普通肝素对脓毒症肠损伤小鼠肠黏膜血红素加氧酶-1表达的影响

Effect of unfractionated heparin on the expression of heme oxygenase-1 in intestinal mucosa of mice with sepsis

目的探讨普通肝素（UFH）对脓毒症肠损伤小鼠肠黏膜血红素加氧酶-1（HO-1）表达的影响。方法将36只雄性C57BL／6J小鼠按随机数字表法分为假手术（Sham）组、盲肠结扎穿孔术（CLP）组、UFH组，每组12只。采用CLP制备脓毒症肠损伤小鼠模型；Sham组开腹暴露盲肠、不结扎。UFH组于术前0．5h和术后12h经尾静脉注射肝素钠注射液各8U。术后4h、24h各取6只小鼠下腔静脉血和回肠末端标本，采用酶联免疫吸附试验（ELISA）测定血清白细胞介素（IL-1β、IL-6）、肿瘤坏死因子-α（TNF—α）含量，采用比色法测定D-乳酸含量；苏木素-伊红（HE）染色后观察回肠组织病理学变化并进行Chiu评分；免疫组化法检测回肠组织HO-1表达。结果Sham组血清IL-1β、IL-6、TNF—α、D-乳酸含量无明显变化；24h肠黏膜结构基本正常，无HO-1阳性细胞。CLP组血清IL-1β、IL-6、TNF—α、D-乳酸含量逐渐升高，且明显高于Sham组[IL-1β（ng／L）：4h为40．87±2．88比22．60±2．05，24h为113．73±3．96比22．07±2．74；IL-6（ng／L）：4h为63．89±3．26比44．89±3．38，24h为176．56±5．45比45．76±4．02；TNF-α（ng／L）：4h为194．62±14．13比152．05±6．22，24h为599．62±10．20比155．90±14．18；D-乳酸（mmol／L）：4h为0．24±0．02比0．19±0．01，24h为0．33±0．04比0．20±0．02，均P〈0．05]；24h肠黏膜水肿、炎性细胞浸润，肠绒毛结构破坏，Chiu评分明显高于Sham组[分：4．5（3．0～5．0）比0（0～1．0），P〈0．05]；肠黏膜局部有少量HO-1阳性细胞。UFH组血清IL-1β、IL-6、TNF—α、D-乳酸含量显著低于CLP组[IL-1β（ng／L）：4h为31．53±2．90比40．87±2．88，24h为61．13±2．80比113．73±3．96；IL-6（ng／L）：4h为51．16±5．68比63．89±3．26，24h为81．16±4．54比176．56±5．45；TNF—α（ng／L）：4h为171．76±5．60比194．62±14．13，24h为328．48±10．79比599．62±10．20；D-乳酸（mmol／L）：4h为0．21±0．01比0．24±0．02，24h为0．24±0．02比0．33±0．04，均P〈0．05]；24h肠损伤明显减轻，Chiu评分较CLP组明显降低[分：1．5（1．0-5．0）比4．5（3．0—5．0），P〈0．05]；肠黏膜HO-1阳性细胞明显增加。结论UFH可通过增强肠黏膜HO-1表达，减少炎性因子释放，减轻肠道炎症反应，从而对脓毒症肠损伤小鼠肠组织起保护作用。

Objective To investigate the effect of unfractionated heparin （UFH） on the expression of heme oxygenase-1 （HO-1） in intestinal mucosa of mice with sepsis. Methods Thirty-six male C57BL/6J mice were randomly divided into sham group, cecal ligation and puncture （CLP） group and UHF group, n = 12 in each group. Model of intestinal injury in sepsis was induced by CLP. In sham group, the mice were exposed without ligation of cecum. In UFH group, the mice were treated intravenously with 8 U of UFH via the tail vein half an hour before the operation and 12 hours after the surgery respectively. Six mice in each group were randomly chosen at 4 hours and 24 hours after operation to collect inferior vena venous blood samples and terminalileum tissues. The serum levels of interleukins （IL-1β, IL-6）, and tumor necrosis factor-ct （TNF-α） were determined by enzyme linked immunosorbent assay （ELISA）. The serum level of D-lactate was determined by colorimetry. Pathological changes of ileum tissue and Chiu score were observed after hematoxylin eosin （HE） staining. The HO-1 expression was detected immunohistochemically. Results In sham group, no significant changes in the serum levels of IL-1β, IL-6, TNF-α and D-lactate were observed. Twenty-four hours after the operation, the structure of intestinal mucosa was basically normal without obvious pathology change and no HO-1 positive cells were found. The serum levels of IL-1β, IL-6, TNF-α, and D-lactate in CLP group were gradually increased, and they were significantly increased as compared with sham group [IL-1β （ng/L）： 40.87±2.88 vs. 22.60±2.05 at 4 hours, 113.73±3.96 vs. 22.07±2.74 at 24 hours; IL-6 （ng/L）： 63.89±3.26 vs. 44.89±3.38 at 4 hours, 176.56±5.45 vs. 45.76±4.02 at 24 hours; TNF-α （ng/L）： 194.62± 14.13 vs. 152.05±4,6.22 at 4 hours, 599.62±10.20 vs. 155.90± 14.18 at 24 hours; D-lactate （mmol/L）： 0.24±0.02 vs. 0.19 ± 0.01 at 4 hours, 0.33±0.04 vs. 0.20 ± 0.02 at 24 hours, all P 〈 0.05]. Twenty-four hours after the operation, edema and inflammation in ileal mueosa, intestinal villi structural damage were observed, the Chiu score was significantly higher than those in the sham group [4.5 （3.0-5.0） vs. 0 （0-1.0）, P 〈 0.05], and a small amount of HO-1 positive cells were localized in the intestinal mucosa. Compared with CLP group, the serum levels of IL-1 15, IL-6, TNF- α, and D-lactate of UFH group were significantly decreased [IL-1β（ng/L）： 31.53 ±2.90 vs. 40.87±2.88 at 4 hours, 61213± 2.80 vs. 113.73±3.96 at 24 hours; IL-6 （ng/L）： 51.16 ± 5.68 vs. 63.89 ± 3.26 at 4 hours, 81.16 ± 4.54 vs. 176.56 ± 5.45 at 24 hours; TNF-α （ng/L）： 171.76±5.60 vs. 194.62 ± 14.13 at 4 hours, 328.48 ± 10.79 vs. 599.62 ± 10.20 at 24 hours; D-lactate （mmol/L）： 0.21±0.01 vs. 0.24±0.02 at 4 hours, 0.24 ±0.02 vs. 0.33 ±0.04 at 24 hours, all P 〈 0.05]. Twenty-four hours after the operation, intestinal injury was ameliorated, the Chiu score was significantly lower [1.5 （1.0-5.0） vs. 4.5 （3.0-5.0）, P 〈 0.05], and HO-1 positive cells in the intestinal mucosa was remarkably increased. Conclusion UFH can enhance the expression of HO-1 in intestinal mucosa, reduce the release of inflammatory factors, ameliorate the intestinal inflammatory response, and thus play a protective role in intestinal tissue in mice with sepsis.