P2X7R/NLRP3 Mediate Anti-retinal Light Damage Mechanism Of Berberine

ObjectiveOn the basis of the previous research of our group,the therapeutic effect of berberine on retinal photodamage was further studied.Whether the purinergic signaling P2X7R was an important target of berberine in improving retinal photodamage was further revealed.Mechanism of action of berberine against retinal photodamage.Moreover,from the P2X7R/NLRP3 signaling pathway,the mechanism of berberine in anti-retinal photodamage was revealed.MethodsPart OneThe C57BL/6J mice were divided into the normal(Ctrl)group,the model(LD)group.The LD group was divided into LD 1d,LD 3d,LD 5d,LD 7d group according to different light times(1 day,3 days,5 days,7 days).Retinal photodamage was induced in mice by 30k lux white LED cold light(4h daily irradiation),The morphology of mouse retina was detected by HE staining.After 1d,3d,5d,and 7d of light irradiation,the optimal light time was selected according to the retinal morphology.According to this light time,the cell damage and inflammatory response after retinal light damage were further evaluated.TUNEL was used to detect cell apoptosis,and Western Blot was used to detect the expression of inflammatory factors(TNF-α,IL-1β).The expression of GFAP in the retina were detected by immunofluorescence.C57BL/6J mice were divided into normal(Ctrl)group,model(LD)group,PBS gavage(PBS)group,and berberine gavage(BBR)group.Except for the normal group,the other three groups were subjected to light stimulation according to the lighting conditions determined in the above experiments.HE and TUNLEL were used to detect retinal morphology and apoptosis,Western Blot to detect the expression of TNF-α and IL-1β,and immunofluorescence to detect the expression of GFAP,NeuN and c-Fos in the retina.Part TwoThe C57BL/6J mice were divided into Ctrl group,LD group,PBS group and BBR group.Except for the normal group,the other three groups were all illuminated.Western Blot and immunofluorescence were used to detect the protein expression of P2X7R,and to detect the co-labeling of GFAP/P2X7 and NeuN/P2X7.The expression of P2X7R protein in the retina in the PBS and BBR groups was detected one week after the end of light exposure,and the dynamic change trend of berberine in the retinal light damage model was evaluated.Part ThereC57BL/6J mice were divided into Ctrl group,PBS group,BBR group,A438079 group,BzATP group,and BzATP+BBR group.PBS,BzATP and A438079 were all administered by intravitreal injection.The C57BL/6J background mice were combined with the P2X7R gene knockout technology for breeding.Wild-type and gene-knockout mice were selected for grouping:wild-type control(WT-Ctrl)group,wild-type model(WT-LD)group,wild-type drug(WT-BBR)group,gene knockout model(P2X7 KOLD)group,gene knockout drug(P2X7 KO-BBR)group.All groups except the Ctrl and WT-Ctrl groups were illuminated.Western Blot was used to detect the expression of P2X7R protein,and HE and TUNEL were used to detect retinal morphology and cell apoptosis to further verify whether berberine played an anti-retinal photodamage effect by inhibiting the level of P2X7R in the retina.Part FourC57BL/6J mice were divided into Ctrl group,LD group,PBS group and BBR group.Mice with C57BL/6J background combined with P2X7R gene knockout technology were divided into:WT-Ctrl group,WT-LD group,WT-BBR group,P2X7 KO-LD group,and P2X7 KO-BBR group.All groups except the Ctrl and WT-Ctrl groups were illuminated.Western blot and immunohistochemistry were used to detect the levels of NLRP3/IL-1β pathway-related proteins,and to explore whether NLRP3 pathway-related signaling molecules mediate retinal photodamage.In the case of P2X7R gene knockout,the correlation between P2X7R and NLRP3 in retinal photodamage model and the regulatory effect of berberine on P2X7/NLRP3 were investigated.ResultsIn the first part,under the illumination condition of white LED cold light(30k lux,4h),the damaged cells in the outer nuclear layer of the retina of mice in the LD 7d group were most serious.Compared with the Ctrl group,the retinal outer nuclear layer of the LD group was significantly thinner,and the expressions of TNF-α,IL-1β and GFAP were enhanced,indicating that the retinal light damage model was successfully established.Compared with the Ctrl group,the outer nuclear layer of the retina in the PBS group was thinner,the number of positive cells apoptotic and the expression of cFos were increased,the expressions of TNF-α,IL-1β and GFAP were increased,and the expression of NeuN was decreased,and the differences were statistically significant(p<0.05).There was no statistical difference between LD and PBS group.Compared with PBS group,BBR group increased retinal inner and outer nuclear layer thickness,decreased positive cell apoptosis and c-Fos expression,decreased TNF-α,IL-1β and GFAP expression,and increased NeuN expression,the difference was statistically significant(p<0.05).In the second part,the P2X7R protein level in the retina of the LD and PBS groups was higher than that of the Ctrl group.Compared with the LD and PBS groups,the P2X7R protein level in the retina of the BBR group was decreased,and the difference was statistically significant(p<0.05).Compared with the Ctrl group,the fluorescence co-labeling of GFAP/P2X7 and NeuN/P2X7 in the retina of the LD and PBS groups increased.Compared with the LD and PBS groups,the fluorescence co-labeling of GFAP/P2X7 and NeuN/P2X7 in the BBR group decreased.One week after the end of the light exposure,the P2X7R protein levels in the retinas of the mice in the PBS and BBR groups were close to those in the Ctrl group,and there was no significant difference between the groups(p>0.05).The third part,BzATP increases the expression of P2X7R in the retina,reduces the thickness of the outer nuclear layer,and increases apoptosis,while P2X7R antagonists and BBR inhibit the expression of P2X7R in the retina,increase the thickness of the outer nuclear layer,and reduce apoptosis.Compared with the WT-LD group,the P2X7 KO-LD,P2X7 KO-BBR and WT-BBR groups had significantly increased retinal outer nuclear layer thickness and decreased cell apoptosis,and the differences were statistically significant(p<0.05).Compared with other groups,the thickness of the outer nuclear layer of the retina in the WT-BBR group was decreased,and the apoptosis was more,and the difference was statistically significant(p<0.05).The fourth part,compared with the Ctrl group,the levels of NLRP3/IL-1β-related proteins in the retina of the mice in the LD and PBS groups increased.Compared with the WT-Ctrl group,the expression of NLRP3,IL-1β and Caspase-1 proteins in the retina of the WT-LD group increased.Compared with the WT-LD group,the expressions of NLRP3,IL-1β,and Caspase-1 proteins in the retina of the P2X7R KO-LD and P2X7 KO-BBR groups were decreased,and the difference was statistically significant(p<0.05).Conclusions1.After intragastric administration,berberine can alleviate retinal light damage in AMD model mice,protect the morphological structure of the outer nuclear layer of the retina,and reduce neuronal cell loss and apoptosis rate.2.Berberine can inhibit the release of inflammatory factors TNF-α and IL-1β in the retina after light damage,and inhibit the proliferation of astrocytes.3.In the process of retinal photodamage,the purinergic signaling P2X7R is overexpressed,which is closely related to photoreceptor and neuron cell damage in the retinal photodamage model.Berberine can down-regulate the level of P2X7R protein in the retina and inhibit the excessive activation of P2X7R,thereby inhibiting retinal damage and prevent the development of AMD.4.During retinal light damage,P2X7R is closely related to NLRP3/IL-1β.Berberine targets P2X7R,reduces the level of NLRP3/IL-1β-related proteins,and inhibits the inflammatory response caused by P2X7R activation.The retinal photodamage effect depends on the P2X7/NLRP3 pathway to a certain extent.