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肠道屏障是由肠上皮细胞(intestinal epithelial cells,IECs)之间的紧密连接组成的,是抵御有害物质由肠道入侵机体的关键[1]。在创伤失血性休克(traumatic hemorrhagic shock,THS)中,人体血量的再分配显著减少肠黏膜的血流量,以优化心脏和大脑等重要器官的血供。然而,缺血缺氧性损伤对IECs的另一个后果是ATP减少和紧密连接破裂,进一步导致肠道屏障功能障碍[2]。肠道含有的大量细菌和其他微生物分子从肠道移位到循环系统被认为是该系统介导全身炎症反应发生的重要贡献者[3]。人们逐渐认识到肠屏障功能的损害成为THS诱导多器官功能障碍综合征(multiple organ dysfunction syndrome,MODS)的启动和发展的关键因素[4]。因此,预防或改善肠屏障功能障碍将是预防THS相关MODS发生一个关键的治疗策略。近年来发现,人类疾病与肠道益生菌越来越相关,而乳酸菌(LAB)作为最具有代表性的益生菌研究最为广泛[5]。而目前对于THS在应用新型复苏液醋酸钠林格液复苏基础上应用乳酸菌探究对肠黏膜屏障的保护作用的研究较少。
既往研究[6-7]表明,TLR4-MAPK信号通路与急性肠道/肺损伤密切相关。因此针对TLR4-MAPK信号通路研究醋酸钠林格液复苏联合乳酸菌对THS大鼠肠黏膜屏障的保护作用是可行的。本研究通过前期应用乳酸菌素片饲养SD大鼠,后期通过建立THS模型,探究醋酸钠林格液复苏基础上应用乳酸菌对THS大鼠肠黏膜屏障作用机制。
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经单因素方差分析,3组大鼠体质量、基础MAP差异均无统计学意义(P>0.05)(见表 1)。
分组 n 体质量/g 平均动脉压/mmHg THS组 10 271.04±10.23 103.12±4.35 AR组 10 273.28±9.71 104.09±6.05 AL组 10 269.25±11.75 102.94±3.20 F — 0.36 0.18 P — >0.05 >0.05 MS组内 — 3 033.431 591.412 表 1 3组大鼠体质量和基础平均动脉压比较(x±s)
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与THS组相比,AL组大鼠外周血TNF-α、IL-6含量降低,IL-4、IL-10含量升高,AR组TNF-α含量降低,差异均有统计学意义(P<0.05);与AR组比较,AL组大鼠外周血TNF-α含量降低,差异有统计学意义(P<0.05),其余各细胞因子水平AR组和AL组比较差异无统计学意义(P>0.05)(见表 2)。
分组 n TNF-α IL-6 IL-4 IL-10 THS组 10 185.98±15.43 80.54±13.77 24.54±7.97 38.64±10.43 AR组 10 143.09±13.08* 70.35±11.54 27.77±6.76 40.03±13.08 AL组 10 122.06±14.66*△ 62.76±7.73* 33.46±8.07* 50.874±9.55* F — 50.99 6.24 3.50 3.63 P — <0.01 <0.01 <0.05 <0.05 MS组内 — 5 617.439 3 442.526 1 572.004 3 339.051 q检验:与THS组比较*P<0.05;与AR组比较△P<0.05 表 2 3组大鼠外周血细胞因子含量比较(x±s; pg/mL)
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各组回肠组织,与THS组相比,AR组大鼠外周血TLR4、P-P38、P-JNK含量降低,AL组ZO-1、Claudin-1含量升高,差异均有统计学意义(P<0.05);与AR组相比,AL组ZO-1、Claudin-1含量升高,TLR4、P-JNK含量降低,差异均有统计学意义(P<0.05)(见图 1、表 3)。
分组 n ZO-1 Claudin-1 TLR4 P-P38 P-JNK THS组 10 0.32±0.10 0.21±0.08 0.44±0.13 0.72±0.21 0.85±0.10 AR组 10 0.38±0.14 0.28±0.15 0.29±0.09* 0.52±0.18* 0.56±0.14* AL组 10 0.51±0.13*△ 0.41±0.11*△ 0.18±0.13*△ 0.47±0.16* 0.41±0.12*△ F — 5.97 8.06 12.72 5.50 33.97 P — <0.01 <0.01 <0.01 <0.01 <0.01 MS组内 — 0.435 0.346 0.380 0.886 0.399 q检验:与THS组比较*P<0.05;与AR组比较△P<0.05 表 3 3组大鼠回肠组织ZO-1、Claudin-1、TLR4、p38磷酸化及JNK磷酸化蛋白相对表达水平比较(x±s)
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在光镜下观察:THS组回肠黏膜完整性破坏,黏膜层可见大量中性粒细胞浸润,绒毛黏膜及黏膜下间质水肿明显,间质层可见淤血;AR组较THS组回肠黏膜完整性破坏少,可见中性粒细胞浸润,黏膜下间质水肿减轻;AL组回肠黏膜结构完整,较少中性粒细胞浸润,无间质淤血表现,间质无明显水肿(见图 2)。
醋酸钠林格液复苏联合乳酸菌对创伤失血性休克大鼠肠黏膜屏障的保护作用
Protective effect of sodium acetate Ringer's solution resuscitation combined with lactic acid bacteria on the intestinal mucosal barrier of rats with traumatic hemorrhagic shock
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摘要:
目的探讨醋酸钠林格液复苏基础上应用乳酸菌对创伤失血性休克大鼠肠黏膜屏障的作用。 方法取30只SD大鼠数字法随机分为创伤失血性休克未复苏组(THS组,n=10),醋酸钠林格液复苏组(AR组,n=10),醋酸钠林格液联合乳酸菌复苏组(AL组,n=10),其中AL组大鼠建立休克模型前在正常喂养的基础上加服用乳酸菌素片1周(按照体积面积换算,大鼠每次服用剂量108 mg/kg,每天3次)。THS、AR组及AL组制备成休克模型[平均动脉压维持(35±5)mmHg],AL组及AR组于休克后60 min应用醋酸钠林格液进行30 min液体复苏,复苏后观察4 h取大鼠回肠组织,THS组不予复苏,于休克观察4 h后取回肠组织。利用ELISA检测大鼠外周血肿瘤坏死因子α(TNF-α)、白细胞介素(IL-6)、IL-4、及IL-10含量;应用Western blotting法检测回肠组织ZO-1、Claudin-1、TLR4、p38磷酸化及JNK磷酸化蛋白相对表达水平。 结果与THS组相比,AL组大鼠外周血TNF-α、IL-6含量降低,IL-4、IL-10含量升高,AR组TNF-α含量降低,差异均有统计学意义(P<0.05);与AR组比较,AL组大鼠外周血TNF-α含量降低,差异均有统计学意义(P<0.05),其余各细胞因子水平AR组和AL组比较差异无统计学意义(P>0.05)。回肠组织中,与THS组相比,AR组大鼠外周血TLR4、P-P38、P-JNK含量降低,AL组ZO-1、Claudin-1含量升高,差异均有统计学意义(P<0.05);与AR组相比,AL组ZO-1、Claudin-1含量升高,TLR4、P-JNK含量降低,差异均有统计学意义(P<0.05);回肠组织病理学结果显示AL组回肠组织损伤程度轻于THS组与AR组。 结论在醋酸钠林格液复苏创伤失血性休克基础上,应用乳酸菌可能进一步抑制TLR4-p38MAPK/JNK炎性信号通路的表达,逆转休克造成的促炎因子和抗炎因子的表达失衡,减轻了创伤失血性休克肠道损伤。 Abstract:ObjectiveTo explore the protective effect of sodium acetate Ringer's solution resuscitation combined with lactic acid bacteria on the intestinal mucosal barrier of rats with traumatic hemorrhagic shock. MethodsThirty SD rats were randomly divided into traumatic hemorrhagic shock without resuscitation group (THS group, n=10), sodium acetate Ringer's solution resuscitation group (AR group, n=10) and sodium acetate Ringer's solution combined with lactic acid bacteria resuscitation group (AL group, n=10).The rats in the AL group were given lactic acid bacteria tablets for 1 week on the basis of normal feeding before the shock model was established (calculated according to the volume area, the rats took 108 mg/kg each time, three times a day).The shock models were established in the THS group, AR group and AL group (average arterial pressure maintained 35±5 mmHg).The AL group and AR group were treated with sodium acetate Ringer's solution for 30 minutes of fluid resuscitation after 60 minutes of shock, and the rats' ileum were observed for 4 hours after resuscitation tissue.THS group was not resuscitated, and ileum tissue was taken 4 hours after shock observation.ELISA was used to detect peripheral blood tumor necrosis factor-α(TNF-α), interleukin(IL)-6, IL-4, and IL-10 concentration.Western blotting method was used to detect the expressions of ileum tissue ZO-1, claudin-1, TLR4, p38 phosphorylation and JNK phosphorylation protein. ResultsCompared with the THS group, the levels of TNF-α and IL-6 in the peripheral blood in the AL group were decreased, and the levels of IL-4 and IL-10 were increased, and the TNF-α concentration in the AR group was decreased, and the differences of which were statistically significant (P<0.05).Compared with the AR group, the level of TNF-α in peripheral blood in the AL group was decreased (P<0.05).There was no statistical difference in the levels of other cytokines between AR group and AL group (P>0.05).Compared with the THS group, the levels of TLR4, P-P38, and P-JNK in the peripheral blood of rats in the AR group were decreased, and the levels of ZO-1 and Claudin-1 were increased in the AL group, and the differences of which were statistically significant (P<0.05).Compared with the AR group, the levels of ZO-1 and Claudin-1 in the AL group were increased, and the levels of TLR4 and P-JNK were decreased (P<0.05).The histopathological results of the ileum showed that the damage of the ileum tissue in the AL group was less than that in the THS group and AR group. ConclusionsOn the basis of sodium acetate Ringer's solution to resuscitate traumatic hemorrhagic shock, the application of lactic acid bacteria may further inhibit the expressions of TLR4-p38MAPK/JNK inflammatory signal pathway and reverse the expression imbalance of pro-inflammatory factors and anti-inflammatory factors caused by shock which reduce the trauma intestinal injury in hemorrhagic shock. -
表 1 3组大鼠体质量和基础平均动脉压比较(x±s)
分组 n 体质量/g 平均动脉压/mmHg THS组 10 271.04±10.23 103.12±4.35 AR组 10 273.28±9.71 104.09±6.05 AL组 10 269.25±11.75 102.94±3.20 F — 0.36 0.18 P — >0.05 >0.05 MS组内 — 3 033.431 591.412 表 2 3组大鼠外周血细胞因子含量比较(x±s; pg/mL)
分组 n TNF-α IL-6 IL-4 IL-10 THS组 10 185.98±15.43 80.54±13.77 24.54±7.97 38.64±10.43 AR组 10 143.09±13.08* 70.35±11.54 27.77±6.76 40.03±13.08 AL组 10 122.06±14.66*△ 62.76±7.73* 33.46±8.07* 50.874±9.55* F — 50.99 6.24 3.50 3.63 P — <0.01 <0.01 <0.05 <0.05 MS组内 — 5 617.439 3 442.526 1 572.004 3 339.051 q检验:与THS组比较*P<0.05;与AR组比较△P<0.05 表 3 3组大鼠回肠组织ZO-1、Claudin-1、TLR4、p38磷酸化及JNK磷酸化蛋白相对表达水平比较(x±s)
分组 n ZO-1 Claudin-1 TLR4 P-P38 P-JNK THS组 10 0.32±0.10 0.21±0.08 0.44±0.13 0.72±0.21 0.85±0.10 AR组 10 0.38±0.14 0.28±0.15 0.29±0.09* 0.52±0.18* 0.56±0.14* AL组 10 0.51±0.13*△ 0.41±0.11*△ 0.18±0.13*△ 0.47±0.16* 0.41±0.12*△ F — 5.97 8.06 12.72 5.50 33.97 P — <0.01 <0.01 <0.01 <0.01 <0.01 MS组内 — 0.435 0.346 0.380 0.886 0.399 q检验:与THS组比较*P<0.05;与AR组比较△P<0.05 -
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