Effect Of Rhein On Diabetic Retinopathy Through Activating Sirt1/PGC-1α Pathway And Its Mechanism

Purpose:Diabetic retinopathy(DR),a devastating vision-threatening condition,is one of the most common diabetic complications.Müller cells are the most dominant glial cells in the retina.It spans the entire width of the retina and is closely related to retinal blood vessels and neurons both structurally and functionally.The main functions of Müller cells include:the uptake of neurotransmitters and ions,the control of retinal metabolism and nutrient supply,and the maintenance of blood-retinal barrier.In the course of DR,neurodegeneration and abnormal regulation of microcirculation occur in the retina,among which Müller cells are important regulatory factors that cannot be ignored.Therefore,in-depth study of Müller cells is helpful for early treatment of DR.Silent information regulator 1(Sirt1)is a highly conserved NAD+-dependent histone deacetylase,which has a regulatory role in numerous physiological and pathological processes by removing acetyl groups from various proteins.Sirt1 controls the activity of numerous transcription factors and cofactors,which impacts the downstream gene expression,and eventually alleviates oxidative stress and associated damage.Therefore,it is speculated that highly effective Sirt1 activators may be candidates for the prevention and treatment of DR.Rhein is a kind of hydroxyanthraquinone compounds in Rhubarb,Senna leaf,Cassia seed and Polygonum Multiflorum.Studies have shown that Rhein has anti-inflammatory and antioxidant activities and can reduce the damage of diabetes mellitus and its complications through the Sirt1/PGC-1α pathway.Consequently,we put forward the following scientific hypothesis:The aim of the present study was to explore the pharmacological effect of Rhein on the oxidative stress and apoptosis in Müller cells cultured under HG conditions,as well as the regulatory role of Sirt1/PGC-1α in this process.Our experimental verification is expected to provide new targets and ideas for the prevention and treatment of DR,and provide theoretical basis for the prevention and treatment of DR with Rhein.Methods:The first part of the experiment:Diabetic mice models were established and divided into three groups:Control group,Diabetic group(DM),and Diabetic+Rhein group(DM+Rhein).The mice were sacrificed 12 weeks later,and the changes of retinal structure were detected by HE staining,and the transcription and expression of retinal inflammatory factors(VEGF,IL-1β,IL-6,TNF-α)were detected by Q-PCR and ELISA.REDOX indicators were determined:malondialdehyde(MDA),catalase(CAT)and superoxide dismutase(SOD)activity,Western blot and Q-PCR were used to detect Bcl-2,Bax and Cleaved caspase-3.The expression of Sirt1 was detected by immunofluorescence and Western blot.The second part of the experiment:establishing high glucose damage of retinal Müller cells model and intervention with Rhein,ELISA and Q-PCR were used to detect transcription and expression level of inflammatory factors:VEGF,IL-6,IL-1β,TNF-α,ELISA was used to detect the levels of CAT,MDA,ROS and SOD activity.TUNEL staining was used to observe apoptosis under fluorescence microscope.Western blot and Q-PCR were used to detect the transcription and expression of Bcl-2,Bax and Cleaved caspase-3.Western blot and Q-PCR were used to observe the effects of Rhein on the expression of Sirt1 signaling pathway related proteins after hyperglycemic stimulation.The transcriptional levels,protein expression and interaction of Sirt1 and PGC-1α were analyzed by ELISA,Western blot and Q-PCR.Reverse validation was performed with a Sirt1 inhibitor(EX-527).The morphological changes of mitochondria were observed by transmission electron microscopy.The contents of ATP and cytochrome C in and outside mitochondria were detected by kit.Results:1.Rhein improved the damage of retinal structure in diabetic mice.2.Rhein reduced VEGF、IL-1β、IL-6、TNF-α level,inhibited inflammation in retina of diabetic mice and Müller cells induced by high glucose.3.Rhein reduced ROS and MDA,increased the activity of SOD,CAT and alleviated oxidative stress in retina of diabetic mice and Müller cells induced by high glucose.4.Rhein attenuated apoptosis,up regulated Bcl-2 and down regulated Bax and Cleaved caspase 3 expression in retina of diabetic mice and Müller cells induced by high glucose.5.Rhein activated Sirt1/PGC-1α signaling pathway,restore mitochondrial morphology,promoted the production of ATP,decreased the release of mitochondrial cytochrome C into cytosol in Muller cells induced by high glucose.Conclusions:These results suggest that Rhein can improve retinal mitochondrial dysfunction,inflammation,oxidative stress and apoptosis induced by hyperglycemia through activating Sirt1/PGC-1α signaling pathway.These findings indicate that Rhein may have potential therapeutic applications for DR.