| This paper focuses on Lycium barbarum and dry age-related macular degeneration as the main research content.the efficacy of Lycium barbarum in the treatment of dry age-related macular degeneration was evaluated from the aspects of visual impairment,retinal pathological damage and photoreceptor cell rhodopsin damage in mice,and its biological mechanism was explored.The research content and main research content of each chapter are summarized as follows:1.LiteratureThe first part of this chapter systematically summarizes the application and research status of Lycium barbarum in ophthalmic diseases,including age-related macular degeneration,glaucoma,diabetic retinopathy and retinitis pigmentosa.The active components in Lycium barbarum were analyzed.Through the comparison of the application and research of Lycium barbarum in the above four diseases,it is found that the research of Lycium barbarum in age-related macular degeneration is still little and lack of depth.In addition,there is a big gap in the research of animal evaluation,detection means and mechanism between Lycium barbarum and age-related macular degeneration,which needs to be filled urgently.What’s more,the records of ophthalmic diseases treated by Lycium barbarum in ancient Chinese medicine books are collated and summarized.Analyzing the differences between modern research and traditional Chinese medicine theory on the effective components of Lycium barbarum,it is found that the current research mainly focuses on the polysaccharides of water extract of Lycium barbarum.On the contrary,the efficacy of Lycium barbarum ethanol extract has not been fully studied and explained,which needs further research and excavation.The second part of this chapter summarizes the research status of age-related macular degeneration systematically.Summarizing the definition of age-related macular degeneration,expounding the incidence of age-related macular degeneration at home and abroad,this part of chapter points out its great harm in the future.The clinical classification of age-related macular degeneration and the characteristics of different stages of disease development were summarized,which laid a foundation for the selection of future research directions.The risk factors leading to the occurrence and development of age-related macular degeneration are described in detail,and it is pointed out that age is the primary influencing factor,which can be used as a reference for improving the animal model buliding.This part of chapter introduces the clinical research status of age-related macular degeneration.By comparing the clinical background of successful drug development of wet age-related macular degeneration with that of dry age-related macular degeneration,dry age-related macular degeneration is selected as the target disease of this study.2.Screening of active sites of wolfberry fruit to interfere with visual impairment in dry miceAs age-related macular degeneration is more common in the elderly,age is the primary risk factor for age-related macular degeneration.Therefore,older animals should be preferred to simulate the elderly’s physiological and pathological conditions in age-related macular degeneration animal construction model.However,in the various animal models induced by sodium iodate reported on the literature,simulating the pathological changes of age-related macular degeneration tends to use adult animals,which is different from the actual situation.Therefore,in this study,the optimal age of mice with dry age-related macular degeneration induced by sodium iodate was screened.Four different age groups of mice aged 6-8 weeks,6 months,12 months and 18 months were injected intraperitoneally with 50mg/kg sodium iodate to construct the model,and 7th day later the mice were sacrificed and their eyeball samples were collected.The overall retinal thickness,the thickness of the outer nuclear layer of the retina,the apoptosis of the whole retina,the apoptosis of the outer nuclear layer of the retina,and the morphology and function of retinal pigment epithelial cells were detected by HE,Tunel and immunofluorescence to screen the mice optimum age of dry age-related macular degeneration model.The results of HE staining showed that:(1)In the all ages mice of control group,only a few outer nuclear layer areas of the peripheral retina of 18-month-old mice were significantly thinner than those of 6-8-week-old mice,while the thickness of the outer nuclear layer of the mouse did not have this phenomenon.The thickness of the rod and cone layer in the central and peripheral retinas of 6-month-old mice did not become significantly thinner,but only slightly decreased,while that of 12-month-old and 18-month-old mice decreased significantly.(2)In the all ages mice of model group,compared with the control group in the same age group,the thickness of the outer nuclear layer of the central retina in the 6-8-week-old model group decreased significantly,while the thickness of the outer nuclear layer of the peripheral retina decreased to a certain extent.The thickness of the outer nuclear layer in the central and peripheral retina of the 6-month-old model group decreased significantly.In the 12-month-old and 18-month-old model group,the thickness of the outer nuclear layer in the central and peripheral retina decreased only sporadically,and the thickness of the outer nuclear layer in most retinas only decreased to a certain extent.The thickness of rod and cone layer in the outer nuclear layer of the central retina was significantly thinner in 6-8-week-old and 6-month-old model mice.In the 12-month-old model group,the thickness of the rod and cone layer of the peripheral retina was significantly thinner,but there was no significant damage to the rod and cone layer of the central retina.There was almost no significant decrease in the thickness of rod and cone layer in both central and peripheral retina of 18-month-old model group.These results suggest that there is no significant difference in the thickness of outer nuclear layer and rod and cone layer between the blank group and the model group after sodium iodate modeling in 12-month-old and 18-month-old mice,which may be caused by aging.The retina of 12-month-old and 18-month-old blank group has thinned spontaneously.Tunel staining is used to reflect the apoptosis of the whole retina and the outer nuclear layer of the retina.Tunel staining results found that:(1)In the all ages mice of control group,no apoptosis of retinal cells(or photoreceptor cells)was found in the 6-8-week-old and 6-month-old control groups.However,apoptosis occurred in retinal cells(mainly photoreceptor cells)in 12-month-old and 18-month-old control mice,and the degree of apoptosis of retinal cells(or photoreceptor cells)in 18-month-old control group was higher than that in 12-month-old control group.(2)Compared with control mice of the same age,the retinal cells(or photoreceptor cells)of the four different age model groups all showed significant damage.The degree of the retinal cells(or photoreceptor cells)apoptosis was related to age that the higher of mice age,the higher of apoptosis percentage.Of all four model groups,the apoptosis degree of retinal cells(or photoreceptor cells)in the 18-month-old model group was the highest.These results suggest that the damage of retinal cells,especially photoreceptor cells,in 12-month-old and 18-month-old mice induced by sodium iodate is more serious.This may be caused by the aging of mice,and the retinal cells of 12-month-old and 18-month-old control mice have apoptosis spontaneously.Immunofluorescence is used to reflect the morphology and function of retinal pigment epithelial cells.The results of immunofluorescence showed that:(1)In the all ages mice of control group,the cumulative fluorescence intensity of RPE65 protein and ZO-1 protein was negatively correlated with age.The cumulative fluorescence intensity of RPE65 protein and ZO-1 protein in 6-8-week-old and 6-month-old blank group was the highest,and there was little difference,followed by 12-month-old blank group,and 18-month-old blank group had the lowest cumulative fluorescence intensity.(2)Compared with the control group in the same age group,the expression of RPE65 protein and ZO-1 protein in the RPE layer of the model group was significantly lower than that of the control group.The above results suggest that the damage of RPE cells in 12-month-old and 18-month-old mice is more serious,which may be caused by senescence,and the retinal pigment epithelial cells in 12-month-old and 18-month-old blank mice have declined spontaneously.The above results showed that the photoreceptor cells and retinal pigment epithelial cells of 12-month-old and 18-month-old mice had spontaneous damage before modeling,and the control group could not be distinguished from the model group after sodium iodate treatment,while sodium iodate could well distinguish the damage of photoreceptor cells and RPE cells of 6-month-old and 8-week-old mice.On the contrary,sodium iodate had a good differentiation on the damage of photoreceptor cells and RPE cells in 6-8-week-old and 6-month-old mice.For this sodium iodate model,6-8-week-old and 6-month-old mice were better than 12-month-old and 18-month-old mice.Because compared with 6-8-week-old mice,6-month-old mice have an age advantage,so 6-month-old mice are selected for the next study.Black-and-white box is a widely reported behavioral method to detect the visual function of mice.By using the habit of darkening and avoiding light in mice,strong light stimulation is applied in the white box to force visually normal and light-sensitive mice to move into the black box.Reduce the cumulative time of mice in the white box,so as to distinguish visually normal mice from visually impaired mice.In this study,according to the literature report,a single-door black-and-white box was built,and two pre-experiments were carried out to evaluate its detection efficiency on sodium iodate model.The results of the first experiment showed that compared with the control group,the proportion of time spent in the white box was significantly increased in the model group.Howere,the results of the second experiment showed that there was no significant difference in the proportion of time spent in the white box between model group and control group.This shows that the stability of the single-door black-and-white box reported in the literature is poor,which may be due to the inclusion of some interference factors in the testing process.The most likely reason is the interference of the strong memory ability of mice to the experiment.In order to eliminate the influence of mouse memory on the test results as much as possible,this study optimized the structure of the black-and-white box,changed the single door of the black-and-white box partition into a double door,and repeated the test twice.The results of the two black-and-white boxes showed that the proportion of time spent in the white box in the model group was significantly higher than that in the control group.The above results suggest that the double-door black-and-white box is better and more stable than the single-door black-and-white box in evaluating the visual function of mice.Therefore,in the next pharmacodynamics experiment,this study will use a double-door black-and-white box for detection.Previous literature investigation found that there are some differences in the understanding of the active components of Lycium barbarum in traditional Chinese medicine classics and modern research.In order to clarify the efficacy of water extract and ethanol extract of Lycium barbarum on dry age-related macular degeneration in further,in this study,we carried out the pharmacodynamic screening and evaluation of water extract and ethanol extract of Lycium barbarum on dry age-related macular degeneration induced by sodium iodate.Mice were pre-protected with high-dose water extract of Lycium barbarum(3g/kg/d)or high-dose ethanol extract of Lycium barbarum(3g/kg/d)for 7 days before modeling.The model was made by intraperitoneal injection with 50mg/kg dose,and then continued to be treated with the dose before modeling for 7 days.During this period,the visual behavior of mice was tested,and the damage of photoreceptor cells was detected by HE staining after the end of the experiment.The results showed that:Compared with the control group,the activity time in the white box was significantly longer in the model group.Compared with the model group,there was no significant change in the activity time in the white box in the high dose Lycium barbarum water extract group,while the activity time of mice in the white box was significantly reduced in the high dose Lycium barbarum ethanol extract group.The results of HE staining showed that compared with the control group,sodium iodate caused extensive and significant damage to the photoreceptor cells of the central and peripheral retina of the model group,and the thickness of the outer nuclear layer and rod and cone layer of the model group was significantly thinner than that of the control group.Compared with the model group,the high dose water extract of Lycium barbarum could significantly increase the thickness of the outer nuclear layer and rod and cone layer of the local central retina.The high-dose ethanol extract of Lycium barbarum could significantly increase the thickness of the central outer nuclear layer and rod and cone layer of the retina,and also thicken the outer nuclear layer and rod and cone layer of the local peripheral retina.Based on the above results,it is suggested that the ethanol extract of Lycium barbarum has a protective effect on photoreceptor cell damage induced by sodium iodate,and this protective effect is stronger than that of water extract of Lycium barbarum.Therefore,in the next study,the pharmacodynamic study of ethanol extract of Lycium barbarum on visual impairment in mice with dry age-related macular degeneration induced by sodium iodate will be carried out.3.Study on the Intervention Effect and Mechanism of the Ethanol Extract of Lycium Barbarum on the Visual Impairment of Mice with Dry Age-Related Macular DegenerationPrevious studies found that the improvement effect of high-dose Lycium barbarum ethanol extract on dry age-related macular degeneration was better than that of high-dose Lycium barbarum water extract.However,the dose-effect relationship of ethanol extract of Lycium barbarum in the treatment of dry age-related macular degeneration is not clear.Therefore,three dose groups were set up-low dose Lycium barbarum ethanol extract group(0.75g/kg),medium dose Lycium barbarum ethanol extract group(1.5g/kg)and high dose Lycium barbarum ethanol extract group(3g/kg).The ethanol extract of Lycium barbarum was given to mice for 7 days before modeling,and the model was made by intraperitoneal injection with the dose of 50mg/kg,and then continued to be treated with the same dose before modeling for 7 days.During this period,the visual behavior of mice was tested,and the damage of photoreceptor cells was detected by HE staining and Tunel test after the end of the experiment.(1)The results of the black and white box showed that compared with the control group,the activity time of the model group mice in the white box was significantly prolonged after being treated with sodium iodate.Compared with the model group,the high and middle dose of Lycium barbarum ethanol extract could significantly reduce the activity time of mice in the white box,but the therapeutic effect of high dose Lycium barbarum ethanol extract was better than that of middle dose Lycium barbarum ethanol extract.The low-dose ethanol extract of Lycium barbarum could only decrease the activity time of mice in the white box,but there was no statistical significance.The above results suggest that the ethanol extract of Lycium barbarum can improve the visual impairment of model mice in a dose-dependent manner.(2)The results of HE staining showed that compared with the control group,sodium iodate significantly damaged the photoreceptor cells in the central and peripheral retina of the model group,and significantly thinned the outer nuclear layer and rod and cone layer of the retina in the model group.Compared with the model group,the high dose ethanol extract of Lycium barbarum significantly increased the thickness of the outer nuclear layer of the central and peripheral retina,and the significant improvement on the thickness of the rod and cone layer was mainly reflected in the central retina.The middle dose of Lycium barbarum ethanol extract significantly increased the thickness of the central retina and part of the peripheral retinal outer nuclear layer,and significantly increased the thickness of the rod and cone layer of part of the central retina and part of the peripheral retina.The low-dose ethanol extract of Lycium barbarum significantly increased the thickness of part of the central retina and a few peripheral retinal outer nuclear layer,and significantly increased the thickness of the rod and cone layer of a few central retina and peripheral retina.The above results suggest that the ethanol extract of Lycium barbarum can improve the photoreceptor cell injury in a dose-dependent manner.(3)The results of tunel experiment showed that compared with the control group,there was significant apoptosis of retinal cells in the model group,mainly in photoreceptor cells.Compared with the model group,both high-dose and medium-dose ethanol extract of Lycium barbarum could significantly inhibit the apoptosis of retinal cells,mainly the apoptosis of retinal photoreceptor cells.however,the inhibitory effect of high-dose Lycium barbarum ethanol extract was better than that of medium-dose Lycium barbarum ethanol extract.Low-dose ethanol extract of Lycium barbarum could only down-regulate the apoptosis of mouse retinal cells and photoreceptor cells,but there was no statistical significance.The above results suggest that the ethanol extract of Lycium barbarum can improve the apoptosis of retinal cells of model mice in a dose-dependent manner,mainly photoreceptor cells.The results of the above visual behavior experiments,HE staining and Tunel experiments all suggest that the ethanol extract of Lyciun barbarum can reduce the visual damage caused by sodium iodate in mice through the protective effect of sodium iodate on photoreceptor cells in a dose-dependent manner.Rod cells are the main photoreceptor cells in mouse retina.Previous studies have confirmed that ethanol extract of Lycium barbarum can protect the morphology of rod cells.Therefore,it is necessary to further detect the function of rod cells.The rod cells rely on the pigment rhodopsin for their photosensitive function,so the expression level of rhodopsin in the rod cells can reflect the photosensitive function of the rod cells.The results of immunofluorescence showed that compared with the control group,the expression level of rhodopsin in the rod cells of the model group was significantly lower than that of the control group.Compared with the model group,the high dose ethanol extract of Lycium barbarum could significantly increase the expression level of rhodopsin.The medium-and low-dose of Lycium barbarum ethanol extract can up-regulate the expression level of rhodopsin,which are no statistical significance.The regulation effect of medium-dose Lycium barbarum ethanol extract on rhodopsin is stronger than that of low-dose Lycium barbarum ethanol extract.The expression of rhodopsin in rod cells is regulated by a series of enzymes in the visual cycle such as LRAT,RPE65 and IRBP,so the expression of these enzymes was detected in this study.The results showed that compared with the control group,sodium iodate significantly inhibited the expression of LRAT,RPE65 and IRBP protein in the model group.High dose ethanol extract of Lycium barbarum can significantly up-regulate the expression of LRAT,RPE65 and IRBP protein.Both medium-dose of Lycium barbarum ethanol extract and low-dose of Lycium barbarum ethanol extract can up-regulate the expression of LRAT,RPE65 and IRBP protein,while there are no statistical significance.The upregulation effect of middle dose Lycium barbarum ethanol extract is better than that of low-dose Lycium barbarum ethanol extract.These results suggest that Lycium barbarum can enhance the expression of rhodopsin by regulating the expression of LRAT,RPE65 and IRBP proteins in the visual circulation to protect the function of rod cells.Based on the above results,the following conclusions can be drawn:(1)In the screening experiment of 50mg/kg sodium iodate model on four different age groups of mice,6-month-old mice are better than other age groups of mice.(2)In the visual behavior experiment,the stability and repeatability of double-door black-and-white box is better than that of single-door black-and-white box.(3)In the activity screening experiment of Lycium barbarum,the therapeutic effect of high-dose ethanol extract of Lycium barbarum on dry age-related macular degeneration induced by sodium iodate was better than that of high-dose aqueous extract of Lycium barbarum.(4)In the pharmacodynamic evaluation experiment of ethanol extract of Lycium barbarum,the ethanol extract of Lycium barbarum could slow down the retinal damage caused by sodium iodate in a dose-dependent manner.(5)Lycium barbarum can enhance the expression of rhodopsin in rod cells and protect the function of rod cells by regulating the expression of LRAT,RPE65 and IRBP protein in a dose-dependent manner to delay visual impairment. |
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