Effects And Mechanisms Of The Inhibitory Co-stimulatory Factor B7-H3 On UVA-induced Human Skin Photoaging

Background:UVA irradiation activates relevant molecular signaling pathways in skin cells through divers ways,such as generating ROS and damaging DNA,etc.,causing damage to macromolecular components such as proteins,nucleic acids and biofilms,thus leading to skin photoaging and photoaging-related dysfunction.In addition,senescent skin cells also provide a biological basis for the occurrence of cutaneous malignancy.B7-H3 belongs to the family of inhibitory co-stimulatory molecules,which plays a variety of functions in vivo,including the regulation of the appearance,progression and prognosis of various tumors.Our previous studies found that B7-H3 was highly expressed in both photoaging-related skin cancer and precancerous tissue.However,whether B7-H3 is involved in and regulates UVA-induced skin photoaging process has not been reported yet.Objective:This study aims to investigate whether B7-H3,as an inhibitory co-stimulatory factor,could regulate the oxidative damage and photoaging of human dermal fibroblasts induced by UVA radiation,and to explore its potential molecular mechanisms.Methods:We first detected the expression of B7-H3 in skin tissues of different clinical populations,and analyzed its correlation with age and location.Next,we isolated and cultured primary human skin fibroblasts(FBS),and established stable B7-H3 knockout cell lines by lentivirus transfection.We irradiated FBs with 0,20 J/cm2,40J/cm2 UVA(0～40J/cm2)respectively to induce photoaging.The following tests were performed simultaneously:②cell senescence was detected by SA-β-Gal staining;②a CCK8 kit was used to detect cell proliferation;③B7-H3 expression,ROS content,mitochondrial membrane potential level changes were measured by flow cytometry;④Real-Time PCR was performed to test mRNA expression of collagen,TIMPs,MMPs,TERT,P21 and other photo-damage markers;⑤Western blotting was used to detect the expression levels of B7-H3,aging and autophagy related proteins;⑥the activities of SOD and CAT were determined by ELISA.Results:The results of immunehistochemical test showed that the expression of B7-H3 in the skin tissue was positively correlated with age and UV exposure.24h after UVA(0～40J/cm2)irradiation,the expression of B7-H3 in fibroblasts also experienced a significant increase,and simultaneously a strong photo-damage response and senescence occurred,which were manifested by enhanced activity of senescence-associated galactosidase(SA-β-Gal).In addition,the content of ROS was up-regulated.The expression of photo-damage related genes MMPs and p21 increased while the expression levels of TIMPs and COL1A1 were inhibited,which promote dermal collagen deposition.The mitochondrial membrane potential,SOD and CAT activities decreased,and autophagy was inhibited.Silencing B7-H3 significantly improved UVA-induced senescence.The changes of the above photoaging related indicators were alleviated to varying degrees,and the level of autophagy increased.Conclusion:The expression of B7-H3 in the tissue of clinical skin specimens was positively correlated with the degree of photoaging.B7-H3 konckout has significant antagonistic effects on UVA-induced photoaging of fibroblasts,including reducing intracellular ROS production and accumulation of photo-damage markers,inhibiting oxidative damage of cells and promoting autophagy.However,the regulatory mechanism of B7-H3 on photoaging process needs to be further studied.