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

1	Srisawat N, Kellum JA. The role of biomarkers in a-cute kidney injury[J]. Crit Care Clin, 2020, 36 (1): 125-140.
2	Mercado MG, Smith DK, Guard EL. Acute kidney in-jury: diagnosis and management[J]. Am Fam Physician, 2019, 100 (11): 687-694.
3	Fan H, Zhao Y, Sun M, et al. Urinary neutrophil ge-latinase-associated lipocalin, kidney injury molecule-1, N-acetyl-β-D-glucosaminidase levels and mortality risk in septic patients with acute kidney injury[J]. Arch Med Sci, 2018, 14 (6): 1381-1386.
4	樊恒，乐健伟，孙敏，等. N-乙酰半胱氨酸上调细胞FLICE抑制蛋白表达对脓毒症小鼠急性肾损伤的保护作用及其机制研究[J/CD]. 中华危重症医学杂志（电子版），2018,11(4):250-255.
5	Skube SJ, Katz SA, Chipman JG, et al. Acute kidney injury and sepsis[J]. Surg Infect (Larchmt), 2018, 19 (2): 216-224.
6	Wang K, Xie S, Xiao K, et al. Biomarkers of sepsis-induced acute kidney injury[J]. Biomed Res Int, 2018 (2018): 6937947.
7	张建楠，刘文，昌广平，等. 核苷酸结合寡聚化结构域样受体蛋白3炎性小体在脓毒症急性肾损伤大鼠肾脏组织的表达及其影响[J/CD]. 中华危重症医学杂志（电子版），2020,13(1):55-59.
8	Lebourgeois S, González-Marín MC, Antol J, et al. E-valuation of N-acetylcysteine on ethanol self-administration in ethanol-dependent rats[J]. Neuropharmacology, 2019 (150): 112-120.
9	Palli E, Makris D, Papanikolaou J, et al. The impact of N-acetylcysteine and ascorbic acid in contrast-induced nephropathy in critical care patients: an open-label randomized controlled study[J]. Crit Care, 2017, 21 (1): 269.
10	Niu X, Yao Q, Li W, et al. Harmine mitigates LPS-induced acute kidney injury through inhibition of the TLR4-NF-κB / NLRP3 inflammasome signalling pathway in mice[J]. Eur J Pharmacol, 2019 (849): 160-169.
11	Shen WC, Liang CJ, Huang TM, et al. Indoxyl sulfate enhances IL-1β-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS / MAPKs / NFκB / AP-1 pathway[J]. Arch Toxicol, 2016, 90 (11): 2779-2792.
12	Fan H, Le JW, Zhu JH. Protective effect of N-ace-tylcysteine pretreatment on acute kidney injury in septic rats[J]. J Surg Res, 2020 (254): 125-134.
13	Li C, Xie N, Li Y, et al. N-acetylcysteine ameliorates cisplatin-induced renal senescence and renal interstitial fibrosis through sirtuin1 activation and p53 deacetylation[J]. Free Radic Biol Med, 2019 (130): 512-527.
14	Gu Y, Huang F, Wang Y, et al. Connexin32 plays a crucial role in ROS-mediated endoplasmic reticulum stress apoptosis signaling pathway in ischemia reperfusion-induced acute kidney injury[J]. J Transl Med, 2018, 16 (1): 117.
15	Fox BM, Gil HW, Kirkbride-Romeo L, et al. Meta-bolomics assessment reveals oxidative stress and altered energy production in the heart after ischemic acute kidney injury in mice[J]. Kidney Int, 2019, 95 (3): 590-610.
16	Gong X, Duan Y, Zheng J, et al. Nephroprotective e-ffects of N-acetylcysteine amide against contrast-induced nephropathy through upregulating thioredoxin-1, inhibiting ASK1 / p38MAPK pathway, and suppressing oxidative stress and apoptosis in rats[J]. Oxid Med Cell Longev, 2016 (2016): 8715185.
17	Rousta AM, Mirahmadi SM, Shahmohammadi A, et al. Protective effect of sesamin in lipopolysaccharide-induced mouse model of acute kidney injury via attenuation of oxidative stress, inflammation, and apoptosis[J]. Immunopharmacol Immunotoxicol, 2018, 40 (5): 423-429.
18	Fan H, Zhao Y, Zhu JH. S-nitrosoglutathione protects lipopolysaccharide-induced acute kidney injury by inhibiting toll-like receptor 4-nuclear factor-κB signal pathway[J]. J Pharm Pharmacol, 2019, 71 (8): 1255-1261.
19	Vasco CF, Watanabe M, Fonseca CDD, et al. Sepsis-induced acute kidney injury: kidney protection effects by antioxidants[J]. Rev Bras Enferm, 2018, 71 (4): 1921-1927.
20	Shimizu MH, Gois PH, Volpini RA, et al. N-acety-lcysteine protects against star fruit-induced acute kidney injury[J]. Ren Fail, 2017, 39 (1): 193-202.
21	Xia Q, Liu C, Zheng X. N-acetylcysteine ameliorates contrast-induced kidney injury in rats with unilateral hydronephrosis[J]. Mol Med Rep, 2018, 17 (2): 2203-2210.

[1]	韩圣瑾, 周正武, 翁云龙, 黄鑫. 碳酸氢钠林格液联合连续性肾脏替代疗法对创伤合并急性肾损伤患者炎症水平及肾功能的影响[J]. 中华危重症医学杂志(电子版), 2023, 16(05): 376-381.
[2]	张秋彬, 张楠, 林清婷, 徐军, 朱华栋, 姜辉. 急性胰腺炎合并急性肾损伤患者的预后评估[J]. 中华危重症医学杂志(电子版), 2023, 16(05): 382-389.
[3]	韩媛媛, 热孜亚·萨贝提, 冒智捷, 穆福娜依·艾尔肯, 陆晨, 桑晓红, 阿尔曼·木拉提, 张丽. 组合式血液净化治疗对脓毒症患者血清炎症因子水平和临床预后的影响[J]. 中华危重症医学杂志(电子版), 2023, 16(04): 272-278.
[4]	张晓燕, 肖东琼, 高沪, 陈琳, 唐发娟, 李熙鸿. 转录因子12过表达对脓毒症相关性脑病大鼠大脑皮质的保护作用及其机制[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 540-549.
[5]	姚咏明. 如何精准评估烧伤脓毒症患者免疫状态[J]. 中华损伤与修复杂志(电子版), 2023, 18(06): 552-552.
[6]	李青霖, 宋仁杰, 周飞虎. 一种重型劳力性热射病相关急性肾损伤小鼠模型的建立与探讨[J]. 中华肾病研究电子杂志, 2023, 12(05): 265-270.
[7]	任加发, 邬步云, 邢昌赢, 毛慧娟. 2022年急性肾损伤领域基础与临床研究进展[J]. 中华肾病研究电子杂志, 2023, 12(05): 276-281.
[8]	李金璞, 饶向荣. 抗病毒药物和急性肾损伤[J]. 中华肾病研究电子杂志, 2023, 12(05): 287-290.
[9]	程庆砾. 新冠病毒感染与肾脏[J]. 中华肾病研究电子杂志, 2023, 12(04): 240-240.
[10]	宋艳琪, 任雪景, 王文娟, 韩秋霞, 续玥, 庄凯婷, 肖拓, 蔡广研. 间充质干细胞对顺铂诱导的小鼠急性肾损伤中细胞铁死亡的作用[J]. 中华肾病研究电子杂志, 2023, 12(04): 187-193.
[11]	朱泽超, 杨新宇, 李侑埕, 潘鹏宇, 梁国标. 染料木黄酮通过SIRT1/p53信号通路对蛛网膜下腔出血后早期脑损伤的作用[J]. 中华神经创伤外科电子杂志, 2023, 09(05): 261-269.
[12]	王淑友, 宋晓晶, 贾术永, 王广军, 张维波. 肝脏去唾液酸糖蛋白受体靶向活体荧光成像评估酒精性肝损伤肝脏功能的研究[J]. 中华消化病与影像杂志(电子版), 2023, 13(06): 443-446.
[13]	易成, 韦伟, 赵宇亮. 急性肾脏病的概念沿革[J]. 中华临床医师杂志(电子版), 2023, 17(08): 906-910.
[14]	谭睿, 王晶, 於江泉, 郑瑞强. 脓毒症中高密度脂蛋白、载脂蛋白A-I和血清淀粉样蛋白A的作用研究进展[J]. 中华临床医师杂志(电子版), 2023, 17(06): 749-753.
[15]	蔡荇, 郑瑞强. 肝素结合蛋白在脓毒症中的应用及研究进展[J]. 中华临床医师杂志(电子版), 2023, 17(04): 487-490.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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