| Objective:To explore the molecular mechanism of the effect of Yijing Tongluo prescription on axial length elongation and myopia progression in highly myopic guinea pigs from the perspective of scleral extracellular matrix remodeling to provide theoretical basis for the prevention and control of myopia with Traditional Chinese Medicine.Methods:l.Modeling method:The right eye of guinea pigs was covered by a poly-methyl methacrylate(PMMA)lens with a diopter of-10.0D to establish defocus-induced high myopia(DIHM)model.2.Grouping and Intervention:30 healthy tricolor guinea pigs were randomly and equally divided into 3 groups:TCM group,model control group and normal control group.TCM group:the right eye of guinea pigs was covered by lens with a diopter of10.0D,and they were given high-dose Yijing Tongluo Decoction for intragastric administration with volume of 10ml/(kg·d);Model control group:The right eye of guinea pigs was covered by lens with a diopter of-10.0D,and the same volume of normal saline was given for intragastric administration.Normal control group:the guinea pigs were given the same volume of normal saline for intragastric administration without any treatment for their right eyes.Intervention was performed once a day for 5 weeks.3.The measurement of Diopter and Axial Length:before intervention,3 weeks and 5 weeks after intervention,the guinea pigs in each group received cycloplegic retinoscopy(with Compound Topicamide eye drops)and a-ultrasound measurement of axial length.4.Histopathological observation:Sclera and choroid of guinea pigs were observed by HE staining.Five weeks after the intervention,the guinea pigs were sacrificed under excessive anesthesia,and the eyeball tissue of the right eye was removed to make an eye cup,and frozen sections were made for HE staining.The sclera and choroid tissue morphology of different groups were observed under light microscope,and the sclera and choroid thickness of different groups were measured by"Image J" Image processing system.5.Molecular biological detection:Western Blot was used for protein detection.5 weeks after the intervention,the guinea pigs were sacrificed under excessive anesthesia.The eyeball tissue of the right eye was removed and fresh scleral tissue was isolated and stored at-80℃ for future use.The expression levels of a smooth muscle actin(αSMA)and matrix metalloproteinase-2(MMP-2)in sclera of guinea pigs were detected by Western Blot.Results:1.Effects of Yijing Tongluo Prescription on eye parameters:DIHM guinea pig model was successfully established 5 weeks after intervention.Comparison between groups:before intervention,there were no significant differences in the diopter and axis length of their right eyes between the TCM group,the model control group and the normal control group(P>0.05).3 weeks and 5 weeks after intervention,there were statistically significant differences in the diopter and axis length of their right eyes among 3 groups(P<0.05).The diopter and axial length in the model control group were significantly higher than those in the TCM group and the normal control group(P<0.05),while the diopter and axial length in the normal control group were significantly lower than those in the TCM group(P<0.05).Comparison within groups:Before intervention,3 weeks and 5 weeks after intervention,there were statistically differences in diopter and axial length among the 3 groups(P<0.05).In the normal control group,the diopter of guinea pigs gradually changed from hyperopia to emmetropia,while in the other two groups,it gradually developed from hyperopia to emmetropia,low and moderate myopia,and reached high myopia at the fifth week.2.Effects of Yijing Tongluo Prescription on ocular histomorphology of guinea pigs:5 weeks after intervention,HE staining showed that sclera and choroid tissues in normal control group,TCM group and model control group became thinner successively.Microscopically,the choroid vessels in the normal control group were orderly in structure,with higher capillary density and wider diameter than those in the other two groups.The nucleus of sclera tissue is blue and there are many cells,and the closer it is to the choroid layer,the more cells are distributed.The structure of collagen fiber is regular and compact.Compared with the normal control group,the choroid vascular layers in the model control group were loose and irregular,with relatively lower capillary density and smaller diameter.The number of scleral tissue cells also decreased,the arrangement of collagen fibers was sparse and loose,and the gap between the fibers increased.The tissue morphology of sclera and choroid in the TCM group was between the normal control group and the model control group,and the structure of each vessel layer in the choroid was slightly loose,and the capillary density and diameter width were lower than that in the normal control group,but higher than that in the model control group.The number of scleral tissue cells decreased slightly,and collagen fibers were sparsely arranged,but the fiber gap was smaller than the model control group.There were statistically differences in choroid and sclera thickness among the three groups(P<0.05).The choroid and sclera thickness in the model control group were significantly higher than those in the TCM group and the normal control group(P<0.05),while the choroid and sclera thickness in the normal control group were significantly lower than those in the TCM group(P<0.05).3.Effects of Yijing Tongluo Prescription on the expression of α-SMA and MMP-2 in sclera:5 weeks after intervention,the relative expression of α-SMA and MMP-2 in sclera of guinea pigs between the TCM group,the model control group and the normal control group was significantly different(P<0.05)._The scleral α-SMA and MMP-2 content of guinea pigs in the TCM group was significantly lower than that in the model control group(P<0.05).The content of MMP-2 in sclera of guinea pigs in model control group was significantly higher than that in normal control group(P<0.05).There was no significant difference in the content of α-SMA and MMP-2 in sclera between the TCM group and the normal control group(P>0.05).Conclusion:Yijing Tonglu Prescription can inhibit the remodeling of extracellular matrix(ECM)by inhibiting the transdifferentiation of scleral fibroblasts into myofibroblasts and inhibiting the degradation of extracellular matrix(ECM)in DIHM guinea pigs,furthermore,it inhibited the increase of diopter,axial length elongation,choroid and sclera thinning,and finally controlled the progression of myopia. |
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