西维来司钠对猪心肺复苏后肾肠损伤作用的研究

doi:10.3877/cma.j.issn.1674-6880.2023.06.001

中华危重症医学杂志(电子版) ›› 2023, Vol. 16 ›› Issue (06) : 441 -447. doi: 10.3877/cma.j.issn.1674-6880.2023.06.001

论著

西维来司钠对猪心肺复苏后肾肠损伤作用的研究

周腾达, 陈庆丽, 杨雪林, 陈琪, 徐杰丰, 周光居, 张茂^†(

)

310009　杭州，浙江大学医学院附属第二医院急诊医学科/浙江省严重创伤与烧伤诊治重点实验室/浙江省急危重症临床医学研究中心；现工作单位为宁波市第二医院急诊医学科
323000　浙江丽水，丽水市人民医院急诊医学科
323000　浙江丽水，丽水市中心医院急诊医学科
311400　杭州，杭州市富阳区第一人民医院重症医学科
310009　杭州，浙江大学医学院附属第二医院急诊医学科/浙江省严重创伤与烧伤诊治重点实验室/浙江省急危重症临床医学研究中心

收稿日期:2023-06-16 出版日期:2023-12-31
通信作者: 张茂
基金资助:
国家自然科学基金项目(82072126); 浙江省重点研发计划项目(2021C03073、2021C03036)

Effect of sivelestat on renal and intestinal injuries after cardiopulmonary resuscitation in pigs

Tengda Zhou, Qingli Chen, Xuelin Yang, Qi Chen, Jiefeng Xu, Guangju Zhou, Mao Zhang^†()

Department of Emergency Medicine, the Second Affiliated Hospital, Zhejiang University School of Medicine / Zhejiang Provincial Key Laboratory for Diagnosis and Treatment of Severe Trauma and Burn/Zhejiang Provincial Acute and Critical Care Clinical Medical Research Center, Hangzhou 310009, China
Department of Emergency Medicine, Lishui People's Hospital, Lishui 323000, China
Department of Emergency Medicine, Lishui Central Hospital, Lishui 323000, China
Department of Intensive Care, the First People's Hospital of Fuyang District, Hangzhou 311400, China

Received:2023-06-16 Published:2023-12-31
Corresponding author: Mao Zhang

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引用本文：

周腾达, 陈庆丽, 杨雪林, 陈琪, 徐杰丰, 周光居, 张茂. 西维来司钠对猪心肺复苏后肾肠损伤作用的研究[J]. 中华危重症医学杂志(电子版), 2023, 16(06): 441-447.

Tengda Zhou, Qingli Chen, Xuelin Yang, Qi Chen, Jiefeng Xu, Guangju Zhou, Mao Zhang. Effect of sivelestat on renal and intestinal injuries after cardiopulmonary resuscitation in pigs[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2023, 16(06): 441-447.

目的

通过建立猪心肺复苏（CPR）模型，探讨在猪复苏成功后使用西维来司钠对肾肠损伤的保护效果及机制。

方法

将25头健康白猪分成假手术组（n = 6）、CPR组（n = 10）和西维来司钠组（n = 9）。假手术组只进行实验造模准备，另两组经右心室放电诱导心室颤动持续9 min后开始CPR 6 min以建立动物模型。复苏成功后5 min，西维来司钠组经股静脉泵入西维来司钠10 mg/kg持续1 h，其余两组泵入等量等渗NaCl溶液。在诱颤前及复苏后1、2、4、24 h依次采血检测血清肌酐、尿素氮（BUN）、肠型脂肪酸结合蛋白（iFABP）以及二胺氧化酶（DAO）含量。在复苏后24 h将动物安乐死后立即取肾肠组织标本，测量组织中NOD样受体蛋白3（NLRP3）、裂解的半胱氨酸天冬氨酸蛋白酶1（C-CAS-1）、消皮素D（GSDMD）蛋白表达情况，以及组织中白细胞介素18（IL-18）和IL-1β炎症因子水平。

结果

诱颤前，3组动物间血清肌酐、BUN、iFABP、DAO比较，差异均无统计学意义（F = 0.474、0.722、0.249、1.465，P = 0.629、0.497、0.781、0.253）。复苏后1、2、4、24 h，3组间血清肌酐（F = 405.699、485.730、1 696.551、2 055.637，P均< 0.001）、BUN（F = 98.617、354.974、451.838、1 139.201，P均< 0.001）、iFABP含量（F = 49.349、115.131、283.316、510.195，P均< 0.001）及DAO活性（F = 35.020、105.490、378.235、543.949，P均< 0.001）比较，差异均有统计学意义。CPR组及西维来司钠组血清肌酐、BUN及DAO活性复苏后1、2、4、24 h较假手术组均明显升高，且CPR组更高（P均< 0.05）。而西维来司钠组仅在复苏后2、4、24 h的血清iFABP含量较CPR组均明显降低（P均< 0.05）。同时，复苏后3组动物肾肠组织NLRP3（F = 13.937、47.761，P均< 0.001）、C-CAS-1（F = 64.209、48.423，P均< 0.001）、GSDMD（F = 14.583、93.754，P均< 0.001）蛋白表达水平及IL-1β（F = 566.930、171.428，P均< 0.001）、IL-18（F = 619.730、84.092，P均< 0.001）含量的比较，差异均有统计学意义。且进一步两两比较发现，肾肠组织中西维来司钠组动物的NLRP3、C-CAS-1、GSDMD蛋白表达水平及IL-1β与IL-18含量均显著低于CPR组（P均< 0.05）。

结论

在猪CPR后早期给予西维来司钠对肾肠组织损伤有保护作用，可能与减少肾肠组织细胞焦亡有关。

关键词: 心肺复苏, 西维来司钠, 缺血预处理, 再灌注损伤, 细胞焦亡

Objective

To investigate the protective effect and mechanism of sivelestat on renal and intestinal injury after cardiopulmonary resuscitation (CPR) in pigs.

Methods

A total of 25 healthy white pigs were randomly divided into a sham group (n = 6), a CPR group (n = 10) and a sivelestat group (n = 9). The pigs in the sham group were only used for experimental preparation; the pigs in other two groups were given ventricular fibrillation induced by right ventricular discharge for 9 min and then CPR for 6 min. At 5 min after successful resuscitation, the pigs in the sivelestat group were given 10 mg/kg sivelestat through the femoral vein for 1 h, and the other two groups were given the same amount of isotonic NaCl solution. Blood samples were collected before ventricular fibrillation and at 1, 2, 4 and 24 h after resuscitation to detect serum creatinine, blood urea nitrogen (BUN), intestinal fatty acid-binding protein (iFABP) and diamine oxidase (DAO). At 24 h after resuscitation, the pigs were euthanized and the tissue samples of kidney and intestine were taken immediately to measure the protein expression levels of NOD like receptor protein 3 (NLRP3), cleaved caspase-1 (C-CAS-1) and Gasdermin D (GSDMD), as well as the levels of interleukin-18 (IL-18) and IL-1β.

Results

There were no significant differences in the serum creatinine, BUN, iFABP and DAO among the sham group, CPR group and sivelestat group before ventricular fibrillation (F = 0.474, 0.722, 0.249, 1.465; P = 0.629, 0.497, 0.781, 0.253). The serum levels of creatinine (F = 405.699, 485.730, 1 696.551, 2 055.637; all P < 0.001), BUN (F = 98.617, 354.974, 451.838, 1 139.201; all P < 0.001), iFABP (F = 49.349, 115.131, 283.316, 510.195; all P < 0.001) and DAO (F = 35.020, 105.490, 378.235, 543.949; all P < 0.001) were significantly different among the three groups at 1, 2, 4 and 24 h after resuscitation. The serum levels of creatinine, BUN and DAO in the CPR group and sivelestat group were significantly higher than those in the sham group at 1, 2, 4 and 24 h after resuscitation, and they were highest in the CPR group (all P < 0.05). However, the serum iFABP levels in the sivelestat group were significantly lower than those in the CPR group only at 2, 4 and 24 h after resuscitation (all P < 0.05). Meanwhile, the protein expression levels of NLRP3 (F = 13.937, 47.761; both P < 0.001), C-CAS-1 (F = 64.209, 48.423; both P < 0.001), GSDMD (F = 14.583, 93.754; both P < 0.001) and the contents of IL-1β (F = 566.930, 171.428; both P < 0.001), IL-18 (F = 619.730, 84.092; both P < 0.001) in renal and intestinal tissues among the three groups all showed significant differences after resuscitation. Further pairwise comparison found that the protein expression levels of NLRP3, C-CAS-1, GSDMD and the contents of IL-1β, IL-18 in the sivelestat group were significantly lower than those in the CPR group (all P < 0.05).

Conclusion

Early application of sivelestat after CPR in pigs can inhibit pyroptosis reaction and reduce the damage of kidney and intestine.

Key words: Cardiopulmonary resuscitation, Sivelestat, Ischemic preconditioning, Reperfusion injury, Pyroptosis

表1 各组动物诱颤前及复苏后各时间点血清肌酐和BUN水平的比较（ ± s）

组别	头数	肌酐（μmol/L）					BUN（mmol/L）
组别	头数	诱颤前	CPR后1 h	CPR后2 h	CPR后4 h	CPR后24 h	诱颤前	CPR后1 h	CPR后2 h	CPR后4 h	CPR后24 h
假手术组	6	61.3 ± 4.0	60.2 ± 1.8	62.7 ± 4.1	61.5 ± 1.9	62.4 ± 2.3	7.97 ± 0.52	8.02 ± 0.77	8.12 ± 0.58	8.00 ± 0.46	8.08 ± 0.61
CPR组	9	59.6 ± 1.8	122.2 ± 6.1^a	166.0 ± 6.6^a	212.4 ± 5.0^a	261.8 ± 8.7^a	7.79 ± 0.34	12.07 ± 0.25^a	15.56 ± 0.37^a	18.90 ± 0.56^a	21.99 ± 0.49^a
西维来司钠组	7	59.5 ± 5.4	85.7 ± 2.0^ab	119.4 ± 7.3^ab	163.1 ± 6.4^ab	204.3 ± 3.5^ab	7.74 ± 0.30	10.57 ± 0.38^ab	12.92 ± 0.65^ab	15.38 ± 0.96^ab	17.61 ± 0.50^ab
F值		0.474	405.699	485.730	1 696.551	2 055.637	0.722	98.617	354.974	451.838	1 139.201
P值		0.629	< 0.001	< 0.001	< 0.001	< 0.001	0.497	< 0.001	< 0.001	< 0.001	< 0.001

表1 各组动物诱颤前及复苏后各时间点血清肌酐和BUN水平的比较（ ± s）

组别	头数	肌酐（μmol/L）					BUN（mmol/L）
组别	头数	诱颤前	CPR后1 h	CPR后2 h	CPR后4 h	CPR后24 h	诱颤前	CPR后1 h	CPR后2 h	CPR后4 h	CPR后24 h
假手术组	6	61.3 ± 4.0	60.2 ± 1.8	62.7 ± 4.1	61.5 ± 1.9	62.4 ± 2.3	7.97 ± 0.52	8.02 ± 0.77	8.12 ± 0.58	8.00 ± 0.46	8.08 ± 0.61
CPR组	9	59.6 ± 1.8	122.2 ± 6.1^a	166.0 ± 6.6^a	212.4 ± 5.0^a	261.8 ± 8.7^a	7.79 ± 0.34	12.07 ± 0.25^a	15.56 ± 0.37^a	18.90 ± 0.56^a	21.99 ± 0.49^a
西维来司钠组	7	59.5 ± 5.4	85.7 ± 2.0^ab	119.4 ± 7.3^ab	163.1 ± 6.4^ab	204.3 ± 3.5^ab	7.74 ± 0.30	10.57 ± 0.38^ab	12.92 ± 0.65^ab	15.38 ± 0.96^ab	17.61 ± 0.50^ab
F值		0.474	405.699	485.730	1 696.551	2 055.637	0.722	98.617	354.974	451.838	1 139.201
P值		0.629	< 0.001	< 0.001	< 0.001	< 0.001	0.497	< 0.001	< 0.001	< 0.001	< 0.001

表2 各组动物诱颤前及复苏后各时间点血清iFABP含量和DAO活性的比较（ ± s）

组别	头数	iFABP（ng/L）					DAO（U/L）
组别	头数	诱颤前	CPR后1 h	CPR后2 h	CPR后4 h	CPR后24 h	诱颤前	CPR后1 h	CPR后2 h	CPR后4 h	CPR后24 h
假手术组	6	430 ± 17	432 ± 34	430 ± 23	419 ± 26	423 ± 32	5 622 ± 832	6 148 ± 716	6 479 ± 720	6 032 ± 521	5 995 ± 725
CPR组	9	424 ± 30	565 ± 21^a	681 ± 35^a	762 ± 28^a	868 ± 21^a	6 218 ± 472	10 486 ± 1 298^a	13 122 ± 1 086^a	16 133 ± 849^a	18 797 ± 392^a
西维来司钠组	7	435 ± 44	552 ± 27^a	630 ± 34^ab	703 ± 30^ab	763 ± 25^ab	6 030 ± 758	8 441 ± 772^ab	10 922 ± 627^ab	12 732 ± 598^ab	14 910 ± 914^ab
F值		0.249	49.349	115.131	283.316	510.195	1.465	35.020	105.490	378.235	543.949
P值		0.781	< 0.001	< 0.001	< 0.001	< 0.001	0.253	< 0.001	< 0.001	< 0.001	< 0.001

表2 各组动物诱颤前及复苏后各时间点血清iFABP含量和DAO活性的比较（ ± s）

组别	头数	iFABP（ng/L）					DAO（U/L）
组别	头数	诱颤前	CPR后1 h	CPR后2 h	CPR后4 h	CPR后24 h	诱颤前	CPR后1 h	CPR后2 h	CPR后4 h	CPR后24 h
假手术组	6	430 ± 17	432 ± 34	430 ± 23	419 ± 26	423 ± 32	5 622 ± 832	6 148 ± 716	6 479 ± 720	6 032 ± 521	5 995 ± 725
CPR组	9	424 ± 30	565 ± 21^a	681 ± 35^a	762 ± 28^a	868 ± 21^a	6 218 ± 472	10 486 ± 1 298^a	13 122 ± 1 086^a	16 133 ± 849^a	18 797 ± 392^a
西维来司钠组	7	435 ± 44	552 ± 27^a	630 ± 34^ab	703 ± 30^ab	763 ± 25^ab	6 030 ± 758	8 441 ± 772^ab	10 922 ± 627^ab	12 732 ± 598^ab	14 910 ± 914^ab
F值		0.249	49.349	115.131	283.316	510.195	1.465	35.020	105.490	378.235	543.949
P值		0.781	< 0.001	< 0.001	< 0.001	< 0.001	0.253	< 0.001	< 0.001	< 0.001	< 0.001

图1 Western-blotting法检测各组动物肾肠组织NLRP3、C-CAS-1、GSDMD蛋白表达情况注：NLRP3. NOD样受体蛋白3；C-CAS-1.裂解的半胱氨酸天冬氨酸蛋白酶1；GSDMD.消皮素D；GAPDH. 3-磷酸甘油醛脱氢酶；CPR.心肺复苏

表3 各组动物复苏后肾肠组织NLRP3、C-CAS-1、GSDMD蛋白表达水平比较（ ± s）

组别	头数	肾组织			肠组织
组别	头数	NLRP3	C-CAS-1	GSDMD	NLRP3	C-CAS-1	GSDMD
假手术组	6	0.68 ± 0.11	0.67 ± 0.15	0.45 ± 0.19	0.76 ± 0.12	0.61 ± 0.09	0.90 ± 0.04
CPR组	9	1.22 ± 0.10^a	1.37 ± 0.09^a	1.07 ± 0.12^a	1.42 ± 0.13^a	1.47 ± 0.15^a	1.35 ± 0.08^a
西维来司钠组	7	0.93 ± 0.27^b	0.98 ± 0.06^ab	0.80 ± 0.26^ab	0.95 ± 0.11^ab	1.06 ± 0.19^ab	1.08 ± 0.04^ab
F值		13.937	64.209	14.583	47.761	48.423	93.754
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表3 各组动物复苏后肾肠组织NLRP3、C-CAS-1、GSDMD蛋白表达水平比较（ ± s）

组别	头数	肾组织			肠组织
组别	头数	NLRP3	C-CAS-1	GSDMD	NLRP3	C-CAS-1	GSDMD
假手术组	6	0.68 ± 0.11	0.67 ± 0.15	0.45 ± 0.19	0.76 ± 0.12	0.61 ± 0.09	0.90 ± 0.04
CPR组	9	1.22 ± 0.10^a	1.37 ± 0.09^a	1.07 ± 0.12^a	1.42 ± 0.13^a	1.47 ± 0.15^a	1.35 ± 0.08^a
西维来司钠组	7	0.93 ± 0.27^b	0.98 ± 0.06^ab	0.80 ± 0.26^ab	0.95 ± 0.11^ab	1.06 ± 0.19^ab	1.08 ± 0.04^ab
F值		13.937	64.209	14.583	47.761	48.423	93.754
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表4 各组动物复苏后肾肠组织IL-1β与IL-18含量的比较（ng/L， ± s）

组别	头数	肾组织		肠组织
组别	头数	IL-1β	IL-18	IL-1β	IL-18
假手术组	6	434 ± 52	522 ± 56	530 ± 44	674 ± 96
CPR组	9	1 572 ± 51^a	2 670 ± 77^a	2 170 ± 187^a	3 276 ± 467^a
西维来司钠组	7	1 135 ± 72^ab	1 643 ± 157^ab	1 794 ± 201^ab	2 190 ± 372^ab
F值		566.930	619.730	171.428	84.092
P值		< 0.001	< 0.001	< 0.001	< 0.001

表4 各组动物复苏后肾肠组织IL-1β与IL-18含量的比较（ng/L， ± s）

组别	头数	肾组织		肠组织
组别	头数	IL-1β	IL-18	IL-1β	IL-18
假手术组	6	434 ± 52	522 ± 56	530 ± 44	674 ± 96
CPR组	9	1 572 ± 51^a	2 670 ± 77^a	2 170 ± 187^a	3 276 ± 467^a
西维来司钠组	7	1 135 ± 72^ab	1 643 ± 157^ab	1 794 ± 201^ab	2 190 ± 372^ab
F值		566.930	619.730	171.428	84.092
P值		< 0.001	< 0.001	< 0.001	< 0.001

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Effect of sivelestat on renal and intestinal injuries after cardiopulmonary resuscitation in pigs

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Effect of sivelestat on renal and intestinal injuries after cardiopulmonary resuscitation in pigs