Investigation Of RhoA/ROCK1 Signaling Pathway On The Mechanism And Therapy Of Diabetic Induced Retinal Vascular Endothelial Cell Dysfunction

Diabetic retinopathy(DR)is one of the most common microvascular complications of diabetes.Retinal endothelial cells(RECs)is exposed to high glucose environment for a long time,with increased inflammation and apoptosis and decreased cell activity,resulting in the destruction of inner blood-retinal barrier(iBRB).Microvascular leakage is enhanced,and a series of pathological changes such as microaneurysm,cotton wool spots and hard exudation,which leads to the decline of visual acuity,seriously affects the quality of life and increases the burden on families.RhoA/ROCK(Ras homolog gene family member a/Rho associated coiled coil containing protein kinase)and connexin43(Cx43)are ubiquitous in various tissues and organs of the human body,participate in a variety of key biological processes,and play a vital role in maintaining homeostasis of the homeostasis.However,their abnormal activation in diseases states will trigger a series of pathological changes and promote the development of diseases.This is the first time to prove the regulatory mechanism of RhoA/ROCK signaling on Cx43 in RECs and the protective effect of ROCK inhibitor on RECs,indicating that the activation of RhoA/ROCK signaling pathway under hyperglycemia breaks the barrier function of iBRB and destroy gap junction intercellular communication(GJIC).Meanwhile,it provides a theoretical basis for ROCK inhibitor to alleviate DR induced microvascular damages.Purpose:To investigate the molecular mechanism of destruction of REC and iBRB by hyperglycemia in diabetic retinopathy(DR),as well as the protective role of ROCK inhibitor,so as to provide theoretical basis for the early treatment of DR.Methods:1.To explore the effect and mechanism of high glucose on the damage of mRECs.Group:control group(5.5 mM D-glucose),high glucose group(30,60,90 mM D-glucose),Y-27632 treated group(10 μM),fasudil treated group(30μM),RhoA overexpression group(3μg).(1)control group:mRECs were treated in 5.5 mM D-glucose for 24h or 48h.(2)High glucose group:mRECs were treated with different concentration of glucose(30,60,90 mM D-glucose)for 24h or 4 8h.(3)Y-27632 group:mRECs were pretreated with Y-27632(10 μM)for 30 min and then treated with 30 mM D-glucose for 24h or 48h.(4)Fasudil group:mRECs were treated with 30 mM D-glucose and fasudil(30 μM)for 24h or 48h.(5)RhoA overexpression group:mRECs were treated in 5.5 mM D-glucose with RhoA overexpression plasmid 3μg for 24h or 48h.qRT-PCR and Western blot were used to detect the mRNA and protein expression of RhoA,ROCK1,ROCK2 and Cx43.ELISA was used to detect the release of inflammation cytokines TNF-α,IL-6、IL-1β in medium supernatant.Flowcytometry was used to detect the apoptosis of mRECs.CCK-8 assay was used to evaluate the cell viability of mRECs.Immunohistochemistry was used to observe the distribution of Cx43.2.To observe the intervention effect of ROCK inhibitors on early DR in vivo.Group:Control group,diabetes mellitus mouse model,intervention model.(1)Control group:healthy male C57BL/6 mice have no treatment.(2)Diabetes mellitus mouse model:8-week-old healthy male C57BL/6J mice were intraperitoneal injection of streptozotocin(STZ,50 mg/kg)-citric acid buffer for 5 days consecutively.7 days after the last STZ injection,mice with fasting blood glucose levels>16.7 mmol/L were regarded as diabetic state.(3)Intervention model:The diabetic mice were received fasudil(50mg/kg/qd)by intraperitoneal injection.qRT-PCR and Western blot were used to detect the mRNA and protein expression of RhoA,ROCK1,ROCK2 and Cx43.The expression of Cx43 in retina was detected by immunofluorescence staining.HE staining was used to compare the retinal thickness of each group.Retinal trypsin digestion and PAS staining were used to observe the number of endothelial cells and acellular capillaries.Evans blue leakage was used to evaluate the vascular permeability.Results:1.High glucose activates RhoA/ROCK1 signaling pathway to promote the development of diabetic retinopathy.In cell experiments,The expression of RhoA and ROCK1 was increased in mRECs treated with different concentrations of high glucose(p<0.05),while the ROCK2 expression was unchanged.The release of inflammatory factors TNF-α,IL-6 and IL-1β and cell apoptosis were significantly increased(p<0.05)and the cell viability was decrease(p<0.05).These data revealed that high glucose activated RhoA/ROCK1 signaling to promote the inflammatory response and apoptosis of mRECs.In diabetic mice retina,the expression of RhoA、ROCK1 and ROCK2 was consistent with that in mRECs(p<0.05).The decrease of retinal thickness(p<0.05)and the number of endothelial cells(p<0.05)and the increase of acellular vessels(p<0.05)and vascular leakage(p<0.05)suggested that high glucose can activate RhoA/ROCK1 signaling to aggravate diabetic retinopathy.2.RhoA/ROCK1 aggravates diabetic retinopathy by regulating the expression and location of Cx43.In high glucose or RhoA overexpression plasmid treated mRECs,the expression of Cx43 was significantly decreased(p<0.05).In the normal glucose group,Cx43 was mainly expressed in the plasma membrane between adjacent cells.In the high glucose group,Cx43 was mainly expressed in the cytoplasm.Compared to the high glucose group,the Y-27632 and fasudil groups showed enhanced Cx43 staining,especially in cell membrane.While the expression of Cx43 in diabetic mice retina was significantly increased(p<0.05).3.ROCK inhibitors significantly alleviated retinal dysfunctions.In vitro,the release of inflammatory cytokines TNF-α,IL-6 and IL-1β and cell apoptosis were significantly decreased after Y-27632 or fasudil treatment(p<0.05),and the cell viability was increased(p<0.05),which indicated that ROCK inhibitors can reduce the inflammatory response and apoptosis of REC induced by high glucose.In vivo,after 12 weeks of continuous injection of fasudil in diabetic mice,retinal thickness(p<0.05)and the number of retinal vascular endothelial cells was increased(p<0.05),while the number of acellular vessels(p<0.05)and vascular leakage was decreased(p<0.05).It is suggested that ROCK inhibitors could protect the blood-retinal barrier and alleviated diabetic retinopathy.Conclusions:Our results illustrated that high glucose could activate RhoA/ROCK signaling,which targets the expression and localization of Cx43 and is responsible for cell viability,apoptosis and inflammation,resulting in retinal endothelial cell injury and increased vascular permeability.ROCK inhibition significantly attenuated these detrimental effects induced by hyperglycemia.Therefore,our study provides clues to the pathogenesis of diabetic retinopathy and provides a theoretical basis for the discovery of potentially effective drugs to treat early diabetic retinopathy.