The Effect Of ALA-PDT On The Activity Of SZ95 Sebocytes And The Apoptosis Signaling Pathway Of Endoplasmic Reticulum Stress

Acne is a chronic inflammatory skin disease of the sebaceous glands,which is one of the most common skin diseases.Predilection in young people,the incidence of up to 80%.Acne is the most common disfigurement of skin disease,for the patient’s physical and mental effects are larger.Sebaceous gland inflammation and excessive sebum secretion is an important link in the pathogenesis of acne.ALA-PDT has been used in clinic for treatment of acne many years,but its mechanism is not very clear.In view of this,this study used SZ95 sebocytes As research object.As the cells have similar biological characteristics with normal human sebaceous gland cells.Endoplasmic reticulum stress(ERS)induced by beta carotene(TG),and with GRP78,CHOP,Caspase12 as the testing index.Objective to study the effects of ALA-PDT(5-Aminolevulinic acid combined with red light)on the activity of SZ95 cells and the apoptosis pathway of endoplasmic reticulum stress.The purpose of this study was to clarify the effect of ALA-PDT on sebaceous gland cells,and to further elucidate the mechanism of ALA-PDT in the treatment of acne.The results will provide theoretical basis for the treatment of acne with ALA-PDT.Objective:1.To determine the safe and effective ALA concentration and red light irradiation dose of ALA-PDT in SZ95 sebocytes.Effects of ALA-PDT on the detection of ERS marker GRP78 and apoptosis related marker molecules CHOP and Caspase-12.2.To determine the safe and effective concentration of TG to induce ERS.ERS marker GRP78 and apoptosis related marker CHOP and Caspase-12 were detected to prove that TG induced SZ95 sebocytes were induced ERS.3.To determine the effect of ALA-PDT on the activity of SZ95 sebocytes after TG,and the effects of Caspase-12 on the expression of ERS marker molecule GRP78 and apoptosis related marker molecule CHOP.Methods:1.MTT method was used to screen the safe and effective concentration of the ALA and the dose of red light.And the ALA-PDT’s concentration of the ALA and the dose of red light.ALAand red light experiment were divided into blank control group and experimental group,while set blank wells all of them(only added medium).Group ALA were given different concentrations of ALA solution;The red groups were given different doses of red light irradiation,the time were20 min,the control group and the blank wells were only added to the culture medium.The ALA experiment groups and the blank wells were incubated with none light for 2h;The red light group was treated with different doses of red light irradiation,The other groups did not change the treatment and just replacement with medium,and then continued to cultivate 24 h.ALA-PDT experiment was divided into two groups: blank control group and ALA-PDT group,while set blank wells.The ALA-PDT group was incubated with ALA at the corresponding concentration of 2h and avoiding light,then the corresponding dose of red light irradiation was given to 20 min,and the control group and the blank wells were not treated with special treatment.24 h was added to the culture medium after irradiation.Finally,MTT was used to determine the safe and effective concentration of ALA and the safe and effective dose of red light.ALA was 25μg/mL+red light for 5J/cm2 composed of ALA-PDT experimental group,acting on cells after 24 h,extraction of mRNA.The expression of GRP78,CHOP and Caspase-12 m RNA were detected by qRT-PCR.2.MTT method for screening the safe and effective concentration of TG.The experiment was divided into two groups: experimental group and blank control group,while set blank wells.Selection of safe and effective TG concentration by MTT with different concentrations of TG acting on cell 24 h.Acting on cells with 0.5μg/m LTG,and qRT-PCR and WB were used to detect the expression of GRP78 and CHOP,Caspase-12 mRNA and protein in RNA and protein after 24 h.3.MTT method for screening the safe and effective concentration and dosage of ALA-PDT after the TG acting on cells.Divided into TG control group(only with TG)and ALA-PDT group,while set blank wells.Each group of cells were cultured with 0.5μg/mLTG for 24 h,the ALA-PDT group was incubated with ALA at different concentrations for 2h,and then irradiated with red light(The dose were 5J/cm2).The other group with the former disposal.MTT was used to screen the safe and effective dose and dose of in the ALA-PDT group after irradiation for 24 h.0.5μg/mL TG acted on cells after 24 h,The cells of ALA-PDT group,which was composed of the concentration of ALA2μg/mL+the red light 5J/cm2,was used to extract RNA and protein from the cells above24 h.GRP78,CHOP and Caspase-12 mRNA and protein expression were detected by qRT-PCR and WB respectively.Results:1.Compared with the blank control group,there was no significant difference in the survival rate between the ALA group and the red light group,(P>0.05).The results indicated that ALA alone and individual red light had no significant effect on the survival rate of SZ95 human sebaceous gland cells.Compared with the blank control group,the survival rate of ALA-PDT inthe experimental group was 200,400 and 800μg/mL ALA,respectively,and the survival rate was significantly decreased after each dose of red light irradiation,(P<0.05).When the solution ALA was 100μg/mL,≥2.5J/cm2 by red light irradiation,the cell survival rate decreased significantly after 24 h,(P<0.05).When the solution ALA was 50μg/mL,≥5J/cm2 by red light irradiation,the cell survival rate decreased significantly after 24 h,(P<0.05).When the ALA solution is 25μg/mL,≥10J/cm2 red after irradiation,the cell survival rate decreased significantly,(P<0.05).The other group ALA-PDT after treatment after the cell survival rate was not significantly changed,(P>0.05).These results indicate that ALA-PDT has a significant effect on the concentration of ALA and the dose dependence of red light.Because of the 25μ g/mL ALA+5J/cm2 red light,the ALA-PDT group acted on the cell,and the survival rate was not significantly changed compared with the blank control group.The condition for the critical value of cell viability.The expression of GRP78,CHOP and Caspase-12 m RNA were significantly increased after the cells detected by this condition,there was statistical significance,(P<0.05).2.Compared with the blank control group,TG were 1,2,4,8μg/mL,the effect of cell survival rate decreased significantly after 24 h,(P<0.05);When the concentration of TG were 0.06,0.125,0.25,0.5μ g/mL,cell survival rate was not significantly different from the control group(P>0.05).When the TG concentration is 0.5μg/mL,the GRP78,CHOP and Caspase-12 mRNA and protein expression were significantly up-regulated,(P<0.05).Therefore,this study selected 0.5g/mLTG as the experimental concentration.3.Compared with TG control group when ALA is 0.25,0.5,1,4,7μg/mL composed ALA-PDT group,the cell survival rate had no significant difference,(P>0.05);When ALA was12.5,25μg/mL composed ALA-PDT experimental group,cell survival rate decreased significantly,(P<0.05).When the ALA is 2μg/mL the survival rate of ALA-PDT cells was significantly up-regulated,(P<0.05).The survival rate of SZ95 sebocytes ERS induced by TG was significantly increased by ALA 2μg/mL+5J/cm2 binding ALA-PDT.The condition of ALA-PDT was selected to act on TG induced ERS cells.The results compared with TG group,the CHOP GRP78,Caspase-12 mRNA and protein expression were significantly reduced,(P<0.05).Conclusion:1.There was no significant effect on the survival rate of SZ95 sebocytes with only ALA or only red light;For ALA-PDT,when the ALA concentration >25μ g/mL and red dose >5J/cm2 was composed of ALA-PDT significantly decreased cell survival rate.There was significant ALA concentration and red light dose dependence.ALA-PDT group with ALA concentration of 25μg/mL and red light dose of 5J/cm2,it no significantly effect on cell viability,but increased ERS marker GRP78 and apoptosis related molecules CHOP and Caspase-12;2.When the TG>0.μ5 g/mL,the cell survival rate decreased significantly.When the TG≤0.5μg/m L the cell survival rate had no significant effect.When TG was 0.5μg/mL,there was no significant effect on cell viability,but it could induce ERS;3.The effect of ALA-PDT on the survival rate of SZ95 sebocytes after 2μg/mL ALA+5J/cm2 red light was significantly increased by TG.These results suggested that ALA-PDT could enhance the activity of SZ95 sebocytes,and had a protective effect on ERS cells induced by TG.The mechanism may be that ALA-PDT can inhibit the expression of GRP78,CHOP and Caspase12 in the ERS pathway.