The Effects And Retinal Remodeling Mechanisms Of Shihu Yeguang Pill On Light-induced Photoreceptor Degeneration

Objectives: This study investigated photoreceptor protective effect and associated retinal remodeling mechanisms of Shihu Yeguang Pill(SYP)to provide experimental and theoretical evidence for the clinical application of SYP in the treatment of retinal degenerative disorders.Methods: 1.BALB/c mice(6 weeks)were randomly divided into normal controls and light-exposed mice.The retinal structure and function were analyzed by optical coherence tomography(OCT)and electroretinogram(ERG)to determine the condition of forming model.2.Bright light-exposed BALB/c mice were treated with vehicle or SYP.Age-matched BALB/c mice without light exposure received vehicle treatment and served as normal controls.The retinal structure and function were analyzed by OCT,immunohistochemistry(IHC)and ERG.3.The expression of genes implicated in the regulation of apoptosis including c-Fos,c-Jun and Bcl2 was analyzed by real-time PCR.TUNEL assay was performed for in situ analysis of retinal cell apoptosis.4.The content of cyclic adenosine monophosphate(c AMP)in the retina was evaluated using an immunocompetitive fluorescence kit.5.IHC was carried out to label retinal bipolar cells,horizontal cells,Müller cells and microglia.The expression level of glial fibrillary acidic protein(GFAP)and tumor necrosis factor-alpha(TNF-α)was analyzed by real-time PCR.Results: 1.OCT and ERG analyses revealed that the disordered retinal retinal structure and the significantly depressed amplitude of electroretinogram(P<0.05)of bright light exposure compared to that from vehicle-treated mice unexposed to light.Modeling of retinal degenerative disorder induced by bright light was successful.2.OCT imaging revealed that compared to the intact retinal structure detected in vehicle-treated mice unexposed to light,markedly diminished outer nuclear layer(ONL)was readily detected in vehicle-treated light-exposed mice.In distinct contrast,significant preservation of ONL was observed in SYP-treated light-exposed mice.ONL thickness measurement revealed that compared to that from vehicle-treated mice unexposed to light,ONL thickness was significantly decreased in vehicle-treated light-exposed mice.However,SYP treatment resulted in significantly increased ONL thickness in light-exposed mice compared to that from vehicle-treated light-exposed mice.IHC examination revealed that compared to that from vehicle-treated mice unexposed to light,the immunopositivity of Rhodopsin and M-opsin was remarkably diminished in vehicle-treated light-exposed mice.In contrast,the immunopositivity of Rhodopsin and M-opsin was evident in SYP-treated light-exposed mice.ERG assessment revealed that the amplitude of electroretinogram in normal control was elevated with the increase of light stimulation,whereas the amplitude was significantly decreased in vehicle-treated light-exposed mice(P<0.05).Compared to that from vehicle-treated light-exposed mice,significantly increased of ERG amplitude was observed in SYP-treated light-exposed mice(P<0.05).3.Real-time PCR analyses showed that compared to that from vehicle-treated mice unexposed to light,the retinal expression of c-Fos and c-Jun was significantly up-regulated(P<0.05).Meanwhile,the retinal expression of Bcl2 was significantly decreased(P<0.05)in the vehicle-treated light-exposed mice.SYP treatment resulted in significantly decreased retinal expression of c-Fos and c-Jun(P<0.05)and increased expression of Bcl2 in the retina(P<0.05).TUNEL assay revealed that there was no TUNEL positive staining in the ONL of vehicle-treated mice unexposed to light.However,massive TUNEL positivity was observed in the ONL of light-exposed vehicle-treated mice.SYP treatment partially mitigated TUNEL positivity in the retina.4.The measurement of retinal c AMP content showed that compared to that from vehicle-treated mice unexposed to the light,the retinal content of c AMP was significantly elevated in light-exposed vehicle-treated mice(P<0.05).In contrast,the retinal content of c AMP was significantly decreased in SYP-treated light-exposed mice(P<0.05).5.IHC evaluation showed that the immunopositivity of markers labeling bipolar cells and horizontal cells was significantly weakened in the retina of light-exposed vehicle-treated mice compared to that from the vehicle-treated mice unexposed to light.However,compared to that from light-exposed vehicle-treated mice,the immunopositivity of aforementioned neuronal markers was evidently enhanced in the retinas of SYP-treated light-exposed mice.Meanwhile,compared to that from the vehicle-treated mice unexposed to light,exaggerated immunopositivity of GFAP and Iba1 was observed in the retinas of light-exposed vehicle-treated mice,indicating reactive gliosis of Müller cells and activation of microglia.In contrast,the immunopositivity of GFAP and Iba1 was evidently decreased as a result of SYP treatment.Real-time PCR analyses showed that compared to that from the vehicle-treated mice unexposed to light,the m RNA expression of GFAP and TNF-α was significantly increased in the retinas of light-exposed vehicle-treated mice(P<0.05).However,decreased retinal expression of GFAP and TNF-α was observed in SYP-treated light-exposed mice compared to that from light-exposed vehicle-treated mice(P<0.05).Conclusions: 1.SYP treatment significantly protects against bright light-induced structural and functional degeneration of photoreceptor cells.2.The protective effects of SYP on photoreceptor cells might be associated with the following mechanisms: inhibiting genes expression of c-Fos and c-Jun,promoting the expression of Bcl2,which resisting photoreceptor cell apoptosis partly;modulating the level of Gs and/or Gi-coupled GPCR signaling under photo-oxidative stress conditions thereby reducing the retinal content of c AMP;promoting reparative retinal remodeling during photoreceptor cell degeneration.