参附注射液对严重脓毒症大鼠促胃生长激素干预作用的机制研究
삼부주사액대엄중농독증대서촉위생장격소간예작용적궤제연구
An observation on interference mechanism of Shenfu injection on ghrelin in rats with severe sepsis
  • 万方数据
    中国中西医结合急救杂志 중국중서의결합급구잡지
    Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
    2015年 6期 596-600 ,共5页

    伍万%江荣林%欧阳侃

    오만%강영림%구양간

    参附注射液%脓毒症%促胃生长激素%胃泌素 參附註射液%膿毒癥%促胃生長激素%胃泌素
    삼부주사액%농독증%촉위생장격소%위비소
    Shenfu injection%Sepsis%Ghrelin%Gastrin
    目的：观察参附注射液对脓毒症大鼠肠功能的影响。方法将40只大鼠按随机数字表法分为假手术组、脓毒症模型组、参附低浓度和高浓度组，每组10只。采用盲肠结扎穿孔术（CLP）制备脓毒症大鼠模型，假手术组大鼠仅开腹而不行CLP。参附低、高浓度组大鼠于制模后10 min经尾静脉推注参附注射液5 mL/kg和10 mL/kg；模型组则推注生理盐水10 mL/kg。12 h后取血并处死大鼠，采用酶联免疫吸附试验（ELISA）检测血清促胃生长激素（Ghrelin）、胃泌素及炎症介质肿瘤坏死因子-α（TNF-α）、高迁移率族蛋白-B1（HMGB1）和髓过氧化物酶（MPO）、二胺氧化酶（DAO）活性；采用蛋白质免疫印迹法（Western Blot）法检测Ghrelin、胃泌素受体（GHSR）蛋白水平；光镜下观察肠黏膜组织病理学改变并行Chiu评分；采用原位末端缺刻标记法（TUNEL）检测肠黏膜上皮细胞凋亡指数（AI）。结果与假手术组比较，模型组血清Ghrelin、胃泌素含量明显减少〔Ghrelin（ng/L）：121.23±3.53比146.28±5.43，胃泌素（ng/L）：81.78±3.27比102.78±4.07〕， TNF-α、HMGB1含量明显增加〔TNF-α（mg/L）：93.71±3.66比11.69±1.44，HMGB1（mg/L）：76.25±4.12比22.41±3.08〕；肠组织DAO活性降低〔DAO（U/mL）：14.64±0.68比25.13±1.98〕，MPO活性升高〔MPO （mg/L）：175.98±6.95比45.64±4.48〕，Ghrelin、GHSR蛋白表达水平明显降低〔Ghrelin（灰度值）：0.12±0.02比0.23±0.04，GHSR（灰度值）：0.18±0.02比0.32±0.03〕；肠黏膜Chiu评分及回肠黏膜上皮细胞AI均明显升高〔Chiu评分（分）：3.90±0.52比0.30±0.30，AI：29.31±1.65比5.45±1.35，均P＜0.01〕。与模型组比较，参附低、高浓度组血清Ghrelin含量和肠组织中Ghrelin、GHSR蛋白表达水平均显著增加（P＜0.05或P＜0.01），肠组织DAO活性、Chiu评分及AI显著降低，且参附高浓度组的变化较参附低浓度组更显著〔Ghrelin（ng/L）：143.54±3.89比136.58±4.91，TNF-α（mg/L）：75.13±4.69比83.70±4.40，HMGB1（mg/L）：57.47±4.53比65.41±4.63，Ghrelin蛋白（灰度值）：0.18±0.03比0.15±0.03，GHSR蛋白（灰度值）：0.28±0.03比0.23±0.03， MPO（mg/L）：154.05±5.75比162.64±5.73，DAO（kU/L）：19.70±1.51比16.67±0.92，Chiu评分（分）：2.30±0.52比3.20±0.48，AI：20.38±1.34比26.40±1.32，均P＜0.05〕；而参附低、高浓度组血清胃泌素含量较模型组有所升高，但差异均无统计学意义（83.59±3.24、86.54±5.93比81.78±3.27，均P＞0.05）。光镜下可见模型组组肠黏膜绒毛破损、肿胀明显，有溃疡形成，血管周围出血明显，有中性粒细胞浸润，基底层断裂；参附组肠绒毛少量缺损，局限性坏死，少量出血，有少量中性粒细胞浸润。结论参附注射液可明显改善脓毒症模型大鼠异常的血清Ghrelin表达，降低血清TNF-α、HMGB1水平，减少肠组织MPO活性、提高DAO活性，减轻回肠黏膜的病理改变，减少回肠黏膜上皮细胞凋亡，且此作用强度与参附注射液剂量成正比。
    목적：관찰삼부주사액대농독증대서장공능적영향。방법장40지대서안수궤수자표법분위가수술조、농독증모형조、삼부저농도화고농도조，매조10지。채용맹장결찰천공술（CLP）제비농독증대서모형，가수술조대서부개복이불행CLP。삼부저、고농도조대서우제모후10 min경미정맥추주삼부주사액5 mL/kg화10 mL/kg；모형조칙추주생리염수10 mL/kg。12 h후취혈병처사대서，채용매련면역흡부시험（ELISA）검측혈청촉위생장격소（Ghrelin）、위비소급염증개질종류배사인자-α（TNF-α）、고천이솔족단백-B1（HMGB1）화수과양화물매（MPO）、이알양화매（DAO）활성；채용단백질면역인적법（Western Blot）법검측Ghrelin、위비소수체（GHSR）단백수평；광경하관찰장점막조직병이학개변병행Chiu평분；채용원위말단결각표기법（TUNEL）검측장점막상피세포조망지수（AI）。결과여가수술조비교，모형조혈청Ghrelin、위비소함량명현감소〔Ghrelin（ng/L）：121.23±3.53비146.28±5.43，위비소（ng/L）：81.78±3.27비102.78±4.07〕， TNF-α、HMGB1함량명현증가〔TNF-α（mg/L）：93.71±3.66비11.69±1.44，HMGB1（mg/L）：76.25±4.12비22.41±3.08〕；장조직DAO활성강저〔DAO（U/mL）：14.64±0.68비25.13±1.98〕，MPO활성승고〔MPO （mg/L）：175.98±6.95비45.64±4.48〕，Ghrelin、GHSR단백표체수평명현강저〔Ghrelin（회도치）：0.12±0.02비0.23±0.04，GHSR（회도치）：0.18±0.02비0.32±0.03〕；장점막Chiu평분급회장점막상피세포AI균명현승고〔Chiu평분（분）：3.90±0.52비0.30±0.30，AI：29.31±1.65비5.45±1.35，균P＜0.01〕。여모형조비교，삼부저、고농도조혈청Ghrelin함량화장조직중Ghrelin、GHSR단백표체수평균현저증가（P＜0.05혹P＜0.01），장조직DAO활성、Chiu평분급AI현저강저，차삼부고농도조적변화교삼부저농도조경현저〔Ghrelin（ng/L）：143.54±3.89비136.58±4.91，TNF-α（mg/L）：75.13±4.69비83.70±4.40，HMGB1（mg/L）：57.47±4.53비65.41±4.63，Ghrelin단백（회도치）：0.18±0.03비0.15±0.03，GHSR단백（회도치）：0.28±0.03비0.23±0.03， MPO（mg/L）：154.05±5.75비162.64±5.73，DAO（kU/L）：19.70±1.51비16.67±0.92，Chiu평분（분）：2.30±0.52비3.20±0.48，AI：20.38±1.34비26.40±1.32，균P＜0.05〕；이삼부저、고농도조혈청위비소함량교모형조유소승고，단차이균무통계학의의（83.59±3.24、86.54±5.93비81.78±3.27，균P＞0.05）。광경하가견모형조조장점막융모파손、종창명현，유궤양형성，혈관주위출혈명현，유중성립세포침윤，기저층단렬；삼부조장융모소량결손，국한성배사，소량출혈，유소량중성립세포침윤。결론삼부주사액가명현개선농독증모형대서이상적혈청Ghrelin표체，강저혈청TNF-α、HMGB1수평，감소장조직MPO활성、제고DAO활성，감경회장점막적병리개변，감소회장점막상피세포조망，차차작용강도여삼부주사액제량성정비。
    Objective To observe the effect of Shenfu injection on intestinal function in rats with sepsis. Methods Forty Sprague-Dawley (SD) rats were randomly divided into four groups: sham operation, sepsis model, low and high concentration Shenfu injection groups, each groupn = 10. The sepsis model was replicated by cecal ligation and puncture (CLP), while the rate in sham operation group just underwent abdominal incision without CLP. Ten minutes after CLP, the low and high dose Shenfu injection groups were given 5 mL/kg and 10 mL/kg Shenfu intravenous injection via a tail vein respectively. The rats in the model group were treated by intravenous injection of 10 mL/kg normal saline through a tail vein in 10 minutes after CLP. Twelve hours later, the rats were sacrificed. The levels of Ghrelin, Gastrin, tumor necrosis factor-α (TNF-α), high mobility group B1 protein (HMGB1), myeloperoxidase (MPO) and diamine oxidase (DAO) activity in serum were detected by enzyme linked immunosorbent assay (ELISA). The levels of protein of Ghrelin and gastrin receptor (GHSR) were detected by Western Blot. Under light microscope, the histopathological changes in intestinal mucosa were investigated, and Chiu score was determined, and the apoptosis index (AI) of intestinal mucosal epithelial cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).Results Compared with sham operation group, in model group, the levels of Ghrelin and Gastrin in serum were significantly decreased [Ghrelin (ng/L): 121.23±3.53 vs. 146.28±5.43, Gastrin (ng/L): 81.78±3.27 vs 102.78±4.07], the serum levels of TNF-α and HMGB1 were markedly increased [TNF-α (mg/L): 93.71±3.66 vs. 11.69±1.44, HMGB1 (mg/L): 76.25±4.12 vs. 22.41±3.08], the DAO activity and protein expressions of Ghrelin and GHSR of intestinal tissue were obviously decreased [DAO (U/mL): 14.64 ±0.68 vs. 25.13±1.98, Ghrelin (grey value): 0.12±0.02 vs. 0.23±0.04, GHSR (grey value): 0.18±0.02 vs. 0.32±0.03], the MPO activity in intestinal tissue, Chiu score of intestinal mucosa and AI of ileum mucosal epithelial cells were remarkably increased [MPO (mg/L): 175.98±6.95 vs. 45.64±4.48, Chiu score: 3.90±0.52 vs. 0.30±0.30, AI: 29.31±1.65 vs. 5.45±1.35, allP < 0.01]. Compared with model group, in low and high Shenfu injection groups, the levels of Ghrelin in serum and protein expressions of Ghrelin and GHSR in intestinal tissues were significantly increased (P < 0.05 orP < 0.01), the activity of DAO of intestinal tissues, the Chiu score and AI were significantly decreased, the degrees of changes being more significant in high Shenfu injection group than those in low Shenfu injection group [Ghrelin (ng/L): 143.54±3.89 vs. 136.58±4.91, TNF-α (mg/L): 75.13±4.69 vs. 83.70±4.40, HMGB1 (mg/L): 57.47±4.53 vs. 65.41±4.63, protein expression of Ghrelin (grey value): 0.18±0.03 vs. 0.15±0.03, protein expression of GHSR (grey value): 0.28±0.03 vs. 0.23±0.03, MPO (mg/L): 154.05±5.75 vs. 162.64 ±5.73, DAO (kU/L): 19.70±1.51 vs. 16.67±0.92, Chiu score: 2.30±0.52 vs. 3.20±0.48, AI: 20.38±1.34 vs. 26.40±1.32, allP < 0.05]. The levels of serum Gastrin in low and high Shenfu injection group were higher than those in model group, but no statistically significant differences were found (83.59±3.24, 86.54±5.93 vs. 81.78±3.27, bothP > 0.05). Under light microscope, the pathological changes were seen as follows: destruction and obvious edema of intestinal mucosal villi, ulcer formation, significant perivascular hemorrhage, presence of neutrophil infiltration and fracture of basement membrane in model group, while in low and high Shenfu groups, the intestinal villi had little defect, focal necrosis, small amounts of hemorrhage and neutrophil infiltration. Conclusions Shenfu injection can significantly improve the abnormal expressions of serum Ghrelin, reduce the levels of serum TNF-α and HMGB1, lowered MPO activity and enhance DAO activity in intestinal tissue, alleviate pathological changes in ileum mucosa, and decrease AI of ileum mucosal epithelial cells in rats with sepsis. And the degree of therapeutic effect is proportional to the Shenfu injection dose.
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