OBJECTIVETo discover and identify differentially expressed genes associated with colorectal adenoma formation and the role of RegIV in colorectal adenoma differentiation.

METHODSA subtracted cDNA library was constructed with cDNAs that were isolated from either the normal mucosa or adenoma tissue of a single patient. Suppressive subtractive hybridization (SSH) combined with virtual northern blotting was used to characterize differentially expressed genes and contigs were assembled by electronic cloning (in silico cloning) with the EST database. Semi-quantitative RT-PCR was performed in 9 colorectal adenomas.

RESULTSThe amino acid sequence was determined with open reading frame (ORF) prediction software and was found to be 100% homologous to the protein product of RegIV (a novel gene isolated from a large inflammatory bowel disease library). RegIV was found to be highly expressed in all of the adenoma samples (9/9) compared with the normal mucosa samples, while 5/6 cases showed RegIV to be more strongly expressed in adenocarcinoma.

CONCLUSIONRegIV may play an important role in the initiation of colorectal adenoma differentiation, and its detection may be useful in the early diagnosis of colorectal adenoma formation.

Adenoma ; genetics ; metabolism ; Blotting, Northern ; Colorectal Neoplasms ; genetics ; metabolism ; Humans ; Lectins, C-Type ; biosynthesis ; genetics ; Nucleic Acid Hybridization ; Pancreatitis-Associated Proteins ; Prognosis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the chemical constituents of the extract of Galium verum.

METHODThe compounds were isolated by chromatography and identified by spectral data.

RESULTThe eleven compounds obtained were identified as (+)-pinoresinol 4,4'-O-bis-beta-D-glucopyranoside (1), epipinoresinol (2), (+) -medioresinol (3), isorhamnetin (4), isorhamnetin 3-O-alpha-L-rhamnopyranosyl-(1-6)-beta-D-glucopyranoside (5), diosmetin (6), diosmetin 7-O-beta-D-glucopyranoside (7), quercetin-3-O-beta-D-glucopyranoside (8), ursolic acid (9), ursolic aldehyde (10) and rubifolic acid (11).

CONCLUSIONCompounds 1-5 and 9-11 were isolated from this genus for the first time.

Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Galium ; chemistry

Objective:To conduct a glucagon like peptide-1(GLP-1)controllability model rat by chemical genetics, and observe the impact of GLP-1 neuron excitability on appetite.Methods:Fifteen rats were evenly divided into Green fluorescent protein(GFP)group, HM3D group, and HM4D group. Various combinations of adeno-associated virus(rAAV)were injected into the nucleus tractus solitarius(NTS). rAAV-GLP-1-cre and rAAV-GFP-dio were administered in rats of GFP group. The rats of HM3D group were injected with rAAV-GLP-1-cre and rAAV-HM3D-mCherry-dio while rAAV-GLP-1-cre and rAAV-HM4D-mCherry-dio were injected in rats of HM4D group . The optimal dose of clozapine N-oxide(CNO)was selected based on feeding behavior and body weight changes of rats after intraperitoneal injection of different doses of CNO. The controllability of GLP-1 neurons was confirmed by comparing with intraperitoneal injection of saline. The number of activated GLP-1 neurons in the NTS area and the expression of POMC neurons in the hypothalamus were detected 30 minutes after CNO injection.Results:GLP-1 neurons in the NTS area of rats were successfully labeled. The rat of HM3D group revealed a decrease in food intake( P=0.021)while the rat of HM4D group showed an increase( P=0.002), when given 1 mg/kg of CNO, no changes at the dose of 0.5 mg/kg and 3.0 mg/kg. Immunofluorescence showed that the activity of GLP-1 neurons in NTS of GFP group was lower than that of HM3D group( P=0.022), and higher compared with that of the HM4D group( P=0.049). The expression of GLP-1 neurons in NTS and POMC neurons in the hypothalamus of the HM3D group after intraperitoneal injection of CNO was also higher than that in the HM4D group( P=0.003). Conclusion:Using chemical genetics technology, GLP-1 controllability model rat could be successfully established via injecting varying combinations of rAAV into the NTS area of rat. Injection of 1 mg/kg CNO can effectively activate or inhibit the neuron to regulate appetite.

Abstract: Flubromazolam (Flub) is a novel psychoactive substance of benzodiazepines and the mechanism underlying its addiction still remains elusive. This study investigated the reward effect of Flub using conditioned place preference (CPP) mouse model. The neuronal activity was evaluated by c-Fos expression, and the neural circuit was tracked by virus tracing. This study also investigated the regulatory effect of neural circuits on Flub-induced reward effects through chemogenetic approach. The results showed that, at the dose of 3 mg/kg, Flub significantly increased CPP score and c-Fos expression in dopaminergic (DA) neurons of ventral tegmental area (VTA). Inhibition of VTA dopaminergic neuron activity dramatically decreased Flub-induced CPP score. Virus tracing verified GABAergic neuronal projection of medial rostrum tegmental nucleus (RMTg) to VTA dopaminergic neurons. Activation of RMTgGABA→VTADA circuit or blockade of benzodiazepine receptors (BZR) in RMTg significantly decreased Flub-induced CPP score. These results indicate that Flub produced reward effect via BZR-mediated RMTgGABA→VTADA circuit.

Objective:To investigate the effect of electroacupuncture (EA) on synaptic plasticity and the expression of autophagy-related proteins in the hippocampus in Alzheimer′s disease (AD). To explore how EA might improve cognition in AD.Methods:Healthy male Sprague-Dawley rats were randomly divided into a sham operation group, a model group and an EA group. The rat model of AD was established by injecting Aβ1-42 into the bilateral CA1 area of the hippocampus. The sham operation group was injected with an equal amount of normal saline at the same site. Starting the day after the successful modelling, the EA group received 20 minutes of EA treatment at the Baihui (DU20) and bilateral Shenshu (BL23) acupoints once a day, 6 times a week for 2 weeks. The rats′ learning and memory were then tested using a Morris water maze. The long-term potentiation (LEP) in the hippocampus was assessed using a MED64 microelectrode array and any ultrastructural changes of autophagosomes were detected using an electron microscope. The expression of the autophagy-related proteins Beclin-1 and microtubule associated protein light chain 3 (LC3) in the hippocampus were determined using western blotting.Results:The escape latency was significantly shorter and the times crossing the platform increased significantly in the EA group compared with the model group. The average amplitude of the postsynaptic excitatory field potentials in the EA group was significantly higher than among the model group. There were many autophagosomes in the hippocampal neurons of the model group, significantly more than in the EA group. The LC3II/LC3I ratio and Beclin-1 protein expression decreased significantly in the EA group compared to the model group.Conclusions:EA can improve learning and memory and restore LEP in the hippocampus of rats modeling AD. The mechanism may be related to its regulation of autophagy in hippocampal neurons.

Objective:To establish an animal model of chronic systemic inflammation with long-term high expression of circulating IL-6 by introducing exogenous IL-6 gene transfer vector.Methods:Recombinant murine IL-6-encoding adeno-associated virus (AAV-IL-6) was constructed. Twenty-one 24-week-old male C57BL/6J mice were randomly divided into three groups with seven in each group: AAV-IL-6 group, vector control (AAV-ctrl) group and blank control group. At 0, 8 and 16 weeks of intervention, the mice in the three groups were injected with AAV-IL-6 (100 μl 0.5×10 10 vp/ml), unloaded AAV (100 μl 0.5×10 10 vp/ml) and the same volume of saline in the tail vein, respectively. IL-6 levels in mouse serum were measured by ELISA. The general condition of mice was observed and blood routine tests were performed. Changes in blood biochemical parameters and C-reactive protein (CRP) levels were detected. At the end of 24-week intervention, the mice were sacrificed and the myocardium, liver, spleen, quadriceps femoris, knee joint and middle femur were taken for HE staining. Results:At 4, 8, 16 and 24 weeks after intervention, serum IL-6 levels were (75.41-169.28) pg/ml in the AAV-IL-6 group, while in the two control groups, the levels were below the lower limit of detection (7.8 pg/ml). At 24 weeks after intervention, the body weight of mice in the AAV-IL-6 group was significantly lower than that of mice in the two control groups; the neutrophil counts and CRP level in the AAV-IL-6 group were higher than those in the two control groups, while the levels of albumin, creatinine, triglyceride and cholesterol were lower than those in the two control groups. There were no differences in the aforementioned parameters between the two control groups. Compared with the blank control group, both AAV-IL-6 and AAV-ctrl groups showed increased lymphocyte counts. All mice had normal liver and kidney functions at the end of intervention. Histopathological findings indicated that the mice in the AAV-IL-6 group had focal infiltration of lymphocytes in the central venous area of the liver and around the myocardial and the skeletal muscle fibers, diffuse infiltration of multinucleated giant cells in the spleen, atrophic skeletal muscle, disorganized growth plate, reduced chondrocyte hypertrophic zone, thinner bone cortex and trabecular, and reduced osteoid. There were no histopathological changes in mice of the two control groups.Conclusions:Repeated tail vein injection of AAV-IL-6 could achieve long-term high expression of circulating IL-6 in mice, which manifested the phenotype of chronic systemic inflammation in preliminary detection and provided a safe, effective and simply accessible animal model for related studies.

BACKGROUND: Glutamine synthetase (GS) and arginase 1 (Arg1) are widely used pathological markers that discriminate hepatocellular carcinoma (HCC) from intrahepatic cholangiocarcinoma; however, their clinical significance in HCC remains unclear. METHODS: We retrospectively analyzed 431 HCC patients: 251 received hepatectomy alone, and the other 180 received sorafenib as adjuvant treatment after hepatectomy. Expression of GS and Arg1 in tumor specimens was evaluated using immunostaining. mRNA sequencing and immunostaining to detect progenitor markers (cytokeratin 19 [CK19] and epithelial cell adhesion molecule [EpCAM]) and mutant TP53 were also conducted. RESULTS: Up to 72.4% (312/431) of HCC tumors were GS positive (GS+). Of the patients receiving hepatectomy alone, GS negative (GS-) patients had significantly better overall survival (OS) and recurrence-free survival (RFS) than GS+ patients; negative expression of Arg1, which is exclusively expressed in GS- hepatocytes in the healthy liver, had a negative effect on prognosis. Of the patients with a high risk of recurrence who received additional sorafenib treatment, GS- patients tended to have better RFS than GS+ patients, regardless of the expression status of Arg1. GS+ HCC tumors exhibit many features of the established proliferation molecular stratification subtype, including poor differentiation, high alpha-fetoprotein levels, increased progenitor tumor cells, TP53 mutation, and upregulation of multiple tumor-related signaling pathways. CONCLUSIONS GS- HCC patients have a better prognosis and are more likely to benefit from sorafenib treatment after hepatectomy. Immunostaining of GS may provide a simple and applicable approach for HCC molecular stratification to predict prognosis and guide targeted therapy.
Humans ; Carcinoma, Hepatocellular/metabolism* ; Sorafenib/therapeutic use* ; Liver Neoplasms/metabolism* ; Glutamate-Ammonia Ligase/metabolism* ; Hepatectomy ; Retrospective Studies ; Prognosis ; Neoplasm Recurrence, Local/surgery*