乌司他丁对脓毒症大鼠急性肺损伤的保护作用及其机制研究

doi:10.3877/cma.j.issn.1674-6880.2017.03.003

目的

研究乌司他丁对脂多糖诱导的脓毒症大鼠急性肺损伤的保护作用及其机制。

方法

将52只大鼠分为乌司他丁组和对照组，每组26只。两组大鼠腹腔注射脂多糖溶液（10 mg/kg）以制备脓毒症模型，分别于建模前18 h和3 h，乌司他丁组大鼠给予乌司他丁（100 000 U/kg），对照组注射等量生理盐水。于造模成功后0.5、2、4、12、24、72 h各时间点采血检测肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、IL-10及肺泡灌洗液蛋白浓度，并通过光学显微镜观察肺组织病理学改变。

结果

两组大鼠血浆TNF-α、IL-6、IL-10及肺泡灌洗液总蛋白浓度各时间点比较，差异均有统计学意义（F = 5.899，P = 0.040；F = 7.932，P = 0.029；F = 18.450，P = 0.005；F = 6.467，P = 0.038），且与对照组相比较，乌司他丁组在0.5、2、4、12、24、72 h各时间点的血浆TNF-α[（21.6 ± 3.8）ng/L vs.（14.8 ± 2.5）ng/L，（69.2 ± 3.2）ng/L vs.（48.8 ± 3.9）ng/L，（74.8 ± 6.9）ng/L vs.（56.3 ± 4.2）ng/L，（120.2 ± 43.8）ng/L vs.（106.4 ± 65.8）ng/L，（190.2 ± 30.4）ng/L vs.（119.8 ± 28.9）ng/L，（159.7 ± 10.5）ng/L vs.（112.3 ± 10.2）ng/L]、IL-6[（145 ± 6）ng/L vs.（119 ± 5）ng/L，（344 ± 19）ng/L vs.（225 ± 22）ng/L，（458 ± 36）ng/L vs.（273 ± 28）ng/L，（949 ± 64）ng/L vs.（328 ± 27）ng/L，（841 ± 61）ng/L vs.（306 ± 6）ng/L，（899 ± 35）ng/L vs.（278 ± 16）ng/L]及肺泡灌洗液总蛋白浓度[（320 ± 8）mg/L vs.（290 ±17）mg/L，（400 ± 7）mg/L vs.（336 ± 9）mg/L，（536 ± 10）mg/L vs.（492 ± 14）mg/L，（806 ± 10）mg/L vs.（608 ± 17）mg/L，（781 ± 17）mg/L vs.（579 ± 16）mg/L，（662 ± 24）mg/L vs.（505 ± 9）mg/L]均明显降低（P均< 0.05），而血浆IL-10浓度[（5.7 ± 0.8）ng/L vs.（11.7 ± 0.9）ng/L，（17.2 ± 1.6）ng/L vs.（31.9 ± 2.6）ng/L，（22.3 ± 2.1）ng/L vs.（54.7 ± 4.8）ng/L，（42.3 ± 4.9）ng/L vs.（80.7 ±1.9）ng/L，（29.7 ± 1.4）ng/L vs.（69.4 ± 3.4）ng/L，（21.4 ± 2.9）ng/L vs.（74.3 ± 5.5）ng/L]均明显升高（P均< 0.05）。对照组大鼠肺泡间隔增厚，间质充血水肿，炎性细胞浸润，肺泡塌陷，而乌司他丁组大鼠上述病理学改变明显减轻。

结论

乌司他丁是通过下调TNF-α、IL-6，上调IL-10水平，从而抑制炎症细胞在肺部的浸润以及肺组织中蛋白酶的表达来保护脂多糖诱导的脓毒症大鼠急性肺损伤。

关键词: 急性肺损伤, 脓毒症, 乌司他丁, 脂多糖类, 大鼠

Objective

To observe the effect and mechanism of ulinastatin on protecting acute lung injury (ALI) in sepsis rats induced by lipopolysaccharide (LPS).

Methods

Fifty-two rats were randomly assigned to the ulinastatin group (n = 26) and control group (n = 26). All the rats were given LPS (10 mg/kg) intraperitoneally, and rats in the ulinastatin group were injected ulinastatin (100 000 U/kg) on 18, 3 h before modeling, and rats in the control group were received equivalence saline. The levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-10 and total protein levels in bronchoalveolar lavage fluid (BALF) were detected at 0.5, 2, 4, 12, 24 and 72 h after modeling. The pathological changes in the lung tissues were observed by optical microscope.

Results

The levels of TNF-α, IL-6, IL-10 and total protein in BALF all showed significant differences between the two groups (F = 5.899, P = 0.040; F = 7.932, P = 0.029; F = 18.450, P = 0.005; F = 6.467, P = 0.038). And compared with the control group, TNF-α [(21.6 ± 3.8) ng/L vs. (14.8 ± 2.5) ng/L, (69.2 ± 3.2) ng/L vs. (48.8 ± 3.9) ng/L, (74.8 ± 6.9) ng/L vs. (56.3 ± 4.2) ng/L, (120.2 ± 43.8) ng/L vs. (106.4 ± 65.8) ng/L, (190.2 ± 30.4) ng/L vs. (119.8 ± 28.9) ng/L, (159.7 ± 10.5) ng/L vs. (112.3 ± 10.2) ng/L], IL-6 [(145 ± 6) ng/L vs. (119 ± 5) ng/L, (344 ± 19) ng/L vs. (225 ± 22) ng/L, (458 ± 36) ng/L vs. (273 ± 28) ng/L, (949 ± 64) ng/L vs. (328 ± 27) ng/L, (841 ± 61) ng/L vs. (306 ± 6) ng/L, (899 ± 35) ng/L vs. (278 ± 16) ng/L] and total protein in BALF [(320 ± 8) mg/L vs. (290 ± 17) mg/L, (400 ± 7) mg/L vs. (336 ± 9) mg/L, (536 ± 10) mg/L vs. (492 ± 14) mg/L, (806 ± 10) mg/L vs. (608 ± 17) mg/L, (781 ± 17) mg/L vs. (579 ±16) mg/L, (662 ± 24) mg/L vs. (505 ± 9) mg/L] were much lower, and the IL-10 levels [(5.7 ± 0.8) ng/L vs. (11.7 ± 0.9) ng/L, (17.2 ± 1.6) ng/L vs. (31.9 ± 2.6) ng/L, (22.3 ± 2.1) ng/L vs. (54.7 ± 4.8) ng/L, (42.3 ± 4.9) ng/L vs. (80.7 ±1.9) ng/L, (29.7 ± 1.4) ng/L vs. (69.4 ± 3.4) ng/L, (21.4 ± 2.9) ng/L vs. (74.3 ± 5.5) ng/L] were much higher in the ulinastatin group at 0.5, 2, 4, 12, 24, and 72 h (all P < 0.05). The optical microscope showed alveolar wall tissue thickening, mesenchyma hyperemia and edema, inflammatory cellular infiltration and alveolar collapse in the control group, and above pathology changes were significantly alleviated in the ulinastatin group.

Conclusion

Ulinastatin can protect sepsis rats with ALI through reducing the levels of TNF-α and IL-6, promoting IL-10 levels and thereby inhibiting the neutrophil infiltration and protease expression in the lungs.

Key words: Acute lung injury, Sepsis, Ulinastatin, Lipopolysaccharides, Rats

表1 两组脓毒症大鼠血浆TNF-α、IL-6和IL-10浓度各时间点的比较（ng/L， ± s）

指标	组别	只数	0.5 h	2h	4 h	12 h	24 h	72 h
TNF-α	乌司他丁组	15	14.8 ± 2.5^a	48.8 ± 3.9^ab	56.3 ± 4.2^abc	106.4 ± 65.8^abcd	119.8 ± 28.9^abcde	112.3 ± 10.2^abcdef
?	对照组	15	21.6 ± 3.8	69.2 ± 3.2^b	74.8 ± 6.9^bc	120.2 ± 43.8^bcd	190.2 ± 30.4^bcde	159.7 ± 10.5^bcdef
IL-6	乌司他丁组	15	119 ± 5^a	225 ± 22^ab	273 ± 28^abc	328 ± 27^abcd	306 ± 6^abcde	278 ± 16^abcdef
?	对照组	15	145 ± 6	344 ± 19^b	458 ± 36^bc	949 ± 64^bcd	841 ± 61^bcde	899 ± 35^bcdef
IL-10	乌司他丁组	15	11.7 ± 0.9^a	31.9 ± 2.6^ab	54.7 ± 4.8^abc	80.7 ±1.9^abcd	69.4 ± 3.4^abcde	74.3 ± 5.5^abcdef
?	对照组	15	5.7 ± 0.8	17.2 ± 1.6^b	22.3 ± 2.1^bc	42.3 ± 4.9^bcd	29.7 ± 1.4^bcde	21.4 ± 2.9^bcdef

表1 两组脓毒症大鼠血浆TNF-α、IL-6和IL-10浓度各时间点的比较（ng/L， ± s）

指标	组别	只数	0.5 h	2h	4 h	12 h	24 h	72 h
TNF-α	乌司他丁组	15	14.8 ± 2.5^a	48.8 ± 3.9^ab	56.3 ± 4.2^abc	106.4 ± 65.8^abcd	119.8 ± 28.9^abcde	112.3 ± 10.2^abcdef
?	对照组	15	21.6 ± 3.8	69.2 ± 3.2^b	74.8 ± 6.9^bc	120.2 ± 43.8^bcd	190.2 ± 30.4^bcde	159.7 ± 10.5^bcdef
IL-6	乌司他丁组	15	119 ± 5^a	225 ± 22^ab	273 ± 28^abc	328 ± 27^abcd	306 ± 6^abcde	278 ± 16^abcdef
?	对照组	15	145 ± 6	344 ± 19^b	458 ± 36^bc	949 ± 64^bcd	841 ± 61^bcde	899 ± 35^bcdef
IL-10	乌司他丁组	15	11.7 ± 0.9^a	31.9 ± 2.6^ab	54.7 ± 4.8^abc	80.7 ±1.9^abcd	69.4 ± 3.4^abcde	74.3 ± 5.5^abcdef
?	对照组	15	5.7 ± 0.8	17.2 ± 1.6^b	22.3 ± 2.1^bc	42.3 ± 4.9^bcd	29.7 ± 1.4^bcde	21.4 ± 2.9^bcdef

表2 两组脓毒症大鼠肺泡灌洗液总蛋白浓度不同时间点的变化（mg/L， ± s）

组别	只数	0.5 h	2h	4 h	12 h	24 h	72 h
乌司他丁组	15	290 ± 17^a	336 ± 9^ab	492 ± 14^abc	608 ± 17^abcd	579 ± 16^abcde	505 ± 9^abcdef
对照组	15	320 ± 8	400 ± 7^b	536 ± 10^bc	806 ± 10^bcd	781 ± 17^bcde	662 ± 24^bcdef

表2 两组脓毒症大鼠肺泡灌洗液总蛋白浓度不同时间点的变化（mg/L， ± s）

组别	只数	0.5 h	2h	4 h	12 h	24 h	72 h
乌司他丁组	15	290 ± 17^a	336 ± 9^ab	492 ± 14^abc	608 ± 17^abcd	579 ± 16^abcde	505 ± 9^abcdef
对照组	15	320 ± 8	400 ± 7^b	536 ± 10^bc	806 ± 10^bcd	781 ± 17^bcde	662 ± 24^bcdef

图1 乌司他丁组和对照组各时间点光学显微镜下观察肺组织情况。注：a、d分别为0.5 h乌司他丁组和对照组，可见肺组织结构完整，肺泡壁结构完整，肺泡内未见水肿液及肺泡萎陷；b为12 h乌司他丁组肺组织结构清晰，肺泡间隔稍增厚，肺泡间质水肿，可见少量炎性细胞浸润；c为72 h乌司他丁组肺泡间隔增厚，肺泡间质水肿，肺泡萎缩塌陷；e为12 h对照组肺泡间隔稍增厚，肺泡内可见红细胞渗出，间质水肿，部分肺泡萎缩塌陷；f为72 h对照组肺泡间隔显著增厚，间质充血水肿，大量炎性细胞浸润，肺泡塌陷，程度明显甚于乌司他丁组（HE染色　× 400）

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Protective effect of ulinastatin on acute lung injury in sepsis rats and its mechanism

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