Inhibition effect of interfering RNA targeting hypoxia inducible factor-1α on vascular endothelial growth factor mRNA expression in the retina of diabetic rats

Objective To observe the inhibition effect of the hypoxia inducible factor-1α(HIF-1α)specific siRNA on the expression of vascular endothelial growth factor(VEGF)mRNA in retinal tissues in diabetic rat.Methods This is a randomized controlled study.HIF-1α specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector.Fifty-four healthy Sprague Dawley(SD)rats were divided into control group(15 rats)and experimental group(39 rats).The experimental rats were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into diabetic retinopathy (DR)group(15 rats),vector group(12 rats)and gene therapy group(12 rats).LipofectamineTM2000mixed with pSilencer2.1-U6neo plasmid or HiF-1α siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively.Nothing was transfected into DR and control group.The expression of VEGF mRNA in retinas was measured by real-time RT-PCR.The inhibition efficiency of VEGFmRNA was calculated at 24,48,72 hours and 1 week after injection respectively.Significant differences between groups were evaluated by one-way analysis and LSD-t analysis.Results HIF-1 α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis.Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group.increased obviously in the DR and vector group,decreased in the gene therapy group.There was no statistically significant between DR and vector group(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980).The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group(t=8.768,13.695,11.285,8.253;P=0.000).The inhibition efficiency of VEGFmRNA was 32.76% ,43.60% ,47.70% ,50.86% at 24,48,72 hours and 1 week after injection.Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1α siRNA recombinant plasmid.     

Inhibition effect of interfering RNA targeting hypoxia inducible factor-1α on vascular endothelial growth factor mRNA expression in the retina of diabetic rats

Objective To observe the inhibition effect of the hypoxia inducible factor-1α(HIF-1α)specific siRNA on the expression of vascular endothelial growth factor(VEGF)mRNA in retinal tissues in diabetic rat.Methods This is a randomized controlled study.HIF-1α specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector.Fifty-four healthy Sprague Dawley(SD)rats were divided into control group(15 rats)and experimental group(39 rats).The experimental rats were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into diabetic retinopathy (DR)group(15 rats),vector group(12 rats)and gene therapy group(12 rats).LipofectamineTM2000mixed with pSilencer2.1-U6neo plasmid or HiF-1α siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively.Nothing was transfected into DR and control group.The expression of VEGF mRNA in retinas was measured by real-time RT-PCR.The inhibition efficiency of VEGFmRNA was calculated at 24,48,72 hours and 1 week after injection respectively.Significant differences between groups were evaluated by one-way analysis and LSD-t analysis.Results HIF-1 α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis.Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group.increased obviously in the DR and vector group,decreased in the gene therapy group.There was no statistically significant between DR and vector group(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980).The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group(t=8.768,13.695,11.285,8.253;P=0.000).The inhibition efficiency of VEGFmRNA was 32.76% ,43.60% ,47.70% ,50.86% at 24,48,72 hours and 1 week after injection.Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1α siRNA recombinant plasmid.     

Sulforaphane modulates TGFβ2-induced conjunctival fibroblasts activation and fibrosis by inhibiting PI3K/Akt signaling

AIM: To examine the effects of sulforaphane on fibrotic changes of transforming growth factor (TGFβ2) induced human conjunctival fibroblast (HConFs). METHODS: HConFs were cultured and divided into control, TGFβ2 (1 ng/mL), sulforaphane and TGFβ2+sulforaphane groups. Cell viability and apoptosis were detected using the MTT and ApoTox-Glo Triplex assay. Cell migration was detected using scratch and Transwell assay. Real-time quantitative PCR method was used to evaluate mRNA expression of TGFβ2, matrix metalloproteinase-2 (MMP2), myosin light chain kinase (MYLK), integrin αV, integrin α5, fibronectin 1 and α-smooth muscle actin (α-SMA). The protein expression of α-SMA, p-PI3K, PI3K, p-Akt, and Akt were detected by Western blot. RESULTS: The proliferation of HConFs was significantly (P<0.05) suppressed by sulforaphane compared to control cells with the increase of the concentration and treatment time. Cell proliferation after 48h incubation was significantly reduced with 100 μmol/L sulforaphane treatment by 17.53% (P<0.05). The Transwell assay showed sulforaphane decreased cell migration by 18.73% compared with TGFβ2-induced HConF (P<0.05). TGFβ2-induced the increasing expression of fibronectin, type I collagen and α-SMA, and the phosphorylation of PI3K and Akt were all significantly suppressed by sulforaphane pretreatment. CONCLUSION: Sulforaphane inhibits proliferation, migration, and synthesis of the extracellular matrix in HConFs, and inhibiting the PI3K/Akt signaling pathway. Sulforaphane could be a potential therapeutic drug for prevention of scar formation in filtering bleb after trabeculectomy.     

Construction of the Enhanced Yellow Fluorescent Protein Expression Vector Carrying IFN—γ Gene

Purpose:To construct the enhanced yellow fluorescent protein (EYFP) vector carrying interferon-γ gene(ifn-γ)in order to provide an ideal reporter in the expression of ifn-γand location of protein in vitro and in vivo.Method:According to the nocleotide sequence of ifn-γ gene,a pair of oligonucleotides was designed as primer whose two end contained nucleotide sequence of EcoR V and Not I restriction endonuclease respectively .The gene encoding for ,inf-γ was amplified using PCR technique After the PCR product was retrieved and purified,itwas digested with EcoR V and Not I restiction endonuclease,and then cloned into the plasmid p IRES-EYFP.The recombinant plasmid p IRES-EYFPIFN-γ was identified by restriction endonuclease enzyme analysis and DNA sequence analysis .Results:The ifn-γ was successfully amplified and verified by partial DNA seuqence analysis.The recombinant plasmid was correctly screened.Conclusion:The EYFP expression vector carrying ifn-γ gene was successfully established.This research work has formed a base for monitoring the ifn-γ gene expression and protein positon in living cells.Eye Science 2001;17:154-157.     

Production of interleukin-1β related to mammalian target of rapamycin/Toll-like receptor 4 signaling pathway during Aspergillus fumigatus infection of the mouse cornea

AIM: To elucidate the effect of rapamycin on regulating the production of interleukin (IL)-1β in Aspergillus fumigatus (A. fumigatus)-induced keratitis and to verify whether the expression of IL-1β in A. fumigatus keratitis is associated with the mammalian target of rapamycin (mTOR)/Toll-like receptor 4 (TLR4) signaling pathway. METHODS: Fungal keratitis mouse models of susceptible C57BL/6 mice were established using A. fumigatus. The mice were subsequently treated with rapamycin. The protein levels of p-mTOR, TLR4, and IL-1β in normal and infected corneal tissue were measured by Western blot. The TLR4 and IL-1β mRNA levels were determined by real-time polymerase chain reaction (PCR). RESULTS: In C57BL/6 mice, rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine, IL-1β. The expression of TLR4, stimulated by A. fumigatus, was reduced as well when the mTOR signaling pathway was suppressed by rapamycin. CONCLUSION: Rapamycin is beneficial for the outcome of fungal keratitis and has an inhibitory effect expression of the inflammatory cytokine IL-1β. The inhibitory effect on IL-1β expression can be associated with the mTOR/TLR4 signaling pathway in A. fumigatus infection in mice.     

Clinical study on interferon treatment of early scarring in filtering bleb

 Purpose: To investigate the effectiveness of needle revision combined with subconjuctival injection of interferon α-2b in reversing early scarring of filtering blebs following trabeculectomy surgery. Methods: Twenty-five  glaucoma patients (31 eyes) who presented with scarred or encapsulated filtering bleb after glaucoma surgery underwent needle revision in combination with subconjuctival injection of interferon α-2b, and were followed for 12 months. Intraocular pressure (IOP) and filtering bleb morphology were observed post treatment. Results: The mean time until scarring occurred was 21.0±7.4 days. The average time between recognition of bleb scarring and completion of needle revision was 2.2±0.8 days. The time interval between surgery and needle revision was inversely correlated with the time until needle revision (r = -0.694, P < 0.001). The mean IOPs before and after needle revision were 24.2±2.7mmHg and 19.6±3.8mmHg, respectively (t = 5.916,P < 0.001). At the 12-month follow-up visit, 18 eyes (58.1%) achieved complete success in IOP control, and 6 eyes (19.4%) had conditional success. The overall success rate for needling was thus 77.4%. Subconjunctival hemorrhage was observed in 4 eyes during the needle revision procedure. Punctate staining was found in the corneal epithelium of 2 eyes. Shallow  nterior chamber (Grade I or II) was identified in 5 eyes.  Conclusion: Slit-lamp needle revision combined with subconjunctival injection of interferon α-2b may be efficacious in the treatment of early scarring of filtering blebs, is easy and safe to perform, and may be considered for more widespread application.     

Construction of the Enhanced Yellow Fluorescent Protein Expression Vector Carrying IFN-γ Gene

Purpose: To construct the enhanced yellow fluorescent protein (EYFP) vector carryinginterferon-γ gene (ifn-γ) in order to provide an ideal reporter in the expression of ifn-γand location of protein in vitro and in vivo.Method: According to the nucleotide sequence of ifn-r gene, a pair of oligonucleotideswas designed as primer whose two end contained nucleotide sequence of EcoR V and NotI restriction endonuclease respectively. The gene encoding for inf-γ was amplified usingPCR technique. After the PCR product was retrieved and purified, it was digested withEcoR V and Not I restriction endonuclease, and then cloned into the plasmidpIRES-EYFP. The recombinant plasmid pIRES-EYFPIFN-γ was identified by restrictionendonuclease enzyme analysis and DNA sequence analysis.Results: The ifn-γ was successfully amplified and verified by partial DNA sequenceanalysis. The recombinant plasmid was correctly screened.Conclusion: The EYFP expression vector carrying ifn-γ gene was successfully established.Thi     

Recombination and identification of human alpha B-crystallin

AIM: To recombine the human alpha B-crystallin (αB-crystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αB-crystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli (E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected. RESULTS: Compared with the gene bank (GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin is successfully constructed, and the recombinant human αB-crystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.     

Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts

AIM: To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon’s capsule, both in vitro and in vivo. METHODS: Human primary Tenon’s capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT) method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase (TIMP-1,2) and proliferating cell nuclear antigen (PCNA). Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d). A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson’s trichrome to compare the neovascularization in the subconjunctiva area. RESULTS: In vitro, cultured Tenon''s capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3rd, 7th, 14th and 21st days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28th day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson’s trichrome observation. CONCLUSION: By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery.     

Effectiveness of conjunctival bleb scarring by knock-down of heat shock protein 47 in rat model

AIM: To evaluate the effectiveness of knock-down of heat shock protein 47 (HSP47) on conjunctival bleb scarring in a rat model and its possible mechanism. METHODS: Male Sprague–Dawley rats were used for glaucoma filtration surgery (GFS) and were treated with either phosphate buffered solution, shControl, mitomycin C, or sh-HSP47 using a microsyringe immediately after GFS. The morphology of filtering blebs was observed postoperatively. The levels of HSP47 were analyzed at 2, 5, 8, and 11d after GFS via realtime quantitative polymerase chain reaction (PCR) and Western blot. The silencing effect of HSP47, the expression of collagen I and III, and the potential signaling pathways of HSP47 during scarification were explored 11d post GFS. The protein levels of transforming growth factor-β1 (TGF-β1), phospho-Smad2 (pSmad2), phospho-Smad3 (p-Smad3), and phospho-p38 (p-p38) were also analyzed using Western blot. RESULTS: Sh-HSP47 treatment significantly prolonged the functional filtration bleb retention. The levels of HSP47 were increased significantly at 5, 8, and 11d postoperatively compared to the control group (P<0.05, P<0.01, and P<0.001). The levels of HSP47 protein at day 11 postoperatively were significantly down-regulated after HSP47 silencing using sh-HSP47 adenovirus transfection (P<0.01). Expression levels of collagen I and III within the blebs were significantly reduced in the absence of HSP47 (P<0.01). Moreover, the protein levels of TGF-β1, p-Smad2/3, and p-p38 were dramatically inhibited after treatment with sh-HSP47 (P<0.01). CONCLUSION: The inhibitory effects of HSP47 knock-down on scarring after GFS have the potential to be an efficacious therapeutic option for the treatment of conjunctival bleb scarring.     

Construction and identification of the eukaryotic expression vector carrying specific siRNA of LEDGF p52 gene

To construct and identify LEDGFp52 eukaryotic expression vector for RNA interference. · METHODS: Recombinants were designed and established by targeting gene LEDGFp52 and plasmid pGensil-1 based on LEDGFp52 cDNA sequences of Genomes. Two pairs of oligonucleotides were synthesized according to the Tuschl principle and inserted into plasmid pGenSil-l to generate siRNA eukaryotic expression vector. DH5α strains were transformed, plasmids were extracted, and recombinant vectors were identified by the restriction map and the sequence analysis. The cultured cells were transfected by the recombinant plasmid (pGensil-1-RNA.LEDGFp52-1). At 48 hours after transfection, the whole cell protein was extracted, and the protein level was detected using Western blotting with mouse anti-human LEDGFp52 monoclonal antibody.　 · RESULTS: Recombinant plasmids completely concord with the designs by the restriction map and the sequence analysis, the protein level of LEDGFp52 was down-regulated at 48 hours after transfecting pGensil-1-LEDGFp52-1 expression vector into HeLa cells, the recombinant eukaryotic expression vectors were successfully constructed. · CONCLUSION: siRNA recombinant can be successfully constructed by RNAi technique to inhibit the expression of LEDGFp52.     

Effect of Tetrandrine on proliferation of human pterygium fibroblasts

AIM: To investigate the effect of Tetrandrine (Tet) on proliferation of human pterygium fibroblasts (HPF) in vitro and to search for a new method to prevent the recurrence after pterygium surgery. METHODS: With different concentrations (0 to 160μmol/L) of Tet acting on HPF cultured in vitro , the impact was observed at 24, 48, 72 hours respectively after Tet intervention. The MTT method was used to assay the biologic activities of Tet and inhibitive rate of cell growth. The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by immunohistochemistry before and after Tet intervention. RESULTS: With different concentrations of 20, 40, 80 and 160μmol/L and acting for 24 to 72 hours, Tet could inhibit the proliferation of HPF in a dose- and time-dependent manner (P <0.05). After the intervention of Tet, the expression of PCNA protein declined. When the concentration of Tet was in the range of 20 to 160μmol/L, it was able to inhibit the expression of PCNA in a concentration-dependent manner (P <0.05). CONCLUSION: Tet can significantly inhibit the proliferation of pterygium fibroblasts, and the inhibitive action is in a dose- and time-dependent manner within a certain range of concentration. But in high concentration (>160μmol/L), Tet will have cytotoxity.     

CCPG1 involved in corneal Aspergillus fumigatus infection

AIM: To investigate whether non-canonical autophagy transport receptor cell cycle progression 1 (CCPG1) is involved in the corneal antifungal immune response. METHODS: Human corneal epithelial cells (HCECs) and human myeloid leukemia mononuclear cells (THP-1) macrophages stimulated by Aspergillus fumigatus (A. fumigatus) were used as cell models. The expression of CCPG1 mRNA was detected by qRT-PCR. Western blot was used to determine the protein expression of CCPG1 and interleukin-1β (IL-1β). The dectin-1 neutralizing antibody was used to detect the association between dectin-1 and CCPG1. Immunofluorescence was used to observe the colocalization of CCPG1 and C-type lectin-like receptor-1 (CLEC-1) in THP-1 macrophages. RESULTS: The expression of CCPG1 started to increase at 4h after infection and increased in a time-dependent manner in HCECs and THP-1 macrophages. With dectin-1 neutralizing antibody pretreatment, the expression of IL-1β was down-regulated. CCPG1 up-regulation in response to A. fumigatus infection was independent of dectin-1. Immunofluorescence showed the colocalization of CCPG1 and CLEC-1 in THP-1 macrophages. CONCLUSION: As a specific autophagy protein of non-canonical autophagy pathway, CCPG1 is involved in corneal infection with A. fumigatus.