Costunolide Protects Myocardial from Ischemia Reperfusion Injury through Nrf2 Activation

doi:10.21203/rs.3.rs-1857993/v1

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Costunolide Protects Myocardial from Ischemia Reperfusion Injury through Nrf2 Activation

https://doi.org/10.21203/rs.3.rs-1857993/v1

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Purpose

Costunolide (Cos) is a naturally occurring sesquiterpene lactone that exhibits anti-oxidative properties. In this study, we demonstrated the protective mechanism of Cos against ischemia/reperfusion (I/R)-induced heart injury.

Methods

C57BL/6 mice were pretreated with Cos (10 mg/kg/day) or 1% CMC-Na for 1 week. The left anterior descending coronary artery (LAD) was ligated for 30 minutes for ischemia, and followed by ligation release for 4 hours for reperfusion. H9c2 cells challenged with tert-butyl hydroperoxide (TBHP) were used for in vitro studies.

Results

Pretreatment of Cos significantly reduced myocardial infarct size, serum CK-MB and LDH level in I/R injured heart. Cos administration significantly attenuated the amount of reactive oxygen species (ROS) and ameliorated the apoptosis both in in vitro and in vivo studies. Further investigation revealed that Cos significantly increased the expression of heme oxygenase 1 (HO-1) and NAD(P)H [quinone] dehydrogenase 1 (NQO-1). Meanwhile Cos increased B-cell lymphoma-2(Bcl-2) and decreased the BCL2-Associated X Protein (Bax) protein level. Silence of nuclear factor erythroid 2-related factor (Nrf2) significantly reverses the protective effect of Cos in TBHP-challenged H9C2 cells.

Conclusion

Our data clearly showed that Cos reduced TBHP challenged H9c2 cells and attenuated myocardial I/R injury through Nrf2 activation. These results indicate that Cos might be benefit in the therapy of myocardial I/R injury.

Cardiac ischemia reperfusion injury

Costunolide

cardiomyocytes

reactive oxygen species

Nrf2

Cardiovascular diseases are the primary factor of death worldwide [1], and myocardial infarction is especially harmful to the health of the people. Cardiac ischemia reduces oxygen and nutrient supply to heart tissues, leading to heart dysfunction and myocardial cell necrosis [2]. Early reperfusion treatment during myocardial ischemia is important for saving the myocardium; however, this strategy can also cause further injury in myocardial cells, known as myocardial ischemia reperfusion injury [3, 4].

The burst of ROS in the myocardial, initiated during ischemia and exacerbated upon reperfusion, is thought to be the main cause of the heart injury [5, 6]. Reperfusion after thrombosis induces the overproduction of ROS by mitochondria, which damages cardiomyocytes [7]; an antioxidant strategy is reported to have beneficial effects in decreasing such I/R injury. Various endogenous antioxidases such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) can eliminate oxidation produces and help decrease the oxidative stress-induced damage [8, 9]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that exist in a variety of cells. Nrf2 promotes many protective genes expression in the maintenance of cellular homeostasis. In the stable state, Nrf2 is anchored by the Keap1-Cul3 ubiquitin E3 ligase complex in the cytoplasm. Under oxidative stress, Nrf2 can quickly separate from the Keap1-Nrf2 complex, transfer to the nuclear, and eventually combine with the antioxidant response elements (AREs) to promote the synthesis of antioxidant enzymes [10].

Costunolide (Cos) is found to be extracted from various medicinal plants, including Costus speciosus [11], Saussurea lappa, and Laurus nobilis [12, 13]. Cos is distinguished by its ability to exhibit various biological effects [14], including anti-cancer [15], antifungal, antimicrobial [16], anti-inflammatory [17], and antioxidant effects [18]. It is known that Cos exerts cytotoxic on various human cancer cells, including human prostate cancer [19], human breast carcinoma [20], renal cell carcinoma [15], and chronic myeloid leukemia [21]. It has been reported that Cos can enhance the chemotherapy effect in the cure of multidrug resistance cancer [22]. A study in our laboratory indicated that Cos can directly bounds to and inhibited the activity of thioredoxin/thioredoxin reductase 1 (TrxR1); thus, Cos administration increases ROS level and activates endoplasmic reticulum stress in colon cancer cells l [23]. In another study, Cos directly targeted AKT and inhibited colon cancer cell growth [24]. Contrarily, a C. speciosus rhizome methanolic extract can significantly clear oxygen free radical and nitric oxide (NO) produces in vitro [25]. In an in vivo experiment, it was found that gavage of Cos in a diabetes rat significantly increased heart glutathione (GSH) content and the activities of SOD [26].

In this study, Cos exhibits protective capability against myocardial I/R injury in vivo and reduces tertiary butyl hydrogen peroxide (TBHP) caused cell apoptosis in vitro. Furthermore, the underlying mechanism of antioxidation by Cos in the signaling pathways responsible for cell apoptosis was elucidated.

Reagents and cell culture

Chemicals. Tert-butyl hydroperoxide (TBHP, cat# A10777) was purchased from XIYA Reagent (Chengdu, Sichuang, China). 2,3,5-triphenyltetrazolium chloride (TTC cat# T8877) and Costunolide (cat# 553-21-9) were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against Lamin B1(cat# SC-374015), GAPDH (Cat# sc-365062), Bax (cat# SC-7480), Bcl2 (cat# SC-7382), NQO-1 (cat# SC-376023) and HO-1 (cat# SC-390991) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against Cleaved Caspase-3(cat# 9664) and Nrf2(cat#12721) were purchased from Cell Signaling Technology (Massachusetts, United States).

Cell culture

H9c2 embryonic rat heart-derived cardiomyocyte line was purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). H9c2 Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco/BRL life Technologies, Eggenstein, Germany) containing 4.5g/L D-glucose. The media was supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 100 U/mL of penicillin, and 100 mg/mL of streptomycin.

Mice and animal experiments

All of the experimental protocols and animal care procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee. (Approval Document No. wydw2021-0124). Six-Seven weeks old male C57BL/6 mice weighting 20–22g were obtained from the Shanghai Laboratory Animal Research Center (Shanghai, China). All mice received humane care according to NIH Guide for the Institutional Animal Care and Use Committee (IACUC). Cos was dissolved in 1% CMC-Na and was given at a dose of 10 mg/kg/day for 7 days by gavage. Mice were given CMC-Na (1%) as vehicle. The mice were anesthetized by 1% pentobarbital sodium (50mg/kg) by intraperitoneal injection before LAD I/R surgical process. The myocardial ischemia reperfusion injury model in C57BL/6 mice was induced as described in our previous study[27].

Myocardial infarct size measurement

The in vivo myocardial infarct size was evaluated using Evans Blue/TTC dual dyeing. Briefly, after retied the LAD, 1% Evans blue dye was injected into the left ventricle. Then we sliced the heart and incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, St. Louis, MO) for 15 minutes. The sliced myocardium was dyed with blue, red and white. The images were taken by a microscopy system (Nikon TE2000-S). The infarct size was calculated use image J as described previously [27].

TUNEL staining

The apoptotic cells in the heart were detected by TUNEL Apoptosis Assay Kit (cat# C1086, Beyotime, China) according to the manufacturer's instruction. For H9c2 cells, cells were fixed by 4% paraformaldehyde, incubated with 0.25% Triton-X100 in PBS, then apoptotic cells were detected with TUNEL Apoptosis Assay Kit (cat# C1086, Beyotime, China).

Heart superoxide production

The frozen slices of the heart were incubated with DHE for 45 min. The images were viewed under the fluorescence microscope (Nikon, Japan). The cellular hydrogen peroxide was detected using 2’,7’- dichlorodihydrofluorescein (DCF) staining.

Determination of MDA and SOD

H9C2 cells or heart tissue were homogenized with sample buffer, and the serums were diluted with sample buffer. The superoxide dismutase (SOD) levels in sample were determined using commercially available kits (cat# S0101S, Beyotime Biotech, Nantong, China) according to the manufacturer's instructions. The malondialdehyde (MDA) were determined using commercially available kits (cat# S0131S, Beyotime Biotech, Nantong, China).

Measurement of CK-MB and LDH

The serum was collected and the CK-MB, and LDH were measured using commercially available assay kits (cat# H197-1-1 and cat# A020-2-2, Jiancheng, China).

Rhodamine-labeled phalloidin staining

To assess cardiomyocyte hypertrophy, H9c2 cells were fixed, permeabilized, then incubated with Rhodamine-labeled phalloidin for 30 min.

Nrf2 silencing by siRNA

Nrf2 was silenced in H9C2 cells by siRNA transfections. siRNA for Nrf2 was designed and synthesized by Gene Pharma Co. (Shanghai, China). siRNA sequences (5’to 3’), GGGUAAGUCGAGAAGUGUUTT and AACACUUCUCGACUUACCCTT, were used. Cells were transfected using Lipofectamine™ 2000 (Invitrogen Ltd., Carlsbad, CA).

Western blot analysis

Lysates from cultured cells or mouse heart tissues were prepared. Samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electro-transferred to nitrocellulose membranes. Membranes were blocked for 2 hour at room temperature in Tris-buffered saline (pH 7.6) containing 0.05% Tween 20 and 5% non-fat milk. Each nitrocellulose membrane was incubated with specific antibodies at 4°C overnight. Secondary antibodies were applied for 1 h at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories). The amounts of the proteins were analyzed using Image J, and normalized to their respective control.

Statistical analysis

All experiments were randomized and blinded, date presented in this study is representative of at least 3 independent experiments and is expressed as Mean ± SEM. Statistical analysis was calculated with GraphPad Prism 8.0 software (San Diego, CA, USA). Differences between groups were examined using the 2-tailed Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test. Details of each experiment can be found in the figure legend. Differences were considered significant at p < 0.05.

Cos limits I/R-induced heart injury

Cos, a natural sesquiterpene lactone (Fig. 1A), has been known to show powerful antioxidative properties. To investigate whether Cos is protective in a hypoxic myocardium, we examined its effect in the in vivo cardiac ischemia reperfusion injury model. C57BL/6 mice were pretreated with Cos or 1% CMC-Na for 1 week. The model of I/R was processed with LAD ligation for 30 minutes, and followed by ligation release for reperfusion. After retied of LAD, the infarct area of the heart was assessed using Evans Blue and TTC dual staining. In Fig. 1B, I/R increased the infarct region, whereas the Cos administration notably reduced I/R-induced myocardial infarction. There is no statistically difference in the ratio of AAR/LV among the three groups as shown in Fig. 1C. While pretreatment of Cos for seven days significantly attenuated the infarct size compared to I/R group (Fig. 1D). Moreover, under myocardial ischemia reperfusion injury, the serum lactate dehydrogenase (LDH) level and creatine kinase M and B isoform (CK-MB) in the mice were significantly increased. Cos downregulated the LDH and CK-MB levels (Figs. 1E and 1F). As presented in Fig. 1G, compared with I/R group, Cos administration reduced the MDA content in the myocardium. Then we measured the SOD levels both in the serum and in the heart tissues. As shown in Fig. 1H, myocardial ischemia reperfusion injury reactively increased the serum SOD activity in both the I/R model group and Cos-pretreated group. Whereas in the myocardial tissue, the Cos-treated I/R group have a higher activity of SOD compare to the sham group. These results demonstrate that myocardial I/R damages the function of the heart, and Cos treatment can ameliorate such injury.

Cos attenuates I/R-induced heart apoptosis

Then we detected the protective effects of Cos on apoptosis in ischemia reperfusion injured mice. As shown in Fig. 2A-2B, the TUNEL-positive cells in the I/R model group were much more compare to the sham group. The apoptotic cells were less in the Cos-treated group than in the I/R model group, indicating that Cos treatment significantly reduced I/R-induced myocardial cell apoptosis. Then were further detected the expression of cleaved caspase-3, Bax, and Bcl-2 protein in each group (Figs. 2C and 2D). I/R mice treated with Cos showed significant downregulation of cleaved caspase-3 and Bax, and a significantly increase in Bcl-2 compared to the I/R group.

Cos reduces I/R-induced hear ROS production

As shown in Figs. 3A and 3B, ROS levels were increased in the I/R group, and Cos treatment decreased myocardial ROS levels under I/R injury. As illustrated in Figs. 3C and 3D, pretreatment with Cos did not increase the expression of Nrf2 protein in the I/R model myocardium, whereas it increased Nrf2 nuclear translocation. As a result, the anti-oxidant protein NQO-1 and HO-1 significantly increased (Figs. 3E and 3F). This further reduced ROS production in the heart of Cos treated mice.

Cos attenuates TBHP-induced apoptosis and ROS levels in H9C2 cells

To investigate the cytoprotective effect of Cos, the H9C2 was exposed to TBHP. As shown in Figs. 4A and 4B, the TUNEL staining indicated that Cos attenuated TBHP-induced cardiac cell apoptosis. In addition, Cos increased the expression of Bcl-2 (Fig. 4C). An increase of the Bax protein was observed in TBHP-treated H9C2 cells; however, Cos significantly reduced such Bax expression. Treatment with 2.5 µM Cos significantly decreased ROS production. To further investigate the antioxidative property of Cos, H9C2 cells were stimulated with TBHP for 24 h. The SOD activity and MDA level were detected. Increasing concentrations of Cos reduced the TBHP-induced MDA level in a dose-dependent manner (Fig. 4D) while maintaining the SOD activity (Fig. 4E). Since the increase in ROS levels is critical in TBHP-induced cardiomyocyte injury, we determined intracellular ROS levels in TBHP-induced H9C2 cells by detecting DCF-positive cells. As evident from Figs. 4F and 4G, DCF staining show that ROS production significantly raised in TBHP-treated group. As shown in Figs. 4H and 4I, pretreatment with Cos decreased TBHP-induced cell morphology changes in H9C2 cells. The results show that Cos pretreatment reduce the ROS levels and apoptosis induced by TBHP.

Cos activates Nrf2 and attenuates TBHP-induced apoptosis

As shown in Figs. 5A, the addition of Cos significantly increased Nrf2 nuclear translocation in TBHP-treated H9C2 cells. Cos also promoted the NQO-1 (Nrf2 target) protein levels in a dose-dependent manner in total cell lysate of TBHP-stimulated H9C2 cells (Fig. 5B). In Fig. 5C, Nrf2 levels were significantly reduced by Nrf2 siRNA transfection. Cos pretreatment decreased the level of the cleaved caspase-3, in TBHP-treated H9C2 cells, whereas Nrf2 silencing increased cleaved caspase-3 accumulation (Fig. 5D). Further analysis of the cell apoptosis pathway revealed that Nrf2 knockdown reversed the protective effect of Cos (Fig. 5E). Furthermore, we also analyzed that Nrf2 silencing reverses NQO-1 overexpression under Cos treatment, using western blotting (Fig. 5F). The changes in the morphology of H9C2 cells induced by Cos were examined using rhodamine-labeled phalloidin staining. As shown in Figs. 5G and 5H, Nrf2 siRNA transfection increased TBHP-induced injury despite the protection by Cos. These results indicate that Cos activates Nrf2 signaling pathway and protects myocytes from TBHP-induced apoptosis in vitro.

The purpose of this research was to expound the mechanism of Cos in protection of myocardial in I/R injured heart. Recent reports have emphasized that oxygen free radicals is the major cause of myocardial apoptosis in I/R injury, and that an antioxidant therapy is an effective approach to reduce cardiac injury [28]. As observed in our study, ischemia reperfusion significantly increased myocardial infarct size, serum CK-MB, ROS level and apoptosis in mice. Interestingly, treatment of Cos attenuated these abnormities, suggesting that Cos exerts myocardial protective activity in I/R mice. The protective effect of Cos was also observed in our in vitro study.

Acute overgeneration of ROS products under pathophysiological states is vital in the development of reperfusion injury after ischemia for thrombolysis or thrombus removal [29]. Mounting evidence indicates that oxidative stress is critical in myocardial apoptosis in the I/R damaged heart [30]. It has been reported that Cos administration decreased doxorubicin-induced cardiorenal injury by suppressing oxidative stress [31]. In another study, Cos attenuated alcohol-induced liver injury by reducing ROS production [32]. Interestingly, Cos obviously increases the intracellular oxidative stress in human U2OS cells and promotes the generation of ROS in esophageal cancer cells, and eventually induces apoptosis of carcinoma cells [33]. Studies have shown that Cos exerts anticancer activity, the diverse mechanism such as the reduction of angiogenic factor signaling pathway [34], inhibition of telomerase activity [20] and accumulation of intracellular ROS [15]. Pretreatment with Cos inhibited TBHP-induced Bax expression and increased the content of the a Bcl-2 in H9C2 cells.

It is known that elimination of oxygen free radicals is a practical strategy to prevent myocardial ischemia [35]. Our study showed that Cos decreased the ROS production induced by I/R injury or TBHP; however, the reason is unknown. As is reported that allicin active Nrf2 and protected the heart from Ang II induced cardiac hypertrophy [36]. Our previous study showed that curcumin and curcumin analogue 14p active Nrf2 pathway and attenuates I/R induced myocardial injury [27]. Nrf2 is the key transcription factor that regulates the antioxidant response, which stimulates the transcription of genes involved in many aspects of cytoprotection, such as those of glutamate cysteine ligase, SODs, HO-1, and NQO-1 [37, 38]. Our results showed that pretreatment with Cos before I/R injury or TBHP challenge did not change total Nrf2 expression but increased Nrf2 nuclear translocation and enhanced the expression NQO1 both in vitro and in vivo. This also indicates that Nrf2 is a feasible strategic target in therapies for myocardial I/R injuries. These results are consistent with previous reports indicating that Cos exerts neuroprotective effects by activating Nrf2 in PC12 cells; however, such prior research was only conducted in vitro [39].

To sum up, our study demonstrates the preventive role of Cos against oxidative stress and apoptosis induced by I/R or TBHP in vitro and in vivo, respectively. The protective mechanism of Cos is schematically shown in Fig. 6. Our results strongly suggest that Cos, a Nrf2 activator, has huge therapeutic potential in the therapy of myocardial ischemia reperfusion injury.

AAR, area at risk; AREs, antioxidant response elements; CAT, catalase; CK-MB, creatine kinase M and B isoform; DHE, dihydroethidium; DCF, 2’,7’-dichlorodihydrofluorescein; GPx, glutathione peroxidase; GSH, glutathione; HO-1, heme oxygenase 1, I/R, ischemia and reperfusion; LDH, lactate-dehydrogenase; MDA, methane dicarboxylic aldehyde; NQO-1, NAD(P)H [quinone] dehydrogenase 1; NO, nitric oxide; Nrf2, NF-E2-related factor 2; ROS, reactive oxygen species; SOD, super oxygen dehydrogenases; TBHP, tert-butyl hydroperoxide; TrxR1, thioredoxin/thioredoxin reductase 1

Authors’ Contributions Guang Liang and Wu Luo contributed to the literature search and study design. Guang Liang, Wu Luo, Siyuan Shen, and Chengyi Dai participated in the drafting of the article. Weixin Li, Yue Luo, Sirui Shen, and Zhuqi Huang carried out the experiments. Guang Liang, Wu Luo, and Weixin Li revised the manuscript. Wu Luo and Weixin Li contributed to data collection and analysis.

Funding This work was supported by the National Key Research Project of China (2017YFA0506000 to G.L.), National Natural Science Foundation of China (82000793 to W.L., and 81930108 to G.L.), Natural Science Foundation of Zhejiang Province (LY22H070004 to W.L.), Zhejiang Provincial Key Research Project (2018C03068 to G.L.), Wenzhou Scientific Project (Y20210213 to W.L.) and Wenzhou Key Scientific Project (2018ZY009 to G.L.).

Data Availability The used and/or analyzed datasets are available from the corresponding author on reasonable request.

Ethics Approval All of the experimental protocols and animal care procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee.

Conflict of Interest The authors declare no conflict of interest.

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Costunolide Protects Myocardial from Ischemia Reperfusion Injury through Nrf2 Activation

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Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Material And Methods

Reagents and cell culture

Cell culture

Mice and animal experiments

Myocardial infarct size measurement

TUNEL staining

Heart superoxide production

Determination of MDA and SOD

Measurement of CK-MB and LDH

Rhodamine-labeled phalloidin staining

Nrf2 silencing by siRNA

Western blot analysis

Statistical analysis

Results

Cos limits I/R-induced heart injury

Cos attenuates I/R-induced heart apoptosis

Cos reduces I/R-induced hear ROS production

Cos attenuates TBHP-induced apoptosis and ROS levels in H9C2 cells

Cos activates Nrf2 and attenuates TBHP-induced apoptosis

Discussion

Conclusion

abbreviations

Declarations

References

Supplementary Files

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