Inhibitory effect of humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma: in vitro and in vivo studies.

OBJECTIVE: To investigate the inhibitory effect of humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma both in vitro and in vivo, which may be a potential agents with sensitivity and targeting ability for human hepatocellular cancer. METHODS: Humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate was previously constructed using ribosome display technology and antibody conjugate technology. In this combined in vitro and in vivo study, the inhibitory effects of anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on tumor growth, invasion, and metastasis was observed with human liver carcinoma cell line Bel7402 and normal cell L02 by MTT assay, Tanswell assay, Hochest33258 staining, and DNA ladder analysis. The anticancer activity and distribution of anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles was then verified in a mouse model of Bel7402 xenografts. RESULTS: Anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles significantly inhibited the proliferation of Bel7402 in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while had almost no effects on L02 cells. And the apoptosis inducing effects were proved by Hochest33258 staining and DNA ladder analysis. Transwell assay found that the drug also inhibited the metastasis ability of tumor cells. Furthermore, anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles significantly delayed the growth of Bel7402 xenografts after administration (92.9%), followed by As2O3-stealth nanoparticles, anti-VEGFR-2 ScFv, and As2O3 (61.4%, 58.8%, 20.5%, P<0.05). The concentration of As2O3 in anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles group was more selectively. CONCLUSIONS: Anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles is a potent and selective anti-hepatocellular carcinoma agent which could inhibit the growth of liver cancer as a targeting agent both in vitro and in vivo and also significantly inhibit angiogenesis.

[Roles of targeting Ras/Raf/MEK/ERK signaling pathways in the treatment of esophageal carcinoma].

Ras is best known for its ability to regulate cell growth, proliferation and differentiation. Mutations in Ras are associated with the abnormal cell proliferation which can result in incidence of all human cancers. Extracellular signal-regulated kinase (ERK) is a downstream effector of Ras and plays important roles in prognosis of tumors. Recently, evidence has gradually accumulated to demonstrate that there are other effectors between Ras and ERK, these proteins interact each other and constitute the thorough Ras/Raf/MEK/ERK signaling pathway. The pathway has profound effects on incidence of esophageal carcinoma and clinical applications of some chemotherapeutic drugs targeting the pathway. Further understanding of the relevant molecular mechanisms of Ras/Raf/MEK/ERK signaling pathway can be helpful for the development of efficient targeting therapeutic approaches which contribute to the treatment of esophageal cancer. In this article, roles of Ras/Raf/MEK/ERK signaling pathway in esophageal carcinoma as well as pharmacological targeting point in the pathway are reviewed.

Nasogastric or nasojejunal feeding in predicted severe acute pancreatitis: a meta-analysis.

INTRODUCTION: Enteral feeding can be given either through the nasogastric or the nasojejunal route. Studies have shown that nasojejunal tube placement is cumbersome and that nasogastric feeding is an effective means of providing enteral nutrition. However, the concern that nasogastric feeding increases the chance of aspiration pneumonitis and exacerbates acute pancreatitis by stimulating pancreatic secretion has prevented it being established as a standard of care. We aimed to evaluate the differences in safety and tolerance between nasogastric and nasojejunal feeding by assessing the impact of the two approaches on the incidence of mortality, tracheal aspiration, diarrhea, exacerbation of pain, and meeting the energy balance in patients with severe acute pancreatitis. METHOD: We searched the electronic databases of the Cochrane Central Register of Controlled Trials, PubMed, and EMBASE. We included prospective randomized controlled trials comparing nasogastric and nasojejunal feeding in patients with predicted severe acute pancreatitis. Two reviewers assessed the quality of each study and collected data independently. Disagreements were resolved by discussion among the two reviewers and any of the other authors of the paper. We performed a meta-analysis and reported summary estimates of outcomes as Risk Ratio (RR) with 95% confidence intervals (CIs). RESULTS: We included three randomized controlled trials involving a total of 157 patients. The demographics of the patients in the nasogastric and nasojejunal feeding groups were comparable. There were no significant differences in the incidence of mortality (RR=0.69, 95% CI: 0.37 to 1.29, P=0.25); tracheal aspiration (RR=0.46, 95% CI: 0.14 to 1.53, P=0.20); diarrhea (RR=1.43, 95% CI: 0.59 to 3.45, P=0.43); exacerbation of pain (RR=0.94, 95% CI: 0.32 to 2.70, P=0.90); and meeting energy balance (RR=1.00, 95% CI: 0.92 to 1.09, P=0.97) between the two groups. Nasogastric feeding was not inferior to nasojejunal feeding. CONCLUSIONS: Nasogastric feeding is safe and well tolerated compared with nasojejunal feeding. Study limitations included a small total sample size among others. More high-quality large-scale randomized controlled trials are needed to validate the use of nasogastric feeding instead of nasojejunal feeding.

[The impact of initial fluid resuscitation with different ratio of crystalloid-colloid on prognosis of patients with severe acute pancreatitis].

OBJECTIVE: To investigate the impact of fluid resuscitation with different ratio of crystalloid-colloid in early resuscitation stage on prognosis of patients with severe acute pancreatitis (SAP). METHODS: A retrospective analysis was made by reviewing clinical data of 47 patients with SAP from January 2001 to December 2011. According to crystalloid-colloid ratio 1.5 or 3, which was the input volume of crystalloid fluid versus colloid fluid in the first 24 hours, patients were divided into low ratio group (crystalloid-colloid ratio <1.5, n=13), middle ratio group (crystalloid-colloid ratio 1.5-3, n=15) and high ratio group (crystalloid-colloid ratio >3, n=19). Among the patients who had been successfully resuscitated, rate of mechanical ventilation, the oxygenation index, intra-abdominal pressure (IAP), and the amount of fluid retention in the third space within the first 24 hours, as well as the parameters of fluid resuscitation and the survival rate within 2 weeks were collected and analyzed. RESULTS: (1) In the first 24 hours, the rate of mechanical ventilation in the high ratio group was significantly higher than that in the middle ratio group and the low ratio group (68.4% vs. 20.0%, 23.1%, both P<0.05); the oxygenation index was significantly lower than that in the middle ratio group and in the low ratio group (180.7±26.3 mm Hg vs. 280.6±24.8 mm Hg, 260.3±25.7 mm Hg, both P<0.05); the IAP was significantly higher than that in the middle ratio group and the low ratio group (16.8±3.6 cm H(2)O vs. 13.4±3.5 cm H(2)O, 13.1±3.3 cm H(2)O, both P<0.05); the amount of fluid retention in the third space was significant higher than that in the middle ratio group and the low ratio group (2834±631 ml vs. 1887±282 ml, 1865±300 ml, both P<0.05). There was no significant difference in above indexes between middle ratio group and low ratio group (all P>0.05). (2) In the first 24 hours, the volume of crystalloid in high ratio group was significantly larger than that in the middle ratio group and the low ratio group (3611±798 ml vs. 2308±416 ml, 2124±477 ml, both P<0.05); and the volume of colloid in high ratio group and middle ratio group was significantly lower than that in the low ratio group (993±233 ml, 948±140 ml vs. 1506±332 ml, both P<0.05); and the mean crystalloid-colloid rate in the high ratio group was significantly higher than that in the middle ratio group and the low ratio group (3.65±0.13 vs. 2.43±0.13, 1.41±0.08, both P<0.05). The volume of infused fluid during the first 72 hours in the high ratio group was significantly higher than that in the middle and low ratio groups (11 941±1161 ml vs. 9036±982 ml, 9400±1051 ml, both P<0.05). (3) The survival rate in the high ratio group (36.8%) was significantly lower than that in the middle ratio group (86.7%, P<0.05) and the low ratio group (61.5%, P>0.05). CONCLUSIONS: A suitable crystalloid-colloid ratio should be considered in the early stage of resuscitation in patients with severe acute pancreatitis, which would result in a decrease in the fluid retention in the third space as well as an improvement of survival rate in return. It is suggested that the middle ratio of crystalloid-colloid fluid resuscitation should be the optimal strategy.

[Comparative observation of microcirculation and tissue healing process in gastrointestinal anastomosis with apposition or inverted suturing].

OBJECTIVE: To compare microcirculation and tissue healing process in gastrointestinal anastomosis with apposition or inverted suture. METHODS: The study was performed in adult rabbits. Animals were randomly assigned to two groups. In group A, small intestine was transected at 20 cm and 40 cm from duodenojejunal ligament and apposition sutures were performed at 20 cm and inverted sutures at 40 cm. In group B, transaction and anastomosis were at the same location with group A while the suturing method was reversed. Anastomotic microcirculation and number of capillary were observed in vivo with naked eyes to evaluate inflammatory response, collagen proliferation and healing of epithelium and smooth muscle in both groups at postoperative day 3, 7, 14 and 28. RESULTS: In group A, tissue layers at the anastomosis were approximated tight and neat with mild inflammation and primary wound healing. In group B, displacement and poor alignment of layers were seen with severe inflammation and secondary wound healing. Low frequency of microcirculation was detected in group A at day 3, and blood flow velocity significantly increased at day 7. Blood flow velocity was close to normal at day 14 and completely restored to the normal level at day 28. Microcirculation was lower in group A than that in group B at each time point. There were more capillaries, lower number of inflammatory cells, less collagen proliferation, and better healing of anastomotic epithelium and smooth muscle in group A than group B. CONCLUSION: Apposition suturing is better than inverted suturing in restoring local anastomotic microcirculation and healing of intestinal tissues.

ShRNA-targeted MAP4K4 inhibits hepatocellular carcinoma growth.

PURPOSE: Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is overexpressed in many types of cancer. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: MAP4K4 expression was examined in 20 fresh HCCs and corresponding nontumor liver tissues. Immunohistochemistry for MAP4K4 was performed on additional 400 HCCs, of which 305 (76%) were positive for hepatitis B surface antigens. The clinical significance of MAP4K4 expression was analyzed. MAP4K4 downregulation was performed in HCC cell lines HepG2 and Hep3B with high abundance of MAP4K4, and the effects of MAP4K4 silencing on cell proliferation in vitro and tumor growth in vivo were evaluated. Quantitative real-time PCR arrays were employed to identify the MAP4K4-regulated signaling pathways. RESULTS: MAP4K4 was aberrantly overexpressed in HCCs relative to adjacent nontumor liver tissues. This overexpression was significantly associated with larger tumor size, increased histologic grade, advanced tumor stage, and intrahepatic metastasis, as well as worse overall survival and higher early recurrence rate. Knockdown of the MAP4K4 expression reduced cell proliferation, blocked cell cycle at S phase, and increased apoptosis. The antitumor effects of MAP4K4 silencing were also observed in vivo, manifested as retarded tumor xenograft growth. Furthermore, multiple tumor progression-related signaling pathways including JNK, NFκB, and toll-like receptors were repressed by MAP4K4 downregulation. CONCLUSIONS: MAP4K4 overexpression is an independent predictor of poor prognosis of HCC patients, and inhibition of its expression might be of therapeutic significance.

[The effect of growth hormone on Clara cell in acute lung injury of rats].

OBJECTIVE: To study the effect of growth hormone (Gh) on Clara cell in acute lung injury (ALI) rats during stress responsive stage. METHODS: One hundred and twelve male Sprague-Dawley (SD) rats were randomly divided into ALI group and recombinant human Gh (rhGh) group. The rats in two groups were subdivided into seven subgroups of different time intervals after lipopolysaccharide (LPS) injection:0 (pre-injection of LPS, acted as control group), 0.5, 1, 2, 4, 6 and 24 hours subgroups respectively. Immunohistochemistry and electron microscope were used to investigate the number and morphological changes in Clara cells in ALI rats. RESULTS: Half of an hour after LPS injection, ALI was induced. The extent of lung injury increased with the time increased after LPS injection,peaked at 6 hours, then the extent of lung injury began to recess. The dynamic changes in lung injury of rats in rhGh group were similar to those in ALI group, but the extent of injury was more severe than that in ALI group at different time intervals. The Clara cell counts decreased significantly 1 hour after LPS injection, reaching the lowest at 6 hours, then began to recover 24 hours after LPS injection with statistically significant difference (all P<0.01). The counts of Clara cells in rhGh group decreased more significantly than that in ALI group at different time intervals after LPS injection, especially at 24 hours.The ultrastructure of Clara cell showed significant damage 24 hours after LPS injection. Compared with ALI group,the ultrastructure of Clara cell in rhGh group were more damaged 24 hours after LPS injection. CONCLUSION: rhGh could deteriorate the damage of Clara cell in acute lung injury rats induced by endotoxemia during stress response stage.

[Effects of different modes of artificial ventilation on lung injury in dog model of acute respiratory distress syndrome].

OBJECTIVE: To investigate the effects of different modes of artificial ventilation on lung injury in a dog model of acute respiratory distress syndrome (ARDS), and to evaluate the protective effect of various parameters in mechanical ventilation on lung injury. METHODS: Thirty-six healthy dogs were randomly divided into normal control group (N group), ARDS group (M group) and ventilation group (A-D groups) based on a series of random number. The ARDS dog model was replicated by intratracheal instillation of hydrochloric acid, and mechanical ventilation was carried out according to the following ventilatory protocols. A group: low V(T) (6 ml/kg) with respiratory rate 30/minutes, low inspiratory flow 6 ml.kg(-1).s(-1). B group: large V(T) (20 ml/kg) with respiratory rate 30/minutes, high inspiratory flow 20 ml.kg(-1).s(-1). C group: large V(T) (20 ml/kg) with respiratory rate 15/minutes, high inspiratory flow 17 ml.kg(-1).s(-1). D group: large V(T) (20 ml/kg) with respiratory rate 15/minutes, low inspiratory flow 10 ml.kg(-1).s(-1). Lung mechanical parameters were recorded at 0, 1, 2 and 4 hours after the change in ventilatory protocol. After 4 hours of mechanical ventilation, animals were sacrificed, and the lung was harvested. Lung wet/dry weight ratio (W/D) was measured. Histopathological changes were observed under light microscope, diffuse alveolar damage (DAD) scores was estimated, and polymorphonuclear leucocytes (PMN) count was done. Nuclear factor-KappaB (NF-KappaB) p65 activity was assessed by flow cytometry. RESULTS: W/D in B group (9.95+/-0.99) was higher than that of A (6.78+/-0.56) and D (7.11+/-0.47) groups (both P<0.01),but there was no significant difference between B and C groups (9.22+/-1.19, P>0.05). DAD scores in B group (12.80+/-1.47) was obvious higher than that of A (7.67+/-1.20) and D (8.83+/-1.17) groups (both P<0.01), but there was no difference compared with C group (11.50+/-1.87, P>0.05). NF-KappaB p65 activity in B group [(33.56+/-2.85)%] was significantly higher than that of A [(10.35+/-0.60)%] and D [(10.79+/-1.02)%] groups, but there was no difference between B and C [(30.87+/-1.16)%] groups. CONCLUSION: Large tidal volumes with high inspiratory flow and high respiratory rate may cause severe ventilator induced lung injury (VILI). Reduction of inspiratory flow and respiratory rate with large tidal volume ventilation may provide pulmonary protection.

[Effect of growth hormone on acute lung injury].

OBJECTIVE: To study the effect of growth hormone (GH) on acute lung injury (ALI) induced by endotoxemia, and its potential therapeutic mechanism. METHODS: One hundred and twelve male Sprague-Dawley rats were randomly divided into ALI group and GH group. The rats in two groups were further divided into seven subgroups determined by the length of interval after lipopolysaccharide (LPS) challenge: 0 (before injection of LPS, served as control group), 0.5, 1, 2, 4, 6 and 24 hours subgroups respectively. Pulmonary alveolar septum area density (PASAD) and the number of neutrophil in lungs of rats were analyzed morphometrically. The levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6) were determined by radioimmunoassay. The expression and activation of nuclear factor-kappaB (NF-kappaB) were analyzed, NF-kappaB positive cells in lungs were counted after immunofluorescence staining, and the levels of NF-kappaB inhibition (I-kappaB alpha) in lung homogenates of rats were assayed by Western blot. RESULTS: Half an hour after intravenous injection of LPS, both the PASAD and the number of neutrophil in lungs of ALI rats began to increase, and peaked at 6 hours post-injury, then began to recover and reached the normal levels at 24 hours. The content of TNF in lung homogenates showed immediate elevation after LPS injection, becoming higher than that of the control after 0.5 hour, reaching the peak value at 1 hour, maintaining high levels until 6 hours, then gradually recovered. The content of TNF in lung homogenates of the GH group increased significantly more than that in the LPS group. The contents of IL-6 in rats' lung homogenates began to increase significantly 1 hour post-injury, peaked at 4 hours, then gradually returned to normal level 6 hours post-injury. The content of IL-6 in the lung homogenates of GH group was higher than that in the LPS group at different time intervals post-injury, showing significant difference at 0.5, 6 and 24 hours (P<0.05 or P<0.01). The number of NF-kappaB positive cells increased dramatically at 0.5 hour post-injury. The intensity of fluorescence was enhanced. Both of them peaked at 4 hours post-injury. The number of NF-kappaB positive cells and the enhanced intensity of fluorescence began to decrease at 6 hours post-injury. But the number of NF-kappaB cells at 24 hours post-injury was still larger than that in the control group. The number of NF-kappaB cells in lungs of GH group was significantly larger than that in the LPS group at different time intervals post-injury (P<0.05 or P<0.01). I-kappaB alpha contents in lung homogenates of ALI rats decreased dramatically 0.5 hour post-injury, nadir at 4 hours, and then began to recover. Correlation analysis indicated that PASAD, the levels of TNF and IL-6 and the extent of neutrophil infiltration in lung were positively correlated to the extent of the expression and the activation of NF-kappaB. CONCLUSION: The application of GH during early stage of endotoxin challenge can deteriorate the lung injury induced by LPS through enhancing the expression and activation of NF-kappaB, thus enhances the inflammatory response in lungs.

[Regulatory effect of clearing-heat secreting-bile regulating-qi flow and activating blood circulation principle on cholecystokinin receptor].

OBJECTIVE: To explore the regulatory effect of clearing-Heat secreting-bile regulating-Qi flow and activating blood circulation (CSRA) principle on cholecystokinin receptor (CCK-R) and its mechanism. METHODS: Cholecystokinin (CCK) in serum of portal venous blood, maximum binding capacity (Bmax) and affinity (Kd) of CCK-R levels in gallbladder of guinea pigs allocated in four groups (control, high cholesterol, natural recovery and treated groups) were determined using radioimmunoassay and radioligand receptor assay (RRA). At the same time, changes of fasting volume (FV) and postprandial volume (PV) of gallbladder, fasting and postprandial bile (FB and PB) in gallbladder, gallbladder contraction rate (GCR) and cholesterol concentration (CC) in bile were observed. RESULTS: Compared with the control group, after two weeks of high cholesterol feeding, increase of FV, FB, PV, PB and CC (P < 0.05), and decrease of GCR (P < 0.01) and Bmax were found in cholesterol group, but with no significant change in Kd and CCK level. The above-mentioned criteria were restored to normal range in the treated group. CONCLUSION: CSRA principle could promote the recovery of gallbladder contraction by regulating CCK-R expression in it, its mechanism is possibly correlated with reduction of cholesterol concentration in bile.