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Objective:To investigate the effect of specnuezhenide on high glucose-induced human retinal microvascular endothelial cells (hRMECs) injury and its mechanism.Methods:The hRMECs were divided into a normal control group cultured in a culture medium containing 5.5 mmol/L glucose, a hypertonic group cultured in a culture medium containing 5.5 mmol/L glucose + 24.5 mmol/L mannitol, a high glucose group cultured in a culture medium containing 30 mmol/L glucose, as well as high glucose+ low-, medium-, and high-dose specnuezhenide groups cultured in culture media containing 30 mmol/L glucose + 25, 50, 100 μmol/L specnuezhenide for 24 hours, respectively.In addition, hRMECs were divided into a high glucose+ small interfering RNA-negative control (si-NC) group cultured in a culture medium containing 30 mmol/L glucose, a high glucose+ si-forkhead box O4 (FOXO4) group cultured in a culture medium containing 30 mmol/L glucose, a high glucose+ specnuezhenide+ pcDNA group cultured in a culture medium containing 100 μmol/L specnuezhenide + 30 mmol/L glucose, and a high glucose+ specnuezhenide+ pcDNA-FOXO4 group cultured in a culture medium containing 100 μmol/L specnuezhenide+ 30 mmol/L glucose for 24 hours after transfection by corresponding reagents.Cell apoptosis was detected by flow cytometry.The malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity in cells were detected by the thiobarbituric acid method and xanthine oxidase method, respectively.The concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the cell culture supernatant were detected by enzyme linked immunosorbent assay.The relative expression level of FOXO4 protein in cells was determined by Western blot.Results:The apoptosis rates of normal control group, hypertonic group, high glucose group, high glucose+ low-, medium- and high-dose specnuezhenide groups were (7.32±0.72)%, (7.44±0.70)%, (23.96±1.32)%, (19.84±1.09)%, (14.13±0.85)% and (9.84±0.70)%, respectively.There were significant differences in cell apoptosis rate, MDA concentration, SOD activity, the concentration of IL-1β, the concentration of TNF-α, and the relative expression level of FOXO4 protein among the six groups ( F=498.545, 1 186.693, 516.629, 654.247, 638.238, 472.655; all at P<0.001). Compared with high glucose group, the apoptosis rate, MDA concentration, IL-1β and TNF-α concentration, FOXO4 protein expression level were significantly decreased in high glucose+ low-, medium- and high-dose specnuezhenide groups, and SOD activity was significantly increased in a dose-dependent manner.Compared with high glucose+ si-NC group, the expression level of FOXO4 protein, cell apoptosis rate, MDA concentration, IL-1β and TNF-α mass concentrations were decreased in high glucose + si-FOXO4 group, while the SOD activity was increased.Compared with high glucose+ specnuezhenide+ pcDNA group, the apoptosis rate, MDA concentration, IL-1β and TNF-α concentrations, FOXO4 protein expression level of hRMECs in high glucose+ specnuezhenide+ pcDNA-FOXO4 group were significantly increased, and SOD activity was significantly decreased (all at P<0.05). Conclusions:Specnuezhenide can protect hRMECs from high glucose-induced apoptosis, oxidative stress and inflammatory response by down-regulating FOXO4.
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Recent studies have shown that the occurrence and development of common liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and liver cancer, are related to liver aging(LA). Therefore, to explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a traditional classic prescription in improving LA with multiple targets, the present study randomly divided 24 rats into a normal group, a model group, a DHZCP group, and a vitamin E(VE) group, with six rats in each group. The LA model was induced by continuous intraperitoneal injection of D-galactose(D-gal) in rats. For the LA model rats, the general situation was evaluated by aging phenotype and body weight(BW). LA was assessed by the pathological characteristics of hepatocyte senescence, hepatic function indexes, the staining characteristics of phosphorylated histone family 2A variant(γ-H2AX), and the expression levels of cell cycle arrest proteins(P21, P53, P16) and senescence-associated secretory phenotype(SASP) in the liver. The activation of the reactive oxygen species(ROS)-mediated phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/forkhead box protein O4(FoxO4) signaling pathway was estimated by hepatic ROS expression feature and the protein expression levels of the key signaling molecules in the PI3K/Akt/FoxO4 signaling pathway. The results showed that after the treatment with DHZCP or VE for 12 weeks, for the DHZCP and VE groups, the characterized aging phenotype, BW, pathological characteristics of hepatocyte senescence, hepatic function indexes, relative expression of ROS in the liver, protein expression levels of key signaling molecules including p-PI3K, p-Akt, and FoxO4 in the liver, staining characteristics of γ-H2AX, and the protein expression levels of P16, P21, P53, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the liver were improved, and the effects of DHZCP and VE were similar. Based on the D-gal-induced LA model in rats, this study demonstrates that DHZCP can ameliorate LA with multiple targets in vivo, and its effects and mechanism are related to regulating the activation of the ROS-mediated PI3K/Akt/FoxO4 signaling pathway in the liver. These findings are expected to provide new pharmacological evidence for the treatment of DHZCP in aging-related liver diseases.
Subject(s)
Animals , Rats , Proto-Oncogene Proteins c-akt/genetics , Phosphatidylinositol 3-Kinases/genetics , Reactive Oxygen Species , Tumor Suppressor Protein p53/genetics , Signal Transduction , Liver , Aging , Cell Cycle Proteins , Interleukin-6ABSTRACT
@#AIM: To explore the effect of dapagliflozin on the apoptosis and oxidative stress of high glucose-induced human retinal vascular endothelial cells and its regulatory effect on forkhead FOXO4. <p>METHODS: High glucose-induced human retinal vascular endothelial cells(HRVECs)were used to establish a cell injury model(high glucose group). Experimental groups include high glucose+dapagliflozin low-dose group(1ng/L dapagliflozin), high glucose+dapagliflozin medium-dose group(5ng/L dapagliflozin), high glucose+dapagliflozin high-dose group(10ng/L dapagliflozin), high glucose+dapagliflozin high-dose+pcDNA group, high glucose+dapagliflozin high-dose+pcDNA-FOXO4 group, and normal sugar group(5.5mmol/L D-glucose). Flow cytometry was used to detect the apoptosis rate. The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were tested with corresponding kits. Western blot assay was used to detect the protein level of FOXO4. <p>RESULTS: Compared with the normal sugar group, the apoptosis rate(<i>P</i><0.05), the level of MDA(<i>P</i><0.05)and FOXO4(<i>P</i><0.05)were increased, but the level of SOD was decreased(<i>P</i><0.05)in high-glucose group. Compared with the high glucose group, cell apoptosis rate(<i>P</i><0.05), the level of MDA(<i>P</i><0.05)and the protein level of FOXO4 were decreased(<i>P</i><0.05), but the level of SOD was increased(<i>P</i><0.05)in high glucose+medium-dose dapagliflozin group and high glucose+high-dose dapagliflozin group. Compared with high glucose+dapagliflozin high-dose+pcDNA group, the apoptosis rate(<i>P</i><0.05)and the level of MDA(<i>P</i><0.05)were increased, but the level of SOD was decreased(<i>P</i><0.05)in high glucose+dapagliflozin high-dose+pcDNA-FOXO4 group(<i>P</i><0.05). <p>CONCLUSION: Dapagliflozin could inhibit oxidative stress and cell apoptosis in high glucose-induced HRVECs by down-regulating FOXO4, thereby reducing cell damage.
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@#AIM: To investigate the effect of microRNA-96-5p(miR-96-5p)on proliferation and apoptosis of rat retinal vascular endothelial cells induced by high glucose and to explore its mechanism. <p>METHODS: SD rat retinal vascular endothelial cells(RRVEC)were cultured and the RRVEC was divided into control group(NG)and high glucose group(HG). The high glucose-induced RRVECs were harvested separately or co-transfected with miR-96-5p mimic, miR-NC, si-FOXO4, si-NC. The expression of miR-96-5p and FOXO4 was detected by qRT-PCR and Western blotting, respectively. MTT assay was used to detect the proliferation activity. Flow cytometry was used to detect the apoptosis rate. The dual luciferase reporter assay validated the target gene of miR-96-5p. Western blotting was used to detect the expression of CyclinD1, p21, p27, Bcl-2, Bax and cleaved-caspased-3. <p>RESULTS:The expression levels of miR-96-5p, CyclinD1 and Bcl-2 in RRVEC were significantly decreased after high glucose treatment, and the expression levels of FOXO4, p21, p27, Bax and cleaved-caspased-3 were significantly increased, inhibiting cell proliferation activity, but promoting apoptosis. Overexpression of miR-96-5p and inhibition of FOXO4 expression increased the expression levels of CyclinD1 and Bcl-2, inhibited the expression of p21, p27, Bax, cleaved-caspased-3, enhanced cell proliferation and inhibited apoptosis. Dual luciferase reporter assay demonstrated that FOXO4 was a target gene for miR-96-5p. Overexpression of FOXO4 reversed the effect of miR-96-5p overexpression on high glucose-induced proliferation and apoptosis of RRVEC. <p>CONCLUSION:miR-96-5p inhibits high glucose-induced apoptosis of rat retinal vascular endothelial cells and promotes cell proliferation by targeting FOXO4.
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Objective@#To explore the effects of FOXO4 D-retro-inverso peptide (FOXO4-DRI) on the radiosensitivity of non-small cell lung cancer (NSCLC) cells.@*Methods@#To detect the effect of FOXO4-DRI on NSCLC cells, H460 and A549 human lung cancer cells were divided into four groups, including untreated control, FOXO4-DRI, γ-ray irradiation and FOXO4-DRI + γ-ray groups. A sigle dose rate of 0.99 Gy γ-rays was used for radiation. H460 cells were administered with 6 μmol/L FOXO4-DRI and A549 cells were adiminstered with 30 μmol/L FOXO4-DRI at 10 min before radiation. Cell viability and survival were detected by CCK-8 assay and colony formation assay, respectively. Cell migration was detected by wound healing assay. Apoptosis and cell cycle arrest were detected with flow cytometry.@*Results@#FOXO4-DRI inhibited growth of H460 and A549 cells (t=1.06-50.75, P<0.05), and decreased cell mobility (t=33.37-139.10, P<0.05) and colony formation (t=5.20-93.48, P<0.05). FOXO4-DRI also increased apoptosis (t=2.95-42.00, P<0.05) and caused a cell cycle arrest at G0/G1 phase accompanied with a decreased proportion of G2/M phase (t=3.50-31.59, P<0.05). Furthermore, FOXO4-DRI increased radiosensitivity of both H460 cells and A549 cells (t=2.94-23.40, P<0.05), caused a Further Decrease of radiation-mediated mobility (t=5.25, 7.56, P<0.05) and colony formation (t=8.43-34.00, P<0.05) and a more increase of radiation-induced apoptosis (t=9.20-11.52, P<0.05). FOXO4-DRI also further decreased the proportion of G2/M phase cells but increased the proportion of S phase cells (t=3.85-17.62, P<0.05).@*Conclusion@#FOXO4-DRI increases radiosensitivity of NSCLC cells by inducing apoptosis and suppressing cell proliferation.
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Objective To explore the effects of FOXO4 D-retro-inverso peptide (FOXO4-DRI)on the radiosensitivity of non-small cell lung cancer (NSCLC) cells.Methods To detect the effect of FOXO4-DRI on NSCLC cells,H460 and A549 human lung cancer cells were divided into four groups,including untreated control,FOXO4-DRI,γ-ray irradiation and FOXO4-DRI + γ-ray groups.A sigle dose rate of 0.99 Gy γ-rays was used for radiation.H460 cells were administered with 6 μmol/L FOXO4-DRI and A549 cells were adiminstered with 30 μmol/L FOXO4-DRI at 10 min before radiation.Cell viability and survival were detected by CCK-8 assay and colony formation assay,respectively.Cell migration was detected by wound healing assay.Apoptosis and cell cycle arrest were detected with flow cytometry.Results FOXO4-DRI inhibited growth of H460 and A549 cells (t =1.06-50.75,P＜ 0.05),and decreased cell mobility (t =33.37-139.10,P＜0.05) and colony formation (t =5.20-93.48,P＜0.05).FOXO4-DRI also increased apoptosis (t=2.95-42.00,P＜0.05) and caused a cell cycle arrest at G0/G1 phase accompanied with a decreased proportion of G2/M phase (t =3.50-3 1.59,P＜0.05).Furthermore,FOXO4-DRI increased radiosensitivity of both H460 cells and A549 cells (t =2.94-23.40,P＜0.05),caused a Further Decrease of radiation-mediated mobility (t =5.25,7.56,P＜0.05) and colony formation (t =8.43-34.00,P＜ 0.05) and a more increase of radiation-induced apoptosis (t =9.20-11.52,P ＜0.05).FOXO4-DRI also further decreased the proportion of G2/M phase cells but increased the proportion of S phase cells (t =3.85-17.62,P ＜ 0.05).Conclusion FOXO4-DRI increases radiosensitivity of NSCLC cells by inducing apoptosis and suppressing cell proliferation.
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@#AIM: To study the effects of FOXO4 on oxidative stress and apoptosis of retinal vascular endothelial cells under high glucose environmental conditions. <p>METHODS: Human retinal vascular endothelial cells were cultured with high glucose medium. Real-time PCR and Western blot were used to detect the expressions of FOXO4 within cells; meanwhile, both FOXO4 RNAi lentivirus and control vector lentivirus were infected the retinal vascular endothelial cells cultured in high sugar culture medium. Real-time PCR and Western blot techniques were used to detect the interference efficiency. After collection of supernatant and cells treated with various interferences, the SOD activity, MDA content in the supernatants and ROS level within cells were detected. Flow cytometry was used to determine the changes of cell apoptosis. Western blot was used to detect the expressions of apoptotic protein cleaved Caspase-3 and cleaved Caspase-9 within cells. <p>RESULTS: The expression of FOXO4 was increased in retinal vascular endothelial cells after treatment with high glucose medium. FOXO4 RNAi lentivirus infection reduced the expression level of FOXO4 in retinal vascular endothelial cells under high glucose environmental conditions. By contrast, control vector lentivirus had no effect on FOXO4 expression in cells. High glucose induced elevated levels of ROS in retinal vascular endothelial cells, reduced the activity of SOD in cell culture medium, increased the content of MDA, elevated the rate of apoptosis, and promoted the expressions of cleaved Caspase-3 and cleaved Caspase-9 proteins in cells. After down-regulation of FOXO4 expression, retinal endothelial cells were induced by high glucose, the activity of SOD in the cell culture medium increased, the levels of MDA, ROS, apoptosis, and the levels of cleaved Caspase-3 and cleaved Caspase-9 proteins decreased in cells as compared with those of retinal vascular endothelial cells. Moreover, compared with those did not interfere with FOXO4 expression, there was statistically significant differences(<i>P</i><0.05). <p>CONCLUSION: High glucose induces the expression of FOXO4 in retinal vascular endothelial cells. Knocking-down of FOXO4 expression reduces oxidative stress and apoptosis induced by high glucose medium.
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Objective To explore significance and impact of hypoxia inducible factor 1α,metastasis associated with colon cancer 1 and transcription factor FOXO4 in gastric carcinoma and clinical pathological characteristics and prognosis .Methods 110 samples diagnosed as gastric carcinoma and 46 samples diagnosed as pericarcinomatous tissue which was 5 cm far away from cancer were collected in first affiliated hospital of Anhui medical university from December 2006 to May 2008 .Immunohistochemical stai-ning was performed in these samples to detect the expressions of HIF-1α,MACC1 and FOXO4 .And the correlation between devel-opment of gastric carcinoma and expressions of HIF-1α,MACC1 and FOXO4 was analyzed with the help of patients′messages .Ka-plan-Meier method and Cox regression were used to analyze patients′5 years survival rate and the factors .Results The expression levels of HIF-1α(P<0 .001) and MACC1(P<0 .001) in gastric carcinoma tissues were significantly higher than pericarcinomatous tissues ,however the expression levels of FOXO4(P<0 .001) was significantly decreased in gastric carcinoma tissues .Statistical a-nalysis revealed that the expressions of HIF-1αand MACC1 were apparently increased with the lower degree of cell differentiation , the greater degree of tumor infiltration ,accompanied with lymph node metastasis and higher TNM scores ,but lowered the expres-sion of FOXO4 factor .There were significant correlations between expressions of the three factors in gastric carcinoma .The expres-sions of HIF-1α,MACC1 and FOXO4 had an apparent influence on the patients′5 years survival rate .And they were independent factors on the prognosis of gastric carcinoma .Conclusion HIF-1α,M ACC1and FOXO4 are abnormal expression in gastric cancer tissue ,and the clinical pathological features of gastric cancer and the prognosis of patients with gastric cancer are closely related .