Investigate A Role Of PI3K? In Proliferative Vitreoretinopathy And Explore Novel Therapeutic Approaches To Retinopathy

Proliferative vitreoretinopathy(PVR)is a clinical syndrome related to retinal traction and detachment.The proliferation and contraction of cells with proliferation potential on the surface of the retina and vitreous cavity are the main reasons for their formation.In most cases,surgical reposition of the retina related to PVR can be achieved,but the visual effect is still not satisfactory.It is found in up to 10%of retinal detachments that most patients will still have low vision despite complicated surgery.Diabetic retinopathy,especially its proliferative stage(proliferative diabetic retinopathy,PDR),is related to pathological angiogenesis and is the second leading cause of blindness.Currently,PDR treatment methods usually use VEGF antibodies(rannibizumab or bevacizumab)or recombinant proteins composed of antibody Fc fragments fused to the extracellular domain of VEGF receptors to neutralize vascular endothelial growth factors in the vitreous.However,drug resistance has been observed in many PDR patients,and new treatments are urgently needed.This thesis is based on the CRISPR-Cas9 gene editing technology to study the mechanism of PI3K?in the formation of PVR,explore new ways of PVR gene therapy;and screen potential lead compounds or drugs for the treatment of retinopathy(such as PVR and PDR)from natural monomer compounds,Provide new treatment methods for the treatment of retinal diseases.The main research contents and results of this paper are as follows:(1)Using CRISPR-Cas9 gene editing technology to study a role of phosphoinositide 3-kinase(PI3K)in vitreous-induced Akt/Mdm2/p53activation and retinal pigment epithelial cell migrationPhosphoinositide 3-kinase(PI3K)is a protein kinase of the lipid family that plays a key role in transmitting signals from cell surface molecules to intracellular protein effectors.The key PI3Ks include PI3K?,PI3K?and PI3K?.PI3Ks play an important role in experimental proliferative vitreoretinopathy(PVR),but it is not clear which PI3K subtype is involved in PVR.Experiments have found that p110?is specifically and highly expressed in RPEs cells.In order to clarify the molecular mechanism of PI3K subtypes in the formation of PVR,CRISPR-Cas9 gene editing technology was used to construct a p110?-knockdown cell line,and the specific inhibitor of p110?,Idelalisib,was used for molecular and pharmacology inactivation of p110?.We found that inactivation of p110?can prevent the activation of the Akt/Mdm2/p53 pathway induced by vitreous and the inherent cellular response of the cell in the formation of PVR.Therefore,these methods of genome editing and pharmacology(Idelalisib)targeting PI3K?have great potential to treat RPE(such as PVR)related diseases.(2)To study a role of PI3K?signal transduction loop induced by TGF-?2 in human retinal pigment epithelial cellsThe PI3K/Akt signaling pathway plays a key role in experimental PVR,and studies based on(1)found that the PI3K?subtype plays a major role in the activation of the vitreous-induced Akt.In the vitreous of patients,the level of transforming growth factor(TGF)-?2 is closely related to the severity of PVR.Therefore,this section mainly clarifies that the inactivation of PI3K?through CRISPR/Cas9 or pharmacology(Idelalisib inhibitor)can effectively block TGF-?2-induced Akt activation and epithelial-mesenchymal cell transformation(EMT).Idelalisib's inhibitory effect on PI3K?can also prevent PVR formation in mice.Using CRISPR/Cas9 and pharmacological methods,it was further discovered that the PI3K?/Akt/Mdm2/NF-?B pathway induced by TGF-?2 controls the expression of PI3K's catalytic subunit p110?and generates a positive feedback loop.The discovery of this new pathway paves a way to the treatment of PVR.(3)Chalcomoracin prevents vitreous-induced Akt activation and retinal pigment epithelial cell migrationIn order to explore new ways to treat retinopathy,this research uses the natural monomer compound Chalcomoracin(CMR)prepared in the laboratory,a new compound purified from ultraviolet-induced mulberry leaves,to study the inhibition of vitreous-induced signal transduction events and PVR Inherent cellular response.Studies have shown that the IC₅₀ of CMR for ARPE-19 cells is35.5?M at 72 hours,while 5?M CMR can significantly inhibit the activation of Akt and p53 inhibition induced by the vitreous.In addition,it was found that the chemical substance effectively blocked the proliferation,migration and contraction of ARPE-19 cells stimulated by the vitreous,indicating that CMR is a promising PVR preventive agent.(4)Capilliposide B blocks VEGF-induced angiogenesis in human primary retinal microvascular endothelial cellsAbnormal angiogenesis is associated with proliferative diabetic retinopathy(PDR).The main purpose of this research is to explore a new way to treating PDR.Capilliposide B(CPS-B)is a new olean fruit triterpene saponins derived from Lysimachia capillipes Hemsl,which can inhibit vascular endothelial growth factor(VEGF)-induced angiogenesis signaling pathway and cellular response of cells(human retinal microvascular endothelium,HREC)in PDR.Studies have shown that CPS-B has an IC₅₀ of 8.5?M for HREC cells at 72 hours,while 1?M CPS-B specifically inhibits the activation of VEGFR2 and its downstream signal transduction enzymes Akt and Erk induced by VEGF.In addition,the chemical was found to effectively block the proliferation,migration and angiogenesis of HREC stimulated by VEGF,which indicates that CPS-B is an effective preventive agent for preventing angiogenesis-related diseases(such as PDR).