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NLRP3 inflammasome pathway
International Immunopharmacology 82 (2020) 106316
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Hydroxysafflor yellow A inhibits hypoxia/reoxygenation-induced cardiomyocyte injury via regulating the AMPK/NLRP3 inflammasome pathway
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Jing-xue Yea, Min Wanga, Rui-ying Wanga, Hai-tao Liua, Yao-dong Qia, Jian-hua Fub, ⁎ ⁎ ⁎ Qiong Zhangb, , Ben-gang Zhanga, , Xiao-bo Suna, a Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China b Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hydroxysafflor yellow A Cardioprotection NLRP3 inflammasome AMPK
Hydroxysafflor yellow A (HSYA) is an effective therapeutic agent that alleviates myocardial ischaemia/reperfusion injury (MIRI), but the exact mechanisms remain elusive. The aim of this study was to investigate the potential protective effect of HSYA against MIRI through mechanisms related to NLRP3 inflammasome regulation. In this study, hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocytes were treated with HSYA or the AMPK inhibitor, compound C (CC). Our results showed that HSYA pretreatment improved cardiomyocyte viability, maintained mitochondrial membrane potential, reduced apoptotic cardiomyocytes, decreased caspase3 activity, and inhibited NOD-like receptor 3 (NLRP3) inflammasome activation during H/R injury. Moreover, the inhibition of AMPK activation by the CC inhibitor partially abolished the effects of HSYA treatment, including suppressing the upregulation of NLRP3 inflammasome components (NLRP3, caspase-1 and interleukin1β) and promoting autophagy (LC3-II/LC3-I and p62). In conclusion, the protective mechanism of HSYA in H/Rinduced cardiomyocyte injury is associated with inhibiting NLRP3 inflammasome activation through the AMPK signalling pathway.
1. Introduction Myocardial infarction (MI) is one of the major causes of death worldwide. Rapid diagnosis and reperfusion are essential for myocardial salvage. However, this treatment may cause augmented cardiovascular dysfunction and further cell death known as ischaemia/reperfusion (I/R) injury [1]. Emerging evidence has shown that the inflammasome plays an irreplaceable role in myocardial I/R injury [2]. The NOD-like receptor family pyrin containing 3 (NLRP3) inflammasome is the most common inflammasome, and it consists of nucleotidebinding oligomerization domain-like receptor with a pyrin domain (NLRP3), apoptosis-associated speck-like protein (ASC) and caspase-1. Previous evidence has indicated that NLPR3 accumulates in the heart during myocardial infarction and promotes myocardial injury and apoptosis [3,4]. Moreover, activation of the NLRP3 inflammasome promotes further myocardial damage after I/R via inducing the
production of potent inflammatory cytokines, including IL-1β and IL18, as well as caspase-1-dependent programmed cell death [5]. However, NLRP3 inflammasome inhibition reduces infarct size, attenuates adverse cardiac remodelling, and preserves cardiac function in animal models of MI [6]. Therefore, suppressing NLRP3 inflammasome activities may be a promising target for pharmaceutical interventions to treat I/R-induced myocardial injury. Hydroxysafflor yellow A (HSYA) is the main bioactive component of Carthamus tinctorius L., which has been used for hundreds of years in traditional Chinese medicine for the treatment of cardiovascular disease [7]. Previous studies have suggested that HSYA may improve myocardial I/R injury by decreasing oxidative stress, restoring mitochondrial energy, promoting angiogenesis, and inhibiting apoptosis [8–10]. Recently, several reports have shown that HSYA exhibits promising anti-inflammatory properties in myocardial I/R associated with hyperlipidaemia, ischaemic stroke and acute lung injury models [11,12].
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Corresponding authors at: Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, PR China (Q. Zhang); Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, PR China (B.G. Zhang and X.-B. Sun). E-mail addresses: [email protected] (Q. Zhang), [email protected] (B.-g. Zhang), [email protected] (X.-b. Sun). https://doi.org/10.1016/j.intimp.2020.106316 Received 1 December 2019; Received in revised form 23 January 2020; Accepted 12 February 2020 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
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then reacted with 2,4-dinitrophenylhydrazine to produce a brownish red colour in basic solution. After reaction, the absorbance of each sample was read at wavelength 440 nm, and the results were expressed as U/L.
Moreover, HSYA markedly inhibits LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and preventing mature IL-1β formation from pro-IL-1β in RAW264.7 macrophages [13]. However, the effect of HSYA and the association between HSYA and NLRP3 in cardiomyocytes following I/R are still unknown. In the present study, we investigated the effects of HSYA on the NLRP3 pathway in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injuries and further explored the underlying mechanisms of HSYA involved in the AMPK signalling pathway. Our results provide novel insight into the mechanism of HSYA in the regulation of myocardial I/R injury.
2.5. Determination of mitochondrial transmembrane potential (ΔΨm) JC-1 was used to analyse the changes in mitochondrial transmembrane potential as previously reported [18]. After treatments, cells were incubated with JC-1 (2 µM final concentration) at 37 °C in the dark for 30 min. Cells were then washed three times with PBS and observed using fluorescence microscopy (EVOSR FL Colour, Life Technologies).
2. Materials and methods 2.6. Flow cytometric detection of cell apoptosis rate 2.1. Reagents The percentages of early apoptosis and necrosis were detected using an Annexin V FITC/PI apoptosis kit according to the manufacturer’s instructions. Following drug treatment, cells were harvested, washed twice with cold PBS, and incubated in the dark with 5 μL of FITC–Annexin V and 1 μL of PI working solution (100 μg/mL) for 15 min at room temperature. Cellular fluorescence was measured by a FACS Calibur flow cytometer (BD Biosciences, CA, United States). The early apoptosis rate was expressed as the ratio of Annexin V-positive/ PI-negative cells to total cells [18].
HSYA (purity > 98%) was obtained from Shanghai Winherb Medical S&T Development (Shanghai, China, cat. no. Q-0293). Cell culture products were purchased from Gibco BRL (Grand Island, NY, USA). The Annexin V/propidium iodide (PI) apoptosis detection kit was obtained from Invitrogen (Eugene, USA, cat. no. V13242). The JC-1 fluorescent dye was purchased from Sigma–Aldrich (St. Louis, MO, USA, cat. no. 420200). The caspase-1 activity assay kit (cat. no. C1102) and the caspase-3 activity assay kit (cat. no. C1115) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). All antibodies were purchased from Abcam (Cambridge, England), and chemical reagents were of at least analytical grade.
2.7. Analysis of caspase-1 activity The activity of caspase-1 was detected by a kit following the manufacturer’s instructions. This assay was based on the ability of caspase-1 to change acetyl-Tyr-Val-Ala-Asp p-nitroaniline (Ac-YVAD-pNA) into pnitroaniline (pNA), a yellow formazan product. The production of pNA per minute in tested samples was used as a measure of the level of caspase-1 activity and inflammasome activation. The results were expressed as fold increase in caspase-1 activity [19].
2.2. Cell culture and pharmacological treatments Rat embryonic cardiomyoblast-derived H9c2 cardiomyocytes (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured as previously described [14,15]. For all experiments, cells were plated at an appropriate density according to the experimental design and were grown for 24 h to reach 70% to 80% confluency before experimentation. The H/R model was generated according to previously published methods [16]. H9c2 cardiomyocytes were cultured under hypoxia for 6 h and then removed from the anaerobic glove box (TYPE C, Coy Laboratory, CA, USA) to a regular incubator with the medium replaced by normal medium to mimic reperfusion. In the HSYA-treated group, H9c2 cardiomyocytes were pretreated with HSYA for 4 h prior to H/R. In the inhibitor-treated group, cells were pre-incubated with 10 μM compound c (CC) for 1 h before they were treated with HSYA. The appropriate concentration of CC was determined based on data found in the literature and our preliminary experiments (Supplementary Fig. 1).
2.8. Analysis of caspase-3 activation Caspase-3 activity was measured using a colorimetric caspase-3 assay kit according to the manufacturer’s instructions. Briefly, cell lysates were prepared after different treatment. Assays were carried out on 96-well plates by incubating 10 μL protein of cell lysate per sample in 80 μL reaction buffer containing 10 μL caspase-3 substrate (AcDEVD-pNA, 2 mM). Lysates were incubated at 37 °C for 2 h. Samples were measured at a wavelength of 405 nm using a microplate reader (Infinite M1000, TECAN, Hombrechtikon, Switzerland) [20].
2.3. Cell viability analysis
2.9. Western blot analysis
Cell viability was determined using a MTT assay as previously described [14]. H9c2 cells were seeded at a density of 1 × 104 cells/well in 96-well plates. After different treatments, 20 µL of MTT (5 mg/mL) was added to each well and incubated for 4 h. The supernatant was then removed, and the coloured formazan crystals were dissolved in dimethyl sulfoxide. Absorbance was detected at 570 nm using a microplate reader (Infinite M1000, TECAN, Switzerland).
Protein expression was analysed by Western blotting as previously described [21]. After the designated treatment, cardiomyocytes were lysed in cell lysis buffer containing 1% phenylmethylsulphonyl fluoride. Equal amounts of protein from each sample were separated by SDS–PAGE and then transferred onto a nitrocellulose membrane. After being blocked with 5% (w/v) non-fat milk powder, membranes were incubated overnight at 4 °C with appropriate primary antibodies: rabbit anti-AMPK (Abcam, cat. no. ab80039), rabbit anti-p-AMPK (Abcam, cat. no. ab23875), rabbit anti-NLRP3 (Abcam, cat. no. ab214185), rabbit anti-IL-1 beta (Abcam, cat. no. ab9722), rabbit anti-LC3B (Abcam, cat. no. ab192890), rabbit anti-p62 (Abcam, cat. no. ab109012), and rabbit anti-β-actin (Abcam, cat. no. ab8227). After washing, membranes were incubated for 1 h with respective horseradish peroxidase-conjugated secondary antibodies at room temperature. Finally, membranes were developed by enhanced chemiluminescence using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA).
2.4. Lactate dehydrogenase release assay For the detection of cytotoxicity in H9c2 cells, lactate dehydrogenase (LDH) production was measured using an LDH cytotoxicity Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China, cat. no. A020-2-2) according to the manufacturer’s protocol [17]. Briefly, cell medium was removed for the activity analysis of extracellular LDH, which catalyses the conversion of lactate to pyruvate, and 2
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Fig. 1. Effects of HSYA on H/R-induced cell injury in H9c2 cells. (A) Cells were incubated with different concentrations (6.25, 12.5 and 25 μM) of HSYA for different times (4, 12, and 24 h) before hypoxia for 6 h. Cell viability was detected by MTT assay. (B) Effect of HSYA on the level of extracellular LDH leakage. HSYA, Hydroxysafflor yellow A. H/R, hypoxia/reoxygenation. Data are presented as the means ± SD from three independent experiments. ##P < 0.01 versus control; *P < 0.05 versus H/R-treated cells; **P < 0.01 versus H/R-treated cells.
3.4. HSYA mitigates H/R-induced NLRP3 inflammasome via activating AMPK
2.10. Statistical analysis Results were expressed as the mean ± standard deviation (SD) of three independent experiments. Comparisons between different groups were performed using Student’s t-test or one-way ANOVA followed by post hoc analysis with Tukey’s multiple comparison test using Prism 5.00 software. Statistical significance was set at P < 0.05.
Emerging evidence has revealed that AMPK is involved in the regulation of inflammatory response [22]. As shown in Fig. 4A and supplementary Fig. 2, p-AMPK/AMPK was reduced in H/R-treated cardiomyocytes, whereas it was improved after HSYA treatment. To verify the effect of AMPK activation on the inhibition of NLRP3 inflammasome activation after HSYA treatment, the AMPK inhibitor, compound C (CC), was used. Fig. 4A shows that CC effectively inhibited the protein expression ratio of P-AMPK/AMPK compared to the H/R + HSYA group. In addition, co-treatment with the AMPK inhibitor and HSYA abrogated the protective effect of HSYA on the NLRP3 pathway during H/R as shown by increased expression levels of NLRP3, IL-1β, and caspase-1 activity (Fig. 4B and C). Furthermore, it has been reported that autophagy activated by AMPK is closely related to the NLRP3 inflammasome [23]. Therefore, we measured the protein expression levels of LC3 and p62. The LC3II/I protein expression ratio was significantly reduced in H/R-treated cardiomyocytes but were improved after HSYA treatment. However, these HSYA-induced improvements were abolished after treatment with CC. Further, CC increased the expression level of p62 (Fig. 4D). These results confirmed that HSYA activates p-AMPK and autophagy, thus inhibiting the NLRP3 inflammasome.
3. Results 3.1. HSYA ameliorates H/R-induced cytotoxicity in H9c2 cardiomyocytes The protective effect of HSYA against H/R-induced cell death was detected using a MTT assay. Fig. 1A demonstrates that HSYA pretreatment alleviated the H/R-induced reduction of cell viability in a dose-dependent manner. Moreover, 12.5 μM HSYA treatment for 4 h exhibited the maximal protective effect. As an indicator of cell injury, LDH levels were measured. As shown in Fig. 1B, HSYA decreased LDH levels in culture medium in a concentration-dependent manner. This effect was consistent with its protective effects on cell viability as assessed by a MTT assay. 3.2. HSYA inhibits H/R-induced apoptosis in H9c2 cells Disruption of mitochondrial membrane potential (ΔΨm) is an early event in the apoptotic cascade [15]. Thus, we assessed the potential effect of HSYA on ΔΨm by JC-1 staining (Fig. 2A). Pretreatment with HSYA reduced ΔΨm depolarization as indicated by a significant decrease in the ratio of red/green fluorescence intensity (Fig. 2C). The anti-apoptotic effect of HSYA was further corroborated through FITC–Annexin V/PI double staining (Fig. 2B). The apoptosis rate significantly increased in the H/R group, while HSYA treatment effectively alleviated H/R-induced early apoptosis (Fig. 2D). Caspases play a key role in regulating the apoptosis cascade. As shown in Fig. 2E, H/R increased the caspase-3 activation, and this change was reversed by HSYA treatment. Together, these results indicated that HSYA protects cardiomyocytes against H/R injury partly by attenuating apoptosis.
4. Discussion HSYA has been widely used in the treatment of cardiovascular diseases in China for a long time. In the present study, HSYA significantly reduced NLRP3 inflammasome activation and subsequent injury in H9c2 cells exposed to H/R stimulation. Moreover, p-AMPK and autophagy were activated by HSYA. However, the AMPK inhibitor, CC, blocked the effects of HSYA on autophagy and NLRP3 levels. Our findings indicated that the inhibitory effects of HSYA on NLRP3 inflammasome activation may be dependent on the AMPK pathway in I/R injury. Our study provided insight into a new mechanism that is responsible for the cardioprotective function of HSYA. Although reperfusion therapy is successful in preventing heart damage, which occurs when blood re-supply evokes an intense and highly specific inflammatory response, it mediates further myocardial damage and dysfunction. Increasing evidence has demonstrated that the NLRP3 inflammasome plays an essential role in detecting cell damage and regulating inflammatory response after myocardial I/R damage [3]. Activation of the NLRP3 inflammasome promotes the recruitment of ASC and caspase-1 activation, which contributes to the maturation and secretion of IL-1β as well as the promotion of pyroptotic cell death, leading to the increase of infarct size and myocardial dysfunction in I/R
3.3. HSYA inhibits H/R-induced NLRP3 inflammasome activation in H9c2 cells To evaluate the effects of HSYA on the NLRP3 pathway, we measured the level of NLRP3, the release of IL-1β into culture medium, and caspase-1 activity. As shown in Fig. 3, HSYA treatment decreased NLRP3 expression, and IL-1β levels and caspase-1 activity in H9c2 cells compared to the H/R group. 3
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Fig. 2. Effects of HSYA on H/R-induced apoptosis. (A) Effect of HSYA on mitochondrial transmembrane permeability transition. ΔΨm was assessed by fluorescence microscopy with JC-1 staining. (B) Scatter diagram of Annexin V–PI double staining as measured using flow cytometry. (C) Quantitative analysis of mitochondrial membrane potential (ratio of red fluorescence obtained at 590 nm to green fluorescence at 530 nm) in H9C2 cells. (D) Quantitative analysis of apoptosis rate. (E) Caspase-3 activity was measured using a fluorometric assay. HSYA, Hydroxysafflor yellow A. H/R, hypoxia/reoxygenation. Data are presented as the means ± SD from three independent experiments. #P < 0.05 versus control, ##P < 0.01 versus control; *P < 0.05 versus H/R-treated cells, **P < 0.01 versus H/R-treated cells.
Fig. 3. HSYA attenuates activation of the NLRP3 inflammasome and upregulates AMPK activation induced by H/R in H9c2 cells. (A) Western blot analysis was performed to assess the protein expression of NLRP3. β-actin expression was utilized as the protein loading control. (B) IL-1β content in culture supernatant was detected by ELISA. (C) Caspase-1 activity was measured by caspase-1 activity assay kit. HSYA, Hydroxysafflor yellow A. H/R, hypoxia/reoxygenation. Data are presented as the means ± SD from three independent experiments. #P < 0.05 versus control, ##P < 0.01 versus control; *P < 0.05 versus H/R-treated cells, **P < 0.01 versus H/R-treated cells. 4
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Fig. 4. AMPK inhibitor (compound c) blocks the suppressive effect of HSYA on the NLRP3 pathway in cardiomyocytes. H9c2 cells were pretreated with CC or CC + HSYA before exposure to H/R. (A) Expression levels of p-AMPK and AMPK. (B) Caspase-1 activity. (C) Expression levels of NLRP3, and IL-1b in cell lysates were determined by Western blotting. (D) Expression levels of LC3 and p62 in H9C2 cells were assayed by Western blot analysis. β-actin expression was utilized as the protein loading control. HSYA, Hydroxysafflor yellow A. H/R, hypoxia/reoxygenation. CC, compound c. Data are presented as the means ± SD from three independent experiments. #P < 0.05 versus control, ##P < 0.01 versus control; *P < 0.05 versus H/R-treated cells, **P < 0.01 versus H/R-treated cells. & P < 0.05 versus H/R + HSYA-treated cells, and &&P < 0.01 versus H/R + HSYA-treated cells.
Among several upstream elements affecting NLRP3 inflammasome under I/R, we investigated the pathway associated with HSYA. AMPK functions as a cell energy sensor and controls a variety of pathophysiological mechanisms, such as autophagy, apoptosis and inflammation [26]. Activation of the AMPK signalling pathway during myocardial I/R has been considered to be an endogenous compensatory mechanism to protect against myocardial injury [27]. Previous studies have shown that hispidulin protects against cerebral I/R injury through suppressing the NLRP3 inflammasome by modulating the AMPK signalling pathway [28]. In this study, HSYA increased the ratio of P-AMPK/T-AMPK concomitant with the inhibition of the NLRP3 inflammasome in H/Rinduced cardiomyocytes. However, compound c, an inhibitor of AMPK, suppressed HSYA-induced inhibition of the NLRP3 inflammasome, indicating the essential role of AMPK signalling in regulating inflammasome activity. In addition, accumulating evidence has indicated that autophagy decreases NLRP3 inflammasome levels via the AMPK signalling
injury [24]. Similarly, our research confirmed that H/R remarkably increases NLRP3 expression and apoptosis in cardiomyocytes. In addition, previous studies have reported the beneficial effects of strategies blocking the activation of the NLRP3 inflammasome in acute myocardial infarction [25], suggesting a potential therapeutic target for preventing myocardial damages. HSYA is a monomer compound extracted from the flower of the safflower plant (Carthamus tinctorius L.). The protective efficacy of HSYA against myocardial I/R injury has been validated previously [9]. However, the exact underlying mechanisms of HSYA remain largely unknown, and evidence is lacking regarding its association with inflammasome regulation. In this study, we demonstrated that treatment with HSYA decreased NLRP3 protein expression, cleaved caspase-1 protein expression and IL-1β secretion, suggesting that HSYA may mitigate H/R-induced inflammation in H9c2 cells. These results suggested that HSYA protects myocardial cells against H/R injury via mitigation of NLRP3 inflammasome activation. 5
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pathway [23]. Although HSYA has been shown to inhibit apoptosis, it remains unclear if autophagy is involved in HSYA-triggered cardioprotection. Microtubule-associated protein 1 light chain 3 (LC3) is known as a key marker for autophagosome. LC3 exists in two forms: LC3-I is cytosolic, whereas LC3-II is membrane bound. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes. Therefore, the expression level of LC3-II can be used to represent the volume of autophagy [29]. Besides, p62 can interact with LC3 on autophagic membranes and make contact with ubiquitin, playing a critical role in autophagy through p62 degradation by the autophagy-lysosome [30]. Interestingly, our data confirmed that autophagy is activated by HSYA as manifested by an increased ratio of LC3-II/LC3-I and decreased p62 level in the H/R group. Our study also showed that AMPK inhibition counteracts HSYA-induced autophagy activation. Previous research has shown that autophagy is enhanced in H9c2 cardiomyocytes following AMPK/mTOR activation and has protective effects in cells during H/R injury [31]. Our finding agreed with that of previous studies, showing that AMPK-regulated autophagy in cardiomyocytes actively participates in H/R injury. Therefore, our findings indicated that HSYA possesses cardioprotective effects at least in part by activating AMPK/autophagy and subsequently inhibiting the NLRP3 inflammasome in myocardial injury. Collectively, our findings suggested that HSYA suppresses activation of the NLRP3 inflammasome via the AMPK pathway in H/R-induced cardiomyocytes. Our study provides insights into the therapeutic mechanisms of HSYA and the novel therapeutic agents targeting the NLRP3 inflammasome during MI.
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Funding This work was supported by the National Natural Sciences Foundation of China (Grant No. 8167141813), the Natural Sciences Foundation of Beijing (Grant No. 7172191), the State Administration of Traditional Chinese Medicine of the People’s Republic of China (Grant No. ZYBZH-C-JIN-44), and the CAMS Innovation Fund for Medical Sciences (CIFMS) (Grant No. 2016-I2M-1-012). CRediT authorship contribution statement Jing-xue Ye: Conceptualization, Investigation, Validation. Min Wang: Writing - original draft. Rui-ying Wang: Investigation, Formal analysis. Hai-tao Liu: Investigation. Yao-dong Qi: Investigation. Jianhua Fu: Resources. Qiong Zhang: Conceptualization, Funding acquisition. Ben-gang Zhang: Supervision. Xiao-bo Sun: Writing - review & editing. Declaration of Competing Interest The authors declare no conflicts of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2020.106316. References [1] V. Sharma, R.M. Bell, D.M. Yellon, Targeting reperfusion injury in acute myocardial infarction: a review of reperfusion injury pharmacotherapy, Expert Opin. Pharmacother. 13 (8) (2012) 1153–1175. [2] Z. Guo, S. Yu, X. Chen, R. Ye, W. Zhu, X. Liu, NLRP3 Is Involved in Ischemia/ Reperfusion Injury, CNS Neurol. Disord.: Drug Targets 15 (6) (2016) 699–712. [3] Z. Meng, M.Y. Song, C.F. Li, J.Q. Zhao, shRNA interference of NLRP3 inflammasome alleviate ischemia reperfusion-induced myocardial damage through autophagy activation, Biochem. Biophys. Res. Commun. 494 (3–4) (2017) 728–735. [4] X. Zhang, Q. Du, Y. Yang, J. Wang, S. Dou, C. Liu, J. Duan, The protective effect of Luteolin on myocardial ischemia/reperfusion (I/R) injury through TLR4/NFkappaB/NLRP3 inflammasome pathway, Biomed. Pharmacother. 91 (2017)
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[31] M. Zhao, L. Sun, X.J. Yu, Y. Miao, J.J. Liu, H. Wang, J. Ren, W.J. Zang, Acetylcholine mediates AMPK-dependent autophagic cytoprotection in H9c2 cells during hypoxia/reoxygenation injury, Cell. Physiol. Biochem. 32 (3) (2013) 601–613.
Y.D. Yoo, J. Hwang, T. McGuire, S.M. Shim, H.D. Song, S. Ganipisetti, N. Wang, J.M. Jang, M.J. Lee, S.J. Kim, K.H. Lee, J.T. Hong, A. Ciechanover, I. Mook-Jung, K.P. Kim, X.Q. Xie, Y.T. Kwon, B.Y. Kim, p62/SQSTM1/Sequestosome-1 is an Nrecognin of the N-end rule pathway which modulates autophagosome biogenesis, Nat. Commun. 8 (1) (2017) 102.
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Hydroxysafflor yellow A inhibits hypoxia/reoxygenation-induced cardiomyocyte injury via regulating the AMPK/NLRP3 inflammasome pathway
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Jing-xue Yea, Min Wanga, Rui-ying Wanga, Hai-tao Liua, Yao-dong Qia, Jian-hua Fub, ⁎ ⁎ ⁎ Qiong Zhangb, , Ben-gang Zhanga, , Xiao-bo Suna, a Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China b Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hydroxysafflor yellow A Cardioprotection NLRP3 inflammasome AMPK
Hydroxysafflor yellow A (HSYA) is an effective therapeutic agent that alleviates myocardial ischaemia/reperfusion injury (MIRI), but the exact mechanisms remain elusive. The aim of this study was to investigate the potential protective effect of HSYA against MIRI through mechanisms related to NLRP3 inflammasome regulation. In this study, hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocytes were treated with HSYA or the AMPK inhibitor, compound C (CC). Our results showed that HSYA pretreatment improved cardiomyocyte viability, maintained mitochondrial membrane potential, reduced apoptotic cardiomyocytes, decreased caspase3 activity, and inhibited NOD-like receptor 3 (NLRP3) inflammasome activation during H/R injury. Moreover, the inhibition of AMPK activation by the CC inhibitor partially abolished the effects of HSYA treatment, including suppressing the upregulation of NLRP3 inflammasome components (NLRP3, caspase-1 and interleukin1β) and promoting autophagy (LC3-II/LC3-I and p62). In conclusion, the protective mechanism of HSYA in H/Rinduced cardiomyocyte injury is associated with inhibiting NLRP3 inflammasome activation through the AMPK signalling pathway.
1. Introduction Myocardial infarction (MI) is one of the major causes of death worldwide. Rapid diagnosis and reperfusion are essential for myocardial salvage. However, this treatment may cause augmented cardiovascular dysfunction and further cell death known as ischaemia/reperfusion (I/R) injury [1]. Emerging evidence has shown that the inflammasome plays an irreplaceable role in myocardial I/R injury [2]. The NOD-like receptor family pyrin containing 3 (NLRP3) inflammasome is the most common inflammasome, and it consists of nucleotidebinding oligomerization domain-like receptor with a pyrin domain (NLRP3), apoptosis-associated speck-like protein (ASC) and caspase-1. Previous evidence has indicated that NLPR3 accumulates in the heart during myocardial infarction and promotes myocardial injury and apoptosis [3,4]. Moreover, activation of the NLRP3 inflammasome promotes further myocardial damage after I/R via inducing the
production of potent inflammatory cytokines, including IL-1β and IL18, as well as caspase-1-dependent programmed cell death [5]. However, NLRP3 inflammasome inhibition reduces infarct size, attenuates adverse cardiac remodelling, and preserves cardiac function in animal models of MI [6]. Therefore, suppressing NLRP3 inflammasome activities may be a promising target for pharmaceutical interventions to treat I/R-induced myocardial injury. Hydroxysafflor yellow A (HSYA) is the main bioactive component of Carthamus tinctorius L., which has been used for hundreds of years in traditional Chinese medicine for the treatment of cardiovascular disease [7]. Previous studies have suggested that HSYA may improve myocardial I/R injury by decreasing oxidative stress, restoring mitochondrial energy, promoting angiogenesis, and inhibiting apoptosis [8–10]. Recently, several reports have shown that HSYA exhibits promising anti-inflammatory properties in myocardial I/R associated with hyperlipidaemia, ischaemic stroke and acute lung injury models [11,12].
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Corresponding authors at: Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, PR China (Q. Zhang); Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, PR China (B.G. Zhang and X.-B. Sun). E-mail addresses: [email protected] (Q. Zhang), [email protected] (B.-g. Zhang), [email protected] (X.-b. Sun). https://doi.org/10.1016/j.intimp.2020.106316 Received 1 December 2019; Received in revised form 23 January 2020; Accepted 12 February 2020 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
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then reacted with 2,4-dinitrophenylhydrazine to produce a brownish red colour in basic solution. After reaction, the absorbance of each sample was read at wavelength 440 nm, and the results were expressed as U/L.
Moreover, HSYA markedly inhibits LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and preventing mature IL-1β formation from pro-IL-1β in RAW264.7 macrophages [13]. However, the effect of HSYA and the association between HSYA and NLRP3 in cardiomyocytes following I/R are still unknown. In the present study, we investigated the effects of HSYA on the NLRP3 pathway in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injuries and further explored the underlying mechanisms of HSYA involved in the AMPK signalling pathway. Our results provide novel insight into the mechanism of HSYA in the regulation of myocardial I/R injury.
2.5. Determination of mitochondrial transmembrane potential (ΔΨm) JC-1 was used to analyse the changes in mitochondrial transmembrane potential as previously reported [18]. After treatments, cells were incubated with JC-1 (2 µM final concentration) at 37 °C in the dark for 30 min. Cells were then washed three times with PBS and observed using fluorescence microscopy (EVOSR FL Colour, Life Technologies).
2. Materials and methods 2.6. Flow cytometric detection of cell apoptosis rate 2.1. Reagents The percentages of early apoptosis and necrosis were detected using an Annexin V FITC/PI apoptosis kit according to the manufacturer’s instructions. Following drug treatment, cells were harvested, washed twice with cold PBS, and incubated in the dark with 5 μL of FITC–Annexin V and 1 μL of PI working solution (100 μg/mL) for 15 min at room temperature. Cellular fluorescence was measured by a FACS Calibur flow cytometer (BD Biosciences, CA, United States). The early apoptosis rate was expressed as the ratio of Annexin V-positive/ PI-negative cells to total cells [18].
HSYA (purity > 98%) was obtained from Shanghai Winherb Medical S&T Development (Shanghai, China, cat. no. Q-0293). Cell culture products were purchased from Gibco BRL (Grand Island, NY, USA). The Annexin V/propidium iodide (PI) apoptosis detection kit was obtained from Invitrogen (Eugene, USA, cat. no. V13242). The JC-1 fluorescent dye was purchased from Sigma–Aldrich (St. Louis, MO, USA, cat. no. 420200). The caspase-1 activity assay kit (cat. no. C1102) and the caspase-3 activity assay kit (cat. no. C1115) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). All antibodies were purchased from Abcam (Cambridge, England), and chemical reagents were of at least analytical grade.
2.7. Analysis of caspase-1 activity The activity of caspase-1 was detected by a kit following the manufacturer’s instructions. This assay was based on the ability of caspase-1 to change acetyl-Tyr-Val-Ala-Asp p-nitroaniline (Ac-YVAD-pNA) into pnitroaniline (pNA), a yellow formazan product. The production of pNA per minute in tested samples was used as a measure of the level of caspase-1 activity and inflammasome activation. The results were expressed as fold increase in caspase-1 activity [19].
2.2. Cell culture and pharmacological treatments Rat embryonic cardiomyoblast-derived H9c2 cardiomyocytes (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured as previously described [14,15]. For all experiments, cells were plated at an appropriate density according to the experimental design and were grown for 24 h to reach 70% to 80% confluency before experimentation. The H/R model was generated according to previously published methods [16]. H9c2 cardiomyocytes were cultured under hypoxia for 6 h and then removed from the anaerobic glove box (TYPE C, Coy Laboratory, CA, USA) to a regular incubator with the medium replaced by normal medium to mimic reperfusion. In the HSYA-treated group, H9c2 cardiomyocytes were pretreated with HSYA for 4 h prior to H/R. In the inhibitor-treated group, cells were pre-incubated with 10 μM compound c (CC) for 1 h before they were treated with HSYA. The appropriate concentration of CC was determined based on data found in the literature and our preliminary experiments (Supplementary Fig. 1).
2.8. Analysis of caspase-3 activation Caspase-3 activity was measured using a colorimetric caspase-3 assay kit according to the manufacturer’s instructions. Briefly, cell lysates were prepared after different treatment. Assays were carried out on 96-well plates by incubating 10 μL protein of cell lysate per sample in 80 μL reaction buffer containing 10 μL caspase-3 substrate (AcDEVD-pNA, 2 mM). Lysates were incubated at 37 °C for 2 h. Samples were measured at a wavelength of 405 nm using a microplate reader (Infinite M1000, TECAN, Hombrechtikon, Switzerland) [20].
2.3. Cell viability analysis
2.9. Western blot analysis
Cell viability was determined using a MTT assay as previously described [14]. H9c2 cells were seeded at a density of 1 × 104 cells/well in 96-well plates. After different treatments, 20 µL of MTT (5 mg/mL) was added to each well and incubated for 4 h. The supernatant was then removed, and the coloured formazan crystals were dissolved in dimethyl sulfoxide. Absorbance was detected at 570 nm using a microplate reader (Infinite M1000, TECAN, Switzerland).
Protein expression was analysed by Western blotting as previously described [21]. After the designated treatment, cardiomyocytes were lysed in cell lysis buffer containing 1% phenylmethylsulphonyl fluoride. Equal amounts of protein from each sample were separated by SDS–PAGE and then transferred onto a nitrocellulose membrane. After being blocked with 5% (w/v) non-fat milk powder, membranes were incubated overnight at 4 °C with appropriate primary antibodies: rabbit anti-AMPK (Abcam, cat. no. ab80039), rabbit anti-p-AMPK (Abcam, cat. no. ab23875), rabbit anti-NLRP3 (Abcam, cat. no. ab214185), rabbit anti-IL-1 beta (Abcam, cat. no. ab9722), rabbit anti-LC3B (Abcam, cat. no. ab192890), rabbit anti-p62 (Abcam, cat. no. ab109012), and rabbit anti-β-actin (Abcam, cat. no. ab8227). After washing, membranes were incubated for 1 h with respective horseradish peroxidase-conjugated secondary antibodies at room temperature. Finally, membranes were developed by enhanced chemiluminescence using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA).
2.4. Lactate dehydrogenase release assay For the detection of cytotoxicity in H9c2 cells, lactate dehydrogenase (LDH) production was measured using an LDH cytotoxicity Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China, cat. no. A020-2-2) according to the manufacturer’s protocol [17]. Briefly, cell medium was removed for the activity analysis of extracellular LDH, which catalyses the conversion of lactate to pyruvate, and 2
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Fig. 1. Effects of HSYA on H/R-induced cell injury in H9c2 cells. (A) Cells were incubated with different concentrations (6.25, 12.5 and 25 μM) of HSYA for different times (4, 12, and 24 h) before hypoxia for 6 h. Cell viability was detected by MTT assay. (B) Effect of HSYA on the level of extracellular LDH leakage. HSYA, Hydroxysafflor yellow A. H/R, hypoxia/reoxygenation. Data are presented as the means ± SD from three independent experiments. ##P < 0.01 versus control; *P < 0.05 versus H/R-treated cells; **P < 0.01 versus H/R-treated cells.
3.4. HSYA mitigates H/R-induced NLRP3 inflammasome via activating AMPK
2.10. Statistical analysis Results were expressed as the mean ± standard deviation (SD) of three independent experiments. Comparisons between different groups were performed using Student’s t-test or one-way ANOVA followed by post hoc analysis with Tukey’s multiple comparison test using Prism 5.00 software. Statistical significance was set at P < 0.05.
Emerging evidence has revealed that AMPK is involved in the regulation of inflammatory response [22]. As shown in Fig. 4A and supplementary Fig. 2, p-AMPK/AMPK was reduced in H/R-treated cardiomyocytes, whereas it was improved after HSYA treatment. To verify the effect of AMPK activation on the inhibition of NLRP3 inflammasome activation after HSYA treatment, the AMPK inhibitor, compound C (CC), was used. Fig. 4A shows that CC effectively inhibited the protein expression ratio of P-AMPK/AMPK compared to the H/R + HSYA group. In addition, co-treatment with the AMPK inhibitor and HSYA abrogated the protective effect of HSYA on the NLRP3 pathway during H/R as shown by increased expression levels of NLRP3, IL-1β, and caspase-1 activity (Fig. 4B and C). Furthermore, it has been reported that autophagy activated by AMPK is closely related to the NLRP3 inflammasome [23]. Therefore, we measured the protein expression levels of LC3 and p62. The LC3II/I protein expression ratio was significantly reduced in H/R-treated cardiomyocytes but were improved after HSYA treatment. However, these HSYA-induced improvements were abolished after treatment with CC. Further, CC increased the expression level of p62 (Fig. 4D). These results confirmed that HSYA activates p-AMPK and autophagy, thus inhibiting the NLRP3 inflammasome.
3. Results 3.1. HSYA ameliorates H/R-induced cytotoxicity in H9c2 cardiomyocytes The protective effect of HSYA against H/R-induced cell death was detected using a MTT assay. Fig. 1A demonstrates that HSYA pretreatment alleviated the H/R-induced reduction of cell viability in a dose-dependent manner. Moreover, 12.5 μM HSYA treatment for 4 h exhibited the maximal protective effect. As an indicator of cell injury, LDH levels were measured. As shown in Fig. 1B, HSYA decreased LDH levels in culture medium in a concentration-dependent manner. This effect was consistent with its protective effects on cell viability as assessed by a MTT assay. 3.2. HSYA inhibits H/R-induced apoptosis in H9c2 cells Disruption of mitochondrial membrane potential (ΔΨm) is an early event in the apoptotic cascade [15]. Thus, we assessed the potential effect of HSYA on ΔΨm by JC-1 staining (Fig. 2A). Pretreatment with HSYA reduced ΔΨm depolarization as indicated by a significant decrease in the ratio of red/green fluorescence intensity (Fig. 2C). The anti-apoptotic effect of HSYA was further corroborated through FITC–Annexin V/PI double staining (Fig. 2B). The apoptosis rate significantly increased in the H/R group, while HSYA treatment effectively alleviated H/R-induced early apoptosis (Fig. 2D). Caspases play a key role in regulating the apoptosis cascade. As shown in Fig. 2E, H/R increased the caspase-3 activation, and this change was reversed by HSYA treatment. Together, these results indicated that HSYA protects cardiomyocytes against H/R injury partly by attenuating apoptosis.
4. Discussion HSYA has been widely used in the treatment of cardiovascular diseases in China for a long time. In the present study, HSYA significantly reduced NLRP3 inflammasome activation and subsequent injury in H9c2 cells exposed to H/R stimulation. Moreover, p-AMPK and autophagy were activated by HSYA. However, the AMPK inhibitor, CC, blocked the effects of HSYA on autophagy and NLRP3 levels. Our findings indicated that the inhibitory effects of HSYA on NLRP3 inflammasome activation may be dependent on the AMPK pathway in I/R injury. Our study provided insight into a new mechanism that is responsible for the cardioprotective function of HSYA. Although reperfusion therapy is successful in preventing heart damage, which occurs when blood re-supply evokes an intense and highly specific inflammatory response, it mediates further myocardial damage and dysfunction. Increasing evidence has demonstrated that the NLRP3 inflammasome plays an essential role in detecting cell damage and regulating inflammatory response after myocardial I/R damage [3]. Activation of the NLRP3 inflammasome promotes the recruitment of ASC and caspase-1 activation, which contributes to the maturation and secretion of IL-1β as well as the promotion of pyroptotic cell death, leading to the increase of infarct size and myocardial dysfunction in I/R
3.3. HSYA inhibits H/R-induced NLRP3 inflammasome activation in H9c2 cells To evaluate the effects of HSYA on the NLRP3 pathway, we measured the level of NLRP3, the release of IL-1β into culture medium, and caspase-1 activity. As shown in Fig. 3, HSYA treatment decreased NLRP3 expression, and IL-1β levels and caspase-1 activity in H9c2 cells compared to the H/R group. 3
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Fig. 2. Effects of HSYA on H/R-induced apoptosis. (A) Effect of HSYA on mitochondrial transmembrane permeability transition. ΔΨm was assessed by fluorescence microscopy with JC-1 staining. (B) Scatter diagram of Annexin V–PI double staining as measured using flow cytometry. (C) Quantitative analysis of mitochondrial membrane potential (ratio of red fluorescence obtained at 590 nm to green fluorescence at 530 nm) in H9C2 cells. (D) Quantitative analysis of apoptosis rate. (E) Caspase-3 activity was measured using a fluorometric assay. HSYA, Hydroxysafflor yellow A. H/R, hypoxia/reoxygenation. Data are presented as the means ± SD from three independent experiments. #P < 0.05 versus control, ##P < 0.01 versus control; *P < 0.05 versus H/R-treated cells, **P < 0.01 versus H/R-treated cells.
Fig. 3. HSYA attenuates activation of the NLRP3 inflammasome and upregulates AMPK activation induced by H/R in H9c2 cells. (A) Western blot analysis was performed to assess the protein expression of NLRP3. β-actin expression was utilized as the protein loading control. (B) IL-1β content in culture supernatant was detected by ELISA. (C) Caspase-1 activity was measured by caspase-1 activity assay kit. HSYA, Hydroxysafflor yellow A. H/R, hypoxia/reoxygenation. Data are presented as the means ± SD from three independent experiments. #P < 0.05 versus control, ##P < 0.01 versus control; *P < 0.05 versus H/R-treated cells, **P < 0.01 versus H/R-treated cells. 4
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Fig. 4. AMPK inhibitor (compound c) blocks the suppressive effect of HSYA on the NLRP3 pathway in cardiomyocytes. H9c2 cells were pretreated with CC or CC + HSYA before exposure to H/R. (A) Expression levels of p-AMPK and AMPK. (B) Caspase-1 activity. (C) Expression levels of NLRP3, and IL-1b in cell lysates were determined by Western blotting. (D) Expression levels of LC3 and p62 in H9C2 cells were assayed by Western blot analysis. β-actin expression was utilized as the protein loading control. HSYA, Hydroxysafflor yellow A. H/R, hypoxia/reoxygenation. CC, compound c. Data are presented as the means ± SD from three independent experiments. #P < 0.05 versus control, ##P < 0.01 versus control; *P < 0.05 versus H/R-treated cells, **P < 0.01 versus H/R-treated cells. & P < 0.05 versus H/R + HSYA-treated cells, and &&P < 0.01 versus H/R + HSYA-treated cells.
Among several upstream elements affecting NLRP3 inflammasome under I/R, we investigated the pathway associated with HSYA. AMPK functions as a cell energy sensor and controls a variety of pathophysiological mechanisms, such as autophagy, apoptosis and inflammation [26]. Activation of the AMPK signalling pathway during myocardial I/R has been considered to be an endogenous compensatory mechanism to protect against myocardial injury [27]. Previous studies have shown that hispidulin protects against cerebral I/R injury through suppressing the NLRP3 inflammasome by modulating the AMPK signalling pathway [28]. In this study, HSYA increased the ratio of P-AMPK/T-AMPK concomitant with the inhibition of the NLRP3 inflammasome in H/Rinduced cardiomyocytes. However, compound c, an inhibitor of AMPK, suppressed HSYA-induced inhibition of the NLRP3 inflammasome, indicating the essential role of AMPK signalling in regulating inflammasome activity. In addition, accumulating evidence has indicated that autophagy decreases NLRP3 inflammasome levels via the AMPK signalling
injury [24]. Similarly, our research confirmed that H/R remarkably increases NLRP3 expression and apoptosis in cardiomyocytes. In addition, previous studies have reported the beneficial effects of strategies blocking the activation of the NLRP3 inflammasome in acute myocardial infarction [25], suggesting a potential therapeutic target for preventing myocardial damages. HSYA is a monomer compound extracted from the flower of the safflower plant (Carthamus tinctorius L.). The protective efficacy of HSYA against myocardial I/R injury has been validated previously [9]. However, the exact underlying mechanisms of HSYA remain largely unknown, and evidence is lacking regarding its association with inflammasome regulation. In this study, we demonstrated that treatment with HSYA decreased NLRP3 protein expression, cleaved caspase-1 protein expression and IL-1β secretion, suggesting that HSYA may mitigate H/R-induced inflammation in H9c2 cells. These results suggested that HSYA protects myocardial cells against H/R injury via mitigation of NLRP3 inflammasome activation. 5
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pathway [23]. Although HSYA has been shown to inhibit apoptosis, it remains unclear if autophagy is involved in HSYA-triggered cardioprotection. Microtubule-associated protein 1 light chain 3 (LC3) is known as a key marker for autophagosome. LC3 exists in two forms: LC3-I is cytosolic, whereas LC3-II is membrane bound. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes. Therefore, the expression level of LC3-II can be used to represent the volume of autophagy [29]. Besides, p62 can interact with LC3 on autophagic membranes and make contact with ubiquitin, playing a critical role in autophagy through p62 degradation by the autophagy-lysosome [30]. Interestingly, our data confirmed that autophagy is activated by HSYA as manifested by an increased ratio of LC3-II/LC3-I and decreased p62 level in the H/R group. Our study also showed that AMPK inhibition counteracts HSYA-induced autophagy activation. Previous research has shown that autophagy is enhanced in H9c2 cardiomyocytes following AMPK/mTOR activation and has protective effects in cells during H/R injury [31]. Our finding agreed with that of previous studies, showing that AMPK-regulated autophagy in cardiomyocytes actively participates in H/R injury. Therefore, our findings indicated that HSYA possesses cardioprotective effects at least in part by activating AMPK/autophagy and subsequently inhibiting the NLRP3 inflammasome in myocardial injury. Collectively, our findings suggested that HSYA suppresses activation of the NLRP3 inflammasome via the AMPK pathway in H/R-induced cardiomyocytes. Our study provides insights into the therapeutic mechanisms of HSYA and the novel therapeutic agents targeting the NLRP3 inflammasome during MI.
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Funding This work was supported by the National Natural Sciences Foundation of China (Grant No. 8167141813), the Natural Sciences Foundation of Beijing (Grant No. 7172191), the State Administration of Traditional Chinese Medicine of the People’s Republic of China (Grant No. ZYBZH-C-JIN-44), and the CAMS Innovation Fund for Medical Sciences (CIFMS) (Grant No. 2016-I2M-1-012). CRediT authorship contribution statement Jing-xue Ye: Conceptualization, Investigation, Validation. Min Wang: Writing - original draft. Rui-ying Wang: Investigation, Formal analysis. Hai-tao Liu: Investigation. Yao-dong Qi: Investigation. Jianhua Fu: Resources. Qiong Zhang: Conceptualization, Funding acquisition. Ben-gang Zhang: Supervision. Xiao-bo Sun: Writing - review & editing. Declaration of Competing Interest The authors declare no conflicts of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2020.106316. References [1] V. Sharma, R.M. Bell, D.M. Yellon, Targeting reperfusion injury in acute myocardial infarction: a review of reperfusion injury pharmacotherapy, Expert Opin. Pharmacother. 13 (8) (2012) 1153–1175. [2] Z. Guo, S. Yu, X. Chen, R. Ye, W. Zhu, X. Liu, NLRP3 Is Involved in Ischemia/ Reperfusion Injury, CNS Neurol. Disord.: Drug Targets 15 (6) (2016) 699–712. [3] Z. Meng, M.Y. Song, C.F. Li, J.Q. Zhao, shRNA interference of NLRP3 inflammasome alleviate ischemia reperfusion-induced myocardial damage through autophagy activation, Biochem. Biophys. Res. Commun. 494 (3–4) (2017) 728–735. [4] X. Zhang, Q. Du, Y. Yang, J. Wang, S. Dou, C. Liu, J. Duan, The protective effect of Luteolin on myocardial ischemia/reperfusion (I/R) injury through TLR4/NFkappaB/NLRP3 inflammasome pathway, Biomed. Pharmacother. 91 (2017)
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