Effect of Cinnamaldehyde on Glucose Metabolism and Vessel Function.

Background

As a major threat to human health, diabetes-related macrovascular and microvascular disease can cause serious complications, including coronary heart disease, stroke, renal failure, foot gangrene, and blindness, leading to high mortality and morbidity [1]. In patients with diabetes, factors such as long-term glucose and lipid metabolism disorders, insulin resistance, and oxidative stress can lead to vascular endothelial dysfunction, which refers to endothelial cell damage due to physical and biological changes such as ischemia and hypoxia, lipid deposition, and hemodynamics [2-4]. Oxidative stress is defined as the disturbance in the balance of antioxidants and pro-oxidants in the body. Specifically, it mainly involves the pathogenesis of complications through 5 major pathways: polyol pathway flux, increased formation of advanced glycation end-products (AGEs), increased expression of the receptor for AGEs and its activating ligands, activation of protein kinase C (PKC) isoforms, and overactivity of the hexosamine pathway [5-7]. Usually, the secretion of vasodilator and contraction factors in endothelial cells maintains a steady state [8]. The loss of balance caused by metabolic disorders and oxidative stress greatly influences endothelium-dependent vasodilatation and vascular permeability, leading to pathophysiological changes such as vascular inflammation, platelet aggregation, and atherosclerosis [9,10]. As a central part of diabetic vascular complications, vascular endothelial dysfunction plays an important role in diabetic cardiovascular complications [11,12].

At present, clinical and animal experiments have confirmed that cinnamaldehyde has obvious anti-diabetic effects such as blood glucose and lipid regulation, insulin resistance improvement, and antioxidation [13]. Nikzamir found that cinnamaldehyde down-regulates blood glucose by upregulating the expression of GLUT4 gene levels in mouse skeletal muscle [14]. Saifudin reported that cinnamaldehyde inhibited protein tyrosine phosphatase-1B (PTP-1B), which helps prevent type 2 diabetes and obesity [15]. Cinnamaldehyde also enhanced the antioxidant defense of reactive oxygen species produced in hyperglycemia, thereby protecting pancreatic β cells and having hypoglycemic effects [16]. In addition, cinnamaldehyde inhibited the biological effects of diabetic nephropathy induced by advanced glycation end- (AGE) products through inhibition of the NO pathway activated by the JAK2-STAT1/STAT3 pathway [17]. However, few reports have been published on the effect of cinnamaldehyde in the treatment of diabetic vascular endothelial dysfunction and prevention of diabetic vascular complications.

In this work, we explored the effect and mechanism of cinnamaldehyde on glucose metabolism and vessel function in db/db mice in order to provide references for further application of cinnamaldehyde in clinical trials.

Material and Methods

Animals and grouping

Ten healthy male C57BL/KsJ mice and 20 db/db mice (8 weeks of age, body weight 16~18 g) were provided by Zhongshan School of Medicine, Sun Yat-sen University (Certificate No. SYXK-2015-0107). With C57BL/KsJ mice as a control group, db/db mice were divided into a normal diet group (db/db ND, n=10) and a 0.2% cinnamaldehyde group (db/db CA, n=10) [18]. The animals were kept in an SPF-grade facility with food and water ad libitum. The intervention started after feeding for 1 week. Food intake was observed and recorded starting 10 days before the experiment. Fasting blood glucose was monitored weekly, while body weight and tail blood pressure were monitored monthly during the intervention time for 12 weeks. Mice were used for all experiments, and all procedures were approved by the Animal Ethics Committee.

Drugs and reagents

Drugs and reagents included: chloral hydrate solution (Shanghai Chem., China); potassium chloride (Sigma-Aldrich, USA); N-Nitroarginine Methyl Ester (Sigma-Aldrich, USA); Acetylcholine (Sigma-Aldrich, USA); Nitroglycerin (Nanjing Jiancheng Bioengineering Institute, China); hematoxylin dye (Nanjing Jiancheng Bioengineering Institute, China); Eosin dye(Nanjing Jiancheng Bioengineering Institute, China); hydrochloric acid (Sinopharm, China); xylene (Shanghai Lingfeng, China); anhydrous ethanol (Shanghai Chem., China); neutral gum (Shanghai Chem., China); paraformaldehyde (Shanghai Health, China); Embedding agent (Leica, Germany); mouse insulin antibody (Santa Cruz, USA); ordinary insulin (Lilly, USA); Alexa Fluor 594 Goat Anti-Mouse IgG Antibody (Invitrogen, USA); Alexa Fluor 488 Goat Anti-Mouse IgG Antibody (Invitrogen, USA); anti-fluorescent quenching seal liquid (Pik Wan Tian, China); D-hanks solution (Beijing Huayue Biotechnology); and DAF-2DA dye (Sigma-Aldrich, USA)

Insulin sensitivity monitoring

Mice received intraperitoneal injection 0.75 U/Kg regular insulin, followed by tail blood glucose measurement at 0 min, 15 min, 30 min, 45 min, and 60 min. The percentage of the value measured at each point to initial value was recorded as the final calculation.

H&E staining of pancreas islets

Mice were sacrificed and pancreatic tissue was treated with paraffin embedding. After paraformaldehyde fixation, tissue slides were then stained by hematoxylin and eosin (H&E). The dry slides, sealed by neutral gum, were observed under an upright microscope.

Fluorescence staining of pancreas islets

Pancreatic tissue was collected and treated with embedding agent at −20°C for 10 min. After paraformaldehyde fixation for 40 min, tissue slides were steeped in hydrogen peroxide/methanol solution for 30 min. Then, fetal calf serum was applied as a blocking agent for 30 min, followed by the addition of anti-insulin antibody or PBS (as control) with appropriate proportion, and left overnight. Goat anti-mouse IgG antibody labeled by FITC was then added and the slides were observed under an inverted fluorescence microscope.

Measurement of superoxide anion level in vascular tissue

Tissues were collected and treated with Krebs solution. DHE mother solution was added into Krebs solution for staining (30 min). Then, after washing 3 times with Krebs solution, the slides were observed under a fluorescence microscope.

NO level measurement of vascular tissues

After the mice were anesthetized with 4% chloral hydrate, the aorta and mesenteric artery were removed after separating the abdominal skin. DAF-2DA dye was added to the final concentration of 5 mmol/L and incubated at 37°C for 30 min. Samples were observed and photographed after washing 3 times with D-hanks solution.

Evaluation of vascular circle function

After preparation of the experimental solution (Krebs solution) and separation of aorta and mesenteric artery, vessel samples were fixed in a bath at 37°C. Tension was adjusted to 1 mN for 30 min. Then, KCl solution (60 mmol/L) was used for the priming experiment. After that, Phe (10−5 mol/L) was used for vasoconstriction and Ach solutions at different concentrations (10−9–10−5 mol/L) added when the reaction reached stationary stage in order to observe Ach-induced endothelium-dependent vasodilatation. Similarly, TG solutions at different concentrations (10−9–10−5 mol/L) were added when the reaction reached stationary stage in order to observe the effect of TG on vascular circle vasodilatation.

H&E staining of aorta and mesenteric artery

Aorta and mesentery with intestinal loop were separated and embedded with embedding agent. After fixation with paraformaldehyde, the slices were stained by hematoxylin and eosin (H&E). The condition of the slices was observed under an upright microscope.

Statistical method

SPSS 19.0 software was employed for statistical analysis. Measurement data are presented as mean ± standard deviation (SD). The t test was used to compare groups. Statistical significance was defined as p<0.05. All statistical results were plotted with GraphPad Prism 5.0 software.

Results

Effect of cinnamaldehyde on food intake

We observed and recorded the daily food intake of mice during consecutive 10 days from cinnamaldehyde intervention. Compared to the C57 Cont group, the db/db ND group had significantly increased daily food intake. No significant difference in daily food intake was observed between the db/db ND group and db/db CA group (Figure 1).

Figure 1

Effects of cinnamaldehyde on food intake in db/db mice (n=10). ** p<0.01 compared to C57 Cont group.

Effect of cinnamaldehyde on body weight

We compared the effects of cinnamaldehyde on body weight of the C57BL/KsJ group and db/db group. Currently, reported results of cinnamaldehyde on the body weight of db/db mice are controversial. Several studies showed that cinnamaldehyde reduced the body weight of db/db mice, while in recent years it has been revealed that cinnamaldehyde improved the weight of end-stage diabetic mice. In our work, after 12-week cinnamaldehyde intervention, body weight in the db/db CA group had a slight decrease comparing to the db/db ND group (p>0.05, Figure 2). Compared to the C57 Cont group, the db/db ND group had significant differences in body weight (p<0.01, Figure 2).

Figure 2

Effects of cinnamaldehyde on body weight in db/db mice. ** p<0.01 compared to C57 Cont group (n=10).

Effect of cinnamaldehyde on fasting blood glucose

In the monitoring of blood glucose of enrolled mice, we found that there were significant differences in fasting blood glucose between the db/db ND group and db/db CA group after the 8th week. Between the 9th and 12th weeks, fasting blood glucose showed significant differences between the 2 groups (p<0.05), indicating that cinnamaldehyde effectively reduced the fasting blood glucose of db/db mice (Figure 3).

Figure 3

Effects of cinnamaldehyde on fasting blood glucose of db/db mice. * p<0.01 compared to C57 Cont group; # p<0.05 compared to db/db CA group (n=10).

Effect of cinnamaldehyde on insulin sensitivity

Currently, it is widely believed that the pathological basis of type 2 diabetes is insulin sensitivity decrease and relatively low insulin secretion. In our work, we found that the insulin sensitivity of db/db mice increased after cinnamaldehyde intervention. There were significant differences (p<0.05) between the db/db ND group and db/db CA group, and there were significant differences (p<0.01) between the C57/BL/KsJ group and db/db CA group (Figure 4).

Figure 4

Effects of cinnamaldehyde on insulin sensitivity of db/db mice. * p<0.01 compared to C57 Cont group; # p<0.05 compared to db/db CA group (n=10).

Effect of cinnamaldehyde on the function and morphology of pancreatic islets

We used immunofluorescent staining and H&E staining of the pancreas to observe the effects of cinnamaldehyde on the function and morphology of pancreatic islets, respectively. Compared to the C57 Cont group, db/db mice had hypertrophic pancreas, disordered islet cells, and progressively damaged islet function. Immunofluorescence showed that the insulin content of pancreas islets in db/db mice was lower than that in the C57 Cont group. However, after cinnamaldehyde intervention, islet cell hypertrophy, cell disorder, and insulin level were improved in db/db mice (Figures 5, 6).

Figure 5

H&E staining of islet morphology in db/db mice (n=10).

Figure 6

Immunofluorescence staining of islet insulin levels in db/db mice (n=10).

Effect of cinnamaldehyde on superoxide anion and NO level in vascular tissues

ROS is a key factor caused diabetic vascular injury but NO has a protective effect on blood vessel endothelium. In our work, we found that compared to the C57BL/KsJ group, db/db mice had significantly increased superoxide anion level but decreased NO level. After the 12-week cinnamaldehyde intervention, the superoxide anion level significantly decreased while the level of NO significantly increased (Figures 7–14).

Figure 7

Effect of CA on mesenteric artery superoxide anion in db/db mice (n=10).

Figure 8

Effects of cinnamaldehyde on mesenteric artery NO level in db/db mice (n=10).

Figure 9

Effect of CA on mesenteric artery superoxide anion in db/db mice. * p<0.01 compared to C57 Cont group; # p<0.05 compared to db/db ND group (n=10).

Figure 10

Effect of CA on mesenteric artery NO level in db/db mice. ** p<0.01 compared to C57 Cont group; # p<0.05 compared to db/db ND group (n=10).

Figure 11

Effect of CA on thoracic aorta superoxide anion level in db/db mice (n=10).

Figure 12

Effect of CA on thoracic aorta NO level in db/db mice (n=10).

Figure 13

Effects of cinnamaldehyde on thoracic aorta superoxide anion level in db/db mice. ** p<0.01 compared to C57 Cont group; ## p<0.01 compared to db/db ND group (n=10).

Figure 14

Effect of cinnamaldehyde on thoracic aorta NO level in db/db mice. * p<0.05 compared to C57 Cont group; ## p<0.01 compared to db/db ND group (n=10).

Effect of cinnamaldehyde on blood pressure

With the application of non-invasive sphygmomanometer, the results of blood pressure indicated that cinnamaldehyde reduced systolic blood pressure and diastolic blood pressure, but without a significant difference, in db/db mice (p>0.05). However, there were significant differences between the db/db ND group and C57 Cont group (p<0.05, Figure 15).

Figure 15

Effect of cinnamaldehyde on blood pressure in db/db mice. * p<0.05 compared to C57 Cont group (n=10; SBP – systolic blood pressure; DBP – diastolic blood pressure).

Effect of cinnamaldehyde on endothelium-dependent diastolic function

In our experiment, we found that cinnamaldehyde improved acetyl choline-induced endothelium-dependent vasodilatation function in db/db mice. There were significant differences between db/db the ND group and db/db CA group, as well as between the db/db CA group and C57 Cont group (p<0.05, Figure 16).

Figure 16

Effect of cinnamaldehyde on vasodilator response induced by acetylcholine in db/db mice. ## p<0.01 compared to db/db ND group; ** p<0.01 compared to C57 Cont group (n=10).

Effect of cinnamaldehyde on endothelium-independent diastolic function

The vascular function experiment showed that, compared to normal-diet groups, cinnamaldehyde had no significant effect on nitroglycerin-induced endothelium-independent vasodilatation function (p>0.05, Figure 17).

Figure 17

Effect of cinnamaldehyde on vasodilator response induced by nitroglycerin in db/db mice (n=10).

Effect of cinnamaldehyde on vascular remodeling

We used H&E staining of the aorta to observe effects of cinnamaldehyde on intima-media thickness in db/db mice. We found that cinnamaldehyde caused a slight reduction of intima-media thickness in the db/db CA group compared to the db/db ND group. We found significant differences between the db/db ND group and the C57 Cont group (p<0.01, Figures 18, 19).

Figure 18

Effect of cinnamaldehyde on aortic intima-media thickness in db/db mice (n=10).

Figure 19

Effect of cinnamaldehyde on aortic intima-media thickness in db/db mice. **p<0.01 compared to C57 Cont group (n=10).

Discussion

In our work, we observed the effect of cinnamaldehyde on the general condition of db/db mice, including food intake, body weight, blood pressure, and glucose and insulin sensitivity. On this basis, we focused on the influence of cinnamaldehyde on the morphology and function of pancreas islets, thoracic aorta, and mesenteric artery in db/db mice. As one of the classic agonists of TRPA1, cinnamaldehyde is believed to reduce the secretion of Ghrelin by activating TRPA1, which further inhibits gastric emptying and reduces food intake [18]. However, research also showed that cinnamaldehyde only reduced appetite in the early phase, while long-term intervention had little effect on food intake [19]. In our experiment, we observed that cinnamaldehyde decreased food intake in mice, but the difference compared to controls was not significant. For mouse body weight, previous studies on cinnamaldehyde lacked uniform conclusions [19,20]. During our intervention period, cinnamaldehyde caused a decreasing trend of body weight in mice, but without a significant difference. In addition, we found that cinnamaldehyde decreased blood pressure and improved acetylcholine-induced endothelium-dependent diastolic function, which was consistent with the literature [20]. With a remarkable reduction in fasting blood glucose, cinnamaldehyde increased insulin sensitivity of db/db mice [13]. A further study showed that cinnamaldehyde improved the morphology and function of pancreas islets, stimulating insulin secretion [21]. In addition, cinnamaldehyde decreased intima-medium thickness of thoracic aorta in db/db mice, but without a significant difference.

Decreased glucose-lipid metabolism and reduced insulin sensitivity are the main metabolic features of type 2 diabetes. In recent years, many studies confirmed that cinnamaldehyde has several functions, such as glucose reduction, lipid regulation, insulin resistance improvement, and antioxidant stress [13]. Our previous cell-level experiments demonstrated that cinnamaldehyde reduces ROS generation and increases NO levels in endothelial cells. In this study, we found that cinnamaldehyde reduced the generation of superoxide anions with antagonism of NO level decrease in aortic and mesenteric arterial tissue. Based on the effect of cinnamaldehyde on improving endothelium-dependent diastolic function and protecting the morphology and function of islets, we hypothesized that the antioxidant properties of cinnamaldehyde led to its protective action on diabetic vascular complications. Additionally, its direct antioxidant properties may be the secondary mechanism to inhibit the generation of ROS and protect blood vessels, while its effect on the expression of endogenous antioxidant stress factors is the primary mechanism. It has been found that several endogenous antioxidant stress factors such as Nrf2, UCP2, AMPK, and PKA are involved in the pathophysiological processes of human diseases [22,23]. Cinnamaldehyde can prevent the formation and differentiation of adipocytes by activating the AMPK pathway and upregulating the expression of phase II detoxification enzyme in Hepg2 cells by promoting the translocation of Nrf2 [24]. Subash found that cinnamaldehyde promoted the expression of anti-oxidases (CAT, GPx, and SOD) to antagonize the injury of pancreatic beta cells induced by STZ in diabetic rats [25]. Therefore, we speculate that cinnamaldehyde plays a protective role against diabetes and related vascular complications by activating endogenous anti-oxidative stress factors.

Conclusions

In this work, we found that cinnamaldehyde significantly reduced fasting glucose levels, increased insulin sensitivity, and improved islet morphology and function in db/db mice. Experiments showed that cinnamaldehyde effectively improved vascular endothelium-dependent diastolic function with antihypertensive trend, which provides experimental evidence for further cinnamaldehyde clinical use.