The Effect Of Gypenosides-containing Serum On The P38mapk Signal Pathway In Photoaging Human Skin Keratinocytes

Sking aging mainly includes chronological aging and photoaging. Chronological aging represents the physiological changes in skin that occur over time,resulting in fine facial wrinkles and is easily injured. Photoaging results fromthe damaging effects of solar Ultraviolet (UV) radiation,inducing the skin agingin advance. The characteristics of photoaging are deep facial wrinkles, dermatochalasis, dullness,pachulosis and irregular melanin pigmentation.People paymore and more attention to the skin damaging and prodtective devices of photoaging and the mechanism of photoaging has become a research focus in recent years.From the point of view of Traditional Chinese Medicine(TCM),the etidogiesand pathogenesis of photoaging is due to the low body resistance,low skin resistance, improper diet,inducing deficiency of qi and blood,stagnation of phlegm-blood stasis,skin dystrophy,“healthy energy is asthenic and evil sthenic”,respenting withered and senescence after a long time by subjected to the sunlighttoxicity. In recent years, Fiveleaf Gynostemma Herb,a traditional chinese medicine was listed to the team of anti-photoaging,many researchers pay more attention to this chinese herb. Fiveleaf Gynostemma Herb is the rhizome and wholegrass of Cucurbitaceae,it has sweet,bitter,cold flavour and blonging into kidney,heart,lung meridian. It has the effect of replenishing qi to invigoratethe spleen, clearing lung and expelling phlegm,heat-clearing and detoxifying.In our previous vivo experiments,we have shown that the Fiveleaf Gynostemma Herbhas the effect of anti-photoaging. Modern pharmacological studies have shownthat it contains Gynostemma pentaphyllum gypenoside, Gynostemma pentaphyllumpolyoses, water-solubility amine acid, flavonoid, multiple vitamins, microe lement, mineral matter. Gypenosides（GPs）is the main active ingredient ofFiveleaf Gynostemma Herb,it has the effect of lowing blood pressure,lowing bloodfat,lowing blood sugar, staying caducity, anti-fatigue,anti-anoxia, anti-hightemperature, anti-low temperature, anti-hypothermia, longevity,advancing SODactivity, enhancing immunity,et al.In the UV radiation, both of Ultraviolet Radiation A (320～400nm) and Ultraviolet Radiation B(290～320nm)could cause photoaging damage, most of Ultraviolet Radiation B were absorbed by keratinocytes. The molecular mechanism ofphotoaging is not completely cleared, in the recent years,the research resultshave shown that UV radiation activated Nuclear Factor κB (NF-κB) and up-regulated c-Jun through Mitogen-Activated Protein Kinase (MAPK) signal pathway,c-Jun and c-Fos of constitutive expression were integrated into activatedprotein1(AP-1) in keratinocytes. Both of AP-1and NF-κB could damage dermalexcellular matrix (collagen fibers) by activating the expression of Matrix Metalloproteinase(MMP) and cytokines.The activated NF-κB could further up-regulatethe expression of inflammatory cytokines,then these inflammatory cytokines could promote the activation of AP-1and NF-κB through cell surface receptors，these forming a vicious cycle and amplifying the effect of UV radiation.MAPKsignal pathway played a important role in the process of photoaging, p38MAPKpathway of MAPK pathway was most closely associated with the activation of cytokine receptors and cellular stress, including UV radiation.Objective:This study simulated the environment of sunlight and human skin,took the p38MAPK signal pathway in photoaging keratinocytes as the research coreto clarify the mechanism of gypenosides on anti-photoaging by immunology andprotocols in molecular biology in vitro,in order to research the effect ofgypenosides-containing serum on photoaging human skin fibroblasts in the futureand to provide the new thread and science theory basis on how to preventing andcuring photoaging by traditional chinese medicine.Material and method: In the first part: The experiment raised the Human immortal skin keratinocyteline (HaCaT) in a routine way,drawed the growth curve of HaCaT cells;observedthe form of HaCaT cells by inverted microscope,detected the expression of keratinous in HaCaT cells by immunocytochemical method,appraised the human skinkeratinocytes; the rats were given gypenosides by gavage, prepared the differentconcentrations of gypenosides-containing serum, detected the HaCaT cells cytotoxicity after interferenced with gypenosides-containing serum by MTT;Irriatedthe HaCaT cells with UVB303nm,detected the HaCaT cells proliferative activityby MTT, determined the UVB dose of photoaging human skin keratinocytes model.In the second part: The method of dividing groupe and interferencing the cells:control group(groupⅠ),normal HaCaT cells without any interferences;UV modelgroup(groupⅡ),the HaCaT cells were radiated by UVB of the dose of35mJ/cm2withnew culture medium;blank serum group(group Ⅲ), the HaCaT cells were interfereenced with rat serum after radiated by UVB of the dose of35mJ/cm2;low dose ofgypenosides-containing serum group(group Ⅳ), the HaCaT cells were interfereenced with low dose of gypenosides-containing serum after radiated by UVB ofthe dose of35mJ/cm2; middle dose of gypenosides-containing serum group(groupⅤ), the HaCaT cells were interferenced with middle dose of gypenosides-containing serum after radiated by UVB of the dose of35mJ/cm2; high dose of gypenosides-containing serum group(group Ⅵ), the HaCaT cells were interferenced withhigh dose of gypenosides-containing serum after radiated by UVB of the dose of35mJ/cm2;p38MAPK inhibitor group(groupⅦ), the HaCaT cells were interferencedwith p38MAPK inhibitor SB203580(concentration of5μmol/L) after radiated byUVB of the dose of35mJ/cm2.Detected the expression of p38MAPK，MMP-1，c-Jun andc-Fos mRNA in earch group cells by the method of RT-PCR; Detected the expressionof p38MAPK，MMP-1，c-Jun and c-Fos protein in earch group cells by the methodof Western blot.In the third part: The method of dividing groupe and interferencing the cells:control group(groupⅠ),normal HaCaT cells without any interferences;UV modelgroup(groupⅡ),the HaCaT cells were radiated by UVB of the dose of35mJ/cm2with new culture medium;blank serum group(group Ⅲ), the HaCaT cells were interferenced with rat serum after radiated by UVB of the dose of35mJ/cm2;low dose ofgypenosi des-containing serum group(group Ⅳ), the HaCaT cells were interferenced with low dose of gypenosides-containing serum after radiated by UVB of thedose of35mJ/cm2; middle dose of gypenosides-containing serum group(group Ⅴ),the HaCaT cells were interferenced with middle dose of gypenosides-containingserum after radiated by UVB of the dose of35mJ/cm2; high dose of gypenosides-containing serum group(group Ⅵ), the HaCaT cells were interferenced with highdose of gypenosides-containing serum after radiated by UVB of the dose of35mJ/cm2;p38MAPK inhibitor group(groupⅦ), the HaCaT cells were interferenced withp38MAPK inhibitor SB203580(concentration of5μmol/L) after radiated by UVBof the dose of35mJ/cm2. Detected the content of IL-1β，IL-6and TNF-αof cellculture supernate in each group by the method of ELISA;Detected the expressionof NF-κB mRNA in earch group cells by the method of RT-PCR; Detected the expression of NF-κB protein in earch group cells by the method of Western blot.Results:In the first part:1. The HaCaT cells growth curve shows: the growing periods were the first dayto the forth day,getting into plateau phase from the forth day.2. The HaCaT cells adhered to the plate,shaped round or oval and closely arranged,forming arrangement of the typal slabstone by inverted microscope.3. Cytokeratin were expressed stably in the cells, the expression of anti-keratinose5in HaCaT cells were staining positive, brown colored positive granuleswere shown in the endochylema;no expression in the negative control group.4. The HaCaT cells multiplication activity in each group by interfered withgypenosides-containing serum was show: the cell multiplication activity hasno significant differences with the increased concentration of gypenosides-containing serum (P＞0.05), gypenosides-containing serum has no apparenteffect of inhibiting or promoting growth on the HaCaT cells. 5. HaCaT cells were growed downward and round gradually, has enlarged intercellular space obviously, arranged confused and increased float cells with theincrease time(dose) of UVB radiation by inverted microscope. The HaCaT cellsmultiplication activity in each group by UVB radiation was shown: the cellcytoactive was decreased obviously with the the increase time(dose) of UVBradiation(P＜0.05),during the first minute of UVB radiation,the cell cytoactive changed most obviously,although after then there was still changed,decreased obviously and mild gradually.In the second part:1. The expressions of p38MAPK,c-Jun mRNA and protein in each group cell shown:the expressions of p38MAPK,c-Jun mRNA and protein were low in the controlgroup, the expressions of p38MAPK,c-Jun mRNA and protein in UV model groupwas increased obviously, compared with the control group,it has statisticalsignificance（P＜0.01）; compared with the UV model group, the expressionsof p38MAPK,c-Jun mRNA and protein were obviously decreased in the p38MAPKinhibitor group,low,middle and high dose of gypenosides-containing serumgroup,there was statistical significance（P＜0.01）;compared with the highdose of gypenosides-containing serum group, the expressions of p38MAPK,c-JunmRNA and protein in middle and low dose of gypenosides-containing serum groupwere on the high level relatively, there was statistical significance（P＜0.05or P＜0.01）.2. The expressions of MMP-1,c-Fos mRNA and protein in each group cell shown:the expressions of MMP-1mRNA and protein in each group were extremely low,there was no statistical significance in each group（P＞0.05）; the expressions of c-Fos mRNA and protein in each group were average,there was no statistical significance of group comparison（P＞0.05）.In the third part:1. The expressions of IL-1β，IL-6and TNF-αof cell supernatant in each groupshown: the expressions of IL-1β，IL-6and TNF-αof cell supernatant in control group were low,compared with the controul group, the expressions of IL-1 β，IL-6and TNF-αof cell supernatan in UV model group increased obviously,there was statistical significance（P＜0.01）; comparing with the UV modelgroup, the expressions of IL-1β，IL-6and TNF-αof cell supernatant in thep38MAPK inhibitor group,low,middle and high dose of gypenosides-containingserum group were decreased obviously,there was statistical signi ficance（P＜0.01）; compared with the high dose of gypenosides-containing serum group,the expressions of IL-1β，IL-6and TNF-αof cell supernatant in middle andlow dose of gypenosides-containing serum group were on the high level relatively, there was statistical significance（P＜0.01）.2. The expressions of NF-κB mRNA and protein of cell in each group shown: theexpressions of NF-κB mRNA and protein were low in the control group, theexpressions of NF-κB mRNA and protein in the UV model group were increasedobviously, there was statistical significance comparing with the controlgroup（P＜0.01）;comparing with the UV model group, the expressions of NF-κBmRNA and protein in the p38MAPK inhibitor group,low,middle and high dose ofgypenosides-containing serum group were decreased obviously, there wasstatistical significance（P＜0.01）.Conclusion：1. The gypenosides-containing serum has no apparent toxic effect on HaCaT cells.2. The method of establishing the model of photoaging human skin keratinocytesby UVB radiating HaCaT cells,the dose of UVB35mJ/cm2is suitable.3. UVB radiation induce the photoaging damage of human skin keratinocytes through the p38MAPK signal pathway possibly.4. The gypenosides could protect and treat photoaging,the mechanism is gypenosides-containing serum has good photoprotection on the photoaging human skinkeratinocytes by inhibiting the p38MAPK signal pathway possibly.