Mechanism of Resveratrol on autophagy mediated by Mst1/Sirt3 signaling pathway in diabetic cardiomyopathy

Zhen-Wang Ma, De-You Jiang, Xing-Xing Yuan, Zhen-Yu Li, Mei Wang, Jun Duan,Shao-Jie Cai

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

2. Heilongjiang Academy of Traditional Chinese Medicine,Harbin 150006,China

Keywords:Resveratrol Autophagy Mst1/Sirt3 signaling pathway Diabetes Myocardial injury

ABSTRACT Objective: To observe the effects of Resveratrol on myocardial cell injury and Mst1/Sirt3 signaling pathway mediated autophagy in type 2 diabetic mice. Methods: The mice were allocated to normal control group, model group, Resveratrol group, and melbine group,with 10 mice per group. Resveratrol group and melbine group were treated with resveratrol and metformin, respectively. The NC and the model group were treated with equal volume of normal saline by gavage for 8 consecutive weeks. HE staining, transmission electron microscopy and immunofluorescence were used to observe the pathological morphology,ultrastructure and apoptosis levels of myocardial tissues. RT-qPCR method was used to detect the expression levels of apoptosis genes Bax and Bcl-2 in myocardial tissues, and Western-blot method was used to detect the expression levels of autophagy proteins (LC3 and p62), Mst1 and Sirt3 proteins in myocardial tissue; Results: Compared with the model group, Resveratrol can significantly reduce the body weight, blood glucose level and serum CK and LDH levels of db/db mice, and the difference is statistically significant (P＜0.05 and P＜0.01). Meanwhile, after Resveratrol treatment, myocardial inflammation score, apoptosis rate, Bax mRNA expression level and Bax/Bcl-2 ratio in myocardial tissue were significantly reduced, and Bcl-2 mRNA expression level was significantly increased, and the difference was statistically significant (P＜0.01). In addition, compared with the model group, the expression level of p62 and p-Mst1 protein in the myocardial tissue of the Resveratrol group was significantly reduced, and the expression level of Sirt3 protein and the ratio of LC3II/LC3I were significantly increased, and the difference was statistically significant (P＜0.01). Conclusion: Resveratrol promotes the autophagy level of cardiomyocytes by activating the Mst1/Sirt3 signaling pathway and inhibits cardiomyocyte apoptosis to play a protective role in diabetic cardiomyopathy.

1. Introduction

Diabetic cardiomyopathy (DCM) is a type of cardiomyopathy characterized by extensive focal necrosis of the myocardium on the basis of metabolic disorders and cardiac microangiopathy[1].As an important complication of diabetes, DCM can cause cardiac dysfunction and decrease of left ventricular compliance, leading to arrhythmia, heart failure and sudden cardiac death. It has become one of the important causes of death in diabetes mellitus [2].Continuous high glucose stimulation can induce cardiomyocyte hypertrophy, apoptosis, collagen secretion and fibrosis, and then damage the diastolic and systolic functions of the heart [3].Nevertheless, the pathogenesis of DCM has not been fully clarified,so there is a lack of effective treatment.

Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenol compound derived from mulberry, Polygonum cuspidatum,raspberry and grape. It has become an important hypoglycemic active ingredient. Resveratrol has been shown to effectively improve islets β Cell function and promote insulin secretion, and reduce the level of inflammation and oxidative stress through a variety of ways,so as to improve myocardial ischemia-reperfusion injury in rats[4-7]. However, the role of resveratrol in DCM needs to be further discussed. In this study, we observed the changes of cardiomyocyte apoptosis and autophagy after resveratrol treatment, and the effect of resveratrol on Mst1 / SIRT3 signaling pathway, so as to clarify its therapeutic effect on DCM and its specific molecular mechanism.

2. Materials and methods

2.1 Experimental animal

C57BL/KSJ db/db mice (male, 8 weeks old, 18-20g) and C57BL/KSJ db/M mice (male, 8 weeks old, 18-20g) were purchased from Shanghai Jiesijie experimental animal Co., Ltd. Laboratory animal production license No.: SCXK (Shanghai) 2018-0004. Animal feeding environment: room temperature 25-28 ℃, humidity 60%,12h-12h cyclic light, free drinking and feeding. Animal welfare and experimental procedures shall strictly abide by the code of ethics for experimental animals of Heilongjiang University of traditional Chinese medicine.

2.2 Reagents and instruments

Resveratrol was purchased from Shanghai Haohong Biomedical Technology Co., Ltd. (CAS: 501-36-0, purity 98%); Metformin hydrochloride tablets were purchased from Shenzhen Zhonglian Pharmaceutical Co., Ltd. (Medicon, approval No.: gyzz h44024853,0.25g); Hematoxylin and eosin (HE) and one-step TUNEL apoptosis detection kit (green fluorescence) were purchased from Shanghai biyuntian Biology (Article No.: c0105m and c1088); Rabbit anti mouse LC3, p62, Mst1, p-Mst1, SIRT3 and β- Actin Yikang was purchased from cell signaling technology (article numbers 4599,39786, 14946, 49332, 2627 and 4970 respectively); HRP labeled Goat anti rabbit secondary antibody was purchased from fluorescent Abcam company (batch No.: ab6721); RNA extraction kit, revertaid first strand cDNA synthesis kit and faststart universal SYBR Green master were purchased from thermo company (article numbers 12183016, k1621 and 14001014 respectively). Desktop highspeed refrigerated centrifuge was purchased from Hunan Michael Experimental Instrument Co., Ltd. (model: mk650r); The double plate vertical electrophoresis instrument was purchased from Beijing Liuyi Biotechnology Co., Ltd. (model: dycz-24ks), the ultra micro spectrophotometer and fluorescence quantitative PCR instrument were purchased from thermo company (model: nanodrop2000 and abi7500), and the automatic chemiluminescence / fluorescence image analysis system was purchased from Shanghai Tianneng Technology Co., Ltd. (model: tanon 5200 multi).

2.3 Animal grouping and drug intervention

One week after adaptive feeding, db/db mice were randomly divided into model group, resveratrol group and Western medicine group, with 10 mice in each group, and 10 dB / M mice were taken as the blank control group. After grouping, mice in each group were given corresponding drugs for intervention. Referring to the dose in reference [8], mice in resveratrol group were given resveratrol 5mg /kg by gavage, mice in western medicine group were given metformin 400mg / kg by gavage, and mice in blank group and model group were given equal volume of normal saline by gavage once a day for 8 weeks. During the intervention period, the weight of mice in each group and the blood of tail vein were weighed every 2 weeks, and the blood glucose was measured. Fasting for 12 hours after the last treatment, inhalation anesthesia with 3% isoflurane, blood collection from abdominal aorta and heart tissue were collected for index detection.

2.4 Index detection

2.4.1 Serum biochemical indexesAbdominal aortic blood was centrifuged at 3000 rmb/min at 4 ℃for 15min, and the contents of CK and LDH in serum were detected by enzyme-linked immunosorbent assay. The detection method refers to the instructions of the kit.

2.4.2 Histopathological observation of heart

Myocardial tissue was embedded in paraffin, sliced with a microtome, fixed with 4% paraformaldehyde solution, 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.

2.4.3 Ultrastructural observation of heart tissueMyocardial tissue was fixed with 3% glutaraldehyde and 1% osmic acid, dehydrated with gradient alcohol and acetone, embedded with epoxy resin, and sliced at 50 nm. The sections were placed behind the copper mesh, and then double stained with 3% lead citrate and uranyl acetate. The stained copper mesh was placed under the transmission electron microscope to observe the ultrastructure and autophagy of myocardial tissue.

2.4.4 Detection of cardiomyocyte apoptosisTake frozen myocardial slices, add 3% triton-x100 solution, and then add goat serum to block. Add 30 drops μ Ltunel reaction solution,add 30 drops after PBS cleaning μ L DAPI dye. After PBS cleaning,seal the film, observe the apoptosis of cardiomyocytes in each group under fluorescence microscope, and calculate the apoptosis rate, in which the apoptosis rate = (number of apoptotic cells / total number of cells) × 100%.

2.4.5 RT-qPCR

Appropriate amount of myocardial tissue was added to Trizol to extract RNA, gel electrophoresis and spectrophotometer were used to detect RNA molecular weight and purity. Take 8 μ L total RNA was incubated at 42 ℃ for 60 min after adding reverse transcription reaction solution to synthesize cDNA. Amplify with cDNA as template, mix all reagents according to the instructions of SYBR premix ex Taq kit, and then run the machine for detection. The sequence of PCR primers was as follows: Bax:upstream: 5'-GGATCGAGCAGAGAGGATGG-3', downstream: 5'-TGGTGAGTGA GGCAGTGAGG-3', primer length: 464bp; Bcl-2:upstream: 5'-CTGGTGGACA ACATCGCTCTG-3', downstream:5'-GGTCTGCTGACCTCACTTGTG-3', primer length: 227bp; β-Actin: upstream: 5'-GATGGTGGGTATGGGTCAGAAGGAC-3',downstream: 5'-GCTCATTGCCGATAGTGATGACT-3', primer length: 630bp. Amplification conditions: 90 ℃ 30s, 94 ℃ 15s, 65 ℃30s, a total of 35 cycles. with β- Actin as an internal reference gene,the target gene and 2-ΔΔCtratio was used to calculate the expression of each gene.

2.4.6 Western blotAn appropriate amount of myocardial tissue was lysed on ice,and the protein concentration was measured by BCA method.Electrophoresis, membrane transfer, skimmed milk sealing. Add diluted LC3, p62, P - Mst1, Mst1, SIRT3 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 fixing, gel imaging system to take pictures. with β- Actin is an internal reference protein, and the ratio of target protein to internal reference protein is taken as the relative expression of protein.

2.5 Statistical analysis

The data of this study were analyzed by SPSS 22. The data were expressed as mean ± standard deviation(±s ). One way ANOVA was used for comparison between groups. The minimum significant difference method was used for comparison between two groups.The difference was statistically significant with P ＜ 0.05.

3. Result

3.1 Effects of resveratrol on body weight and blood glucose level of mice in each group

Compared with the blank control group, the body weight and blood glucose level of model mice in 2w, 4w, 6w and 8w increased significantly (P ＜ 0.01). Resveratrol and metformin significantly decreased body weight and blood sugar level in diabetic mice, and the difference was statistically significant compared with the model group (P ＜ 0.05 and P ＜ 0.01). See Table 1 and table 2

3.2 Effects of Resveratrol on serum CK and LDH levels in mice

Compared with the blank group, the levels of serum CK and LDH in the model group increased significantly (P ＜ 0.01). Resveratrol and metformin significantly decreased serum CK and LDH levels in diabetic mice, and the difference was statistically significant compared with the model group (P ＜ 0.01). See Table 3

Table 1 Effect of resveratrol on body weight of mice(g,±s)

Table 1 Effect of resveratrol on body weight of mice(g,±s)

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

Groupn0w2w4w6w8w Control1019.21±1.3221.12±1.1222.15±1.6424.35±1.5425.27±1.67 Model 1019.35±1.0628.14±2.17**39.05±2.51**47.06±3.15**54.27±3.67**Resveratrol 1020.11±1.2326.34±2.3331.05±2.41#39.64±3.43##45.21±2.98##Metformin1019.87±1.0125.04±1.7429.61±2.04#37.05±2.78##44.01±2.81##Statistical valueF=1.737F=25.566F=123.941F=112.125F=156.601 P value0.1770.0000.0000.0000.000

Table 2 Effect of resveratrol on blood glucose in mice(mmol/L,±s)

Table 2 Effect of resveratrol on blood glucose in mice(mmol/L,±s)

Groupn0w2w4w6w8w Control105.31±0.375.41±0.365.29±0.415.42±0.345.39±0.44 Model 108.83±0.8511.62±1.06**14.52±1.98**15.84±2.61**16.15±3.17**Resveratrol 108.75±0.699.82±0.92#10.62±1.58##11.23±2.31##11.62±2.74##Metformin108.91±0.789.62±0.86#10.11±1.03##10.65±2.44##10.16±2.69##Statistical valueF=46.005F=97.785F=70.462F=29.032F=55.575 P value0.0000.0000.0000.0000.000

Table 3 Effects of resveratrol on serum CK and LDH in mice(±s)

Table 3 Effects of resveratrol on serum CK and LDH in mice(±s)

GroupnCK(U/mL)LDH(U/L)Control1049.12±4.24121.36±15.34 Model 10131.29±10.44**438.45±39.85**Resveratrol 1078.31±6.38##214.35±18.62##Metformin1082.49±5.98##231.08±19.27##Statistical valueF=279.130F=372.287 P value0.0000.000

3.3 Effects of resveratrol on myocardial pathological morphology and apoptosis in mice

The cardiomyocytes in the blank control group were closely arranged and regular, and there was no infiltration of inflammatory cells. Compared with the blank control group, the arrangement of cardiomyocytes in the model group was significantly disordered,enlarged, the number of cells decreased, focal necrosis and obvious inflammatory cell infiltration were observed, and the inflammatory score increased significantly (P ＜ 0.01). The arrangement and necrosis of cardiomyocytes in resveratrol group and Western medicine group were significantly improved compared with the model group, and the inflammatory score was significantly lower than that in the model group (P ＜ 0.01). See Table 4 and figure 1A

Immunofluorescence results showed that a very small amount of apoptosis was observed in the myocardium of mice in the blank control group. Compared with the blank control group, the apoptosis rate of cardiomyocytes in the model group increased significantly(P ＜ 0.01). Resveratrol and metformin significantly decreased the myocardial cell apoptosis in diabetic mice, and the difference was statistically significant compared with the model group (P ＜ 0.01).See Table 5 and figure 1B

Table 4 Effects of resveratrol on myocarditis level and apoptosis in mice(±s)

Table 4 Effects of resveratrol on myocarditis level and apoptosis in mice(±s)

GroupnInflammation scoreApoptosis rate Control100.00±0.001.16±0.11 Model 104.80±0.42**10.41±1.05 Resveratrol 101.90±0.57##3.17±0.19 Metformin101.90±0.74##4.20±0.38 Statistical valueF=150.255F =493.366 P value0.0000.000

Figure 1 He and TUNEL staining were used to observe the pathological morphology and apoptosis level of myocardial tissue

3.4 Effect of resveratrol on myocardial ultrastructure in mice

The results of transmission electron microscope showed that the cardiomyocytes of the blank control group were normal,accompanied by a large number of autophagosomes. In the model group, the cells were disordered with cell swelling, and the number of autophagosomes decreased significantly. The arrangement of cardiomyocytes in resveratrol group and Western medicine group was better than that in model group, and the number of autophagosomes increased significantly. See Figure 2.

Figure 2 Ultrastructure of myocardium of mice in each group was observed by transmission electron microscope( × 5000 times)

3.5 Effect of resveratrol on the expression of Bax and Bcl-2 mRNA in mouse myocardium

Compared with the blank control group, the expression level of Bax mRNA and the ratio of Bax/Bcl-2 increased significantly, and the expression level of Bcl-2 mRNA decreased significantly (P ＜ 0.01).Compared with the model group, the expression level of Bax mRNA and the ratio of Bax/Bcl-2 in myocardium of resveratrol group and Western medicine group decreased significantly, and the expression level of Bcl-2 mRNA increased significantly (P ＜ 0.01). See Table 5 3.6 Effect of resveratrol on autophagy protein in mouse myocardium Compared with the blank control group, the expression level of p62 protein increased significantly, and the ratio level of LC3II/LC3I decreased significantly (P ＜ 0.01). Compared with the model group,the expression level of p62 protein in myocardium of resveratrol group and Western medicine group decreased significantly, and the ratio level of LC3II/LC3I increased significantly (P ＜ 0.01). See Table 6 and Figure 3.

Table 5 Effect of resveratrol on the expression levels of Bax and Bcl-2 mRNA in mouse myocardium(±s)

Table 5 Effect of resveratrol on the expression levels of Bax and Bcl-2 mRNA in mouse myocardium(±s)

GroupnRelative expression level of BaxRelative expression level of Bcl-2Bax/Bcl-2 Ratio Control101.00±0.011.00±0.021.00±0.01 Model 103.26±0.46**0.30±0.02**10.90±2.25**Resveratrol 101.82±0.18##0.77±0.17##2.47±0.60##Metformin101.89±0.34##0.68±0.34##2.82±0.68##Statistical valueF=96.220F =74.145F =136.181 P value0.0000.0000.000

Table 6 Effect of resveratrol on autophagy related protein expression in myocardial tissue(±s)

Table 6 Effect of resveratrol on autophagy related protein expression in myocardial tissue(±s)

GroupnP62/β-acitnLC3II/LC3I Control100.28±0.031.04±0.06 Model 101.25±0.08**0.18±0.07**Resveratrol 100.72±0.03##0.99±0.07##Metformin100.75±0.06##0.92±0.05##Statistical valueF=161.472F =124.815 P value0.0000.000

Figure 3 Western blot was used to detect the expression of p62 and LC3 proteins in myocardium of mice in each group

3.7 Effect of resveratrol on the expression of Mst1 and SIRT3 proteins in mouse myocardium

Compared with the blank control group, the expression of p-mst1 protein was significantly increased and the expression of SIRT3 was significantly decreased in the model group (P ＜ 0.01). However,there was no significant change in the expression of Mst1 protein in myocardial tissue of model group mice, and there was no significant difference compared with blank control group mice (P ＞ 0.05). The expression of p-mst1 protein in myocardium of resveratrol group and Western medicine group decreased significantly, and the expression of SIRT3 increased significantly. Compared with the model group,the difference was statistically significant (P ＜ 0.01). However,there was no significant change in the expression of Mst1 protein in resveratrol group and Western medicine group, and there was no significant difference compared with the model group (P ＞ 0.05).See Table 7 and figure 4.

Table 7 Effect of resveratrol on the expression of Mst1 and SIRT3 proteins in myocardial tissue(±s)

Table 7 Effect of resveratrol on the expression of Mst1 and SIRT3 proteins in myocardial tissue(±s)

Groupnp-Mst1/ Mst1 Mst1/β-acitnSirt3/β-acitn Control100.21±0.061.16±0.291.33±0.06 Model 100.96±0.05**1.11±0.090.23±0.07**Resveratrol 100.47±0.06##1.12±0.240.49±0.08##Metformin100.55±0.05##1.07±0.090.42±0.07##Statistical valueF=106.117F =0.505F =149.163 P value0.0000.6900.000

Figure 4 Western blot was used to detect the expression of Mst1 and SIRT3 proteins in myocardium of mice in each group

4. Discussion

DCM usually induces the biological changes of cardiomyocytes and abnormal myocardial function due to metabolic disorder, which leads to heart failure. The ability of cardiac myocytes is mainly derived from fat metabolism and glycolysis, while diabetes leads to impaired glucose utilization in cardiomyocytes. Therefore, the oxidation of non-esterified fatty acid (NEFA) causes large amounts of intermediate metabolites such as uncoupling protein 3, two glycidyl and ceramide, and a large number of Reactive oxygen species(ROS). Thus, it causes inflammatory cell infiltration, cardiomyocyte necrosis and myocardial dysfunction [9]. High glucose status is the core of inducing DCM, which can not only cause the activation of renin angiotensin aldosterone system, increase the production of terminal products of advanced glycosylation and abnormal secretion of cytokines, but also closely relate to intracellular oxidative stress response and apoptosis [10].

Apoptosis, also known as programmed cell death, is the formation of cell death regulated by a variety of genes. Bcl-2 gene family is the first discovered gene family involved in apoptosis regulation, in which Bax and Bcl-2 are two key apoptosis regulation genes of Bcl-2 gene family [11]. Bcl-2, located at 18q21 of human chromosome,is an endogenous anti apoptotic gene, which is expressed in endoplasmic reticulum, mitochondria and nuclear membrane. In the early stage of apoptosis, Bcl-2 plays an anti-apoptotic role mainly by inhibiting the cleavage of DNA, the production of cell shrinkage and the concentration of chromatin [12]. Bax is a pro apoptotic gene in Bcl-2 gene family, which usually exists in the cytoplasm in the form of monomer. When Bax exists in the form of Bax/Bax homodimer,it induces apoptosis, and when Bax combines with Bcl-2 to form heterodimer, it inhibits the process of apoptosis [13].

Autophagy is a non-specific material degradation process in eukaryotic cells, which plays an important role in maintaining cell function. In the process of autophagy, autophagic vesicles wrap damaged organelles and unwanted lipids or proteins, combine with lysosomes to form autophagic lysosomes, and degrade the contents of autophagic vesicles, so as to realize the renewal of organelles and cell self-metabolism [14-15]. Autophagy plays an important role in maintaining the structure of the heart and the integrity of cardiomyocytes. The destruction and disorder of autophagy can lead to heart failure [16-17]. It was found that promoting the level of cardiomyocyte autophagy in DCM model can significantly inhibit its apoptosis [18]. P62, also known as sqstm1, is an autophagy marker protein together with LC3. LC3I is transformed into LC3II in lipid form during autophagy and stably expressed on autophagosome membrane. P62 is also called selective autophagy receptor. After autophagy activation, p62 protein is degraded in the cytoplasm,while inhibiting the level of autophagy leads to the accumulation of p62 protein.

Mst1 is one of the important components of Hippo signaling pathway, which has the functions of regulating cell proliferation,apoptosis and inhibiting tumor growth [19]. Overexpression of Mst1 can promote apoptosis by activating Yap Hippo signal axis and affect the level of autophagy by regulating the interaction between Bcl-2 and Beclin1 [20]. SIRT3 is a member of the silencing information regulator family. It enhances its transcriptional activity by deacetylating FoxO1, and then participates in the process of cell metabolism and autophagy to improve mitochondrial oxidative stress response [21]. In this study, we found that resveratrol can significantly reduce the body weight and blood glucose level of db/db mice,which is consistent with the results of previous studies [22]. On this basis, by detecting the changes of serum CK, LDH and pathological morphology, it was confirmed that resveratrol can significantly improve DCM myocardial injury and inflammatory cell infiltration.TUNEL staining and RT qPCR results showed that resveratrol could significantly inhibit the level of cardiomyocyte apoptosis, which may be an important way of its anti-myocardial injury. Further studies showed that resveratrol could promote the formation of autophagic lysosomes, up regulate the level of myocardial autophagy in db/db mice, increase the expression of p-MST1 and inhibit the expression of SIRT3 protein in myocardial tissue.

In conclusion, resveratrol has a protective effect on DCM, and its mechanism of improving cardiomyocyte injury is mainly to inhibit cardiomyocyte apoptosis by activating Mst1/SIRT3 signal pathway and then up regulating cardiomyocyte autophagy level.

Author's Contribution

The experiment was designed by Ma ZHENWANG and directed by Jiang Deyou. Cai Shaojie and Li Zhenyu completed experimental modeling and drug intervention; Yuan Xingxing is responsible for index detection; Wang Mei and Duan Jun 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.