Zhen-Wang Ma, De-You Jiang, Bing-Cheng Hu, Xing-Xing Yuan, Shao-Jie Cai, Jing Guo

1. Heilongjiang University of traditional Chinese medicine, Harbin 150040, China

2. Heilongjiang Academy of traditional Chinese medicine, Harbin 150006, China

ABSTRACT Objective: To observe the protective effect of hesperidin on myocardial ischemia/ reperfusion injury in type 2 diabetes mellitus and its effect on SIRT1/Nrf2/HO-1 signaling pathway.Methods: 50 Sprague-Dawley (SD) rats were randomly assigned to the normal control group(NC), model group, ischemia-reperfusion group (IR), hesperidin group, SIRT1 inhibitor group and hesperidin plus SIRT1 inhibitor group. In addition to NC, the rats in the remaining groups were replicated by intraperitoneal of high-fat diet combined with injection of streptozotocin for type 2 diabetic rats. After then, the myocardial ischemia/reperfusion injury (MIRI) rat model was established by LAd for 30 minutes with 2 hours reperfusion. He staining was used to observe the pathological changes of myocardial tissue, and the levels of serum LDH, CKMB and SOD, GSH and MDA in myocardial tissue were detected by kit methods, and the expression abundance of related proteins in 4-HNE and SIRT1/Nrf2/HO-1 signal pathway were detected by immunohistochemistry and Western blot; Results: Hesperidin could significantly inhibit cardiomyocyte necrosis and inflammatory cell infiltration, reduce LDH activity, CKMB and MDA level, and increase SOD activity, GSH and 4-HNE level, the differences were statistically significant when compared with IR group (P＜0.01). In addition, compared with the ischemia-reperfusion group, the expressions of SIRT1, Nrf2 and HO-1 proteins in hesperidin group were significantly up-regulated, the differences were statistically significant (P＜0.01);Conclusion: Hesperidin inhibits oxidative stress by activating SIRT1/Nrf2/HO-1 signaling pathway, and play a protective effect of myocardial ischemia reperfusion injury in diabetic rats.

Keywords:Hesperidin Type 2 diabetes mellitus Ischemia/reperfusion Myocardial injury SIRT1/Nrf2/HO-1 signaling pathway

1. Introduction

The incidence rate and mortality of ischemic heart disease (IHD),especially acute myocardial infarction, are increasing year by year. It has become one of the main causes of death [1]. Timely and effective percutaneous coronary intervention or thrombolysis can effectively restore myocardial blood flow and save ischemic myocardial tissue. However, more and more studies have confirmed that reperfusion will be accompanied by damage to myocardial structure and function [2]. Therefore, reducing myocardial ischemiareperfusion injury and protecting myocardial tissue structure are of great significance to improve the prognosis of IHD.

Diabetes mellitus (DM), as one of the most common metabolic disorders in clinical practice, has been proved to be an independent risk factor for IHD [3]. Studies have shown that the prevalence of IHD and reperfusion injury after acute myocardial infarction in DM patients increased significantly compared with non-diabetic patients[4]. In addition, DM can also eliminate the protective effects of various drugs or ischemic preconditioning on ischemic myocardium[5]. Hesperidin is a derivative of dihydroflavones, which has the effects of antioxidation, improving glucose and lipid metabolism and protecting cardiovascular system [6]. Therefore, in this study, the effects of hesperidin on myocardial tissue, oxidative stress index and SIRT1/Nrf2/HO-1 signal pathway were analyzed in order to further clarify its protective effect and molecular mechanism on myocardial ischemia-reperfusion injury in DM.

2. Materials and methods

2.1 Experimental animal

Sprague Dawley rats (Body weight 220-280g, male, clean grade)were purchased from Charles River company in Beijing (Production license for experimental animals: SCXK (Beijing) 2016-0011).Feeding conditions of experimental animals: room temperature 25-28 ℃, humidity 60%, 12h-12h circulating light, free drinking and feeding. The experimental process is implemented according to the guiding opinions on treating experimental animals well [7].

2.2 Reagent

Hesperidin was purchased from sigma Aldrich company (Article No: H5254, Purity＞80%); Streptozotocin was purchased from Beijing Solabio Technology Co., Ltd. (Article No.: S8050,Purity＞95%); Ex-527 was purchased from Beijing baiaolaibo Technology Co., Ltd. (Article No. m00182, Purity ＞ 99%); High fat feed was purchased from Nanjing synergy pharmaceutical Bioengineering Co., Ltd. (composed of 60% fat, 20%carbohydrate and 20% protein); He staining kit was purchased from bode Bioengineering Co., Ltd. (art. No. ar1180); Rat lactate dehydrogenase (LDH), rat creatine kinase MB isoenzyme (CK-MB)and malondialdehyde (MDA) detection kits were purchased from Wuhan eliret Biotechnology Co., Ltd. (article numbers are e-bck046-m, e-el-r1327c and e-bc-k025-s respectively); ELISA kits for rat superoxide dismutase (SDO) and rat reduced glutathione (GSH)were purchased from Jiangsu enzyme immunity Industry Co., Ltd.(mm-0386r2 and mm-0602r2); 4-hydroxynonenal (4-HNE) was purchased from Shanghai Yaji Biotechnology Co., Ltd. (Article No.: ihc0100542); Silencing signal regulatory factor 1 (sirtuin 1),nuclear factor E2 related factor 2 (NF-E2 — related factor 2, Nrf2)and heme oxygenase 1 (HO-1) and β- Actin monoclonal antibodies were purchased from Abcam company in the UK (article numbers:ab238538, ab110304, ab137550, ab5294 and ab822 respectively);HRP labeled Goat anti rabbit IgG secondary antibody was purchased from Shanghai biyuntian Biotechnology Co., Ltd. (Article No.A0208).

2.3 Method

After 1 week of adaptive feeding, rats were randomly divided into blank group, model group, ischemia-reperfusion group, hesperidin group, SIRT1 inhibitor group and hesperidin + SIRT1 inhibitor group, with 10 rats in each group. In addition to the blank group, the remaining 5 groups of rats were treated with high-fat diet combined with 35 mg / kg intraperitoneal injection of 1% streptozotocin according to the method in literature [8]. The success of the model was judged by blood collection from tail vein and fasting blood glucose ＞ 16.7 mmol / L. the rats in the blank group were fed with ordinary diet. After modeling, hesperidin group and hesperidin +SIRT1 inhibitor group were given 200 mg / kg hesperidin by gavage once a day, and the remaining rats in each group were given the same volume of Shengli saline by gavage for 4 weeks. After the last gavage, SIRT1 inhibitor group and hesperidin + SIRT1 inhibitor group were given ex-527 10mg / kg intraperitoneal injection.

After 4 weeks, in addition to the blank group and the model group, the remaining 4 groups of rats were ligated with the anterior descending branch of the left coronary artery for 30 minutes and reperfusion for 2 hours to construct the myocardial ischemia /reperfusion injury model [9]. After reperfusion, the myocardial color turns red, and the ST-T segment returns to the pre ischemic level,indicating that the reperfusion is successful. After the experiment, the rats were anesthetized with 3% isoflurane, blood was collected from abdominal aorta and heart tissue was collected for index detection.

2.4 Index detection

2.4.1 Myocardial pathomorphological observation

Take an appropriate amount of myocardial tissue from ischemic area and embed it with stone wax for 10 minutes μM slice. The sections were fixed with 4% paraformaldehyde solution for 24 hours and then stained with he. He staining was performed according to the instructions of the kit, and the pathological changes of myocardial tissue were observed under the optical microscope.

2.4.2 LDH and CK-MB detection

Abdominal aortic blood was centrifuged at 3000 rpm for 15min at 4 ℃, and the contents of LDH and CK-MB in serum were detected by enzyme-linked immunosorbent assay. The detection method is in accordance with the instructions of the kit.

2.4.3 Detection of oxidative stress index

Myocardial tissue was taken, homogenized in ice bath, centrifuged at 3000 rpm at 4 ℃ for 15min, and the supernatant was taken. The activity of SOD and the contents of MDA and GSH in myocardial tissue were detected by colorimetry. Refer to the instructions of the kit for the detection method.

2.4.4 The expression of 4-HNE was detected by immunohistochemistry

Take the paraffin section of myocardial tissue, dewax to water,repair the antigen, add the diluted 4-HNE primary antibody,incubate overnight at 4 ℃, add the secondary antibody, and continue to incubate at room temperature for 40 minutes. DAB staining,hematoxylin re staining, neutral gum sealing after gradient alcohol dehydration. The integral optical density of positive expression was analyzed by image pro plus.

2.4.5 Western blot detection

Take an appropriate amount of myocardial tissue, lyse it on ice, and determine the protein concentration by BCA method.Electrophoresis, membrane transfer, skimmed milk sealing. Add diluted SIRT1, Nrf2, HO-1 and β- Actin rabbit monoclonal antibody was incubated at 4 ℃ overnight, the secondary antibody was added, and the incubation was continued at room temperature for 40 minutes. ECL development, gel imaging system analysis,calculate the relative expression of target protein bands.

2.5 Statistical method

The data were expressed as mean ± standard deviation(±s) , and statistical analysis was carried out by one-way variance (ANOVA).Student's t test was used for pairwise comparison between groups,and P＜0.05 showed that the difference was statistically significant.

3. Result

3.1 Effect of hesperidin on pathological morphology of ischemic myocardium

During the experiment, two rats in the ischemia-reperfusion group died, three rats in the inhibitor group died, one rat in the hesperidin group and the hesperidin combined inhibitor group died respectively, and no death was found in the remaining two groups.It can be seen from Figure 1 that the myocardial fibers of rats in the blank group are arranged orderly, and no inflammatory cells are found. Compared with the blank group, the myocardial fibers in the model group were arranged orderly, and only a small amount of inflammatory cell infiltration was observed. Compared with the blank group, the myocardial fibers in the ischemia-reperfusion group were disordered, the cells were swollen and necrotic, and a large number of inflammatory cells were observed. Compared with the ischemia-reperfusion group, the arrangement of myocardial fibers,cell necrosis and inflammatory cell infiltration in hesperidin group were significantly improved, while the myocardial cell necrosis and inflammatory cell infiltration in inhibitor group were significantly aggravated. Compared with the ischemia-reperfusion group, the arrangement of myocardial fibers, cell necrosis and inflammatory cell infiltration in the hesperidin combined inhibitor group were disordered, which was between the hesperidin group and the inhibitor group.

Figure 1 HE staining was used to observe the pathological morphology of ischemic myocardium

3.2 Effects of hesperidin on serum LDH and CK-MB in diabetic rats with myocardial ischemia-reperfusion

Compared with the blank group, the serum LDH activity and CKMB level in the model group increased slightly, and the difference was not statistically significant (P ＞ 0.05). Compared with the model group, the activity of serum LDH and the level of CK-MB in the ischemia-reperfusion group were significantly higher (P ＜ 0.01).Compared with the ischemia-reperfusion group, the activity of serum LDH and the level of CK-MB in hesperidin group decreased significantly, while the activity of serum LDH and the level of CKMB in inhibitor group increased significantly (P ＜ 0.01). Compared with the inhibitor group, the activity of serum LDH and the level of CK-MB in the hesperidin + inhibitor group were significantly lower(P ＜ 0.01). See Table 1.Group n LDH(U/L) CK-MB(ng/mL)

Table 1 Comparison of serum LDH activity and CK-MB level in each group(±s)

Table 1 Comparison of serum LDH activity and CK-MB level in each group(±s)

Note: compared with the control group, △△P＜0.01; Compared with ischemia-reperfusion group, □P＜0.05,□□P＜0.01; Compared with the inhibitor group, ●●P＜0.01. The same below.

Control group 10 923.12±23.15 60.21±5.61 Model group 10 934.44±31.25 61.74±6.92 IR group 8 2414.51±163.25△△ 184.39±16.36△△Hesperidin group 9 1291.67±104.67□□ 91.82±12.14□□Inhibitor group 7 2937.77±214.64□□ 225.38±17.21□□Hesperidin + inhibitor group 8 2312.34±193.54●● 178.95±9.68●●Statistical value F=274.320 F=337.116 P value 0.000 0.000

3.3 Effects of hesperidin on SOD, MDA and GSH in ischemic myocardium

Compared with the blank group, the SOD activity and GSH level of myocardial tissue in the model group decreased slightly, and the MDA level increased slightly. There was no significant difference between the groups (P ＞ 0.05). Compared with the model group, the myocardial SOD activity and GSH level in the ischemia-reperfusion group decreased significantly, and the MDA level increased significantly (P ＜ 0.01). Compared with the ischemia-reperfusion group, the SOD activity and GSH level in hesperidin group increased significantly and the MDA level decreased significantly (P ＜ 0.01),while the SOD activity and GSH level in inhibitor group decreased significantly and the MDA level increased significantly (P ＜ 0.01).Compared with the inhibitor group, the SOD activity and GSH level in hesperidin + inhibitor group increased significantly, and the MDA level decreased significantly (P ＜ 0.01). See Table 2.

Table 2 Comparison of SOD, MDA and GSH in myocardial tissue of rats in each group(±s)

Table 2 Comparison of SOD, MDA and GSH in myocardial tissue of rats in each group(±s)

Group n SOD(U/mg) MDA(μmol/L) GSH(μmol/L)Control group 10 21.34±3.11 1.51±0.24 11.02±1.41 Model group 10 20.08±2.75 1.55±0.19 10.38±1.28 IR group 8 10.76±1.32△△2.23±0.28△△4.17±0.29△△Hesperidin group 9 16.27±2.69□□1.73±0.18□□7.93±1.17□□Inhibitor group 7 7.82±1.22□□ 2.51±0.31□□3.22±0.21□□Hesperidin + inhibitor group 8 10.22±1.52●●2.27±0.24●●4.09±0.71●●Statistical value F=51.875 F=33.045 F=114.140 P value 0.000 0.000 0.000

3.4 Effect of hesperidin on the expression of 4-HNE in ischemic myocardium

Compared with the blank group, the expression of 4-HNE protein in myocardial tissue of model group increased slightly,and there was no significant difference between groups (P ＞ 0.05).Compared with the model group, the expression of 4-HNE protein in myocardial tissue of mice in ischemia-reperfusion group increased significantly (P ＜ 0.01). Compared with ischemia-reperfusion group,the expression of 4-HNE protein in hesperidin group decreased significantly (P ＜ 0.01), while the expression of 4-HNE in inhibitor group increased significantly (P ＜ 0.01). Compared with the inhibitor group, the expression of 4-HNE in hesperidin + inhibitor group decreased significantly (P ＜ 0.01). See Figure 2 and table 3.

Figure 2 Effect of hesperidin on the expression of 4-HNE in myocardial tissue(×200)

Table 3 Comparison of 4-HNE expression levels in myocardium of rats in each group(±s)

Table 3 Comparison of 4-HNE expression levels in myocardium of rats in each group(±s)

Group n 4-HNE(IOD)Control group 10 0.093±0.012 Model group 10 0.102±0.015 IR group 8 0.281±0.029△△Hesperidin group 9 0.161±0.024□□Inhibitor group 7 0.329±0.031□□Hesperidin + inhibitor group 8 0.278±0.026●●Statistical value F=180.284 P value 0.000

3.5 Effect of hesperidin on SIRT1/Nrf2/HO-1 signaling pathway in ischemic myocardium

Compared with the SIRT1, Nrf2 and HO-1 protein in the blank group, the expression of SIRT1, Nrf2 and HO-1 protein in the model group had no significant change, and there was no significant difference between the groups (P ＞ 0.05). Compared with the model group, the expression of SIRT1, Nrf2 and HO-1 protein in myocardial tissue of mice in ischemia-reperfusion group were significantly down regulated (P ＜ 0.01). Compared with ischemiareperfusion group, the expression of SIRT1, Nrf2 and HO-1 protein in hesperidin group was significantly up-regulated (P ＜ 0.01), while the expression of SIRT1, Nrf2 and HO-1 protein in inhibitor group was significantly down regulated (P ＜ 0.01). Compared with the inhibitor group, the expressions of SIRT1, Nrf2 and HO-1 proteins in hesperidin + inhibitor group were significantly up-regulated (P ＜0.01). See Figure 3 and table 4

Table 4 Comparison of SIRT1, Nrf2 and HO-1 protein expression in myocardial tissue of rats in each group(±s)

Table 4 Comparison of SIRT1, Nrf2 and HO-1 protein expression in myocardial tissue of rats in each group(±s)

Group n SIRT1/β-actin Nrf2/β-actin HO-1/β-actin Control group 10 1.21±0.15 1.28±0.21 1.25±0.22 Model group 10 1.22±0.16 1.30±0.23 1.27±0.19 IR group 8 0.52±0.06△△ 0.55±0.09△△ 0.61±0.12△△Hesperidin group 9 0.92±0.13□□ 1.02±0.16□□ 1.04±0.17□□Inhibitor group 7 0.26±0.02□□ 0.31±0.05□□ 0.29±0.03□□Hesperidin + inhibitor group 8 0.56±0.13●● 0.57±0.11●● 0.60±0.09●●Statistical value F=96.502 F=42.957 F=66.843 P value 0.000 0.000 0.000

Figure 3 Comparison of SIRT1, Nrf2 and HO-1 protein expression in myocardial tissue of rats in each group

4. Discussion

A large number of reactive oxygen species (ROS) will be produced during myocardial ischemia / reperfusion. At the same time, DM can aggravate the structural and functional damage of cardiovascular system through metabolic disorder, inflammation, oxidative stress and apoptosis. Oxidative stress plays a core role in DM myocardial ischemia / reperfusion injury [10]. Generally, ROS in cardiomyocytes is at a low level. A large number of released peroxides reduce the activity of cell membrane by destroying membrane protease,membrane receptor and ion channels, so as to induce cardiomyocyte injury [11]. SOD and GSH are important antioxidant enzymes in vivo, which can scavenge free radicals. Sod catalyzes the generation of superoxide free radicals to eliminate harmful substances in the metabolic process, while GSH, as the substrate of GSH Px, can combine with oxygen free radicals through non enzymatic reaction to achieve the effects of anti-oxidation, aging and scavenging free radicals [12]. MDA and 4-HNE are the main products of lipid peroxidation caused by free radical attack on biofilm and important indicators to measure free radical and lipid peroxidation [13].

SIRT1, a member of sirtuins family, plays an important role in many processes such as mitochondrial function, apoptosis, inflammatory response and oxidative stress by participating in the acetylation reaction of related proteins [14]. SIRT1 is an important therapeutic target for cardiovascular diseases, and has a high expression level in normal myocardial tissue. It can protect myocardium and resist arteriosclerosis. Studies have confirmed that the myocardial infarction area of SIRT1 gene knockout mice is significantly higher than that of normal mice, and up regulating the expression of SIRT1 gene can reduce the myocardial infarction area and myocardial injury after reperfusion [15-16]. Nrf2, as an endogenous antioxidant factor, plays a role in protecting cardiomyocytes from injury by regulating cardiomyocyte apoptosis, autophagy, antioxidation,endoplasmic reticulum stress and mitochondrial function [17]. Under normal conditions, Nrf2 binds to Keap1, locates in the cytoplasm and is inactivated. A large amount of ROS produced during acute myocardial ischemia promotes the separation of Nrf2 from nrf2-keap1 complex and into the nucleus, promotes the transcription of downstream target genes and induces cells to produce antioxidant enzymes by combining with the antioxidant response element on the target gene promoter to resist oxidative stress response [18-19].In addition, studies have confirmed that Nrf2 can also participate in myocardial ischemia-reperfusion injury in DM rats by promoting iron death [20]. HO-1 is a target gene downstream of Nrf2 and an important antioxidant kinase. Under oxidative stress, free Nrf2 enters the nucleus and binds HO-1 in the form of dimer with specific macrophage arming factor (SMAF), thus promoting the transcription level of HO-1 [21]. Studies have confirmed that up regulation of HO-1 expression can improve myocardial ischemia-reperfusion injury, while knockout of HO-1 can aggravate myocardial ischemiareperfusion injury in DM by increasing the level of myocardial oxidative stress [22].

Hesperidin is a natural flavonoid composed of a molecule of rutose and hesperidin, which mainly exists in the peel of citrus fruits. Studies have confirmed that hesperidin can regulate lipid metabolism and insulin signaling pathway through AMPK, so as to reduce glucose, lipid and improve insulin resistance [23]. In addition,studies have shown that hesperidin can induce mitochondrial dependent apoptosis of human gastric cancer AGS cells by activating MAPK signaling pathway [24]. In this study, a rat model of type 2 diabetes mellitus was induced by intraperitoneal injection of highfat diet combined with streptozotocin. The myocardial ischemia /reperfusion injury model was established by ligation of 30min and 2h in the left anterior descending coronary artery. The results showed that the levels of serum LDH and CK-MB increased significantly in the ischemia-reperfusion group, suggesting that the model was successful. Hesperidin can significantly reduce the levels of serum LDH and CK-MB, indicating that hesperidin pretreatment can significantly reduce the degree of myocardial injury and improve the metabolic level of cardiomyocytes. At the same time, hesperidin can also significantly increase the activity of antioxidant SOD and the level of GSH, reduce the expression of MDA and 4-HNE, and play an antioxidant role. In addition, the results of this study showed that the expression of SIRT1, Nrf2 and HO-1 protein in myocardial tissue of mice in ischemia-reperfusion group were significantly down regulated, down regulated the expression of SIRT1, inhibited the activation of SIRT1/Nrf2/HO-1 signal pathway, significantly aggravated the level of oxidative stress and myocardial injury, and further confirmed the important role of SIRT1/Nrf2/HO-1 signal pathway in myocardial ischemia-reperfusion of DM. Hesperidin can significantly up regulate the expression of SIRT1, Nrf2 and HO-1 protein, while hesperidin can significantly inhibit the regulation of SIRT1, Nrf2 and HO-1 protein and antioxidant stress under the action of SIRT1 inhibitor, which proves that SIRT1/Nrf2/HO-1 signal pathway may be an important way for hesperidin to treat myocardial ischemia-reperfusion in DM.

In summary, the results of this study indicate that hesperidin can inhibit oxidative stress in myocardial injury and improve myocardial ischemia reperfusion injury in diabetic rats. This effect of hesperidin may be closely related to its activation of SIRT1 / Nrf2 / HO-1 signaling pathway.

Author's contribution

The experiment was designed by Ma ZHENWANG and directed by Jiang Deyou. Cai Shaojie and Guo Jing completed experimental modeling and drug intervention; Yuan Xingxing is responsible for index detection; Hu Bingcheng reviewed and statistically analyzed the experimental data. The paper was written by Ma ZHENWANG and reviewed by Jiang Deyou. All authors declare that there is no conflict of interest.