A Network Pharmacology Approach to Uncover the Molecular Targets and Associated Potential Pathways of Lycii Fructus for the Treatment of Retinitis Pigmentosa

Objective To explore the molecular targets and associated potential pathways of Lycii Fructus(LF,Gou Qi Zi,枸杞子)in the treatment of retinitis pigmentosa(RP)by the approaches of network pharmacology and bioinformatics.Methods The potential blood-entry active ingredients and targets of LF were retrieved by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).RP-related gene targets were retrieved through disease comprehensive databases.Protein-protein interaction(PPI)network of LF component-targets and RP disease-targets was constructed by STRING,and the intersection of the 2 networks was extracted.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of theintersection network were conducted by Database for Annotation,Visualization and Integrated Discovery(DAVID).CytoHubba was used to screen the key targets.Results A total of 188 chemical constituents related to LF was retrieved from TCMSP database.45 active ingredients were screened according to pharmacokinetic parameters oral bioavailability(OB)and drug similarity(DL).36 active ingredients were further screened and 201 targets related to these constituents were obtained.206 target genes directly related to RP were obtained from the disease comprehensive databases,and 89 genes were obtained from the intersection of componenttarget and disease-target PPI network.These genes were mainly involved in intracellular signal transduction,GTPase activity regulation,cell morphology regulation,and other biological processes.Molecular functions were mainly related to Rho guanine nucleotide exchange factor activity,GTPase activator activity,receptor signal protein serine/threonine kinase activity and so on.They were enriched in the cytoplasm,cell membrane,Golgi apparatus,and other regions.The mechanism was related to cell cycle pathways,neurotrophin signaling pathways,Ras signaling pathways,and so on.10 key gene targets of LF in the treatment of RP were screened.Conclusions The material basis for LF to exert its pharmacodynamic effect is 36 active ingredients such as cycloartenol,mandenol,and so on.The key targets of LF in the treatment of RP include 10 genes,such as Rho,PAK,and so on.The main mechanism is related to the regulation of the Ras signaling pathway,neurotrophin signaling pathway,cell cycle related pathway,and other signaling networks.

Visual Prostheses: Technological and Socioeconomic Challenges

Visual prostheses are now entering the clinical marketplace. Such prostheses were originally targeted for patients suff ering from blindness through retinitis pigmentosa(RP). However, in late July of this year, for the first time a patient was given a retinal implant in order to treat dry agerelated macular degeneration. Retinal implants are suitable solutions for diseases that attack photoreceptors but spare most of the remaining retinal neurons. For eye diseases that result in loss of retinal output, implants that interface with more central structures in the visual system are needed. The standard site for central visual prostheses under development is the visual cortex. This perspective discusses the technical and socioeconomic challenges faced by visual prostheses.

Effects of Lycii Fructus and Salviae Miltiorrhizae on the Syndrome of Deficiency with Blood Stasis in RCS(rdy-/-, p-/-) Rats with Retinitis Pigmentosa: An Intervention Study

Objective To investigate the effects of Lycii Fructus(LF,Gou Qi Zi,枸杞子)and Salviae Miltiorrhizae Radix Ex Rhizoma(SM,Dan Shen,丹参)on the syndrome of deficiency with blood stasis in the RCS(rdy-/-,p-/-)rats with retinitis pigmentosa(RP).Methods A total of 32 RCS(rdy-/-,p-/-)rats were divided into 4 groups(equal amounts of female and male rats in each group):model group treated with 0.9%normal saline,LF group treated with LF formula granules,SM group treated with SM formula granules,and LF and SM(L·S)group treated with LF and SM formula granules.Eight RCS(rdy+/+,p+/+)rats(4 males and 4 females)were treated with 0.9%normal saline to serve as blank group.The contents of E2,PG,P-Selectin,plasma viscosity,whole blood relative index of the high shear rate and fibrinogen content in plasma,and the content of cAMP and cGMP in retinal homogenate were detected.The retina was evaluated by hematoxylin-eosin staining.Results The contents of E2,PG,P-Selectin,plasma viscosity,whole blood relative index of the high shear rate,and fibrinogen content in the plasma of L·S group significantly differed from those of model group(P<0.01),but were similar to those of blank group.The contents of cAMP and cGMP in the retinal homogenate of L·S group significantly differed from those in model group(P<0.01)but were similar to those in blank group(P>0.05).Conclusions LF and SM can effectively treat retinitis pigmentosa by ameliorating the syndrome of deficiency with blood stasis.

A COMPLETE SCREEN FOR MUTATIONS OF THE RHODOPSIN GENE IN A PANEL OF CHINESE PATIENTS WITH AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA

Objective To evaluate the prevalence of rhodopsin (RHO) mutations and the genotype-phenotype relationships in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) by conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. Methods We have screened the five coding exons and splice sites of RHO gene in 27 probands who had no relativity from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using CSGE and direct DNA sequencing. Family members of some probands with disease-associated mutations were also genotyped to determine whether the RHO mutations segregated with retinitis pigmentosa (RP) in their families. Results Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified separately in two families, thus the frequency of RHO mutations among this set of Chinese ADRP families is about 7.4% (2/27). Pro347Leu mutation was found in one ADRP proband as well as three her children who also had RP. She had relatively early onset at about 17 years. The only one child without this mutation had no symptom or sign of RP at age of 34. Pro327 (1-bp del) was identified in a late-onset ADRP patient, who appeared night blindness around 30 years old and in her fifties electroretinogram (ERG) has been flat in both scotopic and photopic phases. Family analysis showed that this mutation also existed in her younger dau-ghter and her elder sister, both of them also had RP. Three other family members were genotypically and phenotypically normal. Neither of the two mutations was detected in 100 normal controls.Conclusions The frequency of RHO mutations in Chinese patients was lower than that in Europe and North America. The phenotype of the patients with Pro347Leu corresponded to type 1 ADRP, with severe rod degeneration and some cone preservation later, while the phenotype of the patients carrying Pro327 (1-bp del) corresponded to type 2 ADRP, with a concomitant loss of rod and cone visual function. CSGE was found to be a sensitive, simple, and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.

Mass genetics study of rhodopsin point mutations in retinitis pigmentosa

Objective: To evaluate the incidence and pattern of rhodopsin (RHO) mutations in Chinese patients with retinitis pigmentosa (RP). Methods: Conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing were applied to detect point mutations that occurred in the five coding exons and splice sites of RHO gene in 98 index patients with RP. Results: Four patients of one ADRP family were found to have a missense mutation at codon 347, Pro347Leu. One late-onset RP patient and her daughter, without clinical expression at present, were discovered to have a novel frameshift mutation at codon 327, Pro327 (1-bp del) . Neither of the two mutations was found in 100 normal controls. Ala299Ser was found in one RP patient. Two control subjects also had Ala299Ser, suggesting its nonpathogenicity and just single nucleotide polymorphism (SNP). Conclusion: Two RP patients had rhodopsin mutations, thus the expected frequency of RHO mutations in RP is about 2.0% (95% confidence interval: 0.3%-4.4%). A highly conserved C-terminal sequence QVS(A) PA was altered due to Pro347Leu and thereby misdirecting rhodopsin to incorrect subcellular location. Loss of all phosphory-lation sites at the C-terminus and a highly conserved sequence QVS(A)PA may occur because of Pro327(1-bp del) . To elucidate the predominant biochemical defects in such mutant, transgenic mice and transfected culture cells carrying Pro327(1-bp del) would be of great value.

Two novel mutations of the retinitis pigmentosa GTPase regulator gene in two Chinese families with X-linked retinitis pigmentosa

Objective To detect mutations of the retinitis pigmentosa GTPase regulator (RPGR) gene in two Chinese X-linked retinitis pigmentosa families. Methods Fragments of exons 1-19 of the RPGR gene were amplified with intronic primers, using genomic DNA as template. The polymerase chain reaction (PCR) products were analysed by single-strand conformation polymorphism (SSCP) and direct sequencing. Mutations were identified by comparing DNA sequences of the patients with those of the normal controls.Results Two novel mutations, c1536delC and E332X, were identified in exons 12 and 9 of the RPGR gene in both families. Each mutation was the first mutation found in their respective exons. Both mutations were predicted to cause premature termination, which resulted in truncated proteins without normal functions of the RPGR products.Conclusions Both mutations are the genetic basis of the pathogenesis in the respective families. Our data might be helpful in analysing the function of the RPGR protein.

Dawn of ocular gene therapy:implications for molecular diagnosis in retinal disease

Personalized medicine aims to utilize genomic information about patients to tailor treatment. Gene replacement therapy for ra- re genetic disorders is perhaps the most extreme form of personalized medicine, in that the patients＇ genome wholly determines their treatment regimen. Gene therapy for retinal disorders is poised to become a clinical reality. The eye is an optimal site for gene therapy due to the relative ease of precise vector delivery, immune system isolation, and availability for monitoring of any potential damage or side effects. Due to these advantages, clinical trials for gene therapy of retinal diseases are currently underway. A necessary precursor to such gene therapies is accurate molecular diagnosis of the mutation（s） underlying disease. In this review, we discuss the application of Next Generation Sequencing （NGS） to obtain such a diagnosis and identify disease causing genes, using retinal disorders as a case study. After reviewing ocular gene therapy, we discuss the application of NGS to the identification of novel Mendelian disease genes. We then compare current, array based mutation detection methods against next NGS-based methods in three retinal diseases： Leber＇s Congenital Amaurosis, Retinitis Pigmentosa, and Stargardt＇s disease. We conclude that next-generation sequencing based diagnosis offers several advantages over array based methods, including a higher rate of successful diagnosis and the ability to more deeply and efficiently assay a broad spectrum of mutations. However, the relative difficulty of interpreting sequence results and the development of standardized, reliable bioinforrnatic tools remain outstanding concerns. In this review, recent advances NGS based molecular diagnoses are discussed, as well as their implications for the development of personalized medicine.

Generation of nonhuman primate retinitis pigmentosa model by in situ knockout of RHO in rhesus macaque retina

Retinitis pigmentosa(RP)is a form of inherited retinal degenerative diseases that ultimately involves the macula,which is present in primates but not in the rodents.Therefore,creating nonhuman primate(NHP)models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future.Here we applied adeno-associated virus(AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque(Macaca mulatta)to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP.Through a series of studies,we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression.More importantly,the mutant macaque retinae displayed clinical RP phenotypes,including photoreceptor degeneration,retinal thinning,abnormal rod subcellular structures,and reduced photoresponse.Therefore,we suggest somatic editing of the RHO gene is able to phenocopy RP,and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.