Cinnamaldehyde and allopurinol reduce fructose-induced cardiac inflammation and fibrosis by attenuating CD36-mediated TLR4/6-IRAK4/1 signaling to suppress NLRP3 inflammasome activation

Fructose consumption induces metabolic syndrome to increase cardiovascular disease risk. Cinnamaldehyde and allopurinol possess anti-oxidative and anti-inflammatory activity to relieve heart injury in metabolic syndrome. But the mechanisms of fructose-induced cardiac injury, and cardioprotective effects of cinnamaldehyde and allopurinol are not completely understood. In this study, fructose-fed rats displayed metabolic syndrome with elevated serum ox-LDL, cardiac oxidative stress, inflammation and fibrosis. Scavenger receptor CD36, Toll-like receptor 4 (TLR4), TLR6, IL-1R-associated kinase 4/1 (IRAK4/1), nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome, interleukin-1β, transforming growth factor-β (TGF-β), drosophila mothers against DPP homolog (Smad) 2/3 phosphorylation and Smad4 were increased in animal and H9c2 cell models. These pathological processes were further evaluated in ox-LDL or fructose-exposed H9c2 cells pretreated with ROS scavenger and CD36 specific inhibitor, or IRAK1/4 inhibitor, and transfected with CD36, NLRP3, or IRAK4/1 siRNA, demonstrating that NLPR3 inflammasome activation through CD36-mediated TLR4/6-IRAK4/1 signaling may promote cardiac inflammation and fibrosis. Cinnamaldehyde and allopurinol reduced cardiac oxidative stress to suppress NLPR3 inflammasome activation and TGF-β/Smads signaling by inhibiting CD36-mediated TLR4/6-IRAK4/1 signaling under fructose induction. These results suggest that the blockage of CD36-mediated TLR4/6-IRAK4/1 signaling to suppress NLRP3 inflammasome activation by cinnamaldehyde and allopurinol may protect against fructose-induced cardiac inflammation and fibrosis.

Scientific RepoRts | 6:27460 | DOI: 10.1038/srep27460 significantly increased (Fig. 2C-F), accompanying with high heart hydroxyproline levels ( Table 2) in this animal model. To address this, H9c2 cells were incubated with 1 mM fructose for 24 h. Cellular TG and TC levels, as well as TGF-β , p-Smad2/3 and Smad4 protein levels were significantly increased in fructose-exposed H9c2 cells ( Fig. 3A-F). These data further demonstrate cardiac hypertrophy and fibrosis in fructose-induced metabolic syndrome of animals 5 .
In the present study, cinnamaldehyde at 30 and 40 μ M and allopurinol at 30 μ M attenuated fructose-induced elevation of TG and TC levels in H9c2 cells (Fig. 3A,B). 20 μ M cinnamaldehyde only reduced TC levels in this cell model (Fig. 3B). Cinnamaldehyde and allopurinol both at 30 μ M restored fructose-induced changes of TGF-β , p-Smad2/3 and Smad4 protein levels in H9c2 cells ( Fig. 3C-F). These results indicate that cinnamaldehyde and allopurinol may have the potential to protect against fructose-induced cardiac hypertrophy and fibrosis in metabolic syndrome of rats.
To investigate whether oxidative stress contributed to cardiac CD36 up-regulation under fructose induction, H9c2 cells were pretreated with NAC (1 mM) or SSO (0.4 mM) for 0.5 or 1 h, respectively, and then co-incubated with 1 mM fructose for other 24 h. NAC prevented fructose-induced CD36 over-expression (Fig. 5A), ROS overproduction and TXNIP over-expression, but not NADPH oxidase and XOD hyperactivity (Fig. 5B-E) in H9c2 cells. SSO failed to affect fructose-induced ROS overproduction and TXNIP over-expression, as well as NADPH oxidase and XOD hyperactivity in H9c2 cells (Fig. 6A-D). In fact, ROS overproduction and TXNIP over-expression were observed in CD36 siRNA-transfected H9c2 cells exposed to 1 mM fructose (Fig. 6E,F). Thus, fructose-driven ROS may mainly contribute to CD36 up-regulation in myocardial cells.
Ox-LDL can enhance ROS generation in endothelial cells 44,45 and macrophages 46,47 . Fructose consumption induces high serum ox-LDL levels observed in adult or children subjects 2,7 , as well as in rats in the present study   (Table 2). To further address above findings, H9c2 cells were exposed with ox-LDL (25 and 50 μ g/mL), in the presence or absence of NAC (1 mM). Ox-LDL (25 and 50 μ g/mL) induced ROS overproduction (Fig. S1A,B) and TXNIP over-expression (Fig. S1C,D) in H9c2 cells, which were attenuated by NAC. NAC and SSO also prevented ox-LDL-induced CD36 up-regulation in H9c2 cells (Fig. S2A,B). These observations further demonstrate that cardiac ROS may cause CD36 up-regulation in myocardial cells under fructose induction.
cardiac oxidative stress and ROS to block CD36-mediated NLRP3 inflammasome activation in fructose-induced myocardial cell inflammation.

Discussion
This study for the first time demonstrated that fructose induction increased cardiac oxidative stress and ROS to up-regulate CD36, and subsequently provoked NLRP3 inflammasome in a TLR4/6-IRAK4/1-dependent manner, promoting cardiac inflammation and fibrosis in metabolic syndrome of rats with high serum ox-LDL levels. Furthermore, cinnamaldehyde and allopurinol reduced oxidative stress and ROS to down-regulate CD36, and then mediated TLR4/6-IRAK4/1 signaling to suppress NLRP3 inflammasome activation in fructose-fed rats. This study revealed that anti-oxidants cinnamaldehyde and allopurinol protected against fructose-induced cardiac inflammation and fibrosis.
Ox-LDL is a common risk factor for cardiovascular diseases 49 . CD36 as a signaling molecule binds ox-LDL and functions ox-LDL uptake. Of note, clinical study shows that high serum ox-LDL level remarkably correlates with total fructose intake in children with non-alcoholic fatty liver disease at baseline, and it is significantly reduced after low-fructose diet education for six months 7 . Patients with CD36 deficiency may be susceptible to myocardial damage with abnormal myocardial long-chain fatty acid metabolism 50 . TXNIP up-regulation is observed in cardiomyocyte impairment of diabetic mice 15 , and closely correlates with oxidative stress initiated by CD36 in ceramide-induced pancreatic β -cell dysfunction 17 . In this study, cardiac CD36 protein levels were increased in fructose-induced metabolic syndrome of rats, being consistent with elevation of serum ox-LDL levels under fructose induction. Furthermore, cardiac accumulation of ROS, TXNIP, H 2 O 2 , O 2 ·− , ·OH and MDA were observed in fructose-fed rats with cardiac NADPH oxidase and XOD hyperactivity. Anti-oxidant enzymes SOD and CAT, as well as GSH are known to scavenge ROS. In this animal model, heart SOD and CAT activity, and GSH/GSSG ratio were significantly decreased. In fact, ox-LDL or fructose could induce ROS overproduction, TXNIP over-expression and CD36 up-regulation in H9c2 cells, which were attenuated by ROS scavenger NAC. Moreover, SSO significantly blocked ox-LDL or fructose-induced CD36 over-expression, but failed to affect fructose-induced ROS overproduction, TXNIP over-expression, NADPH oxidase and XOD hyperactivity in H9c2 cells. In CD36 siRNA-transfected H9c2 cells, ROS overproduction and TXNIP over-expression induced by fructose were not reduced. These observations indicate that fructose-induced cardiac oxidative stress and ROS may closely promote myocardial cell CD36 up-regulation in high ox-LDL-associated cardiovascular diseases.
High ox-LDL level is closely linked to cardiac structural and functional injury in response to inflammation 6 . CD36-deficient macrophages reduce IL-1β release by activating NLRP3 inflammasome 13 . Under fructose or ox-LDL induction, NLRP3 inflammasome was activated with IL-1β secretion in rat heart and/or H9c2 cells. More importantly, CD36-specific inhibitor SSO blocked these changes in H9c2 cells. Smad2/3 phosphorylation and fibrosis in response to TGF-β 1 is attenuated in Nlrp3 −/− H293T cells 51 . NLRP3-deficient cardiac fibroblasts impair differentiation and R-Smad activation in response to TGF-β 21 . Subsequently, the present study found that fructose induced high TGF-β , p-Smad2/3 and Smad4 levels in rat heart and H9c2 cells. SSO also blocked fructose-induced elevation of TGF-β , p-Smad2/3 and Smad4 in H9c2 cells. CD36 siRNA suppressed fructose-induced change of NLRP3 and TGF-β in H9c2 cells. Additionally, NLRP3 siRNA attenuated TGF-β change but not ROS overproduction in this cell model. These observations indicate that ROS-triggered CD36 may activate NLRP3 inflammasome to promote cardiac inflammation and fibrosis in fructose-induced heart injury.
Cinnamaldehyde improves metabolic disorders in rodents, decreases ROS production and IL-1β secretion in lipopolysaccharide-stimulated murine J774A.1 macrophages, suppresses plasma TLR4 expression in myocardium of viral myocarditis mice, and alleviates ischemic myocardial injury of rats, exhibiting its anti-oxidative and anti-inflammatory property 32,34-36 . Allopurinol retards NLRP3 inflammasome activation in fructose-induced metabolic syndrome of rats 39,40 and ameliorates high-fat and high-fructose diet-induced oxidative stress, inflammation and cardiomyocyte hypertrophy in mice 41 . In the present study, cinnamaldehyde dose-dependently significantly alleviated oxidative stress, ROS over-production and TXNIP over-expression in fructose-fed rat heart and H9c2 cells. It could down-regulate CD36 protein levels in these animal and cell models. Allopurinol also reduced oxidative stress and CD36 in fructose-fed rat heart and H9c2 cells. In ox-LDL-exposed H9c2 cells, cinnamaldehyde and allopurinol reduced over-production of ROS, and over-expression of TXNIP, CD36, NLRP3 and IL-1β . Furthermore, cinnamaldehyde and allopurinol remarkably abolished fructose-induced ROS overproduction and TXNIP over-expression, but not NADPH oxidase and XOD hyperactivity in NAC-pretreated H9c2 cells. In CD36 siRNA-transfected or CD36 specific inhibitor-pretreated H9c2 cells, cinnamaldehyde and allopurinol reduced fructose-induced ROS overproduction, TXNIP over-expression, NADPH oxidase and XOD hyperactivity. These results indicate that cinnamaldehyde and allopurinol may reduce oxidative stress and ROS, and subsequently decrease CD36 expression in myocardial cells under fructose induction.
NLRP3 inflammasome inhibitor and NLRP3 mutant procedure alleviate cardiac injury in mice 20 . In this study, cinnamaldehyde and allopurinol inhibited NLRP3 inflammasome activation to reduce IL-1β release and TGF-β /Smads signaling in animal and cell models. Of note, they only reduced fructose-induced ROS and CD36 in NLRP3 siRNA-transfected H9c2 cells. These results suggest that cinnamaldehyde and allopurinol may reduce cardiac oxidative stress and ROS to block CD36-mediated NLRP3 inflammasome activation in fructose-induced myocardial cell inflammation and fibrosis.
Scientific RepoRts | 6:27460 | DOI: 10.1038/srep27460 inhibitor I-pretreated H9c2 cells. Cinnamaldehyde and allopurinol also reduced fructose-induced ROS, TXNIP and CD36 in IRAK4 or IRAK1 siRNA-transfected H9c2 cells. Our findings suggest that the blockage of oxidative stress and ROS to suppress NLRP3 inflammasome activation by cinnamaldehyde and allopurinol may be dependent on the suppression of CD36-mediated TLR4/6-IRAK4/1 signaling in fructose-induced cardiac inflammation and fibrosis.
Fructose consumption is closely associated with the development of cardiovascular disease, due to its induction of XOD hyperactivity-mediated intracellular and serum uric acid 41,42 . Allopurinol suppresses XOD and protects heart against ROS, inflammation and fibrosis in high fat and high fructose diet-fed rats 41 . It also alleviates low grade inflammation and cardiac ischemia in fructose-induced hyperuricemia of rats 42 . Allopurinol lowers serum uric acid levels, resulting in the improvement of blood pressure in fructose-induced metabolic syndrome in patients 52 , and reduces free radical production in type 1 diabetes patients with a potentially higher oxidant buildup as a result of increased cardiovascular risk 53,54 . It also reduces serum SBP and ox-LDL in cardiac surgery patients with hyperuricemia, and alters expression of inflammatory markers in patients with acute ischemic stroke 55,56 . Thus, allopurinal is clinically suggested as cardioprotectant [57][58][59] . Cinnamaldehyde is a major and important compound from cinnamon. It exhibits potent XOD inhibitory activity to reduce serum uric acid levels in hyperuricemic mice 60 . Cinnamaldehyde can reduce serum TG, TC and LDL-c levels in patients with type 2 diabetes 32 , improve glucose tolerance in high fat diet-induced obese mice 61 and protect against cerebral ischaemia  = 7), respectively. Cellular protein levels of NLRP3 (D) and IL-1β (E) were determined in SSO-pretreated H9c2 cells co-incubated with fructose, cinnamaldehyde and allopurinol (n = 7), respectively. Cellular NLRP3 protein levels were determined in CD36 siRNA-transfected H9c2 cells co-incubated with fructose, cinnamaldehyde and allopurinol (n = 7) (F). Cellular TGF-β protein levels were determined in NLRP3 siRNA-transfected H9c2 cells co-incubated with fructose, cinnamaldehyde and allopurinol (n = 7) (G). Cellular protein levels of TGF-β (H), p-Smad2/3 (I,J) and Smad4 (K) were determined in SSO-pretreatedH9c2 cells co-incubated with fructose, cinnamaldehyde and allopurinol (n = 7), respectively. Cellular TGF-β protein levels (L) were determined in CD36 siRNA-transfected H9c2 cells co-incubated with fructose, cinnamaldehyde and allopurinol (n = 7). The relative protein levels of CD36, TXNIP, NLRP3, IL-1β , TGF-β and Smad4 were normalized to GAPDH or β -actin, respectively. Relative protein levels of p-Smad2/3 were normalized to Smad2/3, respectively (n = 7). Data were expressed as the mean ± SEM. ## P < 0.01, ### P < 0.001 vs normal cell control group; * P < 0.05, * * P < 0.01, * * * P < 0.001 vs fructose-vehicle cell group, or fructose-vehicle + inhibitor/siRNA control cell group.
Scientific RepoRts | 6:27460 | DOI: 10.1038/srep27460 injury in mice by reducing IL-1β production 62 . Therefore, the attenuation of oxidative stress and ROS to suppress CD36-mediated TLR4/6-IRAK4/1 signaling and NLRP3 inflammasome activation by cinnamaldehyde and allopurinol may be a promising therapeutic strategy for the treatment of metabolic syndrome-associated heart disease. High fructose triggers sophisticated systemic and cardiac oxidative stress, which may be a risk factor in heart injury of metabolic syndrome. More investigations are needed to determine the role of fructose-induced oxidative stress in heart injury, and explore in anti-oxidant mechanisms of cinnamaldehyde and allopurinol in alleviating cardiac inflammation and fibrosis under fructose induction.
In conclusion, this study demonstrates that cinnamaldehyde and allopurinol reduce oxidative stress and ROS to alleviate heart injury in fructose-induced metabolic syndrome of rats. Furthermore, they suppress NLPR3 inflammasome activation to reduce IL-1β and TGF-β /Smads signaling via CD36-mediated TLR4/6-IRAK4/1 signaling in animal and cell models, exhibiting the alleviation of fructose-induced cardiac inflammation and fibrosis. These results suggest that the inclusion of cinnamon in the diet or the treatment of allopurinol in subjects with fructose-induced metabolic syndrome may reduce risk factors associated with heart diseases.

Materials and Methods
Animals. Male Sprague-Dawley rats aged from 8 to 10 weeks (200-220 g) were purchased from the Experimental Animal Centre of Nanjing Medical University (Nanjing, China) (Production license: SCXK2008-0004). They were housed at 22 ± 2 °C under a relative humidity of 55 ± 5% and a normal 12-h light/dark cycle with the lights on at 6:00 a.m. Rats were given a standard chow and water ad libitum for the study and one week for acclimatization before the experiment. Each rat was given 100 mL drinking water or drinking water containing 10% fructose (wt/vol) (Jiakangyuan Science and Technology Co., Ltd., Beijing, China) and standard chow for 10 weeks. After 5-week fructose feeding, rats were randomized into five subgroups (n = 15/group), receiving drinking water, 20, 40 and 80 mg/kg cinnamaldehyde (95% purity) and 5 mg/kg allopurinol (98% purity), (Sigma, St. Louis, MO, USA) for additional 5 weeks, respectively. All tested samples were given orally once daily at 2:00-3:00 p.m..
It is reported that cinnamaldehyde alleviates hyperlipidemia in C57BLKS/J db/db mice at 20 mg/kg 31 , reduces oxidative stress in myocardial tissue in a rat model of ischemic myocardial injury at 22.5, 45 and 90 mg/kg 36 , and protects against cerebral ischaemia injury of mice at 25, 50 and 75 mg/kg 62 . Our previous study showed that allopurinol at 5 mg/kg reduced oxidative stress and NLRP3 inflammasome activation in the liver and kidney of fructose-fed rats 39,63 . Accordingly, based on our preliminary work and these reports, the doses of 20, 40 and 80 mg/kg cinnamaldehyde, as well as 5 mg/kg allopurinol were used in the present study.
Additionally, 15 rats were remained on regular chow for 10 weeks to serve as normal control. Animal body weight was detected weekly. Animal welfare and experimental procedures were carried out in accordance with the recommendations in the guidelines of the Ministry of Science and Technology of China (2006) and the related and Smad4 (F) were determined in NAC-pretreated H9c2 cells co-incubated with fructose, cinnamaldehyde and allopurinol, respectively. The relative protein levels of NLRP3, IL-1β , TGF-β and Smad4 were normalized to GAPDH or β -actin, respectively. Relative protein levels of p-Smad2/3 were normalized to Smad2/3, respectively (n = 7). Data were expressed as the mean ± SEM. # P < 0.05, ## P < 0.01, ### P < 0.001 vs normal cell control group; * P < 0.05, * * P < 0.01 vs fructose-vehicle cell group, or fructosevehicle + NAC control cell group.
ethnical regulations of Nanjing University [SYXK (SU) 2009-0017]. All experimental protocol involving animals were approved by the Institutional Animal Care and Use Committee of Nanjing University. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Body weight and 24-h food intake.
At the end of 10 weeks, rat body weight and 24-h food intake were detected in a metabolic cage as previously described, respectively 43 . SBP. SBP was measured by the tail-cuff system (Softron BP-98A; Softron, Tokyo, Japan) at the end of 10 weeks and the rats were conscious. Data were averaged for six-seven consecutive measurements.
OGTT and ITT. During the last week of feeding period, OGTT and ITT were performed as described before 43 .
Blood and tissue samples collection. After OGTT and ITT, all animals were allowed 3 days to recover wounds. Then, animals were anesthetized intraperitoneally using 50 mg/kg sodium pentobarbital and decapitated at 9:00-10:00 a.m. after a 16-h fast. Blood samples were centrifuged for 10 min to get the serum stored at − 80 °C for biochemical assays. Heart tissue samples were rapidly dissected on ice. Parts of them were immediately fixed for oil red O or Masson trichrome staining, while others were stored at − 80 °C for biochemical and Western blot analysis. (H) were assayed, and relative protein levels were normalized to GAPDH or β -actin (n = 7), respectively. Data are expressed as the mean ± SEM. ## P < 0.01, ### P < 0.001 vs normal animal control group or normal cell control group; * P < 0.05, * * P < 0.01, * * * P < 0.001 vs fructose-vehicle animal group or fructose-vehicle cell group.
Oil red O and Masson trichrome staining analysis. Rat heart tissues were fixed for one day at room temperature in Carnoy's fixative (ethanol: chloroform: acetic acid = 6:3:1) and preserved in 70% ethanol. Cardiac biopsies were dehydrated with a graded series of alcohol and embedded in paraffin. Specimens were cut in 4 μ m-thick sections on a rotary microtome and mounted on 3-aminopropyltriethoxysilane-coated glass slides. Each section was washed by distilled water and then stained with oil red O reagent (Jiancheng Biotechnology Co., Ltd., Nanjing, China) for 5-10 min. After washed with 60% isopropyl alcohol, the sections were deparaffinized in xylene, rehydrated in decreasing concentrations of alcohol in water, and re-stained with Masson trichrome reagent (Google Biological Technology Co., Ltd., Wuhan, China), respectively. The slides were mounted with neutral balsam.
Cinnamaldehyde, allopurinol, SSO or IRAK1/4 inhibitor I dissolved in DMSO, NAC dissolved in ultrapure water at the respective stock concentrations, were directly added to cell culture medium. The final concentration of DMSO in culture medium was maintained at 0.1%. The selected concentrations and incubation time of these reagents were referred to preliminary experiments and other reports [65][66][67][68][69] . Cell culture supernatants were collected. Cell lysates were obtained by cell lysis buffer, and total cellular proteins were extracted, respectively. These samples were stored at − 80 °C before biochemical and Western blot analysis.

RNA isolation and qRT-PCR analysis.
Total RNA was isolated from H9c2 cells using Trizol reagent (Invitrogen) according to the manufacturer's instructions, respectively. The reverse transcription reaction of mRNAs has been previously published 62 . The primers used were listed in Supplementary Table S1. The reverse transcription reaction products were amplified by qRT-PCR with iTaq TM Universal SYBR ® Green Supermix (Bio-Rad) and respective primers. Specificity of the amplification was confirmed using a melting curve analysis. Data were collected and recorded by CFX Manager Software (Bio-Rad), and expressed as a function of threshold cycle (Ct). The samples for qRT-PCR analysis were evaluated using a single predominant peak as a quality control. Relative expressions of target genes were determined by the Ct (2 −ΔΔCt ) method. mRNAs were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively.
Assay of TG, TC, LDL-c and ox-LDL concentrations. Lipids were extracted from rat serum, heart tissue and H9c2 cells by liquid phase extraction using chloroform/methanol (2:1) as previously described 43 . TG, TC and LDL-c levels were determined with standard diagnostic kits (Jiancheng Biotechnology Co., Ltd., Nanjing, China), respectively. Rat heart tissue samples were homogenized in lysis buffer and then centrifuged at 12000× g for 15 min at 4 °C. Ox-LDL concentrations in serum was determined by ELISA kit (Shanghai Lianshuo Biological Technology Co., Ltd., Shanghai, China).
Determination of oxidative stress. Heart tissue was homogenized in PBS and centrifuged (10,000× g, 4 °C) for 15 min. H9c2 cells were detached from the wells by 0.25% trypsin digestion. Total ROS assay (Beyotime Institute of Biotechnology, Haimei, China) was performed with cell-containing aliquots (transferred to 96-well plate at 1 × 10 4 cells/well), while NADPH oxidase and XOD activity assays were performed on cell lysates using a  = 7), respectively. The relative protein levels were normalized to GAPDH or β -actin. Data are expressed as the mean ± SEM. ## P < 0.01 vs normal cell control group; * P < 0.05, * * P < 0.01 vs fructose-vehicle cell group, or fructose-vehicle + NAC control cell group, respectively.