Effects Of P2X4 And TRPV1 Receptor On Berberine Protecting Against Retinal Light Degeneration

The study of this paper is divided into three parts:the pathological study of mouse retinal light degeneration,the mechanism of P2x4 receptor in BBR anti-retinal light degeneration and the effect of TRPV1 receptor on berberine-induced apoptosis in retinal light injury cells.Part A:The pathological study of mouse retinal light degenerationPurposes:In the early study found that berberine has anti-mouse retinal light damage effects.So we basede on pre-experimental model,analysis C57 mouse retinal light lesions after pathological changes.Methods:C57BL/6J mice were randomly divided into control group and LD group.LD group were given 10000 ± 1500 lux and 4 hours of light in a self-made light-damaged box.After 48 hours of modeling,the right eye was fixed in 4%paraformaldehyde fixative,and the left eye was removed and the retina was extracted.Hematoxylin-eosin(HE)staining was used to observe the thickness of retinal ONL layer,and the effect of light injury on retinal morphology was observed.In situ cell apoptosis(Tunel-POD)was used to detect the apoptosis of ONL layers in both groups.Result:1.The results showed that the retinal morphology of the control group was normal,the structure of the layers was clear:the nucleus of the ganglion was neatly arranged;the nuclear layer and the ONL were closely arranged and the nuclei were homogeneous and the nuclei were intact.The photoreceptor cells Inside and outside the section of the rules,clear boundaries.The nucleus of the nucleus of the nucleus of the dorsal nucleus of the LD group was narrowed and stained,and the nucleus was arranged closely and neatly.The nucleus of the outer nuclear layer was disturbed,and some nuclei were concentrated,aggregated and stained.The photoreceptor cells,As the bar outside the arrangement disorder,membrane disk part of the gap.LD group retinal ONL layer thinning,but no significant difference(P>0.05).2.The expression of ONL layer in the retina of mice was observed by TUNEL:no apoptosis was observed in the retina of the blank group.A large number of apoptotic cells appeared in the retinal tissue of the LD rats after 48 hours of light exposure,the difference was statistically significant P<0.05).Part B:The mechanism of P2x4 receptor in BBR anti-retinal light degeneration Purposes:Based on previous experiments,the mechanism of berberine anti-retinal light damage effects,was further revealed from the perspective of purine signal P2x4 receptor,which provided scientific basis for the treatment of retinal light injury caused by senile macular degeneration by clinical berberine.Methods:C57BL/6J mice were randomly divided into three groups:control group,LD group and BBR group,6 rats in each group.BBR group mice were given 200mg/Kg concentration of BBR solution,group and LD group given PBS every day.Seven days later,in addition to blank group,LD group and BBR group were given 10000 ±1500Lux,4 hours of light.After irradiation for 48 hours,the right eye was fixed in 4%paraformaldehyde fixative,and the left eye was removed and the retina was extracted to extract RNA.The expression of P2x4 in the retina of mice was detected by Q-PCR.The positive expression of P2x4 was detected by immunofluorescence double labeling in the ONL layer of mouse retina.Result:1.Compared with the control group,the expression of P2rx4 mRNA in the retina of LD group was significantly lower than that of the control group(P<0.05).The expression of P2rx4 mRNA in the retina of BBR group was statistically significant(P<0.05)<0.05).Compared with LD group,the expression of P2rx4 mRNA in the retina of BBR group was up-regulated,the difference was statistically significant(P<0.05).The thickness of ONL layer was less than that of wild type mice(P>0.05).There was no significant effect of knockout on the thickness of ONL layer.2.The number of microglia and positive expression of P2x4 in retina of ONL were observed by immunofluorescence.The results showed that microglia(green fluorescence)and P2x4 marker(red fluorescence)were co-located in the retina,but the number Less,each field only a few.(P<0.05).The number of glial cells in ONL layer of BBR group was slightly lower than that of control group(P>0.05),but there was no significant difference(P>0.05)(P<0.05).Compared with LD group,the number of microglia in BBR group was significantly lower than that in LD group(P<0.05).Compared with the control group,the expression of P2x4 in the ONL layer of LD group was significantly higher than that in the blank group(P<0.05),but there was no significant difference(P>0.05)LD group,the expression of P2x4 in the retina of BBR group was decreased,the difference was statistically significant(P<0.05).Part C:The effect of TRPV1 receptor in BBR anti-retinal light degenerationPurposes:On the basis of the previous experiment,the effect of berberine on retinal light injury was explored from the perspective of TRPV1 receptor,which provided scientific basis for the treatment of retinal light injury caused by senile macular degeneration by clinical berberine.Methods:(TRPV1 KO)mice were randomly divided into three groups:control group,LD group and BBR group,each group of mice Each of six.BBR group mice were given 200mg/Kg concentration of BBR solution,control group and LD group given PBS every day.Seven days later,in addition to control group,LD group and BBR group were given 10000 ± 1500Lux,4 hours of light.After the irradiation for 48 hours,the right eye was removed and fixed in the 4%paraformaldehyde fixative.The left eye was removed and the retina was extracted.The thickness of ONL layer and the apoptosis of retina were observed by HE and TUNEL.The expression of TRPV1 in the retina of C57BL/6 mice was detected by Q-PCR.Result:1.The results showed that the retinal morphology of the control group was normal,and the structure of the layers was clear:the nucleus of the ganglion was neatly arranged;the nucleus of the nucleus and the outer nuclear layer were closely arranged and the staining was uniform,The nucleus morphology is complete;photoreceptor cells inside and outside the band arrangement rules,clear boundaries.In the LD group,the nuclei of the ganglion cells were narrowed and stained,and the nuclei were arranged closely and neatly.The nucleus of the outer nuclear layer was disordered,and some of the nuclei were concentrated,aggregated and stained.The photoreceptors were arranged in the outer part of the photoreceptors,As the bar outside the arrangement disorder,membrane disc gap part of the fracture.In the BBR group,the retinal morphology was slightly abnormal and the structure was clear:the nucleus of the ganglion nucleus was slightly sparse.The nucleus and outer nucleus of the nucleus were arranged closely,and the nuclei were intact.The nuclei were intact.The thickness of the outer nuclear layer in the BBR group was lower than that in the LD group.Compared with the control group,LD group ONL layer was thinner,BBR group ONL layer slightly thin.There was no significant difference between the two groups(P>0.05).Compared with the model group,the thickness of the outer nuclear layer increased by 17.35%compared with the model group.The thickness of the TRPV1 WT mice model group was 10.39%lower than that of the normal model group.The thickness of the TRPV1 KO mice model group was 10.39%lower than that of the normal group.The thickness of the outer nuclear layer increased by 17.35%compared with the model group after BBR treatment.The thickness of retinal ONL layer in TRPV1 KO mice was thinner than that in TRPV1 WT mice,but there was no significant difference between the two lines(P>0.05).2.The expression of TUNEL-positive cells in the retina of mice was observed in the retina of mice in the control group at 48h after irradiation.The expression of TUNEL positrive cells in the retina of mice was observed in TUNEL.Shrinkage;BBR group of mouse retinal tissue see a small amount of apoptosis.Compared with the control group,the number of apoptotic cells in the ONL layer of LD group was significantly higher than that in the blank group(P<0.05).The number of apoptotic cells in the ONL layer of BBR group was significantly higher than that in the blank group(P>0.05).Compared with LD,the number of apoptotic cells in the ONL layer of BBR group was decreased(P<0.05).However,the apoptotic rate of knockout mice in BBR group was higher than that in wild type mice,but there was no significant difference between the two groups(P>0.05)Statistical differences(P<0.05).Gene knockout has an effect on ONL layer cell apoptosis.3.The expression of TRPV 1 mRNA in the retina of LD group was down-regulated compared with the control group(P<0.05).The expression of TRPV 1 mRNA in the retina of LD group was not statistically significant(P<0.05),but the difference was not statistically significant Gt;0.05).Compared with the model group,the expression of TRPV1 mRNA in the mice was up-regulated and the difference was statistically significant(P<0.05).Conclusion:1.BBR has an effective anti-retinal light damage funtion.2.P2x4 receptor may be involved in the regulation of BBR against retinal light damage by modulating microglia.3.TRPV1 receptor has a certain role in retinal protection,being an important pathological mechanism of retinal light injury,and may be involved in BBR anti-retinal light damage.