Effects Of Gynostemma Pentaphyllum Saponins On Apoptosis Signal Pathway In Human Skin Fibroblasts

Purposes:The overall purpose of this study is to find out the molecular mechanismsand the drug targets of Gyp working on human skin fibroblast in vitro inprotecting HSF cells from pathological apoptosis to serve the future newanti-UVA-injury drug development.After the modeling of UVA-induced photoagedHSF cells, Gyp containing serums of different densities were used to intervenecells in each group. The Amplification of HSF cells, ROS production, the numberof apoptosis cells and the apoptotic-related cytokines, namely Bcl-2，Bax，Caspase-3protein as well as their mRNA expression were detected to investigatethe protective effect of gypenosides (Gyp) on the apoptosis of the injured humandermal fibroblasts induced by ultraviolet A radiation. The related cellsignaling pathways were also focused on in the study, in order to reveal certainmicroscopic mechanisms in genetic and protein levels.Materials and methods:Materials:20healthy male SD rats (Weight200-250g);Human Skin Fibroblast (HSF);Gyp(MUST-11031401);DMEM High Glucose Medium;Fetal Bovine Serum(FBS); Trypsin;PBSBuffer;Dimethyl sulfoxide (DMSO);UVA light (20W);UV photometer; Chloralhydrate;Green/streptomycin;Absolute ethanol;75%ethanol;MTT; apoptosis-situdetection kit; Rabbit anti-human Bax,Bcl-2,and Caspase-3; Internal referenceprimer;β-actin antibody;HRP-conjugated goat anti-rabbit secondary antibody;Trizol;PCR,Electrophoresis;Gel imaging analysis system; Cell proteinextraction reagent;BCA Protein Assay Kit; ECL-emitting fluid; ECL emittingkit;Electric heated oscillator;The Reader;Horizontal shaker; Vertical slabelectrophoresis device;Electrophoresis; Transfer electrophoresis tank, and soon. Methods:PartⅠ Preparation of UVA-induced photoaged HSF model cells and Gyp interveningPreparation of Gypenosides containing serum (high dose, medium dose and lowdose serum).The culture of HSF and grouping procudures, including cell passaging,cell cryopreservation, cell recovery and cell grouping(6groups, namely groupⅠ-Blank control group, groupⅡ-UVA model group, group Ⅲ-blank serum, groupⅣ-groupⅤ-medium dose serum group and group Ⅵ-high doses serum group.HSF photoaged cell modeling.Each group of HSF cells except groupⅠwere madeinto photoaging cells by UVA light, with radiation dose of36J/cm2.Cells in group Ⅰand Ⅱ were let alone without any drug intervening.Cellsin group Ⅲ were intervined by blank serum, while Cells in group Ⅳ, ⅤandⅥ were intervined by low, medium and high Gyp containing serum respectively.Detecting and measuring cell proliferation activities in each group with MTTassay.The cell suspension in each bottle of each and every group is taken.Con-Awas added into the positive stimulai hole, while the RPMI-1640culture solutionwas added into the blank stimuli hole on plate and culture got continued. MTTwas added into each and every hole for the last4hour’s culture. Then dimethylsulfoxide (DMSO) was added into each of them. When the crystalline material gotfully dissolved, the optical density value (OD) was detected and observed at490nm wavelength.Part Ⅱ Dectecting the ROS content and apoptosis number of HSF cellsThe detection of ROS in photoaged HSF cellsin each group.DCFH-DA was usedto detect all kinds of ROS. Twenty hours after cell-seeding at the96well-platewith15thoudsand cells in each hole, the cells were washed by earle’s sugarliquid twice and get cultured in DCFH-DA incubator with25μM of its finalconcentration for30min. Then cells were washed by earle’s sugar liquid twice.The fluorescence detecting was carried out by FLUO Star OPTIMA Microplate Reader,with488nm excitation wavelength and525nm emission wavelength.The detection of apoptosis phenomena of HSF in each group.The apoptosis of HSF in each group was detected with Tunel assay. Cell samples were washed twicein PBS and the rounding area of the cell samples got dried with absorbent paper.50μl TDT enzyme reaction solution was added to each and every cell sample. Onehour after reaction with humidity retention in37℃in darkness, the cells werewashed three times in PBS. The apoptosis condition was detected with Tunel assayusing apoptosis-situ detection kit. Cells colored by FITC fluorescence wereobserved. The cells’ morphological change was observed with a fluorescencemicroscopy. Five sufficiently colored fields of view of the cells are selectedon each cell-climbing film and the apoptosis cell number was counted in eachfield and the total apoptosis cell number was worked out based on it.Part Ⅲ Detecting the mRNA and protein expressions of Bcl-2, Bax and Caspase-3Detecting and measuring the mRNA expression of Bcl-2, Bax and Caspase-3ineach group of HSF cells with RT-PCR assay respectively. The detection processincluded the extraction of the total RNA of Bcl-2, Bax and Caspase-3, first strandcDNA synthesis, polymerase chain reaction, agarose gel electrophoresisexperiment, and polyacrylamide gel electrophoresis, etc.The protein expression of Bcl-2, Bax and Caspase-3in each group of HSF cellswere detected and measured with Western Blotting assay respectively, includingdeterminating the protein concentration of Bcl-2and Bax, rabbit anti-humanbcl-2and bax dilution, goat anti-rabbit secondary antibody dilution, shakingin the horizontal shaker at room temperature for1h, the gray value of internalreference protein and Bcl-2, Bax and Caspase-3protein were measured using ECLemitting detection kit for statistical analysis.Results:1.Compared with the blank control group, the cell proliferation activityin cells of UVA model group decreased significantly (P＜0.01). Compared withUVA model group, the cell proliferation activity in Group Ⅳ, Ⅴ and Ⅵincreased significantly (P＜0.05), with the high dose serum increased mostapparently.The cell proliferation activity in Group Ⅴ was significantly higher than that in Group Ⅲ（P＜0.01）.2.Compared with the blank control group, ROS content in cells of UVA modelgroup increased signficantly（P＜0.01）. Compared with UVA model group, thecell ROS content in Group Ⅳ, Ⅴand Ⅵ decreased significantly (P＜0.01), withthe high dose serum decreased most apparently. The ROS content in Group Ⅴ cellswas significantly lower than that in Group Ⅲ（P＜0.01）.3.With electron microscope, the cell apoptosis in each group wereobserved.Compared with the blank control group, the cell apoptosis number ofUVA model group increased significantly （P＜0.01）. Compared with UVA modelgroup, the cell apoptosis number in Group Ⅳ, Ⅴand Ⅵ decreased significantly(P＜0.05，or P＜0.01), with the high dose serum decreased most apparently.The cell apoptosis number in Group Ⅴ cells was significantly fewer than thatin Group Ⅲ（P＜0.01）.4.Compared with the blank control group, the mRNA and protein expressionof Bcl-2in cells of UVA model group decreased signficantly（P＜0.01）. Comparedwith UVA model group, the mRNA and protein expression of Bcl-2in Group Ⅳ, Ⅴand Ⅵ increased significantly (P＜0.01), with the high dose serum increasedmost apparently. The mRNA and protein expression of Bcl-2in Group Ⅴ cells wassignificantly higher than that in Group Ⅲ（P＜0.01）.5.Compared with the blank control group, the mRNA and protein expressionof Bax in cells of UVA model group increased signficantly（P＜0.01）. Comparedwith UVA model group, the mRNA and protein expression of Bax in Group Ⅳ, Ⅴand Ⅵ decreased significantly (P＜0.01), with the high dose serum decreasedmost apparently. The mRNA and protein expression of Bax in Group Ⅴ cells wassignificantly lower than that in Group Ⅲ（P＜0.01）.6. Compared with the blank control group, the mRNA and protein expressionof Caspase-3in cells of UVA model group increased signficantly（P＜0.01）.Compared with UVA model group, the mRNA and protein expression of Caspase-3inGroup Ⅳ, Ⅴand Ⅵ decreased significantly (P＜0.01), with the high dose serumdecreased most apparently. The mRNA and protein expression of Caspase-3in Group Ⅴ cells was significantly lower than that in Group Ⅱand Ⅲ（P＜0.01）.Conclusions:1. Gyp is capable of increasing the proliferation activity of UVA-inducedphotoaged HSF cells to protect cells form UVA injury.2. Gyp is capable of antagonizing HSF cells’oxidative stress responseinduced by UVA radiation and has a protective effect on HSF model cells.3. Gyp is capable of decreasing the number of HSF cells’ apoptosis inducedby UVA.4. Gyp has protective effects on HSF by helping balance the expression ofBcl-2and Bax both in mRNA and protein level, and eventually suppressing themitochondria-related apoptosis cell signaling induced by UVA.5. Gyp is capable of suppressing the mRNA and protein expression of Caspase-3,and works effectively in protecting HSF from apoptosis, by suppressing theactivation of Caspase-3.