Protective effects of naringin phospholipid complex on oxidative injury in ARPE-19 cells associated with activation of Nrf2 pathway / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the protective effects of naringin(Nar)phospholipid complex(NPC)on oxidative injury in retinal pigment epithelium cells(ARPE-19 cells)induced by tert-butyl hydroperoxide(t-BHP)and elucidate the underlying mechanism.<p>METHODS:The NPC was prepared by solvent method. Experimental cells are divided into seven groups: control group \〖cultured with dimethylsulfoxide(DMSO)\〗, model group(intervention with 200μmol/L t-BHP), nuclear factor erythroid 2-related factor 2(Nrf2)-siRNA group(cell transfection for Nrf2 gene), naringin group(add 200μmol/L t-BHP after pretreatment with 200μmol/L naringin medium), NPC group(add 200μmol/L t-BHP after pretreatment with 200μmol/L NPC medium), Nrf2-siRNA+ naringin group(after 200μmol/L naringin pretreatment, Nrf2 gene interference, then add 200μmol/L t-BHP)and Nrf2-siRNA+ NPC group(after 200μmol/L NPC pretreatment, Nrf2 gene interference, then add 200μmol/L t-BHP). The intracellular levels of superoxide dismutase(SOD), malondialdehyde(MDA)and total antioxidant capacity(T-AOC)were detected, intracellular level of reactive oxygen species(ROS)was detected by DCFH-DA staining method. The mRNA and protein expressions of HO-1, NQO-1, GCL and Nrf2 were detected by real-time PCR and western blot, respectively. <p>RESULTS:NPC more significantly increased the levels of SOD and T-AOC, reduced the contents of ROS and MDA than naringin in t-BHP-treated ARPE-19 cells. After naringin and NPC pre-protected ARPE-19 cells, the relative expression and protein expression of Nrf2, HO-1, NQO-1 and GCL mRNA were higher than those of the model group and Nrf2-siRNA group. There were statistically significant differences in the relative expression of 4 genes and the expression levels of 4 proteins in the naringin group and the NPC group, the Nrf2-siRNA+naringin group and the Nrf2-siRNA+NPC group. The expression of Nrf2, HO-1 and NQO-1 protein in the Nrf2-siRNA+naringin group was not significantly different than that in the Nrf2-siRNA group. Compared with the Nrf2-siRNA group, the expression of 4 proteins in the Nrf2-siRNA+NPC group was statistically significant, and the effect of NPC was significantly stronger than that of naringin.<p>CONCLUSION: After naringin forms a phospholipid complex, it can significantly increase the antioxidant capacity in cells and reduce the oxidation level. It up-regulates the expression of Nrf2 and its downstream antioxidant enzymes and phase Ⅱ detoxification enzymes by activating the Nrf2/ARE antioxidative stress pathway to better protect ARPE-19 cells from oxidative damage.

Dipenyleneiodonium Induces Growth Inhibition of Toxoplasma gondii through ROS Induction in ARPE-19 Cells

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.

Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

Protective effect of Bu Shen Yang Xue Ming Mu Formula on oxidative injury in ARPE-19 cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the effect of Bu Shen Yang Xue Ming Mu (BSYXMM) Formula on hydroquinone-induced oxidative stress injury in ARPE-19 cells.METHODS: The oxidative injury model of ARPE-19 cell was induced by exposure to various concentrations of hydroquinone (HQ) to determine the optimal concentration.Intestinal absorption solutions of BSYXMM Formula were prepared.Effect of intestinal absorption solutions of BSYXMM Formula on the cell viability was detected by CCK-8 assay,and the percentage of apoptotic cells was measured by TUNEL assay.The levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in ARPE-19 cells were detected by means of chemical colorimetry.RESULTS: It was found that ARPE-19 cell viability significantly decreased when the concentration of HQ was higher than 90μmol/L.Compared with the model group,1% and 2% intestinal absorption solutions in the pre-treatment group could significantly alleviate HQ-induced injury (P<0.01) and 0.5% and 5% intestinal absorption solutions in the pre-treatment group could alleviate the injury in certain degree(P<0.05).While in the treatment group 1% and 2% intestinal absorption solutions could alleviate the injury to some extent (P<0.05).TUNEL results showed that the apoptosis rate decreased significantly in the pre-treatment group (P<0.01)and to some extent in the treatment group (P<0.05)compared with the model group.It was shown that both levels of SOD and GSH-Px in pre-treatment group and treatment group were markedly higher than that of model group(P<0.05),and pre-treatment group had more significant effect (P<0.01,P<0.05).CONCLUSION: BSYXMM Formula could protect against HQ-induced oxidative stress injury in ARPE-19 cells,which may be related with the increasing of antioxidant enzyme in the cells.

Effects of the prescription of reinforcing kidney, nourishing blood, improving eyesight on ARPE - 19 cells induced by acrolein / 国际眼科杂志(Guoji Yanke Zazhi)

?AlM: To explore the effects of the prescription of reinforcing kidney, nourishing blood, improving eyesight on the oxidative stress model of ARPE-19 cells induced by acrolein. ?METHODS:SD rats serum containing the prescription of reinforcing kidney, nourishing blood, improving eyesight and the content of distilled water in serum were prepared. The effects of the prescription and distilled water in serum at different concentration ( 2. 5%, 5%, 10%, 20% and 40%) on cell vitality was observed by cell counting kit ( CCK-8 ) assay. the logarithmic phase of ARPE-19 cells were pretreated by different concentrations (1. 25%, 2. 5%and 5%) of the prescription serum and distilled water in serum for 24h. Then it was treated with 75μmol/L acrolein for 24h. Cell vitality was observed by CCK-8 assay. The change of cell nucleus was detected by DAPl staining . ?RESULTS: 2. 5% and 5% serum had no effect on cell viability (P>0. 05), while 10%, 20%, 40% serum could inhibit cell viability (P ?CONCLUSlON: The prescription of reinforcing kidney, nourishing blood, improving eyesight has the protective effect on ARPE-19 cell damage induced by acrolein.

Anti-apoptosis effect of lipoic acid-niacin diad on acrolein-induced apoptosis in ARPE-19 cells / 实用医学杂志

Objective To investigate the regulations of Bax , Bcl-2 in the protection of lipoic acid-niacin diad in acrolein-induced apoptosis in ARPE-19 cells. Methods The ARPE-19 cells were cultured in medium containing 10% fetal bovine serum , at 37 ℃ with 5% CO2. The ARPE-19 was transferred to 6-well plate after reaching to 70% confluence. After starvation for 24 h , the cells in 6-well plates were divided into three groups , including the blank control group , the acrolein treatment group with 50 μmol/L acrolein for 24 h , and the protection group with 100 μmol/L lipoic acid-niacin diad for 24 h and with the acrolein for another 24 h. The apoptotic cells were detected by flow cytometry assay , and expressions of Bcl-2 , Bax protein were detected by Western Blot assay. Results The percentages of normal healthy cells were 94.8%, 60.98%, and 91.34% in the blank control group , 50 μmol/L acrolein group and 100 μmol/L diad contained of lipoic acid and niacin group , respectively. The ratios of Bax/Bcl-2 protein expression were 0.293 9, 1.389 2, and 0.555 8 in the blank control group, 50 μmol/L acrolein group and 100 μmol/L diad contained of lipoic acid and niacin group, respectively. Conclusion The protective effect of lipoic acid-niacin diad on acrolein-induced apoptosis in ARPE-19 cell through promoting Bcl-2 expression and inhibiting Bax expression.