| Ultraviolet(UV)is an important environmental factor,which could lead to the accumulation of intracellular reactive oxygen species(ROS)and inflammation,directly or indirectly affect the stability of the genome.It could cause sunburn and even induce skin malignant tumors,such as melanoma and basal cell carcinoma.Heme oxygenase-1(HO-1),as an important multifunctional protein,plays an anti-inflammatory,antioxidant and anti-apoptotic role in response to ultraviolet radiation and catalyzing the degradation of heme.Therefore,exploring the regulatory effect of UV on HO-1 in skin cells,which will help to reveal its specific mechanism in photo-damage protection.As an electromagnetic wave,ultraviolet radiation is accompanied with energy transmission and owns thermal effect.Ultraviolet rays can cause skin sunburn and it is often classified as skin I-II degree burns,both sunburn and scald are classified as burns.Scald could induce typical skin inflammation and the healing of scald is a long-term and complex repairing process,which accompanied with cell proliferation,migration and tissue reconstruction.It has been found that HO-1 could be rapidly induced around the injured tissue.Overexpression of HO-1 promote cell proliferation and wound healing.It also plays an important role in angiogenesis and vascularization,which is very important for tissue repair process.As a pentacyclic triterpenoid compound,AKBA used to regulate cellular inflammatory response and reduce the accumulation of ROS.It is also an inducer of HO-1.In view of the important role of HO-1 in wound healing,exploring the role of AKBA in scald will helpful to reveal the its regulatory relationship.In addition,the relationship between HO-1 and HO-2 in UVA induced photo-damage is still unclear.DNA damage of melanocytes leads to carcinogenesis is the basic cause of melanoma formation.Excessive UV exposure and sunburn history are the main causes of melanoma.However,the exact cause of melanoma formation is still unclear.Malignant melanoma has been recognized as the most challenging subject in clinical cancer treatment due to its high metastatic ability,chemotherapy resistance and radiation resistance.It has been found that HO-1,as a highly expressed protein in tumor cells,owns anti-apoptosis,promoting cell proliferation and migration.Meanwhile,high expression of HO-1 may be related to the anti-chemotherapy and anti-radiotherapy.Therefore,it is very important to find an effective drug to treat melanoma.Apigenin(4',5,7-trihydroxyflavone),as a low toxicity dietary flavonoid,has a wide range of anti-cancer effects.The anti cancer properties of apigenin is mainly rely upon anti-proliferative and apoptosis induction to halt cancer growth among a variety of cancers,including breast,liver,pancreatic cancers.Apigenin could decrease the mice skin carcinogenesis that induced by UVR.The mechanism of apigenin regulate HO-1 is still unclear in melanoma and melanocytes.Therefore,elucidating the potential role of HO-1 and its upstream signaling pathways,such as endoplasmic reticulum stress pathway and Nrf2/HO-1,in skin photo-damage,scald and melanoma will help to reveal the mechanism of protective molecule HO-1 in different degrees of skin photochemical and thermodynamic damage,which provide research basis for the treatment and prevention of related skin diseases.In the present paper,the relationship between HO-1 and HO-2 in UVA induced photo-damage of HaCaT cells and the photo-protective effect of AKBA were systematically described.Meanwhile,the potential role of AKBA in the repair of scalded mice was also explored.In addition,we investigated the effects of UVA and apigenin on endoplasmic reticulum stress pathway and HO-1 gene expression in melanoma and melanocytes.Based on the above research purposes,this paper mainly includes the following contents:1.The role of HO that participates in photo-damageIn HaCaT cells,both HO-1 and HO-2 were interfered simultaneously,UVA irradiation could alter cell morphology,cause cell shrinkage,cell volume reduction,decreased intercellular contact and significantly reduced cell viability.Reduction of HO,UVA could induce LDH leakage.UVA irradiation increased the number of apoptosis and ROS level.Therefore,it is speculated that reduction of HO increase the sensitivity to UVA radiation and enhance UVA induced photo-damage.In addition,UVA and AKBA could enhance the phosphorylation of eIF2?,respectively.High dose of AKBA could increase the mRNA expression of HO-1 and HO-2 and reduce the ROS accumulation that induced by UVA.It is speculated that AKBA mediated the expression of HO-1 and HO-2 that may related to the photo-protection.2.AKBA mediated HO-1 expression in cutaneous thermal injuryLow-dose of AKBA could improve the acute thermal injury and increase STAT3 expression in JB6 cells.while 15 ?M of AKBA significantly inhibited the mRNA of STAT3 and EGFR,HO-1 can be induced by high concentration AKBA,and AKBA treatment can significantly reduce UVA induced ROS accumulation and it could not affect cell migration significantly.The expression of HO-1,ATF4,STAT3 and MMP-13(MMP-1)increased after acute scald.Immunohistochemistry results showed that HO-1,ATF4 and Nrf2 were highly expressed in the early stage of skin scald and gradually decreased with the healing of scald.The co-localization of STAT3 and HO-1 protein was found in skin tissues and cells,suggesting that the interaction between STAT3 and HO-1 that may plays an important role in the process of tissue repair.3.Apigenin mediated HO-1 expression in melanoma3.1 Apigenin dose-dependent affected ER stress signaling and HO-1 expressionThe protein and mRNA expression of PERK,HO-1 and STAT3 were decreased after(0-500 KJ/m2)UVA irradiation post 6 h and after 12 h apigenin(0-50 ?M)treatment.The mRNA expressions of ATF4,Nrf2,PERK and HO-1 were up-regulated post 12 h UVA irradiation.It is suggested that HO-1 may participate in endoplasmic reticulum stress pathway.3.2 Apigenin time-dependent affected ER stress signaling and HO-1 expressionAfter 0-24 h apigenin treatment,it was found that apigenin could also inhibit the activation of downstream Nrf2 and eIF2?/ATF4 signaling of PERK,thus inhibiting the expression of HO-1 and STAT3.It is speculated that the anti-tumor mechanism of apigenin that participate in inhibiting the proliferation of melanoma cells.3.3 Apigenin and UVA affected cell cycle and apoptosis in A375 cellsUVA irradiation could down regulate HO-1 expression post 6h and 12h,but it up-regulate the mRNA expression of ATF4 and CHOP.However,after the combination of UVA and apigenin,the expressions of ATF4,CHOP and PERK were significantly down-regulated and DR5 expression was up-regulated.The inhibitory effect of UVA and apigenin on HO-1 in melanoma cells can reduce its antioxidant protection effect and increase the cumulative toxicity of ROS,which is also confirmed by ROS detection.The up regulation of ATF4,CHOP and DR5 may be related to the occurrence of apoptosis.Subsequent apoptosis detection also confirmed that UVA and apigenin can increase the early apoptosis rate of melanoma cells.CyclinD1 was up-regulated 3 or 12 h after apigenin or/and UVA irradiation,and significantly down regulated post 6 h after apigenin and apigenin combined with UVA irradiation.The expression of p21 was significantly down regulated after UVA irradiation post 3 h with apigenin.P21 and CyclinD1 are cell cycle related genes.Cell cycle detection also confirmed that UVA and apigenin could increase cell cycle arrest in S phase.These results suggest that UVA combined with apigenin has synergistic effects on the survival of melanocytes and inhibition of melanoma cells in the aspects of ROS accumulation,apoptosis increase and cell proliferation arrest.3.4 ATF4 interacts with Nrf2 and regulates HO-1 expression in A375 cellsOverexpression of ATF4 could induce the expression of wild-type HO-1 and truncated HO-1,which means that endoplasmic reticulum stress pathway may regulate HO-1 expression through ATF4.Meanwhile,truncated HO-1 may be associated with DNA damage and tumorigenesis.Overexpression of Nrf2 increased the expression of HO-1 and ATF4.Co-IP showed that Nrf2 may interacted with ATF4,HO-1 may binds to STAT3.It is suggested that the interaction between Nrf2 and ATF4 may be related to the HO-1 expression in melanoma.4.The effect of apigenin on melanocytes4.1 UVA induced HO-1 and its regulators expression in a dose dependent mannerHO-1 mRNA was significantly up-regulated after UVA irradiation post 3,6,and 12 h.Bachl is a negative regulators of HO-1.The combination of Apigenin and UVA could reduce Bachl expression.UVA could increase Bach2 mRNA level,but UVA combined with apigenin could inhibit the expression of Bach2.STAT3 is also a negative regulator of HO-1.UVA combined with apigenin and UVA irradiation could reduce STAT3 expression post 3 and 6 h.It is suggested that inhibition of Bachl and STAT3 may be related to up regulation of HO-1 expression.ATF4 and Nrf2 are upstream regulators of HO-1.ATF4 was up-regulated by apigenin combined with UVA and post 3 h UVA irradiation.Apigenin combined with post 12 h UVA could up regulate ATF4 and Nrf2 mRNA expression,which suggested that it may be involved in the up regulation of HO-1 expression.The expression of HO-1,ATF4 and Nrf2 mRNA was gradually increased post 6 h UVA irradiation(0-500 KJ/m2).STAT3 and HO-1 were co-located in melanocytes.It is speculated that the different responses to UVA and apigenin treatment of HO-1 and regulatory factors expression in normal human melanocytes and melanoma cells,which may suggest that apigenin and UVA have selective effects on melanoma cells,it may have protective effects on normal human melanocytes.4.2 Apigenin could suppress HO-1 expression in melanocytesApigenin could reduce the expression of STAT3,HO-1,ATF4 and Nrf2,which suggested that apigenin reduced the expression of HO-1 and related regulatory factors in both melanoma and melanocyte cells.It is also helpful to further clarify the potential function of apigenin in melanocytes and the regulatory relationship between apigenin and HO-1. |
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