Aims: Urotensin II UII is a vasoactive neuropeptide involved in migration and invasion in various cell types. However, the effects of Ull on human hepatoma cells still remain unclear. The aim of this study was to investigate the role and mechanism of UR on migration and invasion in human hepatoma cells.Methods: Migration was measured by wound healing assays and a Transwell (R) methodology, and invasion was analyzed using Matrigel (R) 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 Ull. UlI also increased matrix metalloproteinase-2 (MMP2) expression in a time-independent manner. Furthermore, Ull markedly enhanced ROS generation and NADPH oxidase subunit expression, and consequently facilitated the phosphorylation of c-Jun N-terminal kinase (JNK). The UII 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 UM Ull 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. UII played an additional role in regulating hepatoma cells migration and invasion. Thus, our data suggested an important effect of UII in hepatocellular carcinoma metastasis. (C) 2016 Elsevier Inc. All rights reserved.

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-3β (GSK-3β), 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-3β (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-3β (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.

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 (ISC) 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 ISC and decreases the transepithelial resistance. Pretreatment with epithelial Na+ channel blocker did not affect the ISC responses elicited by HQS, but removal of extracellular Cl− or pretreatment with Cl− channel or Na+-K+-2Cl− cotransporter blocker inhibited HQS-elicited ISC 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.

目的 探讨活性氧自由基对骨髓源性肝前体细胞恶性转化的影响.方法 取经过鉴定的3代骨髓间充质干细胞(BMSCs)分为3组:对照组(BMSCs),生长因子诱导组(BMSCs+ HGF+ EGF),过氧化氢刺激组(BMSCs+HGF+EGF+ H2O2).分别在细胞培养的第7天、14天、21天、28天用RT-PCR方法检测各组细胞的白蛋白(ALB)、甲胎蛋白(AFP)、角蛋白18 (CK18) mRNA的表达.用免疫细胞化学方法检测细胞γ-谷氨酰转酞酶(γ-GT)的表达,用Schiff染色方法检测细胞DNA含量,用流式细胞仪检测非整倍体细胞.结果 生长因子诱导组及过氧化氢刺激组细胞有ALB mRNA、AFP mRNA及CK18 mRNA的表达,过氧化氢刺激组细胞AFP mRNA、CK18mRNA、γ-GT的表达、DNA含量及非整倍体细胞均高于生长因子诱导组(P＜0.05).结论 骨髓间充质干细胞在体外诱导成为肝前体细胞,活性氧自由基可促使肝前体细胞过度增殖并恶性转化.

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.

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 distribu-tion 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.

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.

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.

目的总结和探讨严重会阴部损伤的诊治方法。方法回顾性分析我院收治的42例严重会阴部损伤患者的临床资料。结果合并失血性休克35例,骨盆骨折37例,肛管直肠损伤26例。全部病例均行会阴部伤口清创术,伤口填塞压迫止血39例,其中37例止血满意,2例行髂内动脉结扎与栓塞。伤口一期缝合3例。直肠肛管一期修补22例,暂时性结肠转流手术39例。39例会阴部伤口2周~3个月后痊愈,死亡3例,均死于失血性休克。结论治疗成功的关键是积极抢救失血性休克、及时止血、会阴部彻底清创、放置引流管冲洗、尽早联合使用抗生素。大多数患者有必要行转流性结肠造瘘术,以单口造瘘为宜。

对37例原发性甲状旁腺功能亢进(PHPT)患者临床资料进行回顾性分析。发现37例患者中血清碱性磷酸酶升高36例(97.3%),血钙升高33例(89.2%),血磷降低26例(70.3%),28例尿羟脯氨酸与肌酐比高于正常值。X线检查有骨质疏松者35例(94.6%),纤维囊性骨炎21例,骨膜下骨吸收30例,病理性骨折14例;27例行骨密度测定,26例(96.3%)有腰椎和左髋骨量减少或骨质疏松;所有病例经手术和病理证实为甲状旁腺腺瘤。提示重视病史的采集,常规血清碱性磷酸酶和钙磷结果分析并结合骨病X线特点,可大大提高PHPT检出率。

Objective:To investigate the effect of Huqisan(HQS)on the chondriosome structure in hepatic precancerosis of rats.Methods:A Solt-Farber two-step test model of prehepatocarcinoma was established by diethylnitrosamine(DEN)and 2-acetylaminofluorene(2-AAF)in rats at dose of 200mg/kg body weight for one time.The normal controls were injected with physiologic saline.Hepatic precancerosis rats were simultaneously treated by different dose of HQS as the hepatic precancerosis.The hepaticultra structure of all rats was observed by electronmicroscope.Results:The change of hepaticultra structure could be observed in precancerosis rats.The ultra structure changes in clued swollen mitochondria,enlargement of endoplasmic reticulum,condensation and lipid droplet in cytoplasm.HQS could reduce formation of lipid droplet,oliferation of endoplasmic reticulum,swelling of chondriosome and proliferation of interstitial collagen in hepatic cells of precancerosis rats.Conclusion:HQS could relieve the damage of hepatic chondriosome structure in precancerosis rats.

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.

患者女,55岁.因右下腹隐痛5年,加重1个月于2006年4月25日入院.曾诊断慢性阑尾炎,予头孢呋辛钠及甲硝唑治疗无缓解,无其他症状.查体:右中下腹可触及约10 cm×8 cm包块,界较清,不活动,有深压痛,无反跳痛.实验室常规检查及肿瘤标志物测定均未见异常.

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.

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.

Objective To evaluate the role of radionuclide imaging in diagnosis of hyperfunctioning parathyroid tumor and early etiology ascertaining of bone pain and pathologic fracture. Methods Retrospectively studied on the data of radionuclide imaging using 99Tcm-methylene diphosphonic acid (MDP) and 99Tcm-methoxyisobu-tylisonitrile (MTBI). The data was collected from 26 cases with hyperfunctioning parathyroid glands by orthopedics department in this hospital .during the last 11 years. Results Operations and pathologic studies had been performed on all of the 26 cases, including 2 cases with adenoma and 1 case with carcinoma. All cases showed obvious signs of general skeletal metabolic change of hyperparathyroidism oh bone scintigraphy. All the metabolic indexes were ≥5. The features of typical appearance were the ' black skull' and extraordinarily clear bone images. Total 26 cases underwent ultrasonography of the neck and 18 cases of them had double-phase radionuclide parathyroid imaging. All of the 18 cases had positive results and showed accurate position of glands in double-phase hy-perparathyroid radionuclide imaging. Conclusion Signs of bone metabolic change of hyperparathyroidism in bone scintigraphy for the patients with bone pain and pathologic fracture are necessary for the early etiological ascertaining; the hyperfunctioning parathyroid glands can be showed accurately in the parathyroid radionuclide imaging, and that is very important for diagnosing and locating hyperfunctioning parathyroid tumors.

Objective To study the changes of endothelin (ET) in plasma and kidney tissue of Wistar rats with obstructive jaundice (OJ) and investigate the relationship between ET and renal functional impairment in OJ. Methods By using specific radioimmunological method, the levels of plasma ET and kidney ET were measured in rats with common bile duct ligation (n=32) and those with sham operation (n=32) for 5, 10, 15 and 20 days after operation. Meanwhile, the levels of serum bilirubin (DB) and BUN and Cr were also determined in all the rats. Renal histological changes were observed with optical microscopy. Results The levels of ET in plasma and renal tissue as well as those of serum Cr, BUN and DB in all OJ subgroups were continually elevated. The levels of ET in plasma and renal cortex were closely related to serum Cr and BUN. Furthermore, the former was closely related to DB. Conclusions The elevated ET in plasma and renal cortex can lead to impairment of renal filtration function and that in the renal medulla might be one of the factors causing abnormality of renal concentration function in the early stage of OJ. Hyperbilirubinemia might be one of the causes leading to increase of plasma ET level.

ObjectiveTo study the etiology, preoperative diagnosis and treatment of nonocclusive mesenteric ischemia(NOMI). MethodsClinical data of 3 cases of NOMI from 1992 to 1998 were analyzed retrospectively. ResultsMajor clinical presentations were abdominal pain, gastrointestinal bleeding and abdominal irritation. None was correctly diagnosed preoperatively. Partial ileectomy in 2 cases and right colectomy in 1 case was performed with a mortality of 2/3. Conclusion Early diagnosis and accurate assessment of the bowel viability in exploration were quite difficult. Resection of the infarcted bowel or ′second-look procedure′ were recommended if necessary.