Protective Effect Of Aronia Melanocarpa Anthocyanins On Oxidative Damage Of Retinal Pigment Epithelial Cells

Aronia melanocarpa,commonly known as Black fruit mandshurica,is a genus of Rosaceae.It is rich in flavonol,phenolic acid,anthocyanins and other functional components.Its anthocyanins have many biological functions,such as anti-aging,anti-cancer,anti fatigue and so on.In recent years,although China’s research in its functional field has increased,the research in the field of visual protection is relatively scarce.With the increasing trend of population aging,the gradual rise of the prevalence of AMD and the extreme desire of patients for treatment,more and more authoritative people and researchers begin to pay attention to this problem.In this paper,the optimal extraction process of anthocyanins from Aronia melanocarpa was discussed.The oxidative stress model of ARPE-19 cells induced by H₂O₂was established in vitro to explore the protective effect of anthocyanins on it,and the model of retinopathy induced by sodium iodate was established to further explore the protective effect of anthocyanins on retina in vivo.The test methods and conclusions are as follows.Based on the analysis of single factor test results,taking the volume fraction of acidified ethanol,ultrasonic time,liquid-solid ratio and ultrasonic temperature as independent variables and the extraction amount of anthocyanins from Aronia melanocarpa as dependent variables,the response surface test was carried out.Finally,it was concluded that the final conditions were acidified ethanol volume fraction of 65%（p H=2）,ultrasonic time of 44 min,liquid-solid ratio of18:1（ml/g）and ultrasonic temperature of 38℃.Under these conditions,three parallel confirmatory tests were carried out.The average extraction amount of anthocyanins from Aronia melanocarpa reached 4.569 mg/g,and the deviation from the predicted value was about 1.78%.In vitro,ARPE-19 was treated with hydrogen peroxide to successfully simulate oxidative stress injury.The fluorescence intensity of ROS,apoptosis rate and mitochondrial membrane potential were measured by flow cytometry;MTT assay was used to detect the light absorption value of cells;The protein expressions of Cyt C and cleaved caspase 3 were detected by Western blot.The results showed that the anthocyanins of Aronia melanocarpa could inhibit the damage of ARPE-19 cells induced by H₂O₂,and the cell survival rate increased from 60.80%to 86.75%.The addition of Aronia melanocarpa anthocyanins could improve the scavenging ability of ROS,promote the recovery of mitochondrial membrane potential,inhibit the release of Cyt C in cytoplasm,and reduce the protein expression of cleaved caspase 3.At the same time,the apoptosis rate of ARPE-19 cells decreased from 36.84%to 11.84%,which was significantly different from the model group（p<0.01）.The results showed that the anthocyanins of Aronia melanocarpa could reduce the apoptosis of ARPE-19 cells and inhibit the oxidation of H₂O₂.The rat retinal oxidative injury model induced by Na IO₃was successfully established,and the structural changes of rat retina were observed by HE staining and immunohistochemical staining;The expression of apoptosis related protein cleaved caspase 3 was detected by IHC and Western blot.The results showed that Aronia melanocarpa anthocyanins could improve the damage of rat retinal tissue caused by Na IO₃injection,and the protein expression levels of Cyt C and apoptosis protein cleaved caspase 3 in cytoplasm also decreased relatively,reducing retinal cell apoptosis.When the dose of Aronia melanocarpa anthocyanins reached 500 mg/（kg?d）,it had the best effect on the recovery of rat retinal structure damage and could basically return to normal state.The results showed that the anthocyanins of Aronia melanocarpa could prevent retinal pigment epithelium detachment in rats,which opened up a new way for the prevention and treatment of AMD in the future.The rat retinal oxidative injury model induced by Na IO₃was successfully established,and the structural changes of rat retina were observed by HE staining and immunohistochemical staining;The expression of apoptosis related protein cleaved caspase 3 was detected by IHC and Western blot.The results showed that Aronia melanocarpa anthocyanins could improve the damage of rat retinal tissue caused by Na IO₃injection,and the protein expression levels of Cyt C and apoptosis protein cleaved caspase 3 in cytoplasm also decreased relatively,reducing retinal cell apoptosis.When the dose of Aronia melanocarpa anthocyanins reached 500 mg/（kg?d）,it had the best effect on the recovery of rat retinal structure damage and could basically return to normal state.The results showed that the anthocyanins of Aronia melanocarpa could prevent retinal pigment epithelium detachment in rats,which opened up a new way for the prevention and treatment of AMD in the future.