A Glucose-Responsive Hydrogel Inhibits Primary and Secondary BRB Injury for Retinal Microenvironment Remodeling in Diabetic Retinopathy.

Current diabetic retinopathy (DR) treatment involves blood glucose regulation combined with laser photocoagulation or intravitreal injection of vascular endothelial growth factor (VEGF) antibodies. However, due to the complex pathogenesis and cross-interference of multiple biochemical pathways, these interventions cannot block disease progression. Recognizing the critical role of the retinal microenvironment (RME) in DR, it is hypothesized that reshaping the RME by simultaneously inhibiting primary and secondary blood-retinal barrier (BRB) injury can attenuate DR. For this, a glucose-responsive hydrogel named Cu-PEI/siMyD88@GEMA-Con A (CSGC) is developed that effectively delivers Cu-PEI/siMyD88 nanoparticles (NPs) to the retinal pigment epithelium (RPE). The Cu-PEI NPs act as antioxidant enzymes, scavenging ROS and inhibiting RPE pyroptosis, ultimately blocking primary BRB injury by reducing microglial activation and Th1 differentiation. Simultaneously, MyD88 expression silence in combination with the Cu-PEI NPs decreases IL-18 production, synergistically reduces VEGF levels, and enhances tight junction proteins expression, thus blocking secondary BRB injury. In summary, via remodeling the RME, the CSGC hydrogel has the potential to disrupt the detrimental cycle of cross-interference between primary and secondary BRB injury, providing a promising therapeutic strategy for DR.

The effects of urotensin II on migration and invasion are mediated by NADPH oxidase-derived reactive oxygen species through the c-Jun N-terminal kinase pathway in human hepatoma cells.

AIMS: Urotensin II (UII) is a vasoactive neuropeptide involved in migration and invasion in various cell types. However, the effects of UII on human hepatoma cells still remain unclear. The aim of this study was to investigate the role and mechanism of UII on migration and invasion in human hepatoma cells. METHODS: Migration was measured by wound healing assays and a Transwell® methodology, and invasion was analyzed using Matrigel® invasion chambers. Reactive oxygen species (ROS) levels were detected using a 2', 7'-dichlorofluorescein diacetate probe, and flow cytometry, and protein expression levels were evaluated by western blotting. Cell proliferation and actin polymerization were examined using cell proliferation reagent WST-1 and F-actin immunohistochemistry staining. RESULTS: Exposure to UII promoted migration and invasion in hepatoma cells compared with that in cells without UII. UII also increased matrix metalloproteinase-2 (MMP2) expression in a time-independent manner. Furthermore, UII markedly enhanced ROS generation and NADPH oxidase subunit expression, and consequently facilitated the phosphorylation of c-Jun N-terminal kinase (JNK). The UT antagonist urantide or the antioxidant/NADPH oxidase inhibitor apocynin decreased UII-induced ROS production. JNK phosphorylation, migration, invasion, and MMP9/2 expression were also reversed by pretreatment with apocynin. Urantide and JNK inhibitor SP600125 abrogated migration, invasion, or MMP9/2 expression in response to UII. UII induced actin polymerization and fascin protein expression, and could be reversed by apocynin and SP600125. CONCLUSIONS: Exogenous UII induced migration and invasion in hepatoma cells that mainly involved NADPH oxidase-derived ROS through JNK activation. UT played an additional role in regulating hepatoma cells migration and invasion. Thus, our data suggested an important effect of UII in hepatocellular carcinoma metastasis.

Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells.

AIM: To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved. METHODS: Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2',7'-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3ß (GSK-3ß), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91(phox), p67(phox), p47(phox), p40(phox), and p22(phox) were evaluated by Western blot. RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3ß (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3ß (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0.05), and insulin resistance (P < 0.05) in HepG2 cells. CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG2 cells.

Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation.

Urotensin II (UII), a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC), and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs) are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE) or 2', 7'-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu) incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC.

Huqi San-Evoked Rat Colonic Anion Secretion through Increasing CFTR Expression.

Huqi San (HQS) is a Chinese herbal preparation of eight medicinal herbs that promote diuresis, detoxification, blood circulation, and cholestasis. Defects in transporter expression and function can cause cholestasis and jaundice. However, the mechanism of the cholestasis underlying HQS effects, especially on the gastrointestinal tract ion secretion, has not been elucidated. Real-time RT-PCR and Western blotting were used to study the expression and localization of cystic fibrosis transmembrane conductance regulator (CFTR) and α-ENaC in rat alimentary tract, and then the effect of HQS on the ion transport in rat distal colon mucosa was investigated using the short-circuit current (I SC) technique. The results showed that pretreatment with HQS significantly enhanced mRNA transcripts and protein content of CFTR in liver and distal colon but not α-ENaC in alimentary organs. HQS increases I SC and decreases the transepithelial resistance. Pretreatment with epithelial Na(+) channel blocker did not affect the I SC responses elicited by HQS, but removal of extracellular Cl(-) or pretreatment with Cl(-) channel or Na(+)-K(+)-2Cl(-) cotransporter blocker inhibited HQS-elicited I SC responses. These findings demonstrated that HQS, RA, and RP can stimulate Cl(-) secretion in the distal colon by increasing the mRNA transcripts and protein content of CFTR in liver and distal colon.

Up-regulation of urotensin II and its receptor contributes to human hepatocellular carcinoma growth via activation of the PKC, ERK1/2, and p38 MAPK signaling pathways.

Urotensin II (UII) and its receptor (UTR) have mitogenic effects on tumor growth. Our previous study demonstrated that the UII/UTR system is up-regulated in dithyinitrosamine-induced precancerous rat liver lesions. However, its role in human hepatocellular carcinoma remains unknown. In this study, the mRNA and protein expression of UII and its receptor (UTR) in human hepatocellular carcinoma samples and in the BEL-7402 human hepatoma cell line were evaluated. In addition, the effect of exogenous UII on the pathways that regulate proliferation in BEL-7402 cells in vitro were determined. Liver sections were subjected to immunohistochemical staining. mRNA expression was detected by real-time polymerase chain reaction analysis, and protein levels were evaluated by western blotting. Proliferating cells were detected by BrdU incorporation. The expression of UII/UT mRNA and protein significantly increased in human hepatocellular carcinoma samples, and in BEL-7402 cells. Administration with UII increased the phosphorylation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK). Furthermore, GF109203x, PD184352, and SB203580 partially abolished UII-induced proliferation of BEL-7402 cells. These results provide the first evidence that up-regulation of the UII/UT system may enhance proliferation of the human hepatoma cell line at least in part via PKC, ERK1/2, and p38 MAPK signaling pathways, and may provide novel therapeutic targets for inhibiting human hepatocellular carcinoma.

Oleanolic acid and ursolic acid inhibit proliferation in transformed rat hepatic oval cells.

AIM: To investigate H2O2-induced promotion proliferation and malignant transformation in WB-F344 cells and anti-tumor effects of ursolic acid (UA) and oleanolic acid (OA). METHODS: WB-F344 cells were continuously exposed to 7 x 10(-7) mol/L H2O2 for 21 d. Observations of cell morphology, colony formation rates, flow cytometric analysis of cell cycle changes and aneuploidy formation indicated that H2O2 was able to induce malignant transformation of WB-F344 cells. We treated malignantly transformed WB-F344 cells with 4 µmol/L OA or 8 µmol/L UA for 72 h and analyzed the cell cycle distribution by flow cytometry. RESULTS: MTT assay showed that 7 x 10(-7) mol/L H2O2 decreased G1 phase subpopulation from 73.8% to 49.6% compared with the control group, and increased S phase subpopulation from 14.5% to 31.8% (P < 0.05 vs control group). Cell morphology showed that nucleus to cytoplasm ratio increased, many mitotic cells, prokaryotes and even tumor giant cells were shown in H2O2-induced WB-F344 cells. Fluorescence activated cell sorting analysis showed that WB-F344 cell aneuploidy increased to 12% following H2O2 treatment. Flow cytometric analysis of the transformed WB-F344 cells following treatment with OA (4 µmol/L) and UA (8 µmol/L) showed that OA increased G1 subpopulation to 68.6%, compared to 49.7% in unexposed cells. UA increased G1 subpopulation to 67.4% compared to 49.7% in unexposed cells (P < 0.05 vs H2O2 model group). CONCLUSION: H2O2 causes the malignant transformation of WB-F344 cells. OA and UA exert anti-tumor effects by inhibiting the proliferation in malignantly transformed WB-F344 cells.

Activation of apoptosis in hepatocellular carcinoma by the Chinese traditional medicine Hu Qisan.

To investigate the effects of Hu Qisan (HQS) on apoptosis in diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC), a Solt-Farber two-step test model of precancerous liver lesions was established in rats using a previously described method. HQS (4 and 8 g/kg body weight/day) was administered for 4 weeks, after the majority of the liver was removed. HepG2 cells were used to detect the HtrA serine peptidase 2 (HtrA2/Omi) release from mitochondria and caspase-3 activation promoted by HQS. Exposure of the rats to DEN for 6 weeks induced hepatic carcinogenesis. HQS (4 and 8 g/kg body weight/day) markedly induced cell apoptosis. The protective effects against hepatic carcinogenesis were mediated by multiple mechanisms, including the reduction of DEN-induced γ-GT-positive cell proliferation, mitochondrial morphological changes, HtrA2/Omi release from mitochondria and the activation of caspase-3. In conclusion, HQS is a potential anti-carcinogenic agent that may induce apoptosis by reducing the inhibitory effects of X-linked inhibitor of apoptosis protein (XIAP) on caspase-3. Thus, HQS should be further explored as a potentially promising new therapeutic agent against human hepatic cancer.

Modulation of energy metabolic enzyme expression in N-nitrosodiethylamine-mediated hepatocarcinogenesis by Chinese herbs, Huqi San.

AIM: To elucidate the anticancer mechanism of Huqi San by assessing the expression of G-6-Pase, SDH, ATPase and AFP in N-nitrosodiethylamine-mediated hepatocarcinogenesis in rats. METHODS: A Solt-Farber two-step test model of hepatocarcino genesis was established by diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) in rats to investigate the modifying effects of expression of 6-glucosephosphatase (G-6-Pase), succinodehydrogenase (SDH), adenosine triphosphatase (ATPase) in N-nitrosodiethylamine-mediated hepatocarcinogenesis. Hu Qisan compounded by eight medicinal herbs was prepared in glycoprival granules with wich 0.38 g crude herbs/mL solution was prepared for administration. gamma-Glutamy-transpeptidase (gamma-GT), G-6-Pase, SDH and ATPase were immunohistochemically determined. The activity of alpha-fetoglobulin (AFP) in the livers was measured with Immunofluorescence. RESULTS: Huqi San treated rats showed significant decrease in areas of gamma-GT positive foci (P< 0.001). On the other hand, the expression of G-6-Pase, SDH and ATPase has obviously altered in Huqi San treated group. The activity of AFP also significantly decreased after the treatment with Huqi San (8 g/kg body weight or 4 g/kg body weight) or total alkali of mistletoe (0.12 g/kg body weight). CONCLUSIONS: Huqi San can obviously increase these activities of G-6-Pase, SDH and ATPase, and at the same time significantly decrease the expression of gamma-GT and AFP. Therefore, it can obstruct or inhibit the rat's liver preneolastic lesion induced by DEN.

Effect of Qi-protecting powder (Huqi San) on expression of c-jun, c-fos and c-myc in diethylnitrosamine-mediated hepatocarcinogenesis.

AIM: To study the inhibitory effect of Huqi San (Qi-protecting powder) on rat prehepatocarcinoma induced by diethylinitrosamine (DEN) by analyzing the mutational activation of c-fos proto-oncogene and over-expression of c-jun and c-myc oncogenes. METHODS: A Solt-Farber two-step test model of prehepatocarcinoma was induced in rats by DEN and 2-acetylaminofluorene (AAF) to investigate the modifying effects of Huqi San on the expression of c-jun, c-fos and c-myc in DEN-mediated hepatocarcinogenesis. Huqi San was made of eight medicinal herbs containing glycoprival granules, in which each milliliter contains 0.38 g crude drugs. gamma-glutamy-transpeptidase-isoenzyme (gamma-GTase) was determined with histochemical methods. Level of 8-hydroxydeoxyguanosine (OHdG) formed in liver and c-jun, c-fos and c-myc proto-oncogenes were detected by immunohistochemical methods. RESULTS: The level of 8-OHdG, a mark of oxidative DNA damage, was significantly decreased in the liver of rats with prehepatocarcinoma induced by DEN who received 8 g/kg body weight or 4 g/kg body weight Huqi San before (1 wk) and after DEN exposure (4 wk). Huqi San-treated rats showed a significant decrease in number of gamma-GT positive foci (P < 0.001, prevention group: 4.96 +/- 0.72 vs 29.46 +/- 2.17; large dose therapeutic group: 7.53 +/- 0.88 vs 29.46 +/- 2.17). On the other hand, significant changes in expression of c-jun, c-fos and c-myc were found in Huqi San-treated rats. CONCLUSION: Activation of c-jun, c-fos and c-myc plays a crucial role in the pathogenesis of liver cancer. Huqi San can inhibit the over-expression of c-jun, c-fos and c-myc oncogenes and liver preneolastic lesions induced by DEN.

Mistletoe alkali inhibits peroxidation in rat liver and kidney.

AIM: To explore the antioxidant and free radical scavenger properties of mistletoe alkali (MA). METHODS: The antioxidant effect of mistletoe alkali on the oxidative stress induced by carbon tetrachloride (CCl4) in rats was investigated. The rats were divided into four groups (n = 8): CCl4-treated group (1 mL/kg body weight), MA -treated group (90 mg/kg), CCl4+MA-treated group and normal control group. After 4 wk of treatment, the level of malondialdehyde (MDA), a lipid peroxidation product (LPO) was measured in serum and homogenates of liver and kidney. Also, the level of glutathione (GSH), and activities of glutathione reductase (GR), glutathione peroxidase (GSPx), superoxide dismutase (SOD), and glutathione-S-transferase (GST) in liver and kidney were determined. Scavenging effects on hydroxyl free radicals produced in vitro by Fenton reaction were studied by ESR methods using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap reagent and H2O2/UV as the OH. source. Urinary 8-hydroxydeoxyguanosine (8-OHdG) was determined by competitive ELISA. RESULTS: In CCl4-treated group, the level of LPO in serum of liver and kidney was significantly increased compared to controls. The levels of GSH and enzyme activities of SOD, GSPx and GR in liver and kidney were significantly decreased in comparison with controls. In CCl4+MA-treated group, the changes in the levels of LPO in serum of liver and kidney were not statistically significant compared to controls. The levels of SOD, GSPx and GR in liver and kidney were significantly increased in comparison with controls. There was a significant difference in urinary excretion of 8-OHdG between the CCl4-treated and MA-treated groups. CONCLUSION: Oxidative stress may be a major mechanism for the toxicity of CCl4. MA has a protective effect against CCl4 toxicity by inhibiting the oxidative damage and stimulating GST activities. Thus, clinical application of MA should be considered in cases with carbon tetrachloride-induced injury.