调胃承气汤灌胃与灌肠对肠源性脓毒症大鼠肠上皮细胞occludin和ZO-1蛋白表达的影响

Effects of Tiaowei Chengqi Decoction by Gavage and by Enema on Expression of Occludin and ZO-1 in Enterocytes of Gut-origin Sepsis Rat Model

【目的】观察和比较调胃承气汤灌胃与灌肠对肠源性脓毒症(GOS)大鼠肠黏膜屏障的影响。【方法】将48只大鼠随机分为假手术组,模型组,中药灌胃高、低剂量组(分别给予调胃承气汤9.450、4.725 g/kg灌胃),中药灌肠高、低剂量组(分别给予调胃承气汤9.450、4.725 g/kg灌肠),每组8只。采用盲肠结扎穿刺法(CLP)复制GOS大鼠模型。各组在造模后2、10、24 h分别对应给药。采用酶联免疫吸附分析(ELISA)检测血清降钙素原(PCT)、D-乳酸(D-LA)水平,逆转录实时定量PCR(RT-qPCR)法检测小肠组织occludin、闭锁小带蛋白(ZO-1)基因及肝脏组织肿瘤坏死因子α(TNF-α)、干扰素γ(INF-γ)基因表达水平,免疫组化法检测小肠组织occludin、ZO-1蛋白的表达,苏木素—伊红(HE)染色观察小肠病理组织结构的变化。【结果】与假手术组比较,模型组大鼠小肠组织结构有明显损伤,血清PCT和D-LA水平显著升高(P <0.01),小肠组织occludin、ZO-1基因相对表达水平明显下调(P <0.01),肝脏组织TNF-α、INF-γ基因相对表达水平显著上调(P <0.01),小肠组织occludin、ZO-1蛋白免疫组化阳性表达明显减少(P <0.05);与模型组比较,中药各治疗组能明显改善小肠的损伤程度,显著降低血清PCT和D-LA水平(P <0.05或P <0.01),上调小肠组织occludin和ZO-1基因相对表达水平,下调肝脏组织TNF-α和INF-γ基因相对表达水平(P <0.05或P <0.01),并且能提高小肠组织occludin和ZO-1蛋白免疫组化阳性表达(P <0.05或P <0.01);与灌胃组比较,中药灌肠组的治疗效果更为明显(P <0.05或P <0.01)。【结论】调胃承气汤可上调GOS模型大鼠肠上皮细胞紧密连接蛋白occludin、ZO-1及下调体内炎性因子TNF-α、INF-γ的表达,从而修复肠黏膜屏障,治疗GOS,且灌肠疗法要优于灌胃疗法。

Objective To observe and compare the effect of Tiaowei Chengqi Decoction by gavage and by enema on intestinal mucosal barrier of gut-origin sepsis(GOS)rat model. Methods Forty-eight rats were randomly divided into 6 groups,including sham surgery group,model group,high-and low-dose Chinese medicine gavage groups(given gavage with Tiaowei Chengqi Decoction at the dosage of 9.450,4.725 g/kg,respectively)and high-and low-dose Chinese medicine enema groups(given enema with Tiaowei Chengqi Decoction at the dosage of 9.450,4.725 g/kg,respectively), 8 rats in each group. At 2, 10, 24 h after modeling, each group was given corresponding medication, respectively. The serum levels of procalcitonin(PCT)and D-Lactate(D-LA)were detected by enzyme-linked immunosorbent assay(ELISA). The gene expression levels of occludin, zonula occluden1(ZO-1)in small intestinal tissues as well as tumor necrosis factor α(TNF-α),interferon γ(INF-γ)in liver tissues were determined by reverse transcription qPCR(RT-qPCR). The protein expression levels of occludin,ZO-1 in small intestinal tissues were observed by immunohistochemistry. And the pathological features of small intestinal tissues were observed by hematoxylin-eosin(HE)staining. Results Compared with the sham surgery group,the impair in small intestinal tissues was obvious in the model group,the serum levels of PCT and D-LA were increased(P < 0.01),the relative gene expression levels of occludin and ZO-1 in small intestinal tissues were down-regulated(P < 0.01),the relative gene expression levels of TNF-α and INF-γ in liver tissues were up-regulated(P < 0.01),and the positive immunohistochemical expression of occludin and ZO-1 in small intestinal tissues were also decreased(P < 0.05). Compared with the model group,the damage degree of small intestinal tissues was relieved in various Chinese medicine treatment groups, the serum levels of PCT and D-LA were decreased(P < 0.05 or P < 0.01),the relative gene expression levels of occludin and ZO-1 in small intestinal tissues were up-regulated,the relative gene expression levels of TNF-α and INF-γ in liver tissues were downregulated(P < 0.05 or P < 0.01),and the positive immunohistochemical expression levels of occludin and ZO-1 in small intestinal tissues were also increased(P < 0.05 or P < 0.01). And compared with the Chinese medicine gavage groups, the therapeutic effect in Chinese medicine enama groups was stronger(P < 0.05 or P < 0.01).Conclusion Tiaowei Chengqi Decoction is effective for down-regulating expression of inflammatory factors,and up-regulating expression of occludin and ZO-1 in enterocytes of GOS rats,thus to repair the barrier of intestinal mucosa to cure GOS. And the enema therapy is superior to the gavage therapy.