Regulation Of Semen Coicis Extraction On Expression Of Aquaporin 3 Induced By Ultraviolet B Irradiation In HaCaT Cells

IntroductionSkin is the largest human organ directly exposed to the environment and therefore vulnerable to ultraviolet irradiation(UVR) from the sun.Both ultraviolet A and B play a role in the pathogenesis of skin photoaging process.Skin photoageing is the gradual deterioration of cutaneous structure and function following long-term recurrent exposure to UVR.These events are superimposed on intrinsic skin ageing.Depending on the wavelength of UV,the epidermis is affected principally by UVB.Cutaneous fine and coarse wrinkling,dryness,coarseness,telangiectasia,yellowness,mottled pigmentation,laxity,loss of tensile strength and comedones are characteristics of photoagng.UV-irradiated keratinocytes release pre-inflammation cytokines,such as IL-1α, IL-1β,IL-6 and TNF.These cytokines activate AP-1 and NF-κB through cell surface receptors and amplify the UV-irradiation response.MMPs were upregulated in dermal fibroblasts,and responsible for degeneration and inhibition of synthesis of collagenous extracellular matrix of dermis.Aquaporins(AQPs),the water channel proteins,have been focused on since 2003. AQPs mediate the efficient movement of water across the cell membrane.Up to now, thirteen isoforms of aquaporins,AQP0-12 have been identified.Distribution specialty of AQPs in tissues cells has been demonstrated.Only AQP3 was expressed by basal cells in normal epidermis.AQPs not only plays a major role in the maintenance of water and homeostasis in normal physiologic stage,But also facilitate trans-epithelial fluid transport(involving water,glycerol,urea),are involved in swelling of tissues under stress,facilitate cell migration and neural signal transduction.In our study UV-irradiated keratinocvtes down regulated the expression of AQP3;but the mechanism remained unknown. Adequate photo protection is essential to prevent UV-related disorders.Besides currently used photo protective means,several botanical agents,such as polyphenols and baicalin have been demonstrated effective in photo protection via influence on signal transduction pathways.KLT is an extraction from semen coicis,a Chinese herb, which has been effective in anti-cancer and inhibiting COX and NF-κB.In our study,we investigated the photo protective effects and the molecular transduction mechanisms of KLT on HaCaT cells induced by UVB irradiation,and found the promising effective photoprotection measures.MethodsCell cultureHaCaT cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum,100U/ml penicillin and 100U/ml streptomycin and in a humidified atmosphere of 5%CO₂ at 37℃.Assay for cell proliferation by MTTMTT was performed to investigate the effect of KLT on the proliferation of HaCaT cells.RT-PCR to assess AQP3 mRNA expression in HaCaT cells(1) To assess the effects of UVB-irradiation on mRNA expressions of AQP3 in HaCaT cells;(2) To assess the effects of KLT treatment on mRNA expressions of AQP3 in UVB-irradiated HaCaT cells;(3) To assess the effects of IL-1αon mRNA expressions of AQP3 in HaCaT cells. Western blot to detect the expression of AQP3 in HaCaT cells(1) To detect the effects of UVB-irradiation on the protein expressions of AQP3 in HaCaT cells;(2) To detect the effects of KLT treatment on the protein expressions of AQP3 in UVB-irradiated HaCaT cells.(3) To detect the effects of IL-1αon the protein expressions of AQP3 in HaCaT cells.Results The results of MTT assayKLT(120μl/ml) exhibited no inhibitory effect on the proliferation of HaCaT cells (p＞0.05).The results of RT-PCRThe results with relative quantification analysis exhibited that AQP3 mRNA decreased after UVB-irradiation in a dose-dependent manner,which begin at 6 hour and most significantly at 24 hour after treatment.KLT treatment exhibited up-regulation of AQP3 mRNA substantially in a dose-and time-dependent manner than that of the base material(p＜0.001).AQP3 mRNA decreased after IL-1αinduced HaCaT cells in a dose-and time-dependent manner,and most significantly at 12 hour(p＜0.001).The results of western blotThe protein levels of AQP3 decreased significantly after 12 hours exposing to UVB-irradiation or IL-1αinduction of HaCaT cells in a dose-and time-dependent manner.KLT treatment exhibited up-regulation of AQP3 substantially in a dose-and time-dependent manner,which is more prominent than that of the base material.ConclusionsUVB-irradiation and IL-1αinduction decreased the expression of AQP3 at mRNA and protein levels.KLT treatment up-regulated the UVB-irradiation and IL-1αinduced decrement of AQP3 expression in HaCaT cells.KLT exhibited the protection effect on UVB induced acute photo damaging.