Effect Of Artesunate On Retina Autophagy Of Diabetic Rats And The Underlying Mechanism Study

Objective:Autophagy is a complex physiological process,which is responsible for degrading intracellular components and maintaining intracellular homeostasis in response to stress conditions.Autophagy plays an important role in the process of diabetic retinopathy.Artesunate(ART)is a water-soluble derivative extracted from Artemisia artemisinus,a water-soluble derivative of artemisinin.It was approved to be marketed by the Ministry of Health in 1987.Artesunate is currently used clinically as an anti-malarial drug,and it also shows anti-tumor and anti-inflammatory effects,which make it also have a role in autoimmune diseases and diabetes mellitus and their complications.ART has also been found to act as an autophagy inducer to inhibit fibroblast proliferation and surgery-induced epidural fibrosis.Hyperglycemia is a recognized risk factor for the development and progression of diabetic retinopathy.Alterations in biochemical pathways,such as increased glycolylation end product/receptor flux,polyol pathway,protein kinase C activation,and hexosamine pathway,induce oxidative stress and,in particular,contribute to the induction of inflammatory intermediates production.Laboratory and clinical evidence suggests that inflammation and oxidative stress begin to damage the retina in the early stages of diabetic retinopathy,prior to and accompanied by microvascular changes.Free radical release and pro-inflammatory cytokines lead to increased vascular permeability,breakdown of the blood-retinal barrier and loss of capillary pericytes,leading to diabetic retinal injury.Oxidative stress and inflammation play an important role in a series of pathological changes of DR.Autophagy mediates regulation of oxidative stress and inflammation in DR.The search for autophagy regulating drugs has become a hotspot of DR research.In this study,artesunate was used to investigate the changes in oxidative stress and inflammatory response as well as the AMPK/SIRT1 signaling pathway in diabetic retinopathy.We confirmed the protective effect of artesunate on retinal tissue in diabetic rats by experimental study.The study was divided into three parts.First,we established a STZ-induced diabetic rat model and conducted an in vivo study to determine whether artesunate treatment improved the leakage of the retinal barrier in diabetic rats,and to examine oxidative stress and inflammatory responses in retinal tissues.Subsequently,we examined the expression of autophagy-related proteins and the activation of AMPK/SIRT1 signaling pathway in diabetic rat models.Meanwhile,in order to further confirm the inhibitory effect of artesunate on diabetic retinopathy,we also conducted in vitro experiments.The effects of artesunate on oxidative stress and inflammatory response of retinal pigment epithelial cells cultured under high glucose conditions,as well as the activation of autophagy related proteins and AMPK/SIRT1 signaling pathway were examined.Methods:1.SD rats were randomly divided into four groups: control group,diabetes group,and low-dose and high-dose artesunate treatment groups.In addition to the control group,rats were intraperitoneally injected with STZ,and the treatment group was treated with low and high doses of artesunate.2.Histomorphological changes of the retina of rats were detected by HE staining.3.Evens Blue was used to detect the leakage of the retinal barrier in rats.4.Lectin staining was used to detect leukocyte adhesion in retinal vessels.5.Retinal microglia cells were labeled by IBA-1 immunohistochemistry.6.The expression levels of IL-1? and IL-6 in the retinal tissues of rats were detected by Real-time PCR,and the expression of MCP-1 in the retinal tissues of rats was detected by ELISA.7.Changes of ROS levels in the retina of rats were detected by DHE staining.8.The expression levels of Beclin-1,LC3II/I,P62,AMPK,p-AMPK,SIRT1,p-SIRT1 were detected by Western blot.9.Arpe-19 cells were treated with high glucose and high glucose addition.CCK-8 assay was used to detect the changes of proliferative activity of retinal pigment epithelial.10.Flow cytometry was used to detect the effects of artesunate on cell apoptosis and mitochondrial membrane potential under high glucose.11.Evaluate oxidative stress: MDA and SOD biochemical tests,and ROS levels were detected by fluorescence staining.12.The expression levels of IL-1?,IL-6 and MCP-1 in ARPE-19 cells were detected by ELISA.13.Autophagosome formation was detected by MDC staining.14.The effects of artesunate on Beclin-1,LC3II/I,P62 and AMPK/SIRT1 activation in retinal pigment epithelial cells were detected by Western blot.15.Finally,the effect of si-SIRT1 transfection on the effect of artesunate in an in vitro model of diabetic retinopathy was detected.Results: 1.Results showed that retinal thickness was markedly increased in diabetic rats,accompanied by the breakdown of the blood-retinal barrier.2.ART administarion suppressed inflammatory response in the retinas of diabetic rats by quantitating vascular leukocyte adherence,microglial density and the expression of related markers,including interleukin(IL)-1?,IL-6 and monocyte chemoattractant protein-1(MCP-1).3.In addition,ROS production was also decreased with ART treatment.4.The results of western blotting revealed that ART up-regulated Beclin-1 expression and LC3II/I ration as well as down-regulated p62 expression.Further evidence found that treatment with ART induced the occurrence of autophagy by activating the AMP-activated protein kinase(AMPK)/sensor class III histone deacetylase sirtuin 1(SIRT1)signaling pathway in DR rats.5.Artesunate improved the proliferation activity,mitochondrial membrane potential and apoptosis of retinal pigment epithelial cells induced by high glucose.6.Artesunate can effectively improve the oxidative stress of retinal pigment epithelial cells induced by high glucose and reduce the expression of proinflammatory factors.7.Artesunate can enhance autophagy deficiency induced by high glucose and activate AMPK/SIRT1 signaling pathway.8.SIRT1 plays an important role in the role of artesunate in the in vitro model of diabetic retinopathy.Conclusion: 1.Artesunate showed significant therapeutic effect on diabetic retinopathy.2.Artesunate can reduce diabetic retinal inflammatory response and oxidative stress,increase the activation inhibition of AMPK/SIRT1 signaling pathway in diabetic retinal,and enhance the role of autophagy in the treatment of diabetic retinopathy.3.Artesunate can reduce oxidative stress of retinal pigment epithelial cells induced by high glucose and protect cells by SIRT1-dependent autophagy.