| Background:The retina is one of the most oxygen-consuming tissues in the body,where retinal oxygen balance is essential for retinal homeostasis.Recent studies had found that disruption of oxidative balance in the retina might lead to further progress in the loss of photoreceptor cells.Hydrogen sulfide?H2S?,as a small gas signaling molecule,could regulate the body's antioxidant and anti-inflammatory abilities through the Nrf2 pathway,while the use of H2S as a retina protective agent had few studies,and no specific mechanism of using H2S to play a protective role through the Nrf2 pathway had been reported.Objective:Based on the existing literature and the findings of previous work in our laboratory,relevant studies had been carried out on whether using H2S could protect retinal cells from oxidative damage,involved in the Nrf2 pathway as a signal molecule target and what is its mechanism of action.Methods:1.In vivo model:we chose Sprague-Dawley?SD?rats as the research object,by intraperitoneal injection of N-methyl-N-nitrosourea?MNU?and Sodium hydrosulfide hydrate?NaHS?to deal with rats.Eyes,kidneys and liver were tested by HE staining and microscope injury,then the retina injury animal models were constructed,which were examined the protective effects of H2S on retinal damage in the body;We detected changes in MDA and SOD in vivo using MDA and SOD detection kits,and detected the changes of IL-1?and TNF-?inflammatory factors in vivo by ELISA;Finally,the expression of Nrf2 protein was detected by immunohistochemistry.2.In vitro model:human retinal pigment epithelial ARPE-19cells were selected as the research object,and hydrogen peroxide?H2O2?and sodium hydrogen sulfide?NaHS?to build a cell model of retinal damage,which were screened the appropriate concentration of retinal damage and protection by MTT method,and used annexin V/PI staining to detect the effect of retinal cell apoptosis by flow cytometry,in addition to DCFH probes?ROS probes?were used to detect the effects of changes in ROS levels by flow cytometry,ELISA was used to detect the effects of changes in inflammatory factors,and q-PCR technology was used to initially explore the changes in Nrf2 mRNA,and Western blot was used to detect the Nrf2 protein expression;Finally,ML385?Nrf2 inhibitor?was used to test the protective effect induced by H2S.Results:1.In the rat retinal damage model constructed by MNU,the results can be found to reduce the thinning of the MNU-induced retinal thinning by H2S through HE staining,indicating that H2S reduced the death of retinal photoreceptor cells induced by MNU,and H2S and MNU have no obvious effect on liver and kidney,showing the rationality of modeling;2.H2S inhibited the increase of MDA level and SOD level in rat retina induced by MNU through the kit,indicating that H2S can inhibit MNU-induced the increase of the oxidation level;3.H2S can reduce the production of TNF-?and IL-1?in the rat retina induced by MNU through ELISA test,indicating that H2S can reduce the production of MNU-induced inflammation;4.It can be seen that H2S can increase the expression of Nrf2 protein and enhance the antioxidant capacity of the rat retina by immunohistochemistry;5.In the ARPE-19cell damage model constructed by H2O2,it was found that H2S decreased the cell survival rate of ARPE-19 treated with H2O2 by MTT test,and H2S reduced apoptosis rate of ARPE-19 treated with H2O2 by Annexin V/PI staining;6.H2S reduced the ROS level of ARPE-19 cells treated with H2O2 by DCFH probe,and H2S decreased the SOD level of ARPE-19 cells treated with H2O2 through kit test,indicating that H2S effectively reduced oxidative damage to retinal cells caused by H2O2;7.H2S can reduce the increase of inflammatory factors?IL-1?and TNF-??caused by H2O2through ELISA test,and reduced the inflammation of retinal cells;8.It was found that H2O2 will inhibit the Nrf2 mRNA expression by q-PCR,and H2S will release the inhibition of H2O2,and H2O2 will inhibit the expression of Nrf2 protein,and H2S can increase the expression of Nrf2 protein by Western Blot detection,thereby increasing the antioxidant capacity of retinal cells.9.Finally,the role of Nrf2 in the protection of retinal cells in H2S was detected by using ML385 inhibitors,and it was found that Nrf2 was involved in H2S to reduce H2O2-induced apoptosis,inflammation and increased ROS levels.Significance:This study not only confirmed that H2S could protect the retina from oxidative damage,but also this protective effect could enhance the antioxidant capacity of the retina through the Nrf2 pathway.Therefore,the results of this study laid establish a theoretical basis for H2S as a prevention and treatment of retinitis pigmentosa,and provide a certain scheme for the application of H2S as an antioxidant in clinical protection and treatment of retinal diseases. |
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