探讨益生菌制剂对脓毒症小鼠生存情况的影响及作用机制。

60只雄性C57BL6小鼠随机分为三组，分别为假手术组（20只）、对照组（20只）和实验组（20只）。在饲养2 d后，实验组小鼠每天给予200 μL的益生菌制剂，对照组和假手术组小鼠每天给予200 μL等渗NaCl溶液，连续灌胃4周。分别对实验组小鼠与对照组小鼠进行盲肠结扎穿孔术（CLP），而假手术组小鼠不予盲肠穿刺，其余步骤同CLP。每组10只用于观察小鼠活动状态及7 d存活情况；每组剩余10只，术后24 h取血液、结肠组织。采用酶联免疫吸附实验法检测各组小鼠血清中炎症因子白细胞介素22（IL-22）、IL-2及肿瘤坏死因子α（TNF-α）的表达水平，苏木素-伊红（HE）染色法观察各组小鼠结肠组织形态，免疫组织化学法观察各组小鼠肠黏膜抗紧密连接相关蛋白（Occludin）表达。

假手术组小鼠生长状态良好；对照组小鼠则蜷缩在笼角，浑身战栗；实验组小鼠较对照组活跃，活动较多，未见明显战栗。术后第7天，对照组无小鼠存活，实验组小鼠仍有3只存活，予安乐死，其7 d存活情况显著高于对照组小鼠（P = 0.020）；而假手术组小鼠10只均存活，予安乐死。对照组与假手术组比较，小鼠血清IL-22 [（103 ± 23）ng/L vs.（27 ± 9）ng/L，t = 7.590，P < 0.001]、IL-2[（328 ± 27）ng/L vs.（77 ± 21）ng/L，t = 21.368，P < 0.001]及TNF-α[（94 ± 22）ng/L vs.（56 ± 9）ng/L，t = 4.734，P < 0.001]表达水平比较，差异均有统计学意义；实验组与假手术组小鼠血清IL-22[（75 ± 33）ng/L vs.（27 ± 9）ng/L，t = 3.755，P = 0.001]、IL-2[（217 ± 30）ng/L vs.（77 ± 21）ng/L，t = 10.850，P < 0.001]及TNF-α[（107 ± 20）ng/L vs.（56 ± 9）ng/L，t = 5.956，P < 0.001]表达水平比较，差异亦均有统计学意义；与对照组相比，实验组小鼠血清IL-22[（103 ± 23）ng/L vs.（75 ± 33）ng/L，t = 2.185，P = 0.042]及IL-2 [（328 ± 27）ng/L vs.（217 ± 30）ng/L，t = 8.371，P < 0.001]表达水平差异均有统计学意义，而TNF-α[（94 ± 22）ng/L vs.（107 ± 20）ng/L，t = 1.363，P = 0.188]表达水平比较，差异无统计学意义。假手术组小鼠的结肠黏膜上皮完整，腺体排列规则，少许或无炎症细胞浸润；对照组小鼠则出现结肠黏膜上皮腺体排列紊乱、变形、缺失，肠上皮细胞间紧密连接结构模糊，以及炎症细胞广泛浸润现象，有些伴有隐窝脓肿形成；而实验组小鼠结肠上皮基本完整，未有糜烂缺失，腺体排列基本正常，炎症细胞浸润程度也较对照组小鼠有所减轻。免疫组织化学法检测结果显示，假手术组小鼠结肠上皮细胞腺泡结构完整，Occludin蛋白表达较多；对照组小鼠结肠上皮细胞腺泡结构破坏、消失，炎症细胞浸润，Occludin蛋白表达较少；实验组小鼠结肠上皮细胞腺泡结构尚完整，腺泡间间隙增宽，Occludin蛋白表达较对照组有所增加。

益生菌可通过抑制肠上皮细胞间紧密连接相关蛋白的减少，稳定肠道黏膜屏障结构，有效提高脓毒症小鼠的存活情况。

To investigate the effects of probiotics on the survival of sepsis mice and their mechanism.

A total of 60 male C57BL6 mice were randomly divided into 3 groups, respectively, the sham operation group (n = 20), the control group (n = 20) and the experimental group (n = 20). After feeding 2 d, mice in the experimental group were given 200 μL probiotics solution daily; mice in the control and sham operation groups were given 200 μL NaCl solution, with continuous intragastric administration for 4 weeks. Cecal ligation and puncture (CLP) was performed on the mice in the experimental and control groups, while mice in the sham operation group were performed with the same procedures of CLP except for cecal puncture. Ten mice in each group were used to observe their activity and survival of 7 d, and the blood and colon tissues were taken in the other 10 mice in each group after 24 hours. The expression levels of serum inflammatory factor interleukin 22 (IL-22), IL-2 and tumor necrosis factor alpha (TNF-α) were detected by enzyme-linked immunosorbent assay, the colon tissues were measured by the hematoxylin and eosin (HE) staining method, and the expression of colonic mucosa (Occludin) in each group was observed by the immunohistochemical method.

Mice in the sham operation group grew well, mice in the control group curled up in a corner of the cage and trembled, and mice in the experimental group were more active than the experimental group, without obvious tremor. At 7 d, there were still 3 survived mice in the experimental group which were given euthanasia; the 7 d survival was significantly higher than that of the control group (P = 0.020). At 7 d, 10 mice in the sham operation group all survived and were given euthanasia. The expressions of serum IL-22 [(103 ± 23) ng/L vs. (27 ± 9) ng/L, t = 7.590, P < 0.001], IL-2 [(328 ± 27) ng/L vs. (77 ± 21) ng/L, t = 21.368, P < 0.001] and TNF-α [(94 ± 22) ng/L vs. (56 ± 9) ng/L, t = 4.734, P < 0.001] in the control group were significantly different as compared to the sham operation group. Meanwhile, the expressions of serum IL-22 [(75 ± 33) ng/L vs. (27 ± 9) ng/L, t = 3.755, P = 0.001], IL-2 [(217 ± 30) ng/L vs. (77 ± 21) ng/L, t = 10.850, P < 0.001] and TNF-α [(107 ± 20) ng/L vs. (56 ± 9) ng/L, t = 5.956, P < 0.001] in the experimental and sham operation groups all showed statistically significant differences. Compared with the control group, the expressions of serum IL-22 [(103 ± 23) ng/L vs. (75 ± 33) ng/L, t = 2.185, P = 0.042] and IL-2 [(328 ± 27) ng/L vs. (217 ± 30) ng/L, t = 8.371, P < 0.001] in the experimental group were significantly different, while the TNF-α [(94 ± 22) ng/L vs. (107 ± 20) ng/L, t = 1.363, P = 0.188] expression showed no statistically significant difference. In the sham operation group, the colonic mucosa was intact and the glands were regularly arranged with little or no inflammatory cell infiltration. However, mice in the control group appeared derangement, deformation and lack of colonic mucosal epithelial glands, fuzzy connected structures of enterocytes, and extensive infiltration of inflammatory cells some of which had crypt abscess. The colonic epithelia of mice in the experimental group were basically complete without erosion and loss, glands were normally arranged, and the infiltration of inflammatory cells decreased more as compared to the control group. The results of immunohistochemistry showed that mice in the sham operation group had complete acinar structures of colonic epithelial cells and more Occludin proteins. The acinar structures of colonic epithelial cells in the control group were destructed and disappeared, and there were infiltration of inflammatory cells and less Occludin proteins. The acinar structures of colonic epithelial cells in the experimental group were complete with widened interacinar gaps, and the expression of Occludin protein increased as compared to the control group.

Probiotics can inhibit the reduction of Occludin of intestinal epithelial cells and stabilize barrier structures of intestinal mucosas, thus effectively improving the survival of septic mice.