Role of GDC-0449 in a rat model of liver fibrosis induced by carbon tetrachloride combined with 2-acetylaminofluorene / 临床肝胆病杂志

Objective To investigate the intervention effect of GDC-0449, a hedgehog signaling pathway inhibitor, on rats with liver fibrosis induced by carbon tetrachloride (CCl 4 ) combined with 2-acetylaminofluorene (2-AAF). Methods A total of 18 female Fisher344 rats were randomly divided into normal group, CCl 4 /2-AAF group, and GDC-0449 group, with 6 rats in each group. The rats in the CCl 4 /2-AAF group and the GDC-0449 group were given subcutaneously injected 30% CCl 4 -olive oil solution at a dose of 2 mL/kg twice a week for 6 weeks to induce liver fibrosis; since week 7, in addition to the injection of CCl 4 -olive oil solution, the rats in these two groups were given 2-AAF (100 mg/kg/d) by gavage, and the rats in the GDC-0449 group were given GDC-0449 (25 mg/kg/d) by gavage, while those in the normal group were given an equal volume of olive oil solution by injection and normal saline by gavage. All rats were sacrificed at the end of week 9, and related samples were collected. HE staining and sirius red (SR) staining were used to observe the changes in liver histopathology and collagen deposition, and the semi-quantitative analysis of SR-positive area and Ishak score were used to evaluate fibrosis degree; the alkaline hydrolysis method was used to measure the level of hydroxyproline (Hyp) in liver tissue; immunohistochemistry, Western blot, and qRT-PCR were used to measure the expression of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col-Ⅰ), type Ⅳ collagen (Col-Ⅳ), cytokeratin 19 (CK19), cytokeratin 7 (CK7), the epithelial cell adhesion molecule Epcam, and the hedgehog signaling pathway in liver tissue; double immunofluorescence staining was used to observe the colocalization of CK19 and the oval cell marker OV6. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the normal group, the CCl 4 /2-AAF group had marked inflammatory cell aggregation and collagen deposition in liver tissue, with the formation of a pseudolobular structure, as well as significant increases in Hyp level and collagen positive area ratio in liver tissue ( P < 0.05), Ishak score ( P < 0.05), and the expression of α-SMA, Col-Ⅰ, Col-Ⅳ, Epcam, CK19, CK7, the transmembrane transporter Smoothened (Smo), Hedgehog ligand Desert Hedgehog (Dhh), the Indian Hedgehog membrane-binding receptor Patched (Ptch2), and glioma-related oncogenes Gli1, Gli2, and Gli3 (all P < 0.05); double immunofluorescence staining showed that CK19-positive cells also expressed OV6 in the liver tissue of rats in the CCl 4 /2-AAF group, with a significant increase compared with the normal group. Compared with the CCl 4 /2-AAF group, the GDC-0449 group had significant reductions in inflammatory cell aggregation and collagen deposition in liver tissue, Hyp level and collagen positive area ratio in liver tissue ( P < 0.05), Ishak score ( P < 0.05), and the expression of α-SMA, Epcam, CK19, CK7, Smo, Ptch2, Gli1, Gli2, and Gli3 (all P < 0.05); double immunofluorescence staining showed a significant reduction in the number of cells with co-expression of OV6 and CK19 in liver tissue. Conclusion The Hedgehog signaling pathway inhibitor GDC-0449 can significantly inhibit the progression of liver fibrosis induced by CCl 4 /2-AAF in rats, possibly by inhibiting hepatic stellate cell activation, collagen deposition, activation and proliferation of hepatic progenitor cells, and differentiation of hepatic progenitor cells into biliary epithelial cells.

Comparison of Changes in the Interstitial Cells of Cajal and Neuronal Nitric Oxide Synthase-positive Neuronal Cells With Aging Between the Ascending and Descending Colon of F344 Rats

BACKGROUND/AIMS: Neuronal degeneration and changes in interstitial cells of Cajal (ICCs) are important mechanisms of age-related constipation. This study aims to compare the distribution of ICCs and neuronal nitric oxide synthase (nNOS) with regard to age-related changes between the ascending colon (AC) and descending colon (DC) in 6-, 31-, and 74-week old and 2-year old male Fischer-344 rats. METHODS: The amount of fecal pellet and the bead expulsion times were measured. Fat proportion in the muscle layer of the colon was analyzed by hematoxylin and eosin staining. Proto-oncogene receptor tyrosine kinase (KIT) and neuronal nitric oxide synthase (nNOS) expression were analyzed with Western blotting and immunohistochemistry. Isovolumetric contractile measurements and electrical field stimulation were used to assess smooth muscle contractility. RESULTS: Colon transit and bead expulsion slowed with senescence. Fat in the muscle layer accumulated with age in the AC, but not in the DC. The proportion of KIT-immunoreactive ICCs in the submucosal and myenteric plexus was higher in the DC than in the AC, and it declined with age, especially in the AC. In contrast, the proportion of NOS-immunoreactive neurons in the myenteric plexus was higher in the AC than in the DC, and both decreased in older rats. Nitric oxide levels declined with age in the DC. Muscle strip experiments showed that the inhibitory response mediated by nitric oxide in the circular direction of the DC was reduced in 2-year old rats. CONCLUSION: The AC and DC differ in their distribution of ICCs and nNOS, and age-related loss of nitrergic neurons more severely affects the DC than the AC.

Establishment of chronic ocular hypertension rat model by transgoniscope laser photocoagulation to trabecular meshwork and its comparison with translimbal laser photocoagulation / 中华实验眼科杂志

Background The establishment of chronic ocular hypertension is a basis for the research of glaucoma.Previous laser photocoagulation method to establish ocular hypertension model showed obvious fluctuation of intraocular pressure (IOP) and complications and need repeatedly photocoagulation.Improvement of modeling method is of important significance for glaucoma.Objective This study was to establish chronic ocular hypertension rat models by transgoniscope laser photocoagulation to trabecular meshwork and to compare this method with previous translimbal laser photocoagulation.Methods Thirty-six 8 to 12-week-old clean grade Fischer344 rats were collected and divided into normal control group,translimbal laser photocoagulation group and transgoniscope laser photocoagulation group,12 rats for each group.Five hundred and thirty-two nm YAG laser was used to photocoagulate trabecular meshwork translimbally in the right eyes of rats in the translimbal laser photocoagulation group,with the laser power 440-500 mW and spots 40-60,and the photocoagulation was perfored transgoniscopely in the right eyes of rats in the transgoniscope laser photocoagulation group,with the laser power 800-850 mW and spots 100-120.IOP was measured by using Tonolab tonometer in all the rats after modeling.The rats were sacrificed 3 weeks after modeling and retinas were isolated,the Tuj-1 positive retinal ganglion cells (RGCs) were counted by immunofluorescence technology.The use and care of the animals followed the Statement of ARVO.Results The successful rate of establishement of models was 75％ in the translimbal laser photocoagulation group and 100％ in the transgoniscope laser photocoagulation group.The mean IOP was (11.0±1.3),(23.4±12.6) and (25.3± 4.9) mmHg,and the peak IOP was (12.3 ± 1.0),(50.5 ± 7.3) and (44.3 ± 12.3) mmHg in the normal control group,transgoniscope laser photocoagulation group and translimbal laser photocoagulation group,respectively,with significant differences among the groups (F=25.496,80.762,both at P＜0.001),and the mean IOP was significantly higher in the transgoniscope laser photocoagulation group and translimbal laser photocoagulation group than that in the normal control group (all at P＜0.001),and no significant differences in the mean and peak IOP between transgoniscope laser photocoagulation group and translimbal laser photocoagulation group (P=1.000,P=0.195).The numbers of Tuj-1 positive RGCs in the retinas were (2 048.2± 148.5),(645.2 ± 177.1) and (1 223.7 ± 148.6)/mm2 in the normal control group,transgoniscope laser photocoagulation group and translimbal laser photocoagulation group,showing a significant difference among the groups (F=98.767,P＜0.001).The number of Tuj-1 positive RGCs was considerably reduced in the transgoniscope laser photocoagulation group and translimbal laser photocoagulation group compared with the normal control group and the number of Tuj-1 postive RGCs was low in the translimbal laser photocoagulation group compared with the transgoniscope laser photocoagulation group (all at P＜0.001).Conclusions Transgoniscope laser photocoagulation targeting trabecular meshwork can induce chronic ocular hypertension and RGCs losing.However,its pattern is different from translimbal laser photocoagulation.Transgoniscope laser photocoagulation has a higher successful rate of chronic ocular hypertention than that of translimbal laser photocoagulation.

Analysis of Gene Expression through RNA Sequencing in Prefrontal Cortex of Nicotine-Treated Rats / 天津医药

Objective To investigate the effects of nicotine on the gene expression profile in prefrontal cortex (PFC) region of rats. Methods Twenty-four adult male F344 rats were randomly divided into treatment group and control group, with 12 animals in each group. Animals in treatment group were injected with nicotine solution while those in control group were given physiological saline for 17 days. At the end of drug administration, the tissue of PFC region was sliced from the brain of each animal. RNA sequencing (RNA-Seq) was utilized to analyze the gene expression profile in PFC of each animal. Bioinformatics tools including Bowtie, Tophat and Cufflinks were used to map the sequencing reads to the reference genome of rat, and to measure the relative expression level of each transcript. Genes whose expression levels were significantly differ-ent between the two groups were identified. The functional categories and the biochemical pathways associated with these genes were further analyzed. Results By comparing the expression level of each transcript between two groups, 885 signifi-cantly differentially expressed genes were identified. These genes were enriched in multiple Gene Ontology Biological Pro-cess categories (e.g., G protein coupled receptor protein signaling pathway, protein ubiquitination, and neurotransmitter up-take) and biological pathways (e.g., insulin signaling pathway, Alzheimer's disease, and long term potentiation). Conclu-sion Nicotine treatment may regulate signaling transduction, energy metabolism and other biological processes in PFC of rat brain.

Effect of chronic cortisone use in rats strain susceptible to ligature-induced periodontitis / Efeito do uso crônico da cortisona em ratas de linhagem susceptível à periodontite induzida por ligadura

Objective: The present study assessed the effect of prolonged betamethasone use in ligature-induced periodontitis in adult Fischer-344 rats. Methods: Thirty-six Fisher rats were randomly assigned to three groups: group B (betamethasone); group S (sham) and group C (control). The animals in group B were given intramuscular betamethasone daily, those in group S were given saline injections daily, and those in group C were not submitted to any intervention. The interventions lasted 60 days, and all groups were submitted to the same environmental conditions. Tendays after starting the injections, periodontal disease was induced in the animals from groups B and S by tying a sterile silk suture threadaround the upper right second molar. Fifty days later, all animals were sacrificed and the connective tissue attachment level (Jac-E) and boneloss (Jac-O) were measured. Results: Jac-E and Jac-O of groups B and S were not significantly different, but they differed significantly from those of group C. Conclusion: Prolonged use of betamethasone did not affect the progression of induced periodontitis in rats.

Objetivo: Avaliar o efeito do uso prolongado da betametasona na periodontite induzida por ligadura, em ratas adultas da linhagem Fischer-344. Métodos: Selecionou-se trinta e seis ratas Fisher, as quais foram divididas aleatoriamente em três grupos: grupo B (betametasona); grupo S (soro) e grupo C (controle). Os animais do grupo B receberam injeções diárias intramuscular de betametasona, o grupo S recebeu injeções diárias de soro fisiológico e o grupo C foi mantido sem nenhuma intervenção. Estes procedimentos duraram 60 dias. Os três grupos foram mantidos nas mesmas condições ambientais. Decorridos dez dias a partir do início das injeções, os animais dos grupos B e S foram submetidos a indução da doença periodontal, através da colocação do fio de seda em volta do segundo molar superior direito. Ao final do período experimental (50 dias) os animais foram submetidos à eutanásia. O nível de inserção histológica (Jac-E) e perda óssea histológica (Jac-O) foram mensurados.Resultados: Entre os grupos B e S, não houve diferenças estatisticamente significativas para Jac-E e Jac-O. Entretanto, notaram-se diferenças significativas em ambos os parâmetros avaliados quando o grupo C foi comparado aos grupos B e S. Conclusão: Pode-se concluir que o uso prolongado de betametasona não modificou a progressão de periodontite induzida.

Efeito da ingestão crônica de álcool na progressão de periodontite induzida em ratos da linhagem Fischer-344 / Effect of chronic alcohol ingestion on the progression of periodontitis induced in Fisher-344 rats

Objetivo: Compreender o efeito do uso crônico de álcool na progressão de periodontite induzida em ratos da linhagem Fischer-344. Métodos: Para o estudo utilizaram-se 22 ratos da linhagem Fischer-344, com dois meses de idade, divididos nos grupos: álcool (n=8), ligadura (n=7) e controle (n=7). No primeiro dia, expuseram-se os animais do grupo álcool à ingestão de solução de água com álcool a 20% (volume/volume), até o dia 90. Decorridos trinta dias do início do experimento, os animais do grupo álcool e do grupo ligadura submeteram-se à colocação de fio de seda em volta do segundo molar superior direito. Nada foi realizado no lado esquerdo, servindo como controle. Todos os grupos submeteram-se à eutanásia, após 60 dias da colocação da ligadura. Para avaliar a destruição da periodontite, usou-se o exame radiográfico, mensurando a destruição da altura óssea. Resultados: Os resultados do estudo evidenciaram que, no lado em que foi induzida a periodontite, o grupo que ingeriu álcool sofreu um aumento de destruição, demonstrando diferenças estatísticas (p < 0,05), se comparado ao grupo ligadura e ao controle. E, maior destruição óssea no grupo ligadura quando comparado ao controle (p < 0,05). Conclusão: Conclui-se, dentro das limitações do estudo, que o consumo crônico de álcool por ratos da linhagem Fischer-344 induziu a uma maior progressão de periodontite induzida.

Objective: Understand the effect of chronic alcohol on the progression of periodontitis induced in Fischer-344 rats. Methods: For the study, 22 Fischer-344 rats, two months old were used, divided into groups: alcohol (n=8), ligature (n=7) and control (n=7). On the first day, the animals in the alcohol group were exposed to ingestion of a water solution containing 20% alcohol (size/size), up to day 90. After thirty days from the beginning of the experiment, the animals in the alcohol group and the ligature group were submitted to the placement of a silk thread around the right maxillary second molar. Nothing was performed on the left side, serving as control. All the groups were submitted to euthanasia 60 days after ligature placement. To assess the destruction of periodontitis, a radiographic exam was used to measure the destruction of bone height. Results: The results of the study showed that on the side in which periodontitis was induced, the group that ingested alcohol suffered an increase in destruction, with statistical differences when compared with the ligature and control groups and increased bone destruction in the ligature group when compared to control. Conclusion: Within the limitations of the study, it was concluded that chronic alcohol consumption by Fischer-344 rats led to greater progression of induced periodontitis.

Rejection of small bowel transplantation in inbred rats / 中国普通外科杂志

Objective Dynamical observation the development law of rejection of small intestinal transplantation(SIT) in inbred rats BN to F334,to study the value of this SIT madel in the rsesarch of rejection. Methods Heterotopic SIT models were established by microsurgical technique according to modified monchik method in inbred strain F344/RT1 l andBN/RT1 n,including isotransplantation (F344→F344, n =8 ,ITx group) and allotransplantation (BN→F344, n =8, ATx group). Results (1) The mean survival time was over 30 days in AT X group, 12 days in IT X group(P