| Diabetes Mellitus(DM)is a chronic metabolic disease characterized by insulin resistance,insufficient insulin secretion,disturbance of glucose and lipid metabolism,and high blood sugar.The increase of glucose concentration in blood can damage the organs and systems of the body,resulting in many complications.Diabetes can cause a variety of physiological and pathological changes in the structure and function of the eye,such as cataract,glaucoma and atrophy of the optic nerve.The pathophysiology of retina in DM patients is an important cause of the loss of visual function.Proliferative diabetic retinopathy will increase the risk of loss of vision.Rhodiola is a kind of perennial herb belonging to Rhodiola in the Crassulaceae family.It is native to the mountains in Southwest China and the Himalayas.Its roots and stems can be used as medicine,so its pharmacological effects are very extensive.The main pharmacological active components isolated from various Rhodiola are salidroside and its aglycone(tyrosol),pyridine and rhodiolin.Salidroside,as one of the main monomers and active components of Rhodiola,has been detected in a large number of cells and animals in vivo and in vitro,and shows strong biological activity.So far,the main effects are as follows: anti hypoxia,anti-fatigue and anti-aging activities,activities on cardiovascular system and central nervous system,antioxidant activities,anti-cancer activities,anti-inflammatory activities,anti-diabetes activities,anti-virus and immune stimulation activities,anti-osteoporosis activities.These pharmacological effects suggest that salidroside may be a valuable treatment option for the prevention and treatment of atherosclerosis,cardiovascular disease,Parkinson’s disease,Alzheimer’s disease,pneumonia and other diseases.Although the previous studies on the pharmacological effects of salidroside,the main active component of salidroside,have been more and more,mainly focusing on the central nervous system,diabetes and cancer,the in vivo and in vitro experimental research on salidroside is not deeply enough.In particular,the pharmacological effect of salidroside on retinopathy caused by diabetes has not been reported.Because of the continuous increase of blood pressure and blood sugar in diabetics,the body will show high oxidative stress.The imbalance between reactive oxygen species(ROS)and active nitrogen(RNS)and antioxidant defense system in the body may lead to increased ROS and RNS levels through various ways.The upstream activation signal of some superoxide products that activate NLRP3 inflammatory corpuscles is ROS.Using chemical scavengers of ROS,inhibiting NADPH oxidase or si RNA interfering with NADPH oxidase subunit p22 phox to inhibit the production of ROS can widely inhibit the activation of NLRP3 inflammatory bodies.Another study showed that ROS can directly promote the binding of thioredoxin interacting protein(TXNIP)to NLRP3,so as to activate NLRP3 inflammatory bodies and promote the occurrence of inflammatory response.ROS / NLRP3 axis is also the focus of inflammation research,which is closely related to the inflammatory response of various diseases.Transcription initiation is a very important regulatory step in eukaryotic gene expression.It involves the assembly of general transcription factors and RNA polymerase II into a functional pre promoter complex at the core promoter.Many transcriptional coactivators are multi subunit complexes,which are organized into different structural and functional modules and carry a variety of regulatory activities.The first identified nuclear histone acetyltransferase is general control non derepressable 5(GCN5).GCN5 is a catalytic subunit of many different co activator complexes with specific functions.GCN5 was recently confirmed as the transcriptional activity of lysine acetyltransferase 2A(kat2a),which is highly conserved in eukaryotes.In humans,acetylation of GCN5 is associated with chromatin regulation,autophagy,neuronal apoptosis,cell proliferation,chromosome agglutination,inflammation,stem cell differentiation,hematopoiesis and oxidative stress.GCN5 has been widely studied for its role in epigenetic regulation and pathogenesis of different types of diseases,such as cancer,diabetes,multidrug resistance,nervous system diseases,infectious diseases,immune disorders and aging.GCN5 is a histone acetyltransferase which acetylates histone H3 on lysine 9,14 and 18 sites,that is H3K9 Ac,H3K14Ac and H3K18 ac.The acetylation of the markers is directly involved in the expansion of higher-order chromatin structure in DNA damage repair.The activation of transcription of genes related to NLRP3 inflammatory pathway is inseparable from the enrichment of H3K9 Ac in the promoter region,which is a histone modification marker of transcriptional activation.GCN5 is the direct regulator of H3K9 Ac.Therefore,GCN5 is selected in this study to study the activation of genes related to NLRP3 pathway,leading to the development of inflammation.Chromatin remodeling is a typical step of all DNA related core cell processes,many of which determine the fate of key cells.By analyzing the interaction protein of GCN5 through string protein,it is found that smarca5 is also a chromatin remodeling factor,which regulates the opening and closing of chromatin.GCN5 / smarca5 jointly regulate the expression of NLRP3 related genes and affect the disease process.Smarca5 is an ATPase in ISWI chromatin remodeling.It is a key regulator of chromatin structure,cell cycle and DNA repair.It helps to reshape chromatin and promote the process of DNA repair.Synergistic activation between ATP dependent chromatin remodeling complex and GCN5 is necessary to promote DSB repair.However,the regulatory relationship between salidroside and GCN5 and smarca5 has not been reported.In this study,quantitative real-time polymerase chain reaction(q RT-PCR),Western blotting,enzyme linked immunosorbent assay(enzyme linked immunosorbent),immunoprecipitation,flow cytometry and cell transfection were used to detect the expression of inflammatory factors in serum and tear of diabetic retinopathy and the protective effect of salidroside on disease.The expression of GCN5 was detected in the blood of patients with proliferative diabetic retinopathy and salidroside treatment group.At the cellular level,the ARPE-19 cell line was pretreated with salidroside to observe the expression of GCN5 and the changes of ARPE-19 cell proliferation,cell cycle and inflammation.The expression of GCN5 was down regulated in ARPE-19 cell line induced by high glucose.The changes of ARPE-19 cell proliferation,cell cycle,apoptosis,inflammation and oxidative stress were observed.The interaction between GCN5 and smarca5 was verified by String database combined with immune-coprecipitation.The expression of smarca5 was down regulated in ARPE-19 cell line induced by high glucose.The changes of ARPE-19 cell proliferation,cell cycle,apoptosis,inflammation and oxidative stress were observed.Finally,through the recovery test,the changes of apoptosis,oxidative stress and inflammation in the ARPE-19 cells of diabetic retinopathy were confirmed by GCN5 and SMARCA5,and the AKT/m TOR pathway was explored.The protective effect and mechanism of salidroside on diabetic retinopathy induced by ROS/ NLRP3 were discussed.Part I The protective effect of salidroside on diabetic retinopathyObjective To investigate the expression of inflammatory factors in serum and tear and the protective effects of salidroside on diabetic retinopathy.Methods Blood and tears of 28 patients with proliferative diabetic retinopathy in our hospital from June 2016 to June-2019 were collected.Blood and tears were collected from 28 healthy persons during the same period as control group.Blood and tear samples were detected in IL-1βand IL-18 and the expression of GCN5 in blood samples.A diabetic rat model was established,and salidroside was intervened.ELISA was used to detect IL-1β and IL-18 expression changes in the blood of rats.The expression of NLRP3 in rat retina was detected by q RT-PCR and Western blotting.The expression of ROS in rat serum was detected using ELISA.Rat serum was purified,and human retinal microvascular endothelial s cells(h RMEC cells)were cultured.The m RNA and protein changes of NLRP3 expression were detected using q RT-PCR and Western blotting and proliferative activity was detected using CCK8 method.Results Compared with the control group,IL-1β and IL-18 protein in blood and tears of diabetic retinopathy patients were significantly upregulated.IL-1β and IL-18 protein in the blood of rats with diabetes mellitus were upregulated,ROS activity was increased,and the expression of NLRP3 in retinal tissue was up-regulated,too.Salidroside could reduce IL-1β and IL-18 expression in rat model blood,ROS activity and NLRP3 expression in retinal tissues.The serum culture of diabetic rats can increase the expression of NLRP3 in h RMECs cells and decrease the proliferative activity of cells.The serum level of salidroside intervened in rats can reduce the expression of NLRP3 and the proliferative activity of h RMECs cells to some extent.Conclusion IL-1β and IL-18 in patients with diabetic retinopathy weresignificantly up-regulated,and this up-regulation may be related to ROS /NLRP3 activation.Salidroside can reduce the expression of IL-1β and IL-18 in blood by reducing ROS / NLRP3 axis and GCN5 m RNA,in order to alleviate the inhibition of retinal cell proliferation.Part II Salidroside regulates the inflammation,apoptosis and oxidative stress of ARPE-19 cells under high glucose environment through GCN5Objective To establish a high glucose ARPE-19 cell model with glucose,and to explore the effects of GCN5 on inflammation,apoptosis and oxidative stress of ARPE-19 cells under high glucose environment,as well as the regulatory effect of salidroside on GCN5 and its protective effect on ARPE-19 cells.Methods ARPE-19 cells were randomly divided into three groups:(1)control group: cells were cultured in medium containing 5.5mmol/l glucose for24h;(2)High glucose model group(model group): cells were cultured in medium containing 50 mmol / L glucose for 24 hours;(3)Salidroside treatment group(SAL group): cells were cultured in medium containing 50 mmol / L glucose + 500 μ Mol / L Sal for 24 h.The expression of GCN5 in ARPE-19 cells was detected by q RT-PCR.The activity of ARPE-19 cells was detected by CCK8 method.Cell cycle and apoptosis were detected by flow cytometry.The level of ROS was detected by chemical fluorescence method(DCFH-DA probe).The expression of related proteins in ARPE-19 cell line was detected by Western blotting;Changes of IL-18 and IL-1β content and GSH level in cell supernatant was detected by ELISA.Results Salidroside decreased IL-1β and IL-18 expression in the supernatant of ARPE-19 cells,increased the proliferation activity of ARPE-19 cells.Salidroside decreased the m RNA expression of GCN5 in human retinal epithelial cell ARPE-19.Furthermore,downregulation of GCN5 using si RNA inhibited the proliferation,apoptosis,inflammatory response and oxidative stress of ARPE-19 cells in high glucose condition.Conclusion Salidroside reduces the expression of GCN5 in human retinal epithelial cell ARPE-19,and then regulates the proliferation,apoptosis,inflammatory response and oxidative stress of ARPE-19 cells induced by high glucose.Part III The mechanism of salidroside in regulating apoptosis and oxidative stress of ARPE-19 cells through GCN5Objective The specific mechanism of salidroside in regulating inflammation,apoptosis and oxidative stress of ARPE-19 cells in high glucose environment through GCN5 is unclear.Methods ARPE-19 cells in normal culture were randomly divided into groups,and the activity of ARPE-19 cells was detected by CCK8 method.The interaction protein with GCN5 was found in string database,and immune-coprecipitation proved that there was interaction between them.Annexin Ⅴ-FITC / PI was used to detect apoptosis.The level of ROS was detected by DCFH-DA probe.The changes of Akt / m TOR signaling pathway in ARPE-19 cell line were detected by Western blotting.Results There was interaction between GCN5 and SMARCA5 in cells.After down-regulated GCN5 and SMARCA5 in ARPE-19 cells,the protein changes of Akt / m TOR signaling pathway were inhibited.Furthermore,down regulation of SMARCA5 inhibited the proliferation,apoptosis and oxidative stress of ARPE-19 cells with high glucose.Salidroside regulates GCN5,SMARCA5 and Akt / m TOR signaling pathways.Salidroside regulates apoptosis and oxidative stress of ARPE-19 cells and Akt / m TOR signaling pathway dependent on GCN5 and SMARCA5.Conclusion Salidroside regulates apoptosis,oxidative stress and Akt /m TOR signaling pathway depending on GCN5 and SMARCA5.The above results shows that salidroside has an obvious protective effect on ARPE-19 cells induced by high glucose,which is closely related to its inhibition of cell inflammation,proliferation,apoptosis and oxidative stress. |
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