Mechanism and Pharmacodynamic Substance Basis of Raw and Wine-Processed Evodia rutaecarpa on Smooth Muscle Cells of Dysmenorrhea Mice.

Objectives: Evodia rutaecarpa (ER) is a well-known herbal Chinese medicine traditionally used for analgesia in dysmenorrhea, headaches, abdominal pain, etc. Notably, the analgesic effect of wine-processed Evodia rutaecarpa (PER) was more potent than that of raw ER. This research aimed to investigate the mechanism and pharmacodynamic substance basis of raw ER and PER on smooth muscle cells of dysmenorrhea mice. Methods: Metabolomics methods based on UPLC-Q-TOF-MS were utilized to analyse the differential components of ER before and after wine processing. Afterwards, the uterine smooth muscle cells were isolated from the uterine tissue of dysmenorrhea and normal mice. The isolated dysmenorrhea uterine smooth muscle cells were randomly divided into four groups: model group, 7-hydroxycoumarin group (1 mmol/L), chlorogenic acid (1 mmol/L), and limonin (50 µmol/L). The normal group consisted of the isolated normal mouse uterine smooth muscle cells, which were repeated 3 times in each group. The cell contraction and the expression of P2X3 and Ca2+ in vitro were determined using immunofluorescence staining and laser confocal; ELISA was used for detection of PGE2, ET-1, and NO content after 7-hydroxycoumarin, chlorogenic acid, and limonin administered for 24 h. Results: The metabolomics results suggested that seven differential compounds were identified in the extracts of raw ER and PER, including chlorogenic acid, 7-hydroxycoumarin, hydroxy evodiamine, laudanosine, evollionines A, limonin, and 1-methyl-2-[(z)-4-nonenyl]-4 (1H)-quinolone. The in vitro results showed that 7-hydroxycoumarin, chlorogenic acid, and limonin were able to inhibit cell contraction and PGE2, ET-1, P2X3, and Ca2+ in dysmenorrhea mouse uterine smooth muscle cells and increase the content of NO. Conclusion: Our finding suggested that the compounds of the PER were different from those of the raw ER, and 7-hydroxycoumarin, chlorogenic acid, and limonin could improve dysmenorrhea in mice whose uterine smooth muscle cell contraction was closed with endocrine factors and P2X3-Ca2+ pathway.

The effects of hypoxia on the expression of AQP-4 in Müller cells / 眼科研究

Background Hypoxia is an important cause resulting in many retinal diseases,such as retinal edema,diabetic retinopathy,proliferative retinopathy and so on.ObjectiveThis study is to investigate the effects of hypoxia on the expression of AQP-4 in Müller cells in vitro.MethodsMüller cells were isolated from New Zealand white rabbits and primarily cultured in DMEM containing 20% fetal bovine serum by the explant culture method.The cells were identified by immunostaining for the glial fibrillary acidic protein(GFAP).Generation 2 of cells was cultivated with the chemical hypoxia inducer,CoCl_2,for 24 hours in the hypoxic group and only with DMEM in the control group.The expression of the AQP-4 protein in Müller cells was detected by immunocytochemistry.The expression of AQP-4 mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).ResultsAbout 90% of Müller cells(generation 2) presented a positive immunoreactivity for GFAP,showing a brown staining in the cytoplasm.Cultured cells displayed the presence of intermediate filaments,microvillus and various cellular organs.The Integralabsorbance of the AQP-4 protein in Müller cells was markedly increased 24 hours after incubation with CoCl_2 in comparison with the control group (t=6.74,P＜0.05).The expression level of AQP-4 mRNA in Müller cells was significantly enhanced 24 hours after incubation with CoCl_2 in comparison with the control group (t=21.79,P＜0.05). ConclusionHypoxia enchances the expression of AQP-4 in Müller cells and further increases fluid accumulation in the retina.These results suggest that Müller cells play an important role in the formation of retinal edema in diabetic retinopathy or proliferative retinopathy.

Small hairpin loop RNA targeting HIF-1alpha down-regulates VEGF and up-regulates PEDF in human retinal pigment epithelial cells under hypoxic condition.

The aim of this study was to explore the effect of small hairpin loop RNA (shRNA) silencing hypoxia-induced factor 1alpha (HIF-1alpha) gene on the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in human retinal pigment epithelium (RPE) cells under hypoxic condition. Two target sites of HIF-1alpha mRNA were chosen and two kinds of shRNA were designed and synthesized against the target sites. Then the two kinds of shRNA were transfected into human RPE cells in vitro, respectively. These cells were cultured under hypoxic condition that was simulated by using 150 mumol/L CoCl(2). The mRNA expressions of HIF-1alpha, VEGF and PEDF were tested by semi-quantitative reverse transcription PCR (RT-PCR). The protein levels of HIF-1alpha, VEGF and PEDF were analyzed by Western blotting. After the two kinds of HIF-1alpha-specific shRNA were transfected into RPE cells respectively, the expression of HIF-1alpha mRNA and the levels of HIF-1alpha protein were decreased significantly in RPE cells under hypoxic condition. The expression of VEGF mRNA and the levels of protein significantly were also decreased. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA showed no significant changes. In conclusion, HIF-1alpha-specific shRNA can effectively silence the HIF-1alpha gene, and consequently down-regulate VEGF and up-regulate PEDF expression against hypoxia. These results reveal that HIF-1 is associated with posttranslational mechanism for down-regulating PEDF under hypoxia and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results also suggest that HIF-1 is one of the key cytokines to retinal neovascularization.

Small Hairpin Loop RNA Targeting HIF-1α Down-regulates VEGF and Up-regulates PEDF in Human Retinal Pigment Epithelial Cells under Hypoxic Condition / 华中科技大学学报（医学）（英德文版）

The aim of this study was to explore the effect of small hairpin loop RNA (shRNA) silencing hypoxia-induced factor 1α (HIF-1α) gene on the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in human retinal pigment epithelium(RPE) cells under hypoxic condition. Two target sites of HIF-1α mRNA were chosen and two kinds of shRNA were designed and synthesized against the target sites. Then the two kinds of shRNA were transfected into human RPE cells in vitro, respectively. These cells were cultured under hypoxic condition that was simulated by using 150 μmol/L CoCl2. The mRNA expressions of HIF-1α, VEGF and PEDF were tested by semi-quantitative reverse transcription PCR (RT-PCR). The protein levels of HIF-1α, VEGF and PEDF were analyzed by Western blotting. After the two kinds of HIF-1α-specific shRNA were transfected into RPE cells respectively, the expression of HIF-1α mRNA and the levels of HIF-1α protein were decreased significantly in RPE cells under hypoxic condition. The expression of VEGF mRNA and the levels of protein significantly were also decreased. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA showed no significant changes. In conclusion, HIF-1α-specific shRNA can effectively silence the HIF-1α gene, and consequently down-regulate VEGF and up-regulate PEDF expression against hypoxia. These results reveal that HIF-1 is associated with posttranslational mechanism for down-regulating PEDF under hypoxia and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results also suggest that HIF-1 is one of the key cytokines to retinal neovascularization.

[Matrix metalloproteinases inhibitor doxycycline inhibits TGF-beta 1-induced RPE cell migration].

OBJECTIVE: To investigate the effects of matrix metalloproteinases (MMP) inhibitor (Doxycycline) on retinal pigment epithelial (RPE) cell migration induced by transforming growth factor beta 1 (TGF-beta 1). METHODS: The third to sixth passage human RPE cells cultures were treated with TGF-beta 1 at different concentrations (0.01, 0.10, 1.00, 10.00 microg/L), and the conditioned media were collected after 36 h of TGF-beta 1 exposure. Gelatinase activities in conditioned media were analyzed by zymography. Migration assay was performed in a Boyden chamber with addition of different concentrations of Doxycycline. The number of migrated cells was counted under light microscopy. RESULTS: RPE cells migration were stimulated by TGF-beta 1 significantly, the number of migrated RPE cells increased about 27% compared with the control (P < 0.01). In the presence of Doxycycline, the RPE cells migration induced by TGF-beta 1 was inhibited and the number of migrated cells was reduced to 50% - 70% compared with control (P < 0.01). TGF-beta 1 also stimulated the secretion of MMP-2 in dose-dependent manner. CONCLUSIONS: This study indicates that Doxycycline can inhibit migration of RPE cells stimulated by TGF-beta 1. RPE migration induced by TGF-beta 1 may act partially through the stimulation of MMP production.

[The change of the level of the vascular endothelial growth factor in aqueous humor of patients with neovascular glaucoma before and after anterior retinal cryotherapy].

OBJECTIVE: To compare the change of the level of the vascular endothelial growth factor (VEGF) in aqueous humor of patients with neovascular glaucoma (NVG) before and after anterior retinal cryotherapy and investigate the effects of the fluctuation of VEGF on the neovascularization of iris. METHODS: 28 patients with neovascular glaucoma were undergone iris fluorescent angiography to identify the area and amount of new vessels before and after anterior retinal cryotherapy. The neovascularization of iris was observed from 7 to 14 days by iris fluorescent angiography to confirm the regression of new vessels in iris before trabeculectomy. Samples of aqueous humor were obtained before anterior retinal cryotherapy and trabeculectomy, and 30 samples of aqueous humor from patients with senile cataract were collected as normal group. The concentrations of VEGF were measured using enzyme linked immunosorbent assay. RESULTS: the concentration of VEGF (2.096 +/- 0.512) ng/ml in aqueous humor from patients with NVG were much higher than the specimen from the patients pre-trabeculectomy (0.478 +/- 0.312) ng/ml, There was a significant difference between the two groups (t = 17.994, P < 0.01). The mean VEGF concentration of the aqueous humor from patients with senile cataract was (0.198 +/- 0.045) ng/ml which was much lower compared with the samples from patients of pre-trabeculectomy (t = 18.453, P < 0.01). CONCLUSIONS: The concentration of VEGF decline after the regression of new vessels in iris. The results suggest that VEGF play an important role in formation of iris neovascularization. Blockage the release of VEGF might reduce the occurrence of neovascular glaucoma.

[Down-regulation of vascular endothelial growth factor in human retinal pigment epithelial cells by small hairpin loop RNA targeting hypoxia inducible factor-1 alpha under hypoxia condition].

OBJECTIVE: To explore the effect of hypoxia inducible factor-1 alpha (HIF-1 alpha) gene on the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (hRPE) cells under hypoxia conditions by using small hairpin loop RNA (shRNA) to silence HIF-1 alpha. METHODS: CoCl(2) (150 micromol/L) was used to simulate the hypoxia environment for hRPE cells. After choosing a target site of HIF-1 alpha mRNA, shRNA was designed and synthesized by this target site. hRPE cells were transfected by this shRNA in vitro. Then, these cells were cultured under hypoxia conditions (150 micromol/L CoCl(2)). The mRNA expression of HIF-1 alpha and VEGF was measured by semi-quantitative reverse transcription PCR (RT-PCR). The protein level of HIF-1 alpha and VEGF was studied by western blot analysis. RESULTS: After hRPE cells were transfected by HIF-1 alpha-specific shRNA, RT-PCR showed that the expression of HIF-1 alpha mRNA was inhibited by 77.1%, and western blot analysis showed that the level of HIF-1 alpha protein was significantly decreased in hRPE cells under hypoxia conditions. Moreover, the expression of VEGF mRNA was inhibited by 27.8% and the level of VEGF protein was also significantly decreased in transfected hRPE cells under hypoxia conditions. CONCLUSIONS: Under hypoxia conditions, HIF-1 alpha-specific shRNA effectively keeps HIF-1 alpha gene silenced, and consequently down-regulates VEGF expression against hypoxia. These results suggest that HIF-1 alpha is one of the most important cytokines for retinal neovascularization.

Inhibition of PCNA antisense oligonucleotides mediated by liposome on mRNA expression and proliferation of h-RPE cells.

The proliferating cell nuclear antigen (PCNA) gene expression was blocked and retinal pigment epithelium (RPE) proliferation was inhibited by using antisense oligonucleotides (AS-ODN) mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy (PVR). RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN (0.07, 0.28 and 1.12 micro mol/L and sense oligonucleotides (S-ODN with the same concentrations as AS-ODN) mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by 3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression of PCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density (AOD) was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN (0.28 and 1.12 micro mol/L) markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant (P < 0.05 and P < 0.01, repectively) as compared with blank control group. AOD was well correlated with CPM (r = 0.975). It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

Up-regulation of HIF-1alpha and VEGF expression by elevated glucose concentration and hypoxia in cultured human retinal pigment epithelial cells.

In order to explore the effect of high glucose concentration and high glucose concentration with hypoxia on the production of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF), human RPE cells were cultured in 5.56 mmol/L glucose (control group), 5.56 mmol/L glucose with 150 micro mol/L CoCl2 (hypoxic group), 25 mmol/L glucose (high glucose group) and 25 mmol/L glucose with 150 micro mol/L CoCl2 (combination group). RT-PCR was used to detect the expression of HIF-1alpha and VEGF mRNAs. Western blot analysis was used to measure the levels of HIF-1alpha and VEGF proteins. Although the small amount of HIF-1alpha protein was able to be detected in high glucose group but not in control group, there was no significant difference between the expression of HIF-1alpha mRNA of RPE cells in high glucose group and that of RPE cells in control group. As compared with RPE cells in control group, the mRNA expression and the protein synthesis of VEGF in high glucose group were up-regulated. As compared with RPE cells in hypoxic group, the expression of HIF-1alpha mRNA of RPE cells in combination group was not different, but the protein synthesis of HIF-1alpha, the mRNA expression and the protein synthesis of VEGF were more obviously up-regulated. In conclusion, high concentration glucose mainly influence the protein synthesis of HIF-1alpha of RPE cell, and HIF-1alpha protein is able to be accumulated in high concentration glucose. Under hypoxia, the HIF-1alpha protein induced by high concentration glucose is more stable, and the expression of VEGF is obviously increased. It is suggested that high concentration glucose may play a role in retinal neovascularization, especially at ischemia stage of diabetic retinopathy.

Modulation of matrix metalloproteinase and TIMP-1 expression by TGF-beta1 in cultured human RPE cells.

In order to investigate the effects of TGF-beta1 on the expression of MMP-2, -9 and TIMP-1 in human retinal pigment epithelial (RPE) cells, the third-sixth passage cultured RPE cells were treated with TGF-beta1 at different concentrations (0.01, 0.1, 1.0, 10 ng/mL), the expression of MMP-2, -9 and TIMP-1 mRNA was detected by semi-quantitative RT-PCR assays. MMP-2, -9 and TIMP-1 mRNA were expressed in the cultured RPE cells. The values of MMP-2/beta-actin in the cells treated with 0.1, 1.0, 10 ng/mL TGF-beta1 were 1.04 +/- 0.04, 1.07 +/- 0.02 and 1.11 +/- 0.03, respectively, significantly higher than in the control group (0.96 +/- 0.03, P < 0.05-0.01). The expression of MMP-2 mRNA could be up-regulated by TGF-beta1, in a dose-dependent manner. The expression of MMP-9 mRNA in the cultured RPE cells was slightly up-regulated by various TGF-beta1 concentrations treatment. The values of TIMP-1/beta-actin in the cells treated with 0.01 and 0.1 ng/ mL TGF-beta1 were 0.85 +/- 0.01 and 0.97 +/- 0.02 respectively, significantly lower than in the control group (1.07 +/- 0.04, P < 0.01), indicating that the expression of TIMP-1 mRNA was down-regulated by TGF-beta1 at low concentrations. But along with the increase of TGF-beta1 concentrations (1.0 and 10 ng/mL), the expression of TIMP-1 mRNA was slightly up-regulated, not significantly different from that in the control group (P > 0.05). It was concluded that TGF-beta1 might play an important role in the up-regulation of the expression of MMP-2 in RPE cells and result in a directional shift in the balance between MMP and TIMP. This may be facilitated for RPE cells to migrate in the pathogenesis of vitreoretinopathy.

Effect of shRNA inhibiting HiF1alpha gene on TIMP1 expression in RPE cells.

Small hairpin RNA (shRNA) was used to silence the HIF1alpha gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2, to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1alpha mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1alpha was interfered in RPE cultured under hypoxia (induced by 150 micromol/L CoCl2). RT-PCR was employed to detect the expression of HIF1alpha and TIMP1. The expression levels of HIF1alpha and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1alpha mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1alpha gene silencing in RPE was 83.4%. Western blotting revealed that the expression levels of HIF1alpha protein was dramatically dropped. In addition. RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9%, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1alpha mRNA could effectively silence the HIF1alpha gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.

[The inhibitory effects of angiostatin on retinal neovascularization induced by oxygen].

OBJECTIVE: To observe the inhibitory effects of angiostatin on microvascular endothelial cells of mouse retina and test the efficacy of native angiostatin in suppressing experimental retinal neovascularization induced by oxygen. METHODS: Angiostatin was purified with L-lysine Sepharose 4B from human plasma. The primary microvascular endothelial cells from rat retina were cultured. Microvascular endothelial cells growth inhibition assay was carried out with MTT method. Mouse models of hyperoxia-induced ischemic retinopathy were established. Angiostatin or normal saline (NS) were injected into the vitreous in 5 groups: normal, control and various doses. The nuclei of new vessel buds extending from the retina into the vitreous in different groups were counted and compared under the light microscope. RESULTS: Angiostatin could inhibit the growth of microvascular endothelial cells from rat retina in vitro. There were plenty of new vessel buds in the eyes of all mice in hyperoxic condition. The number of the nuclei of new vesselbuds in the murine eyes with injection of angiostatin. They were reduced by 42% (P < 0.01), 57% (P < 0.01) and 82% (P < 0.01) respectively. CONCLUSION: Angiostatin can powerfully inhibit growth of microvascular endothelial cells. The proliferation of retinal vessel may be suppressed by using angiostatin.

[Posterior vitreous detachment induced by intravitreal injection of the combination of chondroitinase ABC and matrix metalloproteinase-3].

OBJECTIVE: To investigate the occurrence of enzymatic induction of posterior vitreous detachment (PVD) by the combination of Chondroitinase ABC (CA) and matrix metalloproteinase-3 (MMP-3), so as to seek a noninvasive and effective pharmacologic approach to facilitate and eventually replace the present mechanical vitreous surgery. METHODS: Twenty-four pigmented rabbits were randomly assigned to two groups of twelve each, the experimental group was treated with CA (0.2 U) and MMP-3 (10 ng) combination, the control group was received equivalent dose of balanced salt solution (BSS). Clinical examinations and electroretinography were performed before and after injection. Over a different period of time, the rabbits were euthanized and killed and their eyes were examined histologically. RESULTS: The foci of partial synchisis and clinically named PVD were recognized for the first time three days after injection. Complete liquefaction was found in every eye of experimental group, and in which, 5/8 eyes developed clinically detected PVD one week after injection Histologic section showed PVD with various extent in 3/7, 7/7, 7/7 eyes of experimental group 30 minutes, 60 minutes and one week after injection respectively, and complete PVD in 0/7, 1/7, 5/7 eyes of experimental group at the same periodic intervals as above. By contrast, no vitreous liquefaction was found and only one eye showed confined partial PVD in the control group. Clinic, electrophysiologic and histologic evaluation in all rabbits revealed no evidence of ocular toxicity. CONCLUSIONS: Vitreous 1iquefaction and PVD can be produced shortly after intravitreal injection using a combination of CA and MMP-3, and the two enzymes were cooperative. Synchisis and weakening of vitreoretinal adherence were almost simultaneously. The dose of 0.2 U CA and 10 ng MMP-3 combination proved to be safe and ideal selection to induce PVD.

An experimental rabbit model of rhegmatogenous retinal detachment.

An experimental model of rhegmatogenous retinal detachment (RRD) in rabbits was established to simulate the pathophysiologic condition of human RRD. 24 rabbits were randomly divided into 3 groups and underwent vitrectomy with a vitrector and/or retinotomy with a Charles flute needle, with 12 in group I (vitrectomy and retinotomy), 7 in group I (retinotomy) and 5 in group III (vitrectomy). All animals underwent follow-up examinations with direct and indirect ophthalmoscopy and fundus photography 12 h and day 1, 3, 5, 7, 10, 14, 21, and 28 after the procedure(s). Retinal changes were recorded. As a result, 10 RRDs were successfully established in group I. Direct and indirect ophthalmoscopy and fundus photography demonstrated typical features of RRD. No RRD developed in group II and III. It was concluded that the experimental rhegmatogenous retinal detachment produced in a rabbit model after vitrectomy with retinotomy in this study was a convenient and reliable one. This RRD model mimicked the typical pathophysiological changes in humans.

Effects on telomerase activity and associated-protein of hRPE cells by TGF-beta 1.

PURPOSE: To investigate the effects on telomerase activity, human telomerase reverse transcriptase(hTERT) gene and TEP1mRNA of retinal pigment epithelial(RPE) cells were treated by TGF-beta 1 of different concentration. METHODS: The cultured human RPE cells were treated with TGF-beta 1 at different concentration (0 ng/ml, 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml) for 24 h, then telomerase activity was detected by telomerase repeat amplification protocol (TRAP). The expression of hTERT mRNA and TEP-1mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: TRAP and RT-PCR showed when the concentration of TGF-beta 1 was gradually increased, telomerase activity and the expression of hTERTmRNA were gradually reduced, TEP1mRNA showed no apparent differential expression. CONCLUSION: TGF-beta 1 can down-regulate telomerase activity and the expression of hTERT mRNA, but no effection on TEP-1mRNA, hTERTmRNA expression was in accordance with telomerase activity in hRPE cells, hTERT gene plays a crucial role in the expression of telomerase activity, while TEP1 plays a much smaller role.