| Objective:Through in vitro experiments on human retinal pigment epithelium(ARPE-19)cells culture,to explore the regulation of exosome signaling pathway on the expression of retinal pigment epithelium(RPE)cells autophagy marker protein LC3-? and its mRNA,downstream factor P62 protein,and the expression levels of complement factors C5b-9 protein and C3a mRNA,which are closely related to autophagy,on this basis,the intervention effect of Qihuang granules was observed,furthermore,it provides a more systematic and scientific theoretical basis for Qihuang granules to treat age-related macular degeneration.Methods:1 Extraction and identification of exosomes derived from ARPE-19 cells.Normal ARPE-19 cells are cultured,and ARPE-19 cells that grow well are irradiated with blue light to create an oxidative stress injury model.The ultracentrifugation method was used to separate the normal and blue light-irradiated ARPE-19 cells culture fluid to obtain the exosomes in the supernatant Western blot(WB)was used to detect the expression level of the specific marker protein CD63 on the surface of exosome Identify the source of exosomes.2 Take 20mg/ml,15mg/ml,10mg/ml,8mg/ml,4mg/ml,2mg/ml,Qihuang granule medicinal solution at 6 concentrations.The drug concentration of Qihuang granules was determined by the thiazole blue method(MTT).3 Regulation of ARPE-19 cells-derived exosomes on RPE cell autophagy and interference of Qihuang Granules.Experimental grouping and processing:The experiment is divided into 7 groups.Blank control group(regular culture of normal ARPE-19 cells without any exosomes and drug intervention),normal model group(exosomes of normal ARPE-19 cells+co-culture of normal ARPE-19 cells),light model group(blue light irradiates exosomes of ARPE-19 cells+normal ARPE-19 cells co-culture),drug control group 1(normal model group+lutein solution),drug control group 2(light model group+lutein medicinal solution),experimental drug 1 group(normal model group+Qihuang granule liquid),experimental drug group 2(light model group+Qihuang granule liquid),n=6 in each group.After 24 hours of drug action,live cell counts were performed under the microscope using a cell counting plate,and the cell morphology of each group was observed and photographed.The proliferation activity of ARPE-19 cells was detected by MTT method.4 The expression of autophagy marker protein LC3-? and its downstream factor P62 protein,complement factor C5b-9 protein closely related to autophagy of RPE cells in each group were detected by Western blot.LC3-? and RPE cells autophagy are closely related to the mRNA expression level of complement factor C3a protein using polymerase chain reaction(PCR)).Results1 The expression of CD63,a specific marker protein on the surface of exosomes,can be detected by Western blot,the exosomes were derived from ARPE-19 cells2 According to the MTT method,when the concentration of Qihuang particles is 4.3 mg/ml,the cell viability can reach 85%.3 Results of detection of relative protein expression:(1)P62 protein:Compared with the blank control group,the drug control group 1,and the drug control group 2,the experimental drug group 2 had significantly higher data,with P values of 0.005,0.022,and 0.009,and the P values were less than 0.05,and the differences were statistically significant;Compared with the blank control group and the drug control group 2,the experimental drug group 1 had significantly higher data.The P values were 0.031 and 0.049,respectively,and the P values were less than 0.05.The difference was statistically significant;There was no statistical difference between the other groups.(2)LC3-? protein:The expression of LC3-? protein in the normal model group was higher than that in the drug control group 2 and the experimental drug group 2,with P values of 0.024 and 0.038,and the P values were less than 0.05.The difference was statistically significant.There was no statistical difference between the other groups.(3)C5b-9 protein:The overall test did not show significant differences between samples,and there was no statistical difference between each group.4 The results of relative mRNA expression showed:(1)LC3-? mRNA:Compared with the normal model group,the expression of LC3-? mRNA in the normal model group is higher than that in the light model group,P=0.008<0.05,the difference is statistically significant.There was no significant difference between the other groups.(2)C3a mRNA:The expression of C3a mRNA in the blank control group compared with the normal model group,the drug control group 1,the drug control group 2,the experimental drug group 1,and the experimental drug group 2,P values are 0.00,0.00,0.030,0.00,0.00,P values are less than 0.05,the difference is statistically significant;The expression of C3a mRNA in the normal model group compared with the light model group,the drug control group l,the drug control group 2,the experimental drug group 1,and the experimental drug group 2,the P values were 0.00,0.00,0.00,0.00,0.00,and P values,respectively.All are less than 0.05,the difference is statistically significant;Compared with the drug control group 1,experimental drug group 1 and experimental drug group C3a mRNA expression levels,the light model group had P values of 0.00,0.00,and 0.00,and the P values were less than 0.05,and the differences were statistically significant;The C3a mRNA expression levels of the drug control group 1 compared with the drug control group 2,the experimental drug group 1,and the experimental drug group 2 were P values of 0.00,0.001,and 0.002,the P values were all less than 0.05,and the differences were statistically significant;The C3a mRNA expression levels of the two groups of the drug control group compared with the experimental drug group 1 and the experimental drug group 2 were P values of 0.00 and 0.00,and the P values were less than 0.05,the difference was statistically significant;C3a mRNA expression in experimental drug group 1 compared with experimental drug group 2,P value=0.00,P<0.05,The difference was statistically significant.Conclusion:1 The expression of the surface-specific marker protein CD63 was detected by Western blot,which can prove that the exosomes originated from ARPE-19 cells2 The concentration of Qihuang granule was determined to be 4.3mg/ml by MTT method3 Under oxidative stress conditions,P62 protein is a key regulator of autophagy Upregulated expression of P62 protein can stimulate and promote autophagy activity and protect RPE cells.Exosomes and Qihuang granules can up-regulate the expression of P62 protein,and the efficacy of Qihuang granules is better than lutein,which proves the effectiveness of exosomes and Qihuang granules to up-regulate P62 protein.ARPE-19 cells-derived exosomes have the effect of up-regulating LC3-? protein and LC3-? mRNA,thereby increasing the autophagy level of RPE cells.The high expression of C3a can be deposited in the basal layer of RPE cells and promote the formation of glass warts.Qihuang granules and lutein both have a significant down-regulation effect on C3a mRNA levels,and both drugs can down-regulate C3a mRNA levels under physiological oxidative stress conditions compared to physiological conditions.mRNA down-regulation was superior to lutein. |
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