Preparation and identification of anti-transforming growth factor β1 U1 small nuclear RNA chimeric ribozyme in vitro

AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: Active UlsnRNA chimeric ribozyme (U1Rz803)had the best cleavage activity at 50 °C; at 37 °C, it was active, Km=34.48 nmol/L, Kcat=0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.

Combined gene therapy of endostatin and interleukin 12 with polyvinylpyrrolidone induces a potent antitumor effect on hepatoma

AIM: To study the antitumor effect of combined gene therapy of endostatin and interleukin 12 (IL-12) with polyvinylpyrrolidone (PVP) on mouse transplanted hepatoma.METHODS: Mouse endostatin eukaryotic plasmid (pSecES) with a mouse Igk signal sequence inside and mouse IL-12 eukaryotic plasmid (pmIL-22) were transfected into BHK-22cells respectively. Endostatin and IL-22 were assayed by ELISA from the supernant and used to culture endothelial cells and spleen lymphocytes individually. Proliferation of the latter was evaluated by M-I-r. H22 cells were inoculated into the leg musde of mouse, which was injected intratumorallywith pSecES/PVP, pmIL-12/PVP or pSecES+pmIL-12/PVPrepeatedly. Tumor weight, serum endostatin and serumIL-22 were assayed. Tumor infiltrating lymphocytes, tumormicrovessel density and apoptosis of tumor cells were also displayed by HE staining, CD32 staining and TUNEL.RESULTS: Endostatin and IL-12 were secreted after transfection, which could inhibit the proliferation of endothelial cells or promote the proliferation of spleen lymphocytes.Tumor growth was highly inhibited by 92.8% after injection of pSecES+pmIL-i2/PVP accompanied by higher serum endostatin and IL-22, more infiltrating lymphocytes, fewertumor vessels and more apoptosis cells compared with injection of pSecES/PVP, pmIL-i2/PVP or vector/PVP.CONCLUSION: Mouse endostatin gene and IL-12 gene can be expressed after intratumoral injection with PVP.Angiogenesis of hepatoma can be inhibited synergisticly,lymphocytes can be activated to infiltrate, and tumor cells are induced to apoptosis. Hepatoma can be highly inhibited or eradiated.

Kangxian ruangan keli inhibits hepatic stellate cell proliferation mediated by PDGF

AIM: To investigate the effect of Kangxian ruangan keli (KXR) on hepatic stellate cell (HSC) proliferation mediated by platelet-derived growth factor (PDGF) and the underlying mechanism.METHODS: In a serum-free culture system, HSCs were treated with a KXR preparation for 24 hours, followed by stimulation with PDGF-BB for 24 hours. Then the cells were incubated again in the medium containing KXR for 3 hours stimulated with PDGF-BB for 5 minutes, and collected. The proliferation of HSC was examined using an MTT assay and flow cytometry. Tyrosine phosphorylation was detected with Western blotting and visualized by the enhenced chemiluminescent (ECL) method.RESULTS: The OD values for the HSCs growing in the media without and with addition of PDGF were 0.17±0.06 and0.82±0.05, respectively. The PDGF-induced increase was hindered remarkably by KXR preparation in a dose-dependent manner. The reaction values for the systems with 5mg/mL, 2.5 mg/mL and 1.25 mg/mL of KXR were 0.28±0.03,0.37±0.02 and 0.43±0.04, respectively. Moreover, the percentages of S-phase cells in these KXR-containing culturesystems were 10.95±1.35, 32.76±1.07 and 43.19±1.09,respectively, all of which were significantly lower than that in the culture free of KXR (68.24±2.72). In addition, the values for tyrosine-phosphorylated protein in HSCs treated with 5 mg/mL and 1.25 mg/mL of KXR were 0.1349±0.0072 and 0.1658±0.0025, respectively, which were smaller than that in the cells treated only with PDGF-BB (0.1813±0.0117).CONCLUSION: Within the dose range used in the present study, KXR preparation shows an inhibitory effect on HSC proliferation induced by PDGF. The mechanism of this process may involve interference with tyrosine phosphorylation mediated by PDGF.

Inhibition of hepatitis B virus by a novel L-nucleoside,β-L-D4A and related analogues

AIM: To explore the inhibition of β-L-D4A on hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome.METHODS: 2.2.15 cells were plated at a density of 5×10^4 per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. In the end,5μl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with HindⅢ. Both DNAs were subjected to Southern blot,hybridized with a ^32P-labeled HBV probe and autoradiophed.Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED50 was calculated. Then Hybond-N membrane was washed and rehybridized with a ^32P-labeled mtDNA-specific probe, and effect of β-L-D4A on mitochondrial DNA was studied. 2.2.15 cells were also seeded in 24-well tissue culture plates,and cytotoxicity with different concentrations was examined by MTT method. ID50 was calculated. Structure-activity relationships between D2A and D4A were also studied as above.RESULTS: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNAwas inhibited in a dose-dependent manner. ED50 was 0.2μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID50 at 200 μM.CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxidty and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.

5-aminosalicylicacid is an attractive candidate agent for chemoprevention of colon cancer in patients with inflammatory bowel disease

Inflammatory bowel disease (IBD) is classically subdivided into ulcerative colitis (UC) and Crohn's disease (CD). Patients with IBD have increased risk for colorectal cancer. Because the pathogenesis of colorectal carcinoma has not been entirely defined yet and there is no ideal treatment for colon cancer, cancer prevention has become increasingly important in patients with IBD. The two adopted methods to prevent the development of colon cancer in clinical practice include the prophylactic colectomy and colonoscopic surveillance.But patients and physicians seldom accept colectomy as a routine preventive method and most patients do not undergo appropriate colonoscopic surveillance. Chemoprevention refers to the use of natural or synthetic chemical agents to reverse, suppress, or to delay the process of carcinogenesis.Chemoprevention is a particularly useful method in the management of patients at high risk for the development of specific cancers based on inborn genetic susceptibility, the presence of cancer-associated disease, or other known risk factors. Prevention of colorectal cancer by administration of chemopreventive agents is one of the most promising options for IBD patients who are at increased risks of the disease. The chemopreventive efficacy of nonsteroidal antiinflammatory drugs (NSAIDs) against intestinal tumors has been well established. But with reports that NSAIDs aggravated the symptoms of colitis, their sustained use for the purpose of cancer chemoprevention has been relatively contraindicated in IBD patients. Another hopeful candidate chemoprevention drug for IBD patients is 5-aminosalicylic acid (5-ASA), which is well tolerated by most patients and has limited systemic adverse effects, and no gastrointestinal toxicity. 5-ASA lacks the well-known side effects of longterm NSAIDs use. Retrospective correlative studies have suggested that the long-term use of 5-ASA in IBD patients may significantly reduce the risk of development of colorectal cancer. According to the literature, this agent might well satisfy clinical expectations with respect to a safe and effective chemopreventive agent.

Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro

AIM: To study the preparation and cleavage activity of HpRzdirected against the transcript of HBV core gene in vitro.METHODS: HpRz gene designed by computer targeting thetranscript of HBV core gene was cloned into the vector p1 .5between 5'-cis-Rz and 3'-cis-Rz. 32p-labeled HpRz transcriptproved whether the vector fit for the preparation of hairpinribozyme in vitro. 32p-labeled pKC transcript containing HBVcore region as target-RNA was transcribed using T7 RNApolymerase and purified by denaturing PAGE. Cold HpRztranscript was incubated with 32p-labeled target-RNAs underdifferent conditions and radioautographed after denaturingpolyacrylamide gel electrophoresis.RESULTS: HpRz has the specific ability of cleavage of target-RNA at 37℃ and 12 mM MgCL2. Km = 26.31nmol/L, Kcat = 0.18/min. These results revealed that the design of HpRz wascorrect.CONCLUSION: HpRz prepared in this study possessesspecific catalytic activity from the identification of cleavageactivity. These results indicate that hairpin ribozyme mayintracellularly inhibit the replication of HBV, therefore it maybecome a novel potent weapon for the treatment of hepatitisB.

Association between TRAIL expression on peripheral blood lymphocytes and liver damage in chronic hepatitis B

AIM:To explore a novel mechanism for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), upregulation of CD4+ and CD8+T lymphocytes participating in the patho-physiological process of chronic hepatitis B (CHB). METHODS: The levels of serum soluble TRAIL (sTRAIL), serum IFN-γ and membrane-bound TRAIL expression on peripheral leucocytes from 58 CHB patients were examined by ELISA and flow cytometry respectively. The levels of TRAIL were compared with the baseline levels of 17 healthy controls, and correlation analysis was performed between ALT, TBIL, PT, morphological change in hepatic tissues, and serum IFN-γ. RESULTS: The results showed that TRAIL levels on membranes of CD4+, CD8+ T cells in CHB patients were much higher than those in healthy controls (P<0.001), and were correlated with serum TBIL (r=0.354, P= 0.008 for CD4+ and r= 0.522, P= 0.000 for CD8+, respectively), ALT (r= 0.393, P= 0.003 for CD8+), PT (r = 0.385, P = 0.004 for CD8+) and serum IFN-y level (r = 0.302, P= 0.011 for CD4+ and r= 0.307, P= 0.009 for CD8+). On the contrary to membrane-bound TRAIL expression, serum level of sTRAIL was not correlated with that of TBIL and PT, though it was higher than that of the normal population and was positively correlated with serum HBeAg expression (r= 0.695, P = 0.001). CONCLUSION: The expression level of TRAIL on the membrane of lymphocytes was upregulated and associated with the liver injury in CHB patients. These findings suggest that upregulation of TRAIL expression may be induced by virus antigen and inflammatory cytokine IFN-γ.

Maxizyme-mediated specific inhibition on mutant-type p53 in vitro

AIM: To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG→AGT)in vitro.METHODS: Two different monomers of anti-mtp53maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G5→A5) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR,pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme).Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEN-T vector under the T7 promoter control.The 32p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with 32p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis.RESULTS: In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 ℃ and 25 mM MgCL2. The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either.CONCLUSION: pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good fundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.

Study on the mechanism of epidermal growth factor-induced proliferation of hepatoma cells

AIM: Many growth factors, such as epidermal growth factor(EGF), are associated with the carcinogenesis. EGF plays itsrole in the proliferation of hepatoma cells through bindingwith EGF receptor (EGFR) and a series of signal transduction.But the postreceptor pathway is still not clear. In the presentexperiment, we studied the effect of tyrosine kinase, proteinkinase C, Na+/H+ exchange, calmodulin and voltage-dependent Ca2+ channel on EGF-induced hepatoma cellproliferation.METHODS: Hepatoma cell line SMMC7721 was cultured inRPMI1640 serum-free medium. In order to study the effectof thyrosine kinase, protein kinase C, Na+/H+ exchange,calmodulin and voltage-dependent Ca2+ channel on humanheptoma cell proliferation induced by epidermal growth factor(EGF), DNA synthesis rate of hepatoma cells was measuredby the method of 3H-TdR incorporation.RESULTS: EGF (10-9 M) stimulated the proliferation of heptomacells significantly (3H-TdR incorporation was 1 880+281 cpm/well, P＜0.05), and this effect was significantly inhibited bytyrosine kinase inhibitor genistein (3H-TdR incorporation was808±209 cpm/well, P＜0.001). Calmedulin inhibitor W-7, proteinkinase C inhibitor H-7 and Na+/H+ exchange inhibitor amilorideindividually had significant inhibiting effect on EGF-inducedproliferation of hepatoma cells (3H-TdR incorporation was978±87.3 cpm/well, 1 241+147 cpm/well, 1 380+189 cpm/well, respectivly, P＜0.001, P＜0.01, P＜0.05), but they allhad no effect on the basal level proliferation of culturedhepatoma cells (3H-TdR incorporation was 1 284+260 cpm/well, 1 179+150 cpm/well, 1 392+152 cpm/well, respectivly,3H-TdR incorporation of the control was 1353+175 cpm/well, P＞0.05). Voltage-dependent Ca2+ channel inhibitorverapamil had no inhibition on EGF-induced proliferation ofhepatoma cells (3H-TdR incorporation was 1 637+133 cpm/well, P＞0.05), it also had no effect on the basal levelproliferation of cultured hepatoma cells (3H-TdR incorporationwas 1196+112 cpm/well,P＞0.05).CONCLUSION: Our data suggest that tyrosine kinase, Ca2+-calmodulin-dependent pathway, protein kinase C and Na+/H+ exchange play a critical role in EGF-induced proliferationof hepatoma cells and that the effect of EGF is independentof voltage-dependent Ca2+ channel.

Experimental study of effect of Ganyanping on fibrosis in rat livers

AIM: To observe the effects of Ganyanping on CCl4-induced hepatic fibrosis in rats. METHODS: The rats were separated randomly into five groups. Groups A to group D, each consisting of 15 rats,were for different tests, while 8 rats were used as normal controls (N). For group D, CCl4 was injected subcutaneously,at a dosage of 3 mi/kg for 9 weeks. For group A,Ganyanping was administered via gastric tube at a dosage of L0 mi/kg. For group B, the treatment with Ganyanping was started 4 weeks after CCl4 administration. Tn group C, Ganyanping was administered 8 weeks after the intoxication, and treatment lasted for 4 weeks. Liver tissues were fixed in 10 % formalin and embedded in paraffin. Pathologic changes, particularly fibrosis, were evaluated on the HE and V-G-stained sections. Ten middlepower fields were randomly selected for assessment of collagen deposition. RESULTS: Loss of normal hepatic architecture, some with pseudo-lobule formation, was observed in group D, while hepatocytes steatosis and fibrosis were less pronounced in the animals treated with Ganyanping. Pseudo-lobule formation was not evident in the latter groups. The total collagen area and ratio were 840.23±81.65 and 7.0+0.9,respectively in group D, the ratio being reduced greatly in the Ganyanping-treated groups (148.73+45.89 and 1.16+0.33, respectively). The activities of MAO and ACP were elevated and that of SDH in group D decreased in the hepatic tissue as compared to the control group. The treatment with Ganyanping abrogated these enzymatic changes. CONCLUSION: Our data approved that Ganyanping could improve the microcirculation in the liver, reduce oxygenderived free radicals, and enhance the cellular metabolism and immune function, all resulting in an anti-fibrotic effect.Hence, Ganyanping can protect the liver from fibrosis. It may be a safe and effective preparation for patient with fibrosis.

Apoptosis pathway of liver cells in chronic hepatitis

AIM: To study the pathway of apoptosis in chronic liver disease and the role of mitochondria in programmed cell death.METHODS: Liver biopsy specimens from 72 cases of chronic hepatitis and 29 cases of post hepatitis cirrhosis were studied. The pro-apoptotic protein Fas, FasL, Bax and the anti-apoptotic protein Bcl-2, Bcl-xL, Bcl-2α were studied immunohistochemically by SP method. Specimens from 15 cases of chronic hepatitis and post hepatitis cirrhosis were examined for their ultramicrostructures with special attention to their mitochondrial changes. Specimens from 3 normal adults (demised in traffic accidents) were used as control.RESULTS: The expression of proapoptotic proteins (Fas, FasL, Bax) in hepatocytes was significantly higher in the chronic hepatitis group than in the cirrhosis group (P<0.001). In the study of ultramicrostructure 364 hepatocytes were examined, from 12 cases of chronic hepatitis (including 10 mild cases, 1 moderate case and 1 severe case). Out of 364 hepatocytes 40 (11.0%) hepatocytes were found with various kinds of destruction in their mitochondria. Rupture of the outer membrane of mitochondria and the leakage of matrix from the intermembrane space were definitely demonstrated. The ultramicrostructural changes of mitochondria in the chronic hepatitis group were statistically higher than that in normal adults control group(X^2=4.32, P<0.05). CONCLUSION: The result of the study was in support of the current view that the apoptotic process in chronic hepatitis patients were largely along the intrinsic pathway (mitochondrial pathway), given that the intrinsic and extrinsic pathways could interlinked (converged) at some point on their progression, also it is impossible at present to exclude the possibility that the two pathways could be chosen by hepatocytes in parallel simultaneously.

Multicenter clinical study on Fuzhenghuayu capsule against liver fibrosis due to chronic hepatitis B

AIM: To study the efficacy and safety of Fuzhenghuayu capsule (FZHY capsule, a capsule for strengthening body resistance to remove blood stasis) against liver fibrosis due to chronic hepatitis B. METHODS: Multicenter, randomized, double blinded and parallel control experiment was conducted in patients (aged from 18 to 65 years) with liver fibrosis due to chronic hepatitis B. Hepatic histologic changes and HBV markers were examined at wk 0 and 24 during treatment. Serologic parameters (HA, LM, P-Ⅲ-P, Ⅳ-C) were determined and B ultrasound examination of the spleen and liver was performed at wk 0, 12 and 24. Liver function (liver function and serologic parameters for liver fibrosis) was observedat wk 0, 6, 12, 18 and 24. Blood and urine routine test, renal function and ECG were examined before and after treatment. RESULTS: There was no significant difference between experimental group (110 cases) and control group (106 cases) in demographic features, vital signs, course of illness, history for drug anaphylaxis and previous therapy, liver function, serologic parameters for liver fibrosis, liver histologic examination (99 cases in experimental group, 96 cases in control group), HBV markers, and renal function. According to the criteria for liver fibrosis staging, meanscore of fibrotic stage(s) in experimental group after treatment (1.80) decreased significantly compared to the previous treatment (2.33, P＜0.05), but there was no significant difference in mean score of fibrotic stage(s) (2.11 and 2.14 respectively). There was a significant difference in reverse rate between experimental group (52%) and control group (23.3%) in liver biopsy. With marked effect on decreasing the mean value of inflammatory activity and score of inflammation (P＜0.05), Fuzhenghuayu capsule had rather good effects on inhibiting inflammatory activity and was superior to that of Heluoshugan capsule. Compared to that of pretreatment, there was a significant decrease in HA, LM, P-Ⅲ-P and Ⅳ-C content in experimental group after 12 and 24 wk of treatment. The difference in HA, LM, P-Ⅲ-P and Ⅳ-C content between 12 and 24 wk of treatment and pretreatment in experimental group was significantly greater than that in control group (P＜0.01-0.05). The effect, defined as two of four parameters lowering more than 30% of the baseline, was 72.7% in experimental group and 27.4% in control group (P＜0.01). Obvious improvement in serum Alb, ALT, AST and GGT was seen in two groups. Compared to that of control group, marked improvement in GGT and Alb was seen in experimental group (P＜0.05). The effective rate of improvement in serum ALT was 72.7% in experimental group and 59.4% in control group. No significant difference was seen in blood and urine routine and ECG before and after treatment. There was also no significant difference in stable rate in ALT and serologic parameters for liver fibrosis between experimental group and control group after 12 wk of withdrawal. CONCLUSION: Fuzhenghuayu capsule has good therapeutic effects on alleviating liver fibrosis due to chronic hepatitis B without any adverse effect and is superior to that of Heluoshugan capsule.

Effect of Calmodulin and Voltage-dependent Ca^(2+) Channel on the Proliferation of Heptoma Cells Induced by Epidermal Growth Factor

The effect of thyrosine kinase, calmodulin and voltage-dependent Ca 2+ channel on the proliferation of hepatoma cells induced by EGF was studied. Hepatoma cell line SMMC7721 was cultured in RPMI1640 serum-free medium. DNA synthesis rate of hepatoma cells was measured by 3H-TdR incorporation. 10 -9 mol/L EGF could significantly stimulate the proliferation of hepatoma cells (P<0.05), and this effect might be significantly inhibited by tyrosine kinase inhibitor (P<0.001). Calmodulin inhibitor W-7 had no effect on the basic phase of cultured hepatoma cells (P> 0.05), but it had very significantly inhibitory effect on the proliferation of hepatoma cells induced by EGF (P<0.001). Voltage-dependent Ca 2+ channel inhibitor Varapamil had no inhibition on the proliferation of hepatoma cells induced by EGF (P>0.05). It had no effect on the basic phase of cultured hepatoma cells (P>0.05). It is suggested that tyrosine kinase and Ca 2+-calmodulin-dependent pathway may play a critical role on the proliferation of heptoma cells induced by EGF, and voltage-dependent Ca 2+ channel is independent of the effect of EGF.