原花青素对过氧化氢诱导羊颞下颌关节盘细胞氧化损伤的保护作用研究

The shielding power of proanthocyanidins against oxidative harm caused by hydrogen peroxide in sheep temporomandibular joint disc cells

目的探究原花青素在过氧化氢诱导颞下颌关节盘细胞氧化损伤中的作用。方法分别用浓度为0、100、200、400、800μmol/L过氧化氢处理颞下颌关节盘细胞12、24、48 h,CCK-8检测细胞活力。然后用不同浓度(0、5、10、20、40μmol/L)的原花青素处理颞下颌关节盘细胞24、48 h,CCK-8检测原花青素对颞下颌关节盘细胞活力的影响。将颞下颌关节盘细胞分为对照组、模型组、低浓度组(5μmol/L原花青素)、中浓度组(10μmol/L原花青素)、高浓度组(20μmol/L原花青素),使用CCK-8法检测细胞活力,比色法分别检测丙二醛水平、超氧化物歧化酶活性,DCFH-DA荧光探针检测活性氧水平,流式细胞仪检测细胞凋亡率,定量反转录PCR检测炎症因子、细胞外基质合成和降解因子相关基因的表达水平。结果400μmol/L过氧化氢处理24 h时,颞下颌关节盘细胞存活率为60.57%,与对照组相比,细胞活力降低(P<0.05)。与对照组相比,模型组颞下颌关节盘细胞活力降低,活性氧水平、丙二醛水平升高,超氧化物歧化酶活性下降。与模型组相比,5、10、20μmol/L原花青素处理组可以提高过氧化氢诱导下颞下颌关节盘细胞活力,降低活性氧水平、丙二醛水平,升高细胞内超氧化物歧化酶活性(P<0.05)。流式结果显示模型组较对照组细胞凋亡率升高,而用原花青素干预后细胞凋亡率较模型组降低(P<0.05)。颞下颌关节盘细胞经过氧化氢干预后细胞内炎症因子(白介素-1β、肿瘤坏死因子-α、白介素-6)表达量与对照组相比增加,原花青素预处理后白介素-1β、肿瘤坏死因子-α、白介素-6表达降低(P<0.05)。此外,原花青素增加了过氧化氢诱导下Ⅰ型胶原和聚集蛋白聚糖的表达(P<0.05),同时抑制了基质金属蛋白酶-1,基质金属蛋白酶-3,基质金属蛋白酶-9的表达(P<0.05),抑制了细胞外基质降解。结论建立颞下颌关节盘细胞氧化应激模型的最适作用条件为400μmol/L过氧化氢作用24 h。原花青素逆转了过氧化氢诱导的颞下颌关节盘细胞脂质过氧化、活性氧生成、炎症损伤和细胞外基质降解,通过提高抗氧化酶水平和减轻细胞凋亡来抑制氧化应激损伤。

Objective To investigate the protective power of proanthocyanidins against oxidative harm caused by hydrogen peroxide in temporomandibular joint disc cells.Methods Temporomandibular joint disc cells were treated with concentrations of 0,100,200,400 and 800μmol/L hydrogen peroxide for 12,24 and 48 h,respectively,and cell viability detected by CCK-8.Then temporomandibular joint disc cells were treated with different concentrations of proanthocyanidins(0,5,10,20,40μmol/L)for 24,48 h,and CCK-8 assayed the effect of proanthocyanidins on temporomandibular joint disc cell viability.Then the experiments were divided into the control group,model group,low concentration group(concentration of 5μmol/L proanthocyanidins),medium concentration group(concentration of 10μmol/L proanthocyanidins)and high concentration group(concentration of 20μmol/L proanthocyanidins).Cell viability was detected using the CCK-8 method,while levels of malondialdehyde and superoxide dismutase were detected using colorimetric assay.Reactive oxygen species were detected using DCFH-DA fluorescent probe staining,apoptosis was measured using flow cytometry,and mRNA expression levels of inflammatory factors,extracellular matrix anabolic factors,and extracellular matrix catabolic factors were detected using quantitative reverse transcription PCR.Results Cell viability was 60.57%when temporomandibular joint disc cells were treated with 400μmol/L hydrogen peroxide for 24 h,and compared with the control group,the cell survival rate decreased(P<0.05).Compared with the control group,the model group showed decreased temporomandibular joint disc cells viability,increased levels of reactive oxygen species and malondialdehyde,and decreased activity of superoxide dismutase.Compared to the model group,the 5,10 and 20μmol/L proanthocyanidins treatment groups can increase temporomandibular joint disc cells viability induced by hydrogen peroxide,reduce the levels of reactive oxygen species and malondialdehyde,and increase the activity of intracellular superoxide dismutase(P<0.05).The flow results revealed a higher level of apoptosis in the model group than in the control group,yet this was diminished post-proanthocyanidins pretreatment(P<0.05).Moreover,the expression of intracellular inflammatory factors(interleukin-1β,tumornecrosis factor-α,interleukin-6)was higher in the model group,whereas the expression of interleukin-1β,tumornecrosis factor-α,interleukin-6 was reduced post-proanthocyanidins pretreatment(P<0.05).The expression of typeⅠcollagen and aggrecan was augmented by proanthocyanidins when hydrogen peroxide was induced(P<0.05),while the expression of matrix metalloproteinase-1,matrix metalloproteinase-3 and matrix metalloproteinase-9 was inhibited(P<0.05)and extracellular matrix degradation suppressed.Conclusion The most effective action condition for constructing an oxidative stress model of temporomandibular joint disc cells was 400μmol/L hydrogen peroxide for 24 h.Proanthocyanidins reversed hydrogen peroxide-induced lipid peroxidation,reactive oxygen species generation,inflammatory damage and extracellular matrix degradation in temporomandibular joint disc cells,and inhibited oxidative stress injury by increasing antioxidant enzyme levels and attenuating apoptosis.