Impacts Of Photodynamic Therapy With 5-Aminolevulinic Acid On HaCaT Cells Keratin 17 Expression

OBJECTIVE:Photodynamic therapy has been applied in neoplasm treatment in recent years.The effects of photodynamic therapy depend on the absorption of harmless visible light after systematic or topical application of photosensitizer,which then produces reactive oxygen species(ROS),such as singlet oxygen,that destroy target cells(cancer cells,blood vessels,and pathogenic micro-organisms)or change function.The possible mechanism of photodynamic therapy includes:promoting cell apoptosis/necrosis;damaging vessels in target tissue;participating in local/systematic innate/acquired immunity.As we know keratinocytes overproliferation and differentiation abnormality,persistent inflammation and immunity disfunction mediated by Treg/Thl7 cells.In psoriasis overproliferation of keratinocytes was considered as similar as tumor cells,some researchers tried to treat localized plaque psoriasis with photodynamic therapy and obtained certain effects.They also found PDT had influences on proliferation and differentiation,protein expression in keratinocytes,local or systemic immune status,synthetise and secretion of inflammatory factors/cytokines.But the exact mechanism of photodynamic therapy in treating psoriasis stayed unclear.,the experimental research was stilled needed.In 1960 Rothberg found keratin 17 was overexpressed in psoriasis lesions specifically which related with disease severnty and progression positively.Researchers proposed the keratin 17 as index of disease severity and effects in psoriasis,so keratin 17 was named as "psoriasis relative keratinocyte keratin",and regimens targeting keratin 17 were approved.Some classic therapies for psoriasis(eg acitretin,adalimumab et al)can downregulate the expression of keratin 17 markedly.HaCat cell is human immortalized keratinocyte which was usually used as psoriasis model in vitro.Keratin 17 was the only kereatin induced by IFN-,yin vitro,so we selected HaCat cell induced by IFN-y as subject to investigate the impacts of photodynamic therapy with 5-aminolevulinic acid on K17 expression,and to discuss the possible mechanism of photodynamic therapy treating psoriasis for the purpose of theory evidence.MATERIALS AND METHODS:1.Inducion of K17 expression in HaCaT cells:HaCaT cells were stimulated with IFN-y(250 U/ml)for 48 h,after which the cells were used for proliferation assays;2.Detection on the impacts of ALA-PDT onHaCaT cells proliferation in vitro by MTT Assay:use different concentration ALA(0?0.1?land 10 mM)and same power density(70J/cm2)to intervene HaCat cells,collect cells respectively after 6h?12h?24h and 48h and use the MTT assay to detect the cells proliferation activity;3.Detection on the impacts of ALA-PDT on K17?PAPR?caspase-3?p-p38?p-ERK?p-JNK protein expression in HaCaT cells by Western Blot methods;4.Detection on the impacts of ALA-PDT on K17 mRNA expression in HaCaT cells by Real-time PCR;5.Detection on the impacts of ALA-PDT on HaCaT cells apotosis by Annexin V/7-AAD Staining;6.Statistical analysis:Then we did statistic analysis with SPSS 19.0 software.All the counting data were taken as the standard deviation of the mean.The comparison of the multigroup mean was made by single factor analysis of variance.The difference between the two groups was statistically analysed using t test,P<0.05 was significant difference.RESULTS:1.The absorbance value of keratin 17 in HaCaT cells 48 hours after induction with 250u/ml IFN-? was 0.37±0.03,higher than that in control group(0.09±0.02)(p<0.05);the mRNA level of keratin 17 in experimental group was 3.07±0.13,higher than that in control group(1.00±0.04)(p<0.05);2.The inhibition rates of IFN-y induced HaCat cells were 20.28%,34.87%and 44.37%respectively under 0.1mM,l1mM and lOmM ALA photodynamic treatment,and the inhibition rate was positively correlated with the concentration of ALA in the experimental range;3.The inhibitory rates of 1 mM ALA photodynamic therapy on IFN-y induced HaCat cells were 28.19%,34.86%,42.44%and 50.88%at 6,12,24 and 48 hours after treatment respectively.The inhibitory effects gradually enhanced with time within 48 hours;4.The absorbance value of keratin 17 in HaCat cells treated with 1 mM ALA-PDT was 0.22 ± 0.10,which was significantly lower than that in control group(0.67±0.04).The levels of keratin 17 protein in HaCat cells treated with 1 mM ALA singly were 0.63 ± 0.04 and irradiation at 70 J/cm 2 singly(0.47±0.15)(p<0.05).After 1 mM ALA-PDT the level of keratin 17 mRNA was 0.5010.01,and lower than that in control(1.00±06)(p<0.05),only 1 mALA(0.99±0.03)and only irradiation at 70 J/cm2(0.65±0.03)(p<0.05);5.The absorbance of keratin 17 induced by IFN-? in HaCat cells treated with ImM ALA was 0.62 ± 0.04,0.41 ± 0.03,0.28 ±0.03 and 0.11 ±0.03 respectively at 6,12,24 and 48 hours after treatment,which was significantly lower than that of control 0.7010.02(p<0.05),and the inhibitory effect enhenced gradually within 48 hours;6.The apoptosis rate of HaCat cells induced by IFN-y was 47.83%after 1 mM ALA photodynamic treatment,which was significantly higher than that of the control group 11.60%(p<0.05).The absorbance values of PARP protein and caspase 3 in HaCat cells after photodynamic treatment were 0.36 ±0.02 and 0.49 ± 0.07 respectively,which were significantly higher than those in the control group(0.12 ± 0.01 and 0.21 ±0.03)(P<0.05);7.P-p38(0.47±0.05)and p-JNK(1.07±0.08)of HaCat cells treated with 1 mM ALA PDT were significantly higher than those before treatment(0.12±0.02 and 0.33±0.04)(p<0.05),while p-ERK(0.1 7±0.03)was significantly lower than that before treatment(0.57±0.09)(p<0.05)CONCLUSIONS:ALA photodynamic therapy can significantly inhibit proliferation and promote apoptosis of IFN-y induced HaCat cells,this effect may be achieved by activating p38,JNK signal transduction pathway;ALA photodynamic therapy can significantly inhibit IFN-y induced expression of keratin 17 in HaCat cells,and the effect is positively correlated with concentration and time in a certain range.