The Study Of Raffinose As A Protective Role In The Ultraviolet B-damaged HaCaT Cell

Objective:Autophagy is an important process for maintaining intracellular homeostasis.Our previous study demonstrated that autophagy was down-regulated in ultraviolet B(UVB)-irradiated keratinocytes.Raffinose is a natural oligosaccharide that serves as a novel activator of autophagy and as a balancing agent to regulate the diversity of environmental stress.The natural UVB is the most common envioronmental stress which causes skin damage.However,whether raffinose balances ultraviolet stress through the autophagy activation pathway has yet to be established.In this study,the biological effects of raffinose on UVB-irradiated HaCaT cells were clarified,including cell death,survival and proliferation.Then,we further investigate the autophagy(flux)level and MTOR activity after the raffinose treatment in the condition with or without UVB irradiation.Finally,we performed that autophagy signaling pathway plays a role in the protection of raffinose against UVB stress.Methods:The immortalized human keratinocyte(HaCaT)is the research subject1.Establishing the UVB-damaged model in HaCaT cells,and detecting the UVB-induced DNA mutations through the whole genome sequence(WGS);2.Evalutating the biological effects of raffinose in UVB-damaged HaCaT cells:The effects on cytotoxicity,cell viability,cell proliferation were detected by Lactate dehydrogenase release test(LDH),trypan blue straining and plate cloning test,respectively.In addition,western blot method was used to examined the expression of cleavage of caspased-3,PARP,endonuclease G,apoptosis induced factor(AIF),and Annexin V-EGFP/PI staining method was used to examined the exposed phosphatidylserine(PS),which are the important markers in apoptosis process3.Detection of the autophagy(flux) level and the signal pathway:1)Autophagy(flux)level Autophagy marker molecule LC3 II/I were detected by western blotting in the treatment of different raffinose concentrations and time points;Conbined with autophagy flow analysis tool drug(E64D+pepstatin),the LC3 Ⅱ/Ⅰ detected by western blotting,acidic vesicles in cytoplasm was observed by Acridine Orange(AO)staining,the specific autophagy staining was analyzed by CYTO-ID test2)Autophagy signal pathway:The expression of mammals rapamycin target protein(MTOR),its phosphorylation level at Ser2448,and the downstream of MTOR(p70 S6 Kinase,S6 Ribosomal Protein,4E-BP1)were detected by western blotting to evaluate the MTOR activity4.Detecting the autophagy biological effect of UVB-irradiated HaCaT cells in the present of raffinose:After the autophagy inhibitor wortmannin or small interfering RNA targeting ATG5(siATG5)technology blocking autophagy flow,the cytotoxicity was examined by LDH in UVB-irradiated HaCaT cells.Results:1.UVB irradiation(10 mJ/cm2)induced 655 identical DNA mutations between UVB-24h and UVB-P2.Apoptosis pathway is one of these mutation genes enrichment pathways.Mitochondria have little significant DNA mutations.2.Raffinose treatment inhibited the LDH release and trypan blue staining in UVB-challenged HaCaT,but neither affected plate cloning proliferation nor apoptotic markers(the cleavage of Caspase-3 and PARP,Endonuclease G and AIF).3.100 mM raffinose treatment can increase the expression of autophagy marker protein LC3 Ⅱ,the average intensity of green fluorescence by CYTO-ID staining flow cytometry,and the number of acidic vesicles by AO staining test in HaCaT cells.As well,100 mM raffinose treatment promoted the conversion of LC3 type Ⅰ to type Ⅱ in HaCaT cell.4.100 mM raffinose treatment did not alter the expression of MTOR,p-MTORSer2448 protein,4E-BP1 protein and p-4E-BP1Ser65 in the MTOR signaling pathway;5.Raffinose treatment can also promote the conversion of autophagy marker protein type LC3 Ⅰ to LC3 Ⅱ and increase acridine orange-stained acidic vesicles in UVB-irradiated HaCaT cells.6.The delated effect of the release LDH level in UVB-irradiated HaCaT cells by raffinose treatment was supressed after blocking autophagy flow by wortmannin or siATG5.Conclusion:1.Raffinose can delay UVB-triggered HaCaT cell death but has little effect on the apoptosis;2.Raffinose treatment enhanced autophagy flux in HaCaT cells as well as UVB damaged HaCaT cells.3.Raffinose induced autophagy in an MTOR-independent manner.4.Autophagy plays an important role in the effect on raffinose reducing the UVB-triggered cell death.We demonstrated that raffinose increases MTOR-independent autophagy and reduces cell death in UVB-irradiated keratinocytes.Our study indicated that the natural agent raffinose presents the potential value in opposing photodamage.