Study on improvement mechanism of caudatin on liver injury in rats / 中国药房

OBJECTIVE To investigate the improvement mechanism of caudatin on liver injury of rats. METHODS SD rats were randomly divided into blank group， model group， caudatin low-dose and high-dose groups （25， 50 mg/kg）， with 6 rats in each group. Diethylnitrosamine （DEN） was injected intraperitoneally three times per week for eight weeks to establish liver injury model of rats. At 5th week of modeling， the rats received relevant medicine or 0.5% sodium carboxymethylcellulose intragastrically for 4 weeks. The levels of liver function indexes [alanine transaminase （ALT）， aspartate transaminase （AST）， total protein （TP） and total bilirubin （TBI）] and inflammatory factors [interleukin （IL-6）， tumor necrosis factor α （TNF-α）， IL-1β] in serum were detected； the histopathological morphological changes of rat liver were observed； the positive protein expressions of nuclear factor κB （NF-κB） and 78 kDa glucose regulatory protein （Grp78） in liver tissue were also determined； the expressions of endoplasmic reticulum stress-related protein Grp78， C/EBP homologous protein （CHOP）， activating transcription factor 6 （ATF6） and inositol requiring enzyme 1α （IRE1α） and the level of protein kinase R-like endoplasmic reticulum kinase robertluoyi@126.com （PERK） in liver tissue were detected. RESULTS Compared with blank group， serum levels of ALT， AST， TBI， IL-6， TNF-α and IL-1β and positive expressions of NF-κB and Grp78 in liver tissue as well as protein expressions of Grp78， CHOP， ATF6 and IRE1α， PERK protein phosphorylation level were all increased significantly in model group （P＜0.05）， while the serum level of TP was decreased significantly （P＜0.05）. The disordered structure of liver lobule， swollen liver cells， unclear intercellular boundary were observed and accompanied by inflammatory cell infiltration. Compared with model group， most of the above indexes were significantly reversed in caudatin groups （P＜0.05）； the structure of hepatic lobule was relatively complete and clear， the cells were arranged orderly， and the infiltration of inflammatory cells was also reduced. CONCLUSIONS Caudatin has a significant improvement effect against DEN-induced liver injury in rats， the mechanism of which may be associated with inhibiting endoplasmic reticulum stress and inflammatory reaction.

Resveratrol Inhibits DEN-induced Malignant Proliferation of Rat Hepatocellular Precancerous Stage Through Reprogramming of Glucose Metabolism / 中国生物化学与分子生物学报

Resveratrol (RES) can inhibit the growth and proliferation of liver cancer cells. However, its role in the precancerous stage is still unclear. This paper aims to study the effect and mechanism of RES on the precancerous stage of liver cancer in rats induced by diethylinitrosamine (DEN). SD rats were divided into normal control group, RES treatment group, DEN treatment group and RES-DEN treatment group. The results showed that after the rats were treated with DEN for 8 weeks, the total expression level of proliferating cell nuclear antigen (PCNA) of hepatocytes increased to 2-fold (P<0.05), and the expression level of PCNA protein in the nucleus increased to 3-fold (P<0.001). However, the expression levels of total PCNA (P<0.05) and nuclear PCNA protein (P<0.001) in hepatocytes of rats treated with RES-DEN decreased, suggesting that RES could significantly inhibit the liver malignant proliferation of cells. Through non-targeted metabolomics and KEGG metabolic pathway enrichment analysis, the results showed that the level of glycolysis did not increase significantly in the hepatocytes of RES-DEN-treated rats, although the transition from the pentose phosphate pathway to the glycolysis pathway was enhanced when compared with the DEN group rats. This finding suggested that the metabolic pathway of phosphoenolpyruvate-pyruvate-lactate was inhibited. Further verification found that the protein expression levels of key enzymes M2-type pyruvate kinase (PKM2) and lactate dehydrogenase (LDHA) in this metabolic pathway were inhibited (P<0.05). RES can reprogram glucose metabolism and inhibit DEN-induced excessive proliferation of rat hepatocytes in the precancerous stage of liver cancer, providing an experimental basis for RES to prevent liver cancer.

Aqueous extracts from / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Hepatocellular carcinoma (HCC), which is the most frequent primary liver malignancy, is ranked as the sixth most common cancer and the third leading cause of cancer-related deaths worldwide, with its incidence expected to continue rising. One of the reasons is that most patients are diagnosed at an advanced stage when therapeutic options are ineffective. The development of HCC is attributed to a chronic exposition to either one or a combination of low amounts of different hepatotoxins, such as in hepatitis virus infection, alcohol consumption, aflatoxin from contaminated foods, metabolic factors, and exposure to chemical carcinogens from tobacco smoke (Forner et al., 2018). Integrative studies combining exome sequencing, transcriptome analysis, and the genomic characterization of HCC have shown that these etiological factors may raise the frequency of particular genetic alterations, resulting in intra-tumor heterogeneity that presents a huge challenge for treatment. For example, mutations in the catenin β-1 (CTNNB1) gene (a proto-oncogene in the WNT signaling pathway that encodes the β‍-catenin transcription factor) are strongly associated with alcohol-related HCC, whereas mutations in the telomerase reverse transcriptase (TERT) promoter and tumor protein p53 (TP53) genes are the most commonly observed in hepatitis B virus (HBV)‍-associated HCC (Calderaro et al., 2017; Cancer Genome Atlas Research Network, 2017). The above findings emphasize the molecular diversity of HCC and the associations of different etiologies with distinct mechanisms in HCC progression. Consequently, prevention strategies are still attractive for HCC management.

LIX1-like protein promotes liver cancer progression

Limb and CNS expressed 1 like (LIX1L) is over-expressed in several types of tumors. However, the function of LIX1L in glucose metabolism and hepatocellular carcinoma (HCC) progression remains elusive. Here we report that LIX1L is over-expressed in human HCC tissues, which predicts unfavorable prognosis. LIX1L deficiency

Inhibitory effect of pills against diethylnitrosamine-induced hepatocarcinogenesis in rats / 南方医科大学学报

OBJECTIVE@#To study the inhibitory effect of pills (BJJ) agaisnt diethylnitrosamine (DEN)-induced hepatocarcinogenesis and explore the relation between this effect and the inflammasome signaling pathway.@*METHODS@#Sixty-five male SD rats were randomly divided into control group, DEN model group, and 3 BJJ treatment groups at low, medium and high dose (with daily dose of 0.55, 1.1 and 2.2 g/kg, respectively, for 12 consecutive weeks starting from the 5th week after modeling). The pathological changes of the liver tissue were observed with HE and Masson staining, and serum levels of alanine transaminase (ALT), glutamic oxaloacetic transaminase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) of the rats were detected using ELISA. Oxidation stress in the liver tissue was assessed with ELISA, and Western blotting and ELISA were used to detect the molecular expressions of inflammasome-related pathway.@*RESULTS@#BJJ significantly inhibited tumor growth in the liver of the rats. HE and Masson staining showed that BJJ treatment obviously ameliorated liver fibrosis and reduced cancer cell and inflammatory cell infiltration in the liver. BJJ significantly reduced elevations of serum ALT, AST, ALP and TBIL levels, increased the contents of superoxide dismutase, catalase and glutathione peroxidase in the liver and suppressed malondialdehyde in Den-treated rats. BJJ also dose-dependently decreased the expressions of NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, pro-IL-1β, pro-IL-18, IL-1β and IL-18 in the liver of Den-treated rats.@*CONCLUSIONS@#BJJ treatment can dose-dependently inhibit DEN-induced hepatocarcinogenesis by enhancing antioxidant capacity and down-regulating inflammatory-related pathways in rats.

In vivo Antifibrotic Potential of Extracts of Acanthospermum hispidum DC. Evaluated in Wistar Rats Using Diethylnitrosamine

Aims: Liver fibrosis is a chronic disease of the liver. This disease is a stage of passage to liver cancer. The objective of this work was to evaluate the ability of the ethanolic extract of Acanthospermum hispidumto block the progression of hepatic fibrosis induced in rats using diethylenitrosamine (DEN).Study Design:Study of the antifibrotic potential of extracts of Acanthospermum hispidum.Place and Duration of Study:In vivotests were performed from September 2018 to January 2019. The animal model tests were carried out in the pet shop of the Institute for Health Sciences Research (IRSS) of Burkina Faso and in the Cytogenetics Laboratory (FSS/ISBA) of the Republic of Benin.Methodology:The evaluationof the antifibrotic activity consisted in treating in wistarrats a liver fibrosis induced with the DEN which is a chemical agent whose effect on the liver has already been confirmed. As a result of the treatment, all animals were removed from the liver and blood. The livers were used for macroscopic and microscopic observations. Blood has been used for the evaluation of biochemical parameters in relation to fibrosis.Results:The analysis of the results of the biochemical parameters in relation to the fibrosis showed that the ethanolic extract of Acanthospermum hispidumat the dose of 250 mg / kg made it possible to obtain an improvement of these parameters compared to the other batches of animals. These results have been confirmed by those of the anatomo-pathological studies.Conclusion:The results of biochemical and histological analyzes revealed a capacity of Acanthospermum hispidumextracts to block the evolution of hepatic fibrosis in the rat. These results confirm the hepatoprotective potential of this medicinal plant used in traditional medicine in Burkina Faso

Issues on carcinogen contaminated antihypertensive drugs and constructing drug safety management system

European Medicines Agency withdrew valsartan from European market in July 2018 because it was contaminated with carcinogen, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA). Medicines and Healthcare Products Regulatory Agency also found the same contamination and withdrew it from England market. US Food and Drug Administration followed the action after confirming its contamination. Ministry of Food and Drug Safety (MFDS) conducted testing all the valsartans at Korean market and withdrew some of them from market after confirming the contamination with NDMA. MFDS provided the pharmaceutical companies and laboratory institutions with the manual for testing both NDMA and NDEA and educated relevant personnels. MFDS also evaluated the health impact of the contaminated valsartan on the hypertensive patients who took the valsartan, which was shown to be very low risk of additional cancer incidence. MFDS pronounced strengthening of the safety management for the raw materials of the medicines. For guaranteeing the safety of medicines, more comprehensive drug safety management system from developing new drugs to consuming the medicines should be established. For achieving such a goal, active participation of all the stakeholders of the medicines including governmental agencies including MFDS and Ministry of Health and Welfare, the National Assembly, healthcare professionals, pharmaceutical companies, mass media, and general population including patients should be needed.

Inhibitory effect of fibroblast growth factor-21 on the carcinogenesis of L02 cells induced by diethylnitrosamine / 临床肝胆病杂志

ObjectiveTo investigate the inhibitory effect of fibroblast growth factor-21 (FGF-21) on the carcinogenesis of L02 cells induced by diethylnitrosamine (DEN).Methods L02 cells were cultured and treated with different concentrations of DEN (1,10,20,50,100,and 150 μmol/L).MTT assay was used to measure the influence of DEN on the viability of L02 cells,and an appropriate stimulation concentration of DEN (20 μmol/L) was selected for further study.The malondialdehyde (MDA) and superoxide dismutase (SOD) detection kits were used to measure the levels of MDA and SOD in L02 cells treated by DEN (20μmol/L) and normal L02 cells.Then L02 cells were divided into model control group (treated with 20μmol/L DEN and PBS),low-dose FGF-21 group (20 μmol/L DEN + 1 μmol/L FGF-21),and high-dose FGF-21 group (20 μmol/L DEN +2 μmol/L FGF-21).The levels of MDA and SOD were measured after 12 hours of cell culture.Real-time PCR and Western blot were used to measure the expression of βKlotho (KLB),and Western blot was used to measure the level of phosphorylated protein kinase B (p-AKT).The t-test was used for comparison of continuous data between two groups;an analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results There was a significant increase in the level of MDA and a significant reduction in the level of SOD after L02 cells were treated with DEN (t =9.336 and 16.281,P =0.011 and 0.004).Compared with the model control group,the low-and high-dose FGF-21 groups had a significant reduction in the level of MDA and a significant increase in the level of SOD (P ＜ 0.05),and compared with the low-dose FGF-21 group,the high-dose FGF-21 group had a significantly lower level of MDA and a significantly higher level of SOD (P =0.030 and 0.042),and there was a significant difference between two groups.The high-and low-dose FGF-21 groups had significantly higher mRNA expression of KLB than the model control group (P ＜ 0.001),and the high-dose FGF-21 group had significantly higher mRNA expression of KLB than the low-dose FGF-21 group (P ＜ 0.001),and there was a significant difference between two groups;the protein expression of KLB showed the same trend.The model control group had a significantly higher level of p-AKT than the other two groups (P ＜0.05),and the high-dose FGF-21 group had a significantly lower level of p-AKT than the low-dose FGF-21 group (P ＜ 0.05).Conclusion DEN can increase oxidative stress in L02 cells.By upregulating the expression of KLB,FGF-21 can reduce the level of p-AKT,inhibit oxidative stress in L02 cells induced by DEN,and thus inhibit the development of hepatocellular carcinoma.

Ethanol extract of Phellinus merrillii protects against diethylnitrosamine- and 2-acetylaminofluorene-induced hepatocarcinogenesis in rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study whether the ethanol extract of Phellinus merrillii (EPM) has chemopreventive potential against liver carcinogenesis.</p><p><b>METHODS</b>Thirty male Spraque-Dawley rats were randomly divided into control group, EPM control group, hepatocarcinoma control group, low-dose EPM group and high-dose EPM group, 6 in each group. Using the Solt and Farber protocol in a rat model of hepatocarcinogenesis, the chemopreventive effect of EPM on diethylnitrosamine (DEN)-initiated, 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH)-promoted liver carcinogenesis in rats was evaluated. Basic pathophysiological and histological examinations, together with the serum levels of glutamic oxaloacetic transaminase (sGOT), glutamic pyruvic transaminase (sGPT) and gamma-glutamyl transpeptidase (γ-GT) were measured.</p><p><b>RESULTS</b>Treatment of EPM at the concentration of 2 g/kg body weight in the diet for 8 weeks clearly prevented the development of carcinogenesis and reduced the levels of sGOT, sGPT, and serum γ-GT of rats as compared with the hepatocarcinoma control group (P<0.05 or P<0.01). These phenotypes were accompanied by a significant increase in natural killer cell activity.</p><p><b>CONCLUSION</b>EPM showed a strong liver preventive effect against DEN+2-AAF+PH-induced hepatocarcinogenesis in a rat model.</p>

Effect of different formula diets on mouse model with DEN-induced hepatocellular carcinoma / 中国实验动物学报

Objective To establish a mouse model of diethylnitrosamine(DEN)-induced hepatocellular carcinoma (HCC),and to explore the effects of two different diet formulas on the establishment of DEN-induced HCC model.Methods SPF C57BL/6 mice (8 males and 8 females) were injected intraperitoneally with 25 mg/kg DEN at day 14 to establish a HCC model.The mice were divided into two groups after weaning.One group was fed with the SPF class rodents cereal-based diet,another group was fed with AIN-93G formula diet.The mice were sacrificed at the age of 9 months.The livers were weighed and the growth of liver cancer was observed and recorded.Results All the mice in the cereal-based diet group developed HCC as expected.The body weight and liver mass of the mice in the AIN-93G diet group were significantly lower than that of the cereal-based diet group.The incidence of HCC,and the number and size of tumor nodules were also significantly lower in the AIN-93G diet group than that in the cereal-based diet group.Conclusions DEN-induced HCC model has been successfully established in mice fed with cereal-based diet,while mice fed with AIN93-G diet prevented the development of DEN-induced HCC,and their body weight was decreased significantly,suggesting that dietary factors play a key role in establishment of animal disease models.

Protective Effect of Boiled and Freeze-dried Mature Silkworm Larval Powder Against Diethylnitrosamine-induced Hepatotoxicity in Mice

BACKGROUND: Hepatocellular carcinoma (HCC) is a representative inflammation-associated cancer and known to be the most frequent tumors. HCC may also induce important pro- and anti-tumor immune reactions. However, the underlying mechanisms are unsatisfactorily identified. We investigated the protective effect of boiled and freeze-dried mature silkworm larval powder (BMSP) on diethylnitrosamine (DEN)-induced hepatotoxicity in mice. METHODS: Mice were fed with diet containing BMSP (0.1, 1, and 10 g/kg) for two weeks and DEN (100 mg/kg, intraperitoneally) was injected 18 hours before the end of this experiment. Liver toxicity was determined in serum and histopathological examination was assessed in the liver tissues. Infiltration of immune cells and expressions of inflammatory cytokines and chemokines were also examined. RESULTS: Pretreatment with BMSP reduced necrotic and histopathological changes induced by DEN in the liver. Measurement of serum biochemical indicators, the levels of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase, showed that pretreatment with BMSP also decreased DEN-induced hepatotoxicity. In addition, BMSP inhibited the macrophage and CD31 infiltration in a dose-dependent manner. The expressions of interleukin-1β, IFN-γ and chemokines for T cell activation were decreased in BMSP pretreatment groups. CONCLUSIONS: BMSP may have a protective effect against acute liver injury by inhibiting necrosis and inflammatory response in DEN-treated mice.

Effects on the tumor specific growth factor and tumor necrosis factor α in rats' precancerous lesion of primary hepatocellular carcinoma by direct moxibustion at Ganshu (BL 18) acupoint / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of direct moxibustion at Ganshu (BL18) on the serum concentrations of tumor specific growth factor (TSGF) and tumor necrosis factor α (TNF-α) in a rat model with precancerous lesion of primary hepatocellular carcinoma (HCC), so as to explore the mechanism of moxibustion underlying improvement of HCC.</p><p><b>METHODS</b>Sixty male Wistar rats were randomly divided into control group (n=10), model group (n=20), prevention group 1 (n=15) and prevention group 2 (n=15). The normal rats were injected with physiological saline as blank control. At the same time, the rats of other three groups were injected with diethylnitrosamine to establish the HCC model. Direct moxibustion with grain-sized moxa was applied to bilateral Ganshu acupoint of the rats in the prevention group 1 (1 treatment course, 20 days) and prevention group 2 (2 treatment courses, 40 days), 5 doses for each acupoint, 0.5 mg/dose, once every other day. At each time point (before model establishment, the end of 1st course prevention, the end of 2nd course prevention and the end of model establishment), serum levels of TSGF and TNF-α were detected using enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>Compared with the control group, there was a remarkably increase of serum TSGF and TNF-α contents in the model group at the end of the experiment (P<0.05). At the end of the 1st course of direct moxibustion, the contents of serum TSGF and TNF-α of rats in the prevention group 1 were significantly increased compared with that of the model group (P<0.05). At the end of the 2nd course of direct moxibustion, serum TSGF and TNF-α levels of rats in the model group were higher than the normal group with significantly difference (P<0.05), and the levels of TSGF and TNF-α in the prevention group 2 were significantly reduced in comparison with the model group (P<0.05).</p><p><b>CONCLUSION</b>It was possible that direct moxibustion could inhibit precancerous lesion and postpone hepatocarcinogenesis, and the therapeutic effect of two courses were better than one course.</p>

Relationship between non-alcoholic fatty liver disease and hepatocellular carcinoma / 肿瘤

Objective: To investigate the relationship between non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC), and to explore the role of nuclear factor-kappa B (NF-KB): and interleukin-6 (IL-6): in mechanism of hepatocarcinogenesis. Methods: Mice were divided into vacuity contrast group, normal diet group and high fat diet group. After feeding for 15 weeks, diethylnitrosamine (DEN, 45mg/kg): was weekly administrated by intraperitoneal injection into mice in both normal diet and high fat diet groups to gradually establish a model of HCC. The mice in blank control group were not treated with any drugs through the whole experiment. Livers and serum were individually collected from five mice in normal diet and high fat diet groups, respectively, and three mice in the blank control group in week 15, 20, 25 and 30. All the remaining mice were executed in week 36, and serum and liver pathologic gross were collected. The pathological changes of liver tissues were observed with HE staining. The differences in hepatocarcinogenesis among three groups were also analyzed. The levels of serum triglyceride (TC), cholesterol (TC), alanine aminotransferase (ALT): and aspartate aminotransferase (AST): were detected. The levels of serum NF-KB and IL-6 were assayed by ELISA. Results: Animal models of NAFLD were successfully established after 15 weeks of high-fat diet feeding. In the week 36, no mice in the blank control group developed HCC. Eight of 20 (40.0%): mice in normal diet group developed HCC. Thirteen of 18 (83.3%): mice in high fat diet group developed HCC. The levels of serum NF-KB and IL-6 in high fat diet group were significantly higher than those in the normal diet group in each period during study (all P < 0.01). Conclusion: The results suggest that NAFLD can promote hepatocarcinogenesis in the presence of DEN. The abnormal levels of NF-KB and IL-6 play important roles during the development of HCC.

Inhibition of primary liver cancer by oral javanica oil emulsion / 中国肿瘤临床

Objective: This study aimed to investigate the inhibitory effects of Brucea javanica oil oral emulsion (BJOOE) on primary liver cancer induced by diethylnitrosamine (DEN). Methods:Rats were randomly divided into the control group, model group, and BJOOE group. Rats were given free access to water. DEN was administered intragastrically to induce liver cancer in rats. Five weeks later, rats were intragastrically administered with BJOOE for five times per week. The rats were killed after 14 weeks. Abdominal aortic blood samples were collected. The contents of ALT, AST, ALP, γ-GT, and AFP of serum were detected by an automatic biochemical analyzer. The liver index, spleen index, thymus index, and changes in liver cancer nodules of the surface were observed in rats. Changes in the number of liver cancer nodules of the surface were detected by imaging. Results:Compared with the control group, the liver index, spleen index, and number of nodules of the model group significantly increased, whereas the thymus index significantly decreased (P<0.01). The levels of ALT, AFP, AST, ALP, andγ-GT of serum in the model group were significantly higher than those in the control group (P<0.01). Compared with the model group, BJOOE significantly reduced the liver index, spleen index, and number of cancer nodules, but increased the thymus index in the liver of rats with cancer (P<0.01). The levels of ALT, AFP, AST, ALP, andγ-GT of serum in rats with hepatic carcinoma significantly improved (P<0.01 or P<0.05). Conclusion:BJOOE could inhibit primary liver cancer, and the underlying mechanisms are complex.

Sequential study of liver collagen fibers in diethylnitrosamine-induced hepatocarcinogenesis in rats / 第二军医大学学报

Objective: To observe the sequential changes of collagen fibers in rats with diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC), so as to provide a reference for pathogenesis research of HCC. Methods: Fifty male Wistar rats, weighing 100-120 g, were randomly divided into normal group and HCC model group. The model group was intraperitoneally administered with 50 mg/kg DEN (0.1 mL), twice a week for 4 weeks, then once a week for another 10 weeks. The control group was given normal saline (0.1 mL) in the same manner. Finally the rats were sacrificed; the normal and diseased liver tissues were observed by H-E, Masson and argyrophilic fiber staining. The expression of collagen type I and type III mRNA was detected by fluorogenic quantitative PCR; the expression of matrix metalloproteinase (MMP) was examined by gelatinases spectrometry. Results: Cirrhosiswas found in rats 5 weeks after treatment with DEN and HCC was induced 14 weeks after DEN treatment; collagen deposition in liver tissues increased in a progressive manner, and the collagen contents in the HCC tissues was greatly less than that in the adjacent tissues, showing a decreasing trend. The contents of MMP-2 and MMP-9 in the HCC and adjacent tissues had opposite changes compared with collagen. Conclusion: Collagen deposition in cirrhosis liver tissue is increased during the process of DEN-induced HCC, but it is decreased in HCC tissues in a progressive manner, which indicates that collagen might be degraded during the progression from cirrhosis to HCC.

Expression of CD44 in mouse diethylnitrosamine (DEN)-induced hepatic tumors as well as in DEN-treated embryo stem cells and derived hepatic lineage cells / 동물의과학연구지

To clarify the role of stem cells in hepatocarcinogenesis, CD44 expression was investigated in mouse livers as well as embryonic cell lineages treated with diethylnitrosamine (DEN). Liver tumors induced by DEN were analyzed by immunohistochemisty for CD44. Liver tissues were sampled at 6, 24, and 48 hr after treatment with saline or DEN. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs), and hepatocyte like cells (HCs), representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5, and 15 mM, respectively) for 24 hr, after which CD44 expression levels were examined by relative quantitative real-time PCR. CD44 expression was weakly detected in tumor cells as well as in some hepatocytes surrounding the tumor cells. However, CD44 expression was not detected in liver tissue treated with DEN at early time points. The CD44 mRNA expression level was significantly different among cells treated with 5 mM DEN at day 22 (P<0.01) as well as 1, 5, and 15 mM DEN at day 40 (P<0.01) compared with control. Taken together, CD44 expression slightly increased in mouse DEN-induced tumors. Furthermore, expression of CD44 in embryonic cell lineages treated with various doses of DEN significantly differed among embryo stem cells and derived hepatic lineage cells. This suggests that CD44 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, and its expression may increase in the tumor stage but not during early carcinogenesis.

Differentially expressed proteins in the liver of Gulo-/- mice following treatments with Helicobacter pylori and diethylnitrosamine / 동물의과학연구지

Vitamin C (ascorbic acid) is an essential nutrient of most living tissues. We established a strain of Gulo-/- mice with known deficiency, in which vitamin C intake can be controlled by diet, like humans, and investigated the differentially expressed proteins following treatments with Helicobacter pylori and diethylnitrosamine (DENA) in the liver of Gulo-/- mice using a proteomic approach. Expression of p53, 14-3-3epsilon and 14-3-3delta in Gulo-/- mice liver tissue was analyzed by immunohistochemistry. 2-DE maps constructed from Gulo-/- mice liver and differentially expressed proteins in liver tissue were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/MS). In Gulo-/- mice after H. Pylori infection, followed by treatment with DENA, no differences in p53, 14-3-3epsilon and 14-3-3delta were observed by immunohistochemistry. Proteome analyses using MALDI-TOF/MS resulted in successful identification of 12 proteins (nine proteins were up-regulated and three were down-regulated). Specifically, peroxiredoxin-6 and Alpha-1-antitrypsin 1-4 were up-regulated in liver after H. Pylori infection followed by treatment with DENA. These results indicated that oral supplementation with vitamin C led to rescue of Gulo-/- mice from vitamin deficiency, and protected the liver from H.pylori infection and/or DENA effect, and vitamin C also protected the liver against oxidative stress.

Expressão imunohistoquímica de P53 e Ki-67 na carcinogênese esofágica induzida pela dietilnitrosamina: modelo experimental / Immunohistochemical expression of P53 and Ki-67 in diethylnitrosamine-induced carcinomas esophageal: experimental model

OBJETIVO: Avaliar a expressão imunohistoquímica de p53 e ki-67 na carcinogênese esofágica induzida quimicamente através do uso de dietilnitrosamina, em um grupo de 100 camundongos fêmeas. MÉTODOS: O estudo experimental foi realizado com quatro grupos de animais, onde os grupos I e II foram considerados controles, sendo diferenciados por gavagem esofágica, uma vez semana, com água fria (temperatura ambiente) ou quente (60º-70ºC). E os grupos III e IV foram considerados estudos, os quais receberam dietilnitrosamina por três dias consecutivos semanalmente, também sendo diferenciados por gavagem, uma vez por semana, com água fria ou quente. O estudo apresentou datas progressivas de sacrifícios com coleta de peças esofágicas, que iniciava aos 30 dias de experimento e terminava aos 150 dias. Demonstrou-se que não houve diferença na incidência tumoral quando foi acrescida a variável temperatura da água; provavelmente devido ao episódio único semanal que era adicionado ao animal em experimentação. RESULTADOS: A análise imunohistoquímica do p53 não evidenciou diferença estatística durante a evolução da carcinogênese até 150 dias, porém quando analisado a relação com alterações patológicas demonstra-se que apresenta significância em relação à patologia baixo grau de displasia, alto grau e carcinoma. CONCLUSÃO: A análise imunohistoquímica do ki-67 demonstrou diferença estatística durante a evolução da carcinogênese a partir do dia 120 de experimento e quando analisada a relação com alterações patológicas demonstrou-se que apresenta significância também em relação à lesão intraepitelial de alto grau e carcinoma.

OBJECTIVE:To evaluate the expression of P53 and Ki-67 during esophageal diethylnitrosamine (DEN)-induced carcinogenesis in 100 mice by immunohistochemistry. METHODS: The animals were assigned to 4 groups, receiving water and food ad libitum. Control groups I and II received weekly esophageal gavage with cold (room temperature) or hot (60-70ºC) water, respectively. Experimental groups III and IV were treated with DEN for 3 consecutive days during the week, and one weekly gavage as above. The mice were sacrificed in different periods from day 30 to day 150 after the beginning of the experiment, for collection of esophageal samples which were then submitted to microscopic and immunohistochemical analyses. The temperature of the water administered by gavage was not related to the frequency of esophageal tumors. RESULTS:The expression of Ki-67 was significantly higher in high-grade intraepithelial lesion (I.L.), and the expression of P53 was also higher in low-grade I.L. CONCLUSION:The results emphasize the direct relationship of the carcinogenic process with early cell alterations detected by immunohistochemistry.

Water extracts of cabbage and kale inhibit ex vivo H2O2-induced DNA damage but not rat hepatocarcinogenesis

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito’s hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 ± 7.8 µm) and kale- (110.8 ± 10.0 µm) treated animals compared with control (120.9 ± 12.7 µm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 ± 0.3 µg/g) or kale (4 ± 0.2 µg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 ± 0.07 µg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

Protective effect of Cassia fistula Linn. on diethylnitrosamine induced hepatocellular damage and oxidative stress in ethanol pretreated rats

Diethylnitrosamine (DEN), found in many commonly consumed foods, is widely reported to induce cancer in animals and humans. The aim of the present study was to investigate the hepatoprotective and antioxidant activities of the leaf extract of the medicinal plant Cassia fistula Linn. against diethylnitrosamine induced liver injury in ethanol pretreated rats. Albino Wistar rats, pretreated with ethanol for 15 days, were administered a single dose of DEN. Thirty days after DEN administration, hepatotocellular damage was observed histologically, along with elevated levels of serum AST, ALT, ALP, LDH, γ-GT and bilirubin and a simultaneous fall in the levels of the marker enzymes in the liver tissue. Liver oxidative stress was confirmed by elevated levels of lipid peroxidation (LPO) and a decrease in enzymic and non-enzymic antioxidants activities. Oral administration of the ethanolic leaf extract (ELE) of Cassia fistula for 30 days to ethanol + DEN treated rats significantly improved the above alterations in the markers of hepatotoxicity and oxidative stress, resulting in the reversal of most of the parameters studied and were comparable to the standard hepatoprotective drug silymarin.