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.

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

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