Sanguinarine attenuates hypoxia/reoxygenation-triggered H9c2 cell injury through activation of the Nrf2/NLRP3 pathway

Abstract

Myocardial ischemia/reperfusion injury (MI/RI) is a prevalent condition encountered by many patients with ischemic heart disease, which can badly influence the health of patients and even do harm their lives. Sanguinarine (SA), one active ingredient separated from the poppy family, and exhibits anti-oxidant, anti-tumor, and anti-inflammation properties. However, the precise regulatory impacts and associated mechanisms of SA in the progression of MI/RI remain largely elusive. In this study, firstly, H9c2 cells were treated by hypoxia/reoxygenation (HR) to mimic MI/RI cell model. It was uncovered that SA strengthened HR-mediated cell viability of H9c2 cells. Following HR treatment, there was an increase in the production of inflammatory markers (TNF-α, IL-1β, and IL-6), whereas this effect was mitigated after SA treatment. The oxidative stress was heightened after HR treatment, but this phenomenon was offset after SA treatment. SA activated the Nrf2/NLRP3 pathway and relieved proptosis. At last, through rescue assays, it was demonstrated that SA improved HR-triggered inflammation and oxidative stress through Nrf2 pathway. SA also modulated HR-triggered cell viability, inflammation, and oxidative stress in rat primary cardiomyocytes. In summary, our findings indicate that SA protects against HR-induced H9c2 cell injury through activation of the Nrf2/NLRP3 pathway. This discovery suggests that SA may be one helpful drug for ameliorating MI/RI.

Introduction

Ischemic heart disease is the major reason for global death, approximately 16%.⁽¹⁾ Timely reperfusion is pivotal for relieving the extent of infarction and improving ischemic myocardium.⁽²⁾ However, during reperfusion, one phenomenon may be happened, which is called myocardial ischemia/reperfusion injury (MI/RI).⁽³⁾ MI/RI exhibits higher morbidity and mortality.⁽⁴⁾ Recently studies have illustrated that diversiform pathological changes participate into the pathogenesis of MI/RI, such as mitochondrial dysfunction, inflammation, cell apoptosis, and oxidative stress.^(5,6) Therefore, preventing and mitigating MI/RI have become the key point for clinical and basic researches, and searching effective therapeutic drugs is set as the top priority.

Natural products have become one key source for existing drugs, and own important roles in ameliorating MI/RI progression.⁽⁷⁾ For example, Berberine modulates the RhoE/AMPK pathway to weaken excessive autophagy, thereby ameliorating MI/RI progression.⁽⁸⁾ In addition, Baicalin retards the JAK/STAT pathway to affect macrophages polarization, thereby alleviating MI/RI.⁽⁹⁾ Moreover, Tanshinone IIA strengthens miR-223-5p to heighten the therapeutic effect of exosomes for MI/RI.⁽¹⁰⁾

Sanguinarine (SA), one natural alkaloid, owns benzophenanthridine polycycle skeleton, and is principally derived from the poppy family.⁽¹¹⁾ SA has attracted vast interests from researchers due to its wide and obvious biological activities, including anti-oxidant, anti-tumor, and anti-inflammation properties. For instance, SA can relieve inflammation and owns neuroprotective functions in cerebral ischemia.⁽¹²⁾ Furthermore, SA affects the nuclear factor E2-related factor 2 (Nrf2)/NF-κB pathway to improve indomethacin-triggered small intestine injury.⁽¹³⁾ In nasopharyngeal carcinoma, SA retards mTOR signaling to alleviate cell proliferation and invasion.⁽¹⁴⁾ Besides, SA owns protective effects on ovariectomy-mediated osteoporosis.⁽¹⁵⁾ Importantly, it has been discovered that SA retards the TLR4/NF-κB pathway to mitigate lipopolysaccharide-stimulated inflammation and apoptosis in H9c2 cardiomyocytes.⁽¹⁶⁾ Besides, SA suppresses NF-κB activation to ameliorate pressure overload-triggered cardiac remodeling.⁽¹⁷⁾ However, the regulatory impacts and associated pathways of SA in the progression of MI/RI remain unknown.

In this work, it was disclosed that SA attenuated HR-triggered H9c2 cell injury through activation of the Nrf2/NLRP3 pathway. This work may offer novel opinions of SA on improving MI/RI progression.

Materials and Methods

Cell lines and treatment

H9c2 cells were obtained from ATCC (Manassas, VA), and incubated in DMEM (10% fetal bovine serum) under one incubator (37°C, 5% CO₂). To establish MI/RI cell model, H9c2 cells were subjected to low-oxygen (95% N₂, 5% CO₂) for 6 ‍h, and then under normal-oxygen (95% air, 5% CO₂) for reoxygenation 12 ‍h. In the Control group, H9c2 cells were kept into the normal incubator condition (37°C, 5% CO₂).

Primary cardiomyocytes were taken from neonatal Sprague Dawley (SD) rats (1–3 day old) as reported previously.⁽¹⁸⁾ After being sterilized by 75% ethanol, neonatal rats were decapitated, then the collected hearts were cut and digested with collagenase. Next, cells were put in DMEM for incubation, and then centrifuged. Cells were put into culture flasks, and nonmyocytes were depleted through using bromodeoxyuridine for 48 ‍h. Cardiomyocytes were transferred into DMEM for another 24 ‍h incubation at 37°C with 5% CO₂.

SA (0, 0.25, 0.5, 1, 2, and 4 ‍μM; Shanghai Selleck Chemicals Co., Ltd., Shanghai, China) was utilized to treat H9c2 cells or primary cardiomyocytes.

Small interfering RNA (siRNA) targeting Nrf2 (si-Nrf2) and its negative control (si-NC) purchased from GenePharma (Shanghai, China) were transfected into H9c2 cells or primary cardiomyocytes through Lipofectamine 2000 (Invitrogen, Waltham, MA).

Cell counting kit-8 (CCK-8) assay

In the 96-well plate, H9c2 cells or primary cardiomyocytes (1,000 cells/well) were put into. Next, each well was appended with CCK-8 solution (10 ‍μl; Dojindo Laboratories, Kumamoto, Japan). After 4 ‍h incubation, the absorbance (450 ‍nm) was inspected under one one Microplate Reader (Bio-Rad, Hercules, CA).

Detection of LDH

In the 96-well plate, H9c2 cells were put into. The LDH assay kit (Beyotime, Shanghai, China) was utilized for measuring the LDH level in the medium according to the manufacturer’s specifications. The absorbance (490 ‍nm) was determined under one Microplate Reader (Bio-Rad).

Flow cytometry

In the dark, staining through 5 ‍μl FITC Annexin V (BD Biosciences, Franklin Lakes, NJ) and 5 ‍μl propidium iodide (PI) was performed for H9c2 cells. At last, cell apoptosis was confirmed under flow cytometry (BD Biosciences).

RT-qPCR

The RNAs from H9c2 cells were got through TRIzol reagent (Invitrogen). The transcription of RNA to obtain cDNA was made under the SuperScript^TM II Reverse Transcriptase Kit (Invitrogen). Next, qRT-PCR was carried out under the SYBR Premix Ex Taq^TM Kit (Takara, Shanghai, China). Finally, through the 2^−∆∆Ct method, the mRNA expressions were assessed.

The primer sequences: TNF-α: F: 5'-AGCCGATGGGTTGTACCT-3', R: 5'-TGAGTTGGTCCCCCTTCT-3'; IL-1β: F: 5'-CCAGCTTCAAATCTCACAGCAG-3', R: 5'-CTTCTTTGGGTATTGCTTGGGATC-3'; IL-6: F: 5'-TCCAGTTGCCTTCTTGGGAC-3', R: 5'-GTACTCCAGAAGACCAGAGG-3'; GAPDH: F: 5'-CTGGGCTACACTGAGCACC-3', R: 5'-AAGTGGTCGTTGAGGGCAATG-3'.

ELISA

The commercial ELISA kits of interleukin-6 (IL-6, ab234570; Abcam, Shanghai, China), tumor necrosis factor-α (TNF-α, ab46070) and IL-1β (ab255730) were adopted for measurement.

Detection of ROS

The reactive oxygen species (ROS) fluorescence intensity was evaluated through the ROS kit (E004-1-1; Nanjing Jiancheng Technology Co., Ltd., Nanjing, China) Dichloro-dihydro-fluorescein diacetate (DCFH-DA) and H9c2 cells were mixed for 20 ‍min. Post washing, the ROS fluorescence intensity was determined under the fluorescence microscope (Olympus, Tokyo, Japan).

The mitochondrial ROS level in rat primary cardiomyocytes was evaluated through MitoSOX staining. After washing, primary cardiomyocytes were mixed with 1 ‍μM MitoSOX^TM Red reagent (Cat. No. M36008; Thermo Fisher Scientific, Waltham, MA) for 0.5 ‍h. Next, cardiomyocytes were trypsinized and suspended. Finally, one fluorescence microscope (Olympus) was employed to examine the fluorescent intensity of MitoSOX.

Detection of MDA, SOD and GSH-Px

The commercial kits of malondialdehyde (MDA, ab118970; Abcam), superoxide dismutase (SOD, ab65354) and glutathione (GSH, ab65322) were adopted to examine the levels of MDA, SOD, and GSH in supernatant.

Western blot

The proteins from H9c2 cells or primary cardiomyocytes were got through using the RIPA buffer (Beyotime). The separation of these proteins was done through SDS-PAGE (10%). Afterwards, the metastasis of proteins was made into PVDF membranes (Beyotime). Post blocking (non-fat milk), membranes were appended with the primary antibodies against Nrf2 (1:500; ab313825; Abcam), NLRP3 (1:1,000; ab263899), ASC (1:1,000; ab309497), cleaved caspase1 (1 ‍μg/ml; ab286125), mature IL-18 (0.5 ‍μg/ml; ab191860), mature IL-1β (1:1,000; ab283818), GSMDM-N (1:1,000; ab215203), p-p65 (1:1,000; ab76302), p65 (1:2,000; ab32536), Keap1 (1:500; ab119403), and β-actin (1:2,000; ab8227). After 12 ‍h incubation, the appropriate secondary antibodies (1:1,000; ab7090) were further appended. Eventually, after 2 ‍h, the chemiluminescence detection kit (Thermo Fisher Scientific) was employed for inspecting the protein bands.

Statistical analysis

All results were displayed as mean ± SD with 3 repetitions. Statistical analysis was dealt with GraphPad Prism Software 9 (GraphPad Software, San Diego, CA). The comparisons were made under the one-way analysis of variance (ANOVA). The p<0.05 was deemed as statistically significant.

Results

SA strengthened HR-mediated cell viability of H9c2 cells

The cell viability of H9c2 cells was not changed after SA treatment with concentrations at 0.25, 0.5, and 1 ‍μM, but was reduced after SA treatment with concentrations at 2 and 4 ‍μM (Fig. 1A). Furthermore, cell viability was weakened after HR treatment, but this effect was reversed after SA treatment (0.5 and 1 ‍μM) (Fig. 1B). The LDH level was increased after HR treatment, but this change was offset after SA addition (Fig. 1C). Additionally, cell apoptosis was heightened after HR treatment, but this impact was attenuated after SA treatment (0.25, 0.5, and 1 ‍μM) (Fig. 1D). To sum up, SA strengthened HR-mediated cell viability of H9c2 cells.

Fig. 1.

Sanguinarine strengthened HR-mediated cell viability of H9c2 cells. (A) The cell viability of H9c2 cells was examined through CCK-8 assay with different concentrations of SA (0, 0.25, 0.5, 1, 2, and 4 ‍μM). (B) The cell viability was tested through CCK-8 assay. Groups were separated into the Control, HR, HR + SA (0.25 ‍μM), HR + SA (0.5 ‍μM), and HR + SA (1 ‍μM) group. (C) The LDH level was confirmed through the LDH kit. (D) The cell apoptosis was detected through flow cytometry. Groups were separated into the Control, HR, HR + SA (0.25 ‍μM), HR + SA (0.5 ‍μM), and HR + SA (1 ‍μM) group. ***p<0.001 vs the Control group; ^††p<0.01, ^†††p<0.001 vs the HR group.

SA alleviated HR-stimulated the generation of inflammatory factors

The mRNA expressions of inflammatory factors (TNF-α, IL-1β, and IL-6) were all elevated after HR treatment, but these changes were alleviated after SA treatment (Fig. 2A). Moreover, through ELISA, the levels of TNF-α, IL-1β, and IL-6 had the same changes (Fig. 2B). Taken together, SA alleviated HR-stimulated the generation of inflammatory factors.

Fig. 2.

Sanguinarine alleviated HR-stimulated the generation of inflammatory factors. Groups were separated into the Control, HR, HR + SA (0.25 ‍μM), HR + SA (0.5 ‍μM), and HR + SA (1 ‍μM) group. (A) The mRNA expressions of TNF-α, IL-1β, and IL-6 were confirmed through RT-qPCR. (B) The levels of TNF-α, IL-1β, and IL-6 were verified through ELISA. ***p<0.001 vs the Control group; ^†p<0.05, ^††p<0.01, ^†††p<0.001 vs the HR group.

SA attenuated HR-triggered oxidative stress

The ROS level was strengthened after HR treatment, but this phenomenon was relieved after SA treatment (Fig. 3A). The MDA level was enhanced as well as GSH and SOD levels were reduced after HR treatment, but these impacts were rescued after SA treatment (Fig. 3B). In a word, SA attenuated HR-triggered oxidative stress.

Fig. 3.

Sanguinarine attenuated HR-triggered oxidative stress. Groups were separated into the Control, HR, HR + SA (0.25 ‍μM), HR + SA (0.5 ‍μM), and HR + SA (1 ‍μM) group. (A) The ROS level was assessed through the ROS kit. (B) The levels of MDA, SOD, and GSH were evaluated through the commercial kits. ***p<0.001 vs the Control group; ^†p<0.05, ^††p<0.01, ^†††p<0.001 vs the HR group.

SA activated the Nrf2/NLRP3 pathway and relieved pyroptosis

The Keap1 protein expression was lifted after HR treatment, but this impact was offset after SA treatment (Fig. 4A). The protein expression of Nrf2 was declined and that of NLRP3 was risen after HR treatment, but these influences were counteracted after SA treatment (Fig. 4B). In addition, the protein expressions of ASC, cleaved-caspase1, mature IL-18, and mature IL-1β were all augmented after HR treatment, but these impacts were neutralized after SA treatment (Fig. 4C). In general, SA activated the Nrf2/NLRP3 pathway.

Fig. 4.

Sanguinarine activated the Nrf2/NLRP3 pathway and relieved pyroptosis. Groups were separated into the Control, HR, HR + SA (0.25 ‍μM), HR + SA (0.5 ‍μM), and HR + SA (1 ‍μM) group. (A) The protein expression of Keap1 was assessed through Western blot. (B) The protein expressions of Nrf2 and NLRP3 were tested through Western blot. (C) The protein expressions of ASC, caspase1, IL-18, and IL-1β were determined through Western blot. ***p<0.001 vs the Control group; ^††p<0.01, ^†††p<0.001 vs the HR group.

SA improved HR-triggered inflammation and oxidative stress through Nrf2 pathway

The declined Nrf2 protein expression and the risen NLRP3 protein expression mediated by HR treatment were reversed after SA treatment, but these changes were further offset after ML385 (Nrf2 inhibitor) treatment (Fig. 5A). The heightened levels of TNF-α, IL-1β, and IL-6 stimulated by HR treatment were attenuated after SA treatment, but these impacts were further reversed after ML385 addition (Fig. 5B). Furthermore, the enhanced MDA level as well as the reduced GSH and SOD levels were rescued after SA addition, but these influences were neutralized after SA treatment (Fig. 5C). Additionally, in control H9c2 cells, SA treatment cannot affect the Nrf2 protein expression (Fig. 5D). These data revealed that SA improved HR-induced inflammation and oxidative stress through Nrf2 pathway.

Fig. 5.

Sanguinarine improved HR-triggered inflammation and oxidative stress through Nrf2 pathway. Groups were separated into the Control, HR, HR + SA (1 ‍μM), and HR + SA (1 ‍μM) + ML385 group. (A) The protein expressions of Nrf2 and NLRP3 were inspected through Western blot. (B) The levels of TNF-α, IL-1β, and IL-6 were confirmed through ELISA. (C) The levels of MDA, SOD, and GSH were measured through the commercial kits. (D) The protein expression of Nrf2 was determined in the Control, SA (0.25 ‍μM), SA (0.5 ‍μM), and SA (1 ‍μM) group through Western blot. ***p<0.001 vs the Control group; ^†p<0.05, ^††p<0.01, ^†††p<0.001 vs the HR group; ^#p<0.05, ^##p<0.01, ^###p<0.001 vs the HR + SA (1 ‍μM) group.

SA regulated MI/RI progression through Nrf2 pathway

The weakened cell viability mediated by HR treatment was offset after SA treatment, but this change was further reversed after Nrf2 suppression (Fig. 6A). The lifted LDH level triggered by HR treatment was attenuated after SA treatment, but this impact was further rescued after Nrf2 knockdown (Fig. 6B). Furthermore, the enhanced MDA level as well as the reduced GSH and SOD levels stimulated by HR treatment were reversed after SA addition, but these influences were counteracted after SA treatment (Fig. 6C). In general, SA regulated MI/RI progression through Nrf2 pathway.

Fig. 6.

Sanguinarine regulated Nrf2 pathway to affect MI/RI progression. Groups were separated into the Control, HR, HR + SA (1 ‍μM), and HR + SA (1 ‍μM) + si-Nrf2 group. (A) The cell viability was confirmed through CCK-8 assay. (B) The LDH level was evaluated through the LDH kit. (C) The levels of MDA, SOD, and GSH were tested through the commercial kits. ***p<0.001 vs the Control group; ^†p<0.05, ^††p<0.01, ^†††p<0.001 vs the HR group; ^#p<0.05, ^##p<0.01, ^###p<0.001 vs the HR + SA (1 ‍μM) group.

SA modulated HR-triggered cell viability, inflammation and oxidative stress in rat primary cardiomyocytes

The cell viability was declined after SA treatment with concentrations at 0.25 and 0.5 ‍μM, but was reduced after SA treatment with concentrations at 1, 2, and 4 ‍μM (Supplemental Fig. 1A*). Next, the declined cell viability stimulated by HR was reversed after SA treatment (0.5 ‍μM) (Supplemental Fig. 1B*). The aggrandized levels of TNF-α, IL-1β, and IL-6 triggered by HR were offset after SA treatment (Supplemental Fig. 1C*). Additionally, the augmented oxidative stress mediated by HR was attenuated after SA treatment (Supplemental Fig. 1D*). Moreover, the MitoSOX fluorescence intensity was fortified after HR treatment, but this impact was neutralized after SA treatment, indicating that SA receded the mitochondrial ROS level (Supplemental Fig. 1E*). The decreased Nrf2 protein expression and the increased NLRP3 protein expression evoked by HR treatment were rescued after SA treatment (Supplemental Fig. 1F*). Furthermore, the up-regulated expressions of ASC, GSDMD-N, and cleaved-caspase 1/pro-caspase 1 triggered by HR treatment were lessened after SA treatment (Supplemental Fig. 1G*). The strengthened mature IL-18 and mature IL-1β stimulated by HR were attenuated after SA treatment (Supplemental Fig. 1H*). Besides, the risen p-p65/p65 level mediated by HR was counteracted after SA treatment (Supplemental Fig. 1I*). These data illuminated that SA also modulated HR-triggered cell viability, inflammation, and oxidative stress in rat primary cardiomyocytes.

Discussion

SA exhibits ameliorative properties in various diseases.^(12–17) However, the regulatory impacts and associated pathways of SA in the progression of MI/RI keep dimness. In this study, firstly, it was uncovered that SA strengthened HR-mediated cell viability of H9c2 cells.

Inflammation and oxidative stress are pivotal processes to participate into MI/RI progression, and have attracted more and more investigations from researchers. For instances, phyllanthin relieves oxidative stress and inflammation to against MI/RI mice.⁽¹⁹⁾ Aucubin affects the STAT3/NF-κB/HMGB-1 pathway to alleviate inflammation and oxidative stress in MI/RI.⁽²⁰⁾ Moreover, sevoflurane modulates miR-99a/BRD4 axis to improve inflammation and oxidative stress in MI/RI.⁽²¹⁾ Additionally, miR-30e targets SOX9 to weaken inflammation and oxidative stress as well as accelerate ventricular remodeling via repression.⁽²²⁾ Similar to these previous studies, in this work, it was demonstrated that the generation of inflammatory factors (TNF-α, IL-1β, and IL-6) was strengthened after HR treatment, but this change was alleviated after SA treatment. The oxidative stress was heightened after HR treatment, but this phenomenon was offset after SA treatment.

Pyroptosis is a newly notarized inflammatory form of lytic programmed cell death.⁽²³⁾ During pyroptosis, Gasdermin D (GSDMD) can serve as an executor that trigger plasma membrane rupture.⁽²⁴⁾ Inflammatory caspases can cleave GSDMD and liberate its N-terminal cell death domain (GSDMD-NT).⁽²⁵⁾ Next, GSDMD-NT monomers aggregate in the plasma membrane to form pores, accelerating the secretion of IL-1β and IL-18, stimulating the inflammatory response, and ultimately resulting into cell death.⁽²⁶⁾ Pyroptosis mediated by NLRP3 inflammasome pathway aggravates the progression of ischemic heart disease, and is one indispensable factor in exacerbating MI/RI.⁽²⁷⁾ It has been affirmed that the Nrf2 pathway can restrain NLRP3-mediated pyroptosis in MI/RI.⁽²⁸⁾ Nrf2 is a transcription factor, and cardiomyocytes may experience oxidative stress under hypoxia-reoxygenation conditions, which can lead to the decreased Nrf2 activity.⁽²⁹⁾ Nrf2 pathway is a vital pathway to participate into the progression of MI/RI. For instance, Catalpol stmulates the Nrf2/HO-1 pathway to improve MI/RI progression.⁽³⁰⁾ In addition, Fucoxanthin affects the NRF2 signaling pathway to suppress ferroptosis in MI/RI.⁽³¹⁾ Moreover, Ginsenoside Rh2 modulates the Nrf2/HO-1/NLRP3 pathway to ameliorate MI/RI.⁽³²⁾ In this study, it was discovered that SA activated the Nrf2/NLRP3 pathway and relieved pyroptosis. At last, through rescue assays, it was demonstrated that SA improved HR-triggered inflammation and oxidative stress through Nrf2 pathway. SA also modulated HR-triggered cell viability, inflammation and oxidative stress in rat primary cardiomyocytes.

In conclusion, SA attenuated HR-triggered H9c2 cell injury through activation of the Nrf2/NLRP3 pathway. Nevertheless, in this study, some limitations still exist (such as lacking human samples, animal model, and other phenotypic explorations). Deeply investigations for SA in MI/RI progression will be made in the future.

Author Contributions

All authors contributed to the study conception and design. Material preparation and the experiments were performed by BQ. Data collection and analysis were performed by XL. The first draft of the manuscript was written by WW and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Availability of Data and Materials

All data generated or analyzed during this study are included in this published article.

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Conflict of Interest

No potential conflicts of interest were disclosed.