| (1)Clinical observation of high myopia macular degeneration treated by Huangban Four DecoctionObjective:To observe the effectiveness and safety of Huangban Four Decoction(HBFD)in patients with high myopia macular degeneration.Methods:The subjects included were from the department of ophthalmology of China-Japan Friendship Hospital from September 2017 to September 2019.A total of 49 patients(49 eyes)were selected.Monocular eyes(1 eye was randomly selected from both eyes)were used as the research object.The Chinese medicine compound HBFD decoction was given,and warmed morning and evening,100ml each time,for a total of 6 months.The best corrected visual acuity,diopter,eye length,myopia arc area,choroidal atrophy arc area,macular foveal thickness,lesion height,and macular foveal choroidal thickness were evaluated before treatment,3 months and 6 months after treatment.Results:After 6 months of treatment,the number of ETDRS letters increased significantly(P<0.05),and the area of myopia arc and choroidal atrophy arc decreased significantly(both P<0.05).Compared with before treatment,the macular fovea thickness and lesion height were significantly reduced after 3 and 6 months of treatment(both P<0.05);the macular foveal choroid thickness increased significantly(both P<0.05).During the treatment,there were no treatment-related adverse events.Conclusions:HBFD can reduce the arc area of myopia,the area of choroidal atrophy,reduce the thickness of the macular fovea and the height of the lesion,and effectively delay the occurrence and development of macular lesions in high myopia.Patients’ vision and improvement of visual function have a positive effect,and can be used as a safe and effective auxiliary treatment for high myopia macular degeneration.(2)Establishment of form-deprived high myopia guinea pigs modelObjective:The form deprivation high myopia(FDHM)guinea pig model was prepared by performing monocular shape deprivation on guinea pigs to simulate the state of high myopia.To lay the foundation for further development of the pathogenesis and prevention of high myopia.Methods:Twenty-four two-week-old three-color guinea pigs were randomly divided into a blank control group(12)and a model group(12,of which 6 were in the form of deprivation for 4 weeks,and 6 were in the form of deprivation for 8 weeks).In the model group,the right eye was selected as the experimental eye(form deprivation myopia,FDM),and the opposite eye was used as the control group.Guinea pigs in the control group did not do any treatment.The diopter,anterior chamber depth,lens thickness,vitreous cavity depth,and axial length were measured at three time points before and after guinea pig shape deprivation.At 4 time points and 8 weeks after shape deprivation,6 guinea pigs were killed in each group(3 randomly selected for light and electron microscopy),and the morphology and structure of the retina and sclera of each group were observed.Results:After 4 and 8 weeks of form deprivation,the refractive status of the FDM group gradually changed from hyperopia to myopia.The refractive power of myopia was higher than that of the blank control group and the self-control group,and the differences were statistically significant(both P<0.05).The depth of vitreous cavity and axial length of the FDM group were higher than those of the blank control group and the self-control group,and the differences were statistically significant(all P<0.05).Under light microscope:Compared with the blank control group,after 4 weeks of shape deprivation,the retina and sclera of the FDM group became thinner and the structure was slightly loose;after 8 weeks of shape deprivation,the guinea pig retina of the FDM group became significantly thinner and the tissue structure was loose.Disordered arrangement,ganglion cell layer,inner and outer nuclear layer cells are significantly reduced,the nuclei are small and uneven,the cell arrangement is disordered,the microvilli protruding from the RPE layer are shorter,and even the fusion is broken;the sclera is significantly thinner and fibroblasts The nucleus is distorted,collagen fibers are sparsely distributed,the diameter is significantly reduced,the arrangement is disordered and distorted,there are breaks and separations,the gaps between fibers are increased,and the extracellular matrix isincreased.Under electron microscope:4 weeks after shape-deprivation,swelling and deformation of the retinal membrane disk,irregular shrinkage of inner and outer nuclear layers and RPE cell membranes,irregular aggregation of nuclear chromatin,swelling,deformation and vacuole-like changes of mitochondria,the scleral collagen fiber diameter decreased significantly,and the fiber density decreased.After 8 weeks of shape-deprivation,the above pathological changes were more obvious.Conclusions:The form deprivation method can successfully induce the FDM model.The myopic diopter,vitreous cavity depth,and axial length increase progressively with the extension of form deprivation time.The thickness of the retina and sclera becomes thinner with the extension of form deprivation time.Pathological changes are an ideal animal model for studying FDHM.(3)Experimental Study on HBFD Intervention in Guinea Pig FDHMObjective:To observe the effects of HBFD on retinal and scleral function and morphological changes of FDHM in guinea pigs,and to explore the dose-effect relationship between HBFD and FDHM in traditional Chinese medicine.Methods:Fifty two-week-old three-color guinea pigs were randomly divided into a blank control group,a FDHM group,a high-dose Chinese medicine group,a medium-dose group,and a low-dose group after one week of adaptive breeding.In the last four groups of guinea pigs,the right eye was selected as the experimental eye,the eyelids were sutured,and the shape sense was deprived for 8 weeks.After deprivation of form,the high,middle and low dose groups of Chinese medicine were administered with different concentrations of HBFD decoction.All guinea pigs in the diopter and A-ultrasonic were measured before and at 8 weeks post-modeling.After the examination,guinea pigs were anesthetized and killed,and bilateral eyeballs were taken to prepare retina and sclera tissue sections.The morphology and structure of retina and sclera of guinea pigs were observed by light microscope.Results:The progress of myopic refractive power in the HBFD high,middle and low dose groups was significantly lower than that in the FDHM group(all P<0.05),and the progress of vitreous cavity depth and axial length were significantly lower than those in the FDHM group(all P<0.05).Among them,the increase in myopic refractive power,vitreous cavity depth,and axial length of the HBFD high-dose group was the slowest.The HBFD high-dose retinal and scleral thickness were significantly higher than those in the FDHM group(both P<0.05).Conclusions:1.HBFD can reduce the progression of myopia diopter in guinea pigs,delay the growth of the axis of the eyes,and have an intervention effect on the myopia progression of FDHM guinea pigs.2.It is initially shown that the high-dose HBFD group can significantly reduce the pathological damage to the retina and sclera of FDHM guinea pigs,and has a certain inhibitory effect on the occurrence and development of FDHM,which has clinical promotion value.However,its specific pathway needs further exploration.(4)Effect of HBFD on FDHM in Guinea PigsObjective:Based on the establishment of guinea pig FDHM model,the mechanism of HBFD intervention in FDHM was explored through molecular biology techniques.Methods:Sixty three-week-old guinea pigs were randomly divided into a blank control group,a FDHM model group,and a Chinese medicine group of 20 rats each.The model group and the Chinese medicine group guinea pigs were sutured with the right eyelid.Refraction and eye biometry were performed before and after shape-deprivation.Guinea pigs were sacrificed 8 weeks after shape-deprivation,and sclera and retinal sections were prepared.Protein Kinase C(PKC),the expression of neuronal nitric oxide synthase(nNOS)in the retina was measured by image analysis software and the colorimetric intensity of immunohistochemistry was measured.The colorimetric method was used to detect the superoxide dismutase(SOD)activity and malondialdehyde(MDA)content in the retina;extract the retinal proteins,and analyze the expression of hypoxia-inducible factor-1α(HIF-1α)by Western Blotting Amount of intrascleral protein was extracted,and sclera TGF-β2,MMP-2,TIMP-2 protein expression.Results:1.After the HBFD intervention,the myopia of the Chinese medicine group was lower,the vitreous cavity depth and the eye length were significantly reduced;the retinal tissue arrangement was relatively complete,the phenomenon of vacuolarization of the membrane disc structure was significantly reduced,the scleral thickness increased,the morphology was regular,Fibrous cells are denser.2.Immunohistochemistry:(1)PKC expression:Compared with the blank control group,the positive expression of PKC in the retina of guinea pigs in the FDHM group was significantly increased;compared with the FDHM group,the expression of PKC in the retina of the Chinese medicine group was significantly reduced.(2)nNOS expression:Compared with the blank control group,the positive expression of nNOS protein in the retina of guinea pigs in the FDHM group was significantly increased;compared with the FDHM group,the expression of nNOS protein in the retina of the Chinese medicine group was significantly reduced.3.Changes in MDA and SOD content:Compared with the blank control group,the retinal SOD activity of the FDHM group was reduced;compared with the FDHM group,the SOD activity of the Chinese medicine group increased and the MDA content decreased.4.Western-Blotting:(1)HIF-1α protein expression:Compared with the blank control group,the expression of HIF-1α protein in the retina of guinea pigs in the FDHM group was significantly increased;compared with the FDHM group,the expression of HIF-1α protein in the retina of the Chinese medicine group was significantly reduced.(2)TGF-β2 protein expression:Compared with the blank control group,the expression of TGF-β2 protein in sclera of guinea pigs in FDHM group was significantly reduced;compared with FDHM group,the expression of TGF-β2 protein in sclera of Chinese medicine group was significantly up-regulated.(3)MMP-2 and TIMP-2 protein expression:Compared with the blank control group,the guinea pig sclera MMP-2 protein content in the FDHM group was significantly increased and the TIMP-2 protein content was significantly reduced;compared with the FDHM group,the Chinese medicine group guinea pig sclera sclera MMP-2 protein content decreased significantly,TIMP-2 protein content increased significantly.Conclusions:1.Monocular shape deprivation-induced FDHM guinea pigs may be closely related to oxygen free radical damage and increased expression of HIF-1α caused by hypoxia.2.HBFD can delay the abnormal growth of vitreous cavity depth and axial length,slow down the development of myopia refractive power,and interfere with the progress of guinea pig shape deprivation myopia.3.HBFD inhibits scleral remodeling by regulating the retinal PKC-NO pathway and scleral TGFβ-MMP2/TIMP2.HBFD protects the function of retinal cells by improving microcirculation,reducing oxygen free radical damage,and improving the body’s ability to resist lipid peroxidation.This effect may be achieved by increasing SOD activity and promptly removing excess oxygen free radicals. |
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