The Study Of Protection Effects And Mechanisms Of Rutin Against Ultraviolet-induced Skin Photodamage

PREFACESkin aging includs endogenous aging and extraneous aging. Endogenous aging also called natural aging which is a normal aging process. Endogenous aging is influenced mainly by external environment factors. As the largest human organ, skin is directly exposed to the environment and vulnerable to ultraviolet irradiation. It has been reported that ultraviolet radiation is the main reason to endogenous aging. Ultraviolet in nature mainly comes from the sun, so extraneous aging is also called photo aging. Ultraviolet is an electromagnetic wave. Its wave length ranges from 200nm to 400nm. According to its wave length, it is divided into three parts. Wavelength ranging from 320nm to 400nm is UVA, also called long-wave ultraviolet. Wavelength ranging from 280nm to 320nm is UVB, also called middle-wave ultraviolet. Wavelength ranging from 200nm to 280nm is UVC, also called short-wave ultraviolet. At the same dosage UVC is the most harmful to skin. UVB is the second harmful to skin. Some studies showed that the damage intensity of UVB was 800-1000 times that of UVA at the same dosage. UVA can penetrate into the dermis and UVB can penetrate into skin partly. UVC is almost completerly absorbed by atmosphere, it cannot reach the surface of the earth in normal. So its damage can be ignored. The dosage of UVA is about 91?99% in the nature UV and UVB is modicum. ALL in all, UVB and UVA is the cause of skin damage induced by ultraviolet, and UVB is the main factor. Many studies have indicated that skin damage induced by ultraviolet related with reactive oxygen speecies(ROS). Normally ROS can be transformed and cleared away by antioxidative system in cells. Excessive ROS is produced in human and animal skin after excessive ultraviolet radiation. Then the balance between oxidative system and antioxidative system is destroyed, which causes injury of cells structures and disorder of biological metabolism, resulting in skin photoaging and cutaneous carcinoma. In recent years, skin diseases are increasing because of more and more ultraviolet radiation reaching the surface of the earth which results from the ozonosphere destruction. Scientists have strong interests in how to effectively prevent and treat skin damage caused by ultraviolet.Antioxidant has been suggested as useful substance to protect the skin from ultraviolet radiation. It has been demonstrated that some antioxidative substances can prevent skin from photodamage caused by ultraviolet, such as antiscorbic acid, astragalus saponin I, pomegranate, vitamin E and (3-carotene.Rutin is also called rutoside. It is a sort of flavone compound which widely distributed in vegetables. It has been widely used in vascular diseases in medicine. Rutin is a safe and natural antioxidant. So it is possible to be taken as sun screener. PART ONEThe protection effects and mechanisms of rutin against UVB-induced skin photodamage in HaCat cellsObjective1. Observe the influence of rutin to HaCaT cells viability2. Establish HaCaT cells photodamage model3. Observe the influence of rutin to HaCaT cells viability after UVB radiation4. Observe the influence of rutin to HaCaT cells apoptosis rate after UVB radiation5. Observe the influence of rutin to HaCaT cells secretion levels of IL-6 and TNF-a after UVB radiationMethod1. Detect the influence of rutin to HaCaT cells viability1.1 HaCaT cells cultureHaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100u/ml penicillin and 100u/ml streptomycin and in a humidified incubator containing 5% CO2 at the temperature of 37?.1.2 HaCaT cells GroupsHaCaT cells were divided into seven groups, which were marked group?,?,?,?,?,?and?.Group I was control group, which was not applied rutin and without ultraviolet radiation.Group II was UVB radiation control, which was not applied rutin medium but radiated with ultraviolet.Group III was supplied with vehicle(DMSO) medium.Group IV was supplied with 12.5 umol/l rutin medium.Group V was supplied with 25.0umol/l rutin medium. Group?was supplied with 50.0umol/l rutin medium.Group?was supplied with 100.0umol/l rutin medium.1.3 Detect the viability of HaCaT cellsDiscard the culture medium, washing one time with PBS. HaCaT cells were incubated for 4 hours with 5mg/ml MTT 20?l. The supernatant liquor was discarded. Then add DMSO 200?l to dissolve the crystal. Measure the absorbance value with enzyme immunity instrumentation at 490 nm.2. Establish HaCaT cells photodamage model2.1 Datum of UVB lightThe wave length of UVB lamp ranges from 290nm to 320nm.The peak of wave length is 312 nm. The lamp instrument contains 8 lamps. Each lamp's power is 9W. The vertical distance from the light to the cells was 15cm. The ultraviolet intensity at the distance was 0.75mW/cm2.2.2 UVB radiate HaCaT cellsWhen the cytomixis was to 80?90%, HaCaT cells were taken out from cell incubator. Except for group I, other six groups were radiated with UVB.The radiation dose was 30mJ/cm2 and the vertical radiation distance from the light to the cells was 15cm.3. Detect the viability of HaCaT cells after UVB radiationHaCaT cells were incubated for 24 hours after ultraviolet radiation and add MTT 20ul to continue culture for 4 hours. Discard the supernatant liquor. Then add DMSO 200?l to dissolve the crystal. Measure the absorbance value with enzyme immunity instrumentation at 490nm.4. Detect the apoptosis rate of HaCaT cells after UVB radiationHaCaT cells were incubated for 24 hours after UVB radiation. Then the cells were digested, centrifugated and adjusted to 1×10°ml. Added Annexin V-FITC and PI, analyzed with FCM and measured the rate of apoptosis in HaCaT cells5. Detect the levels of IL-6 and TNF-? HaCaT cells were incubated for 24 hours after UVB radiation. Then they were collected and centrifugated. The supernatant liquor was detected strictly following the instruction of the enzyme-linked immunosorbent assay (ELISA) kit.Results1.The effects of rutin on the HaCaT cells viabilityThe absorbance value of group I and groupI???were similar to each other. The differences between group???and group I were not significant (P>0.05).The study showed that rutin (concentation 12.5-100umol/l) and vehicle (DMSO) exhibitd no significatnt efffect on the HaCaT cells viability2.The effects of rutin on the HaCaT cells viability after UVB radiationThe HaCaT cells viability was significatntly decreased after UVB radiation (p< 0.01).In the range of concentation(25-100umol/l), rutin can significatntly increase the HaCaT cells viability (p<0.01). The medium contains 12.5 umol/l rutin and the medium contains vehicle (DMSO) exhibitd no significatnt efffect on the HaCaT cells viability (p>0.05).After UVB radiation the HaCaT cells viabilities of group???were graduately decreased after incultured for12,24 and 48 hours. The difference between the values of MTT12?MTT24?MTT48 in the same group were significant (P<0.05). The values of MTT12, MTT24, MTT48 in group I were similar to each other (P>0.05).3.The effects of rutin on HaCaT cells apoptosis rateThe HaCaT cells apoptosis rates were significatntly increased after UVB radiation (p<0.01).In the range of concentation(12.5-1 OOumol/l), rutin can significatntly decrease the HaCaT cells apoptosis rates after UVB radiation (p<0.05).The vehicle group (group?) exhibitd no significatnt efffect on the HaCaT cells apoptosis rate after UVB radiation (P>0.05). The apoptosis rate was increased as the concentration of rutin increased(r=-1, P<0.01). The relationship between the apoptosis rate and concentration of rutin was negative correlation.4.The effects of rutin on HaCaT cells secretion levels of IL—6 and TNF-?The levels of IL-6 and TNF-?were all significantly decreased in group???after UVB radiation. The differences between group???and group I were significant (p<0.01).The levels of IL-6 and TNF-?were decreased as the concentration of rutin increased(r=-1, P<0.01). The relationship between the levels of IL-6, TNF-?and concentration of rutin was negative correlation in group???(r=-1, P<0.01).ConclusionsRutin could have protective effects on photodamage induced by UVB radiation in a certain range of concentration. The study showed that rutin may increase HaCaT cells viability, decrease HaCaT cells apoptosis rate and decrease the levels of IL-6 and TNF-?after UVB radiation. Rutin's protective effect against UVB-induced photodamage was increased as the concentration increased. Objective1. Observe the changes of KM mice's skin surface condition after UVA+UVB radiation2. Observe the changes of collagen fibers and elastic fibers in the KM mice dermis after UVA+UVB radiation3. Observe the changes of MDA content and the changes of antioxidative enzyme activity after UVA+UVB radiation4. Observe the changes of MMP-1 mRNA level after UVA+UVB radiation.Method1. KM Mice photodamage models establishment1.1 Animals and animal groupsSixty KM mice were tested in the study, which were 6-8 weeks old,20?25 grams weight, half male and half female. They were supplied by Shandong Chinese Medical University and fed in the common condition. Six mice of same sex were fed in the one cage.Sixty KM mice were divided into 5 groups randomly and each group had 12 mice.Group A was vehicle cream group. The dorsal skin of mice was applied with vehicle cream without radiation.Group B was rutin cream group. The dorsal skin of mice was applied with rutin cream without radiation.Group C was UVA+UVB radiation group.Group D was UVA+UVB radiation+ vehicle cream group. The dorsal skin of mice was applied with vehicle cream and radiated with ultraviolet.Group E was UVA+UVB radiation+rutin cream group. The dorsal skin of mice was applied with rutin cream and radiated with ultraviolet.1.2 Preparation before Ultraviolet radiationSS-04 ultraviolet instrument:supply with 9 UVA lamps, each lamp power was 15W, the wave length ranges from 320nm to 400nm, and the vertical distance of radiation was 15cm.UVB lamp box:supply with 4 UVB lamps, the wave length ranges from 290 to320 nm, and the vertical distance of radiation was 50cm.1.3 Ultraviolet radiationShave off the hair of mice back with baby hair-clippers. The sample (vehicle cream or rutin cream) was applied on mice dorsal skin 1 hour before radiation. Except for the groupA, B, other three groups were radiated with UVA+UVB every other day during the first 3 weeks and every day during the 4-14weeks. The radiation dosage of UVA was 10J/cm2 and UVB was 50mJ/cm2 every time. The total radiation doszge of UVA was 1130J/cm2 and UVB was 5.65J/cm2.2. Evaluation the changes of KM mice skin surfaceObserve the changes of KM mice skin surface after UVA+UVB radiation every week. Evaluate the changes according to experimental evaluation criterion at the end of tset.3. Evaluation the changes of histomorphology and the content of collagen fibers and elastic fibers.After the last UVA+UVB radiation, the KM mice were killed and their dorsal skin was cut off, then the cut sheets were fixated, dehydrated, embeded and stained(HE stained,VG stained or Weigert stained). Observe the the changes of histomorphology and the content of collagen fibers and elastic fibers in the mice dermis.4. Detection the changes of MDA content and the changes of antioxidative enzyme activity4.1 Preparation of tissue homogenateAfter the last UVA+UVB radiation, the KM mice were killed and their dorsal skin was cut off. Then part of the skin were weighed without subcutaneous fat and prepared for 10% tissue homogenate. Take the supernatant liquid of the tissue homogenate to detection after 3000r/min centrifugation.4.2 Detection the changes of MDA contentFollowing the instruction of MDA kit strictly, added reagents to the supernatant liquid respectively. The mixture was warmed for 40 minutes in 95?water and cooled in running water, centrifugated 10 minutes at 3500r/min. The supernatant liquid was taken, and analyzed at 532nm with spectrophotometer.4.3 Detection the changes of SOD activityThe supernatant liquid was processed strictly following the instruction of the SOD kit, and analyzed at 550nm with spectrophotometer.4.4 Detection the changes of GSH-Px activityThe supernatant liquid was processed strictly following the instruction of the GSH-Px kit, and analyzed at 412nm with spectrophotometer.5. Detection the changes of MMP-1 mRNA level with Real-Time Quantitative RT-PCRTotal cellular RNA was extracted from frozen skin tissue homogenate. MMP-1 cDNA was synthesized by reverse transcription. The expression of MMP-1 mRNA in mice skin tissues were analyzed by Real-Time quantitative PCR.Results1. The changes of mice skin surfaceThe mean skin surface scores of group C, D, E after UVA+UVB radiation were higher than group A, B without radiation. The differences between group C, D, E and group A, B were significant (p<0.01).The mean skin surface score of group A was similar to group B. The difference between group A and group B was not significant (p>0.05).The mean skin surface score of group C was similar to group D. The difference between group C and group D was not significant (p>0.05).The mean skin surface scores of group C, D were higher than group E. The differences between group C, D and group E were significant (p<0.01).2. The changes of the collagen fibers and elastic fibers content and histomorphologyGroup A and B:Epidermis layer was thin and integral. Papillar layer was normal. The collagen fibers and elastic fibers in the dermis arranged in bunchy regularly without obvious rupture.Group C and D:Epidermis layers were hyperkeratosis and papillar layers were flatted. The collagen and elastic fibers in the dermis of KM mice became thick, fragmented and disarranged.Group E:the change was between group A, B and group C, D.3. The changes of MDA content in KM mice skin After UVA+UVB radiation, the MDA content in group C, D, E was significant increased. The differences between group C, D, E and group A,B were significant (p <0.01).The MDA content in group C and group D was highest. The differences between group C and group D were not significant (p>0.05).The MDA content in group E was lower than group C, D. The differences between group E and group C, D were significant (p<0.01).The MDA content in group A and group B was similar. The differences between group A and group B were not significant (p>0.05).4. The changes of SOD and GSH-Px activityThe activity of SOD and GSH-Px was significant decreased in group C, D, E after UVA+UVB radiation. The differences between group C, D, E and group A,B were significant (p<0.01).The activity of SOD and GSH-Px in group A and group B was similar. The differences between group A and group B were not significant (p>0.05).The activity of SOD and GSH-Px in group C and group D was similar after UVA+UVB radiation. The differences between group A and group B were not significant (p>0.05).The activity of SOD and GSH-Px in group E was higher than group C, D after UVA+UVB radiation. The differences between group E and group C, D were significant (p<0.01).5. The changes of MMP-1 mRNA level in KM mice skin tissueThe MMP-1 mRNA level was significant increased in group C, D, E after UVA+UVB radiation. The differences between group C, D, E and group A, B were significant (p<0.01).The MMP-1 mRNA level in group C and group D was highest. The differences between group C and group D were not significant (p>0.05).The MMP-1 mRNA level in group E was lower than group C, D after UVA+UVB radiation. The differences between group E and group C, D were significant (p<0.01).The MMP-1 mRNA level in group A and group B was similar. The differences between group A and group B were not significant (p>0.05). ConclusionsLong term UVA+UVB radiation could cause KM mice skin photodamage. External application with 0.5% rutin cream had protectant effects to skin photodamage of KM mice caused by UVA+UVB radiation. The mechanisms may be that rutin is a strong antioxidant which can inhibit lipid peroxidation, decrease MDA content, increase the activity of SOD and GSH-Px, and decrease the level of MMP-1 mRNA in dermis.