiting RhoA/ROCK1/NF-κB signaling pathway and reducing intestinal inflammation in septic rats.