Cryptotanshinone induces apoptosis of activated hepatic stellate cells via modulating endoplasmic reticulum stress.

BACKGROUND: Cryptotanshinone (CPT) has wide biological functions, including anti-oxidative, antifibrosis, and anti-inflammatory properties. However, the effect of CPT on hepatic fibrosis is unknown. AIM: To investigate the effects of CPT treatment on hepatic fibrosis and its underlying mechanism of action. METHODS: Hepatic stellate cells (HSCs) and normal hepatocytes were treated with different concentrations of CPT and salubrinal. The CCK-8 assay was used to determine cell viability. Flow cytometry was used to measure apoptosis and cell cycle arrest. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses were used to measure mRNA levels and protein expression of endoplasmic reticulum stress (ERS) signaling pathway related molecules, respectively. Carbon tetrachloride (CCL4) was used to induce in vivo hepatic fibrosis in mice. Mice were treated with CPT and salubrinal, and blood and liver samples were collected for histopathological examination. RESULTS: We found that CPT treatment significantly reduced fibrogenesis by modulating the synthesis and degradation of the extracellular matrix in vitro. CPT inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in cultured HSCs. Furthermore, we found that CPT promoted apoptosis of activated HSCs by upregulating expression of ERS markers (CHOP and GRP78) and activating ERS pathway molecules (PERK, IRE1α, and ATF4), which were inhibited by salubrinal. Inhibition of ERS by salubrinal partially eliminated the therapeutic effect of CPT in our CCL4-induced hepatic fibrosis mouse model. CONCLUSION: CPT can promote apoptosis of HSCs and alleviate hepatic fibrosis through modulating the ERS pathway, which represents a promising strategy for treating hepatic fibrosis.

[Clinical efficacy of compound glycyrrhizin tablets in the treatment of children with nonalcoholic fatty liver disease].

Department of Pediatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. zhuchuanlong@jsph.org.cn.

Serum levels of tissue inhibitor of metalloproteinase-1 are correlated with liver fibrosis in patients with chronic hepatitis B.

OBJECTIVE: To investigate the relationship between tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and the severity of liver fibrosis in chronic hepatitis B (CHB) patients, and to explore the diagnostic value of serum TIMP-1. METHODS: A total of 159 CHB patients underwent liver biopsy for the analysis of liver fibrosis stages and inflammation. Serum TIMP-1 was determined by ELISA. Hyaluronic acid (HA), procollagen type III (PCIII), collagen type IV (CIV), laminin (LN), and FIB-4 index were determined and calculated, and diagnostic accuracy was analyzed using receiver operating characteristic (ROC) curve. RESULTS: Serum levels of TIMP-1 were associated with the grade of liver inflammation in CHB patients (r = 0.695, P < 0.01), especially in those without fibrosis or with stage 1 fibrosis, and were also positively correlated with liver fibrosis in CHB patients (r = 0.854, P < 0.01), particularly in those with inflammation at grade 1, 2 and 3. The area under the ROC curve (AUROC) of serum TIMP-1 was 0.918 for significant liver fibrosis (≥stage 2), and a sensitivity of 89.4% and specificity of 83.6% were obtained with a cut-off value of ≥174.5 ng/mL of serum TIMP-1, which were higher than that of HA, CIV and FIB-4 index. CONCLUSION: TIMP-1 is a valuable single biomarker for the evaluation of significant fibrosis in CHB patients.

[Determination and clinical significance of tissue inhibitors of metalloproteinase-1 and -2 in serum of children with nonalcoholic fatty liver disease].

OBJECTIVE: To examine serum tissue inhibitors of metalloproteinases (TIMP) -1 and -2 levels in children with nonalcoholic fatty liver disease and to investigate possible roles of the two markers. METHODS: One hundred and five obese children were classified into 4 groups: simple obesity (n=44), simple nonalcoholic fatty liver (SNAFL, n=25), and nonalcoholic steatohepatitis (NASH, n=36). Serum TIMP-1 and -2 levels were measured using ELISA. Serum ALT and gamma-GT levels were measured with totally automatic enzymatic method. RESULTS: Serum levels of TIMP-1 and gamma-GT increased with the disease development from simple obesity to SNAFL and NASH (P<0.05). Both serum TIMP-1 and -2 levels were positively correlated with gamma-GT levels (r=0.534, P<0.01; r=0.351, P<0.05, respectively). Ninety-seven percent of children in the NASH group had serum TIMP-1 levels over 2 standard deviations of healthy controls (83.35 microg/ L) compared with 76% in the SNAFL group (P<0.05). There were no significant differences in the case proportion with TIMP-2 levels over 2 standard deviations of healthy controls between the NASH and the SNAFL groups. CONCLUSIONS: Both TIMP-1 and -2 may reflect the state of liver fibrosis in children with nonalcoholic fatty liver disease, and serum TIMP-1 appears to be more reliable.

Gene therapy for acute liver failure.

Acute liver failure (ALF) is a life-threatening medical emergency and occurs when the liver rapidly loses its function within a short period. ALF can develop secondary to a variety of causes. Currently, the orthotopic liver transplantation is the "Gold Standard" therapy for the disease. However, due to the limited availability of donor organs and rapid progression of the disease, the mortality of ALF remains high. Therefore, it is imperative to develop novel therapeutic reagents for ALF. Gene therapy by delivering a target gene to the patients appears to be a promising approach for the treatment of ALF. Here, we review the recent advance of gene therapy for ALF, focusing on the three technical elements, animal models, vehicles for gene delivery and technique for gene delivery, which are important for the success of gene therapy as well as the potential targets used for the treatment of ALF.

Inhibitory effect of antisense oligonucleotide targeting TIMP-2 on immune-induced liver fibrosis.

INTRODUCTION: We previously reported that both experimental and human studies have shown the importance of TIMP-1 and TIMP-2 in the development of liver fibrosis, a disease mostly caused by HBV and HCV infection in China. Inhibiting the expression of TIMP-1 by an antisense oligonucleotide (ASON) can prevent liver fibrosis through decreasing the deposition of collagen I and III. Whether blocking the expression of TIMP-2 has the same effect on liver fibrosis is not clear. MATERIALS AND METHODS: To interfere with this potentially effective target, we designed and synthesized two different ASON targeting TIMP-2, then mixed and transfected them by hydrodynamic injection into the rat livers with immune-induced liver fibrosis. We isolated HSCs from the HSA-induced rat model with liver fibrosis, and transfected them with ASON or sense oligonucleotide in vitro. RESULTS: We observed that TIMP-2 ASON markedly reduced the expression of TIMP-2 by real-time PCR, Western blot, and enzyme linked immunosorbent assay. However, TIMP-2 ASON had little effect on alpha-SMA expression in vitro by Western blot. Inhibition of the expression of TIMP-2 by TIMP-2 ASON clearly decreased deposition of collagen I and IV, ameliorated liver pathology, and improved the liver function among the rats with immune-induced liver fibrosis. CONCLUSION: The results suggested that TIMP-2 ASON could prevent the progression of liver fibrosis in this rat model. It is possible that this could form the basis for exploration of new liver anti-fibrosis drugs at a genetic level.

[Construction of the p-mfgl2shRNA and its effect on mfgl2 expression in vitro].

OBJECTIVE: To construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro. METHODS: A plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression. RESULTS: Cotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines. CONCLUSIONS: The study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.

Fibrinogen-like protein 2 fibroleukin expression and its correlation with disease progression in murine hepatitis virus type 3-induced fulminant hepatitis and in patients with severe viral hepatitis B.

AIM: To evaluate the expression of fibrinogen-like protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury. RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2) was detected in 21 of 23 patients (91.30%) with severe AOC hepatitis B, while only 1 of 13 patients (7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B. CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.