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中华危重症医学杂志(电子版) ›› 2024, Vol. 17 ›› Issue (04) : 275 -284. doi: 10.3877/cma.j.issn.1674-6880.2024.04.002

论著

紫草素调控乳酸化修饰和线粒体功能改善脓毒症心肌病小鼠的预后
张霞1, 张瑞2, 郑志波3, 张勤1,†()   
  1. 1. 310003　杭州，浙江大学医学院附属第一医院老年医学科、全省理化与增龄损伤性疾病诊治研究重点实验室
    2. 100083　北京，中关村美年健康产业研究院
    3. 310030　杭州，浙江大学医学院附属浙江医院肾脏内科
  • 收稿日期:2024-07-25 出版日期:2024-08-31
  • 通信作者: 张勤
  • 基金资助:
    国家自然科学基金青年科学基金项目(82002013); 浙江省自然科学基金项目(LTGY23H150003); 浙江省医药卫生科技计划项目(2022RC093、2023KY436); 浙江省中医药科技计划项目(2023ZL218)

Shikonin improves prognosis of mice with sepsis-induced cardiomyopathy by modulating lactylation and mitochondrial function

Xia Zhang1, Rui Zhang2, Zhibo Zheng3, Qin Zhang1,†()   

  1. 1. Department of Geriatrics, the First Affiliated Hospital, Zhejiang University School of Medicine; Provincial Key Laboratory of Diagnosis and Treatment of Physical-chemical and Aging Injury Diseases, Hangzhou 310003, China
    2. Meinian Public Health Institute, Beijing 100083, China
    3. Department of Nephrology, Affiliated Zhejiang Hospital, Zhejiang University School of Medicine, Hangzhou 310030, China
  • Received:2024-07-25 Published:2024-08-31
  • Corresponding author: Qin Zhang
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引用本文：

张霞, 张瑞, 郑志波, 张勤. 紫草素调控乳酸化修饰和线粒体功能改善脓毒症心肌病小鼠的预后[J]. 中华危重症医学杂志(电子版), 2024, 17(04): 275-284.

Xia Zhang, Rui Zhang, Zhibo Zheng, Qin Zhang. Shikonin improves prognosis of mice with sepsis-induced cardiomyopathy by modulating lactylation and mitochondrial function[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2024, 17(04): 275-284.

目的

本研究旨在探讨乳酸化修饰在脓毒症心肌病中的作用，并深入研究调控乳酸化修饰对脓毒症心肌病心功能及预后的影响。

方法

将80只C57BL/6小鼠分为等渗NaCl溶液对照组（Ctrl组）、紫草素对照组（Shik组）、模型组[脂多糖（LPS）组]、LPS +紫草素组（LPS + Shik组）和LPS +紫草素+乳酸组（LPS + Shik + Lac组），每组各16只。LPS组、LPS + Shik组、LPS + Shik + Lac组通过腹腔内注射LPS（5 mg/kg）制备脓毒症心肌病小鼠模型；Ctrl组和Shik组小鼠根据体质量分别注射等渗NaCl溶液（5 mg/kg）和紫草素（8 mg/kg）。LPS + Shik组于LPS处理前1 h腹腔注射紫草素（8 mg/kg）；LPS + Shik + Lac组于LPS处理前1 h腹腔注射紫草素（8 mg/kg），并在LPS腹腔给药后6 h给予乳酸（pH 6.8，0.5 g/kg）。LPS注射后16 h，每组5只小鼠用于检测心功能，收取心脏组织进行乳酸化修饰蛋白的免疫印迹分析，检测各组小鼠烟酰胺腺嘌呤二核苷酸氧化态（NAD+）及其还原态（NADH）和三磷酸腺苷（ATP）水平。每组另外11只在LPS给药后7 d内密切观察并记录小鼠死亡情况。此外，将6只C57BL/6小鼠分为Ctrl组和LPS组，每组各3只。在LPS注射后16 h，采集两组小鼠心脏样本分析心肌乳酸化修饰蛋白及位点。

结果

5组小鼠赖氨酸乳酸化（Kla）、左心室射血分数（LVEF）、短轴缩短率（FS）、NAD+、NAD+/NADH及ATP比较，差异均有统计学意义（F = 58.745、10.560、12.372、21.347、16.407、32.163，P均< 0.001）。与LPS组比较，LPS + Shik组Kla水平显著降低，LVEF、FS、NAD+、NAD+/NADH及ATP显著升高；与LPS + Shik组比较，LPS + Shik + Lac组Kla水平显著升高，LVEF、FS、NAD+、NAD+/NADH及ATP则显著降低（P均< 0.05）。LPS给药后7 d，5组小鼠分别死亡0、0、9、3、10只。LPS组、LPS + Shik组和LPS + Shik + Lac组小鼠死亡情况比较，差异具有统计学意义（χ2 = 11.717，P = 0.003）。且LPS + Shik组小鼠的7 d死亡情况显著低于LPS组，LPS + Shik + Lac组小鼠的7 d死亡情况则显著高于LPS + Shik组（P均< 0.017）。与对照组相比，模型组小鼠心脏组织中分别有105个差异乳酸化蛋白（DLPs）和191个修饰位点上调，10个DLPs和10个修饰位点下调，其中68.70%的DLPs定位在线粒体中且主要涉及代谢。

结论

乳酸化修饰在脓毒症心肌病中发挥重要作用，机制可能与线粒体能量代谢功能紊乱有关，乳酸化修饰可能是脓毒症心肌病治疗的潜在靶点。

关键词: 脓毒症, 心肌病, 乳酸化修饰, 紫草素, 乳酸
Objective

To investigate the role of lactylation modification in sepsis-induced cardiomyopathy, and to further explore the impact of regulating lactylation modification on cardiac function and prognosis in sepsis-induced cardiomyopathy.

Methods

Eighty C57BL/6 mice were divided into a isotonic NaCl solution control group (Ctrl group), a shikonin control group (Shik group), a model group [lipopolysaccharide (LPS) group], a LPS + shikonin group (LPS + Shik group), and a LPS + shikonin + lactate group (LPS + Shik + Lac group), with 16 mice in each group. The LPS group, LPS + Shik group, and LPS + Shik + Lac group were injected LPS (5 mg/kg) intraperitoneally to induce septic cardiomyopathy; the Ctrl group and Shik group were injected with isotonic NaCl solution (5 mg/kg) and shikonin (8 mg/kg). The LPS + Shik group was administered shikonin (8 mg/kg) intraperitoneally 1 h before LPS treatment; the LPS + Shik + Lac group was administered shikonin (8 mg/kg) intraperitoneally 1 h before LPS treatment and then given lactate (pH 6.8, 0.5 g/kg) 6 h after LPS administration. At 16 h after LPS injection, cardiac function was assessed by echocardiography in all groups (five mice/group), and cardiac tissue was collected for immunoblot analysis of lactylation-modified proteins, as well as for the detection of nicotinamide adenine dinucleotide-oxidation state (NAD+), nicotinamide adenine dinucleotide-reduction state (NADH), and adenosine triphosphate (ATP) levels in the mice. Their survival was closely observed and recorded for seven days after LPS administration (11 mice in each group). Furthermore, six C57BL/6 mice were divided into a Ctrl group and a LPS group, with three mice in each group. Cardiac samples were collected at 16 h after LPS injection to analyze myocardial lactylation-modified proteins and sites by mass spectrometry.

Results

The lysine lactylation (Kla), left ventricular ejection fraction (LVEF), fractional shortening (FS), NAD+, NAD+/NADH, and ATP among the five groups showed statistically significant differences (F = 58.745, 10.560, 12.372, 21.347, 16.407, 32.163; all P < 0.001). Compared with the LPS group, the LPS + Shik group exhibited significantly lower Kla levels and significantly higher LVEF, FS, NAD+, NAD+/NADH, and ATP levels; compared with the LPS + Shik group, the LPS + Shik + Lac group had significantly increased Kla levels and significantly decreased LVEF, FS, NAD+, NAD+/NADH, and ATP levels (all P < 0.05). Within seven days after LPS administration, 0, 0, 9, 3, and 10 mice died in the five groups. The survival rate of the LPS, LPS + Shik, and LPS + Shik + Lac groups was statistically significantly different (χ2 = 11.717, P = 0.003). The 7-day death of the LPS + Shik group was significantly lower than that of the LPS group, and the 7-day death of the LPS + Shik + Lac group was significantly higher than that of the LPS + Shik group (both P < 0.017). Compared with the Ctrl group, 105 differential lactylation proteins (DLPs) and 191 modification sites upregulated, and 10 DLPs and 10 modification sites downregulated in cardiac tissue of mice in the LPS group; 68.70% of DLPs were localized in the mitochondria and mainly involved in metabolism.

Conclusions

Lactylation modification plays a significant role in sepsis-induced cardiomyopathy, and the mechanism possibly involves the dysregulation of mitochondrial energy metabolism. Lactylation modification may be a potential therapeutic target for sepsis-induced cardiomyopathy.

Key words: Sepsis, Cardiomyopathy, Lactylation, Shikonin, Lactate
图1 紫草素降低脓毒症诱导的心脏组织乳酸化修饰并改善心功能及预后注：LPS.脂多糖；Kla.赖氨酸乳酸化；LVEF.左心室射血分数；FS.短轴缩短率；Ctrl组为等渗NaCl溶液对照组；Shik组为紫草素对照组；LPS组为模型组；LPS + Shik组为LPS +紫草素组；LPS + Shik + Lac组为LPS +紫草素+乳酸组；a图为使用特异性针对乳酸化赖氨酸残基的抗Kla抗体对各组小鼠心脏裂解物进行乳酸化蛋白的免疫印迹分析，显示在LPS给药后，广泛的分子量谱上多个蛋白条带发生了显著变化；b图为5组小鼠Kla蛋白表达情况；c ~ d图分别为5组小鼠LVEF和FS表达水平；与LPS组比较，aP < 0.05；与LPS + Shik组比较，bP < 0.05
图2 各组小鼠心肌NAD+、NAD+/NADH及ATP水平注：NAD+.烟酰胺腺嘌呤二核苷酸氧化态；NADH.烟酰胺腺嘌呤二核苷酸还原态；ATP.三磷酸腺苷；LPS.脂多糖；Ctrl组为等渗NaCl溶液对照组；Shik组为紫草素对照组；LPS组为模型组；LPS + Shik组为LPS +紫草素组；LPS + Shik + Lac组为LPS +紫草素+乳酸组；与LPS组比较，aP < 0.05；与LPS + Shik组比较，bP < 0.05
图3 质谱分析脓毒症心肌小鼠中Kla修饰的位点及蛋白特征注：Kla.赖氨酸乳酸化；PCA.主成分分析；Slc25a5.线粒体内膜蛋白二磷酸腺苷/三磷酸腺苷转位酶2；Sdhb.琥珀酸脱氢酶；Decr1. 2,4-二 烯酰辅酶A还原酶1；Ppa2.无机焦磷酸酶2；Hspa9.热休克蛋白a9；Ckmt2.线粒体肌酸激酶2；Sucla2.琥珀酸辅酶A连接酶β亚单位；Acadm.中链酰基辅酶A脱氢酶；Acaa2.乙酰辅酶A酰基转移酶2；H2bc15. H2B组蛋白15；Ctrl组为等渗NaCl溶液对照组；LPS组为模型组；a图为PCA图，图中的横纵轴分别代表第一主成分（PC1）和第二主成分（PC2）的解释度，数值越大表示解释度越高，组内样本的聚集程度反映了样本的重复性；b图为差异修饰位点热图，每行代表一个差异修饰位点，每列代表一个样本，红色表示高表达，蓝色表示低表达，与Ctrl组相比，LPS组乳酸化修饰上调的显著增多（红色代表），但也有部分乳酸化修饰下调（蓝色代表）；c图为火山图，显示乳酸化修饰上调差异最显著的5个位点（红点指示）和下调最显著的5个位点（蓝点指示），灰色表示无变化
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