Evaluation Of The Protective Effects Of The Extracts From Inonotus Obliquus On UVB-induced Photoaging Of HaCaT Cells

Ultraviolet B（UVB）can penetrate the epidermis and increase reactive oxygen species（ROS）in skin cells,which increases oxidative stress,activates inflammatory reaction,accumulates DNA damage and induces apoptosis.As a result,the skin loses elasticity.The increase of roughness and dryness resulted in irregular pigmentation and deep wrinkles.This process is called skin photoaging.Preventing and delaying skin aging is a hot and difficulty in current medical research.Researches suggested that antioxidants can effectively remove ROS produced by UVB radiation,which could protect against skin aging.Among them,antioxidants from natural products,due to their structural diversity and good biological safety,become the preferred choice to reduce skin photoaging induced by UVB radiation.As a precious edible and medicinal fungus,Inonotus obliquus could effectively regulate blood lipid,reduce blood glucose,inhibit DNA damage and cell premature induced by oxidative stress.However,the effect and mechanism of Inonotus obliquus on skin photoaging remain to be elucidated.Therefore,in this study,different components of Inonotus obliquus were taken as the research object to explore its protective effect on UVB-induced photoaging of human skin keratinocytes（HaCaT）and the research mechanism.This study provided theoretical and experimental basis for the study of preventing skin photoaging by Inonotus obliquus.The main research contents were as follows:（1）Evaluation of antioxidant activity of different components of Inonotus obliquus.The components of Inonotus obliquus with different polarity were extracted by ultrasonic assisted method.The contents of total polyphenols and total flavonoids were determined.The content of total polyphenols and total flavonoids in ethyl acetate was the highest,which were 12.62%and 22.99%,respectively.DPPH and ABTS free radical scavenging experiments showed that polysaccharide component had the highest scavenging activity,followed by ethyl acetate component.FRAP results showed that ethyl acetate component had the highest iron reduction ability.（2）The protective effects of different components of Inonotus obliquus on the oxidative stress induced by UVB.The results suggested that,compared with the normal group,the activity of superoxide dismutase（SOD）in HaCaT cells decreased after UVB irradiation.The polysaccharide component and N-butanol component of Inonotus obliquus could increase the content of SOD in HaCaT cells at high concentration(20μg·m L^-1).The polysaccharide component had better pharmacodynamics than N-butanol component.The results of glutathione（GSH）content detection showed that,compared with the normal group,the GSH content of UVB model group was significantly decreased,and different components of Inonotus obliquus could effectively promote the GSH content to rise at high concentration.The efficacy of polysaccharide component,ethyl acetate component and N-butanol component was significantly better than that of N-hexane component.The results of flow cytometry showed that different components extracted from Inonotus obliquus could significantly reduce ROS production in cells at high concentration,and the polysaccharide component had the best effect,followed by ethyl acetate component,N-butanol component and n-hexane component.Western blot analysis related proteins of Nrf2/HO-1 pathway showed that UVB induced down-regulation of antioxidant stress level,increased expression of antioxidant stress related proteins in a dose-dependent manner,and activated antioxidant signal pathway.（3）The protective effects of different components of Inonotus obliquus on UVB induced senescence of HaCaT cells.After SA-β-gal staining,the effects of different components on the senescence of HaCaT cells induced by UVB were observed.It was found that the number of positive cells increased in the model group,and the different components of Inonotus obliquus could reduce the positive rate of senescence cells.The polysaccharides component was higher than that of ethyl acetate component,N-butanol component and N-hexane component.Western blot analysis was used to detect the senescence related proteins（p16,p21 and p53）in the model group.It was found that different components of Inonotus obliquus significantly reduced the content of senescence related proteins in cells,and the polysaccharide component had the best effect.The results of RT-PCR analysis showed that the contents of MMP-1 and MMP-3increased and the contents of Collagen I and Collagen III decreased after UVB radiation.Different components of Inonotus obliquus could effectively reverse the above signs,especially polysaccharide.（4）The protective effects of different components of Inonotus obliquus on UVB induced apoptosis in HaCaT cells.The results showed that Inonotus obliquus components could significantly inhibit the apoptosis of HaCaT cells induced by UVB radiation.The effect of polysaccharide component was the best.Western blot analysis of apoptosis related proteins（Bcl-2 and Cleaved-caspase 3）showed that UVB radiation increased the content of pro-apoptosis related protein Cleaved-caspase 3 and decreased the content of anti-apoptosis protein Bcl-2.Different components of Inonotus obliquus significantly inhibited cell apoptosis,and the polysaccharide component was the most effective.（5）The protective effect of different components of Inonotus obliquus on UVB induced HaCaT inflammation.ELISA was used to detect the effects of different components of Inonotus obliquus on IL-6 and MMP-1 in photoaging HaCaT cells.RT-PCR was used to detect the contents of IL-6 and TNF-α.It was found that different components of Inonotus obliquus significantly inhibited the increase of IL-6,TNF-αand MMP-1 contents induced by UVB radiation.Western blot analysis showed that different components of Inonotus obliquus inhibited HaCaT inflammation through TLR4 and NF-κB signal pathway.In conclusion,Inonotus obliquus component could alleviate photoaging of HaCaT cells induced by UVB radiation,which may be related to the inhibition of apoptosis,the alleviation of oxidative stress induced by Nrf2,and anti-inflammatory effect induced by TRL4/NF-κB pathway.