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The present study was taken as an effort to assess the effects of paeonol on diabetic cardiomyopathy. Diabetes was induced in separate groups of Sprague-Dawley rats using streptozotocin. Treatment group animals received paeonol (50, 100 or 200 mg/kg body weight/day; orally) 5 weeks after streptozotocin induction for 6 weeks. Paeonol strikingly reduced myocardial apoptosis and improved cardiac function and myocardial architecture. Serum levels of glucose, reactive oxygen species and inflammatory mediators (TNF-α, IL-6 and IL-1β) were significantly reduced with decreased accumulation of collagen in the cardiac tissue. Paeonol modulated p-Akt, glycogen synthase kinase-3β and glycogen synthase, while significantly down-regulated protease-activated receptor-1, caspase-3, TNF-α, NF-κB p65, and p-Iκ-Bα expressions. Paeonol effectively suppressed diabetic cardiomyopathy by improving myocardial function, regulating the inflammatory responses and Akt signalling.

Introduction

Diabetes mellitus, one of the major health problems worldwide, is increasing alarmingly and by 2030 about 439 million people would be diabetics (Shaw et al., 2010). Diabetic cardiomyopathy, one of the major cardiovascular complications in type-2 diabetes mellitus (Mazzone et al., 2008), is characterized by cardiac dysfunction owing to myocardial structural and functional changes eventually leading to heart failure (Sun et al., 2011).

Accumulating evidences suggest varied mechanisms to be associated with diabetic cardiomyopathy (Wen et al., 2013), including excessive generation of reactive oxygen species (ROS) (Kajstura et al., 2001; Pacher et al., 2007; Wang et al., 2009), activation of inflammatory signals, transcription factors as NF-κB (Aragno et al., 2006; Wang et al., 2009), poly (ADP-ribose) polymerase (Pacher et al., 2002), MAPK cascades (Westermann et al., 2006; Thandavarayan et al., 2009), down-regulation of survival pathways as Akt (Van Linthout et al., 2008), alterations in the extracellular matrix composition (Westermann et al., 2007) and activation of cell death pathways (Frustaci et al., 2000).

ROS-induced oxidative stress, a key factor in diabetic cardiomyopathy etiology (Dewanjee et al., 2009; Bhattacharya et al., 2013) perturbs expressions of several transcription proteins including NF-κB (Bhattacharya et al., 2013). Inflammatory responses critically influence progression of diabetic cardiomyopathy (Lorenzo et al., 2011; Teixeira-Lemos et al., 2011) and cytokines as TNF-α, IL-6 and IL-1β are reported to potentially cause cardiac injury (Bhattacharya et al., 2013).

Furthermore, protease-activated receptors (PARs) are crucial modulators of inflammation (Cocks and Moffatt, 2000). PAR1, up-regulated by oxidative stress (Nguyen et al., 2001) has been shown to influence Akt signalling (Latha et al., 2015). Glycogen synthase kinase-3β (GSK-3β), a key effector, downstream of Akt, is the chief regulator of glycogen synthase in heart and skeletal muscle (Markou et al., 2008; Patel et al., 2008). In the presence of insulin, activated Akt phosphorylates and inhibits GSK-3β causing activation of glycogen synthase (Muniyappa et al., 2007). In diabetes, GSK-3β is activated by decreased phosphorylation (Montanari et al., 2005; Lajoie et al., 2004) and it negatively regulates glycogen synthase and inhibits glycogen synthesis, indicating the critical role of GSK-3β. Thus, Akt signalling could be a potential target in diabetic cardiomyopathy.

Paeonol, a major phenolic compound of genus Paeonia, is used as a nutrient supplement and in Chinese medicine (Deng et al., 2006). It possess bioactive properties as anti-inflammatory (Du et al., 2010; Siu, 2010; Himaya et al., 2012; Lin et al., 2015), neuroprotective (Hsieh et al., 2006; Wu et al., 2008; Su et al., 2010) and cardioprotective effects (Li et al., 2012). Considering these effects we investigated if paeonol regulated inflammatory responses and Akt/GSK-3β signalling in diabetic cardiomyopathy.

Materials and Methods

Sprague-Dawley rats weighing 180-220 g (Central South University Animal Centre, China) were used for this study. The animals were kept in standard animal cages on a 12 hours/12 hours dark/light cycle with free access to standard rat chow and tap water.

Antibodies against Akt, p-Akt, GSK-3β, p-GSK-3β,TNF-α, NF-κB p65, p-IκBα, caspase-3 were procured from Santa Cruz Biotechnology, USA. Antibodies against glycogen synthase, p-GS were obtained from Cell Signalling Technology (USA). Paeonol and streptozotocin was obtained from Sigma-Aldrich, MO, USA. All chemicals and reagents used in the study were obtained from Sigma-Aldrich, USA unless otherwise specified.

Diabetes was induced in the animals by a single injection of streptozotocin, intraperitoneally (50 mg/kg) after 8-12 hours of starvation. Streptozotocin was dissolved in 0.1 M sodium citrate buffer, pH 4.5 (Sun et al., 2011). Control rats were not induced with streptozotocin and were administered equal volumes of citrate buffer. After 72 hours of streptozotocin injection, the blood glucose level was measured by tail vein puncture using glucometer (Accu-Chek, Germany). Animal with a random serum glucose level >200 mg/dL was considered as diabetic. Blood glucose level was monitored periodically. Following 5 weeks of diabetic induction the diabetic rats were grouped separately. Treatment groups received paeonol at 50, 100 or 200 mg/kg orally every day starting from 36^th day for a period of 6 weeks. Animals that did not receive paeonol were grouped as diabetic control. At the end of the experimental protocol the cardiac function measurements were obtained, rats were sacrificed under isoflurane anesthesia and their heart tissues were harvested for further analysis. Whole blood from the aorta was collected and serum was separated and used.

On the day one before streptozotocin injection, 5 and 11 weeks after streptozotocin induction cardiac function was assessed by echocardiography using GE Vivid 7 ultrasound with 10-MHz transducer (General Electric, USA) as described by Wen et al. (2013) with slight modifications. The rats were exposed to isoflurane anesthesia (3%) and were placed in the supine position and the left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured on the parasternal left ventricular long axis view. The measurements taken represent the mean of consecutive 5 cardiac cycles. The LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV) and LV fractional shortening (LVFS), LV ejection fraction (LVEF) were calculated by use of computer algorithms of the ultrasound systems. All the measurements were taken by an investigator who was blinded to the experimental grouping.

Heart tissue samples were fixed in 4% buffered paraformaldehyde and were embedded in paraffin. Histological architecture was assessed by staining with hematoxylin and eosin. Tissue sections of 3 µm thickness were fixed on slides and were stained with hematoxylin and eosin and then observed by light microscopy (Nikon Eclipse 400). Collagen content of the cardiac tissues (5 μm section) was assessed using sirius red stain.

Content of collagen I and III and expression of cleaved caspase-3 in the heart tissues were examined by immunohistochemistry. The tissue sections were blocked with 5% bovine serum albumin in tris buffered saline (pH 7.4) for about 2 hours. Separate sections were then incubated with primary antibody (PAR1) (Santa Cruz Biotechnology Inc., USA), cleaved caspase-3 antibody (Cell signalling Technology, USA), antibody against collagen I (Col I; Abcam, USA) or collagen III (Col III; Abcam, USA) overnight at 4°C. Following incubation, the slides were washed and incubated further with biotin-labelled secondary antibody (Vector lab, USA) for 1 hour at room temperature followed by color development with diaminobenzidine solution (DAB) (Vector lab, USA). The positive signals were quantified with Image Pro Plus 6.0 software.

Myocardial apoptosis was determined by TUNEL (Terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labelling) staining. Heart tissue samples were fixed in 4% buffered paraformaldehyde and were embedded in paraffin. The tissue sections were deparrafinized by washing with xylene, rehydrated in graded alcohol series and then immersed with PBS. The assay was carried out according to manufacturer’s instructions (R and D systems, USA). In brief, the deparrafinised tissue sections were incubated with 20 µg/mL proteinase K for 15 min followed by washing with PBS and then incubated with TdT labelling buffer for 5 min. The sections were treated with labelling reaction mix and incubated for about 1 hour. After washing the sections 50 μL of Strep-HRP solution and incubated for 10 min at 37°C. The cells were then stained with 3, 3’-diaminobenzidine (DAB) counter-stained and were visualised.

Proteins were isolated from homogenized heart tissue using Trizol reagent (Invitrogen, USA) as previously described (Gao et al., 2008; Wei et al., 2010). Protein concentrations were determined by Bradford assay using protein assay kit from Bio-Rad (BioRad Laboratories, USA). Equal amount of protein (50 µg) from each group were separated elctrophoretically on a SDS-PAGE. The gel was blotted and transferred on to nitrocellulose membrane and incubated with primary antibodies against Akt, p-Akt (ser 473), p-GSK-3β (ser 9), GSK-3β, p-NF-κB p65 (ser 536), p-IκBα, TNF-α, glycogen synthase and p-GS overnight at 4°C. Membranes were washed thrice with TBST buffer and further incubated with secondary antibodies coupled to horseradish peroxidase (1:1000 dilution, Santa Cruz Biotechnology) for 2 hours at room temperature. The immunoreactive bands were visualized with a chemiluminescene system (Amersham Bioscience, UK). The signals were quantified by densitometry.

Serum concentrations of TNF-α, IL-1β, IL-6 were measured by enzyme-linked immunosorbent assay (ELISA) (R and D systems, USA) in accordance to the manufacturer’s instructions. Serum concentrations of TNF-α, IL-1β, IL-6 were measured by enzyme-linked immunosorbent assay (ELISA) (R and D systems, USA) in accordance to the manufacturer’s instructions. In brief, 50 µL of the serum sample was added to each well and incubated with the assay diluent for about 2 hours following incubation with specific antibody conjugate and incubated further for 2 hours, after washing 100 µL of the substrate solution was added to each well. The color developed was read at 450 nm.

Total ROS in serum were determined using Oxitest^TM ROS/RNS assay kit (Cell Biolabs, USA) according to manufacturer’s instructions. The assay employs dichlorodihydrofluorescein (DCFH), fluorogenic probe. In the presence of ROS, DCFH is rapidly oxidized to highly fluorescent DCF. Fluorescence is read on a standard fluorometric plate reader at 480 nm excitation/530 nm emission. Briefly, 50 µL of the serum sample 96-well plate and 50 µL of the catalyst was added to each well and incubated for 5 min followed by addition of 100 µL of DCFH and incubated further at room temperature for about 30 min and the fluorescence was measured.

The data are presented as mean ± SD from three or six independent experiments. The statistical significance between the groups were analysed by ANOVA (one-way analysis of variance) at p<0.05 followed by Duncan’s multiple range test (DMRT) for post hoc analysis.

Results

Diabetic animals presented elevated (p<0.05) levels of blood glucose. However rats treated with paeonol exhibited decreased blood glucose levels compared to diabetic control animals (Figure 1). In addition, the trend of decline in the blood glucose levels were more in the rats administered with 100 and 200 mg doses of paeonol than 50 mg. A periodic decrease in the levels was observed on paeonol treatment.

Figure 1:Effect of paeonol on serum glucose levels following streptozotocin induction Values are represented as mean ± SD, n=6. a represents statistical significance at p<0.05 compared against respective controls and b-f represents significant difference (p<0.05) between mean values within the groups as determined by one-way ANOVA followed by DMRT analysis

Collagen deposition is a feature observed in cardiomyopathy. Collagen deposition affects the mechanical properties of the myocardium and also affects the performance. In our study, marked changes in the deposition of total collagen were observed through sirius red staining (Figure 2). Significantly increased collagen content in the perivascular and as well in interstitial sites in the diabetic control animals was observed. Further immunohistochemistry studies revealed increased deposition of both collagen I and collagen III content in the cardiac tissue of diabetic rats. Paeonol reduced the deposition of collagen content, thus reducing cardiac fibrosis (Figure 3).

Figure 2:Sirius red staining showing the effect of paeonol on collagen deposition in cardiac tissues. The myocardium of the diabetic control rats presents with raised collagen accumulation (b) as compared against normal control rats (a) Paeonol treatment caused marked decreased collagen deposition in the myocardium as seen by sirius red staining (c-e); (a) Control; b) streptozotocin; c) 50 mg paeonol + streptozotocin; d) 100 mg paeonol + streptozotocin; e) 200 mg paeonol + streptozotocin)

Figure 3: Effect of paeonol on collagen deposition in cardiac tissues
Values are represented as mean ± SD; n=6; a represents statistical significance at p<0.05 compared against respective controls and b-f represents significant difference (p<0.05) between mean values within the groups as determined by one-way ANOVA followed by DMRT analysis

The tissue sectioning of control rats not induced with streptozotocin or treated with paeonol showed normal cardiac tissue with striated branched muscle fibres with homogeneous cytoplasm (Figure 4). Diabetes mellitus lead to a vast alterations in the myocardium. Severe degeneration of cardiac cells with interstitial fibrosis was observed. The sections exhibited irregular orientation of muscle fibres with shruken nuclei and condensed sarcoplasm. However, paeonol treatment substantially reversed the orientation of muscle fibres and improved the architecture of the myocardium. Treatment with paeonol at 200 mg noticeably restored the histology to almost near normal. The results observed were found to be dose-dependent with the highest dose exhibiting the maximum protective effects.

Figure 4:Histological section showing the effect of paeonol restored the normal myocardial architecture. Marked alterations in the myocytes and shrunken nuclei observed in cardiac tissues of diabetes-induced rats (b) as compared to normal control (a). These aberrant alterations were effectively restored on paeonol treatment (c-e). Paeonol treatment normalised the orientation of the cardiac fibrils and reduced fibrosis of the myocardium. (a) Control; b) streptozotocin; c) 50 mg paeonol + streptozotocin; d) 100 mg paeonol + streptozotocin; e) 200 mg paeonol + streptozotocin)

Cardiac functioning was performed at baseline 5 weeks after streptozotocin injection and 6 weeks after paeonol treatment (Table I). The observed results indicated that paeonol improved LVEF and LVFS in diabetic rats compared with the diabetes group. However, paeonol restored the systolic and diastolic diameters (LVESD and LVEDD). It also significantly reduced LVESV and LVEDV. The results suggest that paeonol administration strikingly improved the cardiac function.

	LVEDD (mm)			LVESD (mm)
Groups	Baseline	5 weeks after STZ and prior paeonol treatment	6 weeks after paeonol treatment	Baseline	5 weeks after STZ and prior paeonol treatment	6 weeks after paeonol treatment
Control	2.8 ± 0.1^a	2.8 ± 0.0^b	2.8 ± 0.1^c	1.6 ± 0.1^a	1.6 ± 0.1^b	1.6 ± 0.1^d
STZ Control	2.7 ± 0.1^a	3.4 ± 0.2^a	3.4 ± 0.2^a	1.5 ± 0.1^a	2.3 ± 0.1^a	2.3 ± 0.2^a
Paeonol 50 mg + STZ	2.7 ± 0.1^a	3.4 ± 0.2^a	3.0 ± 0.1^b	1.5 ± 0.1^a	2.2 ± 0.1^a	1.9 ± 0.1^b
Paeonol 100 mg + STZ	2.7 ± 0.1^a	3.3 ± 0.2^a	2.9 ± 0.2^b	1.6 ± 0.1^a	2.2 ± 0.2^a	1.8 ± 0.1^c
Paeonol 200 mg + STZ	2.8 ± 0.2^a	3.4 ± 0.1^a	2.8 ± 0.3^c	1.6 ± 0.2^a	2.3 ± 0.1^a	1.8 ± 0.2^c
	LVESV (mL)			LVEDV (mL)
Groups	Baseline	5 weeks after STZ and prior paeonol treatment	6 weeks after paeonol treatment	Baseline	5 weeks after STZ and prior paeonol treatment	6 weeks after paeonol treatment
Control	0.3 ± 0.1^a	0.3 ± 0.1^b	0.2 ± 0.0^c	1.1 ± 0.1^a	1.1 ± 0.1^c	1.1 ± 0.0^c
STZ Control	0.2 ± 0.1^a	0.4 ± 0.0^a	0.4 ± 0.0^a	1.0 ± 0.0^a	1.2 ± 0.1^a	1.2 ± 0.1^a
Paeonol 50 mg + STZ	0.3 ± 0.0^a	0.4 ± 0.0^a	0.3 ± 0.0^a	1.1 ± 0.1^a	1.2 ± 0.0^a	1.2 ± 0.1^a
Paeonol 100 mg + STZ	0.3 ± 0.0^a	0.4 ± 0.0^a	0.3 ± 0.1^b	1.1 ± 0.1^a	1.2 ± 0.1^a	1.1 ± 0.0^b
Paeonol 200 mg + STZ	0.2 ± 0.0^a	0.4 ± 0.1^a	0.2 ± 0.0^c	1.1 ± 0.1^a	1.3 ± 0.1^b	1.0 ± 0.1^c
	LVEF (%)			LVFS (%)
Groups	Baseline	5 weeks after STZ and prior paeonol treatment	6 weeks after paeonol treatment	Baseline	5 weeks after STZ and prior paeonol treatment	6 weeks after paeonol treatment
Control	76.5 ± 3.4^a	75.3 ± 5.8^a	76.7 ± 3.5^a	45.2 ± 1.5^a	45.2 ± 2.5^a	46.0 ± 1.0^a
STZ Control	78.7 ± 4.8^a	68.3 ± 4.5^b	69.1 ± 4.2^b	44.5 ± 2.1^a	32.8 ± 3.1^b	33.2 ± 3.5^c
Paeonol 50 mg + STZ	77.1 ± 2.2^a	66.1 ± 3.0^b	71.2 ± 2.2^b	43.3 ± 1.2^a	33.7 ± 2.1^b	38.2 ± 1.9^b
Paeonol 100 mg + STZ	76.3 ± 1.4^a	64.1 ± 2.1^b	73.0 ± 6.5^b	44.3 ± 1.9^a	33.5 ± 2.0^b	41.2 ± 2.7^b
Paeonol 200 mg + STZ	76.1 ± 4.5^a	65.9 ± 6.1^b	78.9 ± 7.6^a	45.1 ±2.2^a	32.7 ± 1.5^b	46.0 ± 1.1^a

As represented in Figure 5, paeonol remarkably reduced apoptotic cell counts in the cardiac tissue. TUNEL positive cells were found to be reduced by paeonol in a dose-dependent manner. Further, immunohistochemistry analysis for activated caspase-3 expressions also supported the results. Increase in caspase 3 positive cell counts (p<0.05) in the cardiac tissue of DM control rats were significantly reduced on paeonol treatment, with 200 mg dose exhibiting the maximum anti-apoptotic effects.

Figure 5: Paeonol reduces the intensive apoptosis in diabetic cardiomyopathy following streptozotocin (STZ) induction. Paeonol reduced the apoptotic cell counts markedly as determined by decrease in TUNEL positive cells (A) and also decreased the expression of cleaved caspase-3 (B),
Values are represented as mean ± SD, n=6. a represents statistical significance at p<0.05 compared against respective controls and b-f represents significant difference (p<0.05) between mean values within the groups as determined by one-way ANOVA followed by DMRT analysis

To further elucidate the possible mechanisms involved in the cardioprotective effects of paeonol, we investigated the effects of paeonol on the PI3K/Akt/GSK-3β signalling. The critical role of Akt in regulating glycogen synthesis is well documented, cell growth and survival. GSK-3β, a critical downstream element of Akt that is inhibited by phosphorylation regulates the activity of glycogen synthase, thereby regulating glycogen synthesis. The level of GSK-3β was enhanced in diabetic rats. However, the levels of Akt and p-Akt were supressed in diabetic rats. Enhanced p-GS expression was in line with GSK-3β level, suggesting the inhibition of glycogen synthase activity and glycogen synthesis contributing to enhanced blood glucose level (Figure 6). Paeonol interestingly caused a multifold increase in the phosphorylated level of Akt and GSK-3β with enhanced glycogen synthase expression, thus promoting glycogen synthesis and reduction of blood glucose level. Increased p-Akt caused increased p-GSK-3β, which further resulted in activation of glycogen synthase. These observations suggest the capacity of paeonol in promoting glycogen synthesis and also reducing blood glucose level as well.

Figure 6: Influence of paeonol on the expressions of PI3K/Akt signalling pathway proteins. Paeonol markedly down-regulated active GSK-3β and increased phosphorylated levels of Akt, GS and GSK-3β (A). Relative expressions of proteins (B)
Values are represented as mean ± SD, n=6. a represents statistical significance at p<0.05 compared against respective controls and b-f represents significant difference (p<0.05) between mean values within the groups as determined by one-way ANOVA followed by DMRT analysis (L1-Control; L2-streptozotocin; L3-50 mg paeonol + streptozotocin; L4-100 mg paeonol + streptozotocin; L5-200 mg paeonol + streptozotocin)

Hyperglycemia increases glucose oxidation and generation of ROS. ROS has been suggested to be involved in cardiac dysfunctions in diabetic conditions (Wang et al., 2011). Further, oxidative stress is associated with inflammation (Zhang et al., 2010). Inflammatory responses have been known to play significant influence on the cardiovascular complications in diabetes. We assessed the influence of paeonol on inflammatory markers and ROS levels. Significant (p<0.05) elevation in the expression levels of NF-κB p65 and p-IκBα was observed in diabetic control rats (Figure 7). The transcription factor, NF-κB is well known to play a crucial role in the regulation of various genes involved in inflammatory responses (Sun et al., 2011). Increase in p-IκBα contributes to release of active NF-κB. The serum levels of inflammatory mediators - IL-6, IL-1β and TNF-α were found to be increased several fold in diabetic control rats (Figure 7). Paeonol treatment decreased the expression of NF-κB p65 and p-IκBα and serum levels of inflammatory mediators and raised the level of ROS. Expression of TNF-α protein correlated with the serum levels. TNF-α is an important pro-inflammatory cytokine and also a valuable marker in heart failure (Miettinen et al., 2008; Vaz Perez et al., 2010). Thus, significant reduction in the levels of TNF-α and also ROS levels following paeonol treatment indicates the protective effects of paeonol on cardiac tissues in diabetes.

Figure 7: Antioxidant and anti-inflammatory effects of paeonol. Paeonol effectively down-regulated expressions of NF-κBp65 and p-Iκ-Bα in the cardiac tissues (A and B), reduced serum levels of inflammatory mediators (C) and decreased serum ROS levels (D)
Values are represented as mean ± SD, n=6. a represents statistical significance at p<0.05 compared against respective controls and b-f represents significant difference (p<0.05) between mean values within the groups as determined by one-way ANOVA followed by DMRT analysis (L1-Control; L2-streptozotocin; L3-50 mg paeonol + streptozotocin; L4-100 mg paeonol + streptozotocin; L5-200 mg paeonol + streptozotocin)

As presented in Figure 8, diabetes mellitus-induced rats presented diabetic cardiomyopathy and expressed elevated number of stained PAR1 positive cells (p<0.05) as compared to control rats. Treatment with paeonol considerably reduced the PAR1 expression in a dose-dependent manner. Activation of PAR1 has been reported to mediate inflammatory responses and in contractility (Steinberg, 2005). Thus, down-regulation of PAR1 in paeonol treatment suggests the anti-inflammatory effects.

Figure 8: Effect of paeonol on the expressions of PAR1. Paoenol significantly down-regulated the expressions of PARI in the myocardial tissues
Values are represented as mean ± SD, n=6. a represents statistical significance at p<0.05 compared against respective controls and b-f represents significant difference (p<0.05) between mean values within the groups of same cell line as determined by one-way ANOVA followed by DMRT analysis

Discussion

Paeonol effectively reduced blood glucose levels over the period of treatment, suggesting the antihyperglycemic effects. In addition paeonol significantly attenuated oxidative stress which was evidenced by the observed striking decrease in ROS level and also reduced apoptosis of cardiomyocytes. TUNEL positive cell counts drastically reduced on paeonol treatment. Anti-apoptotic effect of paeonol was also revealed by substantial reduction in cleaved caspase-3 positive cell counts.

Diabetic cardiomyopathy exhibits ventricular dysfunction with increased risk of heart failure (Tarquini et al., 2012). Consistently with previous reports (Sun et al., 2011; Yu et al., 2012), streptozotocin-induced rats exhibited features of diabetic cardiomyopathy with declined diastolic and systolic myocardial performance and excessive accumulation of collagen and severe alteration in cardiac tissue architecture. Perinuclear vacuolization, destruction and loss of myofibrils, shrunken nuceli, irregular orientation of fibrils, swollen mitochondria were observed in the cardiac tissue of streptozotocin-induced diabetes mellitus control rats. Paeonol markedly reversed the orientation and normalized alterations in myofilaments.

Many phytochemicals have the capacity to neutralize free radicals in diabetes (Jovanovic et al., 2001; Rajesh et al., 2010) and also exhibit anti-apoptotic effects (Rajesh et al., 2010; Sun et al., 2011). Oxidative stress due to excessive free radicals has been reported to play crucial roles in the development of cardiac failure and left ventricular dysfunctioning and remodelling in diabetic cardiomyopathy (Rajesh et al., 2010). Hyperglycemia-induced oxidative stress is considered as a major risk factor contributing to the vascular pathogenesis in the diabetic myocardium, that leads to fibrosis, hypertrophy of the cardiac tissue, myocardial cell death and endothelial dysfunction (Cai and Kang, 2003; Fang et al., 2004; Li et al., 2005). Wang et al. (2011) reported exacerbated glucose oxidation in hyperglycemia that leads to generation of ROS, which subsequently causes DNA damage and apoptosis. Earlier reports have demonstrated myocardial apoptosis in diabetic patients (Frustaci et al., 2000) as well in diabetic animal models (Kajstura et al., 2001; Cai et al., 2002). Furthermore, apoptosis associated with raised level of oxidative stress in multiple organ systems in diabetes mellitus has been well documented (Alici et al., 2000; Cai et al., 2000). Thus, striking suppression of ROS levels could have attributed to the decrease in the apoptotic counts suggests the protective effect of paeonol on cardiac tissue.

Immunohistochemical analysis revealed accumulation of collagen I and III, which account for major collagen in the cardiac tissue, resulting in diabetes-induced cardiac fibrosis. Previous studies have shown alterations in the extracellular matrix associated with cardiac fibrosis (Tschope et al., 2004; Westermann et al., 2007; Zhang et al., 2008). Thus, paeonol-induced striking decreases in collagen accumulation of the cardiac tissues, suggests its capacity in inhibiting cardiac fibrosis. In our study, paeonol significantly attenuated the cardiac systolic and diastolic dysfunction in experimental diabetic cardiomyopathy. Paeonol improved left ventricular functioning and restored the systolic and diastolic volume.

Hyperglycemia-induced-ROS generation has been reported to impair vital cell survival pathways as Akt signalling cascades (Van Linthout et al., 2008). In our study, marked decline in active Akt and glycogen synthase levels were observed with increased expression of GSK-3β. Paenol interestingly down-regulated GSK-3β and increased expressions of p-Akt, p-GSK-3β and glycogen synthase. PI3K/Akt signalling is involved in regulating cell survival and apoptosis. Akt promotes cell survival, inhibits proteins involved in apoptotic cascade and additionally regulates cardiovascular functions (Katare et al., 2010). GSK-3β, a major substrate of Akt functions as negative regulator of glycogen synthesis and glucose metabolism and also plays a crucial role in the apoptotic signalling cascades (Muniyappa et al., 2007; Wang et al., 2009; Liu et al., 2010). Our results demonstrated that paeonol possibly exerts its protective effects through modulation of Akt/GSK-3β pathways.

Further, paeonol exerted anti-inflammatory effects by significantly blocking the expression of NF-κBp65, TNFα and p-Iκ-Bα in the cardiac tissues. In streptozotocin-induced diabetic control rats, increased expression of PAR1 also could have contributed to diabetic cardiomyopathy and inflammation. Expression of cardiac inflammatory markers such as TNF-α, IL-1β, and IL-6 are implicated in diabetic cardiomyopathy (Westermann et al., 2006; Thandavarayan et al., 2009). Studies have demonstrated that excessive ROS activate NF-κB (Aragno et al., 2006; Mariappan et al., 2010) which further cause the expression of important pro-inflammatory cytokines. TNF-α has been reported as a strong mediator in cardiac dysfunctioning and failure. TNF-α acts as a signal amplifier and intensifies inflammatory responses and also contributes to myocardial hypertrophy and fibrosis, leading to LV remodelling and dysfunction as well (Sun et al., 2007). Paeonol, administration to the rats for 6 weeks caused a remarkable decrease in the expression of TNF-α, NF-κBp65 and p-Iκ-Bα in the myocardium and also levels of serum IL-6, IL-1β and TNF-α. Thus, paeonol may serve as a potential candidate in preventing LV dysfunction.

PARs have been demonstrated to be linked with regulation of various cellular functions in the cardiovascular system (Shah, 2009). PARs are crucial for normal homeostasis and also are implicated in vascular disorders that are associated with chronic inflammation (Steinberg, 2005). Activation of PAR1 has been shown to mediate inflammatory responses and contractility (Steinberg, 2005). Down-regulation of PAR1 by paeonol appears to be a promising strategy to treat DCM in DM.

Collectively, the results of the study indicate the efficacy of paeonol in improving cardiac function and architecture. Paeonol also exerts cardioprotective effects via inhibiting inflammatory responses, oxidative stress and modulating Akt/GSK-3β signalling.

References

Alici B, Gümüstas MK, Ozkara H, Akkus E, Demirel G, Yencilek F, Hattat H. Apoptosis in the erectile tissues of diabetic and healthy rats. BJU Int. 2000; 85: 326-29.

Aragno M, Mastrocola R, Medana C, Catalano MG, Vercellinatto I, Danni O, Boccuzzi G. Oxidative stress dependent impairment of cardiac specific transcription factors in experimental diabetes. Endocrinology 2006; 147: 5967-74.

Bhattacharya S, Manna P, Gachhui R, Sil PC. D-Saccharic acid 1,4-lactone protects diabetic rat kidney by ameliorating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via NF-κB and PKC signaling. Toxicol Appl Pharmacol. 2013; 267: 16-29.

Cai L, Kang YJ. Cell death and diabetic cardiomyopathy. Cardiovasc Toxicol. 2003; 3: 219-28.

Cai L, Chen S, Evans T, Deng DX, Mukherjee K, Chakrabarti S. Apoptotic germ-cell death and testicular damage in experimental diabetes: Prevention by endothelin antagonism. Urol Res. 2000; 28: 342-47.

Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. Hyperglycemia induced apoptosis in mouse myocardium: Mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes 2002; 51: 1938-48.

Cai L, Wang Y, Zhou G, Chen T, Song Y, Li X, Kang YJ. Attenuation by metallothionein of early cardiac cell death via suppression of mitochondrial oxidative stress results in a prevention of diabetic cardiomyopathy. J Am Coll Cardiol. 2006; 48: 1688-97.

Cocks TM, Moffatt JD. Protease-activated receptors: Sentries for inflammation? Trends Pharmacol Sci. 2000; 21: 103-08.

Deng C, Yao N, Wang B, Zhang X. Development of microwave-assisted extraction followed by headspace single-drop microextraction for fast determination of paeonol in traditional Chinese medicines. J Chromatogr A. 2006; 1103: 15-21.

Dewanjee S, Das AK, Sahu R, Gangopadhyay M. Antidiabetic activity of Diospyros peregrina fruit: Effect on hyperglycemia, hyperlipidemia and augmented oxidative stress in experimental type 2 diabetes. Food Chem Toxicol. 2009; 47: 2679-85.

Dewanjee S, Gangopadhyay M, Sahu R, Karmakar S. Cadmium induced pathophysiology: Prophylactic role of edible jute (Corchorus olitorius) leaves with special emphasis on oxidative stress and mitochondrial involvement. Food Chem Toxicol. 2013; 60: 188-98.

Du Q, Feng GZ, Shen L, Cui J, Cai JK. Paeonol attenuates airway inflammation and hyperresponsiveness in a murine model of ovalbumin-induced asthma. Can J Physiol Pharmacol. 2010; 88: 1010-16.

Fang ZY, Prins JB, Marwick TH. Diabetic cardiomyopathy: Evidence, mechanisms, and therapeutic implications. Endocr Rev. 2004; 25: 543-67.

Frustaci A, Kajstura J, Chimenti C, Jakoniuk I, Leri A, Maseri A, Nadal-Ginard B, Anversa P. Myocardial cell death in human diabetes. Circ Res. 2000; 87: 1123-32.

Gao HK, Yin Z, Zhou N, Feng XY, Gao F, Wang HC. Glycogen synthase kinase 3 inhibition protects the heart from acute ischemia-reperfusion injury via inhibition of inflammation and apoptosis. J Cardio Pharmacol. 2008; 52: 286-92.

Himaya SW, Ryu B, Qian ZJ, Kim SK. Paeonol from hippocampus kuda Bleeler suppressed the neuro-inflammatory responses in vitro via NF-κB and MAPK signaling pathways. Toxicol Vitro. 2012; 26: 878-87.

Hsieh CL, Cheng CY, Tsai TH, Lin IH, Liu CH, Chiang SY, Lin JG, Lao CJ, Tang NY. Paeonol reduced cerebral infarction involving the superoxide anion and microglia activation in ischemia-reperfusion injured rats. J. Ethnopharmacol. 2006; 106: 208-15.

Jovanovic SV, Boone CW, Steenken S, Trinoga M, Kaskey RB. How curcumin works preferentially with water soluble antioxidants. J Am Chem Soc. 2001; 123: 3064-68.

Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A, Anversa P. IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes 2001; 50: 1414-24.

Katare RG, Caporali A, Oikawa A, Meloni M, Emanueli C, Madeddu P. Vitamin B1 analog benfotiamine prevents diabetes-induced diastolic dysfunction and heart failure through Akt/Pim-1-mediated survival pathway. Circ Heart Fail. 2010; 3: 294-305.

Lajoie C, Calderone A, Trudeau F, Lavoie N, Massicotte G, Gagnon S, Beliveau L. Exercise training attenuated the PKB and GSK-3 dephosphorylation in the myocardium of ZDF rats. J Appl Physiol. 2004; 96: 1606-12.

Latha R, Shanthi P, Sachdanandam P. Protective role of kalpaamruthaa in type II diabetes mellitus-induced cardiovascular disease through the modulation of protease-activated receptor-1. Pharmacognosy Magazine 2015; 11: S209-16.

Li X, Xu Z, Li S, Rozanski GJ. Redox regulation of Ito remodelling in diabetic rat heart. Am J Physiol Heart Circ Physiol. 2005; 288: H1417-24.

Li H, Xie Y-H, Yang Q, Wang SW, Zhang BL, Wang JB, Cao W, Bi LL, Sun JY, Miao S, Hu J, Zhou XX, Qiu PC. Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats. PLoS One. 2012; 7: e48872

Lin C, Lin H-Y, Chen JH, Tseng WP, Ko PY, Liu YS, Yeh WL, Lu DY. Effects of paeonol on anti-neuroinflammatory responses in microglial cells. Int J Mol Sci. 2015; 16: 8844-60.

Liu Y, Tanabe K, Baronnier D, Patel S, Woodgett J, Cras-Méneur C, Permutt MA. Conditional ablation of Gsk-3beta in islet beta cells results in expanded mass and resistance to fat feeding-induced diabetes in mice. Diabetologia 2010; 53: 2600-10.

Lorenzo O, Picatoste B, Ares-Carrasco S, Ramírez E, Egido J, Tunon J. Potential role of nuclear factor κB in diabetic cardiomyopathy. Mediators Inflamm. 2011; 2011: 652097

Mariappan N, Elks CM, Sriramula S, Guggilam A, Liu Z, Borkhsenious O, Francis J. NF-kappaB-induced oxidative stress contributes to mitochondrial and cardiac dysfunction in type II diabetes. Cardiovasc Res. 2010; 85: 473-83.

Markou T, Cullingford TE, Giraldo A, Weiss SC, Alsafi A, Fuller SJ, Clerk A, Sugden PH. Glycogen synthase kinases 3alpha and 3beta in cardiac myocytes: Regulation and consequences of their inhibition. Cell Signal 2008; 20: 206-18.

Mazzone T, Chait A, Plutzky J. Cardiovascular disease risk in type 2 diabetes mellitus: Insights from mechanistic studies. Lancet 2008; 371: 1800-09.

Miettinen KH, Lassus J, Harjola VP, Siirila-Waris K, Melin J, Punnonen KR, Nieminen MS, Laakso M, Peuhkurinen KJ. Prognostic role of pro- and anti-inflammatory cytokines and their polymorphisms in acute decompensated heart failure. Eur J Heart Fail. 2008; 10: 396-403.

Montanari D, Yin H, Dobrzynski E, Agata J, Yoshida H, Chao J, Chao L. Kallikrein gene delivery improves serum glucose and lipid profiles and cardiac function in streptozotocin-induced diabetic rats. Diabetes 2005; 54: 1573-80.

Muniyappa R, Montagnani M, Koh KK, Quon MJ. Cardiovascular actions of insulin. Endocr Rev. 2007; 28: 463-91.

Nguyen KT, Frye SR, Eskin SG, Patterson C, Runge MS, McIntire LV. Cyclic strain increases protease activated receptor-1 expression in vascular smooth muscle cells. Hypertension 2001; 38: 1038-43.

Pacher P, Liaudet L, Soriano FG, Mabley JG, Szabo E, Szabo C. The role of poly(ADP-ribose) polymerase activation in the development of myocardial and endothelial dysfunction in diabetes. Diabetes 2002; 51: 514-21.

Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007; 87: 315-424.

Patel S, Doble BW, MacAulay K, Sinclair EM, Drucker DJ, Woodgett JR. Tissue-specific role of glycogen synthase kinase 3beta in glucose homeostasis and insulin action. Mol Cell Biol. 2008; 28: 6314-28.

Rajesh M, Mukhopadhyay P, Bátkai S, Patel V, Saito K, Matsumoto S, Kashiwaya Y, Horváth B, Mukhopadhyay B, Becker L, Haskó G, Liaudet L, Wink DA, Veves A, Mechoulam R, Pacher P. Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy. J Am Coll Cardiol. 2010; 56: 2115-25.

Shah R. Protease-activated receptors in cardiovascular health and diseases. Am Heart J. 2009; 157: 253-62.

Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract. 2010; 87: 4-14.

Siu D. A new way of targeting to treat coronary artery disease. J Cardiovasc Med. 2010; 1: 1-6.

Steinberg SF. The cardiovascular actions of protease-activated receptors. Mol Pharmacol. 2005; 67: 2-11.

Su SY, Cheng CY, Tsai TH, Hsiang CY, Ho TY, Hsieh CL. Paeonol attenuates H₂O₂-induced NF-κB-associated amyloid precursor protein expression. Am J Chin Med. 2010; 38: 1171-92.

Sun M, Chen M, Dawood F, Zurawska U, Li JY, Parker T, Kassiri Z, Kirshenbaum LA, Arnold M, Khokha R, Liu PP. Tumor necrosis factor-alpha mediates cardiac remodeling and ventricular dysfunction after pressure overload state. Circulation 2007; 115: 1398-1407.

Sun D, Shen M, Li J, Li W, Zhang Y, Zhao L, Zhang Z, Yuan Y, Wang H, Cao F. Cardioprotective effects of tanshinone IIA pretreatment via kinin B2 receptor-Akt-GSK-3β dependent pathway in experimental diabetic cardiomyopathy. Cardiovasc Diabetol. 2011; 10: 4

Tarquini R, Lazzeri C, Pala L, Rotella CM, Gensini GF. The diabetic cardiomyopathy. Acta Diabetol. 2012; 48: 173-81.

Teixeira-Lemos E, Nunes S, Teixeira F, Reis F. Regular physical exercise training assists in preventing type 2 diabetes development: focus on its antioxidant and anti-inflammatory properties. Cardiovasc Diabetol. 2011; 10: 12.

Thandavarayan RA, Watanabe K, Ma M, Gurusamy N, Veeraveedu PT, Konishi T, Zhang S, Muslin AJ, Kodama M, Aizawa Y. Dominant-negative p38{alpha} mitogen-activated protein kinase prevents cardiac apoptosis and remodeling after streptozotocin-induced diabetes mellitus. Am J Physiol Heart Circ Physiol. 2009; 297: 911-19.

Tschope C, Walther T, Koniger J, Spillmann F, Westermann D, Escher F, Pauschinger M, Pesquero JB, Bader M, Schultheiss HP, Noutsias M. Prevention of cardiac fibrosis and left ventricular dysfunction in diabetic cardiomyopathy in rats by transgenic expression of the human tissue kallikrein gene. Faseb J. 2004; 18: 828-35.

Van Linthout S, Spillmann F, Riad A, Trimpert C, Lievens J, Meloni M, Escher F, Filenberg E, Demir O, Li J, Shakibaei M, Schimke I, Staudt A, Felix SB, Schultheiss HP, De Geest B, Tschöpe C. Human apolipoprotein A-I gene transfer reduces the development of experimental diabetic cardiomyopathy. Circulation 2008; 117: 1563-73.

Vaz Perez A, Doehner W, von Haehling S, Schmidt H, Zimmermann AV, Volk HD, Anker SD, Rauchhaus M. The relationship between tumor necrosis factor-α, brain natriuretic peptide and atrial natriuretic peptide in patients with chronic heart failure. Int J Cardiol. 2010; 141: 39-43.

Wang Y, Feng W, Xue W, Tan Y, Hein DW, Li XK, Cai L. Inactivation of GSK-3beta by metallothionein prevents diabetes related changes in cardiac energy metabolism, inflammation, nitrosative damage, and remodeling. Diabetes 2009; 58: 1391-402.

Wang J, Wang H, Hao P, Xue L, Wei S, Zhang Y, Chen Y. Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats. Mol Med. 2011; 17: 172-79.

Wei L, Yin Z, Yuan Y, Hwang A, Lee A, Sun D, Li F, Di C, Zhang R, Cao F, Wang H. A PKC-beta inhibitor treatment reverses cardiac microvascular barrier dysfunction in diabetic rats. Microvascular Res. 2010; 80: 58-165.

Wen HL, Liang ZS, Zhang R, Yang K. Anti-inflammatory effects of triptolide improve left ventricular function in a rat model of diabetic cardiomyopathy. Cardiovasc Diabetol. 2013; 12: 50

Westermann D, Rutschow S, Van Linthout S, Linderer A, Bücker-Gärtner C, Sobirey M, Riad A, Pauschinger M, Schultheiss HP, Tschöpe C. Inhibition of p38 mitogen-activated protein kinase attenuates left ventricular dysfunction by mediating pro-inflammatory cardiac cytokine levels in a mouse model of diabetes mellitus. Diabetologia 2006; 49: 2507-13.

Westermann D, Rutschow S, Jager S, Linderer A, Anker S, Riad A, Unger T, Schultheiss HP, Pauschinger M, Tschöpe C. Contributions of inflammation and cardiac matrix metalloproteinase activity to cardiac failure in diabetic cardiomyopathy: The role of angiotensin type 1 receptor antagonism. Diabetes 2007; 56: 641-46.

Wu JB, Song NN, Wei XB, Guan HS, Zhang XM. Protective effects of paeonol on cultured rat hippocampal neurons against oxygen-glucose deprivation-induced injury. J Neurol Sci. 2008; 264: 50-55.

Yu W, Wu J, Cai F, Xiang J, Zha W, Fan D, Guo S, Ming Z, Liu C. Curcumin alleviates diabetic cardiomyopathy in experimental diabetic rats. PLoS One. 2012; 7: e52013.

Zhang L, Cannell MB, Phillips AR, Cooper GJ, Ward ML. Altered calcium homeostasis does not explain the contractile deficit of diabetic cardiomyopathy. Diabetes 2008; 57: 2158-66.

Zhang N, Andresen BT, Zhang C. Inflammation and reactive oxygen species in cardiovascular disease. World J Cardiol. 2010; 2: 408-10.

Zhang J, Yu XH, Yan YG, Wang C, Wang WJ. PI3K/Akt signaling in osteosarcoma. Clinica Chimica Acta. 2015; 444: 182-92.

Zhu Q, Yang J, Han S, Liu J, Holzbeierlein J, Thrasher JB, Li B. Suppression of glycogen synthase kinase 3 activity reduces tumor growth of prostate cancer in vivo. Prostate 2011; 71: 835-45.

| Research | Article |

Protective effects of paeonol on cardiovascular complications in diabetes mellitus involves modulation of PI3K /Akt-GSK-3β signalling, regulation of protease-activated receptor-1 expressions and down-regulation of inflammatory mediators

Abstract

Introduction

Materials and Methods

Results

Discussion

Ethical Issue

Conflict of Interest

References