| ObjectiveTo observe the therapeutic effect of berberine on chronic retinal light injury,and explore the dynamic change trend of P2X7R in mice with chronic retinal light injury relieved by berberine.Methods1.Protective effect of berberine on mice with chronic retinal light injury:90 adult male C57BL/6J mice with a weight of about 20g±2 for 8-10 weeks were selected,and modeling was started after one week of adaptive feeding.Raised in a clean environment,they were randomly divided into three groups:blank control group,LD group(model group)and LD+BBR group(gavage group),of which 10 were in the blank group and 40 were in the LD group and LD+BBR group respectively.In this study,the modeling method was based on the domestic and international models of mice with chronic light injury,and a long-term,low-energy blue light irradiation method was adopted.The specific operation was to give LD group and LD+BBR group mice about 500Lux±5(blue light)from 6:00 to 18:00 every day for 3 months.UT382 industrial-grade illuminometer was used to check whether the light source intensity was 500Lux±5.Irrigation lavage stomach method to continue the previous studies,the drug preparation for specific:ready to 15 ML of PBS buffer,to join 300 mg of berberine powder,vortex finder on shake until the powder dissolves,as the concentration of 200 mg/kg of liquid medicine,the computation formula for lavage:gastric volume(ML)=mice weight(kg)x 200/(300/15)a week at the same time measurement of body weight in mice,along with the change of weight adjustment to the dose.The specific time period of gavage was:on the first day after the beginning of light,the LD group and the LD+BBR group were given the gavage mode of stopping the drug for 4 consecutive days and 3 days every week,respectively.The purpose of stopping the drug was to prevent the intolerance of mice to the drug and the stimulation of gavage.(the LD+BBR group was given the prepared berberine solution by gavage,and the LD group was given PBS slow-release solution by gavage.)When lighting for 3 months after the end of the light after week 0,respectively random gather the blank group,the LD group and LD+BBR group the left eye,6 mice do HE staining detection and then observed the pathological damage of retina in mice and changes,and contrast tissue morphological differences between groups of mice,according to the change of tissue morphology measure of berberine on chronic retinal light damage there is no healing in mice,so as to determine the retinal protection on the chronic of retinal injury mice.2.Effects of berberine on apoptotic cells in mice with chronic retinal light injury:the selection of experimental animals,modeling method,gavage method and feeding conditions were all the same as the research content 1.After lighting for 3 months after the end of the light week 0,gather the blank group,the LD group and LD+BBR group each 6 mice to organize materials in the right eye,do TUNEL apoptotic cells of the retina examination mice,and compare the differences between groups retinal apoptosis cells in mice,statistics of apoptosis of retinal cells apoptosis rate for comparison,the amount of to judge whether berberine can inhibit the chronic retinal light damage of retinal cell apoptosis in mice.3.The dynamic change trend of purine signal P2X7R in berberine to alleviate chronic retinal light injury:the selection of experimental animals,modeling methods,gavage methods and feeding conditions are all the same as the research content 1.3 months after the end of the light,the rest of the group continued to normal conditions in mice fed,respectively,at the end of the light and lavage with three periods,namely the light after 1 week,2 weeks after light,and light after 3 weeks,gather the normal groups at random,LD,LD+BBR group 6 eye,using real-time fluorescent quantitative PCR molecular biological techniques to detect different groups of mice retina purine signal P2X7R relative expression of mRNA,whether P2X7R participated in the berberine ease chronic the pathological changes of the retinal light damage,According to the results of relative expression of P2X7R mRNA in the retina of mice in 3 time periods,the dynamic change trend of P2X7R in berberine relieving chronic retinal light injury was summarized.Results1.HE staining to observe the changes in the form of mouse retina:materials,fixation,dehydration,embedding,and sectioning organization after HE staining detection,blank control group,observed under optical microscope LD group and LD+BBR retinal structure change of each layer,the outer nuclear layer thickness calculation,each group of randomly selected three sections,each section to choose 4 x magnification of 400 field of nonoverlapping(with the optic nerve is a center,flanked by choosing two horizons)film.Image-pro Plus 6.0 Image analysis software was used to count the ONL thickness with a length of 50um in each field of vision and take the average value for statistical analysis.Research proves:retinal morphology of mice in blank control group is intact.The nuclei of the inner and outer layers are in complete morphology,uniform staining and compact arrangement.The inner and outer segment boundaries of the photoreceptor cells were clear and regular,and the thickness of the outer nuclear layer was 62.72±2.86 m.The morphology of the retina in the LD group was abnormal,and the structure of each layer was changed.Some ganglion nuclei were shrunk,stained darker,and arranged loosely.The nuclei in the kernel layer are arranged slightly loosely;The cells in the outer nuclear layer were arranged in a disordered way,and some nuclei were clustered and stained with concentration.The arrangement of the inner and outer segments of the photoreceptor cells was slightly disordered,the outer segment of the optic rod was arranged in a disordered way,some of the membrane-disc gap was broken,and the thickness of the outer nucleus layer was 47.11±2.01m.The morphology of LD±BBR retina was slightly abnormal,and the nuclei of ganglion were slightly sparse.The nuclei of the inner and outer layers are slightly complete,uniform and closely arranged.The inner and outer segments of the photoreceptor cells were slightly disordered,and the thickness of the outer nuclear layer was 54.07±2.05 m.Compared with the blank control group,the thickness of the outer retinal nucleus of LD group was significantly thinner(P<0.01),and the difference was statistically significant.The thickness of the outer retina of the LD+BBR group was slightly thinner(P<0.05),and the difference was statistically significant.Of mice compared with LD group,LD+BBR retinal thickening of the outer nuclear layer thickness,(P<0.01)the difference is statistically significant,indicating light makes LD group of mice after retinal outer nuclear layer thickness thinning,and after berberine lavage treatment of LD+BBR group of mice,the outer nuclear layer thickness of retina obvious thickening in the LD group,showed the berberine to chronic retinal light damage in mice retina has a protective effect.2.TUNEL observed the apoptosis of ONL layer cells in the retina of mice:Will draw materials,fixation,dehydration,embedding,and sectioning TUNEL detection for the organization,using fluorescent inverted microscope to observe the blank control group of mice,the LD group and LD+BBR apoptotic cells in the retina,using ultraviolet light and green light to stimulate respectively,each group of randomly selected three slices,centered on the optic nerve,left and right sides of the selected two horizons,respectively count between the inner and outer nuclear layer view of apoptosis positive cells number and the total number of outer nuclear layer cells,the apoptotic rate(%)=number of apoptosis positive cells/vision with the total number of cells by 100%.The.results showed that the apoptotic cells were purplish red and presented typical positive apoptotic cells such as "ring" and "crescent".No apoptotic cells were observed in the retinal tissue of mice in the blank group.In the LD group,a large number of apoptotic cells appeared in the outer retinal nucleus layer,and the apoptosis rate of the outer nucleus layer was high 71.16±5.99%.The apoptosis rate of LD+BBR group was 16.02±2.68%.The experimental results show that there are more apoptotic cells in the outer nuclear layer of the retina of the LD group.showing a significant statistical difference(P<0.01).Apoptosis in the retinal outer nuclear layer cells of the gavage group was higher than that of the blank group,and the difference was statistically significant.(P<0.05).Compared with the LD group,the number of apoptotic cells in the outer nuclear layer of the LD+BBR group was significantly reduced(P<0.05).The results showed that light induced a large number of apoptotic cells in the outer retinal nucleus of LD group mice,while the apoptotic cells in the outer retinal nucleus of LD+BBR group were significantly reduced after the administration of berberine,indicating that berberine could inhibit the apoptosis of retinal cells in mice with chronic retinal light injury.3.Real-time fluorescence quantitative PCR was used to detect the expression of P2X7R mRNA in the retina of mice:RNA extraction,cDNA preparation and fluorescence quantitative PCR were performed on the collected retinal tissues.The results showed that the relative expression of P2X7R mRNA in the retina of the LD group was up-regulated compared with that of the blank group(P<0.05).The expression of P2X7R mRNA in the retinal mice was down-regulated,and there was no statistical difference between the two(P>0.05).Compared with the blank group,the expression level of P2X7mRNA in the LD+BBR group was down-regulated(P<0.05),but the relative expression level of P2X7R mRNA in the LD group was significantly lower than that in the LD group,and the difference was statistically significant(P<0.05).This indicated that P2X7R was activated in large quantities after illumination,which increased the relative expression of P2X7RmRNA in the retinas of LD group,and decreased the relative expression of P2X7RmRNA in the retinas of LD+BBR group after the intervention of berberine,indicating that berberine had an inhibitory effect on the relative expression of P2X7RmRNA.Three real-time fluorescence quantitative PCR tests were performed within 3 weeks after the end of light 3 months to observe the dynamic change trend of relative expression of P2X7R mRNA in LD group and LD+BBR group.The results showed that:On the basis of the relative expression of P2X7R mRNA in LD+BBR group and LD group,the LD+BBR group showed a slightly upward trend and tended to the relative expression of P2X7R mRNA in the blank control group,while the LD group showed a slightly downward trend,but still higher than the relative expression of P2X7R mRNA in the blank control group,the difference was statistically significant(P<0.05).The above results indicate that P2X7R is involved in related pathological reactions caused by retinal light damage.From the perspective of molecular biology,the up-regulated expression of P2X7R mRNA in the retina of mice in the model group(LD group)after light damage may prove that a large number of P2X7R is activated after light.However,after intervention with berberine,the expression of P2X7R mRNA in the retina of mice in the LD+BBR group was down-regulated,indicating that berberine can inhibit the activation of P2X7R and participate in the related response to prevent the formation of retinal light damage.Conclusions1.Berberine has a protective effect on the retina of mice with experimental chronic retinal light injury,and can improve the pathological damage of the retina caused by external nuclear layer degeneration caused by light injury.2.Berberine can inhibit the apoptosis of retinal cells in mice with experimental chronic retinal light injury.3.The mechanism of berberine in relieving experimental chronic retinal light injury in mice is related to the signaling pathway of purine signal P2X7R,but its exact mechanism still needs further study. |
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