The effects of farm management practices on liver fluke prevalence and the current internal parasite control measures employed on Irish dairy farms

Fasciolosis caused by Fasciola hepatica is responsible for major production losses in cattle farms. The objectives of this study were to assess the effect of farm management practices on liver fluke prevalence on Irish dairy farms and to document the current control measures against parasitic diseases. In total, 369 dairy farms throughout Ireland were sampled from October to December 2013, each providing a single bulk tank milk (BTM) sample for liver fluke antibody-detection ELISA testing and completing a questionnaire on their farm management. The analysis of samples showed that cows on 78% (n = 288) of dairy farms had been exposed to liver fluke. There was a difference (P < 0.05) between farms where cows were positive or negative for liver fluke antibodies in (a) the total number of adult dairy cows in herds, (b) the number of adult dairy cows contributing to BTM samples, and (c) the size of the total area of grassland, with positive farms having larger numbers in each case. There was no difference (P > 0.05) between positive and negative farms in (a) the grazing of dry cows together with replacement cows, (b) whether or not grazed grassland was mowed for conservation, (c) the type of drinking water provision system, (d) spreading of cattle manure on grassland or (e) for grazing season length (GSL; mean = 262.5 days). Also, there were differences (P < 0.001) between drainage statuses for GSL with farms on good drainage having longer GSL than moderately drained farms. The GSL for dairy cows on farms with good drainage was 11 days longer than for those with moderate drainage (P < 0.001). The percentage of farmers that used an active ingredient during the non-lactating period against liver fluke, gastrointestinal nematodes, lungworm, and rumen fluke was 96%, 85%, 77% and 90%, respectively. Albendazole was the most frequently used active ingredient for treatment against gastrointestinal nematodes (57%), liver fluke (40%) and lungworm (47%), respectively. There was a difference (P < 0.05) in the use of triclabendazole and albendazole between positive and negative farms, with triclabendazole use being more common in positive farms. This study highlighted differences in dairy management practices between Irish farms with dairy herds exposed or not exposed to liver fluke and stressed the need of fine-scale mapping of the disease patterns even at farm level to increase the accuracy of risk models. Also, comprehensive advice and professional support services to farmers on appropriate farm management practices are very important for an effective anthelmintic control strategy.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

Effects of RNA interference targeting CDC25agene on proliferation of human liver cancer HepG2 cells

AIM: To investigate the effect of silencing cell division cycle 25a (CDC25a) gene on the proliferation of human hepatoma HepG2 cells. METHODS: CDC25agene in human hepatoma HepG2 cells was silenced by RNA interference. Real-time PCR was applied to detect the expression of CDC25a, cyclin E and CDK2 at mRNA levels in the HepG2 cells. Western blotting was applied to detect the expression of CDC25a at protein level. In addition, MTT assay, Giemsa staining and flow cytometry were used to measure the proliferation of human hepatoma HepG2 cells. RESULTS: The expression of CDC25a at mRNA and protein levels in RNA silence group was lower than those in negative control group and normal control group (P＜0.05). The mRNA expression of cyclin E and CDK2 in silence group was lower than that in negative control group and normal control group (P＜0.05). The cell proliferation in silence group was lower than that in negative control group and normal control group (P＜0.05). The results of flow cytometry revealed that the cells in silence group were blocked in G1 phase. CONCLUSION: Infection of LV-CDC25a-RNAi recombinant to the HepG2 cells effectively inhibits the CDC25agene expression and the proliferation of human hepatoma cells, and arrests the cells in G1 phase, suggesting that CDC25agene may be a key target for the treatment of liver cancer.     

Effect of siRNA-mediated Smad3 silence on proliferation and apoptosis in activated hepatic stellate cells

AIM: To investigate the effects of siRNA-mediated Smad3 silence on proliferation and apoptosis in activated hepatic stellate cells (HSCs).METHODS: HSCs-T6 cells were divided into 3 groups: blank group, negative control group and siRNA-Smad3 transfection group. The siRNA-Smad3 was transfected into HSCs-T6 cells. At different time points after transfection, cell proliferation was measured by CCK-8, cell apoptosis was detected by flow cytometry, and protein levels of P53 and Bcl-2 were determined by immunocytochemistry.RESULTS: HSCs proliferation was significantly inhibited at the time points of 24 h, 48 h and 72 h after transfection. Meanwhile, the apoptosis of HSCs was significantly increased in siRNA-Smad3 transfection group (P<0.01). Compared to the control cells, the protein expression of P53 was significantly increased while Bcl-2 protein was significantly decreased 48 h after transfection in siRNA-Smad3 transfection group (P<0.01).CONCLUSION: The siRNA-mediated Smad3 silence significantly inhibits HSCs proliferation and induces apoptosis by up-regulating the P53 expression and down-regulating the Bcl-2 expression in HSCs.     

Protective role of heat-shock protein 70 in gastric mucosal lesion induced by endotoxin in cirrhotic rats with portal hypertensive gastropathy

AIM: To investigate the protective role of heat-shock protein 70 (HSP70) in the pathogenesis of gastric mucosal damage in cirrhotic rats with portal hypertensive gastropathy (PHG).METHODS: The rat model of liver cirrhosis with PHG was established by injection with tetrachloride.The animals were divided into normal control group, PHG group, PHG+heat treatment group, PHG+BPI21 group and PHG+endotoxin groups.The endotoxin used in the experiment was at the dose of 3 mg/kg and endotoxin antagonist BPI21 was at the dose of 2 mg/kg.HSP70 was induced by pre-treating the animals with mild whole-body heating.The levels of HSP70 and tumor necrosis factor alpha (TNF-α) in the gastric mucosa were measured by ELISA.Furthermore, the pathological changes of the gastric mucosa were observed under microscope with HE staining.RESULTS: Compared with the normal control rats, the rats in PHG group showed obvious gastric pathological lesion, decrease in HSP70 production and increase in TNF-α level in the gastric mucosa, and increased endotoxin concentration in the plasma.Compared with PHG+endotoxin group, the gastric mucosal lesion in PHG+BPI21 group was significantly attenuated, accompanied by the increase in HSP70 production and decrease in TNF-α level in the gastric mucosa.Heat treatment increased HSP70 production and decreased TNF-α concentration in the PHG rats, thus attenuating the gastric mucosal damage.CONCLUSION: HSP70 alleviates the gastric mucosal lesion induced by endotoxin in cirrhotic rats with PHG and decreases the concentration of TNF-α in gastric mucosa, indicating a protective role of HSP70 in the pathogenesis of gastric mucosal damage in PHG.     

Advances in mechanisms of immunity enhancement induced by thermal ablation therapy in hepatocellular carcinoma

Image-guided tumor thermal ablation plays a key role in the management of hepatocellular carcinoma. Thermal ablation for hepatic tumors not only generates directly killing effect on liver tumors, but also improves the level of anti-tumor immunity of the host. Thermal ablation potentially affects the anti-tumor immunity in the following ways: (1) causing necrosis that is a source for intracellular antigens, and also inducing local inflammation, which further stimulates the antigen-presenting cells; (2) removing the tumor burden; (3) undergoing the process of heat shock in surviving tissue, leading to increase in the expression of heat shock proteins; (4) activating and enhancing tumor-specific T-cell responses. This review discusses these possible mechanisms.     

Inhibition of Ang-2 and RGS-5 expression by nanogold results in normalization of vascellum in hepatic tumor

AIM: To observe the vascular normalization effect of nanogold on hepatic tumor by inhibiting angiopoietin-2 (Ang-2), regulator of G-protein signaling-5 (RGS-5) during certain time window. METHODS: H22 cells, the hepatocellular cancer cell line, were subcutaneously injected into the right armpits of 48 BALB/c nude mice. When the size of transplanted tumor reached 3~4 mm, the mice were divided into 2 groups: 36 mice in experiment group and 12 mice in control group. The mice in experimental group underwent injection of nanogold into the tumor once a day, and the mice in control group were injected with normal saline. After continuous treatment with nanogold for 3 days, 7 days and 11 days, the mice were sacrificed, the liver tumors were taken out to measure the size and weight. The expression of Ang-1,Ang-2 and RGS-5 in the tumor was detected by the method of immunohistochemical staining. The normalizing shapes of tumor vessels and the pericytes were observed under electronic microscope. RESULTS: With nanogold treatment for 3 days, 7 days and 11 days, the positive rates of Ang-1 were 16.7％, 50.0% and 16.7%, respectively. The positive rates of Ang-2 were 33.3%, 16.7% and 41.7%, respectively. The expression of Ang-1 in experiment group was higher than that in control group, especially at the 7th day in experiment group. The expression of Ang-2 in experiment group was lower than that in control group (58.3%), especially at the 7th day in experiment group. With nanogold treatment for 3 days, 7 days and 11 days, the positive rates of RGS-5 were 33.3%, 16.7% and 50.0%, respectively. The immature pericyte coverage indexes (IMPI) were 19.6%±4.3%, 32.5%±7.9% and 41.2%±9.1% respectively. At the 7th day in experiment group, the positive rates of RGS-5 and IMPI were lower than those in other experiment groups and control group. After treated with nanogold for 7 days, the pericytes in the parietal wall of the blood vessels in the tumor showed the tendency to grow normally in morphology and were completely covered by endothelial cells. However, the pericytes in the parietal wall of the blood vessels in control group showed differences in size, impaired integrity and only a few of the pericytes covered by endothelial cells. CONCLUSION: During the time window of nangold treatment for 7 days, the chemical can normalize the blood vessels in liver cancer by inhibiting the expression of Ang-2 and RGS-5.     

Age changes in extramural digestive glands of sheep and rabbits in the postembryonic period

Background:The study of the peculiarities of the anatomy of sheep and rabbits’ digestive systems is an important way to improve the efficiency of these animals’ breeding.Aim:The aim of the presented research was to study structural changes of such digestive glands as the liver and the pancreas which occur in the process of ontogenesis in sheep and rabbits.Methods:Sheep of the “Kazakh fat-tailed semi-coarse-wooled” breed (n = 8) raised in the “Sayan” private peasant agriculture and rabbits of the “Grey Giant” breed (n = 8), raised on the mini rabbit farm of the Agriculture Faculty of Shakarim University were used in the research.Two experimental animal groups were formed (of sheep, “Kazakh fat-tailed semi-coarse-wooled” breed, n = 8; rabbits, “Grey Giant” breed, n = 8). The liver and pancreas’ ontogenesis development has been studied in these animals.Results:The study presents a holistic view of the macro-microscopic structure of the liver and the pancreas of animals in crucial age periods, stages, and phases of postembryonic ontogenesis (by the example of sheep and rabbits). The authors have traced age stages of adaptive change and structural-functional change of stromal-parenchymatous structures of the liver in sheep and rabbits taking into account stages and crucial phases of development.Conclusion:Development of the liver and the pancreas are characterized by discontinuous growth in the process of postnatal ontogenesis. A crucially important period is the first months after birth, during which the weight and functionality of these organs grow rapidly.     

红树植物老鼠簕的研究进展

从多糖成分、甾醇成分、生物碱A等方面对近年来红树植物老鼠簕的化学成分研究及在护肝方面临床药理研究进行综述,为进一步开发利用老鼠簕提供依据。     

The protection of the hepatic ischemic preconditioning is concerned with the NO/ET-1 system

AIM: To study the relationship between the disturbance of nitric oxide/endothelin-1(NO/ET-1) and hepatic ischemia/reperfusion(I/R) injury as well as the regulation of NO/ET-1 system by hepatic ischemic preconditioning(IPC). METHODS: The changes of NO/ET-1 system and their relationship with hepatic I/R injury were compared between I/R group and IPC+I/R group in a rat hepatic I/R model. Two hours after reperfusion, the liver tissues were detected by RT-PCR to see whether there was inducible nitric oxide synthase (iNOS) mRNA expression. RESULTS:In the acute phase of hepatic reperfusion, the ratio of NO/ET-1 was reduced, which was due to a significant reduction of NO₂^-/NO₃^- (the metabolic product of NO) and significant elevation of ET-1 in the blood plasma. The content of ALT, AST, LDH and TNF-α in blood plasma, and of MDA in liver tissue were increased but ATP in liver tissue was reduced, the hepatic damage was deteriorated. The protection of the hepatic IPC was concerned with the elevation of the ratio of NO/ET-1 caused by the elevation of NO₂^-/NO₃^-, and reduction of ET-1 as well. There was no iNOS mRNA detected in the liver tissues.CONCLUSION: Hepatic I/R injury is related to the disturbance of NO/ET-1. The protection of the hepatic IPC in the acute phase might be conducted by its regulation of NO/ET-1 system. The cNOS rather than the iNOS generated the NO in this situation.