| PurposeTo establish a high glucose model of rat retinal Müller cells in vitro,and to explore the effect of HSP90 on the activity of Müller cells in high glucose environment and its mechanism.MethodsPrimary rat retinal Müller cells were cultured with 105mmol/L glucose to simulate high glucose environment in vitro.The cells were divided into four groups:control group(105mmol/L glucose),no-carrier virus group(Adeno-associated virus transfected cells without HSP90 gene+105mmol/L glucose),HSP90 overexpression group(Adeno-associated virus transfected cells carrying HSP90 gene+105mmol/L glucose)and HSP90 inhibition group(0.1 μ mol/L 17-AAG+105mmol/L glucose).The proliferation activity of Müller cells were detected by CCK8 and EdU methods;The cell cycle ratio was determined by flow cytometry;The expression of RIPK1,RIPK3,MLKL in necroptosis and the key proteins in proliferation signal pathway NF-κB p65、p-NF-κB p65、p38MAPK、p-p38 MAPK were detected by Western Blot;The ubiquitin level of RIP1,the key factor regulating proliferation pathway,were detected by co-immunoprecipitation(Co-IP).ResultCompared with the control group,the results of CCK8 and EdU showed that the proliferation activity of Müller cells decreased in HSP90 overexpression group and increased in HSP90 inhibition group;The cell cycle results showed that the proportion of G2 phase decreased and G1 phase increased in HSP90 overexpression group,S phase and G2 phase increased in HSP90 inhibition group;Western Blot results showed that the expression of RIPK1,RIPK3,MLKL protein increased in HSP90 overexpression group,and NF-κB p65,NF-κB p65,p38 MAPK and p-p38 MAPK were decreased in HSP90 overexpression group,while the expression of RIPK1,RIPK3 and MLKL decreased in HSP90 inhibition group,the expression of NF-κB p65,NF-κB p65,p38 MAPK and p-p38 MAPK increased in HSP90 inhibition group.Co-IP results showed that the ubiquitination level of RIP1 was decreased in the HSP90 overexpression group and increased in the HSP90 repression group.Compared with the control group,there was no significant difference in all detection indexes between the no-carrier virus group and the control group.ConclusionIn a high glucose environment,overexpression of HSP90 may promote apoptosis by activating the Müller cell necroptosis and inhibiting the cell proliferation signaling pathway.Conversely,inhibition of HSP90 may promote cell proliferation by inhibiting the necroptosis and activating the cell proliferation signaling pathway,and the underlying mechanism may be related to the regulation of RIP1 ubiquitination levels by HSP90. |
