Yellow Wine Polyphenolic Compounds Regulate SIRT3 Expression to Alleviate Doxorubicin-induced Myocardial Injury

doi:10.12114/j.issn.1007-9572.2022.01.605

Chinese General Practice ›› 2022, Vol. 25 ›› Issue (17): 2102-2109.DOI: 10.12114/j.issn.1007-9572.2022.01.605

Special Issue: 心血管最新文章合集

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Yellow Wine Polyphenolic Compounds Regulate SIRT3 Expression to Alleviate Doxorubicin-induced Myocardial Injury

1.The First School of Medicine, Wenzhou Medical University, Wenzhou 325000, China
2.Cardiovascular Department, Taizhou Hospital of Zhejiang Province, Taizhou 318000, China
3.Department of Cardiology, Shaoxing People's Hospital, Shaoxing 312000, China
4.Shaoxing University, Shaoxing 312000, China

Received:2022-02-06 Revised:2022-04-06 Published:2022-04-28 Online:2022-04-28
Contact: Hangyuan GUO
About author:
SUN S M, SUN Z Z, CHI J F, et al. Yellow Wine Polyphenolic Compounds regulate SIRT3 expression to alleviate doxorubicin-induced myocardial injury[J]. Chinese General Practice, 2022, 25 (17) : 2102-2109.

黄酒多酚诱导沉默调节蛋白3表达减轻阿霉素心肌损伤研究

1.325000　浙江省温州市，温州医科大学第一临床医学院
2.318000　浙江省台州市，台州医院心血管内科
3.312000　浙江省绍兴市人民医院心内科
4.312000　浙江省绍兴市文理学院

通讯作者: 郭航远
作者简介:
孙世民，孙珍珠，池菊芳，等.黄酒多酚诱导沉默调节蛋白3表达减轻阿霉素心肌损伤研究[J].中国全科医学，2022，25（17）：2102-2109.[www.chinagp.net] 作者贡献：孙世民设计研究方案，进行细胞实验及后期数据收集、统计学分析，绘制图表等；孙珍珠负责进行动物实验中的造模、取材以及切片染色；池菊芳负责文章的质量控制及审校；郭航远提出研究思路，对文章整体负责、监督管理。
基金资助:
国家自然科学基金面上项目(81873120); 绍兴市人民医院院内青年基金项目(2018YB24)

Abstract

Abstract:

Background

Doxorubicin (DOX) , a common and powerful anthracycline antitumor agent, has been widely used in the treatment of various tumors, especially solid tumors. However, its strong cardiotoxicity-induced irreversible cardiomyopathy and congestive heart failure limit its clinical application. Yellow Wine Polyphenols Compounds (YWPC) are polyphenols extracted from Shaoxing yellow rice wine which have anti-inflammatory and antioxidant activities, and can alleviate DOX-induced myocardial injury, but the mechanism of actions is still unclear.

Objective

To explore the mechanism of YWPC reducing DOX-induced myocardial injury via in vitro and in vivo experiments.

Methods

SD rats were used for an in vivo experiment and divided into four groups: control group (intervened with normal saline) , YWPC group (intervened with YWPC and normal saline) , DOX group (intervened with DOX and normal saline for establishing a DOX-induced myocardial injury model) and DOX+YWPC group (intervened with DOX, YWPC and normal saline for observing the myocardial protective effect of YWPC in a DOX-induced myocardial injury model) . When the experiment ended, all rats were sacrificed and cardiac tissues were taken out for examining myocardial fiber morphology using Masson's trichrome staining, myocardial cell apoptosis using TUNEL assay, pathological changes using immunohistochemical assay, and levels of proteins (Bcl-2 and Bax) involved in apoptosis as well as expression level of SIRT3 using Western blotting. And the serum lactate dehydrogenase (LDH) was also measured. Cardiomyocyte H9C2 cells of rats were used for an in vitro experiment and divided into five groups: control group, YWPC group (intervened with YWPC and normal saline) , DOX group (intervened with DOX for establishing a DOX-induced myocardial injury model within 24 hours) and three DOX+YWPC (1 mg/L, 10 mg/L, 100 mg/L) groups〔first intervened with DOX for establishing a DOX-induced myocardial injury model within 24 hours, then with YWPC for observing the myocardial protective effect of three concentrations of YWPC (1 mg/L, 10 mg/L, 100 mg/L) , respectively〕. Viability of H9C2 cells was measured by CCK-8 assay. Apoptosis, and SIRT3 expressed in H9C2 cells were measured by Western blotting. Then another batch of the same H9C2 cells were took and divided into control group, DOX group, DOX+YWPC group, DOX+3-TYP group and DOX+YWPC+3-TYP group, and SIRT3 inhibitor 3-TYP was used to inhibit the activity of SIRT3 protein in the latter two groups. Then apoptosis level and SIRT3 protein expressed in H9C2 cells were detected by Western blotting for further assessing the effect of SIRT3 in reducing DOX-induced myocardial injury.

Results

In vivo experiment: (1) Under the microscope, Masson's trichrome-stained myocardial fibers of rats in DOX group were disordered and intersected with a large number of blue collagen fibers. The myocardial texture of DOX+YWPC group was partially restored with decreased blue collagen fibers. DOX group had higher proportion of collagen fibers distributed in cardiovascular tissues, and higher serum LDH than control and DOX+YWPC groups (P<0.05) . DOX group had lower ratio of Bcl-2/Bax and level of expression of SIRT3 than control and DOX+YWPC groups (P<0.05) . The apoptotic myocardial cells with a patch distribution appearing as bright green dots were significantly increased in DOX group at first, but were decreased after YWPC treatment. In vitro experiment: (1) The absorbance value, Bcl-2/Bax ratio and expression level of SIRT3 in DOX group were lower than those of control group, DOX+YWPC (1 mg/L) group, DOX+YWPC (10 mg/L) group and DOX+YWPC (100 mg/L) group (P<0.05) . (2) The Bcl-2 /Bax ratio and expression level of SIRT3 in DOX or DOX+3-TYP group were lower than those in control group (P<0.05) . (3) The Bcl-2/Bax ratio in DOX+YWPC+3-TYP group was lower than that in DOX+YWPC group (P<0.05) . (4) The expression level of SIRT3 was similar in DOX+YWPC and DOX+YWPC+3-TYP groups (P>0.05) .

Conclusion

DOX-induced myocardial injury in in vitro and in vivo experiments with rats may be alleviated by YWPC via improving the expression level of SIRT3, and the effect may be reduced if the expression level of SIRT3 protein is inhibited.

Key words: Heart diseases, Doxorubicin, Yellow Wine Polyphenol Compounds, Antineoplastic agents, SIRT3, Apoptosis

摘要：

背景

阿霉素（DOX）是一种常见的强效蒽环类抗肿瘤药物，被广泛应用于各类肿瘤尤其是实体性肿瘤的治疗中。然而，DOX有较强的心脏毒性，临床表现为不可逆性心肌病和充血性心力衰竭。黄酒多酚（YWPC）是从绍兴黄酒中提取的多酚类物质，具有抗炎、抗氧化等生理活性，对DOX所致心肌损伤有一定缓解作用，但其作用机制还不明晰。

目的

通过体内外实验探讨YWPC减轻DOX心肌损伤的作用机制。

方法

动物实验：使用SD大鼠建立DOX心肌损伤模型，分为对照组、YWPC组、DOX组与DOX+YWPC组；取心脏组织进行MASSON染色、原位末端标记（TUNEL）染色及免疫组织化学染色，取血清测定乳酸脱氢酶（LDH）水平，并用动物组织蛋白、Western Blot法检测凋亡水平〔B淋巴细胞瘤-2蛋白（Bcl-2）、Bcl-2相关X蛋白（Bax）〕及沉默调节蛋白3（SIRT3）表达水平变化。体外实验：通过1 μmol/L DOX干预H9C2细胞24 h建立DOX诱导H9C2细胞损伤模型，先将一批H9C2细胞分为对照组、DOX组、DOX+YWPC（1 mg/L）组、DOX+YWPC（10 mg/L）与DOX+YWPC（100 mg/L）5组，采用CCK-8法检测H9C2细胞活力，通过Western Blot法检测凋亡水平及SIRT3表达水平变化；之后将另一批H9C2细胞分为对照组、DOX组、DOX+YWPC组、DOX+SIRT3抑制剂（3-TYP）组及DOX+YWPC+3-TYP组，通过3-TYP抑制SIRT3蛋白活性，通过Western Blot法检测凋亡水平及SIRT3表达水平变化，进一步明确SIRT3在YWPC减轻DOX心肌损伤中的作用。

结果

动物实验中，大鼠心肌切片通过MASSON染色后可见：DOX组大鼠心肌纤维排列紊乱，大量蓝色胶原纤维贯穿心肌纤维；DOX+YWPC组大鼠心肌切片纹理部分恢复，蓝色胶原纤维减少。DOX组胶原纤维占比、血清LDH水平高于对照组、DOX+YWPC组（P<0.05）。DOX组Bcl-2/Bax比值、SIRT3表达水平低于对照组、DOX+YWPC组（P<0.05）；DOX组大鼠心肌切片中代表凋亡的绿色亮点明显增多，呈片状分布，而经YWPC治疗后凋亡亮点减少。体外实验中，DOX组吸光度值、Bcl-2/Bax比值、SIRT3表达水平低于对照组、DOX+YWPC（1 mg/L）组、DOX+YWPC（10 mg/L）组、DOX+YWPC（100 mg/L）组（P<0.05），DOX组、DOX+3-TYP组Bcl-2/Bax比值、SIRT3表达水平低于对照组（P<0.05）；DOX+YWPC+3-TYP组Bcl-2/Bax比值低于DOX+YWPC组（P<0.05），DOX+YWPC组与DOX+YWPC+3-TYP组SIRT3表达水平比较，差异无统计学意义（P>0.05）。

结论

YWPC可以通过调节SIRT3表达水平减轻DOX心肌损伤，抑制SIRT3会降低YWPC的作用。

关键词: 心脏病, 阿霉素, 黄酒多酚, 抗肿瘤药, 沉默调节蛋白3, 细胞凋亡

Shimin SUN,Zhenzhu SUN,Jufang CHI, et al. Yellow Wine Polyphenolic Compounds Regulate SIRT3 Expression to Alleviate Doxorubicin-induced Myocardial Injury[J]. Chinese General Practice, 2022, 25(17): 2102-2109. DOI: 10.12114/j.issn.1007-9572.2022.01.605.
孙世民,孙珍珠,池菊芳等. 黄酒多酚诱导沉默调节蛋白3表达减轻阿霉素心肌损伤研究[J]. 中国全科医学, 2022, 25(17): 2102-2109. DOI: 10.12114/j.issn.1007-9572.2022.01.605.

Figures/Tables 8

Figure 1 Effect of YWPC in alleviating DOX-induced myocardial injury in the in vivo experiment with four groups of rats

Table 1 Results of YWPC in alleviating DOX-induced myocardial injury explored in the in vivo experiment with four groups of rats

组别	只数	胶原纤维占比（%）	LDH（U/L）	Bcl-2/Bax比值	SIRT3表达水平
对照组	6	25.74±0.79^a	3 773.79±201.61^a	1.04±0.10^a	0.99±0.08^a
YWPC组	6	24.73±3.31	4 059.88±35.08	0.90±0.06	0.84±0.05
DOX组	6	38.50±1.19	5 546.91±137.37	0.19±0.01	0.29±0.08
DOX+YWPC组	6	31.68±1.13^a	4 491.68±149.93^a	0.57±0.02^a	0.53±0.04^a
F值		67.76	174.3	244.0	139.7
P值		<0.01	<0.01	<0.01	<0.01

Figure 2 Effect of YWPC in alleviating myocardial cell apoptosis due to DOX-induced myocardial injury explored in the in vivo experiment with four groups of rats

Figure 3 Effect of YWPC in alleviating apoptosis in DOX-injured H9C2 cells explored in the in vitro experiment

Table 2 Results of viability measured by CCK-8 assay，and levels of apoptosis and SIRT3 measured by Western blotting in DOX-injured H9C2 cells

组别	吸光度值	Bcl-2/Bax比值	SIRT3表达水平
对照组	1.93±0.01^a	1.08±0.09^a	1.11±0.18^a
DOX组	0.74±0.03	0.08±0.01	0.47±0.05
DOX+YWPC（1 mg/L）组	0.94±0.04^a	0.27±0.02^a	0.89±0.08^a
DOX+YWPC（10 mg/L）组	1.11±0.05^a	0.30±0.05^a	0.91±0.10^a
DOX+YWPC（100 mg/L）组	1.35±0.02^a	0.50±0.01^a	0.95±0.25^a
F值	605.1	256.2	8.142
P值	<0.01	<0.01	0.004

Figure 4 Expression levels of SIRT3 in DOX-injured myocardial tissues of rat model and DOX-injured H9C2 cell model

Figure 5 Expression levels of apoptosis-related proteins and SIRT3 in DOX-injured H9C2 cells intervened with 3-TYP

Table 3 Changes of expression levels of apoptosis-related protein and SIRT3 in DOX-injured H9C2 cells intervened with 3-TYP

组别	Bcl-2/Bax比值	SIRT3表达水平
对照组	1.06±0.18	1.03±0.08
DOX组	0.15±0.01^a	0.53±0.09^a
DOX+YWPC组	0.56±0.02	0.81±0.03
DOX+3-TYP组	0.14±0.01^a	0.40±0.16^a
DOX+YWPC+3-TYP组	0.08±0.01^b	0.63±0.23
F值	401.6	9.979
P值	<0.01	0.0016

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Yellow Wine Polyphenolic Compounds Regulate SIRT3 Expression to Alleviate Doxorubicin-induced Myocardial Injury

黄酒多酚诱导沉默调节蛋白3表达减轻阿霉素心肌损伤研究

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