腹内高压条件下血红素加氧酶1基因表达对大鼠肠黏膜损伤的影响

Influence of haemoxygenas （ HO-1 ） gene expression on intestinal mucosa injury induced by intra- abdominal hypertension in rats

目的观察促进或抑制血红素加氧酶1（HO．1）搪闪表达对腹内高压（IAH）致肠黏膜损伤的影响。方法（1）促进或抑制HO—l基因表达大鼠模型的建立。选取24只健康成年Wistar大鼠，采用随机数字表法分为HO—l特异性诱导剂钴原卟啉（Co—PP）2．5mg组、PP5．0mg组、HO—l特异性抑制剂锡原卟啉（Sn—PP）2．5mg组、对照组，每组6只。co—PP2．5mg组、s11-PP2．5mg组大鼠分别腹腔注射c0-PP2．5mg／kg、Sn—PP2．5mg／kg，每12小时1次，共持续3（1；Co—PP5．0mg组大鼠腹腔注射c0-PP5．0mg／kg，每24小时1次，共持续3d；对旦c组大鼠同前2组注射等量生理盐水。于注射后第4天处死大鼠，取肠黏膜组织检测HO-1mRNA表达。选取Co—PP最任制地用于后续实验。（2）1AH条件下促进或抑制HO—l基因表达对肠黏膜损伤的影响。另取24只健康成年Wistar大鼠，按随机数字表法分为对照组、IAH组、c0-IP＋IAH组、Sn—PP＋IAH组，每组6只。Co—PP＋IAtt组、Sn—PP＋lAH组大鼠分别以最佳剂量co—IP、Sn—PP2．5mg／kg同前行腹腔注射；对照组同法注射等量生理盐水。注射后第4天，后2组大鼠腹腔允人氮气至腹内压达20mmHg（1iqrlmHg：0．133kPa），持续作用2h。IAH组大鼠同前制作IAH模型。对照组仅行腹腔穿刺置管，不充入氮气。剖腹取空肠段10～15cm，刮取肠黏膜组织榆测HO-1mRNA表达以及二胺氧化酶（DAO）含=}i=}；抽取门静脉血检测D乳酸盐、TNF—d和lI，6含量；取空肠段1～2c㈨行组织病理学观察。对数据进行单因素方差分析及t检验。结果（1）Co—PP2．5mg组大鼠的HO—ltuRNA表达水平明显高于对照组及c0-PP5．0mg组（t值分别为4．756、3．175，P〈0．05或P〈0．01）。Sn—Pt2．5mg组大鼠的HO-1mRNA表达水平明显低于对照组（t=4．880，P〈0．01）。选择2．5mg／kgCo—PP用于后续实验、（2）Co—PP＋IAH组大鼠HO-1tuRNA表达水平为60±5，明显高于IAH组（49±5，t=3．811，P〈0．01）和对照组（39±4，t=8．034，P〈0．001）。IAH组HO-1mRNA表达水平高于对照组（t=3．826，P〈0．01），Sn-PP＋IAH组HO-mRNA的表达水平为29±4，明显低于对照组（t=4．330，P〈0．01）。Co—PP＋IAH组DAO、D乳酸盐含量分别为（0．52±0．05）U／mL、（1．9±0．6）mg／L，显著低于IAH组[（0．88±0．06）U／mL、（4．3±0．7）Ig／t值分别为11．291、6．376，Pfff均小于0．01]；但仍然高于对照组[（0．34±0．04）U／m1、（J．2±0．5）mg／L，t值分别为6，886、2．295，P〈0．05或P〈0．01]。c0-PP＋I-AH组大鼠TNF．d及IL-6分泌水平显著低于IAH组，但仍高于照组（t值为3．781± 8．557，P值均小于0．01）。Sn—PP＋IAH组的DAO、D乳酸盐含量和TNF—d及IL-6的分泌水平均高于其他各组（t值为4．181～32．938，P值均小于0．01）。对照组肠黏膜上皮细胞结构完整，排列整齐。IAH组大鼠肠黏膜组织水肿，肠绒毛顶端糜烂、坏死。Co—PP＋IAH组大鼠肠绒毛损伤减轻。Sn—PP＋IAH组大鼠肠绒毛的损伤加重。结论诱导肠道HO—l基因高表达，可减轻[AH导致的肠道缺血缺氧性损伤，对肠黏膜具有-定的保护作用。

Objective To observe the effects of upor down-regulation of haemoxygenase 1 （HO-I） gene expression on intestinal mueosa injury induced by intra-abdominal hypertension （IAH）. Methods （1） Reproduction of rat model of upor down-regulation of HO-1 gene expression. Twenty-four healthy adult Wistar rats were divided into Co-PP （HO-1 specific revulsive） 2.5 mg, Co-PP 5.0 mg, Sn-PP （ HO-1 specific inhibitor） 2.5 mg, and control groups according to the random number table, with six rats in each group. Rats in groups Co-PP 2.5 mg and Sn-PP 2.5 mg were respectively given Co-PP 2.5 mg/kg and Sn-PP 2.5 mg/kg by intraperitoneal injection, once every 12 hours for 3 days. The rats in group Co-PP 5.0 mg were intraperitoneally injected with Co-PP 5.0 mg/kg, once a day for 3 days. The rats in control group were treated with equal volume of normal saline by intraperitoneal injection. All rats were sacrificed on post injection day （PID） 4, and intestinal mucosa tissues were collected for determination of HO-1 mRNA expression. Optimal dose of Co-PP was chosen for the following experiment. （2） The influence of up- or down-regulation of HO-1 gene expression on intestinal mucosa injury under IAH condition. Another 24 healthy adult Wistar rats were divided into control, IAH, Co-PP ＋ iAH, and Sn-PP ＋ IAH groups according to the random number table, with six rats in each group. The rats in groups Co-PP ＋ IAH and Sn-PP ＋ IAH were intraperitoneally injected with 2. 5 mg/kg Co-PP and 2. 5 mg/kg Sn-PP, once every 12 hours for 3 days. Equal volume of normal saline was intraperitoneally injected into the rats in control group, once ev- ery 12 hours for 3 days. Then, nitrogen gas pneumoperitoneum was used to establish the model of IAH in rats of the latter three groups on PID 4, with IAP at 20 mm Hg （1 mm Hg =0. 133 kPa） , and it was main- tained for 2 hours. Puncture and intubation were performed in rats of control group without inflating nitrogen gas. Jejunal segment in the length of 10-15 cm was harvested for collecting intestinal mucosa tissues to determine the HO-1 mRNA expression and diamine oxidase （DAO） content. Serum obtained from portal vein blood was collected to determine the D-lactate, TNF-a, and IL-6 contents. Another jejunal segment in the length of 1-2 cm was harvested for histopathological examination. Data were processed with one-way analysis of variance and t test. Results （ 1 ） The HO-1 mRNA expression in group Co-PP 2.5 mg was significantly higher than that in control and Co-PP 5.0 mg groups （ with t values respectively 4. 756, 3. 175, P 〈 0.05 or P 〈 0.01 ）. The HO-1 mRNA expression in group Sn-PP 2.5 mg was significantly lower than that in control group （ t = 4. 880, P 〈 0.01 ）. The optimal dose of Co-PP for the following experiment was 2.5 mg/kg. （2） HO-1 mRNA expression in group Co-PP ＋ IAH was 60 5, and it was obviously higher than that of group IAH （49 ＋ 5, t = 3. 811, P 〈 0.01 ） and control group （39 ~ 4, t = 8. 034, P 〈 0. 001 ）. HO-1 mRNA expression was higher in group IAH than in control group （ t =3. 826, P 〈0.01）. HO-1 mRNA ex- pression in group Sn-PP ＋ IAH was 29 - 4, which was obviously lower than that of control group （ t = 4. 330, P 〈0.01）. The contents of DAO and D-lactate in group Co-PP ＋ IAH were （0.52 ±0.05） U/mL and （ 1.9 ±0.6） rag/L, which were significantly lower than those in group IAH [ （0.88 s0.06） U/mL and （4.3 ±0.7） rag/L, with t values respectively 11. 291, 6. 376, P values all below 0.01 ] , but still higher than those in control group [ （0.34 ±0.04） U/mL, （ 1.2 ±0.5 ） rag/L, with t values respectively 6. 886, 2. 295, P 〈0.05 or P 〈0.01 ]. The contents of TNF-a and IL-6 were much lower in group Co-PP ＋ IAH than in group IAH, but still higher than in control group （with t values from 3. 781 to 18. 557, P values all below 0.01 ）. The contents of DAO, D-lactate, TNF-a, and IL-6 in group Sn-PP ＋ IAH were all higher than those in the other 3 groups （ with t values from 4. 181 to 32. 938, P values all below 0.01 ）. Structure of epi- thelial cells from intestinal mucosa was intact and regularly arranged in rats of control group. Intestinal muco- sal tissue was edematous, and the top of villi was anabrofic and necrotic in rats of group IAH. Compared with that of group IAH, the degree of intestinal mucosa injury was alleviated in rats of group Co-PP ＋ IAH, while the pathology was aggravated in rats of group Sn-PP ＋ IAH. Conclusions Up-regulation of HO-1 gene ex- pression can ameliorate intestinal mucosa injury caused by IAH, thus protecting intestinal mucosa tissues.