· AlM:To investigate the effect of compound anisodine on fundus blood circulation after vitrectomy with face-down position.
· METHODS: Sixty patients ( 60 eyes ) with rhegmatogenous retinal detachment received vitrectomy with silicone oil tamponade operation, who were randomized divided into treatment group ( 30 eyes ) and control group ( 30 eyes ) .The patients in the treatment group received the subcutaneously injection of compound anisodine hydrobromide by the superficial temporal artery once daily for 14d since postoperative first day.Retinal microcirculation blood flow parameters were recorded with Heidelberg retinal flowmeter postoperative 1d, 1 and 2wk, and were compared between two groups.
·RESULTS: The blood flow parameters ( Vol, Flw, Vel) of control group postoperative 1 and 2wk were significantly less than those postoperative 1d.Otherwise the parameters of treatment postoperative 1 and 2wk were significantly more than those postoperative 1d. The parameters between two groups were significant different ( P<0.01) .
· CONCLUSlON: Facing down after vitrectomy with
silicone oil tamponade may reduce retinal blood supply, consequently lead to retinal ischemia; compound anisodine can effectively improve the retinal and choroidal microcirculation after vitrectomy with face-down posture, reduce retinal ischemia, and enhance the visual function.

Aged ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Male ; Middle Aged ; Phytotherapy ; Silicosis ; drug therapy ; Treatment Outcome

Background Clinical studies indicated that the pathogenesis of most corneal dystrophy is associated with the mutation of the transforming growth factor beta-induced (TGFBI) gene.However,the molecular mechanism of mutated TGFBI gene in corneal dystrophy is unclear. Objective The present study was to investigate the expression of the TGFBI gene in human corneal tissue and cells in vitro.MethodsHuman corneal epithelial cells and keratocytes were cultured and passaged,and donor corneal tissue was obtained for the section preparation.RT-PCR was used to detect the expression of TGFBI mRNA in human corneal tissue and cells.Immunofluorescence was used to test the expression of the TGFBI protein in the human corneal tissue,and immunohistochemistry was used to test the expression of the TGFBI protein in human corneal epithelial cells and corneal stromal cells.ResultsRT-PCR analysis showed that TGFBI mRNA could be detected as a 1274 bp band in human corneal tissue and corneal stromal cells,but no TGFBI mRNA was observed in corneal epithelial cells.Immunofluorescence assay revealed that corneal stromal cells were positive ly expressed for the TGFBI protein,but the corneal epithelial cells did not express the TGFBI protein.Immunohistochemistry indicated that the expression of TGFBI was detected the red fluoressence in the cytoplasm of corneal stromal cells;however,no positive response was found in corneal epithelial cells.ConclusionsThe expression of the TGFBI gene occurs in human corneal stromal cells but not in the corneal epithelial cells.This result might be of helpful for studying the function and role of TGFBI gene in pathogenesis of corneal dystrophy.

Background The human transforming growth factor beta-induced gene (TGFBI) is the first determined pathogenic gene to corneal dystrophy.But the molecular genetic mechanism is completely unknown.The study of concerning role of TGFBI is very important for us understand the physiological function of cornea,and the pathogenesis of corneal dystrophy.Objective The vector of human transforming growth factor beta-induced gene (TGFBI) in eukaryotic expression was constructed and transfected into the human corneal epithelial cells in order to explore its influence on the growth of human corneal epithelial cells.Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription.TGFBI cDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV,XhoI double restriction endonuclease.The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMVN-HA plasmid group,non-transfected group and pGFP-C2 transfected group.The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein(EGFP) in the cells.The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection.The growth of the transfected cells was assessed by Cell Counting Kit-8.The expressions of matrix metalloproteinase(MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) proteins and their mRNA in transfected cells were detected using SYBR fluorescence realtime PCR analysis and Western blot assay.Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence.EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70％.The expression intensity of TGFBI mRNA was significantly higher in recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group,and TGFBI protein was expressed in recombinant pCMV-N-HA-TGFBI plasmid group.No significant difference was found in the A450value among recombinant pCMV-N-HA-TGFBI plasmid group,pCMV-N-HA plasmid group and non-transfected group ( F=3.34,P＞0.05 ).The mRNA level of MMP1,MMP3in the transfected cells was significant elevated but that of TIMP1 was declined in the recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group (all P ＜ 0.05 ).Meanwhile,the expressions of MMP1,MMP3 and TIMP1 proteins appeared the same tendency( all P＜0.05).Conclusions Eukaryotic expression vector harboring human TGFBI eDNA can be successfully constructed and efficiently overexpressed in human corneal epithelial cells.TGFBI gene is involved in the physical and pathological conditions of human corneal epithelial cells by regulating the activity of MMP1,MMP3 and TIMP1.The results offer a new approach for the study of the role of TGFBI in pathogenesis of corneal transparency.

Along with the increase in fungal infections, Candida albicans prevention and control become the focus of anti-fungal infection at present. This study aims to discuss the effect monomer andrographolide (AG) on C. albicans biofilm dispersion. In the experiment, micro-well plates and medical catheter pieces were used to establish the C. albicans biofilm model. It was discovered by XTT assay and flat band method that 1 000, 500, 250 mg x L(-1) AG could impact the activity of C. albicans biofilm dispersion cells. The morphological structures of residual biofilms on catheter pieces were observed with scanning electron microscopy, which showed that 1 000, 500, 250 mg x L(-1) AG could induce C. albicans biofilm dispersion in a dose-dependent manner, and the dispersed cells were dominated by the yeast phase. According to the real-time fluorescence quantification PCR (qRT-PCR) test, AG could up-regulate HSP90 expression and down-regulate UME6 and PES1 expressions. This study demonstrates that AG could induce C. albicans biofilm dispersion to some extent.
Anti-Inflammatory Agents ; pharmacology ; Biofilms ; drug effects ; Candida albicans ; genetics ; physiology ; ultrastructure ; Diterpenes ; pharmacology ; Dose-Response Relationship, Drug ; Fungal Proteins ; genetics ; Gene Expression Regulation, Fungal ; drug effects ; HSP90 Heat-Shock Proteins ; genetics ; Microscopy, Electron, Scanning ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVETo investigate the effects of ethyl acetate extract of Huanglian Jiedu decoction (EAHD) , alone and in combination with fluconazole (FLZ) on FLZ-resistant Candida albicans.

METHODThe minimum inhibitory concentrations (MIC) and sessile MIC80 (SMIC80) of EAHD and FLZ to FLZ-resistant C. albicans were determined by CLSI M27-A3 microdilution method, and the synergy of EAHD combined with FLZ were examined by the checkerboard microdilution assay. Agar plate-method was adopted to observe the rate of antifungal activity according to time-kill curve. HPLC and qRT-PCR were utilized to evaluate the changes of ergosterol content and expressions of related genes, respectively.

RESULTMICs of EAHD ranged from 156 to 1,250 mg · L(-1), those of FLZ from 256 to above 2,048 mg · L(-1) with FICI approximate 0.066 in combination; SMIC80 of EAHD were higher than 1,250 mg · L(-1), SMIC80 of FLZ were higher than 512 mg · L(-1) and up to above 2,048 mg · L(-1). Combination group also showed synergy effect except one group showing addition effect. The results of T-K experiment also confirmed obviously fungicidal effect when treated for 12 h. When compared with control groups, the ergosterol was reduced 85% and 50% in the treatments of combination and EAHD alone by HPLC, respective- ly. The expressions of ERG1, ERG2, ERG6, ERG7 and ERG11 were upregulated, and ACS1, ACS2, MET6 were downregulated when exposed to FLZ. The expressions of the above genes were downregulated by treatment of EAHD. The expressions of ERG2, ERG6, ERG11 were upregulated, while ERG1, ERG7, ACS1, ACS2, MET6 were downregulated in combination group.

CONCLUSIONThe combination of EAHD and FLZ exhibited synergy against FLZ-resistant C. albicans through decreasing the synthesis of ergosterol, and resulting in the breakage of cell membrane.

Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; growth & development ; metabolism ; Drug Resistance, Fungal ; drug effects ; Drug Synergism ; Drugs, Chinese Herbal ; pharmacology ; Ergosterol ; biosynthesis ; Fluconazole ; pharmacology ; Microbial Sensitivity Tests

OBJECTIVETo investigate the effects of butyl alcohol extract of Baitouweng decoction ( BAEB) on yeast-to-hyphae transition of Candida albicans isolates from vulvovaginal candidiasis (VVC) in alkaline pH.

METHODSerial 2-fold dilution assay was used to determine the minimal inhibitory concentrations (MICs) of Baitouweng decoction extracts against C. albicans isolates from VVC, XTT assay was applied to determine the metabolic activity of C. albicans hypha treated by BAEB for 6 h. The morphological change of C. albicans treated by BAEB was inspected at different pH by inverted microscope, fluorescence microscope, scanning electron microscopy (SEM). Solid agar plate and semi-solid agar were utilized to evaluate colony morphology and invasive growth of C. albicans, respectively. Quantitative Real-time PCR (qRT-PCR) was adopted to observe the expressions of hyphae-specific genes including HWP1, ALS3, CSH1, SUN41 and CaPDE2.

RESULTThe MIC of BAEB against C. albicans is less than that of other extracts; hyphae grow best at pH 8. 0; 512 mg · L(-1) and 1,024 mg · L(-1) BAEB could inhibit formation of hyphae and influence colony morphology. When treated by 512 mg · L(-1) and 1,024 mg · L(-1) BAEB, the colonies became smooth; while by 0 and 256 mg · L(-1) BAEB, the colonies became wrinkled. In semi-solid agar, the length of hyphae decreased steadily as the concentration of BAEB lowered. The expression of HWP1, ALS3, CSHl, SUN41 were downregulated by 5.12, 4.26, 3.2 and 2.74 folds, and CaPDE2 was upregulated by 2.38 fold.

CONCLUSIONBAEB could inhibit yeast-to-hyphae transition of C. albicans isolates from VVC in alkaline pH.

Antifungal Agents ; isolation & purification ; pharmacology ; Candida albicans ; drug effects ; genetics ; growth & development ; Candidiasis, Vulvovaginal ; drug therapy ; microbiology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Humans ; Hydrogen-Ion Concentration ; Hyphae ; drug effects ; growth & development

OBJECTIVETo investigate anti-attachment effect of ethyl acetate extract of Huanglian Jiedu decoction (EAHD) on Candida glabrata.

METHODSerial 2-fold dilution assay was used to determine the minimum inhibitory concentrations MICs of EAHD to C. glabrata. XTT assay was used to evaluate the effect of EAHD against adhesion of C. glabrata. Inverted microscope, scanning electron microscope (SEM) and fluorescein diacetate (FDA) staining were applied to observe the morphological changes of C. glabrata in adhesion. PCR was adopted to inspect the expression of attachment-related genes such as EPA1, EPA6 and EPA7.

RESULTThe MIC of EAHD and fluconazole to C. glabrata were 320 mg · L(-1) and 1 mg · L(-1) respectively. The total cells including budding cells decreased in a dose-dependent manner following EAHD treatment. The expressions of EPA1, EPA6 and EPA7 were downregulated dramatically after EAHD treatment.

CONCLUSIONEAHD could effectively inhibit adherence of C. glabrata.

Acetates ; Candida glabrata ; drug effects ; physiology ; Drugs, Chinese Herbal ; pharmacology ; Fungal Proteins ; genetics ; Lectins ; genetics ; Plant Extracts ; pharmacology

OBJECTIVETo investigate the effect of andrographolide derivative Yanhuning (YHN) on Candida albicans biofilms in rats.

METHODThe rat C. albicans biofilms subcutaneous catheter model was established by intraperitoneally injecting YHN (40, 20, 10, 5, 2.5 mg x kg (-1)), with the FLC (80 mg x kg(-1)) positive group as the control group. After 7 d, CFU counting and XTT assay were used to evaluate the effect of YHN on C. albicans biofllms in vivo. Scanning electron microscopy (SEM) was applied to observe the morphological changes in rat biofilms intervened by YHN. The real-time fluorescence quantification PCR was adopted to detect expressions of C. albicans adhesion-related genes, such as ALS1, ALS3, HWP1, EAP1 and MP65.

RESULTThe YHN group showed much less CFUs on catheter pieces and lower XTT metabolic activity than the blank group, with dosage dependence. SEM also showed that YHN could obviously decrease C. albicans adhesion on subcutaneous catheters in rats. According to qRT-PCR's results, YHN can down-regulate expressions of ALS1, ALS3, HWP1, EAP1 and MP65.

CONCLUSIONYHN could inhibit C. albicans biofilms in rats.

Animals ; Biofilms ; drug effects ; growth & development ; Candida albicans ; cytology ; drug effects ; physiology ; Catheters ; microbiology ; Cell Adhesion ; drug effects ; Diterpenes ; chemistry ; pharmacology ; Dose-Response Relationship, Drug ; Rats

Background The construction of tissue-engineered corneal endothelium needs the functional seeding cells,so how to culture a large amount of functional corneal endothelial cells (CECs) is an urgent problem to be solved.Objective The aim of this study was to evaluate the role of aqueous humor on bovine CECs in vitro.Methods Aqueous humor of 1.2 ml was collected from the anterior chamber of bovine and sterilized,and the liquid supernatant was obtained.The bovine CECs were isolated from bovine cornea and then cultured in low glucose Dulbecco Modified Eagle Medium with 10％ fetal bovine serum (FBS) in vitro.Aqueous humor was added into the medium with the final concentration of 2.5％,5.0％,l0.0％,15.0％ and 20.0％,respectively,and no aqueous humor was added in the control group.Cell counting kit-8 (CCK-8) assay was used to detect the absorbency value of CECs for the evaluation of cell proliferation.Progression of the cell cycle was analyzed by flow cytometry (FCM).After confluence of the cells was reached,1 ml plastic spear tip was used to scratch the cell single layer,and the cells were incubated consequently in medium with 10％ FBS and with or without aqueous humor for 24 hours.Healing area of the cell single layer was measured.The cells were incubated at a density of 6 × 105 cells/ml and cultured using medium with or without 10.0％ aqueous human for 5 days,and the number of the cells was analyzed by DAPI fluorescence technique.Results Under the phase-contrast microscopy,the confluent CECs showed a slabstone-like and hexagonal appearance.CCK-8 assay revealed that the absorbance values of CECs was significantly different among the various culture groups (F=4.051,P =0.007),and the absorbance value in different concentrations of aqueous human culture groups was significantly higher than that in the control group (P ＜ 0.01).FCM showed that the percentage of the cells in S-G2 phases was (34.80-±3.13)％ in the 10.0％ aqueous humors group and (23.06±1.13)％ in the control group,showing a significant difference (t =-5.729,P=0.005).The scratch test showed that the healing area of the cell signal layer was (0.116±0.019) mm2 in the 10.0％ aqueous humors group and (0.358 ±0.049) mm2 in the control group,showing a significant difference (t =13.842,P =0.000).The density of cells in the 10.0％ aqueous humor group was (1439± 1 10)/field,which was more than (1162±45)/field in the control group (t =-11.020,P=0.000).Conclusions Aqueous humor at the concentration of 10.0％ promote the growth and proliferation of bovine CECs.The result suggests that 10.0％ aqueous humor can be used as a promoting agent during the culture of CECs.