二氢杨梅素对高糖诱导的H9C2心肌细胞损伤的影响

doi:10.3877/cma.j.issn.2095-1221.2019.03.006

目的

探讨二氢杨梅素（DHM）对高糖（HG）诱导的心肌细胞H9C2损伤的影响及机制。

方法

细胞处理分为对照组、35 mmol/L HG组、35mmol/L HG+50 μmol/L DHM组及50 μmol/L DHM组。CCK-8法检测细胞活力，化学比色法检测丙二醛（MDA）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）水平，流式细胞术检测ROS水平；荧光定量PCR法及Elisa法分别检测TNFα、IL1β、IL6 mRNA和含量，Western Blotting检测p-IκBα、IκBα蛋白及核蛋白NF-κB p65的表达水平。采用单因素方差分析进行组间比较。

结果

对照组、35mmol/?L HG组、35?mmol/L HG+50?μmol/L DHM组、35?mmol/L HG+100?μmol/L DHM组的细胞活力分别是（100±0.00） ﹪、（52.23±5.69） ﹪、（74.58±6.12） ﹪和（86.04±3.76）﹪，差异具有统计学意义（F?= 40.61，P?< 0.01）。对照组、35?mmol/L HG组和35?mmol/L HG+100?μmol/L DHM组的MDA和ROS水平，SOD和CAT活性分别是（0.44±0.06）?nmol/?ml，（2.33±0.40）?nmol/?ml，（1.48±0.41）?nmol/ml、（156.0±9.00）U/ml，（325.3±10.69）U/ml，（244.0±9.54）?U/ml，（10.62± 1.59）?U/?ml，（5.18±0.34）U/ml，（7.75±0.53）U/ml，（11.31±0.98）?U/ml，（5.20±1.12）?U/?ml和（8.06±0.66）U/ml，差异具有统计学意义（F?= 30.34，29.75，14.72，P均< 0.01）。DHM预处理可明显拮抗HG对H9C2心肌细胞TNFα、IL1β和IL6 mRNA及含量的上调作用，差异存在统计学意义（P?均< 0.01）。DHM可抑制HG对H9C2心肌细胞p-IκBα/?IκBα蛋白和核蛋白NF-κB p65表达的增加作用，差异存在统计学意义（P均< 0.01）。

结论

DHM可拮抗HG诱导的H9C2心肌细胞损伤，这可能与其抑制NF-κB信号通路有关。

关键词: 二氢杨梅素, 高糖, 氧化应激, 炎症反应, 心肌细胞

Objective

To investigate the influence of dihydrobayberry (DHM) on high glucose (HG) -induced cardiomyocytes H9c2 cells damage and its poteintial mechanism.

Methods

Cells were divided into the following groups: control group, 35 mmol/L HG group, 35?mmol/L HG+50?μmol/L DHM group and 50?μmol/L DHM group. CCk-8 assay was used to detect cell viability. The levels of malondialdehyde (MDA) , superoxide dismutase (SOD) and catalase (CAT) were determined by chemical colorimetry. ROS levels were measured by flow cytometry. TNFα, IL1β and IL6 mRNA expression and contents were determined by fluorescence quantitative PCR and Elisa assays respectively. The expression levels of p-IκBα and IκBα proteins, and nucleoprotein NF-κB p65 were detected by Western Blot. Univariate analysis of variance was used for comparison between groups.

Results

Pretreatment with 50 and 100?μmol/L DHM significantly inhibited the reduced cell viability of H9C2 myocardial cells caused by 35?mmol/L HG: The cell viability of the control group, 35?mmol/L HG group, 35?mmol/L HG+50?μmol/L DHM group, 35?mmol/L HG+100?μmol/L DHM group were (100±0.00) ﹪, (52.23±5.69) ﹪, (74.58±6.12) ﹪ and (86.04±3.76) ﹪, respectively (F?= 40.61, P < 0.001) . We also found that pretreatment with DHM (50?μmol/L) significantly inhibited the enhanced MDA and ROS levels, and decreased SOD and CAT activity of H9C2 myocardial cells induced by HG (35?mmol/?L) . The MDA level, SOD and CAT activity of the control group, HG group and HG+ DHM group were (0.44±0.06) ?nmol/?ml, (2.33±0.40) nmol/ml, (1.48±0.41) nmol/ml, (156.00±9.00) U/ml, (325.3±10.69) U/?ml, (244.0±9.54) U/ml, (10.62±1.59) U/ml, (5.18±0.34) U/ml, (7.75±0.53) ?U/?ml and (11.31±0.98) ?U/?ml , (5.20±1.12) U/ml, (8.06±0.66) U/ml, respectively. (F?= 30.34, 29.75, 14.72, P?all < 0.001) . Pretreatment with DHM significantly inhibited the increased expression levels of TNFα, IL1β and IL6 mRNA and their contents caused by HG in H9C2 myocardial cells (P all < 0.001) . DHM significantly inhibited the increased expression of p-IκBα/IκBα and nucleoprotein NF-κB p65 in H9C2 myocardial cells caused by HG (P all < 0.001) .

Conclusion

DHM can antagonize HG-induced H9C2 myocardial cell injury, which may be related to its inhibition of NF-κB signaling pathway.

Key words: Dihydromyricetin, High glucose, Oxidative stress, Inflammatory response, Myocardial cells

表1 DHM对HG处理的H9C2心肌细胞活力的影响（±s）

分组	样本数	细胞相对活力光密度
对照组	3	100.00±0.00
35 mmol/L HG组	3	52.23±5.69^a
35 mmol/L HG+25 μmol/L DHM组	3	61.62±6.47
35 mmol/L HG+50 μmol/L DHM组	3	74.58±6.12^b
35 mmol/L HG+100 μmol/L DHM组	3	86.04±3.76^b
HPG组	3	104.30±8.15
F值	?	40.61
P值	?	< 0.01

表1 DHM对HG处理的H9C2心肌细胞活力的影响（±s）

分组	样本数	细胞相对活力光密度
对照组	3	100.00±0.00
35 mmol/L HG组	3	52.23±5.69^a
35 mmol/L HG+25 μmol/L DHM组	3	61.62±6.47
35 mmol/L HG+50 μmol/L DHM组	3	74.58±6.12^b
35 mmol/L HG+100 μmol/L DHM组	3	86.04±3.76^b
HPG组	3	104.30±8.15
F值	?	40.61
P值	?	< 0.01

表2 DHM对HG处理的H9C2心肌细胞氧化应激的影响（±s）

分组	样本数	MDA （nmol/ml）	SOD （U/ml）	CAT （U/ml）	ROS
对照组	3	0.44±0.06	10.62±1.59	11.31±0.98	156.0± 9.00
35 mmol/L HG组	3	2.33±0.40^a	5.18±0.34^a	5.20±1.12^a	325.3±10.69^a
35 mmol/L HG+50 μmol/L DHM组	3	1.48±0.41^b	7.75±0.53^b	8.06±0.66^b	244.0± 9.54^b
50 μmol/L DHM组	3	0.45±0.07	11.44±0.61	11.02±0.02	143.0± 8.54
F值	?	30.34	29.75	14.72	241.1
P值	?	< 0.01	< 0.01	< 0.01	< 0.01

表2 DHM对HG处理的H9C2心肌细胞氧化应激的影响（±s）

分组	样本数	MDA （nmol/ml）	SOD （U/ml）	CAT （U/ml）	ROS
对照组	3	0.44±0.06	10.62±1.59	11.31±0.98	156.0± 9.00
35 mmol/L HG组	3	2.33±0.40^a	5.18±0.34^a	5.20±1.12^a	325.3±10.69^a
35 mmol/L HG+50 μmol/L DHM组	3	1.48±0.41^b	7.75±0.53^b	8.06±0.66^b	244.0± 9.54^b
50 μmol/L DHM组	3	0.45±0.07	11.44±0.61	11.02±0.02	143.0± 8.54
F值	?	30.34	29.75	14.72	241.1
P值	?	< 0.01	< 0.01	< 0.01	< 0.01

表3 DHM对HG处理的H9C2心肌细胞炎症的影响（±s）

分组	样本数	TNF α mRNA	IL1β mRNA	IL6 mRNA	TNF α	IL1β	IL6
对照组	3	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
35 mmol/L HG组	3	2.59±0.08^a	3.14±0.26^a	3.34±0.68^a	4.39±0.25	5.21±0.20	2.83±0.12
35 mmol/LHG+50 μmol/L DHM组	3	1.51±0.22^b	2.52±0.32^b	2.10±0.24^b	2.65±0.26	3.48±0.17	1.44±0.67
50 μmol/L DHM组	3	1.11±0.13	0.99±0.12	1.08±0.20	1.15±0.03	1.02±0.12	0.93±0.06
F值	?	15.56	75.58	25.92	228.3	602.2	435.2
P值	?	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

表3 DHM对HG处理的H9C2心肌细胞炎症的影响（±s）

分组	样本数	TNF α mRNA	IL1β mRNA	IL6 mRNA	TNF α	IL1β	IL6
对照组	3	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
35 mmol/L HG组	3	2.59±0.08^a	3.14±0.26^a	3.34±0.68^a	4.39±0.25	5.21±0.20	2.83±0.12
35 mmol/LHG+50 μmol/L DHM组	3	1.51±0.22^b	2.52±0.32^b	2.10±0.24^b	2.65±0.26	3.48±0.17	1.44±0.67
50 μmol/L DHM组	3	1.11±0.13	0.99±0.12	1.08±0.20	1.15±0.03	1.02±0.12	0.93±0.06
F值	?	15.56	75.58	25.92	228.3	602.2	435.2
P值	?	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

图1 DHM对HG处理的H9C2心肌细胞NF-κB信号通路的影响

图2 H9C2心肌细胞NF-κB信号通路相关蛋白灰度值的分析结果

[9]	Zhou MQ,Shao L,Wu J, et al. Dihydromyricetin protects against lipopolysaccharideinduced cardiomyocyte injury through the tolllike receptor4/nuclear factorκB pathway[J]. Mol Med Rep, 2017, 16(6):8983-8988.
[10]	Wu B,Lin J,Luo J, et al. Dihydromyricetin protects against diabetic cardiomyopathy in streptozotocin-induced diabetic mice[J]. Biomed Res Int, 2017, 2017:3764370.
[11]	Ogurtsova K,da Rocha Fernandes JD,Huang Y, et al. IDF Diabetes Atlas: Global estimates for the prevalence of diabetes for 2015 and 2040[J]. Diabetes Res Clin Pract, 2017, 128:40-50.
[12]	Bugger H,Bode C. The vulnerable myocardium[J]. Hamostaseologie, 2015, 35(1):17-24.
[13]	Falcão-Pires I,Leite-Moreira AF. Diabetic cardiomyopathy: understanding the molecular and cellular basis to progress in diagnosis and treatment[J]. Heart Fail Rev, 2012,17(3):325-344.
[14]	Liu L,Yin X,Wang X, et al. Determination of dihydromyricetin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study[J]. Pharm Biol, 2017, 55(1):657-662.
[15]	Wold LE,Ren J. Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a p38 map kinase-dependent oxidative stress mechanism[J]. Biochem Biophys Res Commun, 2004, 318(4):1066-1071.
[16]	Koncsos G,Varga ZV,Baranyai T, et al. Diastolic dysfunction in prediabetic male rats: Role of mitochondrial oxidative stress[J]. Am J Physiol Heart Circ Physiol, 2016, 311(4):H927-H943.
[17]	Liang W,Chen M,Zheng D, et al. A novel damage mechanism: Contribution of the interaction between necroptosis and ROS to high glucose-induced injury and inflammation in H9c2 cardiac cells[J]. Int J Mol Med, 2017, 40(1):201-208.
[18]	Domingueti CP,Dusse LM,Carvalho Md, et al. Diabetes mellitus:The linkage between oxidative stress, inflammation, hypercoagulability and vascular complications[J]. J Diabetes Complications, 2016, 30(4):738-745.
[19]	Fei Y,Sun L,Yuan C, et al. CFTR ameliorates high glucose-induced oxidative stress and inflammation by mediating the NF-κB and MAPK signaling pathways in endothelial cells[J]. Int J Mol Med, 2018, 41(6):3501-3508.
[20]	Safi SZ,Batumalaie K,Qvist R, et al. Gelam honey attenuates the oxidative stress-induced inflammatory pathways in pancreatic hamster cells[J]. Evid Based Complement Alternat Med, 2016, 2016:5843615.
[21]	Liang W,Chen M,Zheng D, et al. The opening of ATP-Sensitive K+ channels protects H9c2 cardiac cells against the high glucose-induced injury and inflammation by inhibiting the ROS-TLR4-Necroptosis pathway[J]. Cell Physiol Biochem, 2017, 41(3):1020-1034.
[22]	Nishikido T,Oyama J,Shiraki A, et al. Deletion of apoptosis inhibitor of macrophage (AIM)/CD5L attenuates the inflammatory response and infarct size in acute myocardial infarction[J]. J Am Heart Assoc, 2016, 5(4):e002863.
[23]	Li X,Zhou J,Huang K. Inhibition of the lncrna mirt1 attenuates acute myocardial infarction by suppressing NF-κB activation[J]. Cell Physiol Biochem, 2017, 42(3):1153-1164.
[1]	Jia G,Hill MA,Sowers JR. Diabetic cardiomyopathy: an update of mechanisms contributing to this clinical entity[J]. Circ Res, 2018, 122(4):624-638.
[2]	Rubler S,Dlugash J,Yuceoglu YZ, et al. New type of cardiomyopathy associated with diabetic glomerulosclerosis[J]. Am J Cardiol, 1972,30(6):595-602.
[3]	Marwick TH,Ritchie R,Shaw JE, et al. Implications of underlying mechanisms for the recognition and management of diabetic cardiomyopathy[J]. J Am Coll Cardiol, 2018, 71(3):339-351.
[4]	Boudina S,Abel ED. Diabetic cardiomyopathy revisited[J]. Circulation, 2007, 115(25):3213-3223.
[5]	Liu L,Wan J,Lang H, et al. Dihydromyricetin delays the onset of hyperglycemia and ameliorates insulin resistance without excessive weight gain in Zucker diabetic fatty rats[J]. Mol Cell Endocrinol, 2017, 439:105-115.
[6]	Fan KJ,Yang B,Liu Y, et al. Inhibition of human lung cancer proliferation through targeting stromal fibroblasts by dihydromyricetin[J]. Mol Med Rep, 2017,16(6):9758-9762.
[7]	Chu J,Wang X,Bi H, et al. Dihydromyricetin relieves rheumatoid arthritis symptoms and suppresses expression of pro-inflammatory cytokines via the activation of Nrf2 pathway in rheumatoid arthritis model[J]. Int Immunopharmacol, 2018,59:174-180.
[8]	Jiang B,Le L,Pan H, et al. Dihydromyricetin ameliorates the oxidative stress response induced by methylglyoxal via the AMPK/GLUT4 signaling pathway in PC12 cells[J]. Brain Res Bull, 2014,109:117-126.
[24]	Xu W,Chen J,Lin J, et al. Exogenous H2S protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the activation of the NF-κB and IL-1β pathways[J]. Int J Mol Med, 2015, 35(1):177-186.
[25]	Tang N,Ma J,Wang KS, et al. Dihydromyricetin suppresses TNF-α-induced NF-κB activation and target gene expression[J]. Mol Cell Biochem, 2016, 422(1-2):11-20.

[1]	魏徐, 张鸽, 伍金林. 新生儿脓毒症相关性凝血病的监测和治疗[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(04): 379-386.
[2]	史孟杰, 贺仕才, 刘斐, 闫燕, 代毅, 王辉. 对miR-206在大鼠下肢缺血再灌注损伤过程中炎症反应的影响分析[J]. 中华损伤与修复杂志(电子版), 2023, 18(03): 249-255.
[3]	罗皓天, 陈丹莹, 王伟财, 周晨. 基质细胞衍生因子1/CXC趋化因子受体4轴在骨免疫相关疾病中的研究进展[J]. 中华口腔医学研究杂志(电子版), 2023, 17(03): 218-227.
[4]	钟轼, 李斌飞, 温君琳, 古晨, 廖小卒. 右美托咪定缓解神经病理性疼痛作用机制的研究进展[J]. 中华普通外科学文献(电子版), 2023, 17(03): 237-240.
[5]	张生军, 赵阿静, 李守博, 郝祥宏, 刘敏丽. 高糖通过HGF/c-met通路促进结直肠癌侵袭和迁移的实验研究[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 21-24.
[6]	王可, 范彬, 李多富, 刘奎. 两种疝囊残端处理方法在经腹腹膜前腹股沟疝修补术中的疗效比较[J]. 中华疝和腹壁外科杂志(电子版), 2023, 17(06): 692-696.
[7]	刘骏, 朱霁, 殷骏. 右美托咪定对腹股沟疝手术麻醉效果及安全性的影响[J]. 中华疝和腹壁外科杂志(电子版), 2023, 17(05): 570-573.
[8]	邓春文, 陈嵩, 钟裴, 闵师强, 万健. LncRNA CRNDE通过miR-181a-5p/SOX6轴调节脂多糖诱导人肺泡上皮细胞的炎症反应和细胞凋亡[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(03): 129-136.
[9]	许磊, 孙杰, 陈先志, 张家泉, 李旺勇, 冯其柱, 王琦. 血液净化治疗在高血脂性重症胰腺炎中的应用[J]. 中华肝脏外科手术学电子杂志, 2023, 12(04): 464-468.
[10]	尚慧娟, 袁晓冬. 机械取栓术后应用依达拉奉右崁醇对急性缺血性脑卒中预后的改善[J]. 中华神经创伤外科电子杂志, 2023, 09(05): 295-301.
[11]	阿迪莱·阿卜杜热西提, 费奥, 邢晓雯, 谢胜强, 张睿, 兰晓娟, 程岗. 三种模拟创伤性脑损伤体外细胞模型的损伤特征比较[J]. 中华神经创伤外科电子杂志, 2023, 09(02): 69-75.
[12]	邹勇, 顾应江, 丁昊, 杨呈浩, 陈岷辉, 蔡昱. 基于Nrf2/HO-1及NF-κB信号通路探讨葛根素对大鼠脑出血后早期炎症反应及氧化应激反应的影响[J]. 中华脑科疾病与康复杂志(电子版), 2023, 13(05): 271-277.
[13]	张敏洁, 张小杉, 段莎莎, 施依璐, 赵捷, 白天昊, 王雅晳. 氢气治疗心肌缺血再灌注损伤的作用机制及展望[J]. 中华临床医师杂志(电子版), 2023, 17(06): 744-748.
[14]	张赟辉, 罗军, 刘栗丽, 汪宏, 耿克明. 腹膜透析与血液透析对老年终末期肾病患者营养状况及炎症反应的影响[J]. 中华临床医师杂志(电子版), 2023, 17(04): 419-423.
[15]	买买提·依斯热依力, 依力汗·依明, 王永康, 阿巴伯克力·乌斯曼, 艾克拜尔·艾力, 李义亮, 克力木·阿不都热依木. 氧化应激对3T3-L1前脂肪细胞中GLP-1/DPP-4信号通路以及炎症因子表达的影响[J]. 中华肥胖与代谢病电子杂志, 2023, 09(03): 186-191.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Effect of dihydrobayberry on high glucose-caused injury of H9C2 myocardial cell

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Effect of dihydrobayberry on high glucose-caused injury of H9C2 myocardial cell