Andrographolide protects against atrial fibrillation by alleviating oxidative stress injury and promoting impaired mitochondrial bioenergetics 穿心莲内酯通过缓解氧化应激和促进线粒体能量合成对心房颤动发挥预防作用 1 equal first-author 2 equal 2 1 1 1 1 3 2 1 2 corresp equal Ling ZHANG, ydzhangling@126.com Ling ZHANG, ydzhangling@126.com ydzhangling@126.com https://orcid.org/0000-0002-1532-680X 1 corresp equal Xia SHENG, 3101008@zju.edu.cn Xia SHENG, 3101008@zju.edu.cn 3101008@zju.edu.cn https://orcid.org/0000-0001-7304-7975 浙江大学医学院附属邵逸夫医院， 浙江省心血管干预与再生重点实验室， 中国杭州市， 310016 浙江大学医学院附属邵逸夫医院， 浙江省心血管干预与再生重点实验室， 中国杭州市， 310016 Department of Cardiology, Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China Department of Cardiology, Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China 新疆医科大学第一附属医院， 新疆心电生理与心脏重塑重点实验室， 中国乌鲁木齐市， 830011 新疆医科大学第一附属医院， 新疆心电生理与心脏重塑重点实验室， 中国乌鲁木齐市， 830011 Xinjiang Key Laboratory of Cardiac Electrophysiology and Remodeling, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, China Xinjiang Key Laboratory of Cardiac Electrophysiology and Remodeling, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, China 浙江中医药大学第一临床医学院心内科， 中国杭州市， 310006 浙江中医药大学第一临床医学院心内科， 中国杭州市， 310006 Department of Cardiology, the First School of Clinical Medicine of Zhejiang Chinese Medicine University, Hangzhou 310006, China Department of Cardiology, the First School of Clinical Medicine of Zhejiang Chinese Medicine University, Hangzhou 310006, China Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia seen in clinical settings, which has been associated with substantial rates of mortality and morbidity. However, clinically available drugs have limited efficacy and adverse effects. We aimed to investigate the mechanisms of action of andrographolide (Andr) with respect to AF. We used network pharmacology approaches to investigate the possible therapeutic effect of Andr. To define the role of Andr in AF, HL-1 cells were pro-treated with Andr for 1 h before rapid electronic stimulation (RES) and rabbits were pro-treated for 1 d before rapid atrial pacing (RAP). Apoptosis, myofibril degradation, oxidative stress, and inflammation were determined. RNA sequencing (RNA-seq) was performed to investigate the relevant mechanism. Andr treatment attenuated RAP-induced atrial electrophysiological changes, inflammation, oxidative damage, and apoptosis both in vivo and in vitro. RNA-seq indicated that oxidative phosphorylation played an important role. Transmission electron microscopy and adenosine triphosphate (ATP) content assay respectively validated the morphological and functional changes in mitochondria. The translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the molecular docking suggested that Andr might exert a therapeutic effect by influencing the Keap1-Nrf2 complex. In conclusions, this study revealed that Andr is a potential preventive therapeutic drug toward AF via activating the translocation of Nrf2 to the nucleus and the upregulation of heme oxygenase-1 (HO-1) to promote mitochondrial bioenergetics. 心房颤动是临床上最常见的室上性心律失常，有着较高的发病率和死亡率。然而，目前临床可用的相关抗心律失常药物疗效有限并具有明确的副作用。本研究旨在探讨穿心莲内酯（Andr）对心房颤动的预防作用和潜在作用机制，为临床治疗房颤提供新思路。通过采用网络药理学方法，研究Andr对房颤可能的治疗作用。为了验证Andr在房颤中的作用，在快速电刺激（RES）前用Andr预处理HL-1细胞1小时，在高频心房起搏（RAP）造模前用Andr预处理1天。分别检测细胞凋亡、肌原纤维降解、氧化应激和炎症等相关指标。采用RNA测序（RNA-seq）以研究潜在作用机制。结果表明，在体内体外实验中，Andr治疗减轻了RAP/RES诱导的心房电生理变化、炎症、氧化损伤和细胞凋亡。RNA-seq表明氧化磷酸化起了重要作用。透射电镜和ATP含量测定分别验证了线粒体的形态和功能变化。Nrf2向细胞核的易位和分子对接表明，Andr可能通过影响Keap1-Nrf2复合物发挥治疗作用。综上所述，本研究表明穿心莲内酯是一种潜在的能有效预防心房颤动的药物，可通过激活Nrf2核易位和诱导HO-1的表达，以维持线粒体的形态和功能。 中药 心房颤动 穿心莲内酯 氧化应激 网络药理学 Traditional Chinese medicine Atrial fibrillation Andrographolide Oxidative stress Network pharmacology 1 Introduction 1 Atrial fibrillation (AF) is the most prevalent type of cardiac arrhythmia that occurs in clinical settings, which is associated with substantial rates of mortality and morbidity ( Chugh et al., 2014 Chugh et al., 2014 ; Chung et al., 2020 Chung et al., 2020 ). Various potential risk factors influence AF, combined with complications such as heart failure and thromboembolic events, make treatment challenging. Current managements for AF patients include drug therapy (antiarrhythmic drugs and anticoagulants) ( Dobrev and Nattel, 2010 Dobrev and Nattel, 2010 ) and non-pharmacological therapy (ablation) ( Dobrev and Nattel, 2010 Dobrev and Nattel, 2010 ; Chung et al., 2020 Chung et al., 2020 ). Drugs such as β‍-blockers and non-dihydropyridine calcium-channel blockers are clinically available but have some major adverse effects, including bleeding and proarrhythmic complications ( Dobrev and Nattel, 2010 Dobrev and Nattel, 2010 ; Amin et al., 2016 Amin et al., 2016 ). Although improvements have been made in non-pharmacological therapy, treatment effectiveness remains limited and only a proportion of AF patients respond satisfactorily ( Brunner et al., 2012 Brunner et al., 2012 ; Kloosterman et al., 2020 Kloosterman et al., 2020 ; Ye et al., 2020 Ye et al., 2020 ). Traditional Chinese medicine (TCM) has been applied in Asia, especially China, for nearly 3000 years. To this end, some TCM formulas such as Celastrol ( Ye et al., 2020 Ye et al., 2020 ), Dan hong ( Zhan et al., 2023 Zhan et al., 2023 ), and Suxiao Jiuxin pill ( Ruan et al., 2018 Ruan et al., 2018 ) have been studied in the context of cardiac research. The role of some TCMs in regulating ion channels to control cardiac rate or rhythm has been demonstrated, which indicated their potential for AF treatment ( Dong et al., 2017 Dong et al., 2017 ). Moreover, clustering analysis and meta-analysis showed that TCM improves the clinical treatment efficiency of AF, increasing the success rate of conversion and shortening the conversion time ( Wang et al., 2017 Wang et al., 2017 ; Lei et al., 2021 Lei et al., 2021 ). Andrographis paniculata (Burm. F.) Nees, a well-known medicinal food, is native to Southeast Asia and grows abundantly in China, Indonesia, and Malaysia. It belongs to the Acanthaceae family and is commonly known as “King of the bitters.” Andrographolide (Andr) is one of the major active constituents extracted from the traditional Chinese herb A. paniculata . Reports in the medical literature ascribe Andr potent actions, including anti-hyperglycemic, anti-pyretic, anti-inflammatory, anti-cancer, anti-leishmanial, fertility-boosting, anti-human immunodeficiency virus (HIV), cardiovascular, immunomodulatory, and choleretic activity ( Maiti et al., 2010 Maiti et al., 2010 ). More recently, there have been reports that Andr ameliorated aortic valve incrassation by inhibiting cell proliferation via the mitogen-activated protein kinase (MAPK)‍-extracellular signal-regulated kinase (ERK) signaling pathway ( Huang et al., 2022 Huang et al., 2022 ) and alleviated aortic valve calcification by regulating lipid biosynthesis and glycerolipid metabolism or via the nuclear factor-‍κB (NF‍-‍κB)/serine/threonine kinase (Akt)/ERK pathway ( Huang et al., 2020 Huang et al., 2020 ). In high-fat-diet-induced obese mice, Andr mitigated cardiac apoptosis to provide a cardio-protective function ( Lin et al., 2020 Lin et al., 2020 ). Other research found that, through inhibiting MAPK signaling, Andr blocks the activation of cardiac fibroblasts and protects against aortic banding-induced experimental cardiac hypertrophy ( Wu et al., 2017 Wu et al., 2017 ). As for myocardial infarction (MI), Andr exerted a protective effect against isoproterenol-induced MI through the inhibition of L-type Ca 2+ channels and the increase in cardiac transient outward K + currents ( Elasoru et al., 2021 Elasoru et al., 2021 ). In addition, Andr alleviated adverse cardiac remodeling following MI through the enhancement of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway ( Xie et al., 2020 Xie et al., 2020 ). In other non-heart disease studies, by strengthening intestinal barrier function and shaping the intestinal microbial composition, Andr ameliorated glucose intolerance and insulin resistance, as well as attenuated diabetes-associated redox disturbance and inflammation in db/db diabetic mice ( Su et al., 2020 Su et al., 2020 ); in 3T3-L1 preadipocytes, via the suppression of glutathione peroxidase 1 (GPX1) and glutathione (GSH) depletion, Andr exerted a proliferation inhibitory effect ( Chen et al., 2016 Chen et al., 2016 ). However, some scholars reported that by inhibiting voltage-gated Na + , and Ca 2+ channels, Andr ameliorated the symptoms of aconitine-induced arrhythmia in rabbits ( Zeng et al., 2017 Zeng et al., 2017 ), which result—the downregulation of L-type Ca 2+ current during the development of AF—was against common sense. Hence, we speculate that the above findings might be due to the use of aconitine as an inducer or the fact that the ex vivo experiment cannot reflect the whole-body conditions. Moreover, there may be other potential mechanisms to interpret the therapeutic effect of Andr on AF. There is scope for the treatment of complex diseases by TCM through the synergistic effects of multiple components, which affect many molecular targets in contrast to Western medicine that tends to be directed at one specific target ( Ma et al., 2015 Ma et al., 2015 ). However, this complexity presents a barrier to the pharmacological and mechanistic study of some TCM examples. The development of network pharmacology approaches with access to databases provides a solution to this problem and can be instrumental in predicting the actions and effects of TCM ( Hopkins, 2008 Hopkins, 2008 ). This approach has been widely applied to the study of TCM for the treatment of various diseases, such as hepatocellular carcinoma ( Guo et al., 2019 Guo et al., 2019 ), heart disease ( Tan et al., 2022 Tan et al., 2022 ), and coronavirus disease 2019 (COVID-19) ( Li et al., 2021 Li et al., 2021 ). There is great potential for the application of network pharmacology to orient the study of drugs and aid development. To the best of our knowledge, there have been no reports that employed network analysis to study TCM therapies relevant to AF as a distinct condition from other cardiac diseases. In the current study, we used network databases to cross-reference potential targets with known mediators of AF to investigate the actions of Andrographis with respect to AF. Then, we tested whether Andr actually caused the therapeutic effects in AF. Our results indicated that Andr could inhibit atrial myocyte apoptosis and myolysis through the regulation of the processes of mitochondrial fission and fusion, improving mitochondrial energy metabolism, and in turn reducing the incidence of AF. 2 Results 1 2.1 Results of network pharmacology analysis 2 Considering the similar clinical effects of both Andrographis and Andr and the fact that there are less research data on Andr, we selected Andrographis as the candidate object of network pharmacological analysis to explore the potential effects of Andr. To confirm that Andr was indeed responsible for the therapeutic effects that was obtained from the network pharmacological analysis on Andrographis, experiments on rabbits and HL-1 cells were performed. A total of 49 components of Andrographis were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database (Table S1). After the absorption, distribution, metabolism and excretion (ADME) screening, 24 (48.98%) met the requirements of oral bioavailability ≥30% and drug-likeness index ≥0.18, which were chosen as candidate bioactive components for further analyses. A total of 294 protein targets of bioactive components present in Andrographis were retrieved from the TCMSP database (mainly from DrugBank; details are found in Table S1). After removing the duplicates, 74 protein targets were retained for further analyses. A total of 3211 human genes with relevance to AF were identified via GeneCards (Table S1). The intersection of the two target clusters yielded 51 protein targets of Andrographis components with relevance to AF ( Fig. 1 Fig. 1 a). A protein‍–‍protein interaction (PPI) network was constructed to illuminate the relationships between protein targets ( Kumar et al., 2015 Kumar et al., 2015 ). CytoHubba from Cytoscape was used to determine the top ten hub genes ( Fig. 1 Fig. 1 b). Among these, AKT1 was identified as the most significant target with degree=41, followed by tumor necrosis factor (TNF) (degree=37), interleukin-6 (IL-6) (degree=36), heat shock protein 90α family class A member 1 (HSP90AA1) (degree=32), tumor suppressor p53 (TP53) (degree=32), caspase-3 (CASP3) (degree=32), posttranscriptional gene silencing 2 (PTGS2) (degree=31), estrogen receptor 1 (ESR1) (degree=30), c-Jun (JUN) (degree=30), and nitric oxide synthase 3 (NOS3) (degree=28). Gene ontology (GO) and pathway enrichment analyses were performed via the functional annotation tool of the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources 6.8. A total of 203 biological process (BP), 28 cellular component (CC), and 43 molecular function (MF) terms met the criteria of count ≥2 and expression analysis systematic explorer (EASE) score ≤0.05 (detailed GO information is presented in Table S2). GO analysis demonstrated the involvement of these targets in response to antibiotic, extrinsic apoptotic signaling pathway in the absence of ligand, positive regulation of nitric oxide biosynthetic process, response to drug, and other BPs. Next, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted (Table S2), and the top 15 significantly enriched pathways are shown in Fig. 1 Fig. 1 b. Interestingly, the top four pathways were all involved in phosphoinositide-‍3-kinase (PI3K)‍-‍Akt signaling and apoptosis, which ranked the 5th and 10th, respectively ( Fig. 1 Fig. 1 c). Then, a target-pathway network and a Chord plot were generated based on associated protein targets and enriched pathways. Many high-degree targets (degree≥10) were identified to play roles in apoptosis, inflammation, and metabolism, including AKT1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit γ (PIK3CG), TP53, MAPK14, TNF, JUN, cyclin D1 (CCND1), protein kinase cyclic adenosine monophosphate (cAMP)‍-activated catalytic subunit α (PRKACA), IL-6, glycogen synthase kinase 3β (GSK3B), CASP3, cyclin-dependent kinase inhibitor 1A (CDKN1A), CASP9, C-X-C motif chemokine ligand 8 (CXCL8), calmodulin 1 (CALM1), B-cell lymphoma 2 (BCL2), CDK2, BCL2-associated X (BAX), and NOS2 (Figs. 1 1 d and 1 1 e). In summary, we used network pharmacological analysis to investigate the mechanisms, by which Andrographis might treat or prevent the development and progression of AF. We concluded that components of Andrographis influence cell survival and inflammation through acting via the PI3K-Akt signaling pathway. 2.2 Effects of Andr treatment on myofibril degradation and HL-1 cell apoptosis in vitro 2 Oxidative stress participates in the development and perpetuation of AF and promotes atrial remodeling, both electrically and structurally ( Pauklin et al., 2022 Pauklin et al., 2022 ). To determine a suitable drug concentration of Andr, HL-1 cells were counted using the cell counting kit-8 (CCK8). We tested suitable stimulation frequencies of 0.5, 1, 3, and 5 Hz, and found that both RNA and protein levels responded the most to the 5 Hz signal based on the degradation of L-type calcium channel (LCC), considered as the marker of the successful AF model building (Figs. S1a and S1b). In addition, the dihydroethidium (DHE) staining showed that rapid electronic stimulation (RES) promoted the intracellular reactive oxygen species (ROS) level (Fig. S1c). Thus, we selected 5 Hz, 10 V, and 24 h as our model parameters. Significant cytotoxicity was observed when using 50 µmol/L Andr to treat HL-1 cells for 24 h. Reducing Andr to 25 µmol/L alleviated the cytotoxicity and was selected as the optimal concentration ( Fig. 2 Fig. 2 a). Myofibril degradation is a characteristic of atrial structural remodeling resulting from increased intracellular ROS. Heme oxygenase-1 ( HO-1 ), a powerful anti-oxidative stress gene, plays an important role in AF. Different concentrations of Andr protected the degradation of cardiac troponin I (cTnI) after RES for 24 h ( Fig. 2 Fig. 2 b). The optimal concentration of Andr was confirmed to be 25 µmol/L. Similar results were observed in the immunofluorescence assay of cardiac troponin T (cTnT) ( Fig. 2 Fig. 2 c). The HO-1 levels slightly increased after RES for 24 h via the upregulation of redox balance. Perhaps more remarkably, HO-1 showed an increase dependent on Andr concentration ( Fig. 2 Fig. 2 b). The DHE staining showed that RES promoted the intracellular ROS level, which could be reversed by the use of Andr (Figs. 3 3 a and 3 3 b). Next, we compared the ROS levels within the cell. Compared with total cellular ROS, the levels of mitochondrial ROS were similar (Figs. 3 3 c and 3 3 d). These results were confirmed by the JC-1 mitochondrial membrane potential (MMP) assay (Figs. 3 3 e and 3 3 f). As mitochondria are the major organelles for adenosine triphosphate (ATP) production and the content of cellular ATP can effectively reflect mitochondrial function, we determined the cellular ATP content. We found that Andr could promote the content of cellular ATP, which was downregulated by RES ( Fig. 3 Fig. 3 g). The activity of total-superoxide dismutase (SOD) and Mn-SOD, two key enzymes in oxidative stress control, was reduced by RES, and Andr treatment effectively restored the enzyme activity (Figs. 3 3 h and 3 3 i). 2.3 Effect of Andr treatment on RAP-induced atrial electrophysiological changes in vivo 2 The results of electrophysiological examination revealed that rapid atrial pacing (RAP) for 6 h decreased the heart rate by 24.51% (sham group) vs. 20.01% (Andr group) (Figs. 4 4 a and 4 4 c). These findings are in line with previous reports, indicating that the construction of acute rabbit AF model was successful ( Zhang et al., 2015 Zhang et al., 2015 ). Compared with the sham group, the AF inducibility and duration were significantly increased in the AF group, while the stimulus thresholds were markedly declined. However, the changes above could be reversed by Andr treatment (Figs. 4 4 b, 4 4 d– 4 4 f). After 6 h of RAP, atrial effective refractory period (AERP) was slightly reduced in the AF group compared with the sham group at baseline; meanwhile, the window of vulnerability (WOV) increased, which could be reversed by the use of Andr (Figs. 4 4 g and 4 4 h). The analysis of echocardiography and hemodynamic parameters indicated that cardiac function was not impaired after 6 h of RAP ( Fig. 5 Fig. 5 a). No statistically significant difference was found in left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left atrial (LA) diameter, interventricular septal thickness at diastole (IVSd), left ventricular posterior wall at systole (LVPWs), or left ventricular posterior wall at diastole (LVPWd) (Figs. 5 5 b– 5 5 d, 5 5 i– 5 5 k). However, left ventricular internal dimension at systole (LVIDs), left ventricular internal dimension at diastole (LVIDd), and interventricular septal thickness at systole (IVSs) were reduced after 6 h of RAP. Regardless of RAP for 6 h, the right atrial (RA) diameter slightly increased in the Andr treatment group ( Fig. 5 Fig. 5 e). Finally, Andr significantly alleviated cardiac dysfunction caused by RAP (Figs. 5 5 f– 5 5 h). 2.4 Effect of Andr treatment on RAP-induced atrial inflammation in vivo 2 As reported previously, inflammation plays an important role in AF ( Wang et al., 2022 Wang et al., 2022 ). Herein, the results of network pharmacological analysis also indicated the effect of Andr in anti-inflammatory processes. We next measured the inflammatory response in the RA tissue and serum of each group. Based on immunofluorescence, compared to the sham rabbit, the atrial tissue of the RAP group was positive for TNF-‍α. As expected, Andr treatment prevented the atrial tissue from accumulating TNF-‍α expression ( Fig. 6 Fig. 6 a). Similar results were observed in the enzyme-linked immunosorbent assay (ELISA) of TNF‍-‍α ( Fig. 6 Fig. 6 b). Additionally, the messenger RNA (mRNA) levels of the pro-inflammation-related genes TNF‍-‍α ( Fig. 6 Fig. 6 c) and IL-1β ( Fig. 6 Fig. 6 d) were validated by quantitative real-time polymerase chain reaction (qPCR). 2.5 Effect of Andr treatment on the atrial structural remodeling in vivo and in vitro 2 In addition to inflammation, atrial structural remodeling is regarded as highly important to the development of AF, mainly including fibrosis and atrial myocardial apoptosis ( Wang et al., 2022 Wang et al., 2022 ). Masson staining and Sirius Red staining showed that the atrial tissue of RAP group presented no significant fibrosis ( Fig. 7 Fig. 7 a). Atrial myocardial damage triggered by 6 h of RAP was confirmed by hematoxylin-eosin (HE) staining ( Fig. 7 Fig. 7 b). In the RAP group, cell degeneration, an uneven distribution of cytoplasm, and local rupture of myocardial fibers were observed. Upon terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining of the atrial tissue, a significantly larger number of TUNEL positive cells were detected in the RAP group compared with the sham group ( Fig. 7 Fig. 7 c). In conjunction to these tests, we performed an Annexin V/propidium iodide (PI) assay to validate the effect of Andr on apoptosis (Figs. 2 2 d and 2 2 g). 2.6 Effect of Andr treatment on oxidative stress injury in vivo 2 We employed DHE staining to measure the intracellular ROS levels in rabbit tissues. RAP-induced ROS production was significantly reduced in the Andr treatment group ( Fig. 8 Fig. 8 a). The immunofluorescent staining of atrial sections showed that the expression levels of cTnT and HO-1 were dramatically reduced after RAP 6 h, which could be restored by Andr treatment ( Fig. 8 Fig. 8 b). Additionally, the mRNA levels of the antioxidant-related genes NAD(P)H quinone oxidoreductase 1 ( NQO1 ) ( Fig. 8 Fig. 8 c), Nrf2 ( Fig. 8 Fig. 8 d), SOD1 ( Fig. 8 Fig. 8 e), and SOD2 ( Fig. 8 Fig. 8 f) were validated by qPCR in rabbit tissue. 2.7 Effect of Andr on RAP/RES, inducing atrial myocardial injury by restoring mitochondrial bioenergetics 2 Next, RNA sequencing (RNA-seq) was performed to further explore the pathways and mechanisms, by which Andr inhibited the changes mentioned above. We found 716 upregulated and 745 downregulated differentially expressed genes (DEGs; defined using |log 2 (fold change)| > 1.5 and P .adj < 0.05) (Figs. 9 9 a and 9 9 b). Comparative GO analysis and gene set enrichment analysis (GSEA) indicated that these DEGs were significantly enriched for the generation of precursor metabolites and energy, mitochondrial protein complex, and mitochondrial membrane (Figs. 9 9 c and 9 9 e). Comparative KEGG pathway analysis and GSEA revealed that, apart from the top enriched KEGG pathway, several signaling pathways identified support the conclusions suggested by our data and those found from the network pharmacological analysis results. These include oxidative phosphorylation, MAPK signaling pathway, TNF signaling pathway, and PI3K-Akt signaling pathway (Figs. 9 9 d and 9 9 e). Subsequently, we selected the genes of electron transport chain (ETC) complex subunits from our RNA-seq results and found that the expression of ETC complex genes was markedly upregulated in the Andr group compared to the RES group ( Fig. 10 Fig. 10 a). These genes were strongly correlated with mitochondrial abnormalities. Next, we used transmission electron microscopy to observe potential phenotypic mitochondrial abnormalities in vivo. After RAP for 6 h, we observed abnormal mitochondrial morphology, mitochondrial size, aberrant cristae, and changes in the matrix color. Conversely, these abnormalities were absent in the Andr treatment group (Figs. 10 10 b and 10 10 d). We then assessed the expression levels. However, we did not detect the obvious change of the protein expression of ETC complex as the RNA-seq results in vitro ( Fig. 10 Fig. 10 c). As previously reported, Andr might exert an effect by the nuclear translocation of Nrf2 ( Xie et al., 2020 Xie et al., 2020 ). Thus, we tested the effect of Andr on Nrf2-Keap1 complex formation in HL-1 cells. Immunofluorescence staining revealed that Andr induced the nuclear translocation of Nrf2, the main transcription factor for HO-1 and NQO1 ( Fig. 11 Fig. 11 a). To predict the binding position of Andr on the Nrf2-Keap1 complex domain, AutoDock was used for 50 docking times ( Figs. 11 Figs. 11 b‍–‍11d), and the binding energy of 50 docking times was presented in Table S3. 3 Discussion 1 AF, as one of the most common types of arrhythmias, is often accompanied with a variety of risk factors and complications. Without effective therapies that are readily or widely available in clinical practice, the increased clinical incidence and subsequent mortality associated with complicated treatments have resulted in a heavy medical burden. Therefore, it is important to discover and test new treatment strategies. During its 3000 years of history, TCM has produced unusually brilliant medicines such as artemisinin and celastrol. Recently, a widespread concern surrounding the pharmacological actions of Andr has surfaced. Considering the high cost and difficulties of constructing AF animal models, we took a pharmacological approach to investigate the possible therapeutic effect of Andrographis on AF, with the aim to provide future research directions. Andrographis was selected instead of Andr due to a lack of published data on the latter. The existing mass spectrometry results for Andrographis suggested similar clinical uses for the two. Our data indicated that cell survival and anti-oxidative stress might be the main target of the drug, possibly through modulation of PI3K-Akt signaling pathway and binding to the Nrf2-Keap1 complex to regulate the nuclear translocation of Nrf2. Previous studies indicated that Andr ameliorates the symptoms of aconitine-induced arrhythmia in rabbits by inhibiting Ca 2+ channels. In this study, we validated the therapeutic effect of Andr on AF and explored the possible underlying mechanism by establishing acute AF models at three different levels: molecular, cellular, and systemic. RAP treatment for 6 h significantly reduced the AF-induced threshold, increased the AF inducibility and duration, shortened AERP, and lengthened WOV. These results were similar to the aforementioned report and marked the success of building the acute AF rabbit model. To validate the action of Andr on AF in rabbits, Andr was injected (10 mg/kg, intraperitoneally) one day before RAP. As expected, Andr treatment inhibited the RAP-induced threshold reduction and increased the AF inducibility and duration, suggesting that Andr treatment resulted in an apparent decrease in AF susceptibility. Meanwhile, Andr treatment effectively shortened the WOV and lengthened the AERP, which hinted that Andr might inhibit the formation of substrate important for AF maintenance. Thus, we further detected whether there were any changes on atrial substrate. However, Masson staining and Sirius Red staining revealed no significant fibrosis among the four groups. The lack of fibrosis suggested that Andr may change the expression of connexin 40 (Cx40) and Cx43; therefore, additional testing was required. During atrial structure remodeling, apart from the atrial fibrosis and the change in the expression of Cx40 and Cx43, the apoptosis of atrial myocytes is important. Thus, we used TUNEL assay and Annexin V/PI staining to evaluate apoptosis in rabbit tissue and HL-1 cells, respectively. Moreover, we detected BAX, BCL2, and cleaved-CASP3 protein expression. We performed RES on HL-1 cells and found reduced protein expression of LCCs and an increase in intracellular ROS production via DHE staining. Additionally, we found that Andr could effectively inhibit apoptosis in vivo and in vitro. After confirming the effective therapeutic function of Andr on AF, we performed comprehensive RNA-seq analysis to investigate the possible phenotype, BP, and mechanism of Andr. Surprisingly, we found that genes associated with inflammation and the regulation of calcium homeostasis were upregulated. In support of the RNA-seq findings, we conducted ELISA to detect the secretion of TNF-α and IL-1β, in addition to qPCR analysis. Given that mitochondria are required for the redox balance, intrinsic apoptosis, and intracellular calcium homeostasis, we began to measure the contents of calcium and ROS in the cytoplasm and mitochondria. Transmission electron microscopy analysis suggested that Andr protected atrial myocytes from RAP-induced morphological abnormalities. Furthermore, we carried out proteomics analysis to find that the regulation of many proteins located in the mitochondria were changed significantly. These findings led us to assess the mitochondrial SOD enzymatic activity and total SOD enzymatic activity, and the content of ATP was measured at the cellular level. PGC-1α is a crucial effector of impaired mitochondrial bioenergetics. Once activated, it can immediately promote the expression of mitochondrial oxidative phosphorylation (OXPHOS)‍-related proteins. In addition, PGC-1α activates the expression of mitochondrial transcriptional factor A (Tfam), and triggers the transcription and replication of mitochondrial DNA (mtDNA) by the translocation of Tfam into the mitochondria. Moreover, increasing evidence has suggested that HO-1 can positively regulate PGC‍-‍1α expression. Shi et al. (2021) Shi et al. (2021) confirmed that, by regulating mitochondrial quality control (MQC), HO-1 activation protects from sepsis-induced lung injury. Hull et al. (2016) Hull et al. (2016) validated that HO-1 overexpression regulates MQC and promotes mitochondrial biogenesis by Nrf1, PGC-1α, and Tfam upregulation. Herein, we found that PGC-1α and Tfam were downregulated in our AF models both in vitro and in vivo, which could be reversed by Andr. To the best of our knowledge, our study is the first to explore the effect of Andr drug on mitochondria. The role of PGC-1α and the transcription of HO-1 both depend on transcription factors (TFs). It was reported that by upregulating Nrf2/HO-1, Andr inhibited H 2 O 2 -induced liver cell death ( Mittal et al., 2016 Mittal et al., 2016 ). Moreover, others have validated that Andr protects against post-MI damage by promoting the dissociation of Nrf2 from Keap1 and upregulating the expression of HO-1 ( Xie et al., 2020 Xie et al., 2020 ). We performed a molecular docking study to find possible key residues where Andr might combine to promote the dissociation of Nrf2 from Keap1 ( Fig. 12 Fig. 12 ). Taken together, our results demonstrated that the preventive effects of Andr on the development of AF and the associated mechanism could contribute to the regulation of mitochondria. Thus, Andr might be a potential drug to treat AF, and anti-oxidant stress response could be a potential target in the clinical prevention of AF. 4 Conclusions 1 The present study highlights Andr as a potential preventive therapeutic drug toward AF via activating HO-1 to promote mitochondrial bioenergetics. Considering the deficiency of using TCM in AF patients, our findings provide novel evidence in support of the therapeutic use of Andr as a TCM. Materials and methods Detailed methods are provided in the electronic supplementary materials of this paper. References Amin A , Houmsse A , Ishola A , et al. , 2016 . The current approach of atrial fibrillation management . Avicenna J Med , 6 ( 1 ): 8 - 16 . https://doi.org/10.4103/2231-0770.173580 https://doi.org/10.4103/2231-0770.173580 The current approach of atrial fibrillation management Brunner G , Abboud L , Shaikh KA , et al. , 2012 . Left atrial scar burden determined by delayed enhancement cardiac magnetic resonance at post radiofrequency ablation: association with atrial fibrillation recurrence . J Cardiovasc Magn Reson , 14 ( S1 ): P204 . https://doi.org/10.1186/1532-429x-14-s1-p204 https://doi.org/10.1186/1532-429x-14-s1-p204 Left atrial scar burden determined by delayed enhancement cardiac magnetic resonance at post radiofrequency ablation: association with atrial fibrillation recurrence Chen W , Su HM , Feng LN , et al. , 2016 . Andrographolide suppresses preadipocytes proliferation through glutathione antioxidant systems abrogation . Life Sci , 156 : 21 - 29 . https://doi.org/10.1016/j.lfs.2016.05.030 https://doi.org/10.1016/j.lfs.2016.05.030 Andrographolide suppresses preadipocytes proliferation through glutathione antioxidant systems abrogation Chugh SS , Havmoeller R , Narayanan K , et al. , 2014 . Worldwide epidemiology of atrial fibrillation: a global burden of disease 2010 study . Circulation , 129 ( 8 ): 837 - 847 . https://doi.org/10.1161/CIRCULATIONAHA.113.005119 https://doi.org/10.1161/CIRCULATIONAHA.113.005119 Worldwide epidemiology of atrial fibrillation: a global burden of disease 2010 study Chung MK , Refaat M , Shen WK , et al. , 2020 . Atrial fibrillation: JACC council perspectives . J Am Coll Cardiol , 75 ( 14 ): 1689 - 1713 . https://doi.org/10.1016/j.jacc.2020.02.025 https://doi.org/10.1016/j.jacc.2020.02.025 Atrial fibrillation: JACC council perspectives Dobrev D , Nattel S , 2010 . New antiarrhythmic drugs for treatment of atrial fibrillation . Lancet , 375 ( 9721 ): 1212 - 1223 . https://doi.org/10.1016/s0140-6736(10)60096-7 https://doi.org/10.1016/s0140-6736(10)60096-7 New antiarrhythmic drugs for treatment of atrial fibrillation Dong Y , Liao JQ , Yao KW , et al. , 2017 . Application of traditional Chinese medicine in treatment of atrial fibrillation . Evid Based Complement Alternat Med , 2017 : 1381732 . https://doi.org/10.1155/2017/1381732 https://doi.org/10.1155/2017/1381732 Application of traditional Chinese medicine in treatment of atrial fibrillation Elasoru SE , Rhana P , de Oliveira Barreto T , et al. , 2021 . Andrographolide protects against isoproterenol-induced myocardial infarction in rats through inhibition of L-type Ca 2+ and increase of cardiac transient outward K + currents . Eur J Pharmacol , 906 : 174194 . https://doi.org/10.1016/j.ejphar.2021.174194 https://doi.org/10.1016/j.ejphar.2021.174194 Andrographolide protects against isoproterenol-induced myocardial infarction in rats through inhibition of L-type Ca2+ and increase of cardiac transient outward K+ currents Guo W , Huang JH , Wang N , et al. , 2019 . Integrating network pharmacology and pharmacological evaluation for deciphering the action mechanism of herbal formula Zuojin pill in suppressing hepatocellular carcinoma . Front Pharmacol , 10 : 1185 . https://doi.org/10.3389/fphar.2019.01185 https://doi.org/10.3389/fphar.2019.01185 Integrating network pharmacology and pharmacological evaluation for deciphering the action mechanism of herbal formula Zuojin pill in suppressing hepatocellular carcinoma Hopkins AL , 2008 . Network pharmacology: the next paradigm in drug discovery . Nat Chem Biol , 4 ( 11 ): 682 - 690 . https://doi.org/10.1038/nchembio.118 https://doi.org/10.1038/nchembio.118 Network pharmacology: the next paradigm in drug discovery Huang YM , Zhou XM , Liu M , et al. , 2020 . The natural compound andrographolide inhibits human aortic valve interstitial cell calcification via the NF-kappa B/Akt/ERK pathway . Biomed Pharmacother , 125 : 109985 . https://doi.org/10.1016/j.biopha.2020.109985 https://doi.org/10.1016/j.biopha.2020.109985 The natural compound andrographolide inhibits human aortic valve interstitial cell calcification via the NF-kappa B/Akt/ERK pathway Huang YM , Liu M , Liu CG , et al. , 2022 . The natural product andrographolide ameliorates calcific aortic valve disease by regulating the proliferation of valve interstitial cells via the MAPK-ERK pathway . Front Pharmacol , 13 : 871748 . https://doi.org/10.3389/fphar.2022.871748 https://doi.org/10.3389/fphar.2022.871748 The natural product andrographolide ameliorates calcific aortic valve disease by regulating the proliferation of valve interstitial cells via the MAPK-ERK pathway Hull TD , Boddu R , Guo LL , et al. , 2016 . Heme oxygenase-1 regulates mitochondrial quality control in the heart . JCI Insight , 1 ( 2 ): e85817 . https://doi.org/10.1172/jci.insight.85817 https://doi.org/10.1172/jci.insight.85817 Heme oxygenase-1 regulates mitochondrial quality control in the heart Kloosterman M , Chua W , Fabritz L , et al. , 2020 . Sex differences in catheter ablation of atrial fibrillation: results from AXAFA-AFNET 5 . EP Europace , 22 ( 7 ): 1026 - 1035 . https://doi.org/10.1093/europace/euaa015 https://doi.org/10.1093/europace/euaa015 Sex differences in catheter ablation of atrial fibrillation: results from AXAFA-AFNET 5 Kumar A , Butler BM , Kumar S , et al. , 2015 . Integration of structural dynamics and molecular evolution via protein interaction networks: a new era in genomic medicine . Curr Opin Struct Biol , 35 : 135 - 142 . https://doi.org/10.1016/j.sbi.2015.11.002 https://doi.org/10.1016/j.sbi.2015.11.002 Integration of structural dynamics and molecular evolution via protein interaction networks: a new era in genomic medicine Lei YR , Tang Y , Huang L , et al. , 2021 . Systematic review and meta-analysis on efficacy of traditional Chinese medicine for atrial fibrillation through cluster analysis . Ann Palliat Med , 10 ( 8 ): 8982 - 8990 . https://doi.org/10.21037/apm-21-1785 https://doi.org/10.21037/apm-21-1785 Systematic review and meta-analysis on efficacy of traditional Chinese medicine for atrial fibrillation through cluster analysis Li XL , Qiu Q , Li MY , et al. , 2021 . Chemical composition and pharmacological mechanism of ephedra-glycyrrhiza drug pair against coronavirus disease 2019 (COVID-19) . Aging , 13 ( 4 ): 4811 - 4830 . https://doi.org/10.18632/aging.202622 https://doi.org/10.18632/aging.202622 Chemical composition and pharmacological mechanism of ephedra-glycyrrhiza drug pair against coronavirus disease 2019 (COVID-19) Lin KH , Marthandam Asokan S , Kuo WW , et al. , 2020 . Andrographolide mitigates cardiac apoptosis to provide cardio-protection in high-fat-diet-induced obese mice . Environ Toxicol , 35 ( 6 ): 707 - 713 . https://doi.org/10.1002/tox.22906 https://doi.org/10.1002/tox.22906 Andrographolide mitigates cardiac apoptosis to provide cardio-protection in high-fat-diet-induced obese mice Ma YM , Zhang XZ , Su ZZ , et al. , 2015 . Insight into the molecular mechanism of a herbal injection by integrating network pharmacology and in vitro . J Ethnopharmacol , 173 : 91 - 99 . https://doi.org/10.1016/j.jep.2015.07.016 https://doi.org/10.1016/j.jep.2015.07.016 Insight into the molecular mechanism of a herbal injection by integrating network pharmacology and in vitro Maiti K , Mukherjee K , Murugan V , et al. , 2010 . Enhancing bioavailability and hepatoprotective activity of andrographolide from Andrographis paniculata , a well-known medicinal food, through its herbosome . J Sci Food Agric , 90 ( 1 ): 43 - 51 . https://doi.org/10.1002/jsfa.3777 https://doi.org/10.1002/jsfa.3777 Enhancing bioavailability and hepatoprotective activity of andrographolide from Andrographis paniculata, a well-known medicinal food, through its herbosome Mittal SPK , Khole S , Jagadish N , et al. , 2016 . Andrographolide protects liver cells from H 2 O 2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A 2a receptor signalling . Biochim Biophys Acta , 1860 ( 11 Pt A ): 2377 - 2390 . https://doi:10.1016/j.bbagen.2016.07.005 https://doi:10.1016/j.bbagen.2016.07.005 Andrographolide protects liver cells from H2O2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A2a receptor signalling Pauklin P , Zilmer M , Eha J , et al. , 2022 . Markers of inflammation, oxidative stress, and fibrosis in patients with atrial fibrillation . Oxid Med Cell Longev , 2022 : 4556671 . https://doi.org/10.1155/2022/4556671 https://doi.org/10.1155/2022/4556671 Markers of inflammation, oxidative stress, and fibrosis in patients with atrial fibrillation Ruan XF , Ju CW , Shen Y , et al. , 2018 . Suxiao Jiuxin pill promotes exosome secretion from mouse cardiac mesenchymal stem cells in vitro . Acta Pharmacol Sin , 39 ( 4 ): 569 - 578 . https://doi.org/10.1038/aps.2018.19 https://doi.org/10.1038/aps.2018.19 Suxiao Jiuxin pill promotes exosome secretion from mouse cardiac mesenchymal stem cells in vitro Shi J , Yu TX , Song K , et al. , 2021 . Dexmedetomidine ameliorates endotoxin-induced acute lung injury in vivo and in vitro by preserving mitochondrial dynamic equilibrium through the HIF-1a/HO-1 signaling pathway . Redox Biol , 41 : 101954 . https://doi.org/10.1016/j.redox.2021.101954 https://doi.org/10.1016/j.redox.2021.101954 Dexmedetomidine ameliorates endotoxin-induced acute lung injury in vivo and in vitro by preserving mitochondrial dynamic equilibrium through the HIF-1a/HO-1 signaling pathway Su HM , Mo JL , Ni JD , et al. , 2020 . Andrographolide exerts antihyperglycemic effect through strengthening intestinal barrier function and increasing microbial composition of Akkermansia muciniphila . Oxid Med Cell Longev , 2020 : 6538930 . https://doi.org/10.1155/2020/6538930 https://doi.org/10.1155/2020/6538930 Andrographolide exerts antihyperglycemic effect through strengthening intestinal barrier function and increasing microbial composition of Akkermansia muciniphila Tan YQ , Jin M , He XH , et al. , 2022 . Huoxue Qingre decoction used for treatment of coronary heart disease network analysis and metabolomic evaluation . Front Pharmacol , 13 : 1025540 . https://doi.org/10.3389/fphar.2022.1025540 https://doi.org/10.3389/fphar.2022.1025540 Huoxue Qingre decoction used for treatment of coronary heart disease network analysis and metabolomic evaluation Wang M , Xiong H , Lu L , et al. , 2022 . Serum lipopolysaccharide is associated with the recurrence of atrial fibrillation after radiofrequency ablation by increasing systemic inflammation and atrial fibrosis . Oxid Med Cell Longev , 2022 : 2405972 . https://doi.org/10.1155/2022/2405972 https://doi.org/10.1155/2022/2405972 Serum lipopolysaccharide is associated with the recurrence of atrial fibrillation after radiofrequency ablation by increasing systemic inflammation and atrial fibrosis Wang ZS , Tang Z , Zhu WQ , et al. , 2017 . Efficacy and safety of traditional Chinese medicine on thromboembolic events in patients with atrial fibrillation: a systematic review and meta-analysis . Complement Ther Med , 32 : 1 - 10 . https://doi.org/10.1016/j.ctim.2017.03.006 https://doi.org/10.1016/j.ctim.2017.03.006 Efficacy and safety of traditional Chinese medicine on thromboembolic events in patients with atrial fibrillation: a systematic review and meta-analysis Wu QQ , Ni J , Zhang N , et al. , 2017 . Andrographolide protects against aortic banding-induced experimental cardiac hypertrophy by inhibiting MAPKs signaling . Front Pharmacol , 8 : 808 . https://doi.org/10.3389/fphar.2017.00808 https://doi.org/10.3389/fphar.2017.00808 Andrographolide protects against aortic banding-induced experimental cardiac hypertrophy by inhibiting MAPKs signaling Xie SY , Deng W , Chen JJ , et al. , 2020 . Andrographolide protects against adverse cardiac remodeling after myocardial infarction through enhancing Nrf2 signaling pathway . Int J Biol Sci , 16 ( 1 ): 12 - 26 . https://doi.org/10.7150/ijbs.37269 https://doi.org/10.7150/ijbs.37269 Andrographolide protects against adverse cardiac remodeling after myocardial infarction through enhancing Nrf2 signaling pathway Ye SJ , Luo W , Khan ZA , et al. , 2020 . Celastrol attenuates angiotensin ii-induced cardiac remodeling by targeting STAT3 . Circ Res , 126 ( 8 ): 1007 - 1023 . https://doi.org/10.1161/CIRCRESAHA.119.315861 https://doi.org/10.1161/CIRCRESAHA.119.315861 Celastrol attenuates angiotensin ii-induced cardiac remodeling by targeting STAT3 Zeng ML , Jiang WZ , Tian YJ , et al. , 2017 . Andrographolide inhibits arrhythmias and is cardioprotective in rabbits . Oncotarget , 8 ( 37 ): 61226 - 61238 . https://doi.org/10.18632/oncotarget.18051 https://doi.org/10.18632/oncotarget.18051 Andrographolide inhibits arrhythmias and is cardioprotective in rabbits Zhan SK , Liang JY , Lin HT , et al. , 2023 . SATB1/SLC7A11/HO-1 axis ameliorates ferroptosis in neuron cells after ischemic stroke by Danhong injection . Mol Neurobiol , 60 ( 1 ): 413 - 427 . https://doi.org/10.1007/s12035-022-03075-z https://doi.org/10.1007/s12035-022-03075-z SATB1/SLC7A11/HO-1 axis ameliorates ferroptosis in neuron cells after ischemic stroke by Danhong injection Zhang L , Po SS , Wang H , et al. , 2015 . Autonomic remodeling: how atrial fibrillation begets atrial fibrillation in the first 24 hours . J Cardiovasc Pharmacol , 66 ( 3 ): 307 - 315 . https://doi.org/10.1097/FJC.0000000000000281 https://doi.org/10.1097/FJC.0000000000000281 Autonomic remodeling: how atrial fibrillation begets atrial fibrillation in the first 24 hours Conceptualization: Pengcheng YU; Data curation: Pengcheng YU, Jiaru CAO, Huaxin SUN, and Hangying YING; Formal analysis: Pengcheng YU; Investigation: Jiaru CAO and Xinyu ZHOU; Methodology: Pengcheng YU, Jiaru CAO, Huaxin SUN, Yingchao GONG, Qingbo LV, and Hang YANG; Project administration: Ling ZHANG and Xia SHENG; Resources: Xia SHENG; Software: Huaxin SUN, Yingchao GONG, Hangying YING, Yuxing WANG, Chenyang QI, and Hang YANG; Supervision: Ling ZHANG and Xia SHENG; Visualization: Pengcheng YU, Yingchao GONG, Hangying YING, and Yuxing WANG; Writing‍ ‒ ‍original draft: Pengcheng YU; Writing ‍‒ ‍review & editing: Xia SHENG and Pengcheng YU. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Natural Science Foundation of China (No. 82270317) and the Zhejiang Provincial Natural Science Foundation of China (No. LY19H020011). The authors would like to express gratitude to EditSprings ( https://www.editsprings.com https://www.editsprings.com ) for the expert linguistic services provided. 10.1631/jzus.B2300086 1673-1581（2023）07-0632-18 余鹏程,曹佳如,孙华鑫等.穿心莲内酯通过缓解氧化应激和促进线粒体能量合成对心房颤动发挥预防作用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(07):632-649. 余鹏程,曹佳如,孙华鑫等.穿心莲内酯通过缓解氧化应激和促进线粒体能量合成对心房颤动发挥预防作用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(07):632-649. This work was supported by the National Natural Science Foundation of China (No. 82270317) and the Zhejiang Provincial Natural Science Foundation of China (No. LY19H020011). 2023-02-07 2023-04-09 2023-07-15 2023-07-14 2023-06-20 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 39916357 Pengcheng YU, Jiaru CAO, Huaxin SUN, Yingchao GONG, Hangying YING, Xinyu ZHOU, Yuxing WANG, Chenyang QI, Hang YANG, Qingbo LV, Ling ZHANG, and Xia SHENG declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication No. 85-23, revised 1996). This study was conducted with approval by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xinjiang Medical University (Approval No. IACUC-20170420-03). Compliance with ethics guidelines Supplementary information 1 Tables S1‒S3; Fig. S1; Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 7 24