Research On The Effect And Mechanism Of Alpha 1 Antitrypsin On The Model Of Chronic Photoaging Of Human Fibroblasts

Background:As one of the important components of ultraviolet radiation,UVA causes photoaging by penetrating into the dermal connective tissue.UVA radiation-induced photoaging is related to oxidative stress,matrix metalloproteinase expression and collagen degradation.Alpha-1antitrypsin has the function of resisting a variety of proteases.In addition,it also has a variety of functions such as delaying cell apoptosis,regulating immunity,and resisting inflammation.The previous research of our group has shown that AAT has certain preventive and therapeutic effects on guinea pig skin photoaging model.In order to further verify the effect of AAT on primary photoaging fibroblasts,this experiment was based on the most important part of the photoaging mechanism.The human skin fibroblasts were irradiated with UVA lamp,and the cell aging model was established.The following research was carried out.Objectives:(1)To investigate whether AAT can delay UVA-induced chronic photoaging process of human skin fibroblasts through exerting antioxidant effect.(2)To investigate whether AAT can delay UVA-induced chronic photoaging of human skin fibroblasts by affecting the gene and protein levels of transcription factor active protein-1(AP-1),matrix metalloproteinase-1(MMP-1),matrix metalloproteinase-3(MMP-3)and procollagen type I.Methods:The fibroblasts extracted from the foreskin tissue of healthy children were subcultured.During the study,fibroblasts within six generations were divided into control group,AAT groups,UVA irradiation group and AAT+UVA irradiation groups in a random manner.In the AAT groups and the AAT+UVA irradiation groups,the cells were pretreated with AAT for 24 hours before each UVA irradiation.Fibroblasts was to irradiated by 20 m W/cm~2UVA lamp at the same time every day,with a daily irradiation dose of 8 J/cm~2,a total of 14 times,to establish a chronic photoaging model of fibroblasts.At the end of the last irradiation,the senescent cells were detected byβ-galactosidase staining.To critically assess the protection of AAT on chronic photoaging of fibroblasts,flow cytometry was used to test the production of reactive oxygen species(ROS).RT-PCR was used to detect the gene expression(m RNA)of AP-1(c-Fos/c-Jun),MMP-1,MMP-3 and type I procollagen.ELISA was used for detecting their protein expression.Results:(1)Compared with the blank control group,UVA radiation significantly increased the percentage ofβ-galactosidase staining positive cells.However,the percentage of stained positive cells in fibroblasts pretreated with corresponding doses of AAT(0.5 mg/ml,1.0mg/ml,1.5 mg/ml)decreased approximately by 8.23%,15.61%,and 14.13%,the difference was statistically significant(P<0.001,P<0.001,P<0.001).In addition,compared with the flat and wide fibroblasts in the UVA irradiation group,the appearance of the cells which was pretreated with AAT was slender.(2)Flow cytometry indicated that the production of ROS in the 0.5 mg/ml AAT+UVA irradiation group was reduced by approximately 12.73%compared with UVA irradiation group,and the difference was not statistically significant(P>0.05);In the 1.0 mg/ml and 1.5mg/ml AAT+UVA irradiation groups,the ROS production was reduced by approximately26.06%and 44.24%,the difference was statistically significant(P<0.001,P<0.001).(3)With RT-PCR detection,it was found that the genes(m RNA)expression of c-Fos,c-Jun,MMP-1 and MMP-3 in the AAT+UVA irradiation groups were suppressed by AAT in a dose-dependent manner,compared that of UVA irradiation group.Furthermore,the gene(m RNA)synthesis of type I procollagen was promoted.The difference was statistically significant(P<0.05).(4)With ELISA detection,it was found that AAT suppressed the protein expression of c-Fos,c-Jun,MMP-1 and MMP-3 in a dose-dependent manner,weakened the protein degradation of type I procollagen and promoted its synthesis in the AAT+UVA irradiation groups,compared that of UVA irradiation group.The difference was statistically significant(P<0.05).Conclusion:UVA-induced chronic photoaging of human skin fibroblasts can be delayed by AAT.The potential mechanisms of this protective effect including:(1)AAT reduces the level of intracellular ROS and the damage to the extracellular matrix by exerting an antioxidant effect;(2)AAT at least partially reduces the expression of MMP-1 and MMP-3 by inhibiting the expression of AP-1(c-Fos/c-Jun),thereby reducing the degradation of type I procollagen;(3)The gene expression of type I procollagen is enhanced by AAT.These results provide strong evidence that AAT has a protective effect on skin damage and that AAT is a potential candidate for the prevention and treatment of photoaging.