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Background and purpose:A large number of studies have showed that retinoblastoma gene 1 (RB1) can inhibit the occurrence and development of many tumors, including neuroblastoma, small cell lung cancer, osteosar-coma, pancreatic cancer, breast cancer and so on. RB1 is also closely related to the regulation of cell cycle, differentia-tion, senescence, apoptosis, growth inhibition, etc. The goal of this article is to elucidate whether miR-222 promotes cell proliferation and invasion by targeting RB1, further to explore the molecular mechanism that miR-222 functions as an oncogene in retinoblastoma cells.Methods:miR-222 (miR-222 mimics) and RB1-wt, miR-NC and RB1-wt, miR-222 and RB1-mut, miR-NC (a controlled miR-222 mimics) and RB1-mut were co-transfected into Y79 cells, and luciferase activity was detected by single photon. Retinoblastoma cells were transfected with miR-222 mimics and miR-NC, and the expressions of RB1 protein were detected by Western blot. Retinoblastoma cell proliferation assays were performed by MTS assay when miR-222, miR-NC, RB1 (pcDNA3.1-RB1), vector (pcDNA3.1), miR-222+RB1 and miR-NC+vec-tor were transfected into Y79 cells. The growth and invasion ability of Y79 cells with ectopic expression of miR-222 were evaluated by MTS and Transwell invasion assays.Results:This study demonstrated that miR-222 could promote the luciferase activity of RB1-wt. The expression levels of luciferase reporter gene activity in Y79 cells after transfection with miR-222+RB1-wt were higher than those in the negative control cells (miR-NC+RB1-wt) (P0.05). After transfection with miR-222 mimics for 48 h, Transwell invasion assay showed that the number of cells through the basement membrane was (193±10). Compared with the control group (144±11), it could signiifcantly accelerate the invasion of Y79 cells (P0.05).Conclusion:miR-222 promotes cell proliferation and invasion by targeting RB1 expression in retinoblastoma cells.
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Background Oxydative stress is an important pathogenesis of age-related macular degeneration.Resent evidences indicate that docosahexaenoic acid (DHA) plays an important role during the development of retinal photoreceptor cells and protect the cells against oxydative stress by inducing the expression of heme oxygenase-1 (HO-1).However,whether DHA can induce the expression of HO-1 in human retinal pigment epithelium (RPE) cells is unelucidated.Objective This study was to investigate the effect of DHA on the expression of HO-1 in RPE cells and its molecular mechanism.Methods Human RPE cell line ARPE-19 was cultured in vitro and treated with 30,50,100 and 120 μmol/L DHA for 4 to 24 hours,respectively,and the cells were cultured without DHA as the control group.The cytotoxicity of DHA was detected by lactate dehydrogenase(LDH),and the expression of HO-1 mRNA and protein were detected by real-time PCR and Western blot assay,respectively.The enzymatic activity of HO-1 was detected by colorimetry.The reactive oxygen species (ROS) proportion in the cells was detected using fluorescence probe H2 DCFDA,and immunofluorescence technology was adopted to detect the nuclear translocation of nuclear facotor-E2-related factor 2 (Nrf2).The expression of Nrt2 protein in the cells was detected by Western blot after intervention of ROS inhibitor N-acetylcysteine (NAC) and transfection of Nrf2 small interfering RNA (siRNA).Results The LDH leakage rate was significantly different after 0,3,50,100 and 120 μmol/L DHA treated the cells for 24 hours (F=8.14,P＜0.05),and the LDH leakage rate in the 120 μmol/L DHA group was significantly higher than that of 0,30,50 and 100 μmol/L DHA group (all at P＜0.05).The relative expression levels of HO-1 mRNA and HO-1 protein or HO-1 enzymatic activity in the cells were significantly different among different concentrations of DHA group in 8 hours after treatment (F=16.24,P＜0.05;F=11.34,P＜0.05;F=11.81,P＜0.05),and the expressions of these factors were considerably higher in the 30,50 and 100 μ mol/L DHA group than those in the 0 μmol/L DHA group (all at P＜0.05).The ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were statistically significant among different concentrations of DHA groups (F =11.08,P ＜ 0.05;F=16.42,P＜0.05),and the ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were evidently higher in the 30,50 and 100 μmol/L DHA group than those in the 0 μmol/L DHA group (all at P＜0.05).The relative expression levels of HO-1 protein and the proportion of nuclear Nrf2 positive cells were significantly lower in the NAC pretreated 100 μmol/L DHA group than those in the 100 μmol/L DHA group.In addition,the HO-1 relative expression level and the positive cells proportion of nuclear Nrf2 were significantly lower in the of Nrf2 siRNA transfection group than those in the blank siRNA transfection group (both at P＜0.05).Conclusions DHA with concentration below 100 μ mol/L can protect RPE cells from oxidative stress by inducting the expression of HO-1 in the cells via ROS/Nrf2 pathway.
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AIM:To observe the effect of docosahexaenoic acid ( DHA) on H2 O2-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism .METHODS: Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro, and 12.5 mmol/L H2 O2 was used to mimic the oxidative stress condition .The cells were treated with 30~100μmol/L DHA for 4~24 h.The expression of heme oxygenase-1 (HO-1) at mRNA and protein levels was detected by real-time PCR and Western blot , respectively .The enzymic activity of HO-1 was measured by colorimetry . Production of reactive oxygen species ( ROS) was determined by fluorescent probe .Activation of NF-E2-related factor 2 (Nrf2) was examined by immunofluorescence method .Apoptosis of ARPE-19 cells was analyzed by flow cytometry .RE-SULTS:The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment .Meanwhile , nuclear translocation of Nrf 2 was also observed .Apoptosis appeared and ROS was produced upon H2O2 incubation.In contrast, DHA at 100 μmol/L significantly abrogated H2O2-induced apoptosis and ROS production.Furthermore, silencing of HO-1 by specific siRNA, or treatment with ZnPP, an inhibitor of HO-1, partly counteracted the protective effect against H 2 O2-induced apoptosis and ROS production .CONCLUSION: DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase -1 expression after Nrf2 activation .
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Tetrandrinehasawiderangeofpharmacologicaleffectssuchasanti-inflammation,analgesiceffect,anti-tumor,anti-silicosis, lowering blood pressure and anti-arrhythmia. Up till now, only tablets and ordinary injections are on the market, which is hard to meet the clinical requirements. The paper reviewed the research progress in the new technology and dosage forms of tetrandrine to provide the basis for new dosage form development of tetrandrine.
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Objective To study the protective effect mechanism of Rosa laevigata Michx (RLM) on cardiotoxicity induced by adriamycin in rats. Method 30 SD rats were randomly into control group, doxorubicin group and RLM groups. The control group was injected with normal saline injection, while the model group was injected with adriamycin intraperitoneally at the dosage of 15 mg/kg every other day. For the RLM groups,1~5 g/kg RLM were given after adriamycin injection. The survival rate, plasma BNP was observed. Apoptosis of cardiomyocyte was detected by instituted-labeled DNA (TUNEL). The activity of GSH-PX, CAT and total SOD in the myocardium tissue were also observed. The expression level of CuZn-SOD , bcl-2 and bax were detected by real-time PCR. Results The survival rate was significantly improved in SD rats treated with RLM compared with that in the adriamycin group (P<0.01). The BNP level was increased when treated by adriamycin (P<0.01), and decreased after RLM administration (P<0.01). RLM could also upregulate the expression of the CuZn-SOD mRNA level, and enhance the activity of GSH-PX, CAT and T-SOD compared with that in adriamycin group. Adriamycin could induce myocardial cells apoptosis, as demonstrated by TUNEL. RLM could inhibit adriamycin-induced apoptosis, bax mRNA expression, and increase bcl-2 expression and bcl-2/bax ratio. Conclusion RLM exhibit some antioxidant activity through many stages, and the anti-apoptosis activity may be related to affect the expression of bax and bcl-2 expression.
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<p><b>OBJECTIVE</b>To investigate whether cellular immunity and humoral immunity are involved in trichlorethylene (TCE)-induced mixed allergy, then provide the scientific basis for the mechanism of this disease.</p><p><b>METHODS</b>Guinea pigs and rats were tested for this study by application of guinea pig maximization test (GPMT), the animals were randomly divided into negative control, positive control and TCE treatment groups. Animals of these groups were administrated with olive oil, 2, 4-dinitrochlorobenzene (DNCB), and TCE, respectively, by intradermal injection. After TCE administration, rat peripheral blood samples were collected by flow cytometry to detect lymphocytes CD3⁺, CD4⁺, CD8⁺. Guinea pig peripheral blood samples were collected to detect the levels of IgG, IgA, IgM, C3, C4, and the spleens were taken out from guinea pigs after various treatment, mRNA expression of GATA3, T-bet, CTLA4 and Foxp3 in lymphocytes of guinea pig spleen was detected by real-time fluorescent PCR assay. Additionally, TCE allergic dermatitis patients were selected for the study, the peripheral blood samples were collected from the TCE patients group and control group, quantitative PCR was applied to detect mRNA expression of immune-related genes Foxp3, GATA3, CTLA4, T-bet.</p><p><b>RESULTS</b>TCE induced obvious skin allergic reaction in guinea pigs, the sensitization rate was 83.3%, IgG levels in TCE group and positive control increased significantly. Additionally, mRNA expression levels of GATA3, T-bet, CTLA4 significantly elevated in TCE group and positive control, but Foxp3 mRNA levels decreased. The lymphocytes CD3⁺ ratio in TCE group and positive control of rats was higher than that in negative control, we found that there was no statistical difference of CD4⁺, CD8⁺, CD4⁺/CD8⁺ between TCE group and negative control of rats. The mRNA expression levels of Foxp3, GATA3, CTLA4 in TCE patients increased by 115%, 97%, 241%, respectively as compared with the control, T-bet levels decreased by 47%when compared with the control.</p><p><b>CONCLUSIONS</b>TCE could induce obvious changes of cellular immunity and humoral immunity in guinea pigs, rats, and TCE patients, these findings indicated that TCE-induced immunological disorder belongs to the mixed allergy with involvment of cellular immunity and humoral immunity, the mixed allergy might be type IV and type II allergy.</p>
Subject(s)
Animals , Humans , Rats , Allergens , CTLA-4 Antigen , Guinea Pigs , Hypersensitivity , Immunity, Cellular , Immunity, Humoral , Lymphocytes , RNA, Messenger , Spleen , Trichloroethylene , ToxicityABSTRACT
The recipe-analgesic compound decoction composed of herbs according to the theory of Traditional Chinese Medicine and the author's clinical experience of treating painful diseases. The experimental studies showed that the pain threshold of mice in hot-plate test was raised (p