The Study On The Effect Of Echinacoside In Inhibiting Amyloid Fibrillization And Protecting Skin Against UVB-induced Damage

Objective:1. HEWL and Aβ have been seleeted as an in vitro model to investigate the anti-amyloid property of ECH. 2. To establish UVB- irradiation Ha Ca T cells damage model, investigate the protection of ECH against UVB-induced cell damage, and explore the possible mechanism. 3. To study the preparation and quality evaluation of ECH cream. 4. To establish UVB-irradiation Balb/c mice damage model, investigate the protection of ECH against UVB-induced skin damage.Methods:Part one: Inhibitory effect of ECH against amyloid fibrillization1. HEWL was incubated with different concentrations of ECH in water bath for 12 d ays at 55 °C to form fibrils. Th T fluorescence, Congo red absorption and ANS fluorescence assays were used to probe the presence of cross β-pleated sheet structure and hydrophobic patches associated with amyloid fibrils; the morphology of the HEWLsamples were analyzed by TEM; the alteration in secondary structure is monitored using C D spectra; and MTT and hemolytic assays were employed to evaluate the relevant cytotoxicity and their hemolytic activities. 2. ECH(500 μM) was added to the incubation medium at 0, 3, 7, and 11 day of HEWL incubation, and the HEWL samples were analysed by Th T fluorescence, CR absorption and ANS fluorescence assays; CD spectra; TEM observation; cell viability and hemolysis assay. 3. Aβ25-35 was incubated for 48 h at 37 °C for aggregation, and then acted on PC12 cells with ECH, MTT assay and DCFH-DA fluorescent measurement were used to evaluate the cell viability and intracellular ROS production. 4. Antioxidant potential of EC H was tested by the DPPH and ?HO scavenging activity methods, and Vc was used as a positive control.Part two: Protection of ECH on UVB-induced damage in Ha Ca T cells1. Cells were exposed to UVB at the range of 25 ~ 500 m J/cm2, and the optimum level of UVB irradiation intensity was deter mined by MTT assay. 2. Cells were divided into six groups: control; 50 μM ECH; UVB; 25 μM ECH + UVB; 50 μM ECH + UVB; 100 μM ECH + UVB. Cells were pretreated with various concentrations(25, 50, 100 μM) of ECH. After incubation for 24 h, they were stimulated by UVB, and do correlation tests. 3. MTT and LDH leakage assays were used to evaluate cell viability; analyzing the activities of SOD, CAT, GSH-Px, and the content of MDA; intracellular ROS generation was monitored by flow cytometer and confocal microscopy; 8-OHd G production was decteted by ELISA kit; agarose gel electrophoresis was used to test DNA Fragmentation; apoptosis was measured using TUN EL assay and Flow Cytometry; the protein levels of ATR, Chk1, p53, Caspase-3, PARP, and XPA were also measured by western blot.Part three: Preparation of ECH cream1. The amount of glycerin monostearate, liquid paraffin, vaseline, and azone was chosen as investigating factor, the preparation process was optimized by orthogonal experiment, with the appearance, d uctility, viscosity, and the stability of high temperature, low temperature and room temperature as investigating indexes. 2. According to the optimal prescription to prepare ECH cream, using ultraviolet spectrophotometry to deter mine the content of ECH in the cream.Part four: Protection of ECH on UVB-induced skin damage in Balb/c mice1. ECH creams were prepared according the technological processes, and drug loading capacities of ECH in the creams were 0.5%, 1%, 5%, respectively. 2. The dorsal skin was shaved clean, and divided into six groups(n = 10): control; UVB; vehicle + UVB; 0.5% Echinacoside + UVB; 1% Echinacoside + UVB; 5 % Echinacoside + UVB. 3. Vehicle cream or Echinacoside cream was topically applied to the back of each mice. After 30 min, mice were exposed to UVB with a total dose of 1956 m J/cm2. 4. Mice were sacrificed after 24 h of the last UVB exposure, a part of the dorsal skin was placed for measurement of skin edema; H&E staining; immunohistochemical detection of 8-OHd G; deter mination of SOD, GSH and CAT activities, and MDA level.Result:Part one: Inhibitory effect of ECH against amyloid fibrillization1. The results of ThT fluorescence, Congo red absorption and AN S fluorescence assays indicated that, after 12 days of incubation HEWL + EC H groups showed a significant decrease in Th T fluorescence, ANS fluorescence intensity, and Congo red absorptioncompared to HEWL group; TEM analyse indicated that exposure of HEWL to ECH resulted in attenuation of fibrillization and for ming shorter and thinner fibrils; at the beginning of incubation, the C D spectra of the samples showed a similar spectrum with two absorptions at approximately 208 nm and 222 nm, upon incubation, HEWL alone shows a deep peak at approximately 220 nm, and this negative peaks decreased in the HEWL samples with ECH. C ytotoxicity and hemolysis assays demonstrated that addition of ECH significantly increased the cell viability and reduced hemolysis. 2. ECH not only disrupted pre- formed fibrils, but also transformed amyloid fibrils into amorphous aggregates in various phase of the fibrillation process(at 0, 3, 7, 11 day). And this effect was stronger when ECH was added at earlier time point. 3. ECH was more effective in scavenging DPPH than ?HO. However, the ability of ECH to scavenge these two radicals was weaker than that of Vc. 4. Aβ25–35-induced toxicity and intracellular ROS accumulation in PC12 cells were abrogated by ECH pre-treatment, and this effect was proportional with the drug’s concentration.Part two: Protection of ECH on UVB-induced damage in Ha Ca T cells1. UVB irradiation in the range 25 ~ 500 m J/cm2 reduced the viability of Ha Ca T cells in a dose-dependent manner, and 300 m J/cm2 UVB irradiation induced about 50% of cell viability compared with that in control group without UVB irradiation. 2. Compared with UVB group, ECH pre-treatment ameliorated UVB- induced cytotoxicity; improved the activities of SOD, C AT, GSH-Px; degraded the level of MDA, ROS and 8-OHd G; attenuated DNA fragmentation and apoptosis; normalized the protein levels of p-ATR, p-Chk1(Ser345), p-p53(Ser15), cleaved Caspase-3,cleaved PARP, and XPA. And these effects were positively correlated with the drug’s concentration.Part three: Preparation of ECH cream1. The optimum weight proportion of ECH cream was ECH 5 %、octadecanoic acid 10%、glycerin monostearate 5%、liquid paraffin 5%、vaseline 10%、triethanola mine 4%、azone 3%、ethylparaben 0.1%。 2. The linear range of 1 ~ 20 μg/m L(r = 0.9990), the average rate of recovery was 102.2%(RSD = 1.4%), and the cream was stable.Part four: Protection of ECH on UVB-induced skin damage in Balb/c miceCompared with UVB group, the ad ministration of ECH cream partly improved the skin edema, erythema, wrinkling and hyperplasia; reduce 8-OHd G expression in skin tissue; increased the activities of SOD, CAT, GSH-Px, and degraded the content of MDA.Conlusion:1. ECH dose dependently inhibites HEWL aggregation, suppresses HEWL undergoing α-helix to β-sheet conversion, hampers the exposure of hydrophobic regions, reduces the HEWL-elicited cell toxicity and hemolysis; abrogates Aβ25–35-induced toxicity and intracellular ROS accumulation. And this function is likely related to its antioxidant effect. 2. The preparation process is simple, the cream is homogenous, delicate, stable, easy to apply. And the quality control method is simple, accurate, and reliable.ECH displayes a protective effect against UVB- induced damage in Ha Ca T cells and BALB/c mice, and this effect have been at least partly mediated by its antioxidant and suppression of DNA damage with concomitant decrease in the apoptotic response.