Effects Of Dendrobium Chrysotoxum And Erianin On PERK-eIF2α-CHOP Pathway In Diabetic Retinopathy

Objective:This study aims to observe the influence of Ethanol extract of Dendrobium chrysotoxum（EDC）and its Bioactive ingredient Erianin on diabetic retinopathy by DR model or high glucose induced RF/6A cells in vitro and in vivo models.The engaged the mechanism were further studied by investigating of EDC and Erianin on diabetic retinopathy from the endoplasmic reticulum stress PERK-eIF2α-CHOP pathway.Methods:Experiment in vivo:STZ-induced SD rats were established diabetic model by random number table divided into:Control group,Diabetes group,Diabetes+EDC low dose group[100 mg·kg^-1·d^-1],Diabetic+EDC high-dose group[300mg·kg^-1·d^-1].After 8 weeks of STZ injection,EDC were given interventions.HE staining to observe the retinal tissue structure of each groups.The protein expression of PERK-eIF2α-CHOP pathway induced by endoplasmic reticulum stress in rat retina was detected by Western-Blot.The effect of EDC on the expression of CHOP in rat retinal was observed by immunohistochemistry.TUNEL staining was used to detect retinal cell apoptosis in each group.Experiment in vitro:RF/6A cells cultured in vitro,high glucose induced endoplasmic reticulum stress.RF/6A cells were cultured 24h,48h,72h,CCK-8 kit was used to detect the proliferation of RF/6A cells,the concentration of 0,20,40,60,80,100nM concentrations of erianin were used to determine the optimal drug concentration.According to the results,they are divided into four groups for follow-up experiments.Western Blot method was used to observe the changes of PERK-eIF2α-CHOP pathway protein induced by ER stress in RF/6A cells.Real-time PCR assay was used to detect the effect of erigeron on PERK-eIF2α-CHOP pathway gene expression induced by ER stress in RF/6A cells.Immunofluorescence staining was used to observe the changes of CHOP expression in RF/6A cells.Results:Experiment in vivo:HE staining showed that compared with the Control group,the inner limiting membrane of the retina was incomplete in DM group,,the boundaries of inner and outer nuclear layers were fuzzy,the arrangement of the cells was disordered,and the nerve fiber layer was obviously swollen.While the inner and outer nuclear layer boundaries of different doses of EDC groups clearer than the DM group,the swelling of the nerve fiber layer was improved.Western-Blot results showed that compared with Control group,the protein expression of P-PERK,P-eIF2αin retinal of DM group were increased.Compared with DM group,both of LEDC and HEDC group could significantly reduce the protein expression（both P<0.05）.Immunohistochemistry showed that there was no significant positive expression of CHOP in the Control group,while DM group was significantly expressed in the nerve fiber layer,the inner nuclear layer and the pigment epithelium.LEDC,HEDC group expressed a small amount of nerve fiber layer,pigment epithelium.The TUNEL method was used to observe the positive expression of apoptosis in DM group,while the different dose of EDC group could improve the expression of apoptosis.Experiment in vitro:The experimental results of CCK-8 showed that under the action of 48h,the cells showed higher proliferation;at different concentrations of Erianin at 48h in RF/6A cells,at 40nM have the best cell proliferation.They were divided into NC group（5mM glucose）,Mannitol group（20mM mannitol）,HG group（30mM glucose）,Erianin group（30mM glucose+40nM Erianin）.Western-Blot results showed that compared with NC group,the protein expressions of P-PERK,P-eIF2αin RF/6A cells of HG group are increased.However,theErianin group significantly inhibited the signaling pathway protein expression.Real-Time PCR results showed that the gene expression of ATF4、CHOP in RF/6A cells was significantly up-regulated under high glucose conditions.While Erianin inhibits the activation of the PERK-eIF2α-CHOP pathway in high glucose conditions.The result of immunofluorescence showed that CHOP was obviously expressed in RF/6A cells under high glucose condition,and the expression of CHOP was significantly decreased under the intervention of Erianin.Conclusion:1.Ethanol extract of Dendrobium chrysotoxum could the ER stress-mediated PERK-eIF2α-CHOP pathway.To improve the amelioration on DR.2.Further Results showed that Erianin improved the mechanism of DR and found that Erianin could effectively reduce the PERK-eIF2α-CHOP pathway activation.