Zymography Research Articles

Immune stimulating and galectin-3 inhibitory effects of a hydrophilic polysaccharide nutraceutical from Tinospora cordifolia

Tinospora cordifolia is an important ayurvedic herb known for its immune stimulating effect. In this investigation, we describe the galectin-3 inhibitory and immune stimulating properties of a novel water-soluble nutraceutical made from Tinospora cordifolia stem. This proprietary extract (T2CA) had a total yield of 6% and contained 86.9% carbohydrates with a high proportion of patented (1,4)-α-D-glucan in it. The T2CA activated T-cytotoxic and NK cells significantly contributing to its immune stimulating effect. Moreover, T2CA induced the activation of functional NK cells in human lymphocyte cultures that in turn contributed to the death of K562 leukemic cells in a dose-dependent manner in co-culture experiments. T2CA inhibited the expression of phosphorylated galectin-3 expression in human glioblastoma cell line which in turn inhibits the activity of MMP2 and MMP9 proteins in zymography investigations and inhibits migration of cancer cells in in vitro scratch assay. Western blot hybridization studies showed that T2CA downregulates the expression of total gal 3 proteins and extended the survival of AKR/J mice injected with Ehrlich ascites tumor cells in the Kepler-Meier survival curve analysis. The inhibitory effect of T2CA on phopho-galectin-3 expression and the activation of T-cytotoxic and NK cells suggested its protective effects against pathogenic infections and human malignancies via its immune stimulating mechanisms.

Evaluation of biochemical and immunological assembly line in emergency test

Objective To explore the application value of biochemical and immunological assembly line in emergency test by comparatively analyzing the changes in sample test process and turnaround time (TAT) in emergency test before and after installation of the assembly line. Methods A retrospective study was performed with the run data of the emergency biochemical and immunodetection project in Department of Clinical Laboratory in Tongji Hospital Affiliated to Tongji University from July to September in 2017 and from July to September in 2018. (1)The changes in sample circulation mode and test process were compared in emergency test before and after installation of the assembly line.(2)The changes in TAT of sample test and distribution of time interval in emergency laboratory report were statistically analyzed before and after installation.(3)The number of samples tested between instruments and occurrence of instrument failure were statistically compared before and after installation. (4)Biosafety risk assessment was performed on the sample test process before and after installation to analyze the effects of the assembly line installation on biosafety control in emergency test. Results (1) By applying the assembly line, the sample test process was well controlled and the workload for laboratorians was eased greatly through reduction of manual work.(2) Report TAT was remarkably shortened after installation compared to that before installation; the TAT of dry biochemistry and myocardial zymogram report in emergency test was shorted from 39 min and 48 min to 34 min and 42 min respectively, with a reduction of 12.82% an 12.50% compared to those before installation; the TAT of dry biochemistry and myocardial zymogram report in inpatient emergency was shorted from 37 min and 43 min to 29 min and 35 min respectively, with a reduction of 21.61% an 18.60% compared to those before installation; distribution of report TAT was comparatively analyzed, the percentage of reports in 0-30 min and 31-45 min increased remarkably compared to that before installation, the percentage of TAT of dry biochemistry report falling into 0-45 min increased to 74.58% and 85.66% after installation from 57.91% and 78.28% before installation, respectively in emergency and inpatient emergency. (3)For the model VITROS5600, the percentage of samples tested changed from 30.29% and 69.71% before installation to 47.63% and 52.37% after installation, respectively; for the model DXI800, the percentage of samples tested changed from 28.77% and 71.23% before installation to 53.49% and 46.51% after installation, respectively. After installation, there was a tendency of balance in the number of samples tested between instruments. Instrument failure was significantly reduced: for the model VITROS5600, the frequency of instrument failure decreased from 3.5 times a month/instrument before installation to 2 times a month/instrument after installation on average; for the model DXI800, the frequency of instrument failure decreased from 2.8 times a month/instrument before installation to 1.8 times a month/instrument after installation on average.(4) Installation of the assembly line lowered the risks biosafety events in sample test process in emergency test and the two risks of accidental contact with aerosol in sample tubes before detection and accidental contact with biological specimen due to overturning and spilling during detection were reduced from a high level before installation to a low level after installation. Conclusion In emergency test, biochemical and immunological assembly line is of certain value in process improvement, reduction of workload for laboratorians, improvement of work efficiency, TAT reduction and optimization, as well as control of biosafety conditions. Key words: Biochemical and immunological assembly line; Emergency test; Sample test process; Turn-around time

Diagnostic value of serum NSE, S100B protein and myocardial zymogram in premature infants with intrauterine infection

Objective To explore the diagnostic value of serum NSE, S100B protein and myocardial zymogram in premature infants with intrauterine infection. Methods From January 2016 to December 2017, 60 preterm infants with intrauterine infection in the Integrated Chinese and Western Medicine Hospital of Wenzhou were selected in the study.According to whether brain injury occurred, they were divided into brain injury group (28 cases) and non-brain injury group (32 cases). Serum NSE content was detected by chemiluminescence method, serum S100B protein level was detected by enzyme linked immunosorbent assay (ELISA), and serum CK and HBDH levels were detected by automatic biochemical analyzer.The serum levels of NSE, S100B, CK and HBDH were compared between the two groups, the combined diagnostic efficacy of NSE+ S100B protein+ CK+ HBDH was analyzed, the correlation of serum NSE, S100B protein, CK, HBDH with brain injury wasanalyzed. Results The levels of serum NSE [(2.43±0.54)μg/L] and S 100B [(14.36±3.21)ng/L] in the brain injury group were higher than those in the non-brain injury group [(0.97±0.27)μg/L and (8.10±1.87)ng/L] (t=13.498, 9.370, all P<0.05). The levels of serum CK [(437.64±54.12)U/L] and HBDH [(387.91±56.45)U/L] in the brain injury group were significantly higher than those in the non-brain injury group [(183.54±32.58)U/L and (174.3±26.63)U/L] (t=22.347, 19.126, all P<0.05). The sensitivity and specificity of the combined diagnosis of NSE+ S100B protein and myocardial zymogram were higher than those of each single index.Serum NSE, S100B protein, CK and HBDH were positively correlated with brain injury. Conclusion The elevation of serum NSE, S100B protein and myocardial zymogram in preterm infants with intrauterine infection after birth has certain clinical significance in judging whether brain injury occurs or not. Key words: Intrauterine infection; Premature infants; Brain injury; Neuron-specific enolase; S100B protein; Creatine kinase; Carboxybutyrate dehydrogenase; Early diagnosis

Exopolysaccharides from an Ophiopogon japonicus endophyte inhibit proliferation and migration in MC-4 human gastric cancer cells

Background: Recently, natural products have gained increasing attention. Endophytic bacteria represent a source of natural anticancer products, and are well-known for producing diverse bioactive anti-cancer compounds. Herein, we investigated the antitumor activity of exopolysaccharides (EPSs) extracted from endophytes in Ophiopogon japonicus . Methods: The endophyte bacterium MD4 was isolated from O. japonicus . To identify MD4, a partial sequence of 16S rDNA was analyzed. The phenol-sulfuric acid method was used to measure the EPSs concentration. Cell viability was tested by the MTT assay to quantify the EPS cytotoxicity effect. Wounding healing, gelatin zymography, and ELISA were used to measure cell migration ability. Results: MD4 was identified as a Staphylococcus sp . with 99% similarity. The results of the phenol sulfuric acid method showed that the purified EPS concentration from MD4 was 28.068 µg/µL. There was a concentration-dependent cytotoxic effect of MD4-EPS on MC-4 cells, with IC50 values of 0.891 µg/µL. The results of the MTT assay showed that MD4-EPS effectively inhibited the migration of MC-4 cells as the concentration increased. The gelatin zymography analysis showed that the enzymatic activities of the migration-related MMP2 and MMP9 proteins decreased after EPS treatment. Furthermore, the protein secretion levels of MMP2 and MMP9 after MD4-EPS treatment were significantly lower than those in the control and PBS groups. Conclusions: Our results demonstrate that the EPSs derived from the MD4 endophyte of O. japonicus acted as natural antitumor agents to inhibit the migration of gastric cancer cells.

Effects of PinX1 gene on migration and invasion of colon cancer cells and the molecular mechanisms

Objective To observe the function of telomerase inhibitor 1 (PinX1) gene in cell migration and invasion and its possible molecular mechanisms in the colon cancer cell lines. Methods The PinX1 interfering lentivirus (sh-PinX1) and its negative control (sh-con) were transfected into HCT116 and SW480 colon cancer cells. The protein expression levels of PinX1, matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blotting. The alterations in the ability of the migration and invasion of colorectal cancer cells (HCT116 and SW480) were investigated by Transwell migration and invasion assays. The MMPs activities in the two colon cancer cell lines were examined by gelatin zymography. Results Western blotting showed that PinX1 protein level was decreased by interference of PinX1 gene in colon cancer cell lines: for HCT116 [(40 459±154) vs. (3 135±24), P=0.001]; for SW480 (54 709±202) vs. (6 537±55), (P=0.000). Migration assay showed that the decreased PinX1 increased the ability of migration in colon cancer cells [116-sh-con group (100.0±5.8) vs. 116-sh-PinX1 group (217.3±11.6), P=0.000; 480-sh-con group (193.0±6.6) vs. 480-sh-PinX1 group (407.0±6.6), P=0.004]. Invasion assay showed that decreased PinX1 increased the invasive ability in colon cells [116-sh-con group (71.7±3.3) vs. 116-sh-PinX1 group (170.0±5.8), P=0.000; 480-sh-con group (58.3±4.4) vs. 480-sh-PinX1 (130.0±5.8), P=0.001]. Western blotting showed that MMP-2 protein level was increased by interference of PinX1 gene in colon cancer cells [116-sh-PinX1 group (58 549±220) vs. 116-sh-con group (3 819±31), P=0.000); 480-sh-PinX1 group (56 540±207) vs. 480-sh-con group (2 909±33), P=0.001]. Western blotting showed that PinX1 did not affect the MMP-9 expression [116-sh-PinX1 group (8 499±769) vs. 116-sh-con group (8 499±769, P=0.390); 480-sh-PinX1 group (4 008±290) vs. 480-sh-con group (3 907±256), P=0.808]. Gelatin zymography assay showed that low expression PinX1 suppressed the activity of MMP-9 [116-sh-PinX1 group (12 699±284) vs. 116-sh-con group (5 571±91, P=0.000); 480-sh-PinX1 group (14 418±296) vs. 480-sh-con group (8 704±55), P=0.000]. Conclusion PinX1 could increase the ability of migration and invasion by activating MMP-2 signaling pathway in colon cancer cells. Key words: PinX1; Colon cancer; Migration; Invasion; Matrix metalloproteinase-2

Inhibitory Effect of Hizikia fusiformis Solvent-Partitioned Fractions on Invasion and MMP Activity of HT1080 Human Fibrosarcoma Cells.

Matrix metalloproteinases (MMPs) are endopeptidases that take significant roles in extracellular matrix degradation and therefore linked to several complications such as metastasis of cancer progression, oxidative stress, and hepatic fibrosis. Hizikia fusiformis, a brown algae, was reported to possess bioactivities, including but not limited to, antiviral, antimicrobial, and anti-inflammatory partly due to bioactive polysaccharide contents. In this study, the potential of H. fusiformis against cancer cell invasion was evaluated through the MMP inhibitory effect in HT1080 fibrosarcoma cells in vitro. H. fusiformis crude extract was fractionated with organic solvents, H2O, n-BuOH, 85% aqueous MeOH, and n-hexane (n-Hex). The non-toxicity of the fractions was confirmed by MTT assay. All fractions inhibited the enzymatic activities of MMP-2 and MMP-9 according to the gelatin zymography assay. Cell migration was also significantly inhibited by the n-Hex fraction. In addition, both gene and protein expressions of MMP-2 and -9, and tissue inhibitor of MMPs (TIMPs) were evaluated by reverse transcription-polymerase chain reaction and Western blotting, respectively. The fractions suppressed the mRNA and protein levels of MMP-2, MMP-9 while elevating the TIMP-1 and TIMP-2, with the H2O fraction being the least effective while n-Hex fraction the most. Collectively, the n-Hex fraction from brown algae H. fusiformis could be a potential inhibitor of MMPs, suggesting the presence of various derivatives of polysaccharides in high amounts.

Myocardial protective effect of L-carnitine in children with hand, foot and mouth disease caused by Coxsackie A16 virus

To investigate the myocardial protective effect of L-carnitine in children with hand, foot and mouth disease (HFMD) caused by Coxsackie A16 virus and possible mechanisms. A total of 60 HFMD children with abnormal myocardial enzyme after Coxsackie A16 virus infection were enrolled and randomly divided into L-carnitine group and fructose-1,6-diphosphate group (fructose group), with 30 children in each group. The two groups were given L-carnitine or fructose diphosphate in addition to antiviral and heat clearance treatment. Another 30 healthy children who underwent physical examination were enrolled as control group. The changes in myocardial zymogram, malondialdehyde (MDA), superoxide dismutase (SOD), and apoptosis factors sFas and sFasL after treatment were compared between groups. There was no significant difference in treatment response between the L-carnitine group and the fructose group (P>0.05). One child in the fructose group progressed to critical HFMD, which was not observed in the L-carnitine group. Before treatment, the L-carnitine group and the fructose group had significantly higher indices of myocardial zymogram and levels of MDA, sFas, and sFasL and a significantly lower level of SOD than the control group (P<0.05), while there were no significant differences in these indices between the L-carnitine group and the fructose group (P>0.05). After treatment, the L-carnitine group and the fructose group had significant reductions in the indices of myocardial zymogram and levels of MDA, sFas, and sFasL and a significant increase in the level of SOD (P<0.05); the fructose group had a significantly higher level of creatine kinase (CK) than the control group and the L-carnitine group, and there were no significant differences in other myocardial enzyme indices, MDA, sFas, and sFasL between the L-carnitine group and the fructose group, as well as between the L-carnitine and fructose groups and the control group (P>0.05). SOD level was negatively correlated with aspartate aminotransferase, lactate dehydrogenase (LDH), CK, and creatine kinase-MB (CK-MB) (r=-0.437, -0.364, -0.397, and -0.519 respectively; P<0.05), and MDA level was positively correlated with LDH and CK-MB (r=0.382 and 0.411 respectively; P<0.05). L-carnitine exerts a good myocardial protective effect in children with HFMD caused by Coxsackie A16 virus, possibly by clearing oxygen radicals and inhibiting cardiomyocyte apoptosis.

Effect of CD147 antagonist peptide and doxycycline on gallbladder carcinoma cells

Objective To research the effect of GBC-SD on proliferation, apoptosis, expression of CD147 and activity of matrix metalloproteinase (MMP)-9 in gallbladder carcinoma cells, with single and combination therapy of CD147 antagonist peptide and doxycycline. Methods Gallbladder carcinoma cells were treated with antagonist peptide (AP-9) (50, 100, 200, 400 μg/ml) or DOX (5, 10, 20, 40 μg/ml) alone and combination therapy (AP-9 200 μg/ml + DOX 20 μg/ml) for 12, 24 and 48 h, then the inhibition rate of proliferation was detected by cell counting kit-8 (CCK-8), expression of CD147 was detected by Western blotting, the activity of MMP-9 was detected by gelatin zymography, and the early apoptosis rate was detected by flow cytometry with Annexin V/propidium iodide (PI) staining. Results After treatment with several concentrations of AP-9, the apoptosis rate [(2.13±0.10)% and (2.92±0.19)%] was higher (P=0.002, P=0.001) than control group [(1.58±0.08)%]; the activity of MMP-9 [(320.08±33.90), (249.59±33.63), (259.63±34.08) and (172.31±35.20)] was reduced (P=0.013, 0.002, 0.003 and 0.001 respectively) as compared with that in control group; and cell proliferation was inhibited as compared with that in control group (P=0.001). After treatment with several concentrations of doxycycline, the apoptosis rate [(3.25±0.12)% and (3.63±0.24)%] was higher (P=0.001) than in the control group [(1.58±0.08)%]; the activity of MMP-9 [(203.52±18.25), (175.22±23.30), (147.86±22.83) and (109.99±29.22)] was reduced (P=0.001) as compared with control group [(446.08±42.42)]; cell proliferation was inhibited as compared with control group (P=0.001); the expression level of CD147 [(0.97±0.12), (0.80±0.10), (0.69±0.11) and (0.51±0.12)] was reduced (P=0.001) as compared with cotrol group [(2.04±0.15)]. As compared with treatment alone, cell proliferation [(38.78±0.39)%, (43.76±0.71)% and (52.00±0.91)%] was increased (P=0.001, P=0.003), the activity of MMP-9 [(110.19±24.74)] was reduced (P=0.001, P=0.024), and the apoptosis rate [(3.73±0.29)%] was elevated after combination therapy (P=0.001, P=0.048). Conclusion DOX can down-regulate the expression of CD147. AP-9 and DOX alone or in combination can inhibit the proliferation of gallbladder cancer cells, inhibit the activity of MMP-9, and promote apoptosis, and the inhibitory effect was enhanced after combination therapy. Key words: Gallbladder carcinoma; CD147; Extracellular matrix metalloproteinase-9; Antagonist peptide; Doxycycline

INDUCTION OF ANTIOXIDANT ENZYMES AND ISOZYMES PROFILE OF CHICKPEA GENOTYPES UNDER COMPATIBLE AND INCOMPATIBLE INTERACTIONS WITH FUSARIUM WILT

Chickpea genotypes (ten desi and eight kabuli), differing in their reaction to Fusarium wilt, were grown in Fusarium infested soil and screened for wilt resistance and susceptibility. The root and leaf tissues of highly resistant (desi WR 315 and kabul KAK 2) and highly susceptible (desi JG 62 and kabul L 550) chickpea genotypes were collected during disease development stages under pathogen infestation and non-infestation conditions. They were analyzed for antioxidant enzyme activities and isozymes profiling. The specific activities of ROS scavenging enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX), ascorbate peroxidase (APX), polyphenol oxidase (PPO) and catalase (CAT) were found higher in the root and leaf tissues under compatible interactions (infection) compared with incompatible (non-infested or non-infected?) during disease development stages. The susceptible compatible interactions of desi and kabuli chickpea genotypes induced systemic antioxidant bursts (SOD, GPX, APX, PPO, CAT) efficiently after Fusarium infection. The isozymes profiling of SOD, APX, PPO, CAT agreed with the enzyme activities and also showed differences in GPX zymogram pattern distinctive in the root of kabuli resistant KAK 2 during infection stage (S2). However, nativePAGE electrophoregram revealed distinctive bands for infected root tissues of desi resistant WR 315 (novel) and kabuli resistant KAK 2 (correlated with GPX isoforms) at S2 stage. The SOD, GPX, and PPO isoforms are more diverse and associated with Fusarium interactions during disease development stages. The results explained production of an oxidative burst and related responses during Fusarium wilt infection of desi and kabuli chickpea genotypes.

Effect of thrombin on malignant biological behavior of esophageal cancer cell line Eca109

Objective To study the effect of thrombin on proliferation and invasion of esophageal cancer cell line Eca109, and to explore its possible mechanism. Methods The proliferation and invasion of Eca 109 cells treated with thrombin were detected by MTT and Transwell assay, respectively. The activity of matrix metalloproteinase 2 (MMP-2) and MMP-9 in the supernatant of Eca109 cells was detected by gelatin zymography. Reverse transcription polymerase chain reaction (PCR) and immunocytochemistry were used to study the mRNA expression of protease-activated receptor 1 (PAR-1), the important receptor of thrombin, and subcellular localization of PAR-1 protein in Eca109 cells, respectively. Results Thrombin could promote Eca109 cells proliferation in a dose-dependent manner. Cell proliferative rates of 0.5 U/ml and 1.0 U/ml thrombin were 34.38% and 57.19%, respectively (P < 0.05). Compared to that of control group, the number of Eca109 cells incubated with 1.0 U/ml thrombin invading through the basement membrane of Transwell was increased (303.33±6.66 vs. 116.33±11.51, P < 0.05). When treated with various concentrations of thrombin for 24 h, the activities of MMP-2 and MMP-9, especially MMP-9, in the supernatant of Eca109 cells were increased in a dose-dependent manner. Eca109 cells expressed PAR-1 mRNA, and PAR-1 protein was mainly located on the cellular membrane. Conclusion Thrombin increases proliferation and invasion of esophageal cancer Eca109 cells and enhances the activities of MMP-2 and MMP-9 in cells supernatant, which might be induced by activation of PAR-1 located on cellular membrane. Key words: Esophageal neoplasms; Thrombin; Protease-activated receptor 1

Comparison and analysis of three types of stress urinary incontinence mice models

Objective To compare the 3 kinds of stress urinary incontinence (SUI) mice models. Methods 140 female mice were randomly divided into 4 groups: control group, vaginal distension (VD) group, ovariectomy (OV) group, and VD+ OV group. There were 65 mice in control group, and 25 mice each in the rest three groups. The postoperative infection rate and mortality rate were recorded, and maximum bladder capacity (MBC) and leak point pressures (LPPs) were measured. The contents and activity of matrix metalloproteinase (MMP)-2 and MMP-9 were detected by zymography assay after surgery. The advantages and disadvantages of the three models were comparatively analyzed. Results The MBC/abdominal leak point pressure (ALPP)/bladder leak point pressure (BLPP) in control group, VD group, OV group, and VD+ OV group were respectively (0.101±0.014) ml/(46.420±6.399) cmH2O/(44.180±4.912) cmH2O (1 cmH2O=0.098 kPa); (0.081±0.015) ml/(30.990±5.021) cmH2O /(26.680±6.485) cmH2O; (0.095±0.010) ml/(44.330±6.465) cmH2O /(41.450±6.474) cmH2O and (0.071±0.013) ml/(28.920±5.232) cmH2O /(22.120±7.412) cmH2O, reduced significantly in VD group (P value respectively were 0.000, 0.000 and 0.000) and VD+ OV group (P value respectively were 0.000, 0.000 and 0.000), compared with control group and OV group, but the difference between VD+ OV group and VD group was not notable (P value respectively were 0.051, 0.245 and 0.062), or between OV group and control group (P value respectively were 0.093, 0.238 and 0.064); The expression of MMP-2/MMP-9 in these group were respectively 13.930±0.823/9.610±0.771, 14.990±0.908/11.100±0.735, 13.890±0.707/9.761±0.680, 15.470±1.070/11.240±0.750, increased notably in VD group (P value respectively were 0.003/0.000 and 0.001/0.000) and VD+ OV group (P value respectively were 0.000/0.000 and 0.000/0.000) compared with control grou, but the difference of MMP-2/MMP-9 between the VD+ OV group and VD group was not significant (P value respectively were 0.216/0.604), or between OV group and control group (P value respectively were 0.912/0.573). Conclusion Vaginal distension and vaginal distension with ovariectomy can both establish SUI mice models, but vaginal distension is preferable. Key words: Stress Urinary Incontinence; Vaginal Distension; Ovariectomy; Urine Dynamics Measurement

Inhibitory Effect and Molecular Mechanism of an Anti-tyrosinase in B16F1 Melanoma Cells by α-mangostin, Isolated from Mangosteen Pericarp

Tyrosinase is the key enzyme of melanogenesis which is responsible for hyperpigmentation and food browning. Thus, investigations on natural and safe tyrosinase inhibitors has received massive attention around the globe. Phytochemistry work on Garcinia mangostana L. or locally known as mangosteen using various chromatography techniques led to the isolation of the chief constituent, α-mangostin which showed outstanding anti tyrosinase activity. The compound remarkably showed better inhibition on melanogenesis induced by α-MSH compare to positive control used, arbutin and kojic acid when tested on B16F1 melanoma cell line. The inhibition of intracellular tyrosinase activity was found to be exerted at the protein expression level when analyzed by immunoblot and tyrosinase zymography. The expression level of microphthalmia-associated transcription factor (MITF) was reduced by treatment of α-mangostin. The down-regulation of intracellular tyrosinase expression seemed to correlate with decrease in MITF protein level which is the key factor for the genes involved in melanogenesis. ABTS assay on α-mangostin indicated that the compound possessed moderate antioxidant activity with IC50 value of 28.8.µg/ml. The overall results suggest that α-mangostin has strong and potent anti-melanogenic activity that is exerted by direct inhibition of tyrosinase enzyme activity and by down-regulation of the genes expression involved in the melanogenesis pathways.

Mechansim of resveratrol inhibiting interleukin-6-induced invasion of human gastric cancer SGC7901 cells

Objective To elucidate the inhibitory effect of resveratrol on interleukin (IL)-6-induced invasive ability of SGC7901 cells and the molecular mechanism. Methods The cell viability of cells treated with resveratrol was evaluated by methyl thiazolyltetrazolium (MTT) assay. The SGC7901 cells were treated with IL-6 and non-cytotoxic concentration of resveratrol. The invasion of SGC7901 cells was detected by Boyden chamber assay. Matrix metalloproteinase (MMP)-2 activity was determined by gelatine zymography assay. The changes of the related proteins were detected by Western blotting. Results Resveratrol was not toxic to SGC7901 cells at the concentration under 30 μmol/L. The invasion ability of IL-6-induced SGC7901 cells was decreased about 20% after treatment with 20 μmol/L resveratrol for 24 h, accompanied by the inhibition of MMP-2 secretion for 50%, as well as the levels of phosphorylated extracellular regulated protein kinases 1/2 (p-ERK1/2). Conclusion Resveratrol of 20 μmol/L inhibits invasion of SGC7901 cells possibly through the suppression of the activation of ERK signaling pathway, subsequently exerting inhibitory effect on MMP-2. Key words: Resveratrol; Extracellular signal regulated kinase 1/2; Gastric carcinoma

Localization and identification of thrombin and plasminogen activator activities in model human thrombi by in situ zymography.

Human thrombi vary in their susceptibility to lysis and this is clinically important. Several potential contributory factors were examined in this study by using model thrombi, created under flow; these provide a robust, reproducible and easily-manipulated system. Here we identify the plasminogen activators (PA) active in model thrombi of known age and define the cellular and plasma contribution to activity in different areas. The cell-rich head of model thrombi had strong thrombin and PA activity, with coagulant activity also at the tail. Thrombin activity decreased as model thrombi were aged. PA activity in the thrombus head also decreased on ageing of thrombi but activity emerged around the thrombi, including the tail. Activity in the head of fresh model thrombi was primarily due to uPA, with some contribution from tPA. Experiments with thrombi prepared from platelet-rich plasma and added leucocytes showed that uPA activity at the head of fresh thrombi was derived from PMN. Older thrombi had tPA activity around the tail of the thrombus; this activity occurred in the absence of cells. This study highlights the importance of PMN-derived uPA activity in the lysis of fresh thrombi, with activity originating in the leucocyte-rich head. It also shows that thrombi are dynamic structures in which fibrin can be repeatedly laid down and lysed, observations that are relevant to therapeutic lysis and potential rethrombosis.

INHIBITION OF DIGESTIVE Α-AMYLASE FROM EURYGASTER INTEGRICEPS (SUNN PEST) BY A PROTEINACEOUS EXTRACT FROM WHEAT VARIETIES BEZOSTAYA, GASPARD, AND DARAB

At present time, chemical control is the main method of protection of sunn pests. The aim of the current study was to investigate the effect of seed proteinous extracts from different varieties of wheat ( Triticum aestivum var Bezostaya, Gaspard and Darab) on the α-amylase activity of Sunn Pest. To achieve this goal, adult insects were dissected and each insect's gut was removed and their amylase enzyme was extracted. Proteins of wheat were extracted using 0.1 M NaCl. The effect of proteinaceous extracts on α-amylase activity both as an enzymatic assay and as a gel assay using zymogram was performed. Inhibition percentages were recorded for the Bezostaya and Gaspard and Darab wheat varieties which were (73%), (%63) and (33%), respectively.

Protection against myocardial ischemia -reperfusion injury in rats by a novel selective lymphotoxin binding p55TNFR

Objective To investigate the effect of a novel lymphotoxin with selectively binding to p55 tumor necrosis factor receptor (p55TNFR) on myocardial ischemia -reperfusion injury in rats in order to explore the mechanism. Methods A total of 40 SD rats were randomly (random number) assigned into four groups (n =10 in each) , namely sham operation group (group A), I/R group (group B), wild type rhLTα treatment group (group C), and p55TNFR selective rhLTα (rhLTα-Q107E) treatment group (group D). After I/R model rats were established, various therapeutic agents or saline were given by continuous intravenous infusion for 24 h via a micropump. After 24 hours of treatment, serum myocardial zymogram, such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK), as well as superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) activities were determined. Myocardial infarction size (MIS) was measured by nitro blue tetrazolium chloride (NBT) staining. Results Compared to sham operation group, MIS, AST, LDH, CK, MDA were increased, while the activities of SOD and GSH-Px were decreased. However, all the effects were significantly reversed by treatment with rhLTα-Q107E (P 0.05). Conclusions The rhLTα-Q107E plays a role in the protection against myocardial ischemia -reperfusion injury in rats by the mechanism of scavenging oxygen free radicals and increasing the activity of endogenous antioxidant system. Key words: p55TNFR; Ischemia-reperfusion; Myocardial injury; Oxidative stress

Effect of neuregulin-1 on invasion and migration ability of human glioma cells

Objective To study the relationship between the protein level of Neuregulin-1 (NRG-1) and glioma progression and the effect of NRG-1 on the invasive and migratory ability of glioma cells. Methods The protein expression level of NRG-1 was detected by Western blot (WB) in nontumorous brain tissues and glioma specimens. Next, the effect of NRG-1 down-regulation on the migratory and invasive ability of glioma cells were examined by wound healing assay and matrigel-precoated transwell chambers, respectively. Last, the excretion of MMP2 was examined by gelatin zymography assay after down-regulating the NRG-1. Results The protein level of NRG-1 in human gliomas was considerably higher than that of nontumorous brain tissues. Compared with the control group, the number of cells acrossing the wound edge decreased in NRG-1 down-regulation group (P<0.01). In the matrigel invasion assay, cell number invading through matrigel dramatically decreased in NRG-1-siRNA transfection group (P<0.01). Gelatin zymography revealed that the excretion of MMP2 of NRG-1 down-regulation group was obviously lower than that of the control group (P<0.01). Conclusions The expression level of NRG-1 in human gliomas was higher than that in nontumorous brain tissues, indicating the involvement of NRG-1 in glioma development. Down-regulation of NRG-1 inhibited glioma cell invasion and migration and the excretion of MMP2 in vitro. Key words: Glioma; Neuregulin-1; Cell migration; Neoplasm invasion; Matrix metalloproteinase 2

Expression and significance of SATB1 and wnt/β-catenin signaling molecule in the placenta of preeclampsia

To explore the role of specific AT rich sequence binding protein 1(SATB1) and wnt/β-catenin signaling pathways in the regulation of trophoblast invasion and its effect in the pathogenesis of preeclampsia. From March 2013 to March 2014, 20 cases of human villous tissues (early pregnancy group) from women of 8-10 gestational weeks who received artificial abortion at the First Affiliated Hospital of Chongqing Medical University, 18 cases of placental tissues (mid-pregnancy group) from women of 18-20 gestational weeks who had labor induction by water bag, 20 cases of placental tissues (normal full-term group) from healthy full-term pregnancy women and 20 cases of placental tissues (preeclamptic group) from women with preeclampsia who received elective c-section in were collected. Immunohistochemical SP method was utilized to determine the position of SATB1 and beta-catenin in villous tissues or placental tissues. Western blot was performed to analyze the expression level of SATB1 and beta-catenin in villous tissues or placental tissues. Immunofluorescence assay was used to determine the location of SATB1 and β-catenin in HTR8/SVneo cells. Western blot was performed to detect the expression level of SATB1 and beta-catenin in HTR8/SVneo cells cultured in normoxia and hypoxia reoxygenation (H/R) condition. Co-Immunoprecipitation detection was used to evaluate the interaction between SATB1 and β-catenin in placental tissues in preeclamptic group and HTR8/SVneo cells in H/R group. Gelatin zymography analysis was used to measure the activity of matrix metalloproteinases (MMP)-2 and 9 in placental tissues from preeclamptic group and HTR8/SVneo cells in H/R group. (1) In the normal full-term group, rare syncytiotrophoblastic nodule, less fibrinoid necrosis and abundant numbers of capillary could be observed in placental tissues. In comparison, there were obvious vacuolation in the cytoblast of syncytiotrophoblast, rich fibrinoid necrosis and poor numbers of villous capillary in placental tissues from preeclamptic group. (2) SATB1 could be found by immunochemical staining in placenta or villous tissues from all the groups. The staining intensity of SATB1 were more weakening in preeclamptic group than in the normal full-term group. (3) β-catenin could be found by immunochemical staining in placenta or villous tissues from all the groups. The staining intensity of β-catenin were more weakening in preeclamptic group than in the normal full-term group. (4) The protein expression levels of SATB1 in early pregnancy group, mid-pregnancy group, normal full-term group and preeclamptic group were 0.300 ± 0.009, 0.271±0.015, 0.238±0.018 and 0.153±0.007, respectively. The protein levels of β-catenin among the above groups were 0.743±0.041, 0.648±0.021, 0.549±0.069 and 0.269±0.047, respectively. Both the expression of SATB1 and β-catenin protein were significant decreased in placental tissues from preeclamptic group compared with the other three groups. (5) The SATB1 and β-catenin protein was located in nucleus of trophoblast and a small amount was in the cytoplasm. The fluorescence intensity of both SATB1 and β-catenin in the H/R group were significantly decreasing when compared to the normoxia group. (6) HTR8/SVneo cells in H/R group showed a significant decrease in both SATB1 and β-catenin protein levels when compared to the normoxia group. The protein level of SATB1 in the normoxia group was 0.213±0.005, while was 0.083±0.021 in the H/R group. The protein level of β-catenin in the normoxia group was 0.797±0.081, and was 0.543±0.131 in the H/R group. (7) There was an interaction between SATB1 and β-catenin in placental tissues from the preeclamptic group and HTR8/SVneo cells exposed by H/R. (8) The enzymatic activity of MMP-2 and MMP-9 protein were decreased significantly in placental tissues from the preeclamptic group (2.251±0.310, 1.447±0.102, respectively) when compared to the normal full-term group (7.098±0.451, 5.502±0.197, respectively). MMP-2 and MMP-9 were significantly decreased in the H/R group (0.471±0.104, 0.297±0.103, respectively) when compared to the normoxia group (0.842±0.209, 0.595±0.100, respectively). The expression of SATB1 decreased in the placenta of preeclampsia. This may influence the activity of MMP-2 and 9 by regulating Wnt/β-catenin signaling pathways, affect trophoblast invasion and eventually result in preeclampsia.

Effect of resveratrol on plasmatic molecular indicators of brain tissue response to the hypoperfusion/ reperfusion challenge

It is well-documented that endocannabinoids (eCBs) and congeners show a neuroprotective role in several experimental models of brain injury and that changes in eCB levels in peripheral blood cells may reflect the severity of neurological insult. We have previously shown that the preventive administration of dietary natural compounds may increase the plasmatic levels of palmytoylethanolamide (PEA) and oleoylethanolamide (OEA) following the transient bilateral common carotid artery occlusion (BCCAO)-induced brain tissue challenge (1). Resveratrol (RVT), (3,4’, 5-trihidroxystilbene) is a strong natural antioxidant of polyphenolic structure found in grapes and red wine, with many physiological effects, including the prevention of lipid peroxidation in human LDL, inhibition of arachidonic acid metabolism, and platelet activity. RVT has been further shown to protect cerebral tissue and cardiac muscle from tissue damage caused by oxidative stress triggered by reperfusion (2) and has been proposed as a potential neuroprotective agent in treating acute states in focal cerebral ischemia injury (3). In this line, we intend to evaluate whether exogenous administration of RVT prior to induction of BCCAO followed by reperfusion influences the molecular changes occurring in cerebral cortex and plasma, with particular focus on the eCB system. With this aim, cerebral hypoperfusion was produced by a 30 min BCCAO followed by 60 min reperfusion (BCCAO/R). Animals were starved for 12 hours before surgery and 6 hours prior to ischemia RVT (40 mg/kg/0.45 ml of sunflower oil as vehicle) was administered via gavage. Biological samples of plasma, cerebrospinal fluid (CSF), and brain tissue were examined by HPLC, gel zymography, western blot and immunohistochemistry. Data obtained indicate that RVT appears to influence the outcome of BCCAO/R cerebral injury by modulating changes in levels of lipid hydroperoxides, markers of oxidative stress, eCBs and eCB congeners, expression of CB1 and CB2 receptors, peroxisome proliferator-activated receptor- (PPAR) alpha, ciclooxygenase-2 (COX-2) protein levels and enzymatic activity of matrix-metalloproteinase- 9 (MMP-9). Interestingly, changes in brain of some of these parameters, like lipid hydroperoxides, were also found in plasma. Results obtained suggest that exogenous administration of RVT may modulate the brain tissue compensatory or repair mechanisms triggered by the hypoperfusion/reperfusion and support the possible use of this molecule as treatment to prevent the BCCAO/R-induced brain insult. In addition, the finding that changes in plasma mirrored those found in cerebral tissue, opens to the possibility to test whether RSV exerts its positive activities in humans.

Transforming activities of Chlamydia pneumoniae in human mesothelial cells.

Knowledge in viral oncology has made considerable progress in the field of cancer fight. However, the role of bacteria as mediators of oncogenesis has not yet been elucidated. As cancer still is the leading cause of death in developed countries, understanding the long-term effects of bacteria has become of great importance as a possible means of cancer prevention. This study reports that Chlamydia pneumoniae infection induces transformation of human mesothelial cells. Mes1 cells infected with C. pneumoniae at a multiplicity of infection of 4 inclusion-forming units/cell showed many intracellular inclusion bodies. After a 7-day infection an increased proliferative activity was also observed. Real-time PCR analysis revealed a strong induction of calretinin, Wilms' tumour gene 1, osteopontin, matrix metalloproteinases-2, and membrane-type 1 metalloproteinases gene expression in Mes1 cell, infected for a longer period (14 days). The results were confirmed by western blot analysis. Zymography analysis showed that C. pneumoniae modulated the in-vitro secretion of MMP-2 in Mes1 cells both at 7 and 14 days. Cell invasion, as measured by matrigel-coated filter, increased after 7 and 14 days infection with C. pneumoniae, compared with uninfected Mes1 cells. The results of this study suggest that C. pneumoniae infection might support cellular transformation, thus increasing lung cancer risk.