| Diabetic retinopathy(DR)is a common and specific microvascular complication of diabetes that can cause irreversible damage to a patient’s vision.The pathogenesis of DR is complex,and abnormal microvasculature,neurons,and neuroglial cells in the retina can cause disruption of retinal function and homeostasis.In particular,the neuroinflammatory response is a key participant in the disruption of the intraocular environment in DR,but the exact underlying molecular mechanisms are not fully understood.Müller cells are one of the major neuroglial cells in the retina and play an important role in maintaining normal retinal function.In DR,the proliferation of Müller cells and reactive gliosis are characterized by an increase in the expression of glial fibrillary acidic protein(GFAP)during the progression of diabetes,the increased secretion of inflammatory factors confirmed that Müller cells were involved in the inflammatory process.IL-22 belongs to the IL-10 family and plays an important role in inflammation.Studies have shown that IL-22 acts as an inflammatory cytokine promoting the inflammatory response of experimental autoimmune uveitis(EAU)in the early stages of the disease.Meanwhile,it has been found that IL-22 levels are elevated in the serum of diabetic patients.In addition,it was recently found that IL-22 is a new inflammatory factor secreted by Müller cells,but the source of IL-22 in DR and whether it is involved in the disease process remains unclear.Retinal ganglion cells(RGCs)aggregate at the optic disc to form the optic nerve,which is the only neuron that encodes visual information from light energy into membrane potential and transmits it to the brain.Under DR conditions,RGCs are susceptible to damage by inflammatory cytokines,leading to the apoptosis of metabolically active RGCs,which may be an important factor in the development of DR.However,it is not entirely clear how Müller cells affect RGCs through inflammatory responses in DR.Salidroside(SAL)is the main medicinal ingredient of Rhodiola,which has strong antioxidant,anti-inflammatory and neuroprotective functions.Recent studies have shown that SAL has a positive effect on the treatment of db/db diabetes mice,and confirmed that SAL can show hypoglycemic and hypolipidemic activities in type 2 diabetes,and alleviate insulin resistance.The research team also confirmed that SAL can improve the cognitive dysfunction caused by diabetes.However,it is unclear whether SAL has a protective effect on DR.Therefore,this experiment aims to explore and verify the following hypotheses in three parts: 1.The protective effect of SAL on retinopathy in diabetes mice and its effect on activation of Müller cells;2.Whether Müller cells secrete IL-22 under diabetic conditions and its effect on RGC apoptosis;3.Whether SAL inhibits RGC apoptosis by suppressing the secretion of IL-22 by Müller cells.Purpose: The aim of this study is to investigate the inhibitory effects of SAL on inflammation in Müller cells and RGCs apoptosis in the retina of diabetic mice at both the in vivo and in vitro levels,as well as to elucidate the underlying molecular mechanisms of its protective effects in DR.This will provide a theoretical basis and experimental scientific evidence for further research on DR.The study will involve a series of morphological and molecular biology analyses.Methods:Part Ⅰ: Establishment of mouse DM model by intraperitoneal injection of streptozotocin(STZ).80 C57BL/6J mice were randomly divided into 4 groups according to digital table method: control group(without any treatment),DM group(intraperitoneal injection of STZ),SAL group(gavage with SAL),sham group(gavage with physiological saline),with 20 mice in each group.Changes in retinal morphology were observed through fundus fluorescence angiography(FFA),optical coherence tomography(OCT),and HE staining;the activation of Müller cells in the retina was detected through immunohistochemistry,Western blotting,and ELISA.Part Ⅱ: 80 C57BL/6J mice were randomly divided into 4 groups according to digital table method: control group,DM group,IL-22 BP group(intravitreal injection of IL-22 BP in diabetic mouse model),sham group(intravitreal injection of physiological saline in diabetic mouse model),with 20 mice in each group.The localization and source of IL-22 were detected through immunofluorescence and ELISA;the expression of IL-22-related factors was detected through Western blotting and q RT-PCR;downstream molecules of IL-22 were predicted using bioinformatics and molecular docking technology;the effect of Müller cells on RGCs was observed through Transwell co-culture under the same environment.Part Ⅲ: Müller cells were treated with high glucose,and different treatment groups were divided as follows:(1)the effect of SAL on retinal Müller cell activation: divided into control group(complete DMEM),mannose group(complete DMEM containing 50mmol/L mannose),HG group(complete DMEM containing 50 mmol/L glucose),1 μM SAL group(HG group containing 1 μM SAL),10 μM SAL group(HG group containing10 μM SAL),and 100 μM SAL group(HG group containing 100 μM SAL).(2)The effect of SAL on RGCs through Müller cells: divided into control group(complete DMEM),mannose group(complete DMEM containing 50 mmol/L mannose),HG group(complete DMEM containing 50 mmol/L glucose),SAL+r IL-22 group(HG group containing recombinant IL-22 protein and 10 μM SAL),SAL group(HG group containing 10 μM SAL),Colivelin group(HG group containing IL-22 BP and Colivelin,which is a STAT3 activator),and IL-22 BP group(HG group containing IL-22BP).The effect of different concentrations of SAL on Müller cell activation was validated through CCK-8 and immunofluorescence;RGCs apoptosis was detected through TUNEL staining,flow cytometry,and Western blotting;the binding situation between SAL and IL-22 was validated through molecular docking;the effect of Müller cells treated with r IL-22,IL-22 BP,and Colivelin on RGCs was observed through Transwell co-culture under the same environment.Results:Part Ⅰ: After 12 weeks of diabetes induction,OCT,FFA,and HE staining showed that compared to the control group(P < 0.001),the DM group had a decreased retinal thickness,retinal leakage,neovascularization,and a significant decrease in RGCs(P <0.05).After SAL intervention,retinal thickness increased,retinal leakage and neovascularization were alleviated,and RGCs significantly increased(P < 0.01).Immunohistochemistry and Western blotting results showed that compared to the control group,GFAP expression in the retina of DM mice increased significantly(P < 0.01).After SAL intervention,GFAP expression decreased significantly(P < 0.05).ELISA results showed that compared to the control group,IL-1β,IL-6,and TNF-α levels in the retina of DM mice significantly increased(P < 0.001),while after SAL intervention,these levels significantly decreased(P < 0.01).There was no significant difference between the sham group and DM group(P > 0.05).Part Ⅱ: Immunofluorescence and ELISA results showed that IL-22 was expressed in Müller cells and its secretion significantly increased in high glucose-treated Müller cells(P < 0.01).IL-22Rα1 was co-expressed with RGCs.Western blotting results showed that compared to the control group,IL-22 expression in the DM group significantly increased(P < 0.001),while after SAL treatment,IL-22 expression decreased significantly(P < 0.01).Under the action of IL-22 BP,Western blotting was used to detect p-STAT3 and c-caspase-3 protein expression.Compared to the control group,p-STAT3 and c-caspase-3 in the DM group significantly increased(P < 0.01),while after IL-22 BP treatment,the expression significantly decreased(P < 0.01).The in vitro experiments were consistent with the above results.Part Ⅲ: Under the action of SAL,TUNEL staining was used to detect RGC apoptosis in each group of mice.TUNEL results showed that compared to the control group,the number of TUNEL-positive cells in the DM group significantly increased(P< 0.001),while after SAL intervention,the number of TUNEL-positive cells significantly decreased(P < 0.01).Western blotting and q RT-PCR were used to detect GFAP protein and gene expression in Müller cells.The results showed that compared to the HG group,GFAP expression in the 10 μM SAL group significantly decreased(P < 0.01).After intervention with r IL-22 and 10 μM SAL in the co-culture system,the protective effect of SAL was partially reversed.After intervention with IL-22 BP and Colivelin in the co-culture system,the protective effect of IL-22 BP was partially reversed.Conclusion:1.SAL can inhibit the activation of Müller cells in diabetic mice,alleviate retinal inflammation,increase retinal thickness and the number of RGCs.2.IL-22 secreted by Müller cells promotes apoptosis of RGCs in diabetes mice.3.SAL reduces the phosphorylation level of STAT3 in RGCs by inhibiting IL-22 secreted by Müller cells,thereby inhibiting the apoptosis of RGCs. |
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