Tebbe B, Wu S, Geilen C C, Eberle J, Kodelja V, Orfanos C E
Department of Dermatology, University Medical Center Benjamin Franklin, Free University of Berlin, Germany.
J Invest Dermatol. 1997 Mar;108(3):302-6. doi: 10.1111/1523-1747.ep12286468.
We investigated the antioxidative effect of L-ascorbic acid on lipid peroxidation and on secretion and mRNA expression of IL-1alpha and IL-6 after UVA irradiation (20 J/cm2) in cultured human keratinocytes. Lipid peroxidation was measured by (i) high performance liquid chromatography with UV detection of malondialdehyde (MDA) at 256 nm and (ii) spectrometric measurement of thiobarbituric acid-reactive substances (TBARS). To evaluate UV-induced cytotoxicity, we assessed cell membrane damage by measuring lactate dehydrogenase (LDH) release. UVA-induced lipid peroxidation in cultured human keratinocytes was inhibited by ascorbic acid in a concentration-dependent manner: MDA protein equivalent was reduced by 47% (10(-6)), compared to keratinocytes not exposed to L-ascorbic acid (p < 0.05), and the TBARS showed a concentration-dependent decrease of 49% (10(-6) M) in L-ascorbic acid-supplemented cultures compared to controls (p < 0.05). LDH release was decreased by 45% in L-ascorbic acid-supplemented keratinocyte cultures, indicating protection against cell death (p < 0.05). L-Ascorbic acid was able to downregulate IL-1alpha mRNA expression in both UVA-irradiated and nonirradiated cells; however, IL-6 mRNA expression remained unaffected. The secretion of these cytokines was reduced nearly to normal in the presence of L-ascorbic acid. These findings indicate a major cell-protective effect of L-ascorbic acid on UVA-induced lipid peroxidation and the secretion of pro-inflammatory cytokines by UVA-irradiated human keratinocytes.
我们研究了L-抗坏血酸对体外培养的人角质形成细胞经紫外线A（UVA）照射（20 J/cm2）后脂质过氧化、白细胞介素-1α（IL-1α）和白细胞介素-6（IL-6）分泌及mRNA表达的抗氧化作用。脂质过氧化的检测方法如下：（i）采用高效液相色谱法，在256 nm处通过紫外线检测丙二醛（MDA）；（ii）采用分光光度法测定硫代巴比妥酸反应性物质（TBARS）。为评估紫外线诱导的细胞毒性，我们通过测量乳酸脱氢酶（LDH）释放来评估细胞膜损伤情况。UVA诱导的体外培养人角质形成细胞脂质过氧化受到抗坏血酸的浓度依赖性抑制：与未接触L-抗坏血酸的角质形成细胞相比，MDA蛋白当量降低了47%（10-6）（p<0.05），且在添加L-抗坏血酸的培养物中，TBARS较对照组呈现出49%（10-6 M）的浓度依赖性降低（p<0.05）。在添加L-抗坏血酸的角质形成细胞培养物中，LDH释放降低了45%，表明细胞死亡受到保护（p<0.05）。L-抗坏血酸能够下调UVA照射和未照射细胞中IL-1α的mRNA表达；然而，IL-6的mRNA表达未受影响。在L-抗坏血酸存在的情况下，这些细胞因子的分泌几乎降至正常水平。这些发现表明，L-抗坏血酸对UVA诱导的脂质过氧化以及UVA照射的人角质形成细胞促炎细胞因子的分泌具有主要的细胞保护作用。
