Zhen-WangMa,De-You Jiang, Bing-Cheng Hu, Xing-Xing Yuan, Mei Wang, Hai-Long Li, Shao-Jie Cai, Jing Guo

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

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

ABSTRACT Objective: To observe the effect of resveratrol on the injury of diabetic cardiomyocytes and its effect on HSF1 mediated iron death. Methods: the diabetic cardiomyopathy model was established by high glucose induced H9c2, and H9c2 was exposed to normal glucose concentration as a control. Then the intervention was performed with the corresponding drugs.The cell proliferation level was detected by CCK8 method, the concentrations of LDH, SOD,MDA and iron ions were detected by kit method, and the expression levels of HSF1, apoptotic proteins (Bax and Bcl-2) and iron death marker proteins (ACSL4, GPX4 and SLC7A11)were detected by Western blot; Results: compared with the blank group, the cell activity,SOD activity, the expression of HSF1, Bcl-2, GPX4 and SLC7A11 protein in the model group decreased significantly, and the LDH activity, MDA content, Bax and ACSL4 protein expression, Bax/Bcl-2 ratio and Fe2+ content increased significantly (P＜0.01). Compared with the model group, the activity of H9c2 cells, the activity of SOD, the expression of HSF1, Bcl-2,GPX4 and SLC7A11 protein increased significantly, and the activity of LDH, the content of MDA, the expression of Bax and ACSL4 protein, the ratio of Bax/Bcl-2 and the content of Fe2+decreased significantly in the resveratrol group (P＜0.01). After the intervention, the activity of H9c2 cells, the activity of SOD, the expression of HSF1, Bcl-2, GPX4 and SLC7A11 protein decreased significantly, and the activity of LDH, the content of MDA, the expression of Bax and ACSL4 protein, the ratio of Bax/Bcl-2 and the content of Fe2+ increased significantly in si-hsf1 group (P＜0.01); Conclusion: resveratrol can inhibit cell iron death and improve high glucose induced cardiomyocyte injury by up regulating the expression of HSF1.

Keywords:Resveratrol Diabetic cardiomyopathy Myocardial injury HSF1 Ferroptosis?Corresponding author: JIANG De-you, Professor, Doctoral Supervisor.E-mail: jiangdeyou0001@126.com.

1. Introduction

Diabetic cardiomyopathy (DCM) is a common clinical cardiomyopathy independent of coronary heart disease and hypertension. It has become one of the most important causes of death in diabetes mellitus [1]. The results showed that the prevalence of heart dysfunction in patients with type 1 diabetes and type 2 diabetes was 14.5% and 35% respectively, and the risk of DCM in 1 and 2 diabetes increased 30% and 8%, respectively. It has become a serious public health problem [2-4].High glucose level plays a key role in the pathogenesis of DCM.The accumulation of reactive oxygen species, the increase of apoptosis level and the chemotaxis of inflammatory cells induced by long-term hyperglycemia are the main pathological mechanisms of DCM [5]. However, the mechanism of high glucose induced cardiomyocyte injury is complex. Exploring the key targets and therapeutic drugs of high glucose induced cardiomyocyte injury is of great significance to prevent and improve the prognosis of DCM.Resveratrol (Resveratrol) is a non flavonoid polyphenol compound.It has been proved to be effective in improving cardiac dysfunction and myocardial fibrosis in diabetic cardiomyopathy [6-10]. However,the mechanism of resveratrol inhibiting cardiomyocyte injury is lack of in-depth research. In this study, the project group is to construct a diabetic cardiomyopathy model in vitro through high glucose induced H9c2, and further clarify the mechanism and target of Resveratrol treatment of DCM through the iron death mechanism mediated by heat shock transcription factor 1 (HSF1), transcription.

2. Materials and methods

2.1 materials

The rat H9c2 cell line was purchased from the cell resource center of Shanghai Academy of Biological Sciences, Chinese Academy of Sciences; Fetal bovine serum, DMEM low sugar (5mm) and high sugar (30mm) medium were purchased from GIBCO company of the United States (article numbers 10099141c, 11885084 and 11965084 respectively); Resveratrol was purchased from Shanghai Haohong Biomedical Technology Co., Ltd. (CAS: 501-36-0,Purity≥98%);CCK8 cell viability test kit was purchased from Nanjing Jiancheng Bioengineering Institute (Article No.: g021-1-2); Si-hsf1 and negative control Si NC were purchased from Shanghai gene Pharma Co., Ltd; Lipofectaminetm 3000 transfection kit was purchased from Thermo Fisher company (Article No.: l3000001). HSF1,Bax, Bcl-2 and β-Actin Rabbit anti mouse monoclonal antibody was purchased from American cell signaling technology company(article numbers 12972, 5023, 3498 and 4970 respectively), ACSL4,GPX4, SLC7A11 Rabbit anti mouse monoclonal antibody and iron detection kit were purchased from British Abcam company (article numbers ab155282, ab125066, ab175186 and ab83366 respectively);Horseradish peroxidase labeled Goat anti rabbit IgG secondary antibody was purchased from Shanghai biyuntian Biotechnology Co., Ltd. (Article No. A0208); Rat lactate dehydrogenase (LDH) and malondialdehyde (MDA) test kits were purchased from Wuhan eliret Biotechnology Co., Ltd. (article numbers: e-bc-k046-m and e-bck046-m respectively) E-BC-K025-S); The rat superoxide dismutase(SOD) ELISA kit was purchased from Jiangsu enzyme immune Industry Co., Ltd. (Article No.: mm-0386r2).

2.2 cell grouping and Administration

H9c2 cells were routinely cultured in low sugar DMEM medium containing 10% fetal bovine serum, 10% l-glutamine and 0.5%penicillin/streptomycin. The cells were cultured under the conditions of 5% CO2and 37℃. After 80% adhesion and confluence, they were cultured with serum-free low sugar DMEM for 24 hours. Then the cells were divided into blank group, model group, resveratrol group,Si NC group and Si HSF1 group. The blank group was cultured in low sugar DMEM medium, and the remaining four groups were cultured in high sugar medium for 48h. Then resveratrol group was given 20 μMol/L resveratrol intervened, while SiNC group and Si HSF1 group introduced SiNC and Si HSF1 into cells through liposomes according to the instructions of the kit. After 24 hours,cells were collected for detection.

2.3 cell activity test

After 24 hours, 10 cells were added to each group μL of CCK8 solution, continue to incubate for 4H. Refer to the instructions of the kit and detect the absorbance value of each well at 450 nm.

2.4 Western blot detection

After 24 hours, the cells of each group were collected and lysed on ice, and the protein concentration was measured by BCA method.Electrophoresis, membrane transfer, skimmed milk sealing. Add diluted HSF1, Bax, Bcl-2, ACSL4, GPX4, SLC7A11 and β-Actin Rabbit anti mouse monoclonal antibody was incubated at 4℃overnight, HRP labeled 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 ldh, SOD and MDA detection

After 24 hours, the cell supernatant of each group was collected,and the activity of LDH in the cell supernatant of each group was detected according to the instructions of the kit; At the same time,the collected cells were lysed by repeated freezing and thawing,centrifuged at 3500 rpm for 15min, the supernatant was collected,BCA was quantified, and the MDA content and SOD activity were detected according to the instructions of the kit.

2.6 Determination of iron ion content

After 24 hours, the cells of each group were collected, an appropriate amount of cell lysate was added, centrifuged at 2500 rpm for 10 minutes, and the supernatant was collected. The content of iron ions in cells of each group was detected according to the instructions of the kit.

2.7 Statistical analysis

The experimental data are expressed as mean±standard deviation(±s). Single factor ANOVA test is used for the comparison between multiple groups. LSD method is used when equal variance is assumed, dunnit T3 method is used when equal variance is not assumed, and independent sample t-test is used for the comparison between groups. P＜0.05 indicates that the difference is statistically significant.

3. Results

3.1 Effect of resveratrol on the morphology of H9c2 cells

As shown in Fig. 1, the cells in the blank group have regular morphology, orderly arrangement, spindle shape, clear cell boundary and equal size. The cells in the model group were arranged disorderly, showing a short spindle shape, floating dead cells were seen, and the cell gap was significantly widened. The morphology and arrangement of cells in resveratrol group were significantly improved compared with those in model group and similar to those in blank group, while the morphology and arrangement of cells in SiNC group were not significantly improved compared with those in model group. Compared with the model group, the arrangement of cells in si-hsf1 group was significantly disordered, the cells became round and showed short spindle shape with different sizes, and a large number of cell fragments and dead cells could be seen.

Figure 1 The morphology of H9c2 cells in each group was observed under inverted phase contrast microscope(×200 times)

3.2 Effect of resveratrol on the activity of H9c2 cells

It can be seen from table 1 that compared with the blank group,the cell activity of the model group decreased significantly, and the difference was statistically significant (P＜0.01). Compared with the model group, the activity of H9c2 cells in resveratrol group increased significantly (P＜0.01), while the activity of H9c2 cells in Si NC group did not change significantly (P＞0.05). After intervention, the activity of H9c2 cells in si-hsf1 group was significantly lower than that in model group (P＜0.01).

Table 1 Comparison of H9c2 cell activity in each group (±s)

Table 1 Comparison of H9c2 cell activity in each group (±s)

Note: compared with the blank group, *P＜0.05, **P＜0.01; Compared with the model group, #p＜0.05, ##p＜0.01. Same below

Group n Cytoactive(%)Control 3 94.15±3.24 Model 3 62.15±4.62**Resveratrol 3 81.75±3.67##si-NC 3 62.75±3.98 si-HSF1 3 53.34±3.18##Statistical value F=102.225 P value 0.000

3.3 Effects of resveratrol on LDH, SOD and MDA in H9c2 cells

Compared with the blank group, the activity of LDH and the content of MDA in H9c2 cells in the model group increased significantly,and the activity of SOD decreased significantly (P＜0.01). Compared with the model group, the activity of LDH and the content of MDA in H9c2 cells in resveratrol group decreased significantly, and the activity of SOD increased significantly (P＜0.01). Compared with the model group, there was no significant change in LDH, SOD activity and MDA content of H9c2 cells in SiNC group (P＞0.05). After the intervention, the activity of LDH and the content of MDA in H9c2 cells in si-hsf1 group increased significantly, and the activity of SOD decreased significantly. Compared with the model group, the difference was statistically significant (P＜0.01). See Table 2

Table 2 Comparison of LDH, SOD and MDA of H9c2 cells in each group (±s)

Table 2 Comparison of LDH, SOD and MDA of H9c2 cells in each group (±s)

Group n LDH(U/L) SOD(U/mL) MDA(mmol/mL)Control 3 200.15±13.24 214.61±15.36 146.38±17.92 Model 3 539.15±34.77** 91.52±7.65** 337.15±34.28**Resveratrol 3 295.74±23.05## 176.37±16.37## 197.65±21.06##si-NC 3 528.51±40.57 92.47±7.75 339.47±36.54 si-HSF1 3 728.51±47.43## 53.64±4.78## 482.14±47.28##Statistical value F=90.554 F =581.198 F =20.284 P value 0.000 0.000 0.000

3.4 Effect of resveratrol on the expression of apoptotic protein in H9c2 cells

Compared with the blank group, the expression of Bax protein and the ratio of Bax/Bcl-2 in H9c2 cells in the model group increased significantly, and the expression of HSF1 and bcl-2 protein decreased significantly (P＜0.01). Compared with the model group,the expression of Bax protein and the ratio of Bax/Bcl-2 in H9c2 cells in resveratrol group decreased significantly, and the expression of HSF1 and bcl-2 protein increased significantly (P＜0.01). The expression of HSF1, Bax and bcl-2 protein and the ratio of Bax/Bcl-2 in Si NC group had no significant change compared with the model group (P＞0.05). After the intervention, the expression of Bax protein and Bax/Bcl-2 ratio in H9c2 cells in si-hsf1 group increased significantly, and the expression of HSF1 and bcl-2 protein decreased significantly compared with the model group (P＜0.01).See Table 3 and Figure 2

Table 3 Comparison of HSF1, Bax and bcl-2 protein expression in H9c2 cells of each group (±s)

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Figure 2 Comparison of HSF1, Bax and bcl-2 protein expression in H9c2 cells of each group

3.5 Effect of resveratrol on iron content in H9c2 cells

Compared with the blank group, the content of Fe2+in H9c2 cells in the model group increased significantly (P ＜ 0.01). Compared with the model group, the content of Fe2+in H9c2 cells in resveratrol group decreased significantly (P ＜ 0.01). The content of Fe2+ in H9c2 cells in Si NC group had no significant change compared with the model group (P ＞ 0.05). The content of Fe2+in H9c2 cells in sihsf1 group increased significantly after intervention, which was statistically significant compared with the model group (P ＜ 0.01).See Table 4

Table 4 Comparison of iron ion content in H9c2 cells of each group (±s)

Table 4 Comparison of iron ion content in H9c2 cells of each group (±s)

Group n Fe2+ content(μg/μG protein)Control 3 9.15±2.24 Model 3 30.37±5.62**Resveratrol 3 16.27±3.11##si-NC 3 30.75±4.98 si-HSF1 3 46.29±7.06##Statistical value F=64.239 P value 0.000

3.6 Effect of resveratrol on the expression of iron death related protein in H9c2 cells

Compared with the blank group, the expression of ACSL4 protein in H9c2 cells in the model group increased significantly, and the expression of GPX4 and SLC7A11 protein decreased significantly(P＜0.01). Compared with the model group, the expression of ACSL4 protein in H9c2 cells in resveratrol group decreased significantly, and the expression of GPX4 and SLC7A11 protein increased significantly (P＜0.01). The expression of ACSL4,GPX4 and SLC7A11 protein in H9c2 cells in Si NC group had no significant change compared with the model group (P＞0.05). After the intervention, the expression of ACSL4 protein in H9c2 cells increased significantly, and the expression of GPX4 and SLC7A11 protein decreased significantly in si-hsf1 group. Compared with the model group, the difference was statistically significant (P＜0.01).See Table 5 and figure 3

Table 5 Comparison of ACSL4, GPX4 and SLC7A11 protein expression in H9c2 cells of each group (±s)

Table 5 Comparison of ACSL4, GPX4 and SLC7A11 protein expression in H9c2 cells of each group (±s)

Group n ACSL4/β-actin GPX4/β-actin SLC7A11/β-actin Control 3 0.31±0.06 0.98±0.16 1.13±0.15 Model 3 0.82±0.13 0.51±0.09 0.56±0.12 Resveratrol 3 0.36±0.05 0.96±0.19 0.99±0.13 si-NC 3 0.83±0.12 0.50±0.04 0.55±0.13 si-HSF1 3 1.02±0.16 0.21±0.03 0.33±0.04 Statistical value F=32.809 F =13.231 F =8.206 P value 0.000 0.001 0.003

Figure 3 Comparison of ACSL4, GPX4 and SLC7A11 protein expression in H9c2 cells of each group

4. Discussion

In recent years, with the change of diet structure and living habits, the prevalence of diabetes has been increasing year by year. It has become a serious public health problem and brings enormous social and economic burden. The survey showed that the prevalence of diabetes in adults in China was 10.7%, higher than the global average. The total number is 114 million, accounting for more than 25% of the total number of adults with diabetes in the world, ranking the first [11] in the world. Metabolic disorder can trigger the biological changes of cardiomyocytes, resulting in the abnormality of myocardial function, and then develop to heart failure on the basis of microcirculation disorder, myocardial small vessels and autonomic neuropathy. The toxicity of hyperglycemia to cardiomyocytes caused by impaired glucose clearance and increased gluconeogenesis is the core mechanism of DCM [12]. On the one hand, chronic hyperglycemia can increase ROS in the electron transport chain derived from complex I and complex III, and cause cell damage by inducing cardiomyocyte apoptosis [13]. On the other hand, the activation of ROS induces the cascade of myocardial injury through the increase of advanced glycation end products and aldose reductase, resulting in cardiomyocyte apoptosis [14]. In this study, our results show that resveratrol can significantly improve the activity of high glucose induced H9c2 cells, reduce LDH activity and MDA content, and increase the activity of SOD in H9c2 cells, indicating that resveratrol can effectively improve the injury and oxidative stress level of high glucose induced cardiomyocytes and improve the antioxidant capacity of cardiomyocytes. In addition, the expression levels of apoptotic proteins Bax and Bcl-2 in cardiomyocytes were detected by Western blot. The results showed that resveratrol could significantly reduce the expression of Bax protein and the ratio of Bax/Bcl-2 in H9c2 cells, increase the expression of Bcl-2 protein,and inhibit the apoptosis level of cardiomyocytes.

HSF1 is a member of the heat shock transcription factor family,which is mainly responsible for the transcriptional regulation of heat shock proteins. In addition to degrading, repairing damaged proteins and maintaining newborn protein folding, the latter can also participate in the cellular biological process by regulating embryonic development, cell proliferation, apoptosis and differentiation.Liu Ting et al. [15] confirmed through in vitro experiments that overexpression of HSF1 can enhance the effect of Shenxiong Glucose Injection on oxidative stress injury of cardiomyocytes by up regulating the expression of HSP70 in cells and the ability of antioxidant stress. In addition, HSF1 can also promote the phosphorylation level of Akt and improve pressure overload induced ventricular remodeling and heart failure by regulating the mechanism of angiogenesis [16]. The results of this study showed that the expression level of HSF1 protein was significantly lower in the high glucose induced diabetic cardiomyopathy model in vitro. Interfering with the expression of HSF1, significantly inhibited the cell viability and increased LDH activity of diabetic cardiomyopathy in vitro,further explaining the protective effect of HSF1 on H9c2 cells induced by high glucose. At the same time, inhibiting the expression of HSF1 can also significantly increase the content of MDA in cells and reduce the activity of SOD, increase the expression of Bax protein and the ratio of Bax / Bcl-2, and reduce the expression of Bcl-2 protein, which indicates that the protection of HSF1 against myocardial injury is mainly achieved by promoting the oxidation resistance and apoptosis of cardiomyocytes.

Iron death is a new type of iron dependent programmed cell death.It is a pathological process that exceeds the metabolic capacity of lipid ROS by many ways, such as lipid peroxidation, oxidative stress, glutamate cystine transport and abnormal iron metabolism[17]. Iron is one of the essential trace elements of human body.Iron in the body is transported to cells through transferrin receptor(TfR1) on cell membrane in the form of Fe3+/transferrin (TF)complex. Fe3+is reduced to Fe2+by cytoplasmic reductase. Iron homeostasis plays an important role in maintaining the physiological function of cells. Once the iron metabolism in the body is abnormal(especially the intracellular Fe2+overload), it will produce a large number of ROS and hydroxyl free radicals with strong toxicity to cells through Fenton reaction, so as to destroy cellular DNA and proteins and cause damage to cells and tissues [18]. Different from apoptosis and autophagy, iron death is mainly manifested in the increase of mitochondrial membrane density and the contraction of mitochondrial volume. However, more and more studies have confirmed that there is a close relationship between iron death,apoptosis and autophagy [19]. On the one hand, apoptosis can turn into iron death, and iron death can increase the sensitivity of apoptosis. On the other hand, Fe2+degraded and released by autophagy lysosome further induces oxidative stress, thus promoting the occurrence of iron death [20].

ACSL4, GPX4 and SLC7A11 are marker proteins of iron death. ACSL4 promotes cell iron death through the synthesis of phosphatidylinositol or phosphatidylethanol. Meanwhile, ACSL4 can also induce cell iron death by up regulating the expression of 5-hydroxyeicosapentaenoic acid, which has strong lipotoxicity [21].As the most classical antioxidant enzyme defense pathway, GPX4 inhibits the level of cell iron death by specifically catalyzing lipid peroxidation and losing its oxidative activity. Li Li et al. Confirmed that the expression level of GPX4 protein in myocardial tissue of myocardial ischemia-reperfusion mouse model was significantly reduced. Up regulating the expression of GPX4 protein can significantly inhibit iron death and improve myocardial ischemiareperfusion injury [22]. SLC7A11 is located on human chromosome 4. As a member of slc7 family, SLC7A11 is an important transporter of cystine and glutamate in cells. Under physiological conditions,SLC7A11 can transport cystine to cells and participate in the clearance of ROS, while inhibiting the expression of SLC7A11 can induce iron death by damaging the antioxidant capacity of cells [23].In this study, our results showed that the content of Fe2+in H9c2 cells induced by high glucose increased significantly, the expression of ACSL4 protein increased significantly, and the expression of GPX4 and SLC7A11 protein decreased significantly, indicating that the level of cell iron death increased significantly. Resveratrol can inhibit intracellular Fe2+overload by down regulating the expression of ACSL4 protein and up regulating the expression of GPX4 and SLC7A11 protein. Inhibiting the expression of HSF1 increased the expression of ACSL4 and down regulated the expression levels of GPX4 and SLC7A11 proteins, indicating that HSF1 is an important target for resveratrol to inhibit H9c2 cells.

In conclusion, resveratrol can promote the activity of cardiomyocytes induced by high glucose, inhibit the level of oxidative stress and apoptosis, and protect cardiomyocytes induced by high glucose. The specific mechanism is to inhibit the level of iron death by up regulating the expression of HSF1.