Introduction 介绍

Acute liver failure (ALF) is defined as severe liver dysfunction characterized by an international normalized ratio (INR) of ≥1.5 and altered mental status caused by hepatic encephalopathy in patients with no known liver diseases. The interval between the onset of jaundice and the development of hepatic encephalopathy is usually no more than 26 weeks [1, 2]. ALF has a high mortality rate, leading to significant health and economic burdens worldwide. At present, there is still a lack of effective treatments for ALF [3]. Liver transplantation is the only recommended treatment for ALF. However, liver transplantation is limited by the scarcity of liver transplant resources and organ rejection. Therefore, an in-depth study of the pathophysiological mechanism of ALF may provide new strategies for the clinical treatment of ALF.
急性肝功能衰竭（ALF）被定义为严重的肝功能障碍，其特征是国际标准化比值（INR）≥1.5 和由肝性脑病引起的精神状态改变，患者没有已知的肝病。黄疸发作与肝性脑病发展之间的时间间隔通常不超过 26 周[1,2]。ALF 具有较高的死亡率，给全球带来重大的健康和经济负担。目前，对 ALF 仍缺乏有效的治疗方法[3]。肝移植是唯一推荐的 ALF 治疗方法。然而，肝移植受限于肝移植资源的稀缺性和器官排斥。因此，对 ALF 的病理生理机制进行深入研究可能为 ALF 的临床治疗提供新策略。

Recent studies have reported that pyroptosis, apoptosis, and necroptosis interact with each other. Therefore, a concept of total cell death has been proposed, called PANoptosis, which is characterized by pyroptosis, apoptosis, and necroptosis, but cannot be explained by any of them alone [4]. PANoptosis is regulated by a cascade of molecular signals. The molecules assemble into complexes called PANoptosome. PANoptosis is associated with a variety of diseases, including infectious and oncological diseases [5, 6]. Pyroptosis includes classical signaling pathways mediated by caspase-1 and non-classical signaling pathways mediated by caspase-4, 5, and 11 [7]. The inflammasome binds to caspase-1 and activates it. Activated caspase-1 cleaves interleukin-1β (IL-1β) and IL-18 precursors, facilitating the secretion of IL-1β and IL-18 and inducing cell death [8]. The non-classical pyroptosis pathway is mainly mediated by caspase-4, caspase-5, and caspase-11. After the activation of caspase-4, caspase-5, and caspase-11, Gasdermin-D (GSDMD) is cleaved to initiate pyroptosis [9]. Apoptosis involves both endogenous and exogenous pathways. The endogenous apoptosis pathway is also known as the mitochondrial apoptosis pathway. Apoptosis is characterized by the activation of caspases, nucleus fragmentation, and the formation of apoptotic bodies. Caspase-3 plays a crucial role in apoptosis. Necroptosis is mainly regulated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL). RIPK1 activates the autophosphorylation of RIPK3, which in turn activates MLKL, leading to cell rupture and ultimately cell death [10].
最近的研究报告称，焦亡、凋亡和坏死相互作用。因此，提出了一个名为 PANoptosis 的总细胞死亡概念，其特点是焦亡、凋亡和坏死，但不能单独由任何一种解释。PANoptosis 受一系列分子信号级联调控。这些分子组装成称为 PANoptosome 的复合物。PANoptosis 与多种疾病相关，包括传染性和肿瘤性疾病。焦亡包括由 caspase-1 介导的经典信号通路和由 caspase-4、5 和 11 介导的非经典信号通路。炎症小体结合 caspase-1 并激活它。激活的 caspase-1 裂解白细胞介素-1β（IL-1β）和 IL-18 前体，促进 IL-1β和 IL-18 的分泌并诱导细胞死亡。非经典焦亡途径主要由 caspase-4、caspase-5 和 caspase-11 介导。激活 caspase-4、caspase-5 和 caspase-11 后，Gasdermin-D（GSDMD）被裂解以启动焦亡。凋亡涉及内源和外源途径。内源凋亡途径也称为线粒体凋亡途径。凋亡的特点是 caspase 的激活、核碎裂和凋亡小体的形成。Caspase-3 在凋亡中起着至关重要的作用。坏死主要由受体相互作用丝氨酸/苏氨酸蛋白激酶 1（RIPK1）和混合谱系激酶结构域样（MLKL）调控。RIPK1 激活 RIPK3 的自磷酸化，后者激活 MLKL，导致细胞破裂最终细胞死亡。

The liver is the major organ related to energy metabolism. Hepatocytes are rich in mitochondria, which serve as the main site of metabolism, producing adenosine triphosphate (ATP) to supply energy for the normal functioning of the liver. Many pathogenic factors in the liver, such as viral infections, drugs, and inflammation, can induce mitochondrial damage and dysfunction by interfering with the tricarboxylic acid cycle [11]. Therefore, in the case of liver failure, the oxygen consumption of liver cells is decreased, the expression of genes related to the tricarboxylic acid cycle is reduced, and the energy metabolism is impaired [12, 13]. In our previous study, we utilized proteomics and discovered that the expression of energy metabolic enzymes malate dehydrogenase 1 (MDH1) and isocitrate dehydrogenase 1 (IDH1) decreased in ALF [14]. Subsequent studies revealed that the histone deacetylase (HDAC) inhibitor ACY1215 alleviated ALF by modulating acetylation at MDH1 K118 and IDH1 K93 sites. However, the role of PANoptosis in ALF and its relationship with MDH1 and IDH1 acetylation remain unclear. Therefore, in this study, we explored the role of PANoptosis in ALF and the effects and mechanisms of deacetylated MDH1 and IDH1 on PANoptosis. This research aims to provide theoretical basis for the clinical treatment of ALF.
肝脏是与能量代谢相关的主要器官。肝细胞富含线粒体，线粒体是代谢的主要场所，产生三磷酸腺苷（ATP）以供给肝脏正常功能所需的能量。肝脏中的许多病原因素，如病毒感染、药物和炎症，可以通过干扰三羧酸循环诱导线粒体损伤和功能障碍[11]。因此，在肝功能衰竭的情况下，肝细胞的氧消耗减少，与三羧酸循环相关基因的表达减少，能量代谢受损[12, 13]。在我们之前的研究中，我们利用蛋白质组学发现，能量代谢酶苹果酸脱氢酶 1（MDH1）和异柠檬酸脱氢酶 1（IDH1）在急性肝功能衰竭中表达下降[14]。随后的研究揭示了组蛋白去乙酰化酶（HDAC）抑制剂 ACY1215 通过调节 MDH1 K118 和 IDH1 K93 位点的乙酰化缓解了急性肝功能衰竭。然而，PANoptosis 在急性肝功能衰竭中的作用及其与 MDH1 和 IDH1 乙酰化的关系仍不清楚。因此，在本研究中，我们探讨了 PANoptosis 在急性肝功能衰竭中的作用，以及去乙酰化的 MDH1 和 IDH1 对 PANoptosis 的影响和机制。本研究旨在为急性肝功能衰竭的临床治疗提供理论基础。

Results 结果

PANoptosis was increased in LPS/D-Gal-induced cell injury
PANoptosis 在 LPS/D-Gal 诱导的细胞损伤中增加了

We first assessed the level of PANoptosis during the ALF process. After the cells were stimulated with LPS/D-Gal, cell death and the expression of PANoptosis-related molecules were detected. As shown in Fig. 1A–C, cell viability decreased, lactate dehydrogenase (LDH) release increased, and propyl iodide (PI) staining indicated increased cell death in the LPS/D-Gal group compared with the control group. The levels of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18, and IL-1β were increased in the LPS/D-Gal group compared with the control group (Fig. 1D, E), indicating elevated levels of PANoptosis in ALF.
我们首先评估了 ALF 过程中 PANoptosis 的水平。细胞受到 LPS/D-Gal 刺激后，检测到细胞死亡和 PANoptosis 相关分子的表达。如图 1A-C 所示，与对照组相比，LPS/D-Gal 组的细胞存活率下降，乳酸脱氢酶（LDH）释放增加，丙碘化物（PI）染色表明细胞死亡增加。与对照组相比，LPS/D-Gal 组的 PANoptosis 相关分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的水平增加（图 1D、E），表明 ALF 中 PANoptosis 水平升高。

Fig. 1: PANoptosis was increased in LPS/D-Gal induced hepatocyte injury.
图 1：LPS/D-Gal 诱导的肝细胞损伤中 PANoptosis 增加。
figure 1

A Cell viability, (B) LDH release, and (C) PI staining of AML-12 cells in each group. D The expression of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18, and IL-1β in AML-12 cells was detected by Western blot. E The expression of RIPK1 and GSDMD in AML-12 cells was detected by immunofluorescence. The results are presented as mean ± SD based on three repetitions. * compared with the control group, P < 0.05.
细胞存活率（B）LDH 释放和（C）AML-12 细胞的 PI 染色在每组中。 D 通过 Western blot 检测 AML-12 细胞中 PANoptosis 相关分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的表达。 E 通过免疫荧光检测 AML-12 细胞中 RIPK1 和 GSDMD 的表达。结果以三次重复的平均值±SD 表示。*与对照组相比，P <0.05。

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Deacetylated MDH1 and IDH1 aggravated PANoptosis during ALF
去乙酰化的 MDH1 和 IDH1 在 ALF 期间加重了 PANoptosis

In our previous studies, we found that the acetylation of MDH1 K118 and IDH1 K93 is closely related to the activity and function of MDH1 and IDH1 [15, 16]. Therefore, in this study, we mutated the MDH1 K118 and IDH1 K93 sites (lysine K mutated to arginine R) to simulate deacetylation. We examined the impact of deacetylated MDH1 and IDH1 on PANoptosis in ALF. The results showed that compared with the LPS/D-Gal + empty vector (EV) and LPS/D-Gal + wild-type (WT) groups, the acetylation levels of MDH1 or IDH1 in LPS/D-Gal + MDH1 K118R or LPS/D-Gal + IDH1 K93R group were significantly decreased. Additionally, cell viability decreased, and LDH release increased in the LPS/D-Gal + MDH1 K118R and LPS/D-Gal + IDH1 K93R groups compared with the LPS/D-Gal +EV and LPS/D-Gal + WT groups (Fig. 2A–C). Moreover, the expressions of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18, and IL-1β were increased in the LPS/D-Gal + MDH1 K118R and LPS/D-Gal + IDH1 K93R groups compared with the LPS/D-Gal +EV and LPS/D-Gal + WT groups (Fig. 2D, E). These results indicated that deacetylated MDH1 and IDH1 aggravated PANoptosis in ALF.
在我们之前的研究中，我们发现 MDH1 K118 和 IDH1 K93 的乙酰化与 MDH1 和 IDH1 的活性和功能密切相关。因此，在这项研究中，我们突变了 MDH1 K118 和 IDH1 K93 位点（赖氨酸 K 突变为精氨酸 R）以模拟去乙酰化。我们检查了去乙酰化的 MDH1 和 IDH1 对 ALF 中 PANoptosis 的影响。结果显示，与 LPS/D-Gal +空载体（EV）和 LPS/D-Gal +野生型（WT）组相比，LPS/D-Gal + MDH1 K118R 或 LPS/D-Gal + IDH1 K93R 组中 MDH1 或 IDH1 的乙酰化水平显著降低。此外，与 LPS/D-Gal +EV 和 LPS/D-Gal + WT 组相比，LPS/D-Gal + MDH1 K118R 和 LPS/D-Gal + IDH1 K93R 组中细胞存活率降低，LDH 释放增加（图 2A-C）。此外，与 LPS/D-Gal +EV 和 LPS/D-Gal + WT 组相比，LPS/D-Gal + MDH1 K118R 和 LPS/D-Gal + IDH1 K93R 组中 PANoptosis 相关分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的表达增加（图 2D、E）。这些结果表明，去乙酰化的 MDH1 和 IDH1 加重了 ALF 中的 PANoptosis。

Fig. 2: Deacetylated MDH1 and IDH1 aggravated PANoptosis during ALF.
图 2：去乙酰化的 MDH1 和 IDH1 在 ALF 期间加重了 PANoptosis。
figure 2

A The levels of MDH1 and IDH1 acetylation, (B) cell viability and (C) LDH release of AML-12 cells in each group. D The expression of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18 and IL-1β in AML-12 cells was detected by Western blot. E The expression of GSDMD in AML-12 cells was detected by immunofluorescence. The results are presented as mean ± SD based on three repetitions. * compared with EV group, P < 0.05; # compared with WT group, P < 0.05; & compared with LPS/D-Gal + EV group, P < 0.05; % compared with LPS/D-Gal + WT group, P < 0.05.
A. 每组 AML-12 细胞中 MDH1 和 IDH1 乙酰化水平，B. 细胞存活率和 C. LDH 释放。D. 通过 Western blot 检测 AML-12 细胞中 PANoptosis 相关分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的表达。E. 通过免疫荧光检测 AML-12 细胞中 GSDMD 的表达。结果以三次重复的平均值±标准差表示。*与 EV 组比较，P < 0.05；#与 WT 组比较，P < 0.05；&与 LPS/D-Gal + EV 组比较，P < 0.05；%与 LPS/D-Gal + WT 组比较，P < 0.05。

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Deacetylated MDH1 and IDH1 weakened the inhibitory effect of the HDAC inhibitor ACY1215 on PANoptosis
去乙酰化的 MDH1 和 IDH1 削弱了 HDAC 抑制剂 ACY1215 对 PANoptosis 的抑制作用

In order to further clarify the effect of deacetylated MDH1 and IDH1 on PANoptosis, the HDAC inhibitor ACY1215 was used. The results showed that compared with the LPS/D-Gal group, the acetylation levels of MDH1 or IDH1 in LPS/D-Gal + ACY1215 group were increased. Cell viability was increased, and LDH release was decreased in LPS/D-Gal + ACY1215 group compared with the LPS/D-Gal group. Besides, compared with the LPS/D-Gal + ACY1215 group, the acetylation levels of MDH1 or IDH1 in the LPS/D-Gal + ACY1215 + MDH1 K118R group or LPS/D-Gal + ACY1215 + IDH1 K93R group were decreased. Additionally, cell viability decreased, and LDH release increased in the LPS/D-Gal + ACY1215 + MDH1 K118R group and LPS/D-Gal + ACY1215 + IDH1 K93R group compared with the LPS/D-Gal + ACY1215 group (Fig. 3A–C). We further examined the levels of PANoptosis and found that compared with the LPS/D-Gal group, the levels of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18, and IL-1β were decreased in the LPS/D-Gal + ACY1215 group. Compared with LPS/D-Gal + ACY1215 group, the levels of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18, and IL-1β were increased in LPS/D-Gal + ACY1215 + MDH1 K118R group and LPS/D-Gal + ACY1215 + IDH1 K93R group (Fig. 3D, E). These results indicated that deacetylated MDH1 and IDH1 weakened the inhibitory effect of the HDAC inhibitor ACY1215 on PANoptosis, suggesting that the acetylation of MDH1 and IDH1 influenced PANoptosis.
为了进一步阐明去乙酰化的 MDH1 和 IDH1 对 PANoptosis 的影响，使用了 HDAC 抑制剂 ACY1215。结果显示，与 LPS/D-Gal 组相比，LPS/D-Gal + ACY1215 组中 MDH1 或 IDH1 的乙酰化水平增加。与 LPS/D-Gal 组相比，LPS/D-Gal + ACY1215 组中细胞存活率增加，LDH 释放减少。此外，与 LPS/D-Gal + ACY1215 组相比，LPS/D-Gal + ACY1215 + MDH1 K118R 组或 LPS/D-Gal + ACY1215 + IDH1 K93R 组中 MDH1 或 IDH1 的乙酰化水平降低。另外，与 LPS/D-Gal + ACY1215 组相比，LPS/D-Gal + ACY1215 + MDH1 K118R 组和 LPS/D-Gal + ACY1215 + IDH1 K93R 组中细胞存活率降低，LDH 释放增加（图 3A-C）。我们进一步检测了 PANoptosis 水平，发现与 LPS/D-Gal 组相比，LPS/D-Gal + ACY1215 组中 PANoptosis 相关分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的水平降低。与 LPS/D-Gal + ACY1215 组相比，LPS/D-Gal + ACY1215 + MDH1 K118R 组和 LPS/D-Gal + ACY1215 + IDH1 K93R 组中 PANoptosis 相关分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的水平增加（图 3D、E）。这些结果表明，去乙酰化的 MDH1 和 IDH1 削弱了 HDAC 抑制剂 ACY1215 对 PANoptosis 的抑制作用，表明 MDH1 和 IDH1 的乙酰化影响了 PANoptosis。

Fig. 3: Deacetylated MDH1 and IDH1 weakened the inhibition of HDAC inhibitor ACY1215 on PANoptosis.
图 3：去乙酰化的 MDH1 和 IDH1 削弱了 HDAC 抑制剂 ACY1215 对 PANoptosis 的抑制。
figure 3

A The levels of MDH1 and IDH1 acetylation, (B) cell viability, and (C) LDH release of AML-12 cells in each group. D The expression of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18 and IL-1β in AML-12 cells was detected by Western blot. E The expression of GSDMD in AML-12 cells was detected by immunofluorescence. The results are presented as mean ± SD based on three repetitions. * compared with LPS/D-Gal group, P < 0.05; # compared with LPS/D-Gal + ACY1215 group, P < 0.05.
A. AML-12 细胞中 MDH1 和 IDH1 的乙酰化水平，B. 细胞存活率，以及 C. 每组 AML-12 细胞的 LDH 释放。D. 通过 Western blot 检测 AML-12 细胞中与 PANoptosis 相关的分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的表达。E. 通过免疫荧光检测 AML-12 细胞中 GSDMD 的表达。结果以三次重复的平均值±标准差表示。*与 LPS/D-Gal 组相比，P < 0.05；#与 LPS/D-Gal + ACY1215 组相比，P < 0.05。

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Deacetylated MDH1 and IDH1 could exacerbate liver injury and aggravate PANoptosis in liver tissues of mice with ALF
脱乙酰化的 MDH1 和 IDH1 可能加重小鼠 ALF 肝组织的损伤并恶化 PANoptosis

We further verified these results in mice. MDH1 K118R and IDH1 K93R mutated adeno-associated viruses were injected into mice and the HDAC inhibitor ACY1215 was administered in mice. As shown in Fig. 4A, B, the MDH1 K118R or IDH1 K93R mutations significantly reduced the acetylation levels of MDH1 or IDH1. HE staining and liver function test results showed that liver injury was more severe, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were increased in the LPS/D-Gal + MDH1 K118R group and LPS/D-Gal + IDH1 K93R group compared with the LPS/D-Gal + EV and LPS/D-Gal + WT group. In addition, compared with the LPS/D-Gal + ACY1215 group, liver damage increased, and the levels of ALT, AST, and TBIL were elevated in the LPS/D-Gal + ACY1215 + MDH1 K118R group and LPS/D-Gal + ACY1215 + IDH1 K93R group (Fig. 4C, D). These results indicated that deacetylated MDH1 and IDH1 could exacerbate liver tissue damage in mice with ALF. We further examined the levels of PANoptosis in the liver tissues of mice. The results showed that compared with the LPS/D-Gal + EV and LPS/D-Gal + WT groups, the levels of PANoptosis-related molecules RIPK1, caspase-3, MLKL, IL-18, and IL-1β were increased in the LPS/D-Gal + MDH1 K118R group and LPS/D-Gal + IDH1 K93R group. Besides, compared with LPS/D-Gal + ACY1215 group, the levels of these molecules in LPS/D-Gal + ACY1215 + MDH1 K118R and LPS/D-Gal + ACY1215 + IDH1 K93R groups were significantly increased (Fig. 5A, B). These results suggested that deacetylated MDH1 and IDH1 could elevate the level of PANoptosis in the liver tissues of ALF mice.
我们进一步在小鼠中验证了这些结果。将 MDH1 K118R 和 IDH1 K93R 突变的腺相关病毒注射到小鼠体内，并在小鼠中给予 HDAC 抑制剂 ACY1215。如图 4A、B 所示，MDH1 K118R 或 IDH1 K93R 突变显著降低了 MDH1 或 IDH1 的乙酰化水平。HE 染色和肝功能检测结果显示，与 LPS/D-Gal + EV 和 LPS/D-Gal + WT 组相比，LPS/D-Gal + MDH1 K118R 组和 LPS/D-Gal + IDH1 K93R 组的肝损伤更严重，丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和总胆红素（TBIL）水平增高。此外，与 LPS/D-Gal + ACY1215 组相比，LPS/D-Gal + ACY1215 + MDH1 K118R 组和 LPS/D-Gal + ACY1215 + IDH1 K93R 组的肝损伤加重，ALT、AST 和 TBIL 水平升高（图 4C、D）。这些结果表明，脱乙酰化的 MDH1 和 IDH1 可能加重小鼠 ALF 的肝组织损伤。我们进一步检测了小鼠肝组织中 PANoptosis 的水平。结果显示，与 LPS/D-Gal + EV 和 LPS/D-Gal + WT 组相比，LPS/D-Gal + MDH1 K118R 组和 LPS/D-Gal + IDH1 K93R 组中 PANoptosis 相关分子 RIPK1、caspase-3、MLKL、IL-18 和 IL-1β的水平增加。此外，与 LPS/D-Gal + ACY1215 组相比，LPS/D-Gal + ACY1215 + MDH1 K118R 组和 LPS/D-Gal + ACY1215 + IDH1 K93R 组中这些分子的水平显著增加（图 5A、B）。这些结果表明，脱乙酰化的 MDH1 和 IDH1 可能提高 ALF 小鼠肝组织中 PANoptosis 的水平。

Fig. 4: Deacetylated MDH1 and IDH1 aggravated liver tissue injury in mice.
图 4：去乙酰化的 MDH1 和 IDH1 加重了小鼠肝组织损伤。
figure 4

A Representative fluorescent images of adeno-associated viruses and (B) acetylation levels of MDH1 and IDH1 in liver tissues of mice in each group. C HE staining of mice liver tissues in each group. D Serum ALT, AST, and TBIL levels of mice in each group. The results are presented as mean ± SD based on three repetitions. * compared with EV group, P < 0.05; # compared with WT group, P < 0.05; & compared with EV + LPS/D-Gal group, P < 0.05; % compared with WT + LPS/D-Gal group, P < 0.05; $ compared with EV + LPS/D-Gal + ACY1215 group, P < 0.05; @ compared with WT + LPS/D-Gal + ACY1215 group, P < 0.05.
A 代表性的腺相关病毒荧光图像和（B）小鼠肝组织中 MDH1 和 IDH1 的乙酰化水平。C 每组小鼠肝组织的 HE 染色。D 每组小鼠的血清 ALT、AST 和 TBIL 水平。结果以三次重复的平均值±标准差表示。* 与 EV 组相比，P < 0.05；# 与 WT 组相比，P < 0.05；& 与 EV + LPS/D-Gal 组相比，P < 0.05；% 与 WT + LPS/D-Gal 组相比，P < 0.05；$ 与 EV + LPS/D-Gal + ACY1215 组相比，P < 0.05；@ 与 WT + LPS/D-Gal + ACY1215 组相比，P < 0.05。

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Fig. 5: Deacetylated MDH1 and IDH1 aggravated PANoptosis in liver tissues of ALF mice.
图 5：脱乙酰化的 MDH1 和 IDH1 加重了 ALF 小鼠肝组织中的 PANoptosis。
figure 5

A The expression of PANoptosis-related molecules MLKL, caspase-3, IL-18, and IL-1β in mice liver tissues was detected by Western blot. B The expression of RIPK1 in mice liver tissues was detected by immunofluorescence. The results are presented as mean ± SD based on three repetitions. * compared with EV group, P < 0.05; # compared with WT group, P < 0.05; & compared with EV + LPS/D-Gal group, P < 0.05; % compared with WT + LPS/D-Gal group, P < 0.05; $ compared with EV + LPS/D-Gal + ACY1215 group, P < 0.05; @ compared with WT + LPS/D-Gal + ACY1215 group, P < 0.05.
A 通过 Western blot 检测小鼠肝组织中与 PANoptosis 相关分子 MLKL、caspase-3、IL-18 和 IL-1β的表达。B 通过免疫荧光检测小鼠肝组织中 RIPK1 的表达。结果以三次重复的平均值±标准差表示。*与 EV 组相比，P < 0.05；#与 WT 组相比，P < 0.05；&与 EV + LPS/D-Gal 组相比，P < 0.05；%与 WT + LPS/D-Gal 组相比，P < 0.05；$与 EV + LPS/D-Gal + ACY1215 组相比，P < 0.05；@与 WT + LPS/D-Gal + ACY1215 组相比，P < 0.05。

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Deacetylated MDH1 and IDH1 promoted endoplasmic reticulum stress signaling in vivo and in vitro
脱乙酰化的 MDH1 和 IDH1 在体内和体外促进了内质网应激信号传导

Deacetylated MDH1 and IDH1 deacetylation could aggravate PANoptosis during ALF, but the mechanism is unclear. Studies have shown that endoplasmic reticulum (ER) stress is associated with the occurrence and development of a variety of liver diseases, such as non-alcoholic fatty liver disease, alcohol-related liver disease, viral hepatitis, liver ischemia, and liver cancer [17]. Therefore, we examined the effects of deacetylated MDH1 and IDH1 on ER stress. Results of cell and animal experiments showed that compared with LPS/D-Gal + EV and LPS/D-Gal + WT groups, the expression of ER stress-related molecules activating transcription factor 6 (ATF6), heavy-chain binding protein (BIP), X-box binding protein 1 (XBP1), C/EBP-homologous protein (CHOP) were increased in LPS/D-Gal + MDH1 K118R and LPS/D-Gal + IDH1 K93R groups (Fig. 6A, B). The results suggested that deacetylated MDH1 and IDH1 could promote ER stress signaling in vivo and in vitro.
脱乙酰化的 MDH1 和 IDH1 的脱乙酰化可能在急性肝衰竭期间加重 PANoptosis，但机制尚不清楚。研究表明，内质网（ER）应激与多种肝病的发生和发展相关，如非酒精性脂肪肝病、酒精相关性肝病、病毒性肝炎、肝缺血和肝癌[17]。因此，我们研究了脱乙酰化的 MDH1 和 IDH1 对内质网应激的影响。细胞和动物实验的结果显示，与 LPS/D-Gal + EV 和 LPS/D-Gal + WT 组相比，LPS/D-Gal + MDH1 K118R 和 LPS/D-Gal + IDH1 K93R 组中内质网应激相关分子激活转录因子 6（ATF6）、重链结合蛋白（BIP）、X-box 结合蛋白 1（XBP1）、C/EBP 同源蛋白（CHOP）的表达增加（图 6A，B）。结果表明，脱乙酰化的 MDH1 和 IDH1 可以促进体内和体外的内质网应激信号传导。

Fig. 6: Deacetylated MDH1 and IDH1 promoted ER stress signaling in vivo and in vitro.
图 6：去乙酰化的 MDH1 和 IDH1 在体内和体外促进了内质网应激信号传导。
figure 6

A The expression of ER stress-related molecules BIP, ATF6, XBP1, CHOP in AML-12 cells was detected by Western blot. B The expression of ER stress-related molecules BIP, ATF6, XBP1, CHOP in mice liver tissues was detected by Western blot. The results are presented as mean ± SD based on three repetitions. * compared with EV group, P < 0.05; # compared with WT group, P < 0.05; & compared with EV + LPS/D-Gal group, P < 0.05; % compared with WT + LPS/D-Gal group, P < 0.05.
AML-12 细胞中 ER 应激相关分子 BIP、ATF6、XBP1、CHOP 的表达通过 Western blot 检测。小鼠肝组织中 ER 应激相关分子 BIP、ATF6、XBP1、CHOP 的表达通过 Western blot 检测。结果以三次重复的平均值±SD 表示。*与 EV 组比较，P < 0.05；#与 WT 组比较，P < 0.05；&与 EV + LPS/D-Gal 组比较，P < 0.05；%与 WT + LPS/D-Gal 组比较，P < 0.05。

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Deacetylated MDH1 and IDH1 aggravated PANoptosis during ALF through ER stress signaling
去乙酰化的 MDH1 和 IDH1 通过内质网应激信号加重了 ALF 期间的 PANoptosis

In order to determine if deacetylated MDH1 and IDH1 could aggravate PANoptosis in ALF through ER stress signaling, we intervened ER stress inhibitor 4-PBA in cells. As shown in Fig. 7A, 4-PBA significantly decreased the expression of ER stress-related molecules BIP, ATF6, XBP1, and CHOP. Western blot and immunofluorescence results showed that, compared with the LPS/D-Gal + MDH1 K118R and LPS/D-Gal + IDH1 K93R groups, 4-PBA significantly increased cell viability, decreased LDH release, and reduced the expression of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18 and IL-1β (Fig. 7B–D). The results indicated that 4-PBA weakened the impact of deacetylated MDH1 and IDH1 on PANoptosis. These findings suggested that deacetylated MDH1 and IDH1 may aggravate PANoptosis in ALF through ER stress signaling.
为了确定去乙酰化的 MDH1 和 IDH1 是否通过 ER 应激信号加重 ALF 中的 PANoptosis，我们在细胞中介入了 ER 应激抑制剂 4-PBA。如图 7A 所示，4-PBA 显著降低了 ER 应激相关分子 BIP、ATF6、XBP1 和 CHOP 的表达。Western blot 和免疫荧光结果显示，与 LPS/D-Gal + MDH1 K118R 和 LPS/D-Gal + IDH1 K93R 组相比，4-PBA 显著增加了细胞存活率，降低了 LDH 释放，并减少了与 PANoptosis 相关分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的表达（图 7B-D）。结果表明，4-PBA 削弱了去乙酰化的 MDH1 和 IDH1 对 PANoptosis 的影响。这些发现表明，去乙酰化的 MDH1 和 IDH1 可能通过 ER 应激信号加重 ALF 中的 PANoptosis。

Fig. 7: Deacetylated MDH1 and IDH1 deacetylation aggravated PANoptosis during ALF through ER stress signaling.
图 7：去乙酰化的 MDH1 和 IDH1 去乙酰化通过内质网应激信号在 ALF 期间加重了 PANoptosis。
figure 7

A The expression of ER stress-related molecules BIP, ATF6, XBP1, CHOP in AML-12 cells was detected by Western blot. B Cell viability and LDH release of AML-12 cells in each group. C The expression of PANoptosis-related molecules RIPK1, GSDMD, caspase-3, MLKL, IL-18, and IL-1β in AML-12 cells was detected by Western blot. D The expression of GSDMD in AML-12 cells was detected by immunofluorescence. The results are presented as mean ± SD based on three repetitions. * compared with LPS/D-Gal group, P < 0.05; # compared with LPS/D-Gal + MDH1K118R group, P < 0.05; & compared with LPS/D-Gal + IDH1 K93R group, P < 0.05.
A. 通过 Western blot 检测 AML-12 细胞中与 ER 应激相关分子 BIP、ATF6、XBP1、CHOP 的表达。B. 每组 AML-12 细胞的细胞存活率和 LDH 释放。C. 通过 Western blot 检测 AML-12 细胞中与 PANoptosis 相关分子 RIPK1、GSDMD、caspase-3、MLKL、IL-18 和 IL-1β的表达。D. 通过免疫荧光检测 AML-12 细胞中 GSDMD 的表达。结果以三次重复的平均值±标准差表示。*与 LPS/D-Gal 组相比，P < 0.05；#与 LPS/D-Gal + MDH1K118R 组相比，P < 0.05；&与 LPS/D-Gal + IDH1 K93R 组相比，P < 0.05。

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Discussion 讨论

PANoptosis is an extensive form of cell death characterized by pyroptosis, apoptosis, and necroptosis, and plays a crucial role in the body’s defense mechanisms. There is an inseparable interaction between necroptosis, apoptosis, and pyroptosis. The role of PANoptosis in infectious diseases has been widely reported, including bacterial, viral, and fungal infections [18, 19]. But its relationship with ALF, MDH1 and IDH1 acetylation remains unclear.
PANoptosis 是一种广泛的细胞死亡形式，其特征是焦亡、凋亡和坏死，对机体的防御机制起着至关重要的作用。坏死、凋亡和焦亡之间存在着密不可分的相互作用。PANoptosis 在传染性疾病中的作用已被广泛报道，包括细菌、病毒和真菌感染[18, 19]。但它与 ALF、MDH1 和 IDH1 乙酰化的关系仍不清楚。

Our results showed that the expression of PANoptosis-related molecules RIPK1, GSDMD, MLKL, caspase-3, IL-18, and IL-1β was increased during ALF, indicating an elevated level of PANoptosis during ALF. The caspase family and GSDMD are important molecules that regulate pyroptosis, apoptosis, and necroptosis. Caspase-3 is a crucial regulatory molecule of apoptosis. The inflammasome binds to caspase-1, and the activated caspase-1 cleaves IL-1β and IL-18 precursors, promoting the release of IL-1β and IL-18, and mediating cell death [8]. Activated caspases can also cleave GSDMD to initiate cell death [9]. RIPK1 is a molecule required for the regulation of PANoptosis and inflammatory responses [20]. Loss of RIPK1 can eliminate pyroptosis and apoptosis induced by Yersinia [5]. RIPK1 can recruit NLRP3 and ASC to form a cell death complex, activate the inflammasome, caspase-8, and GSDMD, leading to cell pyroptosis and apoptosis [4, 21]. RIPK1 is also involved in tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-induced PANoptosis driven by the FADD/caspase-8 signaling. Therefore, targeting key molecules of PANoptosis may be a new way to treat ALF.
我们的研究结果显示，在急性肝衰竭期间，PANoptosis 相关分子 RIPK1、GSDMD、MLKL、caspase-3、IL-18 和 IL-1β的表达增加，表明急性肝衰竭期间 PANoptosis 水平升高。Caspase 家族和 GSDMD 是调节焦亡、凋亡和坏死的重要分子。Caspase-3 是凋亡的关键调节分子。炎症小体结合 caspase-1，激活的 caspase-1 裂解 IL-1β和 IL-18 前体，促进 IL-1β和 IL-18 的释放，介导细胞死亡。激活的 caspase 还可以裂解 GSDMD 以启动细胞死亡。RIPK1 是调节 PANoptosis 和炎症反应所需的分子。失去 RIPK1 可以消除 Yersinia 诱导的焦亡和凋亡。RIPK1 可以招募 NLRP3 和 ASC 形成细胞死亡复合物，激活炎症小体、caspase-8 和 GSDMD，导致细胞焦亡和凋亡。RIPK1 还参与肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）诱导的 FADD/caspase-8 信号驱动的 PANoptosis。因此，针对 PANoptosis 的关键分子可能是治疗急性肝衰竭的新途径。

Our results also found that deacetylated MDH1 and IDH1 could enhance the expression of PANoptosis-related molecules such as RIPK1, GSDMD, MLKL, caspase-3, and other molecules in ALF. This suggested that deacetylated MDH1 and IDH1 could aggravate PANoptosis during ALF. MDH1 and IDH1 are two important metabolic enzymes involved in energy metabolism. Studies have reported that the acetylation of MDH1 and IDH1 can affect their activities and function in diseases [22, 23]. In our previous studies, we found that the deacetylation of MDH1 K118 and IDH1 K93 reduced the activity and function of MDH1 and IDH1, respectively [15, 16]. The dysfunction of MDH1 and IDH1 can disrupt energy metabolism. Cell death is closely related to energy metabolism and mitochondria [24]. Necroptosis is characterized by mitochondrial swelling, loss of mitochondrial membrane potential, impaired oxidative phosphorylation, and ATP production [24]. Anoikis is a unique form of apoptosis. Cells undergo drastic metabolic changes in Anoikis, characterized by reduced glucose uptake, glycolytic flux, mitochondrial respiration, and pentose phosphate pathway, as well as decreased ATP and NADPH production [25]. The Bcl-2 family, which regulates apoptosis, is influenced by various metabolic stresses. Glucose metabolism can also affect the activation of the pro-apoptotic molecule Bax [26, 27]. Therefore, cell death is inseparable from the regulation of energy metabolism. In addition to their basic role in energy metabolism, mitochondria also play a crucial role in regulating various forms of cell death, with the most extensively studied being their involvement in apoptosis. In the exogenous apoptotic pathway, ligands bind to receptors, resulting in the formation of a death complex that activates caspase-8. Caspase-8 can activate downstream caspase cascades, including caspase-3, which targets hundreds of substrates, leading to apoptosis. Mitochondria play a role in amplifying the cycle of exogenous apoptosis [28]. Mitochondria play a crucial role in endogenous apoptosis. The endogenous apoptotic pathway is also known as the mitochondrial apoptotic pathway. Signals such as DNA damage, oxidative stress, or endoplasmic reticulum stress act on mitochondria, stimulate the permeabilization of the mitochondrial outer membrane, and lead to the release of pro-apoptotic factors from mitochondria [28,29,30].
我们的研究结果还发现，去乙酰化的 MDH1 和 IDH1 可以增强 ALF 中与 PANoptosis 相关的分子的表达，如 RIPK1、GSDMD、MLKL、caspase-3 等分子。这表明，去乙酰化的 MDH1 和 IDH1 可能在 ALF 期间加重 PANoptosis。MDH1 和 IDH1 是参与能量代谢的两种重要代谢酶。研究表明，MDH1 和 IDH1 的乙酰化可能影响它们在疾病中的活性和功能。在我们之前的研究中，我们发现 MDH1 K118 和 IDH1 K93 的去乙酰化分别降低了 MDH1 和 IDH1 的活性和功能。MDH1 和 IDH1 的功能障碍可能破坏能量代谢。细胞死亡与能量代谢和线粒体密切相关。坏死凋亡的特征是线粒体肿胀、线粒体膜电位丧失、氧化磷酸化受损以及 ATP 产生减少。Anoikis 是一种独特的凋亡形式。细胞在 Anoikis 中经历剧烈的代谢变化，表现为葡萄糖摄取、糖酵解通量、线粒体呼吸和核糖磷酸途径减少，以及 ATP 和 NADPH 产生减少。调节凋亡的 Bcl-2 家族受各种代谢应激影响。葡萄糖代谢也可能影响促凋亡分子 Bax 的激活。因此，细胞死亡与能量代谢的调节密不可分。除了在能量代谢中的基本作用外，线粒体还在调节各种形式的细胞死亡中发挥关键作用，其中最广泛研究的是它们在凋亡中的作用。在外源性凋亡途径中，配体结合受体，形成激活 caspase-8 的死亡复合物。Caspase-8 可以激活下游 caspase 级联，包括作用于数百个底物的 caspase-3，导致凋亡。 线粒体在放大外源性凋亡循环中发挥作用[28]。线粒体在内源性凋亡中起着至关重要的作用。内源性凋亡途径也被称为线粒体凋亡途径。信号如 DNA 损伤、氧化应激或内质网应激作用于线粒体，刺激线粒体外膜通透性增加，并导致线粒体释放促凋亡因子[28, 29, 30]。

Our results also suggest that the mechanism by which deacetylated MDH1 and IDH1 promote PANoptosis during ALF may be through the ER stress signaling. Physiological and pathological stimuli, such as nutritional deprivation, oxidative stress, hypoxia, and genetic mutations, can disrupt ER homeostasis and lead to ER stress [31]. In response to ER stress, cells activate the unfolded protein response (UPR) to adapt to ER stress or undergo cell death. During mild to moderate ER stress, the UPR is activated to eliminate unfolded or misfolded proteins and restore ER homeostasis. However, under severe or persistent ER stress, the UPR can be overactivated, leading to the initiation of cell death [32]. When unfolded or misfolded proteins accumulate inside the cell, they bind to BIP, competitively dissociating BIP from the UPR sensor, causing BIP to activate [33]. After BIP dissociation, IRE1α was autophosphorylated [34]. IRE1α autophosphorylation activates XBP1 and enhances the transcriptional expression of CHOP, thereby promoting cell death. In addition, under ER stress, ATF6 is transported to the Golgi apparatus in a vesicular manner. There, it is cleaved and activated, leading to the transcriptional expression of ER stress genes, such as CHOP. CHOP is considered one of the key factors of ER stress and is widely involved in the pathogenesis of liver diseases [35, 36]. Therefore, excessive ER stress can lead to the activation of cell death. Our results showed that deacetylated MDH1 and IDH1 could impact ER stress signaling, indicating a strong connection between metabolism, mitochondria, and ER stress. MDH1 and IDH1 are two important metabolic enzymes involved in energy metabolism. The dysfunction of MDH1 and IDH1 can disrupt energy metabolism. Studies have shown that the activation level of the UPR pathway changes with metabolic status in various mammalian tissues [37, 38]. Both carbohydrate metabolism and lipid metabolism can influence ER stress. Glucose metabolism is related to protein folding and translocation. Glucose metabolism imbalance can activate ER stress and UPR [39]. Excessive lipid exposure can also lead to ER stress, which, in turn, disrupts lipid metabolism and ultimately compromises the protective mechanisms of the ER [40, 41]. Therefore, the influence of energy metabolism imbalance caused by MDH1 and IDH1 on ER stress may be multifaceted.
我们的研究结果还表明，去乙酰化的 MDH1 和 IDH1 促进急性肝衰竭期间 PANoptosis 的机制可能是通过内质网应激信号传导。生理和病理刺激，如营养匮乏、氧化应激、缺氧和基因突变，可以破坏内质网稳态并导致内质网应激。在内质网应激的响应中，细胞激活未折叠蛋白应答（UPR）以适应内质网应激或进行细胞死亡。在轻度到中度内质网应激期间，UPR 被激活以清除未折叠或错误折叠的蛋白质并恢复内质网稳态。然而，在严重或持续的内质网应激下，UPR 可能过度激活，导致细胞死亡的启动。当细胞内未折叠或错误折叠的蛋白质积累时，它们结合到 BIP，竞争性地使 BIP 与 UPR 传感器解离，导致 BIP 激活。在 BIP 解离后，IRE1α被自体磷酸化。IRE1α的自体磷酸化激活 XBP1 并增强 CHOP 的转录表达，从而促进细胞死亡。此外，在内质网应激下，ATF6 以囊泡方式转运到高尔基体。在那里，它被切割和激活，导致内质网应激基因的转录表达，如 CHOP。CHOP 被认为是内质网应激的关键因子之一，并广泛参与肝病的发病机制。因此，过度的内质网应激可能导致细胞死亡的激活。我们的研究结果显示，去乙酰化的 MDH1 和 IDH1 可能影响内质网应激信号传导，表明代谢、线粒体和内质网应激之间存在密切联系。MDH1 和 IDH1 是涉及能量代谢的两种重要代谢酶。MDH1 和 IDH1 的功能障碍可能破坏能量代谢。研究表明，UPR 途径的激活水平随各种哺乳动物组织的代谢状态而变化。碳水化合物代谢和脂质代谢都可能影响内质网应激。 葡萄糖代谢与蛋白质折叠和转位相关。葡萄糖代谢失衡可能激活内质网应激和 UPR [39]。过度脂质暴露也可能导致内质网应激，进而破坏脂质代谢，最终损害内质网的保护机制[40,41]。因此，由 MDH1 和 IDH1 引起的能量代谢失衡对内质网应激的影响可能是多方面的。

In summary, this study explored the effect and possible mechanism of deacetylated MDH1 and IDH1 on PANoptosis in ALF. The results indicated that deacetylated MDH1 and IDH1 could aggravate PANoptosis during ALF, and the mechanism may involve the ER stress signaling. Targeting MDH1 and IDH1 acetylation modifications, as well as PANoptosis-related molecules, may represent a novel strategy for treating ALF.
综上所述，本研究探讨了去乙酰化 MDH1 和 IDH1 对 ALF 中 PANoptosis 的影响和可能机制。结果表明，去乙酰化的 MDH1 和 IDH1 可能在 ALF 期间加重 PANoptosis，并且机制可能涉及 ER 应激信号传导。针对 MDH1 和 IDH1 的乙酰化修饰，以及与 PANoptosis 相关的分子，可能代表了治疗 ALF 的一种新策略。

Materials and methods 材料和方法

Reagents 试剂

Foetal bovine serum (FBS) and DMEM/F12 medium were obtained from Gibco (USA). D-Gal and LPS were purchased from Sigma-Aldrich (USA). ACY1215 (#HY-16026) and 4-PBA (#HY-A0281) were purchased from MCE (USA). IDH1 (#12332-1-AP), MDH1 (#15904-1-AP), RIPK1 (#17519-1-AP), GSDMD (#20770-1-AP), IL-18 (#10663-1-AP), MLKL (#21066-1-AP), ATF6 (#24169-1-AP), BIP (#11587-1-AP), XBP1 (#24868-1-AP), CHOP (#15204-1-AP), and GAPDH (#60004-1-Ig) specific antibodies were obtained from Proteintech (China). Acetylated lysine (Ac-Lys) antibody (#9441) was purchased from Cell Signaling Technology (USA). The IL-1β antibody (#ab254360) was purchased from Abcam (USA). The caspase-3 antibody (#sc-56053) was purchased from Santa Cruz Biotechnology (USA).
胎牛血清（FBS）和 DMEM/F12 培养基来自 Gibco（美国）。D-Gal 和 LPS 购自 Sigma-Aldrich（美国）。ACY1215（#HY-16026）和 4-PBA（#HY-A0281）购自 MCE（美国）。IDH1（#12332-1-AP）、MDH1（#15904-1-AP）、RIPK1（#17519-1-AP）、GSDMD（#20770-1-AP）、IL-18（#10663-1-AP）、MLKL（#21066-1-AP）、ATF6（#24169-1-AP）、BIP（#11587-1-AP）、XBP1（#24868-1-AP）、CHOP（#15204-1-AP）和 GAPDH（#60004-1-Ig）特异抗体来自 Proteintech（中国）。乙酰化赖氨酸（Ac-Lys）抗体（#9441）购自 Cell Signaling Technology（美国）。IL-1β抗体（#ab254360）购自 Abcam（美国）。caspase-3 抗体（#sc-56053）购自 Santa Cruz Biotechnology（美国）。

Cell culture and treatment
细胞培养和处理

The mouse liver cell line AML-12 was cultured in DMEM/F12 supplemented with 10% FBS, sourced from Wuhan Pinuofei Biological. The source of cell line was recently authenticated and determined to be free of mycoplasma. LPS (100 ng/mL) combined with D-Gal (44 μg/mL) [42] was used to stimulate cells, except for those in the control group. ACY1215 (2.5 μM) was added 2 h before LPS/D-Gal [43]. The cells were harvested 24 h after LPS/D-Gal administration.
小鼠肝细胞系 AML-12 在添加了 10% FBS 的 DMEM/F12 培养基中培养，FBS 来源于武汉品诺菲生物。细胞系来源最近经过鉴定，确认不含支原体。除了对照组外，使用 LPS（100 ng/mL）结合 D-Gal（44 μg/mL）[42]来刺激细胞。ACY1215（2.5 μM）在 LPS/D-Gal 之前 2 小时加入[43]。细胞在 LPS/D-Gal 给药后 24 小时收获。

Plasmid transfection 质粒转染

MDH1 K118R and IDH1 K93R mutants were constructed by Genomeditech (China). Cells were seeded into 6-well plates. Serum-free medium and plasmids were added to a centrifuge tube and mixed. Seru-free medium and Lipofectamine 2000 were added to another centrifuge tube. After 5 min at room temperature, the two tubes were mixed and incubated for 20 min at room temperature. The transfection mixture was added to the cell culture plate and mixed. Cells were intervened after 24–72 h transfection. LPS/D-Gal and ACY1215 were used to stimulate the cells after plasmid transfection, as described above.
MDH1 K118R 和 IDH1 K93R 突变体由 Genomeditech（中国）构建。细胞被播种到 6 孔板中。无血清培养基和质粒被加入离心管中并混合。另一个离心管中加入无血清培养基和脂质体 2000。在室温下静置 5 分钟后，两个管混合并在室温下孵育 20 分钟。转染混合物被加入细胞培养板中并混合。细胞在转染后 24-72 小时进行干预。LPS/D-Gal 和 ACY1215 在质粒转染后用于刺激细胞，如上所述。

Immunofluorescence 免疫荧光

After treatment, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton for 15 min, blocked with 5% bovine serum albumin (BSA) for 30 min, and incubated with primary antibodies (1:100) overnight at 4°C and secondary antibodies (Servicebio, China) for 1 h at room temperature. The results were observed under a fluorescence microscope (Olympus, Japan). After dewaxing and antigen retrieval, tissue slices were blocked with BSA for 1 h, incubated with primary antibodies (1:100) overnight at 4 °C, and then with secondary antibodies for 1 h at room temperature. The results were observed using a fluorescence microscope (Olympus, Japan).
细胞经处理后，用 4%戊醛固定 30 分钟，用 0.2% Triton 渗透 15 分钟，用 5%牛血清蛋白（BSA）阻断 30 分钟，然后在 4°C 孵育一夜，浓度为 1:100 的初级抗体，再在室温下用 1 小时的二级抗体（Servicebio，中国）孵育。结果在荧光显微镜（日本奥林巴斯）下观察。去蜡和抗原检索后，组织切片用 BSA 阻断 1 小时，然后在 4°C 孵育一夜，浓度为 1:100 的初级抗体，再在室温下用 1 小时的二级抗体孵育。结果使用荧光显微镜（日本奥林巴斯）观察。

PI staining PI 染色

After being treated, the cells were washed with phosphate-buffered saline (PBS) and then incubated with a Calcein AM/PI dye solution at 37 °C for 30 min to 1 h, avoiding light. After incubation, the results were observed under a fluorescence microscope (Olympus, Japan).
经过处理后，细胞用磷酸盐缓冲盐水（PBS）洗涤，然后在 37°C 下用 Calcein AM/PI 染料溶液孵育 30 分钟至 1 小时，避光。 孵育后，结果在荧光显微镜（日本奥林巴斯）下观察。

Cell viability 细胞存活率

The CCK-8 kit was used to assess cell viability. After being treated, the cells were washed with PBS and then incubated with CCK-8 testing solution at 37 °C for 1 to 2 h. Following the incubation, the results were measured at 450 nm using a microplate reader (Perkin Elmer, USA).
使用 CCK-8 试剂盒评估细胞存活率。 经过处理后，细胞用 PBS 洗涤，然后在 37°C 下用 CCK-8 测试溶液孵育 1 至 2 小时。 孵育后，结果在 450nm 处使用微孔板阅读器（美国佩金埃尔默）测量。

LDH release LDH 释放

After being treated, the cell supernatant was collected and incubated with LDH testing solution at room temperature for 30 min, avoiding light, according to the instructions. After incubation, the results were detected at 490 nm using a microplate reader (Perkin Elmer, USA).
细胞上清液经处理后，按照说明书，在室温下与 LDH 测试溶液孵育 30 分钟，避光。孵育后，使用微孔板读数器（Perkin Elmer，美国）在 490nm 处检测结果。

Western blotting and immunoprecipitation
蛋白印迹和免疫沉淀

The samples were lysed, incubated with 5 μg of primary antibodies or IgG, followed by incubation with 30 μl of protein A + G agarose. Subsequently, they were washed with immunoprecipitation buffer, resuspended in 40 μl of 1.5× loading buffer, boiled, and centrifuged. The supernatants were collected for Western blot analysis. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) was used to separate the proteins. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Subsequently, the membrane was cut and incubated with specific primary and secondary antibodies. GAPDH was used as a loading control. The results were detected by a chemiluminescence apparatus (Bio-Rad, USA). The Image J software was used to analyze the quantification of blots.
样品被裂解，与 5μg 的初级抗体或 IgG 孵育，然后与 30μl 的蛋白 A+G 琼脂糖孵育。随后，用免疫沉淀缓冲液洗涤，用 40μl 的 1.5×加载缓冲液重悬，煮沸，离心。上清液用于 Western 印迹分析。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离蛋白质。电泳后，将蛋白质转移到聚偏氟乙烯膜（Millipore，美国）。随后，切割膜并与特定的初级和次级抗体孵育。GAPDH 用作加载对照。结果通过化学发光仪器（Bio-Rad，美国）检测。使用 Image J 软件分析印迹的定量。

Animal groups 动物群体

Seventy-two male C57BL/6 mice (6-8 weeks old, 20-25 g) were obtained from the Experimental Animal Center of Wuhan University. MDH1 K118R and IDH1 K93R adeno-associated viruses were constructed for transfection in mice (AAV8, Genomeditech, China). The mice were acclimated for 5 days and then randomly and blindingly divided into twelve groups: EV group, WT group, MDH1 K118R group, IDH1 K93R group, EV + LPS/D-Gal group, WT + LPS/D-Gal group, LPS/D-Gal + MDH1 K118R group, LPS/D-Gal + IDH1 K93R group, EV + LPS/D-Gal + ACY1215 group, WT + LPS/D-Gal + ACY1215 group, LPS/D-Gal + ACY1215 + MDH1 K118R group, and LPS/D-Gal + ACY1215 + IDH1 K93R group. Except for the EV, WT, MDH1 K118R, and IDH1 K93R groups, the remaining mice received intraperitoneal injections of LPS (100 µg/kg) and D-Gal (400 mg/kg) [43]. Adeno-associated viruses were administered via tail vein injection at a dose of 1E11 vg 4 weeks before LPS/D-Gal injection. The ACY1215 (25 mg/kg) was administered via intraperitoneal injection 2 h before the LPS/D-Gal injection [43]. At 24 h after the LPS/D-Gal injection, the mice were sacrificed. The livers and serum were collected for experiments. The animal experiments were conducted in accordance with ARRIVE guidelines and other relevant regulations. Approval was granted by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University.
七十二只 C57BL/6 雄性小鼠（6-8 周龄，20-25 克）来自武汉大学实验动物中心。MDH1 K118R 和 IDH1 K93R 腺相关病毒用于小鼠转染（AAV8，Genomeditech，中国）。小鼠在适应环境 5 天后，随机盲目分为十二组：EV 组、WT 组、MDH1 K118R 组、IDH1 K93R 组、EV + LPS/D-Gal 组、WT + LPS/D-Gal 组、LPS/D-Gal + MDH1 K118R 组、LPS/D-Gal + IDH1 K93R 组、EV + LPS/D-Gal + ACY1215 组、WT + LPS/D-Gal + ACY1215 组、LPS/D-Gal + ACY1215 + MDH1 K118R 组和 LPS/D-Gal + ACY1215 + IDH1 K93R 组。除 EV、WT、MDH1 K118R 和 IDH1 K93R 组外，其余小鼠接受腹腔注射 LPS（100 µg/kg）和 D-Gal（400 mg/kg）[43]。腺相关病毒在 LPS/D-Gal 注射前 4 周以 1E11 vg 的剂量通过尾静脉注射。ACY1215（25 mg/kg）在 LPS/D-Gal 注射前 2 小时通过腹腔注射给予[43]。LPS/D-Gal 注射后 24 小时，小鼠被牺牲。肝脏和血清被收集进行实验。动物实验按照 ARRIVE 指南和其他相关法规进行。获得了武汉大学人民医院动物实验委员会的批准。

Hematoxylin-eosin (HE) staining and detection of ALT, AST, and TBIL
苏木精-伊红染色（HE 染色）和检测 ALT、AST 和 TBIL

Fresh tissues were fixed with 4% paraformaldehyde for 24 h at room temperature, dehydrated through a serial alcohol gradient, embedded in paraffin, processed for sectioning. Before staining, sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. And then stained with hematoxylin and eosin. After staining, sections were dehydrated through increasing concentrations of ethanol and xylene. The results were analyzed under a microscope (Olympus, Japan). ALT, AST, and TBIL levels in mouse serum were tested using a fully automatic biochemical analyzer (ADVIA 2400, Siemens AG).
新鲜组织在室温下用 4%多聚甲醛固定 24 小时，经过一系列酒精梯度脱水，包埋在石蜡中，进行切片处理。在染色之前，切片在二甲苯中脱蜡，通过逐渐降低的乙醇浓度脱水，然后在 PBS 中洗涤。然后用苏木精和嗜酸性染色。染色后，切片通过逐渐增加的乙醇和二甲苯浓度脱水。结果在显微镜下（日本奥林巴斯公司）进行分析。小鼠血清中 ALT、AST 和 TBIL 水平使用全自动生化分析仪（西门子公司 ADVIA 2400）进行测试。

Statistical analysis 统计分析

All data were analyzed using SPSS 25.0. GraphPad Prism 8.0 software was used to generate the figures. The results are presented as mean ± standard deviation. Analysis of variance (ANOVA) followed by a post-test was used to analyze the differences between groups. P < 0.05 was considered statistically significant.
所有数据均使用 SPSS 25.0 进行分析。使用 GraphPad Prism 8.0 软件生成图表。结果以均值±标准差表示。方差分析（ANOVA）后进行后续检验以分析组间差异。P < 0.05 被认为具有统计学意义。