Akt pathway

Accepted Manuscript Celastrol pretreatment attenuates rat myocardial ischemia/ reperfusion injury by inhibiting high mobility group box 1 protein expression via the PI3K/Akt pathway Suiyang Tong, Liangliang Zhang, Jacob Joseph, Xue jun Jiang PII:

S0006-291X(18)30352-8

DOI:

10.1016/j.bbrc.2018.02.121

Reference:

YBBRC 39486

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 10 February 2018 Accepted Date: 13 February 2018

Please cite this article as: S. Tong, L. Zhang, J. Joseph, X.j. Jiang, Celastrol pretreatment attenuates rat myocardial ischemia/ reperfusion injury by inhibiting high mobility group box 1 protein expression via the PI3K/Akt pathway, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/ j.bbrc.2018.02.121. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Celastrol Pretreatment Attenuates Rat Myocardial Ischemia/ Reperfusion Injury by Inhibiting High Mobility Group Box 1 Protein Expression via the PI3K/Akt Pathway Suiyang Tonga,b

Liangliang Zhangb Jacob Josephb Xue jun Jianga

Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei, P.R. China

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Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital

and Harvard Medical School , Boston, Massachusetts.

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Corresponding Author: Xuejun Jiang, E-mail: [email protected]

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a

Abstract

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Background/Aims: Celastrol pretreatment has been shown to protect against myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism is poorly understood. This study aimed to investigate the cardioprotective effects of celastrol pretreatment on I/R injury and to further explore whether its mechanism of action was associated with the inhibition of high mobility group box 1 protein (HMGB1) expression via the phosphoinositide 3-kinase (PI3K)/Akt

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pathway. Methods: In a fixed-dose study, hematoxylin and eosin staining and myocardial enzyme measurements were used to determine the optimal dose of celastrol that elicited the best cardioprotective effects against I/R injury. Furthermore, rats were pretreated with 4mg/kg celastrol,

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and infarct size and the levels of myocardial enzymes, apoptosis, inflammatory and oxidative indices, and HMGB1 and p-Akt expression were measured. Results: Our results indicated that

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celastrol dose-dependently attenuated histopathological changes and the elevation in myocardial enzymes induced by I/R. Moreover, the celastrol pretreatment (4mg/kg) not only significantly decreased infarct size as well as myocardial enzyme levels but also inhibited myocardial apoptosis, inflammatory response and oxidative stress. Additionally, celastrol downregulated HMGB1 expression and upregulated p-Akt expression in the myocardium. LY294002, a specific pI3k inhibitor, partially reversed the decreased HMGB1 expression, increased p-Akt expression induced by celastrol, and abolished the anti-apoptotic, anti- inflammatory and anti-oxidative effects of celastrol. Conclusion: These findings suggest that short-term pretreatment with celastrol protects against myocardial I/R injury by suppressing myocardial apoptosis, inflammatory response and oxidative stress via pI3k/ Akt pathway activation and HMGB1 inhibition.

ACCEPTED MANUSCRIPT Key Words Celastrol, Myocardial ischemia/reperfusion Injury, Protective effect, PI3K/Akt

Introduction

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Coronary heart disease is becoming a major public health burden in both developed and developing countries, with acute myocardial infarction (AMI) as the leading cause of premature mortality [1]. The standard mode of cure is re-establishment of coronary reperfusion by thrombolytic therapy, primary percutaneous intervention or coronary artery bypass grafting. Early

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and successful myocardial reperfusion is the most effective strategy to reduce infarct size and preserve cardiac function after AMI [2]. However, reperfusion itself causes a localized burst of

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oxidative stress and inflammatory response that can lead to additional myocardial damage named “ischemia/reperfusion (I/R) injury” [3]. The mechanism of myocardial I/R injury is very complex, the currently accepted theories include altered energy metabolism, free radicals, calcium overload, neutrophil infiltration, and vascular endothelial dysfunction, eventually resulting in apoptosis or necrosis[4-6].

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Celastrol has been reported to have multiple bioactivities such as anti-inflammatory, anti-oxidative, radioprotective and anticancer effects [7-10]. Recently, Chu et al. [11] showed that the celastrol pretreatment could be useful for preventing I/R-induced renal injury in rats by

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suppressing inflammation and oxidative stress. Meanwhile, Li et al. [12] demonstrated that the celastrol pretreatment attenuated cerebral ischemia in the rat permanent middle cerebral artery

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occlusion. Sarkissian et al. [13] suggested that celastrol treatment promoted cardiomyocyte survival and reduced of injury and adverse remodelling with preservation of cardiac function. Celastrol may represent a novel potent pharmacological cardioprotective agent mimicking ischaemic conditioning that could have a valuable impact in the treatment of myocardial infarction. However, the mechanism underlying celastrol protection against I/R injury is not fully elucidated. High mobility group box 1 protein (HMGB1) is a ubiquitous, non-chromosomal nuclear protein that has been identified as a novel pro-inflammatory mediator in several cardiovascular diseases, including myocardial I/R injury [14-15]. Our prior studies confirmed that the downregulation of HMGB1 by several drugs, such as hesperidin [16] and minocycline [17], can reduce myocardial I/R injury in rats. Phosphoinositide 3-kinases (PI3Ks) are a conserved family of

ACCEPTED MANUSCRIPT signal transduction enzymes which are involved in regulating cellular activation, inflammatory responses, and apoptosis [18-20]. Furthermore, Han et al. [21] reported that the PI3K pathway may be associated with the inhibition of HMGB1 expression in a rat model of myocardial I/R injury.

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Hence, we examined whether celastrol could prevent myocardial I/R injury and whether its mode of action was via PI3/AKt activation and consequent inhibition of HMGB1 expression. Materials and methods Animals

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Eighty male sprague-dawley rats (200-250g) were obtained from the animal experiment center of Wuhan University, china. All experimental protocols conformed to the Guideline for the

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Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Wuhan University (Approval Number: 2015-0027). All rats used in this study were free to eat food and drink water, and housed under the specific pathogen-free condition (12h/12h lighte-dark cycle with humidity of 55±5% at 25±2 ).

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Myocardial I/R model and experimental design

After being anesthetized with an intraperitoneal (i.p.) injection of sodium pentobarbital (45 mg/kg, Sigma, St. Louis, USA) , the rats were ventilated artificially with a volume-controlled

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rodent respirator and monitored with an electrocardiogram using a computer-based EP system (LEAD2000B, jinjiang Ltd. Chengdu, China). Then, the myocardial I/R rat model was established

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with a 30-min left anterior descending coronary artery (LAD) occlusion and a 4-h reperfusion, as previously described [22]. At the end of the reperfusion period, the animals were euthanized, and their blood and hearts were collected for various biochemical analyses. Celastrol (HPLC>98% , Paypay Technologies, Inc, Shenzhen, China) was dissolved in 0.9%

sodium chloride (NaCl) containing 1% dimethyl sulfoxide at a concentration of 1 mg/mL. Different doses of celastrol were administered immediately through intraperitoneal injection after being dissolved in 0.9% NaCl including 1% DMSO 30 min before the myocardial ischemia models were completed [11-12]. To determinate the optimal dose for the celastrol administration, we designed a fixed-dose study in which thirty rats were randomly assigned to one of the following five groups ( n=6 per

ACCEPTED MANUSCRIPT group) (i) Sham-operated (SO): 0.9% NaCl including 1% dimethyl sulfoxide (DMSO); (ii) I/R: 0.9% NaCl including 1% DMSO; (iii) Celastrol (2mg/Kg) +I/R (Cel 2-I/R) ; (iv) Celastrol (4mg/ Kg) +I/R (Cel 4-I/R) ; or (v) Celastrol (6mg/Kg) +I/R (Cel 6-I/R) . To further clarify the protective effects of celastrol on myocardial I/R injury, we conducted

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another experiment in which 48 rats were randomly assigned to one of the four following groups (n=12/group): (i) SO; (ii) I/R; (iii) Cel 4-I/R; (iv) LY294002 + Celastrol (4mg/Kg) + I/R (LY-Cel 4-I/R). After anesthesia, rats in LY-Cel 4-I/R group were treated with LY294002 ( a specific PI3K inhibitor, 0.3mg/Kg, Sigma-Aldrich, St. Louis, USA) via the caudal vein 30 min before LAD

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occlusion [22], and other three groups were subjected to 30 min occlusion and 4 h reperfusion except for the SO group.

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Histopathological examination

Myocardial tissue was fixed in 4% paraformaldehyde and embedded in paraffin. To quantify extent of myocardial damage, paraffin sections of hearts were stained with hematoxylin and eosin (H&E) and 6 fields (×200 magnification) were randomly selected from two sections in each group and scored in a blinded manner by two individuals, as previously described [23]. The scores were

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as follows: 0, no damage; 1 (mild), interstitial edema and focal necrosis; 2 (moderate), diffuse myocardial cell swelling and focal necrosis; 3 (severe), necrosis with the presence of contraction bands and inflammatory cell infiltrate; and 4 (highly severe).

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Assessment of myocardial injury

The serum levels of lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac troponin

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I (cTnI) were determined to assess myocardial injury. Blood samples were collected and centrifuged. Standard techniques using commercialized assay kits according to the manufacturer’ s instructions (Elabscience Biotechnology Co., Ltd, Wuhan, China) were used for the analyses. The values are expressed in international units (U) per liter. Assessment of infarct size Infarct size was determined using 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, USA) staining as previously described [16, 24]. In brief, after reperfusion, 2 ml of 1% Evans blue dye (Sigma-Aldrich, St. Louis, USA) was injected via the femoral vein. Each frozen heart (-80 , 15 min) was then cut (approximately 2 mm) from the apex to the base. The slices were incubated in 1% TTC for 20 min at 37

and then fixed in 4% paraformaldehyde. The infarct

ACCEPTED MANUSCRIPT area (white) and the risk area (red) from each section were measured using image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Silver Spring, USA); six rats in each group were used for myocardial infarct size measurement. Infarct size was expressed as the following percentage: infarct area / (risk + infarct) area.

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Measurement of Oxidative stress-related biochemical index Myocardial superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’ s instructions. These markers were used as indices of oxygen free

Detection of inflammation related factors

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radical and lipid superoxide levels in the myocardium, respectively.

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The expression levels of tumor necrosis factor-α (TNF-α), interferon-γ(INF-γ) and interleukin-6 (IL-6) in myocardial tissue were assessed by a commercial ELISA kit (TNF-α, INF-γ and IL-6: Nanjing Jiancheng Bioengineering Institute, China; TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were

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performed to determine the extent of apoptosis in the myocardium using a commercial kit (Roche Applied Science, Indianapolis, USA) according to the manufacturer’ s instructions. Cells with clear nuclear labeling were defined as TUNEL-positive cells. To determine the extent of

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myocardial apoptosis, 6 fields (×200 magnification) were randomly selected from two sections in each group, and the apoptosis Index (AI) was calculated using image-pro plus 6.0 (AI) as follows:

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AI = number of apoptotic cells / total number of cells counted. Western blot analysis of Bax, Bcl-2 , LC3, Beclin 1, HMGB1, t-Akt and p-Akt (ser473) Pulverized, frozen ischemic areas of the left ventricle samples were analyzed by western

blot analysis, as previously described [25]. The following primary antibodies were used for western blot analysis: anti-Bax (1:1000), anti-Bcl-2 (1:1000), anti-LC3 (1:1000), anti- Beclin 1 (1:1000), anti-t-Akt (1:1000), anti-p-Akt (ser473, 1:2000) (Cell Signaling Technology, Danvers, USA) , and anti-HMGB1(1:200, Boster, Wuhan, China). Five hearts in each group were used for the western blot analysis. Statistical analysis SPSS 17.0 was used for statistical analysis. Data are expressed as the means±SD. The

ACCEPTED MANUSCRIPT one-sample Kolmogorov-Smirnov test was used to check for a normal distribution. Student’ s t-test was used for between-group comparisons. One-way ANOVA or a Welch test was used for comparisons among groups, and Tukey’s post hoc test was used for multiple comparisons. A p value<0.05 was considered statistically significant.

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Results Pretreatment with celastrol attenuated histopathological changes and reduced myocardial injury

As shown in Fig 1A, the myocardial structure in the SO group exhibited a regular

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arrangement, normal cardiac muscle fibers and no necrosis. Compared with the SO group , the I/R group demonstrated ruptured cardiac muscle fibers, necrosis with inflammatory cell infiltration

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edema and hemorrhage. The myocardial damage in the celastrol groups was less than that in the I/R group. In the celastrol groups ,we observed less myocardial fiber disruption, necrosis and inflammatory cell infiltration.

Compared with the SO group, the I/R group exhibited a markedly increased damage score as well as serum CK, LDH and cTnI levels (p<0.05). All groups pretreated with celastrol (2, 4 or 6

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mg/Kg) showed lower damage scores as well as decreased serum CK, LDH and cTnI levels, suggesting that the celastrol pretreatment could attenuate myocardial I/R injury. However, only groups pretreated with 4 or 6 mg/Kg of celastrol showed significant differences compared with the

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I/R group (Fig. 1C and 1D). As no significant differences were observed between the groups receiving 4 and 6 mg/kg of celastrol , the 6 mg/Kg dose was used for subsequent experiments.

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Celastrol pretreatment reduced myocardial injury after I/R Compared with the I/R group, the celastrol group exhibited a significant decrease in infarct

size; However, the addition of LY294002 abolished the celastrol- mediated decrease in infarct size (p<0.05, Fig. 1B and 2E) . Additionally, serum levels of CK, LDH and cTnI were significantly higher in the I/R group than in the SO group (p<0.05, Fig 1F) . Intraperitoneal administration of celastrol significantly lowered the serum levels of CK, LDH and cTnI, which were increased in the I/R group. These results suggested that celastrol could attenuate myocardial I/R injury. However, administration of LY294002 partially reversed the cardioprotective effects of celastrol (p<0.05). Celastrol pretreatment suppressed myocardial apoptosis

ACCEPTED MANUSCRIPT Compared with the SO group, the AI and the level of Bax expression were markedly increased in the I/R group; in addition, both Bcl-2 expression and the Bcl-2/Bax ratio were significantly lower in the I/R group than in the SO group (p<0.05). The celastrol pretreatment group exhibited striking decreases in the AI and Bax expression and increases in the Bcl-2/Bax

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ratio and Bcl-2 expression compared with those of the I/R group (p<0.05), indicating that celastrol could reduce myocardial apoptosis. Conversely, administration of LY294002 partially reversed the anti- apoptotic effects of celastrol (p<0.05, Fig. 2).

Celastrol pretreatment inhibited the oxidative stress and inflammatory response

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Compared with the SO group, SOD activity was significantly decreased and MDA content was significantly increased in the I/R group (p<0.05) . Celastrol treatment markedly inhibited the

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decrease in SOD activity and the increase in MDA content (p<0.05), illustrating the anti-oxidative effects of celastrol; however , LY294002 reversed these effects (p<0.05, Fig. 3A ) Compared with the SO group, the cardiac levels of TNF-α, INF-γ and IL-6 were substantially increased in the I/R group. The levels of TNF-α, INF-γ and IL-6 were significantly lower in the celastrol group than in the I/R group (p<0.05), demonstrating the anti-inflammatory effects of

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celastrol. However, LY294002 completely abolished these effects (p< 0.05, Fig 3B ) Celastrol pretreatment suppressed myocardial autophagy As shown in Fig. 4, compared with the SO group, the level of Beclin-1 and LC3B expression

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were markedly increased in the I/R group (p<0.05). The celastrol pretreatment group exhibited striking decreases in the Beclin-1 and LC3B expression compared with those of the I/R group (p<

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0.05), indicating that celastrol could reduce myocardial apoptosis. Conversely, administration of LY294002 partially reversed the anti-autophagy effects of celastrol (p<0.05, Fig 4B). Celastrol pretreatment activated the PI3K/Akt pathway and suppressed HMGB1 expression Celastrol substantially reduced the I/R-induced HMGB1 signal accumulation , but its effect

was abrogated by LY294002. Meanwhe compared with the SO group, the mean densitometry values and HMGB1 expression were significantl increased and p-Akt expression was markedly decreased in the I/R group (p<0.05). Celastrol markedly suppressed the increased mean densitometry values and HMGB1 expression as well as the decreased p-Akt expression induced by I/R (p <0.05, Fig. 4A and C), indicating that hesperidin could activate the PI3K/Akt pathway and inhibit HMGB1 expression. However, administration of LY294002 dramatically reversed

ACCEPTED MANUSCRIPT these effects (p<0.05). In addition, no significant difference in t-Akt expression was observed among the four groups (p > 0.05) Discussion Celastrol is a pharmacologically active ingredient extracted from the root of traditional

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Chinese herb Tripterygium wilfordii Hook F with potent anti-inflammatory and anti-tumor activities [26]. Der Sarkissian et al. [27]suggested that celastrol may represent a novel potent pharmacological cardioprotective agent mimicking ischaemic conditioning that could have a valuable impact in the treatment of myocardial infarction. Cheng et al. [28]demonstrated that

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celastrol ameliorates myocardial fibrosis and cardiac dysfunction.

In this present study, we first found that low-dose celastrol could attenuate myocardial I/R

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injury as evidenced by the increase in cell viability and decline in the release of LDH, CK and cTnI. Moreover, we also cannot ignore the fact that high-dose celastrol showed no benefcial effect on myocardial I/R injury, and instead led to a decline in cell viability and rise in damage score. The reason we speculated is that high-dose celastrol may induce cell apoptosis. Myocardial I/R injury is a complex pathophysiological process in which myocardial

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apoptosis, inflammatory response and oxidative stress all play important roles. Sharma et al. [29]Show that celastrol significantly recovers myocardial remodeling and cardiomyopathy by diminishing infarct size, apoptosis, and inflammation. Zhang et al. [30] reported that celastrol

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attenuates neuronal apoptosis via inhibiting Akt/mTOR pathway. Consistent with these damage during myocardial I/R injury, suggesting that celastrol could inhibit myocardial apoptosis and

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further attenuate myocardial I/R injury. Ample evidence shows that the injury is in part caused by an excessive generation of

reactive oxygen species or free radicals. Shaker et al. [31] Show that celastrol ameliorates murine colitis via modulating oxidative stress and inflammatory cytokines. Bian et al. [32] reported that celastrol protects mouse retinas from bright light-induced degeneration through inhibition of oxidative stress and inflammation. Similarly, our research showed that celastrol inhibited the infiltration of Inflammatory cells and reduce the levels of pro-inflammatory cytokines, such as TNF-α, INF-γ and IL-6. In addition celastrol alleviated oxidative stress reduced the area of myocardial infarction. These results suggest that the cardioprotective effects of celastrol are partly due to its anti-inflammatory response and anti-oxidative stress effects.

ACCEPTED MANUSCRIPT HMGB1 is a novel pro-inflammatory cytokine, can up-regulate other classic proinflammatory cytokines, such as TNF-α and IL-6, thus aggravating inflammation and myocardial I/R injury. Our previous study demonstrated that HMGB1 may worsen myocardial I/ R injury in a dose-dependent manner, and inhibiting the expression of HMGB1 by hesperidin or interleukin-33

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attenuated inflammatory responses and myocardial I/R injury [16,24]. In the present study, increased HMGB1 expression was detected in the I/R group, thus suggesting that high levels of HMGB1 may contribute to inflammatory response and oxidative stress that occur during myocardial I/ R injury. Our findings suggest that celastrol may attenuate I/R injury by inhibiting

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HMGB1 expression.

Activation of the PI3K/Akt pathway also protects against myocardial I/R injury by

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decreasing oxidative stress, repressing the inflammatory cascade, and inhibiting apoptosis in vivo and in vitro. Celastrol also has been reported to block the endoplasmic reticulum stress and oxidative stress pathway in brain and liver of mice [33, 34]. In the present study, we found that celastrol activated the PI3K/Akt pathway and decreased HMGB1 expression, while the LY294002 treatment partially abrogated the anti-apoptosis, anti-autophagy, anti-inflammatory response and

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oxidative stress effects of celastrol.

In conclusion our present study demonstrated that the pretreatment with celastrol attenuated myocardial I/R injury by reducing myocardial apoptos, myocardial autophagy, the inflammatory

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response and oxidative stress. We also showed that these protective effects of celastrol were

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predominantly due to HMGB1 expression inhibition via PI3K/Akt pathway activation.

Funding: This study was partly supported by grants from the National Natural Science foundation of China (No. 81170307, 81700249 and 81270184), the National Key Basic Research and Development Program (973 Program) (No. 2011cb606202), Fundamental Research Funds for the Central Universities of China (No. 2042014kf0158)

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ACCEPTED MANUSCRIPT inflammation. J Neuroinflammation. 201627;13:50. 33. Yanhua Lia, Dan Hea, Xiangjian Zhanga, Zongjie Liua, Xiaolin Zhanga, Lipeng Donga, Yinxue Xinga, ChaoHui Wanga, Huimin Qiaoa, Chunhua Zhua, Yulin Chen. Protective effect of celastrol in rat cerebral ischemia model: Down-regulating p-JNK, p-c-Jun and NF-κB.

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Type 2 Diabetic Rats. J Diabetes Res. 2016; 2016: 2641248. Figure legends

Fig. 1. Celastrol reduced myocardial I/R injury, but LY294002 partially reversed these effects. (A) Representative

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images of H&E stained samples (×200)demonstrating histopathological changes in the myocardium. (B) Representative images of TTC stained samples showing the infarct area (white) and risk area (red). (C) Damage score (n=6) . (D) The serum level of CK, LDH and cTnI (n=6) . (E) Myocardial infarct size (n =6 ) . (F) The expression of CK, LDH and cTnI in serum (n=6).

a

p < 0.05 vs. the SO group; b p < 0.05 vs. the I/R group

Fig. 2. Celastrol pretreatment suppressed myocardial apoptosis, but LY294002 partially reversed its anti-apoptotic

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effects. (A) Representative images of TUNEL assays. (B) Apoptosis index (AI, n=6) . (C) The expression levels of Bcl-2 and Bax, and the ratio of Bcl-2/Bax (n=6). 0.05 vs. the Cel 4-I/R group

a

p < 0.05 vs. the SO group; b p < 0.05 vs. the I/R group; c p <

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Fig. 3. Celastrol pretreatment ameliorated the oxidative stress and inflammatory response induced by myocardial I/R injury, but LY294002 partially abrogated these anti-oxidative effects. (A) The expression of SOD and MDA a

p < 0.05 vs. the SO group; b p < 0.05 vs. the I/R

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(n=6) . (B) The expression of INF-γ, IL-6 and TNF-α (n=6). group; c p < 0.05 vs. the Cel 4-I/R group

Fig. 4. Celastrol suppressed myocardial autophagy and HMGB1 expression, and activated the PI3K/Akt pathway. But LY294002 partially reversed its anti-apoptotic effects. (A) The expression levels of HMGB1 (n= 6). (B) The expression levels of LC3B and Beclin-1 (n=6). (C) The expression levels of p-Akt and t-Akt (n=6). a p < 0.05 vs. the SO group; b p < 0.05 vs. the I/R group; c p < 0.05 vs. the Cel 4-I/R group