Diagnostic accuracy of magnetically guided capsule endoscopy with a detachable string for detecting oesophagogastric varices in adults with cirrhosis: prospective multicentre study.

OBJECTIVE: To evaluate the diagnostic accuracy and safety of using magnetically guided capsule endoscopy with a detachable string (ds-MCE) for detecting and grading oesophagogastric varices in adults with cirrhosis. DESIGN: Prospective multicentre diagnostic accuracy study. SETTING: 14 medical centres in China. PARTICIPANTS: 607 adults (>18 years) with cirrhosis recruited between 7 January 2021 and 25 August 2022. Participants underwent ds-MCE (index test), followed by oesophagogastroduodenoscopy (OGD, reference test) within 48 hours. The participants were divided into development and validation cohorts in a ratio of 2:1. MAIN OUTCOME MEASURES: The primary outcomes were the sensitivity and specificity of ds-MCE in detecting oesophagogastric varices compared with OGD. Secondary outcomes included the sensitivity and specificity of ds-MCE for detecting high risk oesophageal varices and the diagnostic accuracy of ds-MCE for detecting high risk oesophagogastric varices, oesophageal varices, and gastric varices. RESULTS: ds-MCE and OGD examinations were completed in 582 (95.9%) of the 607 participants. Using OGD as the reference standard, ds-MCE had a sensitivity of 97.5% (95% confidence interval 95.5% to 98.7%) and specificity of 97.8% (94.4% to 99.1%) for detecting oesophagogastric varices (both P<0.001 compared with a prespecified 85% threshold). When using the optimal 18% threshold for luminal circumference of the oesophagus derived from the development cohort (n=393), the sensitivity and specificity of ds-MCE for detecting high risk oesophageal varices in the validation cohort (n=189) were 95.8% (89.7% to 98.4%) and 94.7% (88.2% to 97.7%), respectively. The diagnostic accuracy of ds-MCE for detecting high risk oesophagogastric varices, oesophageal varices, and gastric varices was 96.3% (92.6% to 98.2%), 96.9% (95.2% to 98.0%), and 96.7% (95.0% to 97.9%), respectively. Two serious adverse events occurred with OGD but none with ds-MCE. CONCLUSION: The findings of this study suggest that ds-MCE is a highly accurate and safe diagnostic tool for detecting and grading oesophagogastric varices and is a promising alternative to OGD for screening and surveillance of oesophagogastric varices in patients with cirrhosis. TRIAL REGISTRATION: ClinicalTrials.gov NCT03748563.

Causal associations between gastroesophageal reflux disease and essential hypertension: A bidirectional Mendelian randomization study.

BACKGROUND: Clinical studies have reported that patients with gastroesophageal reflux disease (GERD) have a higher prevalence of hypertension. AIM: To performed a bidirectional Mendelian randomization (MR) analysis to investigate the causal link between GERD and essential hypertension. METHODS: Eligible single nucleotide polymorphisms (SNPs) were selected, and weighted median, inverse variance weighted (IVW) as well as MR egger (MR-Egger) regression were used to examine the potential causal association between GERD and hypertension. The MR-Pleiotropy RESidual Sum and Outlier analysis was used to detect and attempt to reduce horizontal pleiotropy by removing outliers SNPs. The MR-Egger intercept test, Cochran's Q test and "leave-one-out" sensitivity analysis were performed to evaluate the horizontal pleiotropy, heterogeneities, and stability of single instrumental variable. RESULTS: IVW analysis exhibited an increased risk of hypertension (OR = 1.46, 95%CI: 1.33-1.59, P = 2.14E-16) in GERD patients. And the same result was obtained in replication practice (OR = 1.002, 95%CI: 1.0008-1.003, P = 0.000498). Meanwhile, the IVW analysis showed an increased risk of systolic blood pressure (ß = 0.78, 95%CI: 0.11-1.44, P = 0.021) and hypertensive heart disease (OR = 1.68, 95%CI: 1.36-2.08, P = 0.0000016) in GERD patients. Moreover, we found an decreased risk of Barrett's esophagus (OR = 0.91, 95%CI: 0.83-0.99, P = 0.043) in essential hypertension patients. CONCLUSION: We found that GERD would increase the risk of essential hypertension, which provided a novel prevent and therapeutic perspectives of essential hypertension.

Difference between type 2 gastroesophageal varices and isolated fundic varices in clinical profiles and portosystemic collaterals.

BACKGROUND: There is significant heterogeneity between gastroesophageal varices (GOV2) and isolated gastric varices (IGV1). The data on the difference between GOV2 and IGV1 are limited. AIM: To determine the etiology, clinical profiles, endoscopic findings, imaging signs, portosystemic collaterals in patients with GOV2 and IGV1. METHODS: Medical records of 252 patients with gastric fundal varices were retrospectively collected, and computed tomography images were analyzed. RESULTS: Significant differences in routine blood examination, Child-Pugh classification and MELD scores were found between GOV2 and IGV1. The incidence of peptic ulcers in patients with IGV1 (26.55%) was higher than that of GOV2 (11.01%), while portal hypertensive gastropathy was more commonly found in patients with GOV2 (22.02%) than in those with IGV1 (3.54%). Typical radiological signs of cirrhotic liver were more commonly observed in patients with GOV2 than in those with IGV1. In patients with GOV2, the main afferent vessels were via the left gastric vein (LGV) (97.94%) and short gastric vein (SGV) (39.18%). In patients with IGV1, the main afferent vessels were via the LGV (75.61%), SGV (63.41%) and posterior gastric vein (PGV) (43.90%). In IGV1 patients with pancreatic diseases, spleno-gastromental-superior mesenteric shunt (48.15%) was a major collateral vessel. In patients with fundic varices, the sizes of gastric/esophageal varices were positively correlated with afferent vessels (LGVs and PGVs) and efferent vessels (gastrorenal shunts). The size of the esophageal varices was negatively correlated with gastrorenal shunts in GOV2 patients. CONCLUSION: Significant heterogeneity in the etiology and vascular changes between GOV2 and IGV1 is useful in making therapeutic decisions.

Pyrrolizidine alkaloids-induced hepatic sinusoidal obstruction syndrome: Pathogenesis, clinical manifestations, diagnosis, treatment, and outcomes.

Hepatic sinusoidal obstruction syndrome (HSOS) can be caused by the intake of pyrrolizidine alkaloids (PAs). To date, PAs-induced HSOS has not been extensively studied. In view of the difference in etiology of HSOS between the West and China, clinical profiles, imaging findings, treatment, and outcomes of HSOS associated with hematopoietic stem cell transplantation or oxaliplatin might be hardly extrapolated to PAs-induced HSOS. Reactive metabolites derived from PAs form pyrrole-protein adducts that result in toxic destruction of hepatic sinusoidal endothelial cells. PAs-induced HSOS typically manifests as painful hepatomegaly, ascites, and jaundice. Laboratory tests revealed abnormal liver function tests were observed in most of the patients with PAs-induced HSOS. In addition, contrast computed tomography and magnetic resonance imaging scan show that patients with PAs-induced HSOS have distinct imaging features, which reveal that radiological imaging provides an effective noninvasive method for the diagnosis of PAs-induced HSOS. Liver biopsy and histological examination showed that PAs-induced HSOS displayed distinct features in acute and chronic stages. Therapeutic strategies for PAs-induced HSOS include rigorous fluid management, anticoagulant therapy, glucocorticoids, transjugular intrahepatic portosystemic shunt, liver transplantation, etc. The aim of this review is to describe the pathogenesis, clinical profiles, diagnostic criteria, treatment, and outcomes of PAs-induced HSOS.

Unusual cause of lesions in the descending duodenum and liver: A case report and review of literature.

The descending duodenum is rarely involved in Schistosoma japonicum (S. japonicum) infection. Here, we report a case of acute Schistosoma infection, which presented with abdominal pain, abdominal distension and irregular fever. Tumor-like lesions were observed in the descending duodenum. Simultaneously, heterogeneity in hepatic perfusion was demonstrated by dynamic computed tomography scanning. Biopsy of the descending duodenum showed the deposition of Schistosoma eggs. Following administration of the antihelminthic drug praziquantel, the patient showed rapid clinical improvement. In conclusion, we report a patient with acute S. japonicum infection presenting as tumor-like lesions in the descending duodenum and heterogeneity of blood perfusion in liver parenchyma.

CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.

Primary biliary cirrhosis-autoimmune hepatitis overlap syndrome: simplified criteria may be effective in the diagnosis in Chinese patients.

OBJECTIVE: To evaluate the Paris criteria, the revised diagnostic criteria and the simplified diagnostic scoring system in the diagnosis of primary biliary cirrhosis (PBC)-autoimmune hepatitis (AIH) overlap syndrome in Chinese patients. METHODS: Medical records of the patients who were diagnosed with PBC at the Union Hospital and Tongji Hospital, Tongji Medical University, Huazhong University of Science and Technology (Wuhan, Hubei Province, China) from 2003 to 2012 were retrospectively reviewed. The overlap syndrome was diagnosed based on the Paris criteria, the revised criteria and the simplified criteria, respectively. Patients' clinical characteristics, laboratory examination results and histological findings were collected. The sensitivity and specificity of the three criteria for diagnosing PBC-AIH overlap syndrome were calculated. RESULTS: PBC-AIH overlap syndrome was diagnosed in 2, 13 and 10 patients with PBC based on the Paris, the revised and the simplified criteria, respectively. The sensitivity and specificity of the simplified criteria in diagnosing the overlap syndrome was 90.0% and 98.2%, which were the highest among the three criteria, followed by the revised criteria. The Paris criteria showed a high specificity (100%) but a relatively low sensitivity (20.0%). In addition, some patients who did not fulfil the Paris criteria still benefited from the immunosuppressive agents. CONCLUSIONS: For Chinese patients with the PBC-AIH overlap syndrome, the simplified criteria appear to be the most efficacious compared with the Paris criteria and the revised criteria. Further studies should be performed to confirm these observations with respect to long-term outcomes and therapeutic implications.

Persistent effect of IFNAR-1 genetic polymorphism on the long-term pathogenesis of chronic HBV infection.

Genetic polymorphism of IFNAR-1 plays a large role in determining the clearance or chronicity after hepatitis B virus (HBV) exposure. However, it is not clear whether type I interferon receptor-1 (IFNAR-1) variations continuously exert their effects to influence the final outcomes following HBV chronicity, including acute-on-chronic hepatitis B liver failure (ACLF-HBV), chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Here we report that these four potential outcomes of chronic HBV infection are strongly associated with IFNAR-1 polymorphisms through a hospital-based case-control study of 663 cases. ACLF-HBV and HCC were each compared with CHB+LC. In comparison with the G/G genotype, the C/G and C/C genotypes at both single-nucleotide polymorphism (SNP) sites (rs1012335 and rs2257167) showed significant susceptibility to ACLF-HBV (the highest odds ratio [OR] reached 2.374; 95% CI = 1.488, 3.788; p < 0.001 for the C/G genotype at rs2257167), as well as HCC (OR = 2.475; 95% CI = 1.435, 4.426; p < 0.001 for the C/C genotype at rs1012335). The C allele at both loci was a susceptibility allele for ACLF-HBV and HCC, with the highest ORs reaching 1.653 (95% CI = 1.233, 2.216; p < 0.01 at rs1012335) in the ACLF-HBV group, and 1.659 (95% CI = 1.274, 2.159; p < 0.01 at rs1012335) in the HCC group. A strongly linked disequilibrium was also found within these two alleles (p < 0.001). Our research indicates that genetic polymorphisms of IFNAR-1 not only contribute to the determination of clearance or chronicity in the early stages of HBV exposure, but they also persistently influence pathogenesis over the long-term process of chronic HBV infection.

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.

[Insertion of TC motif into hepatitis B virus core protein c/e1 site does not affect the expression of S and e antigen].

OBJECTIVE: To investigate whether insertion of TC motif into hepatitis B virus (HBV) core protein c/e1 site affects the expression of S and e antigen. METHODS: Different oligonucleotides encoding TC motif were inserted into the c/e1 site of the core gene of a 1.3 copy wild-type HBV genome vector. HepG2 cells were divided into several groups of cells to transiently transfect with the wild-type and mutant HBV vectors, respectively. In each group, the expression level of core protein inside cells was detected by western blotting, and the levels of S and e antigen in culture medium were analyzed by ELISA assay. RESULTS: Western blotting showed that these TC-tagged core proteins were expressed at similar level of wild-type one. ELISA assay indicated that the level of S and e antigen in culture medium of different groups were not significantly different. CONCLUSION: Insertion of TC motif into HBV core protein c/e1 site does not interference with the expression of viral protein encoded by HBV genome.

[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.

[Relationship between death receptor 5 and apoptosis in hepatocellular carcinoma].

OBJECTIVE: To investigate the antitumor efficacy of death receptor 5, its ligand (TRAIL) and DR5mAb in human hepatocellular carcinoma. METHODS: Expression of DR5 in the HCC cell lines HepG2, SMMC 7721 and normal human liver cell line LO2 was measured at mRNA and protein level by semi-quantitative RT-PCR and Western blot, respectively. MTT method was used to measure the cell viability and flow cytometry assay was used to detect apoptosis so as to observe the inhibitory effect of TRAIL and DR5mAb on HCC cells. RESULTS: Death receptor 5 was highly expressed in the HCC cell lines, but rarely expressed in normal human liver cell line (P < 0.01). With the increase of TRAIL concentration, the cell viability of HCC cells decreased gradually. However, when the concentration of TRAIL was above 1000 ng/ml, HCC cells were resistant to TRAIL, but still sensitive to DR5mAb. After incubation with DR5mAb (1000 ng/ml) for 24 h, the rate of apoptosis in HCC cells reached to 52.45% +/- 0.57%, which was higher than that incubated with TRAIL under the same condition (14.74% +/- 0.48%) (P < 0.05). The cell viability of normal human liver cell line treated with TRAIL tended to decline with the increase of the concentration, which was significantly different from that of matched control group. But DR5mAb had little effect on normal human liver cell line. CONCLUSION: Death receptor 5 as a target plays an important role in the course of HCC apoptosis induction. Agonistic monoclonal antibody specific for human DR5 can selectively and effectively kill hepatocellular carcinoma cells in vitro, while is not harmful to normal human hepatocytes. It reveals that DR5mAb might provide a new direction in hepatocellular carcinoma treatment research.

[The deletion of La protein binding site in HBV RNA leads to instability of S gene mRNA].

OBJECTIVE: To investigate the effect of deletion of the La protein binding site of HBV RNA, caused by its mutation, on the HBV S-mRNA stability of S gene, to study the role of the site in hepatitis B virus life cycle, and to try to find a new anti-HBV target in the future. METHODS: A HBV vector with mutation related to the La protein binding site was constructed using molecular cloning and PCR based site directed mutagenesis, and the vector was named pHBV-mLa. The HBV RNA secondary structure of the site was calculated using a computer. Normal HBV vectors and mutant vectors were respectively transfected into HepG2 cells by Lipofectamine 2000. HBV S-mRNA levels in the two groups were analyzed using semi-quantitative RT-PCR, and HBsAg secretion into the culture media was tested using ELISA. RESULTS: A HBV vector with mutation related to the La protein binding site was successfully constructed, and it was identified and confirmed using restriction analysis and sequencing. The HBV RNA secondary structure of the mutant vector was completely different to the stem-loop structure of the normal HBV vector. Semi-quantitative RT-PCR and ELISA analyses showed that the level of HBV S-mRNA in the mutant vector group was significantly lower than that in the normal HBV vector group (t'=12.703, P<0.05), and the expression efficacy of HBsAg was reduced in the mutant vector group (t= 44.648, P<0.01). CONCLUSIONS: The change of La protein binding site in the HBV RNA caused by the mutation in HBV DNA disorganizes the stem-loop structure in the HBV RNA site. With the structural change, the La protein cannot bind the site and stabilize the HBV RNA (HBV S-mRNA), as the cleavage site in the upstream of the stem-loop structure is exposed to endoribonuclease. This results in HBV S-mRNA decay and affects the expression of the S gene. This study shows that only the sequence of this site in the HBV DNA is reserved, then the stem-loop structure in the La protein binding site will remain intact, and the disorganization of the stem-loop structure affects the stability of the transcripted HBV RNA. The La protein binding site in HBV RNA and the special secondary structure of the site are crucial to the life cycle of the hepatitis B virus.

[Hammerhead ribozyme-mediated cleavage of transforming growth factor beta1 RNA in a cell-free system and in hepatic stellate cells].

OBJECTIVE: To identify the activity of hammerhead ribozyme against transforming growth factor beta1 (TGFbeta1) in a cell-free system and in activated hepatic stellate cells (HSCs). METHODS: The ribozyme against TGFb1 was designed with computer software. The transcripts of ribozyme, disabled ribozyme and target RNAs were prepared using the RiboMAX large scale RNA production system. The in vitro cleavage reactions were performed through incubation of 32P-labeled target RNAs with ribozyme or disabled ribozyme in different conditions. The eukaryotic expression vector encoding ribozyme and disabled ribozyme were constructed, and then transfected into HSC-T6 cells which exhibited characteristics of activated HSCs. The intracellular activity of the ribozyme was determined by detecting the ribozyme, disabled ribozyme and the TGFbeta1 expression. RESULTS: The ribozyme cleaved target RNAs into anticipated products effectively. As expected, the disabled ribozyme possessed no cleavage activity in vitro. Further study demonstrated that the ribozyme expressed efficiently and inhibited TGFbeta1 expression in activated HSCs, while the disabled ribozyme displayed only a slight effect on TGFbeta1 expression. CONCLUSION: The ribozyme with perfect cleavage activity in the cell-free system used inhibited TGFbeta1 expression effectively in activated HSCs. This ribozyme can provide a potential therapeutic approach for liver fibrosis.

[Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines].

OBJECTIVE: To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines. METHODS: Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot. RESULTS: In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis. CONCLUSION: Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.

Effect of ribozyme against transforming growth factorbeta1 on biological character of activated HSCs.

Transforming growth factorbeta1 (TGFbeta1) is considered to be the principal contributor to liver fibrosis. So in this study the ribozymes against TGFbeta1 were designed. The in vitro cleavage activities of the ribozymes were assayed through incubation of (32)p-labeled target RNAs and (32)p-labeled ribozymes in different conditions. HSC-T6 cells were transfected with the eukaryotic constructs encoding ribozyme and disable ribozyme, then the stable cell clones were used to evaluate its antifibrotic characteristic through the effect of ribozyme on biological character of activated hepatic stellate cells (HSCs). The results demonstrated that two ribozymes (Rz803 and Rz1395) could cleave target RNAs into expected products effectively, Rz803 possessed better cleavage activity in vitro. Stable transfection of Rz803 into activated HSCs reduced TGFbeta1 expression in mRNA and protein level efficiently. The further studies demonstrated that Rz803 reduced deposition of collagen I, suppressed HSC proliferation, but had no effect on HSC activation in transfected HSC-T6 cells. Therefore, it indicated that Rz803 could reverse the character of activated HSCs by down-regulating TGFbeta1 expression efficiently and diminishing TGFbeta1 signaling underlying activation of hepatic stellate cells. As the consequence, it would provide a potential therapeutic approach for liver fibrosis.

Ribozymes against TGFbeta1 reverse character of activated hepatic stellate cells in vitro and inhibit liver fibrosis in rats.

BACKGROUND/AIMS: Transforming growth factor beta (TGFbeta1) is considered the key mediator in the process of liver fibrosis. The purpose of this investigation was to evaluate the activity of ribozymes against TGFbeta1 in a cell-free system and activated hepatic stellate cells (HSCs), and antifibrotic effect in activated HSCs in vitro and in rats. METHODS: Three ribozymes targeting against TGFbeta1 mRNA were designed, and then cloned into the U1 snRNA expression cassette. The chimeric ribozymes were selected for the analysis of their performances in activated HSCs through the detection of their cleavage activities in a cell-free system. After ribozyme-encoding plasmids had been transfected into HSC-T6 cells, the effects of ribozymes on activated HSCs were evaluated through the analysis of proliferation, activation and collagen deposition of HSC-T6. The adenoviral vector expressing the ribozymes was constructed, and then delivered into rat models of hepatic fibrosis induced by carbon tetrachloride. RESULTS: TGFbeta1 expression was efficiently down-regulated in activated HSCs by U1 snRNA chimeric ribozymes which possessed perfect cleavage activity in a cell-free system. Further studies demonstrated that U1 snRNA chimeric ribozymes inhibited the synthesis of collagen I, reduced deposition of collagen I, suppressed BrdU incorporation, but had no effect on desmin and alpha-SMA expression in transfected HSC-T6 cells. Histological analysis demonstrated that the adenoviral vector expressing ribozyme (Rz803) could alleviate fibrotic pathology in rats treated with carbon tetrachloride. CONCLUSIONS: The anti-TGFbeta1 ribozymes could reverse the character of activated HSCs in vitro and improve fibrotic pathology in vivo. It indicated that TGFbeta1 could be considered as a novel candidate for a therapeutic agent against hepatic fibrosis.