Role of hepatitis B virus in intrahepatic TGF-β1/Smads signaling pathway

AIM:To explore the effects of hepatitis B virus (HBV) on intrahepatic expression of transforming growth factor β1（TGF-β1） and Smads. METHODS:The expression of intrahepatic TGF-β1, HBsAg and HBcAg in control group and chronic hepatitis B (CHB) group was detected by immunohistochemical method.The serum HBV DNA content was determined by real-time PCR. The role of HBV in the expression of TGF-β1, Smad3 and Smad7 in human hepatic stellate cell line LX-2 in vitro was observed by cell culture and Western blotting. RESULTS:The average score of intrahepatic TGF-β1 expression in CHB group was higher than that in control group. With the increase in serum HBV DNA content, intrahepatic TGF-β1 expression was also enhanced. In the HBcAg positive hepatic tissue, there was higher TGF-β1 expression than that in the liver tissue of HBcAg negative. Compared with control group and HBV+anti-TGF-β1 group, HBV caused increased expression of TGF-β1 and Smad3 in HBV group in vitro. No difference of Smad7 protein among control group, HBV group and HBV+anti-TGF-β1 group was observed. CONCLUSION: The expression of intrahepatic TGF-β1 is related to serum HBV DNA and hepatocellular HBcAg in the patients with CHB. HBV-induced liver fibrosis mainly relies on positive regulatory mechanisms of Smad3,and the negative regulation by Smad7 almost does not function.     

NK cells from donor liver inhibit NF-κB activity and IL-15 expression after liver transplantation in rats

AIM:To investigate the mechanism that donor liver natural killer (NK) cells alleviate acute rejection after liver transplantation by observing the secretion level of interleukin 15 (IL-15) in peripheral blood, the protein expression of IL-15 in transplanted liver tissues and the activity of NF-κB in spleen tissues in rat acute liver graft rejection model. METHODS:An acute rejection model of liver transplantation in rats was established by the modified two-cuff method, in which Lewis rats were used as donors and BN rats as recipients. The donor leukocytes were depleted by whole body irradiation of ［60Co］ source and the donor liver immunity was reconstituted by transfusion of liver NK cells from the same type of donor (donor type liver NK cells, dtlNKs) via portal vein immediately after grafting the irradiated liver. The rats were divided into the following groups: group A, acute rejection group; group B, BN rats receiving the liver of Lewis rats with ［60Co］ irradiation; group C, BN rats receiving the liver of ［60Co］-irradiated Lewis rats and treated with dtlNKs via the portal vein. The recipients were sacrificed at 1 d, 3 d and 7 d after transplantation. IL-15 level in peripheral blood was detected by ELISA. The expression of IL-15 in the liver grafts was determined by Western blotting. NF-κB activity in the spleen tissues of recipient rats was identified by electrophoretic mobility shift assay. The survival quality and living time in crude survival subgroup were observed. RESULTS:Acute rejection in group B was severer than that in group A and group C. The rats in group B showed significantly shorter average survival time compared with group A and group C. At 3 d and 7 d after transplantation, the IL-15 content in peripheral blood was significantly higher in group B than that in group A and group C. The expression of IL-15 in transplanted liver tissues was significantly higher in group B than that in group A and group C. The activity of NF-κB in the spleen tissues was higher in group B. CONCLUSION: IL-15 might be a significant indicator for monitoring acute rejection after liver transplantation. The donor liver NK cells modulate the immunity of liver transplantation by inhibiting the expression of IL-15 via the suppression of NF-κB activity.     

Expression of calpain 2 and Bax in rat fibrotic liver tissues

AIM：To observe the expression of calpain 2 and Bcl-2-associated X protein (Bax) in rat fibrotic liver tissues and to explore their effects on hepatic fibrosis.
METHODS：Forty male Wistar rats were randomly divided into four groups (each n=10): 4-week control group, 8-week control group, 4-week liver fibrosis group and 8-week liver fibrosis group. Liver fibrosis model was induced by subcutaneous injection of 40% CCl4 (3 mL/kg) every 3 days for 4 or 8 weeks. The apoptosis of hepatocytes was detected by TUNEL. Additionally, the mRNA expression of calpain 2 and bax was determined by real-time PCR, and the protein expression of calpain 2 and Bax was determined by immunohistochemistry and Western blotting. RESULTS：Real-time PCR showed that the mRNA expression of calpain 2 and bax in liver tissues was elevated in 4-week and 8-week liver fibrosis groups. The results of immunohistochemistry and Western blotting revealed that there was no difference of calpain 2 protein expression in liver tissues between 4-week liver fibrosis group and control group, but that in 8-week liver fibrosis group was obviously increased. The protein expression of Bax in 4-week and 8-week liver fibrosis groups was higher than that in control groups. Additionally, the numbers of apoptotic hepatocytes in 4-week and 8-week liver fibrosis groups were obviously increased compared with control groups.CONCLUSION：Calpain 2 and Bax may play important roles in the process of liver fibrosis.     

Expression of ICAM-1 gene in the ischemia-reperfusion liver

AIM: To investigate the expression of ICAM-1 mRNA in hepatic sinusoidal cells (HSCs) during liver ischemia-reperfusion injury and its effects. METHODS: Three groups of rabbit livers experiencing ischemia for 15, 30 and 60 minutes respectively, and reperfusion for 60 minutes were conducted. The hepatic ICAM-1 mRNA expression was observed and leukocytes in hepatic sinus were counted. RESULTS: The lower expression of ICAM-1 mRNA showed in HSCs before reperfusion. Besides, leukocyte counts could no change. However, the more expression of ICAM-1 mRNA was found at the point of 60 minutes reperfusion. Furthermore, the higher expression of ICAM-1 mRNA showed in the more prolonged ischemic liver. Meanwhile, there was significant positive correlation between ICAM-1 mRNA contents and leukocyte counts. CONCLUSION: The high ICAM-1 mRNA expression induced by hepatic ischemia in HSCs increased adherence of HSCs and sequestrated more leukocytes in liver.     

EVALUATION OF EXPERIMENTALLY INDUCED CANINE HEPATIC CIRRHOSIS USING DUPLEX DOPPLER ULTRASOUND

Portal blood flow was measured with duplex Doppler ultrasound in ten normal dogs and in ten dogs with hepatic cirrhosis induced by common bile duct ligation 4 weeks previously. Mean portal blood flow velocity in the 10 dogs with experimentally induced hepatic cirrhosis was markedly reduced (9.2 ± 1.70 cm/sec vs. normal 18.1 ± 7.6 cm/sec, p < 0.01). Mean portal blood flow was also significantly decreased compared to normal (17.2 ± 4.9 cc/min/kg versus normal 31.06 ± 9.1 cc/min/kg, p < 0.01) while portal vein diameter remained unchanged. The dogs with induced hepatic cirrhosis developed extensive extrahepatic portosystemic shunting that was confirmed at necropsy. It was concluded that decreased portal velocity and portal flow which resulted from hepatic cirrhosis was detectable noninvasively with Doppler ultrasound.     

Proteins invoked by vitamin K absence and clotting times in clinically ill cats

The clinical utility of the Thrombotest, a method for determining the prothrombin time that is uniquely sensitive to the presence of proteins invoked by vitamin K absence (PIVKA), was prospectively evaluated and compared to routine coagulation tests in cats with clinically suspected bleeding tendencies. Abnormal PIVKA clotting values were determined by comparison to results of a concurrently evaluated pooled feline plasma sample and by use of an absolute cutoff value of 25.2 seconds. To be recognized as abnormal, PIVKA clotting values had to be >20% of the pooled feline plasma PIVKA clotting time (the "20% rule") or > or =25.2 seconds (mean + 2 standard deviations of 150 different pooled feline plasma samples). Among the disorders in the population examined were 74 cats with liver disease and 19 cats with severe inflammatory bowel disease. Overall, a prolonged PIVKA clotting time based on the 25.2-second cutoff was found in 39.3% of cats, and based on the 20% rule in 40.7% of cats. An abnormal prothrombin time (PT) developed in 5.8% of cats, an abnormal APTT in 14% of cats, subnormal fibrinogen in 8.8% of cats, and thrombocytopenia in 3.3% of cats. Bleeding tendencies were confirmed in 22 cats, of which abnormal PIVKA clotting times were recognized in 95.5%, abnormal PT in 21%, abnormal activated partial thromboplastin time in 25%, hypofibrinogenemia in 16.7%, and thrombocytopenia in 4.5%. Response to treatment with vitamin K was demonstrated in 21 of 24 cats with an abnormal PIVKA clotting time. In these cats, an abnormal PIVKA clotting time normalized within 3 to 5 days of parenteral vitamin K administration. Cats responding to vitamin K administration had hepatic lipidosis (n = 7), severe inflammatory bowel disease (n = 4), severe inflammatory bowel disease associated with cholangiohepatitis (n = 5), and miscellaneous disorders (n = 5). Using either endpoint, the PIVKA clotting time is more sensitive for the detection of cats with coagulopathies than routinely used coagulation assessments in our hospital. Our findings confirm that cats with hepatic lipidosis, severe cholangiohepatitis, and severe inflammatory bowel disease develop coagulopathies responsive to vitamin K administration.     

The action of recombinant human nucleoside diphosphate kinase A (rhNDPK-A) on the growth of S180,H22,Lewis and H460 tumors in vivo

AIM:To study the action of nucleoside diphosphate kinase A (NDPK-A) on the growth of S180,H22,Lewis and H460.METHODS:S180 or H22 cell (5×106) were inoculate subcutaneously into the right armpit of 85 Kunming mice,which were randomized into 8 groups.Lewis lung carcinoma cells (2×105) were inoculate subcutaneously into the right armpit of 85 C57BL/6 mice,which were randomized as Kunming mice.From the 2nd day,the treated groups were given different dose of rhNDPK-A once a day for 8 days (for S180 or H22 by iv) or for 10 days (for Lewis by ip),and the control group was given physiological saline only.H460 tissue pieces about 1.5 mm×1.5 mm×1.5 mm each were inoculated subcutaneously into the armpit of 38 Balb/c/neu mice.After the volume of xenograft become 100 mm×100 mm×100 mm,the nude mice were randomized into 5 groups and given different dose of rhNDPK-A once a day for 17 days.2 days after above treatments,the mice were killed and dissected.The knubs were peeled off and weighted.RESULTS:The growth of S180,H22 and H460 were inhibited by rhNDPK and the growth of H22 was inhibited by rhNDPK at dose of 20 mg/kg combined with cisplatin (0.5 mg/kg).But the growth of Lewis lung cancer was not inhibited.CONCLUSION:rhNDPK-A inhibited the growth of S180,H22 and H460.rhNDPK-A (20 mg/kg) potentiated the antitumor action of cisplatin on H22.     

低温胁迫下黑龙江林蛙肝脏可溶性蛋白电泳图谱分析

初步分析黑龙江林蛙肝脏可溶性蛋白在聚丙烯酰胺凝胶电泳（PAGE）上的分布及低温胁迫下黑龙江林蛙肝脏可溶性蛋白的适应性变化。结果表明：分离胶浓度为6％的肝脏电泳图谱分辨率高、条带多,可分辨出约15～19条谱带;1℃环境温度胁迫条件的黑龙江林蛙肝脏Rf约0．79的可溶性蛋白含量较室温条件下的含量有所增加（P〈 0．05）,而随机选取的三组蛋白含量并无显著性差异（P0.10〉0．05,P0.42〉0．05,P0.84〉0．05）。     

亮菌多糖ATPS-2对小鼠四氯化碳和酒精肝损伤的保护作用

分别采用四氯化碳（CCl4）和北京红星二锅头诱导小鼠肝损伤，比色法测定血清中丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）的含量；肝脏中超氧化物歧化酶（SOD）活力和丙二醛（MDA）浓度，并作肝组织切片病理观察。亮菌多糖ATPS-2（25mg／kg、50mg／kg、100mg／kg）给药明显降低小鼠血清中升高的ALT和AST水平，抑制肝脏中上升的MDA水平和提高过低的SOD活性。病理检查结果显示亮菌多糖ATPS-2有明显的保肝作用。亮菌多糖ATPS-2对小鼠四氯化碳肝损伤和酒精肝损伤均具有显著的保护作用。