Prevalence and associated factors for pterygium in a Chinese rural population with type 2 diabetes in a cross-sectional study: Jiangsu Diabetic Eye Disease Study (JDEDS).

PURPOSE: To investigate the pterygium prevalence and evaluate risk factors of pterygium in rural type 2 diabetic (D2M) patients aged 50 years and above in Funing Country, Jiangsu Province, China. METHODS: A cross-sectional ophthalmic survey was conducted in type 2 diabetes mellitus (D2M) patients aged ≥ 50 years in Funing County, Jiangsu Province, China, which was named Jiangsu Diabetic Eye Disease Study (JDEDS). All participants underwent a comprehensive questionnaire and ocular examination. Pterygium was diagnosed by slit lamp examination. The risk factors associated with pterygium were evaluated with logistic regression models. RESULTS: The prevalence of pterygium was 22.37% (n = 427) and 95% confidence interval (CI) (20.50-24.24%) in D2M patients aged 50 years and above in JDEDS. The prevalence of pterygium was 18.32% (95% CI 15.33-21.32%) in men and 24.43% (95% CI 22.06-26.80%) in women. Women had a higher prevalence than men (p = 0.001). Multivariate analysis showed, for male participants with D2M, pterygium was independently associated with increasing age [70-79 years: OR and 95% CI 2.49(1.20-5.18), p = 0.014; ≥ 80 years: 4.84(2.04-11.47), p < 0.001], while cigarette smoking was the protective factors, especially in current smoker [OR and 95% CI 0.79(0.67-0.92); p = 0.003]. For female participants with D2M, age [60-69 years OR and 95% CI 1.68(1.07-2.62), p = 0.023; 70-79 years: 2.62(1.69-4.06), p < 0.001; ≥ 80 years:3.24(1.70-5.90), p < 0.001], hypertension [OR and 95% CI 1.40(1.05-1.87), p = 0.024], BMI 24-27.9 [OR and 95% CI 1.20(1.00-1.44), p = 0.047], higher HbA1c [(5.6-7.9) % OR and 95% CI 1.42(1.10-1.82), p = 0.006; (8.0-9.9) %: 1.32(1.10-1.58), p = 0.003] were risk factors. CONCLUSIONS: D2M patients aged over 50 years has a high prevalence of pterygium in JDEDS. The pterygium prevalence is higher in female D2M participants. Diabetes and related factors may be risk factors of pterygium in female D2M patients. Further studies are needed to explore the gender difference in the pathogenesis of pterygium in D2M subjects.

Exosomal microRNA-222-3p increases UVB sensitivity of lens epithelium cells by suppressing MGMT.

BACKGROUND: Age-related cataract (ARC) is a leading cause of blindness worldwide with multiple pathogenic factors. Oxidative damage of lens epithelium cells (LECs) is one of the well-accepted pathogenesis of ARC which can be regulated by DNA repair genes (DRGs). The present research aimed to clarify the regulatory mechanism of exosomal microRNAs (miRNAs) on DRGs in LECs. METHODS: The LECs oxidative damage model was established by UVB-irradiation on SRA01/04 (human lens epithelium cell line). Exosomes from UVB-irradiated cells (UVB-exo) and exosomes from normal control cells (NC-exo) were collected from the culture medium. To explore the functions of LECs exosomes, SRA01/04 were incubated with UVB-exo/NC-exo. Then, we detected SRA01/04 proliferation, viability and apoptosis respectively using 5'-ethynyl-2'-deoxyuridine (EdU), cell-counting kit-8 (CCK-8) and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. Next, the miRNA expression profiles of UVB-exo and NC-exo were identified by miRNA microarrays. RNA expression in exosomes, cells, and clinical samples was verified by qRT-PCR. The location and expression of MGMT and CD63 proteins were detected by immunofluorescence and western blot. The 3'UTR regulation of miR-222-3p to MGMT was verified by luciferase analyses. RESULTS: MGMT down-regulated while miR-222-3p up-regulated in LECs sub-central anterior capsule from ARC lenses. MGMT and miR-222-3p expressions in central and peripheral LECs from anterior lens capsules were differential. UVB-exo can transport the up-regulated miR-222-3p from oxidative-damaged LECs to normal LECs, which could suppress MGMT expression and increase UVB sensitivity of LECs. CONCLUSIONS: Findings on exosomal miRNA functions provided novel insights into pathogenesis of ARC. Exosomal miR-222-3p can be a potential target for prevention and cure of ARC.

Geniposide alleviates choroidal neovascularization by downregulating HB-EGF release from RPE cells by downregulating the miR-145-5p/NF-κB axis.

Age-related macular degeneration (AMD), mainly wet AMD, is the major reason for nonreversible vision loss worldwide. Choroidal neovascularization (CNV) is a characteristic pathological manifestation of wet AMD. Stress or injury to the retinal pigment epithelium (RPE) induces proangiogenic factors that drive CNV. An iridoid glycoside extracted from the fruit of gardenia, geniposide (GEN) plays an antiangiogenic role. In this study, GEN inhibited the transcription and expression of heparin-binding epidermal growth factor (HB-EGF), a proangiogenic factor, in hypoxic RPE cells and a mouse laser-induced CNV model. Inhibition of glucagon-like peptide-1 receptor (GLP-1R), a GEN receptor blocker, eliminated the protective effect of GEN. Additionally, GEN decreased the transcription and expression of HB-EGF in hypoxia-exposed RPE cells by downregulating the miR-145-5p/NF-κB axis. Therefore, our research provides a promising novel strategy for wet AMD therapy.

Brivanib, a multitargeted small-molecule tyrosine kinase inhibitor, suppresses laser-induced CNV in a mouse model of neovascular AMD.

In age-related macular degeneration (AMD), choroidal neovascularization (CNV), a major pathologic feature of neovascular AMD (nAMD), affects 10% of patients, potentially causing serious complications, including vision loss. Vascular endothelial growth factor receptor 2 (VEGFR2) and fibroblast growth factor receptor 1 (FGFR1) contribute to the pathogenesis of CNV. Brivanib is an oral selective dual receptor tyrosine kinase (RTK) inhibitor of FGFRs and VEGFRs, especially VEGFR2 and FGFR1. In this study, brivanib inhibited zebrafish embryonic angiogenesis without impairing neurodevelopment. In a mouse CNV model, brivanib intravitreal injection blocked phosphorylation of FGFR1 and VEGFR2 and reduced CNV leakage, area, and formation without causing intraocular toxicity. Moreover, brivanib oral gavage reduced CNV leakage and area. Accordingly, brivanib remained at high concentrations (above 14,000 ng/ml) in retinal/choroidal/scleral tissues following intravitreal injection. Similarly, brivanib remained at high concentrations (over 10,000 ng/ml) in retinal/choroidal/scleral tissues following oral gavage. Finally, in vitro cell experiments demonstrated that brivanib inhibited the proliferation, migration and tube formation of microvascular endothelial cells. In conclusion, our study suggested that brivanib treatment could be a novel therapeutic strategy for nAMD.

Andrographolide attenuates choroidal neovascularization by inhibiting the HIF-1α/VEGF signaling pathway.

Choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), leads to severe vision loss amongst the elderly in the developed countries. Currently, the premier treatment for AMD is anti-VEGF therapy, which has limited efficacy, and is still controversial. Previous studies have showed that Andrographolide (Andro) had various biological effects, including anti-angiogenesis, anti-inflammation, and antioxidant. However, the effect of Andro on the formation of CNV has not been studied thus far. Here our results showed that Andro reduced the expression levels of HIF-1α and VEGF in the RF/6A cells chemical hypoxia model and the laser-induced CNV mouse model. Moreover, Andro inhibited the tube formation activity of RF/6A cells under hypoxic conditions. Furthermore, intraperitoneal injection of Andro reduced the severity of choroidal vascular leakage and the size of CNV in the laser-induced CNV mouse model, indicating that Andro attenuated the development of CNV by inhibiting the HIF-1α/VEGF signaling pathway. These results suggest that Andro could be a potential novel therapeutic agent for AMD.

Long non-coding RNA MEG3 promotes cataractogenesis by upregulating TP53INP1 expression in age-related cataract.

Age-related cataract (ARC) is the leading cause of visual impairment or even blindness among the aged population globally. Long non-coding RNA (LncRNA) has been proven to be the potential regulator of ARC. The latest study reveals that maternally expressed gene 3 (MEG3) promotes the apoptosis and inhibits the proliferation of multiple cancer cells. However, the expression and role of MEG3 in ARC are unclear. In this study, we investigated the effects of MEG3 in ARC and explored the regulatory mechanisms underlying these effects. We observed that MEG3 expression was up-regulated in the age-related cortical cataract (ARCC) lens capsules and positively correlated with the histological degree of ARCC. The pro-apoptosis protein, active caspase-3 and Bax increased in the anterior lens capsules of ARCC tissue, while the anti-apoptotic protein Bcl-2 decreased compared to normal lens. Knockdown of MEG3 increased the viability and inhibited the apoptosis of LECs upon the oxidative stress induced by H2O2. MEG3 was localized in both nucleus and cytoplasm in LECs. MEG3 facilitated TP53INP1 expression via acting as miR-223 sponge and promoting P53 expression. Additionally, TP53INP1 knockdown alleviated H2O2-induced lens turbidity. In summary, MEG3 promoted ARC progression by up-regulating TP53INP1 expression through suppressing miR-223 and promoting P53 expression, which would provide a novel insight into the pathogenesis of ARC.

lncRNA H19 contributes to oxidative damage repair in the early age-related cataract by regulating miR-29a/TDG axis.

Age-related cataract (ARC) is caused by the exposure of the lens to UVB which promotes oxidative damage and cell death. This study aimed to explore the role of lncRNA H19 in oxidative damage repair in early ARC. lncRNAs sequencing technique was used to identify different lncRNAs in the lens of early ARC patients. Human lens epithelial cells (HLECs) were exposed to ultraviolet irradiation; and 8-OHdG ELISA, Cell counting kit 8 (CCK8), EDU, flow cytometry and TUNEL assays were used to detect DNA damage, cell viability, proliferation and apoptosis. Luciferase assay was used to examine the interaction among H19, miR-29a and thymine DNA glycosylase (TDG) 3'UTR. We found that lncRNA H19 and TDG were highly expressed while miR-29a was down-regulated in the three types of early ARC and HLECs exposed to ultraviolet irradiation, compared to respective controls. lncRNA H19 knockdown aggravated oxidative damage, reduced cell viability and proliferation, and promoted apoptosis in HLECs, while lncRNA H19 overexpression led to opposite effects in HLECs. Mechanistically, miR-29a bound TDG 3'UTR to repress TDG expression. lncRNA H19 up-regulated the expression of TDG by repressing miR-29a because it acted as ceRNA through sponging miR-29a. In conclusion, the interaction among lncRNA H19, miR-29a and TDG is involved in early ARC. lncRNA H19 could be a useful marker of early ARC and oxidative damage repair pathway of lncRNA H19/miR-29a/TDG may be a promising target for the treatment of ARC.

NEAT1 induces osteosarcoma development by modulating the miR-339-5p/TGF-ß1 pathway.

The long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) has been recognized as a tumor oncogene involved in the development of multiple cancers. However, the function of NEAT1 and its molecular mechanism in osteosarcoma (OS) remain unclear. First, we detected the NEAT1 expression in OS cell lines by performing quantitative reverse-transcription polymerase chain reaction. Next, the effects of NEAT1 on OS cell growth, apoptosis, migration, and invasion were tested by lentivirus-mediated downregulation. We observed that inhibition of NEAT1 restrained OS cell progression greatly. Interestingly, in the last few years, increasing studies have shown that some lncRNAs can act as miRNA sponges and reduce the amount of the same. Here, we found that NEAT1 can modulate OS development via sponging miR-339-5p. MiR-339-5p was significantly decreased in OS cells, and its overexpression can remarkably repress the OS proliferation. These results indicated that NEAT1 could function as a tumor oncogene in OS by inhibiting miR-339-5p in vitro. Then, the following assays validated that transforming growth factor ß1 (TGF-ß1) can act as a functional target of miR-339-5p in OS cells. Finally, we indicated that NEAT1 could mediate TGF-ß1 expression by competitively sponging miR-339-5p. NEAT1 induced OS cell proliferation and cell mobility by binding to miR-339-5p and increasing TGF-ß1 in OS. It was demonstrated in our study that lncRNA NEAT1 could impede miR-339-5p expression to maintain the expression of TGF-ß1, which led to the development of OS. Our findings implied that the novel identified NEAT1/miR-339-5p/TGF-ß1 axis might be a new molecular pathway or therapeutic target for OS diagnosis and treatment.

Long noncoding RNA glutathione peroxidase 3-antisense inhibits lens epithelial cell apoptosis by upregulating glutathione peroxidase 3 expression in age-related cataract.

Purpose: Age-related cataract (ARC) is the leading cause of visual impairment and blindness worldwide. The apoptosis of lens epithelial cells (LECs) induced by oxidative damage is a major contributing factor to ARC. Long noncoding RNAs (lncRNAs) play important roles in various biologic processes. We aimed to explore the role of glutathione peroxidase 3 (GPX3)-antisense (AS) in ARCs. Methods: We extracted total RNAs from transparent and age-matched cataractous human lenses and detected lncRNA expression profiles using high-throughput RNA sequencing. The expression of GPX3-AS and GPX3 was detected by quantitative real-time PCR (qRT-PCR). Apoptotic proteins were detected by western blot and immunofluorescence. We treated SRA01/04 cells with H2O2 to mimic oxidative stress and induce cell apoptosis, which was analyzed by flow cytometry and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. The cell counting kit-8 (CCK-8) assay was used to detect the viability of SRA01/04 cells. The location of GPX3-AS was determined by fluorescence in situ hybridization (FISH) and cell nuclear and cytoplasmic RNA separation. Results: The lncRNA GPX3-AS, which is located in the nuclei of LECs, was downregulated in cataractous human lenses compared with control lenses, and proapoptotic proteins were expressed at high levels in the anterior lens capsules of ARC tissues. An in vitro study suggested that GPX3-AS inhibited H2O2-induced SRA01/04 cell apoptosis. As GPX3-AS is transcribed from the AS strand of the GPX3 gene locus, we further revealed its regulatory role in GPX3 expression. GPX3-AS was positively correlated with GPX3 expression. In addition, GPX3-AS inhibited H2O2-induced SRA01/04 cell apoptosis by upregulating GPX3 expression. Conclusions: In summary, our study revealed that GPX3-AS downregulated the apoptosis of LECs via promoting GPX3 expression, implying a novel therapeutic target for ARCs.

BLM can regulate cataract progression by influencing cell vitality and apoptosis.

Age-related cataract (ARC) is the most common cause of severe visual impairment and blindness. The precise mechanisms of ARC are not completely understood, but it is well accepted that oxidative damage plays an important role in the disease pathogenesis. BLM, the key enzyme of the double-strand break repair (DSBR) pathway, is part of a family of DNA unwinding enzymes and has a crucial role in multiple steps of the DNA recombination, replication and repair processes. We have recently shown that BLM-rs1063147 is initially associated with nuclear ARC in a cross-section study. Therefore, we wanted to study the effects of BLM on ARC progression. In ARC patients, BLM transcription in lens capsules was decreased, so did the BLM protein, and after UVB irradiation, BLM mRNA and protein levels were increased in SRA01/04 cells. Upon silencing BLM in SRA01/04 cells and rat lens, cell vitality and apoptosis were altered, and the rat lens opacification was considerable. In conclusion, BLM can regulate cataract progression by influencing cell vitality and apoptosis.

Inhibition of YAP ameliorates choroidal neovascularization via inhibiting endothelial cell proliferation.

Purpose: Age-related macular degeneration (AMD) is the leading cause of central visual loss among patients over the age of 55 years worldwide. Neovascular-type AMD (nAMD) accounts for approximately 10% of patients with AMD and is characterized by choroidal neovascularization (CNV). The proliferation of choroidal endothelial cells (CECs) is one important step in the formation of new vessels. Transcriptional coactivator Yes-associated protein (YAP) can promote the proliferation of multiple cancer cells, corneal endothelial cells, and vascular smooth muscle cells, which participate in angiogenesis. This study intends to reveal the expression and functions of YAP during the CNV process. Methods: In the study, a mouse CNV model was generated by laser photocoagulation. YAP expression was detected with western blotting and immunohistochemistry. YAP siRNA and ranibizumab, a VEGF monoclonal antibody, were injected intravitreally in CNV mice. The YAP and VEGF expression levels after injection were detected with western blotting. The incidence and leakage area of CNV were measured with fundus fluorescein angiography, choroidal flat mounting, and hematoxylin and eosin (HE) staining. Immunofluorescent double staining was used to detect YAP cellular localization with CD31 (an endothelial cell marker) antibody. Proliferating cell nuclear antigen (PCNA) expression in CNV mice without or with YAP siRNA intravitreal injection and the colocalization of PCNA and CD31 were measured with western blotting and immunofluorescent double staining, respectively. Results: YAP expression increased following laser exposure, in accordance with vascular endothelial growth factor (VEGF) expression. YAP siRNA and ranibizumab decreased VEGF expression and the incidence and leakage area of CNV. YAP was localized in the vascular endothelium within the CNV site. Additionally, after laser exposure, YAP siRNA inhibited the increased expression of PCNA, which was colocalized with endothelial cells. Conclusions: This study showed that YAP upregulation promoted CNV formation by upregulating the proliferation of endothelial cells, providing evidence for the molecular mechanisms of CNV and suggesting a novel molecular target for nAMD treatment.

Inhibition of RACK1 ameliorates choroidal neovascularization formation in vitro and in vivo.

Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. The receptor for activated C-kinase 1 (RACK1) serves as a scaffold protein which is recently found to promote angiogenesis. However, the impact of RACK1 on the vascular endothelial growth factor (VEGF) expression in endothelial cells and subsequent choroidal angiogenesis formation remains to be elucidated. In this study, we found that RACK1 and VEGF expression increased, and reached the peak at 7d in mouse CNV model by laser application. Furthermore, on RPE/choroid cryosections, RACK1 co-localized with CD31, suggesting that RACK1 was expressed in endothelial cells. In vitro, RF/6A cell hypoxia model showed that RACK1 expression was up-regulated in parallel with hypoxia-induced factor 1 (HIF-1α) and VEGF expression, reaching the peak at 6h. Silencing of RACK1 suppressed the invasion and tube formation activity of RF/6A cells in ARPE-19 and RF/6A co-culture system, possibly through VEGF signal pathway. Overexpression of RACK1 showed the opposite effect. Intravitreal injection of anti-RACK1 monoclonal antibody predominantly decreased RACK1 and VEGF expression in mouse laser-induced CNV model. Meanwhile, anti-RACK1 monoclonal antibody intravitreal injection also decreased incidence of CNV and leakage area. These data indicated that RACK1 promoted CNV formation via VEGF pathway. Additionally, anti-RACK1 monoclonal antibody significantly decreased CNV in mouse model and may have therapeutic potential in human CNV.

Up-Regulation of PKM2 Relates to Retinal Ganglion Cell Apoptosis After Light-Induced Retinal Damage in Adult Rats.

Pyruvate kinase isozyme type M2 (PKM2), a key glycolytic enzyme, which is involved in ATP generation and pyruvate production, participates in tumor metabolism, growth, and other multiple cellular processes. However, one attractive biological function of PKM2 is that it translocates to the nucleus and induces cell apoptosis. Recently, increased PKM2 has been found in age-related macular degeneration (AMD), but little is known regarding its function in the AMD pathophysiology. To investigate whether PKM2 participated in retinal degeneration, we performed a light-induced retinal damage model in adult rats. Western blot and immunohistochemistry analysis showed a significant up-regulation of PKM2 in retinal ganglion cells (RGCs) layer (GCL) after light exposure. Immunofluorescent labeling indicated that PKM2 located mainly in RGCs. Co-localization of PKM2 and active caspase-3 as well as TUNEL in RGCs suggested that PKM2 might participate in RGC apoptosis. In addition, the expression patterns of cyclin D1 and phosphorylated extracellular signal-regulated kinase (p-ERK) were parallel with that of PKM2. Furthermore, PKM2, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of U0126, a highly selective inhibitor of MAPK/ERK kinase. Collectively, we hypothesized that PKM2 might participate in RGC apoptosis after light-induced retinal damage medicated by p-ERK through cycle re-entry mechanism.

DNA damage in lens epithelial cells and peripheral lymphocytes from age-related cataract patients.

BACKGROUND: Oxidative DNA damage may be one of the etiologies of age-related cataract (ARC). We quantified DNA damage in lens epithelial cells (LECs) and peripheral blood lymphocytes of ARC. METHODS: A total of 64 patients with different types of ARC and 23 control subjects were enrolled. Fresh LECs and peripheral lymphocytes were collected and DNA damage was evaluated by alkaline comet assay. The percentage of DNA in the tail of comets (%Tail DNA) and the olive tail moment (OTM) were calculated by CASP software. RESULTS: The results showed the %Tail DNA and OTM in LECs and lymphocytes in the overall cataract patient group were significantly higher than those in the control subjects. The %Tail DNA and OTM of LECs and lymphocytes showed no differences among cortical, nuclear and posterior subcapsular cataracts. The %Tail DNA and OTM in LECs were significantly lower than those in lymphocytes but a significant correlation of the DNA damage was found between them. CONCLUSION: We concluded that DNA damage in lens and peripheral blood lymphocytes increased in ARC. The results imply that local and systemic oxidative DNA damage might play certain roles in ARC pathogenesis.

[Study on gene-gene, gene-environmental interactions of DNA repair genes related with age-related cataract].

OBJECTIVE: To investigate the interaction between polymorphisms and environment factors in age related cataract (ARC). METHODS: Population-based case-control study. All of 448 cataract cases were selected from cataract eye database and 248 controls were recruited from Funing county. All 18 single nucleotide polymorphism (SNPs) of WRN, OGG1, BLM and ERCC6 genes were genotyped using real-time fluorescent quantitative PCR. The possible risk of ARC was estimated by unconditional Logistic regression. The interaction effects between gene-gene and gene-environment were evaluated by use of multifactor dimensionality reduction (MDR). RESULTS: Risk of ARC in illiterate group was significantly increased (OR = 1.53, P < 0.05), compared to no illiterate group. The risk of cataract in farmers was significantly high with ORs of 3.75. Long exposure to sunlight may increase cataract risk. Factors such as smoking, drinking, hypertension had no relationship with ARC (P > 0.05). WRN-rs11574311 was still associated with ARC with statistical significance after Bonferroni correction (OR = 1.98, Pa<0.018). There was no evidence for any association at a significance level of P < 0.05 for the remaining alleles. MDR analysis showed a significant interaction model among the six factors: education, solar radiation, WRN-rs11574311, WRN-rs1801195, WRN-rs4733220 and BLM-rs17273206 (consistency inspection accuracy of 0.683 on average, cross validation at 10/10, P = 0.005). Logistic regression model showed that the "high-risk" individuals had a significantly increased risk of ARC, compared to those "low- risk" individual classified by the MDR mode mentioned above(OR = 5.51, P < 0.05). CONCLUSION: WRN-rs11574311 allele was the risk factor for ARC, and individuals who carried WRN-rs1801195, WRN-rs4733220 and BLM-rs17273206 allele and low level of education, long sunlight time significantly increased the risk of the ARC.

[Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.

Rheum palmatum L. and Salvia miltiorrhiza Bge. Alleviates Acute Pancreatitis by Regulating Th17 Cell Differentiation: An Integrated Network Pharmacology Analysis, Molecular Dynamics Simulation and Experimental Validation.

OBJECTIVE: To identify the core targets of Rheum palmatum L. and Salvia miltiorrhiza Bge., (Dahuang-Danshen, DH-DS) and the mechanism underlying its therapeutic efficacy in acute pancreatitis (AP) using a network pharmacology approach and validate the findings in animal experiments. METHODS: Network pharmacology analysis was used to elucidate the mechanisms underlying the therapeutic effects of DH-DS in AP. The reliability of the results was verified by molecular docking simulation and molecular dynamics simulation. Finally, the results of network pharmacology enrichment analysis were verified by immunohistochemistry, Western blot analysis and real-time quantitative PCR, respectively. RESULTS: Sixty-seven common targets of DH-DS in AP were identified and mitogen-activated protein kinase 3 (MAPK3), Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), protein c-Fos (FOS) were identified as core targets in the protein interaction (PPI) network analysis. Gene ontology analysis showed that cellular response to organic substance was the main functions of DH-DS in AP, and Kyoto Encyclopedia of Genes and Genomes analysis showed that the main pathway included Th17 cell differentiation. Molecular docking simulation confirmed that DH-DS binds with strong affinity to MAPK3, STAT3 and FOS. Molecular dynamics simulation revealed that FOS-isotanshinone II and STAT3-dan-shexinkum d had good binding capacity. Animal experiments indicated that compared with the AP model group, DH-DS treatment effectively alleviated AP by inhibiting the expression of interleukin-1ß, interleukin-6 and tumor necrosis factor-α, and blocking the activation of Th17 cell differentiation (P<0.01). CONCLUSION: DH-DS could inhibit the expression of inflammatory factors and protect pancreatic tissues, which would be functioned by regulating Th17 cell differentiation-related mRNA and protein expressions.

An Ex Vivo Study on Release, Uptake, and miRNA Profile of Exosomes in Rat Lens.

Purpose: To identify the ability of releasing and uptaking exosomes in rat lens and characterize the exosomal microRNA profile of lens-derived exosomes. Methods: The rat lenses were cultured ex vivo and the medium was collected. The exosomes were isolated from medium and measured in size and concentration by nanoflow cytometry (nFCM) and transmission electron microscopy (TEM) and verified with CD63 and TSG101 by Western blot. The miRNAs in exosomes released from lens epithelial cells (LECs) were sequenced. The plasma exosomes labeled by PKH26 were used to verify the exosomes uptake LECs, and their colocalized fluorescence was imaged by confocal microscopy. Results: LECs released numerous exosomes into the medium through the capsule, which contained abundant miRNAs. The most abundant miRNAs included miR-184, let-7c-5p, let-7a-5p, let-7b-5p, let-7f-5p, miR-125a-5p, miR-204-5p, miR-125b-5p, miR-1b, and miR-23a-3p. The LECs but not the lens fibre cells showed exosome uptake. The LECs uptake more PKH26-labeled exosomes at day 7 than day 3 and day 14. Conclusions: Our results suggested that LECs can release and uptake exosomes through the capsule. Exosomes may be an important way for the lens to communicate among LECs, aqueous humour, vitreous body, and other ocular tissues.

XRCC5 downregulated by TRIM25 is susceptible for lens epithelial cell apoptosis.

Exposure of the lens to UVB can lead to oxidative stress, which would result in age-related cataract (ARC) formation. In this study, we investigate the regulatory mechanism of tripartite motif containing 25 (TRIM25) in ARC. The protein level of TRIM25 was elevated in ARC specimens and UVB-exposed SRA01/04 cells. Bioinformatic analysis indicated that X-ray repair cross complementing 5 (XRCC5) might interact with TRIM25, and the interaction was validated via immunoprecipitation. TRIM25 interacted with XRCC5 and ubiquitinated it for degradation. Further studies showed that XRCC5 overexpression notably repressed UVB-induced apoptosis, while XRCC5 knockdown promoted apoptosis. Of note, ubiquitination of XRCC5 mediated by TRIM25 overexpression facilitated apoptosis. Attenuation of XRCC5 ubiquitination by mutant with substitution of lysine residues with arginine residues rescued its anti-apoptosis effect. Moreover, we observed that TRIM25-mediated XRCC5 degradation was reversed by proteasome inhibitor MG-132 or lysosome inhibitor 3-MA. In conclusion, TRIM25 mediates ubiquitination of XRCC5 to regulate the function and degradation of XRCC5, suggesting that interventions targeting TRIM25 might be a promising therapeutic strategy for ARC.

Doxycycline Suppresses Vasculogenic Mimicry in Human Pterygium Fibroblasts.

PURPOSE: To explore the effect of doxycycline on vasculogenic mimicry (VM) formation and the potential mechanism in human pterygium fibroblasts in order to find novel targets for pterygium therapy. METHODS: First, we demonstrate the existence of VM in 73 pterygium specimens by CD31 and periodic acid Schiff (PAS) dual staining. Then we used cell counting kit-8, clone formation assay and flow cytometry to prove the inhibitory effect of doxycycline on cell proliferation and apoptosis. The VM formation was evaluated through wound healing assay, cell transwell assay and three-dimensional cell culture combined with PAS staining. Finally, we used Western blot to testify the correlation of the VM and the factors in protein level preliminarily. RESULTS: Our results showed that VM existed in human pterygium specimens exactly. Otherwise, in human pterygium fibroblasts, doxycycline induced a dose-dependent inhibitory effect on cell proliferation and apoptosis induction. Besides, doxycycline significantly suppressed vasculogenic mimicry tube formation, cell migration and invasion. Furthermore, doxycycline impaired the expression of MMP-9, MMP-2 and VEGF which may related to pterygium VM formation. CONCLUSIONS: Doxycycline decelerated pterygium progression might be through inhibiting VM formation according to the downregulation of MMP-9, MMP-2 and VEGF, which may provide the basis of further studies involving doxycycline for pterygium treatment.