Effect of hepatocyte damage in hepatic fibrogenesis of patients infected with Schistosoma japonicum.

Schistosomiasis is a serious public health problem, and previous studies found that liver function and hepatic cells are damaged. To evaluate the serum parameters of liver function and fibrosis in schistosomiasis patients infected with Schistosoma japonicum (Schistosoma J.) and analyze the correlations between liver function and serum fibrosis markers in patients infected with Schistosoma J., this retrospective study enrolled 133 patients. The study population was divided into four groups: healthy people control group (n = 20), chronic schistosomiasis without liver cirrhosis (CS) group (n = 21), schistosomiasis cirrhosis without hypoalbuminemia (SC-HA) group (n = 68), and schistosomiasis cirrhosis with hypoalbuminemia (SC +HA) group (n = 24). Clinical and laboratory data were collected for analysis. In the multiple comparison of abnormal rates of aspartate aminotransferase (AST) and total bilirubin (TBIL), the abnormal rate of the SC +HA group was significantly higher than that of the other three groups (P < 0.05), and the abnormal rate of γ-GT in the SC +HA group was significantly higher than that in the control group (P < 0.05). Multiple comparison results of serum levels of fibrosis markers showed that the SC group had a significantly higher level of indexes than other groups (P < 0.05). The levels of TGF-ß1 in the CS group, SC-HA group and SC +HA group were significantly higher than those in the control group (P < 0.001). Our study demonstrated that the liver function and hepatic cells were damaged with the progression of liver disease in patients infected with Schistosoma J., and they played an important role in the occurrence and development of liver fibrosis.

NLRP3 inflammasome activation results in liver inflammation and fibrosis in mice infected with Schistosoma japonicum in a Syk-dependent manner.

Granulomatous and fibrosing inflammation in response to soluble egg antigen (SEA) from Schistosoma japonicum (S. japonicum) is the main pathological process of S. japonicum infection. Inflammasome activation has recently been implicated in the pathogenesis of liver disease. However, the role of inflammasome activation in schistosomiasis-associated liver fibrosis (SSLF) has not been extensively studied. In this study, it is demonstrated that the NLRP3 inflammasome is markedly activated in mouse HSCs both in vivo and in vitro during S. japonicum infection. Furthermore, it is demonstrated that inhibition of NLRP3 inflammasome significantly alleviates the liver inflammation and collagen deposition that are induced by infection with S. japonicum. The mechanism of SEA-induced NLRP3 inflammasome activation is studied in isolated, cultured mouse HSCs and it is shown that SEA-induced NLRP3 inflammasome activation in HSCs is dependent upon the activities of spleen tyrosine kinase (Syk), an enzyme usually associated with a pathogen recognition receptor for fungal pathogens. Moreover, it is demonstrated that Dectin-1 and JNK signaling are also involved in SEA-induced NLRP3 inflammasome activation in HSCs. These data shed new light on the mechanisms of NLRP3 inflammasome activation during an infection with S. japonicum, and further characterize its role in schistosomiasis-associated liver fibrosis (SSLF).

MiR-375 and Doxorubicin Co-delivered by Liposomes for Combination Therapy of Hepatocellular Carcinoma.

Doxorubicin (DOX) is one of the most frequently used anti-cancer drugs and the front line option for hepatocellular carcinoma (HCC) treatment. However, the clinical applications of DOX are restricted largely due to its toxicity and chemoresistance. Here, we report that miR-375 and DOX were co-delivered by liposomes (named L-miR-375/DOX-NPs) for combination therapy of HCC and drug resistance reversion of DOX. In vitro, L-miR-375/DOX-NPs could deliver DOX and miR-375 efficiently and simultaneously into HCC cells and ensure the successful release of mature miR-375 and DOX. Then, the released miR-375 suppressed the malignant hallmarks of HCC by significantly decreasing the expression of AEG-1, YAP1, and ATG7, while the released DOX evidently accelerated cell apoptosis and blocked cycle at a G2/M stage by activating the P53/Bax/Bcl-2, caspase-3, and P-JNK, P-P38 pathway. Furthermore, miR-375 dramatically inhibited drug resistance of DOX by reducing the expression of multidrug resistance gene 1 (MDR1). In vivo, L-miR-375/DOX-NPs exhibited enhanced anti-tumor efficiency in xenograft HCC mouse models with mild adverse effects compared with doxorubicin or miR-375 alone. In conclusion, our research demonstrated that L-miR-375/DOX-NPs had significant synergetic anti-tumor effects and added values in overcoming drug resistance, which may represent a promising approach for the therapy of HCC.

Activation of NLRP3 inflammasomes in mouse hepatic stellate cells during Schistosoma J. infection.

The major pathological changes during Schistosoma J. infection are characterized by granulomatous inflammation in the liver, a cellular immune response to schistosomal egg antigens. The molecular mechanisms initiating or promoting this schistosomal granulomatous inflammation remain poorly understood. In the present study, we first demonstrated that in mice infected with Schistosoma J. for 6 weeks exhibited increased levels of IL-1ß in liver, a major product of NLRP3 inflammasomes and collagen deposition around the eosinophilic granuloma with Schistosoma J. eggs, which was substantially attenuated by caspase-1 inhibitor, YVAD. This activation of the NLRP3 inflammasome occurred in hepatic stellate cells (HSCs), as shown by a marked increase in co-localization of IL-1ß with HSCs marker, desmin. Using isolated, cultured mouse HSCs, we further explored the mechanisms by which soluble egg antigen (SEA) from Schistosoma J. activates NLRP3 inflammasomes. SEA induced the formation and activation of NLRP3 inflammasomes, which was associated with both redox regulation and lysosomal dysfunction, but not with potassium channel activation. These results suggest that NLRP3 inflammasome activation in HSCs may serve as an early mechanism to turn on the inflammatory response and thereby instigate liver fibrosis during Schistosoma J infection.

Endocannabinoids signaling: Molecular mechanisms of liver regulation and diseases.

The endocannabinoid system (ECS) includes endocannabinoids (eCBs), cannabinoid (CB) receptors and the enzymes that are responsible for endocannabinoid production and metabolism. The ECS has been reported to be present in both brain and peripheral tissues. Recent studies have indicated that eCBs and their receptors are involved in the development of various liver diseases. They were found to be altered in response to many danger factors. It is generally accepted that eCB may exert a protective action via CB2 receptors in different liver diseases. However, eCBs have also been demonstrated to have pathogenic role via their CB1 receptors. Although the therapeutic potential of CB1 receptor blockade in liver diseases is limited by its neuropsychiatric side effects, many studies have been conducted to search for novel, peripherally restricted CB1 antagonists or CB2 agonists, which may minimize their neuropsychiatric side effects in clinical use. This review summarizes the current understanding of the ECS in liver diseases and provides evidence for the potential to develop new therapeutic strategies for the treatment of these liver diseases.

Efficacy of telbivudine in the treatment of chronic hepatitis b and liver cirrhosis and its effect on immunological responses.

This study was aimed to evaluate the long-term effects of telbivudine (LdT) in the treatment of chronic hepatitis B (CHB) and HBV-related liver cirrhosis (LC) and to observe the changes of immunological responses during LdT treatment. Clinical data of 80 CHB and 28 HBV-related LC patients who were administered with LdT for 108 weeks and followed up were retrospectively analyzed. The liver function indicators including ALT, AST and γ-GT, HBV DNA copy number in serum and the rates of hepatitis B e antigen (HBeAg) seroconversion were analyzed before and 12, 24, 36, 48, 60, 72, 84, 96 and 108 weeks after LdT treatment in CHB and LC groups. Four serum fibrosis-related markers, including hyaluronic acid (HA), human laminin (LN), human type IV collagen (IV-C) and human N-terminal procollagen III peptide (PC-III), were detected before and after LdT treatment in LC group. The results showed favorable viral suppression and biochemical responses after treatment with LdT for 12 weeks, and a high rate of virological and biochemical control was maintained during the course of 108-week treatment in both CHB and LC groups. The four fibrosis-related markers, especially HA and LN, were down-regulated to some degrees in LC group. Moreover, LdT treatment led to the fluctuation of the circulating interferon-γ (IFN-γ) and interleukin-10 (IL-10) levels at different time points in CHB group. It was concluded that LdT could favorably lead to the virological suppression and biochemical remission. Besides, IFN-γ and IL-10 may represent a suitable and effective predictor of responsiveness during LdT therapy.

Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: role of NADPH oxidase.

The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O2(-)) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of O2(-) production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O2(-) production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.

[Effects of anandamide on proliferation of and pErk expression in primary hepatic stellate cells of schistosome-induced liver fibrosis mice].

OBJECTIVE: To investigate the potential therapeutic properties of the endogenous cannabinoid N-arachidonic acid aminoethanols (anandamide, AEA) in liver fibrosis by observing its affects on proliferation of and expression of phosphorylated-Erk (pErk) in primary hepatic stellate cells (HSCs) from a mouse model of schistosome-induced liver fibrosis. METHODS: The schistosome-induced liver fibrosis model was established by attaching cercaria to the skin on the ventral side of the mouse and allowing infection to occur via direct penetration. Six weeks later, the model was confirmed by pathological analysis of liver, with Masson trichrome staining showing collagen fiber deposition around the blood vessels and hematoxylin-eosin staining showing eosinophilic granuloma formation. Primary HSCs were isolated by discontinuous density gradient centrifugation, confirmed by immunofluorescence detection of double-staining for a-smooth muscle actin and desmin (95% purity), and cultured in the presence of absence of various concentrations of AEA. Proliferative ability was evaluated by MTT assay and the expression of pErk was observed by Western blotting. RESULTS: AEA treatment inhibited the proliferation of the primary HSCs in a concentration-dependent manner (AEA: 5 mumol/L, inhibition: 7.68%; 10 mumol/L, 11.65%; 20 mumol/L, 14.70%; 40 mumol/L, 15.07%; 60 mumol/L, 18.18%; 80 mumol/L, 20.26%; 100 mumol/L, 20.17%; 120 mumol/L, 29.24%). AEA treatment increased pERK expression in both a concentration-dependent manner (AEA: 20 mumol/L, average gray value: 39.90+/-4.61; 60 mumol/L, 43.45+/-0.91; 120 mumol/L, 52.91+/-1.97; vs. negative control, all P less than 0.05) and a time-dependent manner (time: 15 min, average gray value: 85.05+/-15.80; 30 min, 103.41+/-11.89; 1 h, 118.02+/-12.24; 3 h, 109.17+/-15.69; 6 h, 100.86+/-10.55; 12 h, 71.70+/-12.87; 24 h, 34.62+/-14.85; 48 h, 22.84+/-11.73; vs. negative control, all except 48 h had P less than 0.05). CONCLUSION: AEA can suppress the proliferative capacity of primary HSCs from schistosome-induced fibrotic livers through activation of the Erk signaling pathway.

Schistosoma japonicum egg antigen up-regulates fibrogenesis and inhibits proliferation in primary hepatic stellate cells in a concentration-dependent manner.

AIM: To investigate the effects of different concentrations of Schistosoma japonicum (S. japonicum) egg antigen on fibrogenesis and apoptosis in primary hepatic stellate cells (HSCs). METHODS: A mouse model of schistosomiasis-associated liver fibrosis (SSLF) was established by infecting mice with schistosomal cercaria via the abdomen. HSCs were isolated from SSLF mice by discontinuous density gradient centrifugation, and their identity was confirmed by immunofluorescence double staining of α-smooth muscle actin (α-SMA) and desmin. The growth inhibitory effect and 50% inhibitory concentration (IC50) of S. japonicum egg antigen for primary HSCs (24 h) were determined using a cell counting kit-8 (CCK-8) assay. The expression levels of α-SMA, matrix metalloproteinase-9 (MMOL/LP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in HSCs in response to different concentrations of S. japonicum egg antigen were detected by Western blotting and real-time reverse transcription-polymerase chain reaction. The levels of phospho-P38 (P-P38), phospho-Jun N-terminal kinase (P-JNK) and phospho-Akt (P-AKT) in HSCs were detected by Western blotting. RESULTS: An SSLF mouse model was established, and primary HSCs were successfully isolated and cultured. S. japonicum egg antigen inhibited HSC proliferation in a concentration-dependent manner. The IC50 of the S. japonicum egg antigen was 244.53 ± 35.26 µg/mL. S. japonicum egg antigen enhanced α-SMA expression at both the mRNA and protein levels and enhanced TIMP-1 expression at the mRNA level in HSCs (P < 0.05), whereas the expression of MMOL/LP-9 was attenuated at both the mRNA and protein levels in a concentration-dependent manner (P < 0.05). A high concentration of S. japonicum egg antigen enhanced P-P38, P-JNK and P-AKT activation (P < 0.05). The changes in α-SMA and MMOL/LP-9 expression induced by S. japonicum egg antigen were closely correlated with P-P38 and P-JNK activation (P < 0.05). The attenuation of MMOL/LP-9 was also correlated with P-AKT activation (P < 0.05), but the increase in α-SMA expression was not. TIMP-1 expression was not correlated with P-P38, P-JNK or P-AKT activation. CONCLUSION: S. japonicum egg antigen promotes fibrogenesis, activates the P38/JNK mitogen-activated protein kinase and AKT/PI3K signaling pathways and inhibits proliferation in primary HSCs isolated from SSLF mice in a concentration-dependent manner.

[Expression of ASMase in alcoholic liver fibrosis in rats].

OBJECTIVE: To investigate the expression of the lysosomal enzyme acid sphingomyelinase (ASMase) in alcohol-induced hepatic fibrosis using a rat model. METHODS: The model of liver fibrosis was induced by administration of alcohol and high fat diet using 20 rats. Six rats given no alcohol and normal diet served as the control group. Real-time PCR, western blotting, and immunohistochemistry were used to evaluate fibrosis-related changes in the mRNA and protein expressions of ASMase. RESULTS: The fibrotic liver tissues of the model rats showed significantly higher expression levels of ASMase than the non-fibrotic liver tissues of the control rats (P less than 0.05). CONCLUSION: Expression of ASMase is increased in the fibrotic liver tissue of an alcohol-induced hepatic fibrosis rat model, suggesting that this lysosomal enzyme may contribute to development of this disease condition.

[Membrane cholesterol mediates the endocannabinoids-anandamide affection on HepG2 cells].

OBJECTIVE: To study the effect of anandamide (AEA) on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer. METHODS: Localization of the fatty acid hydrolytic enzyme (FAAH), cannabinoid receptors 1(CB1) and cannabinoid receptors 2 (CB2) proteins was detected in L02 and HepG2 cells using immunofluorescence. L02 and HepG2 cells were treated with different concentrations of AEA and methyl-beta-cyclodextrin, and the rates of cells necrosis were examined by PI stain. Meanwhile, the expression levels of FAAH, CB1 and CB2 receptor proteins, as well as P38 mitogen-activated protein kinase (p-P38 MAPK) and c-Jun-NH2-terminal kinase (p-JNK) proteins, were analyzed by Western blot. RESULTS: The FAAH, CB1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells. The expression level of FAAH protein was higher in HepG2 than in L02 cells. The expression level of CB1 receptor protein was very low in both L02 and HepG2 cells. The expression level of CB2 receptor protein was high in both L02 and HepG2 cells. AEA treatment induced necrosis in HepG2 cells but not in L02 cells. Methyl-beta-cyclodextrin treatment prevented necrosis in HepG2 cells (t = 3.702; 5.274; 3.503, P less than 0.05). The expression patterns of FAAH, CB1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot, which were consistent with the immunofluorescence results. AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependent manner in HepG2 cells (F = 11.908; 26.054, P less than 0.05) and the increase can be partially by prevented by MCD (t = 2.801; t = 12.829, P less than 0.05). CONCLUSION: AEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.

Hepatic stellate cell-specific gene silencing induced by an artificial microRNA for antifibrosis in vitro.

BACKGROUND: We previously reported that the anti-transforming growth factor-beta1 (TGF-beta1) ribozymes directed by T7 and CMV promoters could reverse the character of activated hepatic stellate cells (HSCs) in vitro and improve fibrotic pathology in vivo. However, nonspecific elimination of the effects of TGF-beta1 without selectivity might have unfavorable consequences, such as overwhelming inflammation, tissue necrosis, etc. AIMS: To establish an activated-HSC-specific gene silencing method and validate its feasibility for antifibrosis in vitro. METHODS: An artificial intronic microRNA (miRNA) expression system was established, containing three parts: (1) a 1,074-bp SM-alpha actin promoter SMP8, which is a kind of RNA polymerase II promoter and has no activity in normal liver-derived cells but is switched on during the activation of HSCs, (2) intron1 modified by inserting an artificial pre-miRNA sequence against TGF-beta1, and (3) report gene enhanced green fluorescent proteins (EGFP). The feasibility of this system for artificial microRNA expression was validated through microRNA detection by real-time polymerase chain reaction (PCR). Alteration of biological characteristics of HSCs with the anti-TGF-beta1 miRNAs was preliminarily evaluated by measuring the expression levels of TGF-beta1 and its downstream molecules, including collagen I, matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP-1), etc. RESULTS: The microRNA expression system could successfully produce mature anti-TGF-beta1 miRNAs in an activated-HSC-specific manner. The microRNA-induced inhibition rate of TGF-beta1 reached 70% and above. Accompanied by TGF-beta1 suppression, its downstream targets such as collagen I, MMP2, TIMP-1, etc. were also significantly downregulated in vitro. CONCLUSIONS: Activated-HSC-cell-specific gene silencing could be induced well by the artificial intronic microRNA expression system to realize antifibrosis in vitro.

Endocannabinoids anandamide and its cannabinoid receptors in liver fibrosis after murine schistosomiasis.

This study examined endogenous cannabinoid (ECB)-anandamide (AEA) and its cannabinoid receptors (CBR) in mice liver with the development of schistosoma japonicum. Mice were infected with schistosoma by means of pasting the cercaria onto their abdomens. Liver fibrosis was pathologically confirmed nine weeks after the infection. High performance liquid chromatography (HPLC) was employed to determine the concentration of AEA in the plasma of mice. Immunofluorescence was used to detect the expression of CBR1 and CBR2 in liver tissue. Morphological examination showed typical pathological changes, with worm tubercles of schistosoma deposited in the liver tissue, fibrosis around the worm tubercles and infiltration or soakage of inflammatory cells. Also, CBR1 and CBR2 were present in hepatocytes and hepatic sinusoids of the two groups, but they were obviously enhanced in the schistosoma-infected mice. However, the average optical density of CBR1 in the negative control and fibrosis group was 13.28+/-7.32 and 30.55+/-7.78, and CBR2 were 28.13+/-6.42 and 52.29+/-4.24 (P<0.05). The levels of AEA in the fibrosis group were significantly increased as compared with those of the control group. The concentrations of AEA were (0.37+/-0.07) and (5.67+/-1.34) ng/mL (P<0.05). It is concluded that the expression of endocannabinoids AEA and its cannabinoid receptor CBR were significantly increased in schistosoma-infected mice. Endogenous endocannabinoids may be involved in the development of schistosoma-induced liver fibrosis.

Endocannabinoids Anandamide and Its Cannabinoid Receptors in Liver Fibrosis after Murine Schistosomiasis / 华中科技大学学报（医学）（英德文版）

This study examined endogenous cannabinoid (ECB)-anandamide (AEA) and its can-nabinoid receptors (CBR) in mice liver with the development of schistosomajaponicum.Mice were infected with schistosoma by means of pasting the cercaria onto their abdomens.Liver fibrosis was pathologically confirmed nine weeks after the infection.High performance liquid chromatography (HPLC) was employed to determine the concentration of AEA in the plasma of mice.Immunofluorescence was used to detect the expression of CBR 1 and CBR2 in liver tissue.Morphological examination showed typical pathological changes,with worm tubercles of schistosoma deposited in the liver tissue,fibrosis around the worm tubercles and infiltration or soakage ofinfiammatory cells.Also,CBRI and CBR2 were present in hepatocytes and hepatic sinusoids of the two groups,but they were obviously enhanced in the schistosoma-infected mice.However,the average optical density of CBR1 in the negative control and fibrosis group was 13.28±7.32 and 30.55±7.78,and CBR2 were 28.13±6.42 and 52.29±4.24 (P<0.05).The levels of AEA in the fibrosis group were significantly increased as compared with those of the control group.The concentrations of AEA were (0.37±0.07) and (5.67±1.34) ng/mL (P<0.05).It is concluded that the expression of endocannabinoids AEA and its cannabinoid receptor CBR were significantly increased in schistosoma-infected mice.Endogenous endocannabinoids may be involved in the development of schistosoma-induced liver fibrosis.

[Effects of norepinephrine on the proliferation and activation of rat hepatic stellate cells].

OBJECTIVE: To elucidate the relationship between rat hepatic stellate cells (HSC) and sympathetic neurotransmitter norepinephrine (NE) during liver fibrosis. METHODS: Using immunofluorescence and RT-PCR, the expressions of a1 and b2-adrenoceptors in activated HSC were detected. Methyl thiazolyl tetrazolium (MTT) was adopted to investigate the effect of NE on the proliferation of HSC. Meanwhile, the expressions of collagen-1, transforming growth factor beta (TGFb) and smooth muscle a-actin (a-SMA) in NE-stimulated HSC were detected by RT-PCR. The contents of NE in HSC were determined by high performance liquid chromatography-electrochemical detector (HPLC-ECD). RESULTS: The a1 and b2-adrenoceptors were expressed in HSC. NE markedly stimulated the proliferation of HSC in a concentration-dependent manner (F = 140.464, P less than 0.05). NE induced the mRNA expressions of collagen-1, TGFb and a-SMA in HSC (t= -4.160; t= -8.763; t= -17.651, P less than 0.05). HSC were synthesizing and releasing NE, especially when stimulated with platelet-derived growth factor (PDGF) (10 ng/ml) (t= -32.907, P less than 0.05). CONCLUSION: Our findings show that HSC are direct targets of NE and HSC are hepatic neuroglial cells that produce and respond to sympathetic neurotransmitter norepinephrine, suggesting that interrupting sympathetic nervous system signaling may be useful in the treatment of liver fibrosis.

[Effects of anandamide on the activation and proliferation of hepatic stellate cells through cannabinoid-2 receptors].

OBJECTIVE: To study the effects of endogenous cannabinoid anandamide (AEA) and its putative endocannabinoid receptors (CBR) on the activation and proliferation of hepatic stellate cells (HSC) and to study the role played by AEA during liver fibrosis. METHODS: By using immunofluorescence and cell culture, the expression of CBR 1 and 2 in the PDGF-stimulated HSCs was investigated. By using PCR and Western-blot, the effects of 10, 20mumol/L AEA and CBR2 antagonist AM630 on the cultured and activated HSC were observed. Methyl thiazolyl tetrazolium and flow cytometry were used to investigate whether AEA induces growth inhibition or apoptosis in the activated HSCs. RESULTS: Both CBR1 and CBR2 receptors were detectable in cultured HSCs with a higher level of CBR2 than CBR1 (F = 116.797, P less than 0.01). When HSCs were stimulated by PDGF, the expression of CBR2 receptors was significantly enhanced (F = 7.878, P less than 0.05). HSC proliferation was dose-dependently inhibited by 10, 20, and 50micromol/L AEA, with the rates of 7.12%+/-0.34%, 12.52%+/-0.78%, 80.13%+/-1.57% respectively (F = 533.41, P less than 0.01). However, it did not induce apoptosis, but necrosis. The expressions of alpha-SMA, TGFb1, a1(I), a1(III) and TIMP-1 were significantly suppressed by 20micromol/L AEA, but CBR2 antagonist AM630 reversed this suppressor action of AEA. CONCLUSIONS: AEA may inhibit activation and proliferation of HSCs; CBR2 receptors mediate AEA-induced inhibitory action on the activation of HSCs. This CBR2 receptor-mediated action and AEA on HSCs could be used as a therapeutic target against liver fibrosis.

[The effects of sympathetic neurotransmitters and adrenergic receptors on liver fibrosis in murine schistosomiasis].

OBJECTIVE: To investigate the effects of sympathetic neurotransmitters and adrenergic receptors on liver fibrosis in murine schistosomiasis. METHODS: Mice were infestated with schistosoma by means of pasting cercariae on their abdomens. Thirty mice were randomly divided into a control group and a model group. Hematoxylin eosin and Van Gieson staining were used to view the histopathology of their livers. Immunofluorescence histochemistry and laser scanning confocal fluorescence microscopy were used to measure the a1A and beta2 adrenergic receptors in livers of the two groups of mice. High performance liquid chromatography-electrochemical detector (HPLC-ECD) was used to determine the concentration of norepinephrine (NE) and dopamine (DA) in the plasma of the mice. RESULTS: Immunofluorescence histochemistry showed that a1A and beta2 receptors were present in hepatocytes and hepatic sinusoids of the livers of the mice of the two groups, but there were many more in the livers of the schistosoma infected mice (t=-2.888; t=-6.648) (P<0.05). The results of HPLC-ECD showed that the levels of NE and DA in the model group were higher than those of the control group (t=-3.372; t=-4.428) (P<0.05). CONCLUSION: Sympathetic neurotransmitters and adrenergic receptors may participate in liver fibrogenesis in mice infected with schistosoma.

[Expression and significance of hypoxia inducible factor-1 alpha in hepatocellular carcinoma tissues].

OBJECTIVES: To investigate the expression and significance of HIF1 alpha in hepatocellular carcinoma (HCC) tissues and in hepatoma carcinoma cell line HepG2. METHODS: The expression of the HIF1 alpha mRNA and protein were detected with immunohistochemistry (IHC), Western blot and RT-PCR techniques in HCC, normal liver tissues and HepG2. Their relationship with the pathological characteristics of the HCC was also analyzed. RESULTS: HIF1 alpha protein was obviously expressed in HCC. The positive rate of HIF1 alpha protein in HCC tissues was 76.4% and was higher than that in normal hepatic tissues. The expression of HIF1 alpha had a correlation to the differentiation degree of HCC tissues and intrahepatic and extrahepatic metastases (P<0.05), but there was no correlation to the existence of portal vein tumor emboli, the status of HBsAg and the prognosis (P<0.05). The results of Western blot and RT-PCR were similar to the results of IHC. The positive rate of HIF1 alpha in HepG2 was 93.6%. The levels of HIF1 alpha protein and mRNA began to increase after being treated two hours with hypoxia or with CoCl(2) (150 micromol/L). CONCLUSIONS: HIF1 alpha protein is obviously expressed in HCC and it is mainly affected by hypoxia. The expression of HIF1 alpha is related to the differentiation of the HCC and its intrahepatic and extrahepatic metastases but has no correlation to the existence of portal vein tumor emboli, the status of HBsAg and the prognosis.

Low-dose ATRA supplementation abolishes PRM formation in rat liver and ameliorates ethanol-induced liver injury.

The effects of all-trans-retinoic acid (ATRA) in low doses supplementation on concentrations of polar retinoid metabolites (PRM) and retinoids in the ethanol-fed rat liver, and on hepatocyte injury were investigated. The rat model of alcoholic liver disease (ALD) was induced by intragastric infusion of ethanol, and then the rats were administrated with ATRA in two different doses (150 microg/kg body weight and 1.5 mg/kg body weight) for 4 weeks. Concentrations of retinoids in rat liver and plasma were determined by using HPLC. Liver tissues pathologic changes were observed under the light microscopy and electron microscopy. The serum transaminases concentrations were measured. The results showed that the HPLC analysis of retinoids revealed that retinoids (vitamin A, RA, retinyl palmitate) concentrations in ethanol-fed rat liver and RA concentration in ethanol-fed rat plasma were markedly diminished (P<0.01) after ethanol feeding for 12 weeks. Furthermore, obvious peaks of PRM were formed in livers of ethanol-fed rats. ATRA 150 microg/kg supplementation in ethanol-fed rats for 4 weeks raised RA concentration in both liver and plasma, and also raised vitamin A concentration in liver to control levels, partially restored retinyl palmitate concentration (P<0.05) in liver. ATRA 1.5 mg/kg supplementation raised not only RA concentrations in liver and plasma but also retinyl palmitate concentrations in liver. However, the vitamin A concentration in liver of ATRA-supplemented rats (1.5 mg/kg) was higher than that of controls (P<0.05). The histologic observation of liver tissues indicated that ATRA treatment notably alleviated hepatocellular swelling, steatosis, the swelling of mitochondria and proliferation of smooth endoplasmic reticulum (SER). ATRA treatment greatly decreased levels of serum transaminases as compared with the only ethanol-fed group (P<0.05). It was concluded that low-dose ATRA treatment could restore retinoids concentrations and abolish the PRM formation in liver of ALD rats, and then ameliorate the injury of liver cells.