高迁移率族蛋白B1 / Toll样受体4信号通路在脓毒症大鼠致急性肺损伤中的作用研究

doi:10.3877/cma.j.issn.1674-6880.2020.06.004

目的

探讨高迁移率族蛋白B1（HMGB1） / Toll样受体4（TLR4）信号通路对脓毒症导致的急性肺损伤大鼠的影响。

方法

将60只清洁级雄性Sprague Dawley大鼠分为假手术组、脓毒症组和实验组，每组各20只。假手术组大鼠麻醉后开腹翻动肠道，随即关腹；脓毒症组和实验组行盲肠结扎穿孔（CLP）术后0.5 h于尾静脉分别注射等渗NaCl溶液（4 mL / kg）及抗HMGB1单克隆抗体（2 mg / kg）。各组分别取10只用于观察大鼠CLP建模后7 d存活情况，其余大鼠于造模后24 h处死并留取肺组织标本。计算各组大鼠的肺损伤Smith评分，并比较各组大鼠HMGB1和TLR4阳性蛋白在肺组织中的表达水平。采用酶联免疫吸附测定检测各组大鼠肺泡灌洗液（BALF）中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、HMGB1和TLR4水平，计算每个巨噬细胞内微球蛋白数以比较各组大鼠肺泡巨噬细胞（AM）的吞噬功能，并采用Western-blotting法测定AM内HMGB1、TLR4蛋白表达水平。

结果

假手术组大鼠建模后7 d全部存活，脓毒症组大鼠仅3只存活，实验组大鼠5只存活。各组大鼠间存活情况的比较，差异有统计学意义（ χ² = 10.833，P = 0.004）；且假手术组大鼠的存活情况明显优于脓毒症组与实验组大鼠（P均< 0.017），而脓毒症组与实验组大鼠间存活情况比较，差异无统计学意义（P = 0.120）。假手术组、脓毒症组及实验组大鼠间肺组织损伤评分[（2.20 ± 0.27）、（8.20 ± 1.27）、（4.25 ± 2.21）分，F = 56.432，P < 0.001]，肺组织HMGB1阳性蛋白[（10.4 ± 1.5）、（34.4 ± 5.0）、（26.6 ± 6.9），F = 35.203，P = 0.003]、TLR4阳性蛋白[（10.6 ± 2.1）、（48.0 ± 5.8）、（38.2 ± 5.3），F = 103.414，P = 0.002]，BALF中TNF-α[（19 ± 4）、（45 ± 4）、（35 ± 4）μg / L，F = 2.749，P < 0.001]、IL-6[（56 ± 19）、（86 ± 15）、（70 ± 19）μg / L，F = 4.648，P = 0.001]、HMGB1[（41 ± 18）、（70 ± 15）、（56 ± 12）μg / L，F = 7.254，P = 0.002]、TLR4[（20.9 ± 1.8）、（51.2 ± 1.6）、（49.8 ± 2.6）μg / L，F = 3.978，P = 0.035]，AM的吞噬功能[（21.8 ± 2.7）、（3.1 ± 1.9）、（12.6 ± 2.2）个，F = 32.821，P = 0.001]及AM中HMGB1蛋白[（11 ± 3）、（40 ± 15）、（24 ± 13），F = 7.253，P < 0.001]、TLR4蛋白[（0.9 ± 0.4）、（1.2 ± 0.6）、（1.1 ± 0.4），F = 3.984，P = 0.028]的比较，差异均有统计学意义。进一步两两比较发现，脓毒症组与实验组大鼠肺组织损伤评分，肺组织HMGB1阳性蛋白、TLR阳性蛋白表达水平，BALF中TNF-α、IL-6、HMGB1水平及AM中HMGB1蛋白表达水平均明显高于假手术组大鼠，且脓毒症组大鼠更高（P均< 0.05）；脓毒症组及实验组大鼠AM的吞噬功能均显著低于假手术组，且脓毒症组更低（P均< 0.05）；脓毒症组及实验组大鼠BALF中TLR4水平及AM中TLR4蛋白表达水平均显著高于假手术组（P均< 0.05），但脓毒症组与实验组间上述指标的比较，差异均无统计学意义（P均> 0.05）。

结论

通过抑制HMGB1 / TLR4信号通路可以缓解脓毒症致肺损伤大鼠肺组织的炎症损伤，抑制炎症介质过度释放，并增强AM的细胞吞噬功能。

关键词: 高迁移率族蛋白质类, Toll样受体4, 脓毒症, 急性肺损伤, 大鼠

Objective

To investigate the effect of high mobility group box 1 (HMGB1) / Toll-like receptor 4 (TLR4) signaling pathway on rats with acute lung injury induced by sepsis.

Methods

A total of 60 clean male Sprague Dawley rats were randomly divided into a sham operation group, a sepsis group and an experimental group, with 20 rats in each group. Rats in the sham operation group received celiotomy, and rats in the sepsis group and experimental group were injected with isotonic NaCl solution (4 mL / kg) and anti-HMGB1 monoclonal antibody (2 mg / kg) respectively via caudal vein 0.5 h after cecal ligation and puncture (CLP). Ten rats in each group were used to observe the survival status 7 d after CLP, and the others were sacrificed to take lung tissue samples 24 h after CLP. The Smith score of lung injury was calculated, and the expression levels of HMGB1 and TLR4 positive proteins in the lung tissue were compared among the three groups. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), HMGB1 and TLR4 in bronchoalveolar lavage fluid (BALF) were measured by the enzyme-linked immunosorbent assay, the phagocytosis of alveolar microphage (AM) was compared by counting the number of microglobulins in each AM, and the expression levels of HMGB1 and TLR4 proteins in AM were determined by Western-blotting.

Results

On 7 d after CLP, rats in the sham operation group all survived, while three rats in the sepsis group and five rats in the experimental group survived. The survival status of rats among the three groups was significantly different ( χ² = 10.833, P = 0.004), and it was better in the sham operation group than in the sepsis group and experimental group (both P < 0.017), but no significant difference was noted between the latter two groups (P = 0.120). The Smith score of lung injury [(2.20 ± 0.27), (8.20 ± 1.27), (4.25 ± 2.21); F = 56.432, P < 0.001], HMGB1 [(10.4 ± 1.5), (34.4 ± 5.0), (26.6 ± 6.9); F = 35.203, P = 0.003] and TLR4 [(10.6 ± 2.1), (48.0 ± 5.8), (38.2 ± 5.3); F = 103.414, P = 0.002] positive proteins in the lung tissue, TNF-α [(19 ± 4), (45 ± 4), (35 ± 4) μg / L; F = 2.749, P < 0.001], IL-6 [(56 ± 19), (86 ± 15), (70 ± 19) μg / L; F = 4.648, P = 0.001], HMGB1 [(41 ± 18), (70 ± 15), (56 ± 12) μg / L; F = 7.254, P = 0.002] and TLR4 [(20.9 ± 1.8), (51.2 ± 1.6), (49.8 ± 2.6) μg / L; F = 3.978, P = 0.035] in BALF, phagocytosis of AM [(21.8 ± 2.7), (3.1 ± 1.9), (12.6 ± 2.2); F = 32.821, P = 0.001], and HMGB1 [(11 ± 3), (40 ± 15), (24 ± 13); F = 7.253, P < 0.001] and TLR4 [(0.9 ± 0.4), (1.2 ± 0.6), (1.1 ± 0.4); F = 3.984, P = 0.028] proteins in AM were significantly different among the sham operation group, sepsis group and experimental group. Further pairwise comparison revealed that the Smith score of lung injury, HMGB1 and TLR4 positive proteins in the lung tissue, TNF-α, IL-6 and HMGB1 in BALF, and HMGB1 protein in AM in the sepsis group and experimental group were higher than those in the sham operation group, and they were highest in the sepsis group (all P < 0.05). The phagocytosis of AM in the sepsis group and experimental group was worse than that in the sham operation group, and it was worst in the sepsis group (all P < 0.05). The levels of TLR4 in BALF and TLR4 protein in AM in the sepsis group and experimental group increased obviously as compared with those in the sham operation group (all P < 0.05), while no difference was noted between the former two groups (both P > 0.05).

Conclusion

Inhibition of the HMGB1 / TLR4 signaling pathway can reduce the inflammatory response, prevent the excessive release of inflammatory mediators, and enhance the phagocytic function of AM in rats with lung injury induced by sepsis.

Key words: High mobility group proteins, Toll-like receptor 4, Sepsis, Acute lung injury, Rats

图1 各组大鼠肺组织病理形态学改变

图2 各组大鼠肺组织HMGB1及TLR4阳性蛋白表达情况

表1 各组大鼠肺组织中HMGB1、TLR4阳性蛋白表达水平的比较（±s）

组别	只数	HMGB1阳性蛋白	TLR4阳性蛋白
假手术组	10	10.4 ± 1.5	10.6 ± 2.1
脓毒症组	10	34.4 ± 5.0^a	48.0 ± 5.8^a
实验组	10	26.6 ± 6.9^ab	38.2 ± 5.3^ab
F值		35.203	103.414
P值		0.003	0.002

表1 各组大鼠肺组织中HMGB1、TLR4阳性蛋白表达水平的比较（±s）

组别	只数	HMGB1阳性蛋白	TLR4阳性蛋白
假手术组	10	10.4 ± 1.5	10.6 ± 2.1
脓毒症组	10	34.4 ± 5.0^a	48.0 ± 5.8^a
实验组	10	26.6 ± 6.9^ab	38.2 ± 5.3^ab
F值		35.203	103.414
P值		0.003	0.002

表2 各组大鼠BALF中TNF-α、IL-6、HMGB1和TLR4水平的比较（μg / L，± s）

组别	只数	TNF-α	IL-6	HMGB1	TLR4
假手术组	10	19 ± 4	56 ± 19	41 ± 18	20.9 ± 1.8
脓毒症组	10	45 ± 4^a	86 ± 15^a	70 ± 15^a	51.2 ± 1.6^a
实验组	10	35 ± 4^ab	70 ± 19^ab	56 ± 12^ab	49.8 ± 2.6^a
F值		2.749	4.648	7.254	3.978
P值		< 0.001	0.001	0.002	0.035

表2 各组大鼠BALF中TNF-α、IL-6、HMGB1和TLR4水平的比较（μg / L，± s）

组别	只数	TNF-α	IL-6	HMGB1	TLR4
假手术组	10	19 ± 4	56 ± 19	41 ± 18	20.9 ± 1.8
脓毒症组	10	45 ± 4^a	86 ± 15^a	70 ± 15^a	51.2 ± 1.6^a
实验组	10	35 ± 4^ab	70 ± 19^ab	56 ± 12^ab	49.8 ± 2.6^a
F值		2.749	4.648	7.254	3.978
P值		< 0.001	0.001	0.002	0.035

图3 各组大鼠AM吞噬功能

表3 各组大鼠AM中HMGB1、TLR4蛋白表达水平的比较（±s）

组别	细胞数（个）	HMGB1	TLR4
假手术组	5 × 10⁶	11 ± 3	0.9 ± 0.4
脓毒症组	5 × 10⁶	40 ± 15^a	1.2 ± 0.6^a
实验组	5 × 10⁶	24 ± 13^ab	1.1 ± 0.4^a
F值		7.253	3.984
P值		< 0.001	0.028

表3 各组大鼠AM中HMGB1、TLR4蛋白表达水平的比较（±s）

组别	细胞数（个）	HMGB1	TLR4
假手术组	5 × 10⁶	11 ± 3	0.9 ± 0.4
脓毒症组	5 × 10⁶	40 ± 15^a	1.2 ± 0.6^a
实验组	5 × 10⁶	24 ± 13^ab	1.1 ± 0.4^a
F值		7.253	3.984
P值		< 0.001	0.028

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Effect of high mobility group box 1 / Toll-like receptor 4 signaling pathway on acute lung injury in septic rats

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Effect of high mobility group box 1 / Toll-like receptor 4 signaling pathway on acute lung injury in septic rats