Protective Effects Of Plant Extracts On Human Dermal Cells InjuryInduced By H₂O₂

Human dermal fibroblasts are the predominant cell type in the dermis, and play animportant role in the aging process because they could regulate skin physiology and pathology.In this paper, we used the H₂O₂-induced oxidative damage in human dermal fibroblasts as amodel to study the protective effects of plant and Chinese herb extrations on H₂O₂-inducedoxidative damage, apoptosis, and examined its underlying mechanism. The main results andconclusion were described as follows:（1） MTT assay was first used to examine the survival rate of H₂O₂-induced humandermal fibroblasts, and established the cell oxidative injury model.Cell viability was assessedusing PI staining and MTT colorimetric assay; damaged cell nuclei, which indicated apoptoticcells, were detected with the Hoechst33342and TUNEL assay. After exposure to H₂O₂,evaluation of fibroblast survival rate and damaged cell number with TUNEL-positive nucleirevealed an increased cell injury. This result indicated that approximately50%of the injuredcells were apoptotic when the cells were treated with0.8mM H₂O₂for6h.（2） Whole-cell currents in human fibroblasts were recorded using a patch-clamptechnique. Cultured human dermal fibroblasts express multiple K+channels. We firstdetermined whether the two main voltage-dependent outward K+currents, transientinactivation outward K+currents （IA） and IKor IK （Ca）, could be recorded with double pulses.The data suggest that H₂O₂treatment significantly modified the voltage-dependencesteady-state activation property of the non-inactivating outward K+current channels. Theapplication of bisindolylmaleimide （Bis）, a membrane-permeable PKC-specific inhibitor,significantly reduced the H₂O₂-induced increase in K+current amplitudes. The effect of10M PMA on the activation properties of K+currents was similar to that induced byapplication of H₂O₂. Dermal fibroblasts possess three types of functional KCachannels, inwhich large conductance Ca2+-activated K+channels （BK channels） might be the predominantKCachannels because the amplitudes of BK channels were much more affected by specificmodulators than that of the other two types of KCachannels. When applying IBTX in theculture medium, the fluorescence imaging revealed that IBTX significantly enhanced thesurvival rate of human dermal fibroblasts treated with H₂O₂and decreased the numbers ofTUNEL-positive nuclei induced by H₂O₂. Hydrogen peroxide enhances Ca2+-activated BKcurrents by PKC pathway and the increased K+currents may be partly related toH₂O₂-induced cell injury in human dermal fibroblasts.（3） Whether the （）-epigallocatechin-3-gallate （EGCG） treated human dermal fibroblastshad the ability to fight against the oxidative stress by using the H₂O₂-induced human dermalfibroblasts apoptotic model was studied. The antioxidant activity of EGCG was detected by ascavenging DPPH radical assay. With200μg/ml EGCG, its effect on radical scavengingreached up to91.42%. Thus, EGCG has significant effects on free radical scavenging. EGCGwas first added with concentrations ranged from0to200μg/ml （0,10,20,50,100and200μg/mL） and tested the survival rate of the cells after H₂O₂treatment with MTT assay. Theresults suggested that EGC effectively improved the survival rate of H₂O₂-treated cells. In particular, they showed significant differences in20μg/ml,50μg/ml and100μg/ml groups （P<0.05）, and the50μg/ml group showed the most prominent effect. The results from TUNELassay experiments showed that by adding EGCG （50μg/ml）, the positive green fluorescencecells are significantly less than the cells treated with H₂O₂only. Apoptotic rate was reducedfrom38.12%to28.05%. EGCG increases SOD, GSH-pxactivity in human dermal fibroblastsand lowers the MDA level, significantly inhibits the lipid peroxidation in the human dermalfibroblasts and shows a clear protective effect against H₂O₂damage. EGCG can increaseendogenous antioxidant enzymes activity （SOD and GSH-px） and enhanced the oxygen freeradical scavenging capacity of human body’s own antioxidant defense system while reducingthe MDA level.（4） EGCG, which has been reported in previous chapter to exhibit certain antioxidantactivity, was used as the positive control. We first determine whether the oat bran and threeChinese herb compound extracts have antioxidant activity using a scavenging DPPH radicalassay. When the concentration of Oatp was0.5mg/ml,29%of the DPPH radicals werescavenged during a20min treatment. And36.7%,42.87%and40.54%of the DPPH radicalswere scavenged with three Chinese herb compound extracts. Pre-incubation of human dermalfibroblasts in Oatp for3h,6h and12h before H₂O₂injury did not significantly increase cellviability in human dermal fibroblasts exposed to0.8mM H₂O₂for6h, except for a slightincrease （8%） in cell viability observed in human dermal fibroblasts pre-incubated in1mg/mlof Oatp for12h. When the pre-incubation time with Oatp was increased to24h before H₂O₂injury, oat bran extracts exhibited a significant effect against H₂O₂-induced cell damage wasobserved （P <0.05）. The effect of three Chinese herb compound extracts on the H₂O₂-inducedhuman dermal fibroblasts damage was similar to that induced by application of H₂O₂.Pre-incubation of human dermal fibroblasts in three Chinese herb compound extracts for3h,6h and12h before H₂O₂injury did not significantly increase cell viability in human dermalfibroblasts exposed to0.8mM H₂O₂for6h. When the pre-incubation time with three Chineseherb compound extracts was increased to24h before H₂O₂injury, oat bran extracts exhibiteda significant effect against H₂O₂-induced cell damage was observed （P <0.05）. Pre-incubationwith1mg/ml oat bran extracts for24h before H₂O₂injury significantly reduced the numbersof TUNEL-positive nuclei of fibroblasts exposed to0.8mM H₂O₂for6h. Statistical analysisindicated that the numbers of TUNEL-positive nuclei were reduced from8.9%to14.5%ofthe control. The apoptotic rate of two extracts of three Chinese herb compound extracts wasreduced to31.98%and33.07%. The experimental results of the SOD activity and the level ofintracellular MDA are consistent with the results observed for cell survival: the effect of Oatpon SOD activity and the level of intracellular MDA occurred only after pretreatment withOatp for24h before H₂O₂injury. It is notable that the activity of another antioxidant enzyme,GSH-px, was not significantly affected by Oatp. It is well known that SOD serves as the firstgatekeeper in the antioxidant defense system designed to scavenge superoxide anions, whileGSH-Px is a catalyzer that activates the reaction of lipid hydroperoxides with reducedglutathione to form glutathione disulfide. Therefore, we presumed that Oatp was able toprevent H₂O₂-induced oxidative stress in human dermal fibroblasts by selectively enhancingSOD activity, rather than GSH-Px activity. These research results enrich the the theory and mechanism of the apoptosis of skin cells,which can provide a good platform for screening antioxidants and researching mechanism.Also these research findings have both theoretical and applicable value in cosmetic field. Inthis paper, we screened the plant extractions which can protect human dermal fibroblastsagainst oxidative damage and poptosis. These results provide proofs and application prospectfor the application of health and skin care products.