OBJECTIVETo observe the contribution of Borneolum syntheticum to the intervention effect of Liuwei Dihuang Pill (, LDP) on experimental retinal degeneration, and initially investigate the mechanism of Borneolum syntheticum as meridian-lead-in drug.

METHODSA total of 180 sodium iodateinduced retinital degeneration rats were randomly divided into three groups, including distilled water group, LDP group, and LDP+Borneolum syntheticum (LDP+BS) group. Twenty normal rats were fed regularly without any treatment as normal control. On day 7 and 14 after treatment, histopathological study and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) test were performed to evaluate the retinopathy. Claudin-5 expression at blood-retina barrier (BRB) was detected by Western blot at different time points from 0.5 to 8 h after gavage.

RESULTSOn day 7 and 14 after treatment, the retinal lesion grades were significantly different among the three groups (P<0.05). The grade in the LDP+BS group was significantly less than the LDP and distilled water groups (both P<0.05), no significant difference was observed between the LDP and distilled water groups (P>0.05). The apoptosis rates in the LDP+BS group was significantly less than the distilled water and LDP groups (both P<0.05), while there was no significant difference between LDP and distilled water groups (P>0.05). Expression of claudin-5 in LDP+BS group was significantly less than the other two groups at 0.5, 1 and 2 h after gavage (P<0.05). There was no apparent difference among the three groups at 4 and 8 h after gavage (P>0.05).

CONCLUSIONBorneolum syntheticum could strengthen the effect of LDP on experimental retinal degeneration, indicated that Borneolum syntheticum might play the role of meridian-lead-in drug in the formula. The mechanism may be due to Borneolum syntheticum could promote the physiologically openness of bloodretina barrier through transiently affecting the expression of claudin-5.

Animals ; Apoptosis ; drug effects ; Claudin-5 ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Rats, Sprague-Dawley ; Retinal Degeneration ; chemically induced ; drug therapy ; pathology ; Retinal Pigment Epithelium ; drug effects ; pathology ; Time Factors

PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.
Anti-Bacterial Agents/pharmacology ; Apoptosis ; Blotting, Western ; C-Reactive Protein/biosynthesis/*genetics ; Cells, Cultured ; Endoplasmic Reticulum Stress/*drug effects/genetics ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Humans ; Polymerase Chain Reaction ; RNA, Messenger/*genetics ; Retinal Pigment Epithelium/*metabolism/pathology ; Serum Amyloid P-Component/biosynthesis/*genetics ; Tunicamycin/*pharmacology

PURPOSE: Chronic use of topical hypotensive agents induces several side effects caused by preservatives. The purpose of this study was to evaluate the effects of prostaglandin analogs with varying concentrations of benzalkonium chloride (BAC), preservative-free (PF), and alternative preservatives on mouse corneal tissue. METHODS: Thirty-five, 8- to 10-week-old female C57BL/6 mice (five mice for each group) were used for this study. To the control group, we applied normal saline, and to each drug-treated group we applied 0.02% BAC, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), travoprost 0.004% (with 0.001% polyquad) or tafluprost 0.0015% with/without 0.001% BAC, once a day (9 p.m.) for 4 weeks. Corneal fluorescein staining was evaluated in all groups. After harvest, the corneal tissues were embedded in paraffin and then Hematoxylin-Eosin stain was performed for histopathological examination. Immunofluorescence staining was done against TNF-alpha, IL-6, HLA DR, pJNK, and pAkt. RESULTS: In corneal fluorescein staining, severe punctate epithelial keratitis was seen in the groups of 0.02% BAC, 0.02% BAC containing bimatoprost 0.01% and latanoprost 0.005%. The surface desquamation, irregular surface, loss of cell borders, anisocytosis and stromal shrinkage were observed in the groups of BAC-containing eye drops. Moreover, the groups treated with BAC-containing eye drops have high inflammatory markers, significantly decreased cell viability-related signal, pAkt, and higher apoptosis-inducing signal, pJNK, than the control group. On the other hand, travoprost 0.004% and PF tafluprost 0.0015% have less cellular morphologic changes, lower inflammation, and higher cellular viability than BAC-containing formulations. CONCLUSIONS: Corneal damage, increased inflammation and apoptosis and low cell viability were observed in BAC-containing groups. PF or alternatively preserved glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC.
Animals ; Cell Survival ; Conjunctiva/drug effects/*pathology ; Disease Models, Animal ; Epithelium, Corneal/drug effects/*pathology ; Female ; Glaucoma/*drug therapy/pathology ; Mice ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; Ophthalmic Solutions ; Preservatives, Pharmaceutical ; Prostaglandins, Synthetic/*administration & dosage

PURPOSE: To evaluate the effects of bevacizumab on expression of B-cell leukemia/lymphoma (Bcl)-2 and apoptosis in retinal pigment epithelial (RPE) cells under oxidative stress conditions. METHODS: RPE cells were treated with H2O2 (0, 100, 200, 300, and 400 microM) and bevacizumab at or above the doses normally used in clinical practice (0, 0.33, 0.67, 1.33, and 2.67 mg/mL). Cell apoptosis was measured using flow cytometry with annexin V-fluorescein isothiocyanate. The expression of Bcl-2 mRNA was determined using reverse transcription polymerase chain reaction. RESULTS: Under low oxidative stress conditions (H2O2 100 microM), cell apoptosis was not significantly different at any concentration of bevacizumab, but Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL). Under moderate oxidative stress conditions (H2O2 200 microM), Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL), but cell apoptosis increased only at 2.67 mg/mL of bevacizumab. Under high oxidative stress (300 microM) conditions, cell apoptosis increased at high concentrations of bevacizumab (1.33 and 2.67 mg/mL), but it did not correlate with Bcl-2 expression. CONCLUSIONS: Withdrawal of vascular endothelial growth factor can lead to RPE cell apoptosis and influences the expression of anti-apoptotic genes such as Bcl-2 under oxidative stress conditions. Since oxidative stress levels of each patient are unknown, repeated injections of intravitreal bevacizumab, as in eyes with age-related macular degeneration, might influence RPE cell survival.
Angiogenesis Inhibitors/*pharmacology ; Apoptosis/*drug effects ; Bevacizumab/*pharmacology ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Gene Expression Regulation/physiology ; Humans ; Hydrogen Peroxide/toxicity ; Oxidative Stress/drug effects ; Proto-Oncogene Proteins c-bcl-2/*genetics ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction ; Retinal Pigment Epithelium/*drug effects/metabolism/pathology ; Vascular Endothelial Growth Factor A/antagonists & inhibitors

PURPOSE: To investigate the effects of resveratrol on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human adult retinal pigment epithelial (ARPE-19) cells, and on experimental choroidal neovascularization (CNV) in mice. MATERIALS AND METHODS: ARPE-19 cells were treated with different concentrations of resveratrol and then incubated under hypoxic conditions with subsequent evaluation of cell viability, expression of HIF-1alpha, and expression of VEGF. The effects of resveratrol on the synthesis and degradation of hypoxia-induced HIF-1alpha were evaluated using inhibitors of the PI3K/Akt/mTOR and the ubiquitin proteasome pathways. In animal studies, CNV lesions were induced in C57BL/6 mice by laser photocoagulation. After 7 days of oral administration of resveratrol or vehicle, which began one day after CNV induction, image analysis was used to measure CNV areas on choroidal flat mounts stained with isolectin IB4. RESULTS: In ARPE-19 cells, resveratrol significantly inhibited HIF-1alpha and VEGF in a dose-dependent manner, by blocking the PI3K/Akt/mTOR signaling pathway and by promoting proteasomal HIF-1alpha degradation. In mice experiments, orally administered resveratrol significantly inhibited CNV growth in a dose-dependent manner. CONCLUSION: Resveratrol may have therapeutic value in the management of diseases involving pathological neovascularization.
Adult ; Animals ; Anoxia/metabolism/physiopathology ; Cell Survival/drug effects ; Choroidal Neovascularization/*metabolism/pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*drug effects/metabolism ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/antagonists & inhibitors/*physiology ; Proteasome Endopeptidase Complex ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*physiology ; Retinal Pigment Epithelium/*drug effects/metabolism ; Signal Transduction ; Stilbenes/administration & dosage/*pharmacology ; TOR Serine-Threonine Kinases/antagonists & inhibitors/*physiology ; Ubiquitin ; Vascular Endothelial Growth Factor A/*drug effects/metabolism

The purpose of this study was to investigate the protective effect of puerarin on retina pigment epithelial (RPE) cells of diabetic rats against apoptosis. One hundred and eight Sprague-Dawley (SD) rats were randomly divided into 3 groups: control group, streptozotocin (STZ) group and puerarin group. STZ and puerarin groups received 3 d of STZ injection (45 mg/kg per day, i.p.). Additionally, puerarin groups were treated with puerarin (140 mg/kg, i.p.) from the 4th day to the end of experiment. The rats from different groups were sacrificed on 20, 40 and 60 d after STZ injection for harvesting RPE cells. Western blot analysis, DNA laddering, RT-PCR and immunohistochemistry were used for determining the expression of nitrotyrosine (NT, the foot print of peroxynitrite), cell apoptosis, iNOS mRNA and Fas/Fas ligand (FasL) signal transduction in RPE cells, respectively. The results showed that control group maintained low apoptosis level and little NT, iNOS mRNA, Fas/FasL protein expressions, as well as normal blood glucose and body weight during 60 d of the experiment. Compared with control group, STZ group showed obvious apoptosis and higher NT, iNOS mRNA, Fas/FasL protein expressions from 20 d after STZ injection. Puerarin relieved apoptosis of RPE cells and decreased NT, iNOS mRNA, Fas/FasL protein expressions in puerarin group 20 or 40 d after STZ injection, compared with STZ group. These results suggest puerarin can decrease RPE cells apoptosis in diabetic rats by reducing peroxynitrite level and iNOS expression, thus being a potential therapeutic agent in controlling of diabetic retinopathy.
Animals ; Apoptosis ; drug effects ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Diabetic Retinopathy ; prevention & control ; Fas Ligand Protein ; metabolism ; Isoflavones ; pharmacology ; Male ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Peroxynitrous Acid ; metabolism ; Protective Agents ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retinal Pigment Epithelium ; pathology ; fas Receptor ; metabolism

OBJECTIVETo observe the clinical effects of Erhuang Powder (EHP) in promoting the wound healing of cervical columnar epithelium ectopy after physiotherapy.

METHODSSixty-four patients with moderate and severe cervical columnar epithelium ectopy accompanied with contact bleeding were assigned to the treatment group (34 cases) and the control group (30 cases) according to table of random digit. Physiotherapy of the cervix was given to all patients. EHP was externally applied to the affected site of patients in the treatment group once every 3 days. Five times consisted as one therapeutic course, totally for 2 courses. The therapeutic effects, the wound healing time, the vaginal fluid quantity, the vaginal fluid discharge time, the wound infection, and adverse reaction were observed in the two groups.

RESULTSThe healing rate of the treatment group was 85.29% (29/34), higher than that of the control group (50%, 15/30) after 8 weeks of treatment, showing statistical difference (P < 0.1). The average wound healing time of the treatment group (3.2 - 0.8 weeks) was shorter than that of the control group (5.3 +/- 0.9 weeks), showing statistical difference (P < 0.5). The vaginal fluid quantity and the vaginal fluid discharge time in the treatment group were superior to those in the control group, showing statistical difference (P < 0.5, P < 0.1). Increased vaginal secretion accompanied with foreign odor or infection occurred in 2 patients of the control group. No adverse reaction or infection occurred in the treatment group.

CONCLUSIONSLocal application of EHP could promote the wound healing of cervical columnar epithelium ectopy after physiotherapy. It could improve its healing rate. Therefore, it was one ideal and reliable method, and worthy of clinical spreading.

Adult ; Cervix Uteri ; pathology ; Drugs, Chinese Herbal ; therapeutic use ; Endometriosis ; therapy ; Epithelium ; drug effects ; pathology ; Female ; Humans ; Middle Aged ; Physical Therapy Modalities ; Wound Healing

PURPOSE: The goal of the present research was to study post-treatment changes in polypoidal choroidal vasculopathy (PCV) shown by optical coherence tomography (OCT). METHODS: The study included 12 patients with naive PCV. Photodynamic therapy and 3 consecutive intravitreal bevacizumab injections at 6-week intervals were given. Best corrected visual acuity, subretinal fluid (SRF), pigment epithelium detachment (PED), central macular thickness (CMT), and total macular volume (TMV) were measured before and after treatment as assessed by Stratus OCT3. RESULTS: After treatment, the SRF height decreased earlier than the PED height. The SRF diameter decreased with statistical significance. However, the PED diameter did not show a statistically significant improvement, persisting at pre-treatment levels. Both CMT and TMV decreased significantly after treatment. CONCLUSIONS: After PCV treatment, SRF and PED stabilized, as shown by OCT. However, the PED treatment response was both delayed and refractory compared to the SRF response. The small change in post-treatment PED diameter may suggest the possibility of PCV recurrence.
Aged ; Choroid/*pathology ; Choroid Diseases ; Choroidal Neovascularization/diagnosis/*drug therapy ; Disease Progression ; Female ; Fluorescein Angiography ; Follow-Up Studies ; Fundus Oculi ; Humans ; Male ; Photochemotherapy/*adverse effects ; Prognosis ; Retinal Detachment/diagnosis/*etiology ; Retinal Pigment Epithelium/*pathology ; Retrospective Studies ; *Subretinal Fluid ; Time Factors ; Tomography, Optical Coherence ; Visual Acuity

OBJECTIVEOxidative stress plays an important role in retinal pigmental epithelium (RPE) death during aging and the development of age-related macular degeneration. Although early reports indicate that reactive oxygen species (ROS) including H₂O₂ can trigger apoptosis at lower concentrations and necrosis at higher concentrations, the exact molecular mechanism of RPE death is still unclear. The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS, especially at higher concentrations.

METHODSCultured ARPE-19 cells were treated with H₂O₂ at different concentrations and cell viability was measured with the MTT assay. Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining. Furthermore, the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG. Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects.

RESULTSH₂O₂ reduced the viability of ARPE-19 cells in a concentration-dependent manner, which was presented as a typical s-shaped curve. Cell death caused by high concentrations of H₂O₂ was confirmed to be programmed necrosis. Morphologically, dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane, positively detected with APOPercentage assay and PI staining. 24-hour treatment with 500 μmol/L H₂O₂ induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells. Moreover, antioxidant treatment using EGCG effectively protected cells from H₂O₂-induced injury, increasing cell viability from 14.17%±2.31% to 85.77%±4.58%. After H₂O₂ treatment, intracellular calcium levels were highly elevated with a maximum concentration of 1200 nM. Significantly, the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway, rescuing the ARPE-19 cell from H₂O₂-induced death.

CONCLUSIONSAt high concentrations, H₂O₂ induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.

Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Cell Culture Techniques ; Cell Line ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Heme Oxygenase-1 ; metabolism ; Humans ; Hydrogen Peroxide ; toxicity ; Necrosis ; drug therapy ; Oxidative Stress ; drug effects ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Retinal Pigment Epithelium ; drug effects ; enzymology ; metabolism ; pathology

AIMTo investigate the role of the Chinese herbal medicine Xianhuayin on the reversal of 7,12-dimethylbenz[a]anthracene (DMBA)-induced premalignant mucosal lesions in the oral buccal pouch of golden hamsters.

METHODOLOGYThe animals were randomly divided into a non-diseased control group (n=5) and an experimental group including 50 animals in which the buccal mucosa had been painted with DMBA (0.5% in acetone) to generate an oral mucosa premalignant lesion. Animals in the experimental group were further divided into Xianhuayin-treated group (n=30), untreated premalignant lesion group (n=10) and normal saline (NS)-treated group (n=10). The cheek (buccal) pouch mucosa of the golden hamsters in each group was observed with light and electron microscopy eight weeks after intragastric administration with NS or Xianhuayin.

RESULTSIn the non-diseased control group, the buccal mucosa was keratinized and stratified squamous epithelium under a light microscope. In the untreated premalignant lesion group, variable degrees of epithelial dysplasia was observed. The irregular epithelial mucosa gradually became distinct in the Xianhuayin-treated group. Scanning electronic microscopic (SEM) analysis showed that surface of the cells exhibited honeycomb structures in the hamster of untreated-group. The cells were morphologically irregular, overlapped and loosened in the untreated premalignant lesion group. Most of the cell surface exhibited honeycomb structure in the Xianhuayin-treated group. Transmission electronic microscopic (TEM) analysis showed that buccal mucosal epithelial cells were morphologically regular in the non-diseased control group. Desmosomes and tonofibrils were reduced and the nucleus was morphologically irregular in the untreated premalignant lesion group. In the Xianhuayin-treated group, the widening intercellular gap was gradually reduced, desmosomes and the cells becoming morphologically regular. No significant difference was observed between the hamsters in NS-treated group and those in the untreated premalignant lesion group. Significant therapeutic efficacy was observed in the group receiving Xianhuayin.

CONCLUSIONXianhuayin is effective in the reversal of DMBA-induced premalignant lesions in the buccal pouch of golden hamsters.

9,10-Dimethyl-1,2-benzanthracene ; adverse effects ; Amomum ; Animals ; Anticarcinogenic Agents ; administration & dosage ; therapeutic use ; Carcinogens ; Carthamus tinctorius ; Cell Nucleus ; drug effects ; Cricetinae ; Desmosomes ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; therapeutic use ; Epithelial Cells ; drug effects ; Epithelium ; drug effects ; Glycyrrhiza ; Hyperplasia ; Intercellular Junctions ; drug effects ; Intermediate Filaments ; drug effects ; Keratins ; Mesocricetus ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Mouth Mucosa ; drug effects ; pathology ; Mouth Neoplasms ; prevention & control ; Philodendron ; Poria ; Precancerous Conditions ; prevention & control ; Random Allocation ; Sodium Chloride