N-乙酰半胱氨酸对脓毒症小鼠急性肾损伤的保护作用及机制研究

doi:10.3877/cma.j.issn.1674-6880.2021.03.002

目的

探讨N-乙酰半胱氨酸（NAC）对脓毒症小鼠急性肾损伤（AKI）的保护作用及其机制。

方法

将60只小鼠分为假手术组、脓毒症AKI组（SAKI组）和SAKI + NAC组，每组各20只。采用盲肠结扎穿刺法（CLP）构建SAKI小鼠模型，其中SAKI + NAC组在使用NAC（50 mg / kg）预处理3 d后行CLP。假手术组小鼠仅进行外科手术，不予CLP。模型构建成功后24 h处死小鼠，采用苏木素-伊红（HE）染色法评估小鼠肾组织病理损伤，比较3组小鼠血清肌酐、血尿素氮、肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、IL-6、病理学评分、丙二醛、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶、肾组织细胞凋亡情况以及Toll样受体4（TLR4）、核因子κB（NF-κB）、Caspase-3、Bax蛋白表达水平。

结果

HE染色结果显示，假手术组小鼠肾组织切片中肾小球、肾小管和血管结构清晰正常；SAKI组小鼠可见肾小球破裂、广泛的肾小管坏死和刷状缘的丧失，间质大量炎症细胞浸润；SAKI + NAC组小鼠肾组织损伤明显减轻，肾小球、肾小管上皮细胞可见充血水肿，间质炎症细胞明显减少。假手术组、SAKI组和SAKI + NAC组小鼠血清肌酐[（39.0 ± 1.8）、（129.6 ± 4.8）、（81.7 ± 2.0）μmol / L]、血尿素氮[（6.65 ± 0.29）、（24.78 ± 0.71）、（15.45 ± 0.40）mmol / L]、TNF-α [（16.8 ± 0.4）、（43.5 ± 1.0）、（22.0 ± 0.6）ng / L]、IL-1β [（15.2 ± 0.7）、（44.8 ± 1.0）、（25.5 ± 0.8）ng / L]、IL-6 [（20.8 ± 0.4）、（70.6 ± 2.0）、（35.8 ± 1.2）ng / L]、病理学评分[（0.65 ± 0.11）、（3.55 ± 0.14）、（2.00 ± 0.15）分]、丙二醛[（25.1 ± 0.7）、（85.3 ± 1.1）、（40.1 ± 1.6）nmol / g]、SOD [（90.4 ± 1.4）、（22.4 ± 0.8）、（70.7 ± 2.5）U / mg]、GSH-Px [（336.2 ± 5.5）、（181.1± 2.5）、（265.0 ± 6.4）U / mg]、过氧化氢酶[（14.9 ± 0.6）、（5.7 ± 0.4）、（8.4 ± 0.4）U / mg]、TUNEL阳性细胞数[（1.35 ± 0.20）、（13.55 ± 0.72）、（8.25 ± 0.42）个]以及TLR4 [（0.14 ± 0.03）、（0.40 ± 0.03）、（0.18 ± 0.03）]、NF-κB [（0.20 ± 0.04）、（0.66 ± 0.05）、（0.16 ± 0.03）]、Caspase-3 [（0.152 ± 0.027）、（0.464 ± 0.061）、（0.147 ± 0.022）]和Bax [（0.14 ± 0.03）、（0.71 ± 0.03）、（0.15 ± 0.04）]蛋白表达水平比较，差异均有统计学意义（F = 204.316、328.913、378.460、327.102、344.226、123.104、709.235、421.732、233.016、104.521、153.920、127.135、260.514、115.960、505.826，P均< 0.001）。进一步两两比较发现，SAKI + NAC组血清肌酐、血尿素氮、TNF-α、IL-1β、IL-6、病理学评分、丙二醛、TUNEL阳性细胞数以及TLR4、NF-κB、Caspase-3和Bax蛋白表达水平均较SAKI组显著降低，SOD、GSH-Px和过氧化氢酶水平均较SAKI组显著升高（P均< 0.05）。

结论

NAC对脓毒症小鼠AKI有显著的保护作用，其具体机制与抑制肾组织细胞炎症反应、抗氧化损伤和抗细胞凋亡相关。

关键词: N-乙酰半胱氨酸, 脓毒症, 急性肾损伤, 小鼠

Objective

To investigate the protective effect and mechanism of N-acetylcysteine (NAC) on acute kidney injury (AKI) in septic mice.

Methods

Sixty mice were divided into a sham operation group (sham group), a septic AKI group (SAKI group) and a SAKI + NAC group, with 20 mice in each group. The SAKI mouse model was constructed by cecal ligation and puncture (CLP), and mice in the SAKI + NAC group were performed CLP after pretreatment with NAC (50 mg / kg) for 3 d. Mice in the sham group only underwent surgery without CLP. All mice were sacrificed when the model was successfully constructed for 24 h, and the renal tissue pathological injury was evaluated by hematoxylin-eosin (HE) staining. The serum creatinine, blood urea nitrogen, tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-6, pathological score, malondialdehyde, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase and renal tissue apoptosis were compared among these three groups, as well as protein levels of Toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), Caspase-3 and Bax.

Results

HE staining showed that the glomerular, tubular and vascular structures were clear and normal in the renal tissue sections of mice in the sham group. The glomerular rupture, extensive tubular necrosis, loss of brush border and a large number of interstitial inflammatory cells were seen in the SAKI group. The renal tissue damage was alleviated, the glomerular and renal tubular epithelial cells revealed hyperemia and edema, and the interstitial inflammatory cells significantly decreased in the SAKI + NAC group. The serum creatinine [(39.0 ± 1.8), (129.6 ± 4.8), (81.7 ± 2.0) μmol / L], blood urea nitrogen [(6.65 ± 0.29), (24.78 ± 0.71), (15.45 ± 0.40) mmol / L], TNF-α [(16.8 ± 0.4), (43.5 ± 1.0), (22.0 ± 0.6) ng / L], IL-1β [(15.2 ± 0.7), (44.8 ± 1.0), (25.5 ± 0.8) ng / L], IL-6 [(20.8 ± 0.4), (70.6 ± 2.0), (35.8 ± 1.2) ng / L], pathological score [(0.65 ± 0.11), (3.55 ± 0.14), (2.00 ± 0.15)], malondialdehyde [(25.1 ± 0.7), (85.3 ± 1.1), (40.1 ± 1.6) nmol / g], SOD [(90.4 ± 1.4), (22.4 ± 0.8), (70.7 ± 2.5) U / mg], GSH-Px [(336.2 ± 5.5), (181.1 ± 2.5), (265.0 ± 6.4) U / mg], catalase [(14.9 ± 0.6), (5.7 ± 0.4), (8.4 ± 0.4) U / mg], TUNEL positive cells [(1.35 ± 0.20), (13.55 ± 0.72), (8.25 ± 0.42) cells], TLR4 protein [(0.14 ± 0.03), (0.40 ± 0.03), (0.18 ± 0.03)], NF-κB protein [(0.20 ± 0.04), (0.66 ± 0.05), (0.16 ± 0.03)], Caspase-3 protein [(0.152 ± 0.027), (0.464 ± 0.061), (0.147 ± 0.022)] and Bax protein [(0.14 ± 0.03), (0.71 ± 0.03), (0.15 ± 0.04)] levels were statistically significantly different in the sham group, SAKI group and SAKI + NAC group (F = 204.316, 328.913, 378.460, 327.102, 344.226, 123.104, 709.235, 421.732, 233.016, 104.521, 153.920, 127.135, 260.514, 115.960, 505.826; all P < 0.001). Further comparisons revealed that the serum creatinine, blood urea nitrogen, TNF-α, IL-1β, IL-6, pathological score, maldialdehyde, TUNEL positive cells and protein levels of TLR4, NF-κB, Caspase-3 and Bax were significantly lower in the SAKI + NAC group than in the SAKI group, and the SOD, GSH-Px and catalase levels were significantly higher (all P < 0.05).

Conclusion

NAC has a significant protective effect on AKI in septic mice, and its specific mechanisms are related to the inhibition of renal cell inflammation, anti-oxidative damage and anti-apoptosis.

Key words: N-acetylcysteine, Sepsis, Acute kidney injury, Mice

表1 3组小鼠肾功能指标和血清促炎症因子表达水平比较（ ± s）

组别	只数	血清肌酐（μmol / L）	血尿素氮（mmol / L）	TNF-α（ng / L）	IL-1β（ng / L）	IL-6（ng / L）
假手术组	20	39.0 ± 1.8	6.65 ± 0.29	16.8 ± 0.4	15.2 ± 0.7	20.8 ± 0.4
SAKI组	20	129.6 ± 4.8^a	24.78 ± 0.71^a	43.5 ± 1.0^a	44.8 ± 1.0^a	70.6 ± 2.0^a
SAKI + NAC组	20	81.7 ± 2.0^ab	15.45 ± 0.40^ab	22.0 ± 0.6^ab	25.5 ± 0.8^ab	35.8 ± 1.2^ab
F值		204.316	328.913	378.460	327.102	344.226
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表1 3组小鼠肾功能指标和血清促炎症因子表达水平比较（ ± s）

组别	只数	血清肌酐（μmol / L）	血尿素氮（mmol / L）	TNF-α（ng / L）	IL-1β（ng / L）	IL-6（ng / L）
假手术组	20	39.0 ± 1.8	6.65 ± 0.29	16.8 ± 0.4	15.2 ± 0.7	20.8 ± 0.4
SAKI组	20	129.6 ± 4.8^a	24.78 ± 0.71^a	43.5 ± 1.0^a	44.8 ± 1.0^a	70.6 ± 2.0^a
SAKI + NAC组	20	81.7 ± 2.0^ab	15.45 ± 0.40^ab	22.0 ± 0.6^ab	25.5 ± 0.8^ab	35.8 ± 1.2^ab
F值		204.316	328.913	378.460	327.102	344.226
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

图1 3组小鼠肾组织病理学改变

表2 3组小鼠肾组织氧化因子表达水平比较（ ± s）

组别	只数	丙二醛（nmol / g）	SOD（U / mg）	GSH-Px（U / mg）	过氧化氢酶（U / mg）
假手术组	20	25.1 ± 0.7	90.4 ± 1.4	336.2 ± 5.5	14.9 ± 0.6
SAKI组	20	85.3 ± 1.1^a	22.4 ± 0.8^a	181.1 ± 2.5^a	5.7 ± 0.4^a
SAKI + NAC组	20	40.1 ± 1.6^ab	70.7 ± 2.5^ab	265.0 ± 6.4^ab	8.4 ± 0.4^ab
F值		709.235	421.732	233.016	104.521
P值		< 0.001	< 0.001	< 0.001	< 0.001

表2 3组小鼠肾组织氧化因子表达水平比较（ ± s）

组别	只数	丙二醛（nmol / g）	SOD（U / mg）	GSH-Px（U / mg）	过氧化氢酶（U / mg）
假手术组	20	25.1 ± 0.7	90.4 ± 1.4	336.2 ± 5.5	14.9 ± 0.6
SAKI组	20	85.3 ± 1.1^a	22.4 ± 0.8^a	181.1 ± 2.5^a	5.7 ± 0.4^a
SAKI + NAC组	20	40.1 ± 1.6^ab	70.7 ± 2.5^ab	265.0 ± 6.4^ab	8.4 ± 0.4^ab
F值		709.235	421.732	233.016	104.521
P值		< 0.001	< 0.001	< 0.001	< 0.001

图2 3组小鼠肾组织细胞凋亡情况

表3 3组小鼠肾组织TLR4、NF-κB、Caspase-3和Bax蛋白表达水平比较（ ± s）

组别	只数	TLR4	NF-κB	Caspase-3	Bax
假手术组	20	0.14 ± 0.03	0.20 ± 0.04	0.152 ± 0.027	0.14 ± 0.03
SAKI组	20	0.40 ± 0.03^a	0.66 ± 0.05^a	0.464 ± 0.061^a	0.71 ± 0.03^a
SAKI + NAC组	20	0.18 ± 0.03^b	0.16 ± 0.03^b	0.147 ± 0.022^b	0.15 ± 0.04^b
F值		127.135	260.514	115.960	505.826
P值		< 0.001	< 0.001	< 0.001	< 0.001

表3 3组小鼠肾组织TLR4、NF-κB、Caspase-3和Bax蛋白表达水平比较（ ± s）

组别	只数	TLR4	NF-κB	Caspase-3	Bax
假手术组	20	0.14 ± 0.03	0.20 ± 0.04	0.152 ± 0.027	0.14 ± 0.03
SAKI组	20	0.40 ± 0.03^a	0.66 ± 0.05^a	0.464 ± 0.061^a	0.71 ± 0.03^a
SAKI + NAC组	20	0.18 ± 0.03^b	0.16 ± 0.03^b	0.147 ± 0.022^b	0.15 ± 0.04^b
F值		127.135	260.514	115.960	505.826
P值		< 0.001	< 0.001	< 0.001	< 0.001

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Protective effect and mechanism of N-acetylcysteine on acute kidney injury in septic mice

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Protective effect and mechanism of N-acetylcysteine on acute kidney injury in septic mice