富氢液对脓毒症小鼠心肌细胞线粒体自噬的影响

doi:10.19586/j.2095-2341.2022.0117

生物技术进展 ›› 2022, Vol. 12 ›› Issue (4): 497-502.DOI: 10.19586/j.2095-2341.2022.0117

富氢液对脓毒症小鼠心肌细胞线粒体自噬的影响

侯凯耀¹(), 张二飞², 郑李娜³, 陈红光⁴, 谢克亮⁵()

^1.延安大学医学院，陕西延安 716000
^2.延安大学附属医院麻醉科，陕西延安 716000
^3.山西省人民医院麻醉科，太原 030012
^4.天津医科大学总医院麻醉科，天津 300052
^5.天津医科大学总医院重症医学科，天津 300052

收稿日期:2022-06-20 接受日期:2022-07-07 出版日期:2022-07-25 发布日期:2022-08-10
通讯作者: 谢克亮
作者简介:侯凯耀 E-mail：daguang521521@163.com；

The Effect of Hydrogen-rich Saline on Mitochondrial Autophagy of Myocardial Cells in Sepsis-induced Mice

Kaiyao HOU¹(), Erfei ZHANG², Lina ZHENG³, Hongguang CHEN⁴, Keliang XIE⁵()

^1.School of Medicine，Yan'an University，Shaanxi Yan'an 716000，China
^2.Department of Anesthesiology，Yan'an University Affiliated Hospital，Shaanxi Yan'an 716000，China
^3.Department of Anesthesiology，Shanxi Provincial People's Hospital，Taiyuan 030012，China
^4.Department of Anesthesiology，Tianjin Medical University General Hospital，Tianjin 300052，China
^5.Department of ICU，Tianjin Medical University General Hospital，Tianjin 300052，China

Received:2022-06-20 Accepted:2022-07-07 Online:2022-07-25 Published:2022-08-10
Contact: Keliang XIE

摘要/Abstract

摘要：

为评价富氢液（hydrogen-rich saline，HRS）对脓毒症小鼠心肌细胞线粒体自噬的调节及其对心功能障碍的治疗作用，选取72只雄性C57BL/6J小鼠作为研究对象，采用随机数字表法分为假手术组（Sham组）、假手术+富氢液组（Sham+HRS组）、脓毒症组（CLP组）、CLP+富氢液组（CLP+HRS组），每组18只。采用盲肠结扎穿孔法建立小鼠CLP模型。Sham+HRS组和CLP+HRS组分别于造模后1、6 h时腹腔注射富氢液10 mL·kg^-1。每组随机取6只小鼠，于造模后24 h时收集小鼠颈动脉血样，采用ELISA法测定血液肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）、白细胞介素（interleukin-1β，IL-1β）、肌钙蛋白I（cardiac troponin I，cTnI）和肌酸激酶同工酶（creatine kinase MB，CK-MB）水平；于造模后24 h时取心肌组织，采用荧光素-荧光酶发光法检测ATP，荧光分光光度法检测线粒体膜电位（mitochondrial membrane potential，MMP）。造模后24 h采用Western blot法测定心肌组织线粒体自噬相关蛋白微管关联蛋白轻链3Ⅱ/轻链3Ⅰ（microtubule-associated protein 1 light/protein 3 light，LC3Ⅱ/LC3Ⅰ）和蛋白62（protein 62，P62）的表达水平。结果显示，与Sham组比较，CLP组血清TNF-α、IL-1β、cTnI和CK-MB水平升高，心肌ATP、MMP水平下降，心肌LC3Ⅱ/LC3Ⅰ表达水平上调，P62表达水平下调，差异有统计学意义（P<0.05）；与CLP组比较，CLP+HRS组血清TNF-α、IL-1β、cTnI和CK-MB含量下降，心肌组织ATP、MMP水平升高，LC3Ⅱ/LC3Ⅰ表达水平进一步上调，P62表达进一步下调（P<0.05）。结果表明，富氢液对脓毒症小鼠心功能障碍的治疗作用可能是通过调节心肌细胞线粒体自噬实现的。研究旨在探讨富氢液对脓毒症小鼠心功能障碍的治疗作用及机制，以期为富氢液的临床转化提供理论依据。

关键词: 富氢液, 脓毒症, 心功能障碍, 线粒体自噬

Abstract:

To evaluate the regulatory effect of hydrogen-rich saline （HRS） on mitochondrial autophagy in myocardial cells of septic mice and its therapeutic effect on cardiac dysfunction， the seventy two male C57BL/6J mice were selected as the research objects and randomly divided into sham operation group （Sham group）， sham operation+hydrogen-rich saline group （Sham+HRS group）， sepsis （CLP group） and CLP+hydrogen-rich saline group （CLP+HRS group）， with 18 mice in each group. CLP model in mice was established by cecal ligation and puncture. Sham+HRS group and CLP+HRS group were intraperitoneally injected with 10 mL·kg^-1 hydrogen rich solution at 1 and 6 hours after modeling， respectively. Six mice in each group were randomly selected， and the mouse carotid blood sample was collected 24 hours after modeling， and the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， cardiac troponin I （cTnI） and creatine kinase MB （CK-MB） in blood were determined by ELISA. Myocardial tissue was taken at 24 hours after modeling， ATP was detected by fluorescein fluorescent enzyme luminescence method， and mitochondrial membrane potential （MMP） was detected by fluorescence spectrophotometry. The expression of microtubule-associated protein 1 light/protein 3 light （LC3Ⅱ/LC3Ⅰ） and protein 62 （P62） in myocardial tissue was measured by Western blot at 24 hours after modeling. Results showed that， compared with Sham group， the levels of serum TNF-α， IL-1β， cTnI and CK-MB in CLP group were increased， the levels of myocardial ATP and MMP were decreased， the expression of myocardial LC3 Ⅱ/LC3 Ⅰ was increased， and the expression of P62 was decreased， the difference was statistically significant （P<0.05）. Compared with CLP group， the levels of serum TNF-α， IL-1β， cTnI and CK-MB in CLP+HRS group decreased， the levels of ATP and MMP in myocardial tissue increased， the expression of LC3Ⅱ/ LC3Ⅰ increased， and the expression of P62 decreased （P<0.05）. The results showed that the therapeutic effect of hydrogen-rich saline on cardiac dysfunction in sepsis mice may be achieved by regulating mitochondrial autophagy of cardiomyocytes. The study amied to explore the therapeutic effect and mechanism of hydrogen-rich saline on cardiac dysfunction in septic mice， and provide a theoretical basis for the clinical transformation of hydrogen-rich saline.

Key words: hydrogen-rich saline, sepsis, cardiac dysfunction, mitochondrial autophagy

中图分类号:

R459.7

侯凯耀, 张二飞, 郑李娜, 陈红光, 谢克亮. 富氢液对脓毒症小鼠心肌细胞线粒体自噬的影响[J]. 生物技术进展, 2022, 12(4): 497-502.

Kaiyao HOU, Erfei ZHANG, Lina ZHENG, Hongguang CHEN, Keliang XIE. The Effect of Hydrogen-rich Saline on Mitochondrial Autophagy of Myocardial Cells in Sepsis-induced Mice[J]. Current Biotechnology, 2022, 12(4): 497-502.

图/表 3

参考文献 28

1	SINGER M, DEUTSCHMAN C S, SEYMOUR C W, et al.. The third international consensus definitions for sepsis and septic shock (Sepsis-3)[J]. JAMA, 2016, 315: 801-810.
2	VIEILLARD-BARON A, CAILLE V, CHARRON C, et al.. Actual incidence of global left ventricular hypokinesia in adult septic shock[J]. Crit. Care Med., 2008, 36: 1701-1706.
3	HAN D, LI X, LI S, et al.. Reduced silent information regulator 1 signaling exacerbates sepsis-induced myocardial injury and mitigates the protective effect of a liver X receptor agonist[J]. Free. Radic. Biol. Med., 2017, 113: 291-303.
4	MARTIN L, PETERS C, HEINBOCKEL L, et al.. The synthetic antimicrobial peptide 19-2.5 attenuates mitochondrial dysfunction in cardiomyocytes stimulated with human sepsis serum[J]. Innate. Immun., 2016, 22: 612-619.
5	SHIRAKABE A, ZHAI P, IKEDA Y, et al.. Drp1-dependent mitochondrial autophagy plays a protective role against pressure overload-induced mitochondrial dysfunction and heart failure[J]. Circulation, 2016, 133: 1249-1263.
6	XIE K, YU Y, PEI Y, et al.. Protective effects of hydrogen gas on murine polymicrobial sepsis via reducing oxidative stress and hmgb1 release[J]. Shock, 2010, 34: 90-97.
7	XIE K, WANG Y, YIN L, et al.. Hydrogen gas alleviates sepsis-induced brain injury by improving mitochondrial biogenesis through the activation of PGC-a in mice[J]. Shock, 2021, 55: 100-109.
8	YAN M, YU Y, MAO X, et al.. Hydrogen gas inhalation attenuates sepsis-induced liver injury in a FUNDC1-dependent manner[J]. Int. Immunopharmacol., 2019, 71: 61-67.
9	刘玲玲,张红涛,于泳浩,等.氢对脓毒症相关性脑病小鼠脑组织Nrf2/HO-1信号通路的影响[J].中华麻醉学杂志,2018,38(1):101-104.
10	杨静华.考马斯亮蓝法测定苦荞麦中可溶性蛋白的含量[J].山西医药杂志,2018,472:207-207.
11	甄恩迪,高阳.紫檀芪对小鼠HT22细胞模拟缺血再灌注损伤的保护作用[J].细胞与自焚免疫学杂志,2017,33(7):915-919.
12	ACUÑA-CASTROVIEJO D, RAHIM I, ACUÑA-FERNÁNDEZ C, et al.. Melatonin, clock genes and mitochondria in sepsis[J]. Cell Mol. Life Sci., 2017, 74: 3965-3987.
13	SINHA K, DAS J, PAL P B, et al.. Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis[J]. Arch. Toxicol., 2013, 87: 1157-1180.
14	LI Y, LIANG P, JIANG B, et al.. CARD9 promotes autophagy in cardiomyocytes in myocardial ischemia/reperfusion injury via interacting with Rubicon directly[J/OL]. Basic Res. Cardiol., 2020, 115: 29 [2022-07-08]. .
15	TAO G, SONG G, QIN S. Molecular hydrogen: current knowledge on mechanism in alleviating free radical damage and diseases[J]. Acta. Biochim. Biophys. Sin., 2019, 51: 1189-1197.
16	LI H, CHEN O, YE Z, et al.. Inhalation of high concentrations of hydrogen ameliorates liver ischemia/reperfusion injury through A2A receptor mediated PI3K-Akt pathway[J]. Biochem. Pharmacol., 2017, 130: 83-92.
17	QIU X, YE Q, SUN M, et al.. Saturated hydrogen improves lipid metabolism disorders and dysbacteriosis induced by a high-fat diet[J]. Exp. Biol. Med. (Maywood), 2020, 245: 512-521.
18	SARAMAGO E A, BORGES G S, SINGOLANI-JR C G, et al.. Molecular hydrogen potentiates hypothermia and prevents hypotension and fever in LPS-induced systemic inflammation[J]. Brain. Behav. Immun., 2019, 75: 119-128.
19	WANG D, WANG L, ZHANG Y, et al.. Hydrogen gas inhibits lung cancer progression through targeting SMC3[J]. Biomed. Pharmacother., 2018, 104: 788-797.
20	SHAO A, WU H, HONG Y, et al.. Hydrogen-rich saline attenuated subarachnoid hemorrhage-induced early brain injury in rats by suppressing inflammatory response: possible involvement of NF-‍κB pathway and nlrp3 inflammasome[J]. Mol. Neurobiol., 2016, 53: 3462-3476.
21	CHEN H, HAN H, LI Y, et al.. Hydrogen alleviated organ injury and dysfunction in sepsis: the role of cross-talk between autophagy and endoplasmic reticulum stress: experimental research[J/OL]. Int. Immunopharmacol., 2020, 78: 106049 [2022-07-08]. .
22	FERDOUS A, BATTIPROLU P K, NI Y G, et al.. FoxO, autophagy, and cardiac remodeling[J]. J. Cardiovasc. Transl. Res., 2010, 3: 355-364.
23	SEBASTIANO S, YASUHIRO M, DANIELA Z, et al.. The role of autophagy in the heart[J]. Annu. Rev. Physiol., 2018, 80: 1-26.
24	XIE K, WANG Y, YIN L, et al.. Hydrogen gas alleviates sepsis-induced brain injury by improving mitochondrial biogenesis through the activation of PGC-α in mice[J]. Shock, 2021, 55: 100-109.
25	CHEN H, LIN H, DONG B, et al.. Hydrogen alleviates cell damage and acute lung injury in sepsis via PINK1/Parkin-mediated mitophagy[J]. Inflamm Res., 2021, 70: 915-930.
26	YAO L, CHEN H, WU Q, et al.. Hydrogen-rich saline alleviates inflammation and apoptosis in myocardial I/R injury via PINK-mediated autophagy[J]. Int. J. Mol. Med., 2019, 44: 1048-1062.
27	LIU Q, WU J, ZHANG X, et al.. Circulating mitochondrial DNA-triggered autophagy dysfunction via STING underlies sepsis-related acute lung injury[J/OL]. Cell Death Dis., 12(7): 673 [2022-07-11]. .
28	CHEN H G, DONG B B, WANG Y Q, et al.. Hydrogen alleviates cell damage and acute lung injury in sepsis via PINK1/Parkin-mediated mitophagy[J]. Inflamm. Res., 2021, 70(8): 915-930.

组别	TNF-α（pg·mL^-1）	IL-1β（pg·mL^-1）	cTnI（pg·mL^-1）	CK-MB（U·L^-1）
Sham组（n=6）	43.17±5.67	31.67±4.72	2.73±0.22	1 450.17±60.48
Sham+HRS组（n=6）	44.01±5.73	30.17±4.79	2.73±0.20	1 464.01±61.56
CLP组（n=6）	184.83±13.11^*	62.67±6.98^*	3.63±0.22^*	3 670.50±404.76^*
CLP+HRS组（n=6）	123.17±8.42^*#	40.17±5.71^*#	3.04±0.28^*#	2 328.67±305.05^*#
F值	366.42	42.67	20.01	99.26
P值	<0.001	<0.001	<0.001	<0.001

组别	TNF-α（pg·mL^-1）	IL-1β（pg·mL^-1）	cTnI（pg·mL^-1）	CK-MB（U·L^-1）
Sham组（n=6）	43.17±5.67	31.67±4.72	2.73±0.22	1 450.17±60.48
Sham+HRS组（n=6）	44.01±5.73	30.17±4.79	2.73±0.20	1 464.01±61.56
CLP组（n=6）	184.83±13.11^*	62.67±6.98^*	3.63±0.22^*	3 670.50±404.76^*
CLP+HRS组（n=6）	123.17±8.42^*#	40.17±5.71^*#	3.04±0.28^*#	2 328.67±305.05^*#
F值	366.42	42.67	20.01	99.26
P值	<0.001	<0.001	<0.001	<0.001

组别	MMP（红/绿强度比值）	ATP（nmol·g^-1蛋白）
Sham组（n=6）	5.02±0.78	6.60±0.67
Sham+HRS组（n=6）	5.02±0.65	6.53±0.69
CLP组（n=6）	3.15±0.50^*	3.32±0.59^*
CLP+HRS组（n=6）	4.44±0.56^*#	6.28±0.67^*#
F值	11.684	34.925
P值	<0.001	<0.001

组别	MMP（红/绿强度比值）	ATP（nmol·g^-1蛋白）
Sham组（n=6）	5.02±0.78	6.60±0.67
Sham+HRS组（n=6）	5.02±0.65	6.53±0.69
CLP组（n=6）	3.15±0.50^*	3.32±0.59^*
CLP+HRS组（n=6）	4.44±0.56^*#	6.28±0.67^*#
F值	11.684	34.925
P值	<0.001	<0.001

组别	LC3Ⅱ/LC3Ⅰ	P62/GAPDH
Sham组（n=6）	1.00±0.14	0.75±0.12
Sham+HRS组（n=6）	0.98±0.13	0.74±0.13
CLP组（n=6）	1.45±0.22^*	0.56±0.10^*
CLP+HRS组（n=6）	1.87±0.29^*#	0.31±0.06^*#
F值	25.430	31.310
P值	<0.001	<0.001

富氢液对脓毒症小鼠心肌细胞线粒体自噬的影响

The Effect of Hydrogen-rich Saline on Mitochondrial Autophagy of Myocardial Cells in Sepsis-induced Mice

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 3

参考文献 28

相关文章 1

编辑推荐

Metrics

本文评价