The Protective Effect And Mechanism Of Acidosasa Longiligula Extract On UVB-induced Skin Damage

Background:Skin,as the outermost organ,is exposed to a wide range of environmental risk factors including ultraviolet(UV).Excessive UV exposure contributes to many disorders,such as photoaging,skin inflammation,and carcinogenesis.UVB is a medium-wave ultraviolet,which is mostly absorbed by keratinocytes.This wavelength is more energetic and has a strong mutagenic capacity.Recently,there has been a focus on a wide range of natural compounds with antioxidant properties that have a high safety profile.Acidosasa longiligula is a genus of plants in the family Gramineae with high nutritional and application value.For centuries,bamboo has been used as a source of medicine for a variety of diseases and has proven its therapeutic potential.Studies have shown that different bamboo products have anti-inflammatory,anti-allergic,antioxidant,anti-aging,and anti-tumor effects.However,the effect of this specific bamboo extract(BEX)in UVB-radiated skin cells has yet to be determined.Objective:To investigate the protective effect of BEX on photodamage caused by UVB radiation by establishing in vitro cellular models and animal models,and to explore the potential mechanisms.To provide a molecular basis and theoretical basis for the development of drugs for the prevention and treatment of photodamage.Methods:Part Ⅰ:Study on the protective effect of BEX on UVB-induced photodamagePrimary human keratinocytes were used as the research object.The cells were treated with different concentrations of BEX(10,30,100,300μg/m L)and cell viability was detected by CCK8 assay so as to evaluate the BEX drug toxicity.After pretreatment of cells with different concentrations of BEX,cells were then radiated with different doses of UVB(5,10,15,20,25 m J/cm~2)and cell viability was detected using the CCK8 assay to select the optimal drug concentration.The optimal concentration of BEX was selected as 100μg/m L and the UVB dose was 20 m J/cm~2 to establish a photodamaged cell model and used for subsequent studies.Cells were given BEX pretreatment followed by UVB radiation.The protein expression level of cleaved caspase3 was detected by western blot,and TUNEL assay was used to assess the level of apoptosis.Cellular oxidative stress levels were assessed by ROS assay,and DNA damage was detected by DNA single-strand breaks analysis.C57BL/6 mice were used as the study subjects.UVB radiation was used to construct a photodamage model.C57BL/6 mice were given a 100 mg/ml BEX topical skin agent to apply to the dorsal skin,and UVB(80 m J/cm~2)was administered 1 hour later for 7 weeks on alternate days.Observe the changes on the dorsal skin of the mice.Mice were sacrificed using the cervical dislocation,dorsal skin tissues were retained,and histopathological changes were assessed after HE staining.Masson staining and elastic fiber staining were applied to detect the content and changes of collagen and elastic fibers in mice skin.Part Ⅱ:Study on the mechanism of TXN system in BEX anti UVB-induced photodamagePrimary human keratinocytes were used as the research object.After pretreatment of cells with BEX(100μg/m L),UVB(20 m J/cm~2)radiation was performed.Cellular TXN and TXNRD activity levels were measured using the TXN Activity Fluorescence Assay Kit and the TXNRD Assay Kit.After treating cells with BEX(100μg/m L),the protein expression levels of TXN family proteins TXN1,TXN2,TXNRD1 and TXNRD2 were detected by western blot.Cells was transfected with sh RNA to knock down cellular TXN1 expression.The transfected cells were pretreated with BEX(100μg/m L),followed by UVB(20 m J/cm~2)radiation.Cell viability was detected using CCK8 assay and apoptosis level was detected using Histone-DNA ELISA assay.Part Ⅲ:Study on the mechanism of NF-κB/p65 signaling in BEX anti UVB-induced photodamagePrimary human keratinocytes were used as the research object.After pretreatment of cells with BEX(100μg/m L),UVB(20 m J/cm~2)radiation was performed.NF-κB DNA binding activity was detected using the NF-κB p65 transcription factor assay kit.Cytoplasm and nuclear proteins were extracted separately,and NF-κB/p65cytoplasm and nuclear protein expressions were detected by western blot.Results:Part Ⅰ:Study on the protective effect of BEX on UVB-induced photodamageBEX had no toxic effect on primary human keratinocytes.UVB radiation of primary human keratinocytes resulted in decreased cell viability and increased apoptosis levels.BEX pretreatment reduced the level of UVB-induced apoptosis,ROS production and DNA single-strand breaks in primary human keratinocytes,and increased cell survival in a dose-dependent manner.The thickness of the epidermis was reduced and inflammatory infiltration was reduced in mice given topical BEX skin agent for treatment compared to UVB-radiated mice alone.In mice skin,UVB radiation reduced the expression of collagen and elastic fibers and disorganized the structure of the mice dermis.In contrast,in the skin of pretreated mice given BEX skin topical agent,the above phenomenon was significantly alleviated.Part Ⅱ:Study on the mechanism of TXN system in BEX anti UVB-induced photodamageBEX pretreatment reduced UVB-induced decrease in TXN and TXNRD activity levels in primary human keratinocytes.BEX treatment of cells altered TXN1 protein expression levels in primary human keratinocytes.After knockdown of TXN1,BEX pretreatment attenuated the BEX-mediated increase in UVB-induced cell viability and decrease in apoptosis levels compared to the non-target control sh RNA cell group.Part Ⅲ:Study on the mechanism of NF-κB/p65 signaling in BEX anti UVB-induced photodamageBEX pretreatment reduced UVB-induced NF-κB DNA binding activity.UVB induced a decrease in cytoplasm NF-κB/p65 protein expression and an increase in nuclear NF-κB/p65 protein expression in primary human keratinocytes.In contrast,BEX pretreatment increased cytoplasm NF-κB/p65 protein expression and decreased nuclear NF-κB/p65 protein expression.Conclusion:BEX inhibits UVB-induced photodamage in primary human keratinocytes and mice skin.BEX exerts its anti-photodamage effects by modulating the TXN system and regulating its family protein TXN1,and by inhibiting the activation of the NF-κB/p65 pathway and the translocation of NF-κB/p65 from the cytoplasm to the nucleus.BEX could be further tested as a novel anti-UV skin protectant.