| Objective: To establish a laser-induced CNV rat model and study the mechanism of Mongolian medicine Mingmu-11 inhibiting choroidal neovascularization in wet age-related macular degeneration,providing experimental evidence for the treatment of wet age-related macular degeneration.content and method:1.Analyze the source,solution and clinical application of Mongolian Medicine Mingmu-11(MM-11)to provide a scientific basis for further development and medical application.2.In order to observe the short-term efficacy of Mongolian medicine Mingmu-11 combined with Compaqcept in the treatment of wet age-related macular degeneration(w AMD),68 cases(68 eyes)were prospectively included from January 2018 to June 2019 in the Affiliated Hospital of Inner Mongolia University for Nationalities.w AMD patients were randomly divided into the combination drug group and the conbercept group only according to the time of admission.All patients were injected with intravitreal injection of 0.5 m L of conbercept in the vitreous cavity.The Mongolian medicine combined with conbercept group was given oral MM-11,2 times a day,13 capsules each time,3 weeks as a course of treatment,and one course per month.All patients were examined before the first injection and 1,3 months after the injection.The best corrected visual acuity(BCVA),central macular thickness(CMT),and choroidal neovascularization(CNV)area were involved to evaluate the efficacy of Mongolian Medicine Mingmu-11.3.Mingmu-11 was used for intragastric administration to laser-induced CNV rat models,and its inhibitory effect on choroidal neovascularization was evaluated by morphological and molecular biological tests.120 BN(Brown Norway)rats(body weight 130-150g)were randomly divided into 6 groups according to body weight,namely,blank group,model group,Mingmu-11 low-dose administration group,middle-dose administration group,high-dose administration group,positive control group,which was the model group + the conbercept group.A 532 nm laser photocoagulator was set at a distance of 2-3PD from the optic papillae to coagulate 8 points in each eye between two blood vessels around the optic papilla.The spot energy output power was 150-200 mw,the diameter was 50 um,and the exposure time was 0.1s.Dosing was started on the first day after modeling.On the 21 st day,fundus fluorescence angiography(FFA)and optical coherence tomography(OCT)were performed and the rats were sacrificed.Blood was taken from the abdominal aorta.The expression of choroidal neovascularization(CNV)-related proteins was analyzed by ELLISA,and CNV leakage area and CNV thickness were detected by HE staining.4.A oxcidative stress model of choroidal microvascular endothelial cells(CECs)was made,and the Mingmu-11 drug-containing serum? Ming Mu-11 methanol extract and 11 screened active drugs were tested by MTT method,and the inhibition rate of CECs was observed,and screen the components with significant activity and evaluate their inhibitory effect on CNV from the cellular level.;MTT assay was used to detect the CEC cell inhibition rate;Matrigel gel was used to detect the CEC cell channel formation ability.5.The RNA-seq sequencing technology of transcriptomics was used to study the inhibitory mechanism of cinnamaldehyde on CEC cell angiogenesis,from the RNA level,to study the inhibitory effect of safranal on CEC cells and providing data support for the functional analysis of Mongolian medicine Ming Mu-11.At the same time,Western bloting was applied to verify the pathway of differential gene enrichment.Result:1.Mongolian medicine Mingmu-11 stems from “Inner Mongolia Mongolian Medicine Preparation Specification”,which was a traditional tested prescription.The prescription is composed of terminalis fruit,saffron,artificial bezoar,clove,Rhezoma Gymnadeniae,talc,vermiculite(manufactured),bear bile powder,cinnabar,white osmanthus,and turmeric and its active chemicals such as crocetin,curcumin,gallic acid can treat macular degeneration.The prescription has the function of clearing heat and improving eyesight and is effective in treating Qisu Xila eye disease,narcoma,red eyes,blurred vision and dry conjunctivitis.The clinical curative effect is exact,and the treatment effect on fundus hemorrhage is remarkable.2.Mongolian medicine Mingmu-11 and anti-VEGF drugs can significantly improve vision and protect visual function in patients with wet AMD;Mongolian medicine Mingmu-11 combined with anti-VEGF drugs can promote bleeding absorption,reduce CNV area of macular area,and combination effect is better than that of anti-VEGF drugs alone.Mongolian medicine Mingmu-11 combined with anti-VEGF is safe and effective in the treatment of wet AMD;combined Mongolian medicine therapy can reduce the number of wet AMD treatments,enhance the effectiveness and persistence of suppressing new blood vessels,and improve visual function at the same time.3.After the laser-induced BN rat CNV model was successfully performed,the area of fluorescence leakage and the thickness of CNV measured by HE staining showed,(1)The model group had significant differences compared with the treatment groups,P <0.05,which was statistically significant.(2)There was no significant difference between the Ming Mu-11 medium-dose treatment group and the positive control group,P > 0.05,there was no significant difference between the two groups in the treatment of CNV,P > 0.05;the positive control group and the Ming Mu-11 medium-dose group were compared respectively with lose-dose and high-dose treatment groups.The difference of each comparison was significant,P< 0.05,(3)which was statistically meaningful.)There was no significant difference between Ming Mu-11 low and high-dose groups,P > 0.05,no statistical significance.The expression of VEGF-165 and integrin ?6?1 in serum by enzyme-linked immunoassay(ELISA)showed,the structure of detecting VEGF-165 was as follows:(1)There was a significant difference in VEGF-165 content between the blank group and the other groups,P <0.05;there was a significant difference between the model group and each treatment group P(2)<0.05;There was no difference between the Ming Mu-11 medium-dose group)and the positive control group,P >0.05.(3)Ming Mu-11 medium-dose group and positive control group were compared respectively with Ming Mu-11 low-dose and high-dose groups and the difference in each comparison was significant,P<0.05.Enzyme-linked immunoassay(ELISA)detection of integrin ?6?1 content was significantly different between the blank group and the model group,the model group and the medium-dose,high-dose,and positive control groups,P <0.05.Comparison between the treatment groups: there was a significant difference between the low-dose group and the positive control group,and between the high-dose group and the positive control group,P <0.05;there was no significant difference between the medium-dose group and the positive control group,P> 0.05.4.A oxcidative stress model of choroidal microvascular endothelial cells(CECs)was made,and the Mingmu-11 drug-containing serum?Ming Mu-11 methanol extract and 11 screened active drugs were tested by MTT method,and the inhibition rate of CECs was observed.Results were as follows: Ming Mu-11 methanol extract and crocetin exhibited noticeably higher inhabitation rate as the drug concentration increased;Matrigel gel was used to test the pipeline formation ability of CECs.Results showed that before angiogenesis induction,crocetin had no effect on cell proliferation and growth,and the cell morphology and density growth of each group tended to be the same.After the induction,safranal exhibited a dose-dependent inhibition of angiogenesis with increasing doses.5.The RNA-seq sequencing technology of transcriptomics was used to study the inhibitory mechanism of cinnamaldehyde on CEC cell angiogenesis,from the RNA level: screening for differentially expressed m RNA expression profiles of the drug group and model group,the results showed that there were 314 m RNAs in the drug group compared with the model group,of which,145 m RNAs were up and 169 m RNAs were down.The m RNAs shared by the model group,the control group,the drug group,and the model group were screened.The GO function analysis showed that the molecular function of GO was enriched to 66 terms,the cellular component was enriched to 75 terms,and the biological pathway was enriched to 411 terms.KEGG pathway enrichment analyzed the pathways in which differential genes may participate,and a total of 7 pathways were enriched.The pathways related to the occurrence and development of AMD disease were PI3 K / Akt pathway and FOXO pathway.Western blot verification of the differentially expressed gene-rich pathway confirmed the IRS-PI3K-PDK1 / 2-AKT-BAD signaling pathway,which can be simply described as the IRS-PI3K-PDK signaling pathway.On this pathway,the original was progressively phosphorylation activated,and finally the cell was induced towards the BAD-mediated apoptosis signal pathway.The verification of western blot proved that Mingmu-11 can regulate this pathway and treat AMD.Conclusion:Mingmu-11 comes from "Inner Mongolia Mongolian Medicine Preparation Specification ",which was a traditional prescription.It consists of terminalia chebula Retz,saffron,artificial bezoar,clove,ginseng,talc,vermiculite(manufactured),bear bile powder,cinnabar,white osmanthus,and turmeric.It has the function of clearing liver heat and improving eyesight.The clinical effect is definite,and the treatment effect on fundus hemorrhage is remarkable.2.Mongolian medicine Mingmu-11 and anti-VEGF drugs can significantly improve the vision of wet AMD patients and protect the visual function of patients;Mongolian medicine Mingmu-11 combined with anti-VEGF drugs can significantly reduce the number of treatments of wet AMD than anti-VEGF drugs alone and it can improve the effectiveness and persistence of inhibiting new blood vessels,while enhancing visual function.3.The laser-induced CNV model of BN rats was successful,and the area of fluorescence leakage and the thickness of CNV measured by HE staining showed that the MM-11 medium-dose group and the positive control group had a significant absorption effect on retinal leakage.The expression of VEGF-165 and integrin ?6?1 in serum was measured by enzyme-linked immunoassay(ELISA).The results showed that: MM-11 and positive control drugs were effective in the treatment of CNV.VEGF-165 and integrin ?6?1 were involved in cell growth and pathological process,and it plays an important role in cell migration and neovascularization.4.MTT method was used and test results were: Mingmu-11 methanol extract and safranal,the active ingredient,significantly inhibited h RMECs cell proliferation.Matrigel gel tested the CECs pipeline formation ability and it showed that before angiogenesis induction,safranal had no effect on cell proliferation and growth;after angiogenesis induction,safranal increased with its dose,showing a dose-dependent inhibition of angiogenesis.5.The RNA-seq sequencing technology of transcriptomics was used to study the inhibitory mechanism of cinnamaldehyde on CEC cell angiogenesis,from the RNA level,to study the inhibitory mechanism of cinnamaldehyde on the angiogenesis of CEC cells,and KEGG pathway enrichment analysis was performed to find out the IRS-PI3K-PDK signaling pathway in which the differential genes may participate,before western blot verification of this pathway was completed.It has been shown that Mingmu-11 can regulate this pathway and treat AMD.The study provides experimental basis for the application of Mongolian medicine Mingmu-11 in the treatment of w AMD and fundus neovascular diseases. |
PDF Full Text Request