Study On Mechanism Of Mingshi Prescription In Preventing And Controlling Myopia Based On Scleral Untargeted Metabolomics

Part ⅠObjectivesIdentification and analysis of Mingshi prescription’s components in blood,to serve a foundation for the study of Mingshi prescription pharmacodynamic substances and mechanism of action.MethodsBased on the previously established in vitro chemical composition sub-library of Mingshi Prescription,the same liquid phase and mass spectrometry conditions were used to detect the components in serum of rats before and after administration,and then the chemical components in blank serum and drug-containing serum were compared with in vitro chemical components to analyze and identify the components of Mingshi prescription’s components in blood.ResultsCompared with blank serum,46 components were identified in serum of Mingshi prescription at 0.5h,1h,2h and 4h.Among them,13 components were derived from Ginseng,mainly ginsenosides;7 species were derived from Salvia Miltiorrhiza,including tanshinone,salvia aldehyde,etc.8 species were derived from Poria cocos,mainly from poria cocos neoacid.Ten species were derived from Radix Polygonae,mainly saponins and oligosaccharides.Three species were derived from Gastrodia elata,mainly gastrodin and parishin.Two components are derived from Chinese yam and one is derived from Polygonatum cyrtonema.ConclusionsAccording to the modern pharmacological studies on Mingshi prescription’s components in blood and the related studies on the mechanism of myopia,it is confirmed that Mingshi prescription could act on multiple links in the occurrence and development of myopia.It is preliminarily speculated that the effect of Mingshi prescription on the prevention and control of myopia may be related to the improvement of blood circulation,angiogenesis,antioxidant,regulation of ECM,protection of the optic nerve and other mechanisms.Part ⅡObjectivesTo observe the effects of Mingshi prescription on diopter,axial length and scleral histopathology in LIM rabbits,and further clarify the effect of Mingshi prescription on prevention and control of myopia,and initially reveal the scientific connotation of the theory of myopic imbalance of Jing and Jin.MethodsThe experimental animals were randomly divided into blank control group,model control group,low-dose group,medium-dose group and high-dose group.Except the blank group,the rabbit myopia model was established using-10.0D concave lens,and different doses of Mingshi prescription were administered by gavage from the first day after modeling for 8 weeks.Before and after administration,the diopter and eye axis were measured by photometry and A-ultrasound,respectively.After administration,the samples were sacrificed,and the changes of scleral morphology and thickness were observed by HE staining.Results1.Diopter:Before intervention,there was no statistically significant difference in diopter among each group(P>0.05).After intervention,there was statistically significant difference in diopter among blank group,model group and Mingjifang intervention group(P<0.01).Compared with model group,there was no statistically significant difference in myopia diopter between low-dose and high-dose groups(P>0.05),while myopia diopter in medium-dose group was lower than that in model group(P<0.05).2.axial length:Before intervention,there was no statistically significant difference in axial length among all groups(P>0.05).After intervention,there was statistically significant difference in axial length among blank group,model control group and Mingshi prescription group(P<0.01),and there was statistically significant difference in axial length between model group and medium dose group of Mingshi prescription(P<0.05).But there was no significant difference in axial length between model group and low-dose and high-dose groups of Mingshi prescription(P>0.05).3.HE staining of sclera:(1)Morphological and structural characteristics of sclera:After intervention,the scleral collagen fibers in the blank control group were uniform in thickness,interwoven in order,and densely distributed.In the model control group,the collagen fibers were uneven in thickness,disordered in arrangement,poorly structured,and the collagen fiber gap increased.The regularity of scleral collagen fibers,the uniformity of collagen fibers and the changes of collagen fiber gap in lowdose,medium-dose and high-dose groups of Mingshi prescription were better than those in model control group,and the morphological and structural changes in the medium-dose group were the least.(2)Scleral thickness:Compared with blank control group,the scleral thickness of model control group and low-dose,mediumdose and high-dose groups of Mingshi prescription were decreased;compared with model group,the scleral thickness of low-dose,medium-dose and high-dose groups of Mingshi prescription were greater than model control group,the difference was statistically significant(P<0.05),while the scleral thickness of medium dose and high-dose groups was not statistically significant(P>0.05).However,the scleral thickness was higher than that in low-dose group(P<0.05).Conclusions1.In this study,the low myopia model of rabbit eyes was successfully induced by the hyperfocal of concave lens at-10.0D,which confirmed that hyperfocal of hyperfocal vision would lead to scleral remodeling,resulting in excessive growth of the eyeball.Moreover,from the aspect of scleral histology,it confirmed the scientific nature of fascia imbalance in the theory of Jing and Jin imbalance.2.It was confirmed that Mingshi prescription under the guidance of the theory of Jing Jin imbalance could improve the scleral remodeling,delay the axial length growth and myopia diopter deepening,and prevent the occurrence and development of myopia.The clinical equivalent dose of Mingshi prescription was preliminarily determined as the best dose to play the curative effect,and its specific mechanism of action still needs further study.Part ⅢObjectivesBased on the non-targeted metabolomics technology of sclera and the network pharmacological analysis of the Mingshi prescription’s components in blood,the key targets and pathways of Mingshi prescription’s action on myopic sclera were studied,and the mechanism of Mingshi prescription’s action in preventing and controlling the occurrence and development of myopia was clarified.Methods1.Rabbit eye LIM model was constructed,and the optimal dose of Mingshi prescription was used for intervention starting from the first day after modeling,lasting for 8 weeks,and the samples were sacrificed after administration.UPLC-QTOF-MS technique was used to collect mass spectrum data of scleral tissues of each group,and metabolites were identified based on KEGG,HMDB and LipidMaps.Partial least squares discriminant analysis(PLS-DA)combined with univariate analysis was used to screen differentially expressed metabolites.Metabolic pathways were analyzed based on KEGG database.2.Obtain drug targets of the Mingshi prescription’s components in blood through STITCH,Swiss Target Prediction,SEA Search Server and PharmMapper,then myopia is the key word to collect myopia related disease targets from the database.In order to explore the potential target of myopia prevention and treatment of Mingshi prescription.The protein interaction network was constructed using STRING software and Cytospace software.Topological parameters including degree were calculated to screen key targets of Mingshi prescription for myopia prevention and control,and then GO enrichment analysis and KEGG pathway enrichment analysis were performed on the selected key targets.Results1.In the non-targeted metabolomics analysis of sclera,105 differential metabolites were screened by comparing the model group and the Mingshi prescription administration group,and the pathway enrichment analysis was conducted on the obtained differential metabolites.It is mainly related to purine metabolism,circadian synergistic signal,taurine and low taurine metabolism,cysteine and methionine metabolism,oxidative phosphorylation,pentose phosphate pathway,tryptophan metabolism,mTOR signaling pathway,cAMP signaling pathway,FoxO signaling pathway,HIF-1 signaling pathway,etc.2.74 core targets of Mingshi prescription related to myopia,such as AKT1,EGFR,VEGFA,FGF2,IGF1,MMP2,MTOR,TGFB2 and BMP2,were screened out through the network pharmacological study on the components of Mingshi prescription into blood.Core targets are involved in PI3K-Akt signaling pathway,MAPK signaling pathway,HIF-1 signaling pathway,JAK-STAT signaling pathway,Rapl signaling pathway,EGFR signaling pathway,cAMP signaling pathway,Ras signaling pathway,mTOR signaling pathway,etc.3.Based on the results of scleral metabolomics and network pharmacology analysis,MMP-2,mTOR and HIF-1 signaling pathways are the intersection targets and signaling pathways of the two.Conclusions1.mTOR signaling pathway,HIF-1 pathway and MMP-2 play a key role in the prevention and treatment of myopia by Mingvision prescription.The prevention and treatment of myopia by Mingvision prescription may be achieved through these targets and pathways.2.In addition,the results of non-targeted metabolomics in the sclera suggest that Mingshi prescription could also prevent and control the occurrence and development of myopia by affecting purine metabolism,circadian synergistic signals,amino acid metabolism,FoxO signaling pathway,drug metabolism-other enzymes and other pathways and signaling pathways in the sclera,and its related mechanism still needs to be further studied.Part ⅣObjectivesThe key targets and pathways of Mingshi prescription for myopia prevention and control were further verified based on molecular biology technology.MethodsThe protein expressions and mRNA contents of COL I,MMP-2,mTOR and HIF1α in sclera of blank group,model group,Mingshi prescription low-dose group,Mingshi prescription medium-dose group and Mingshi prescription high-dose group were detected by Western blot and PCR.Results1.Western blot results showed that there were statistically significant differences in the expressions of mTOR,MMP-2 and COL I among the groups(P<0.01).The relative expression levels of mTOR and MMP-2 in model comtrol group were higher than those in blank group(P<0.05).Compared with model group,the expression of mTOR was decreased in high dose group of Mingshi prescription(P<0.01),but there was no significant difference between low dose group and medium dose group(P>0.05).The expression of MMP-2 in middle-dose and high-dose groups of Mingshi prescription was lower than that in model comtrol group(P<0.01),and there was no statistical significance between the two groups(P>0.05),while there was no statistical significance between low-dose group and model group(P>0.05).The expression of scleral COL I protein in model group was lower than that in blank control group,while the expression of COL I protein in middle-dose and high-dose groups was higher than that in model group(P<0.01,P<0.05),and there was no statistical significance between medium-dose and high-dose groups(P>0.05).2.RT-qPCR results showed that the relative expression levels of mTOR,HIF-1α,MMP-2 and COL I mRNA were significantly different among groups(P<0.01).The mRNA relative expressions of mTOR,HIF-la and MMP-2 in model control group were higher than those in blank control group(P<0.05).Compared with model control group,mTOR mRNA expression in each group of Minshi prescription was decreased,and was dose-dependent(P<0.05).The mRNA expression of HIF-la in medium dose group and high dose group was decreased compared with model control group,the difference was statistically significant(P<0.05),but there was no statistically significant difference between the two groups(P>0.05).The mRNA relative expression level of MMP-2 in the medium-dose and high-dose groups was lower than that in the model control group(P<0.01),and the high-dose group was lower than that in the medium dose group(P<0.05).There was no statistical significance between the low-dose and model control group(P>0.05).In addition,the relative expression of COL I mRNA in the model control group was lower than that in the blank control group(P<0.05),while the expression of COL I mRNA in the lowdose,medium-dose and high-dose groups of Mingshi prescription was higher than that in the model control group(P<0.01),and the medium-dose group was>highdose group>low-dose group,with statistical significance(P<0.05).ConclusionsBy regulating mTOR/HIF-1α signaling pathway,Mingshi prescription can improve scleral hypoxia,inhibit MMP-2 overexpression,increase scleral type I collagen content,and delay scleral remodeling,so as to play a role in controlling axial growth,deepening myopia diopter,and preventing the occurrence and development of myopia.