Hepatoprotective activity of Sapindus mukorossi and Rheum emodi extracts： In vitro and in vivo studies

AIM： To study the hepatoprotective capacity of Sapindus mukorossi （S. mukorossi） and Rheum emodi （R. emodi） extracts in CCl4 treated male rats. METHODS： The dried powder of S. mukorossi and R. emodi was extracted successively with petroleum ether, benzene, chloroform, and ethanol and concentrated in vacuum. Primary rat hepatocyte monolayer cultures were used for in vitro studies. In vivo, the hepatoprotective capacity of the extract of the fruit pericarp of S. mukorossi and the rhizomes of R. emodi was analyzed in liver injured CCl4-treated male rats. RESULTS： In vitro： primary hepatocytes monolayer cultures were treated with CCl4 and extracts of S. mukorossi ＆ R. emodi. A protective activity could be demonstrated in the CCl4 damaged primary monolayer culture. In vivo ： extracts of the fruit pericarp of S. mukorossi （2.5 mg/mL） and rhizomes of R. emodi （3.0 mg/mL） were found to have protective properties in rats with CCl4 induced liver damage as judged from serum marker enzyme activities. CONCLUSION： The extracts of S. mukorossi and R. emodi do have a protective capacity both in vitro on primary hepatocytes cultures and in in vivo in a rat model of CCl4 mediated liver injury.     

Helicobacter pylori lipopolysaccharide： Biological activities in vitro and in vivo, pathological correlation to human chronic gastritis and peptic ulcer

AIM: To determine the biological activity of Helicobacter pylori(H pylori) lipopolysaccharide (H-LPS) and understand pathological correlation between H-LPS and human chronic gastritis and peptic ulcer.METHODS: H-LPS of a clinical Hpylori strain and LPS of Escherichia coli strain O55:B5(E-LPS) were extracted by phenol-water method. Biological activities of H-LPS and E-LPS were detected by limulus lysate assay, pyrogen assay,blood pressure test and PBMC induction test in rabbits, cytotoxicity test in NIH 3T3 fibroblast cells and lethality test in NIH mice. By using self-prepared rabbit anti-H-LPS serum as the first antibody and commercial HRP-labeled sheep anti-rabbit sera as the second antibody, H-LPS in biopsy specimens from 126 patients with chronic gastritis (68 cases) or gastric ulcer (58 cases) were examined by immunohistochemistry.RESULTS: Fibroblast cytotoxicity and mouse lethality of H-LPS were weaker than those of E-LPS. But the ability of coagulating limulus lysate of the two LPSs was similar (+/0.5 ng/mL).At 0.5 h after H-LPS injection, the blood pressures of the 3 rabbits rapidly declined. At 1.0 h after H-LPS injection, the blood pressures in 2 of the 3 rabbits fell to zero causing death of the 2 animals. For the other one rabbit in the same group, its blood pressure gradually elevated. At 0.5 h after E-LPS injection, the blood pressures of the three rabbits also quickly declined and then maintained at low level for approximately 1.0 h. At 0.5 hafter injection with H-LPS or E-LPS, PBMC numbers of the rabbits showed a remarkable increase. The total positivity rate of H-LPS from 126 biopsy specimens was 60.3%(76/126). H-LPS positivity rate in the biopsy specimens from chronic gastritis (50/68, 73.5%) was significantly higher than that from gastric ulcer (26/58, 44.8%) (χ^2=10.77,P＜0.01). H-LPS positivity rates in biopsy specimens from chronic superficial gastritis (38/48, 79.2%) and chronicactive gastritis (9/10, 90.0%) were significantly higher than that of the patients with atrophic gastritis (3/10, 30.0%)(χ^2=7.50-9.66,P＜0.01). CONCLUSION: The biological activities of H-LPS were weaker than those of E-LPS, the activities of H-LPS of lowering rabbit blood pressure and inducing rabbit PBMC were relatively stronger. H-LPS may play a critical role in inducing inflammatory reaction in human gastritis.     

Antimicrobial activity of Sapindus mukorossi and Rheum emodi extracts against H pylori： In vitro and in vivo studies

INTRODUCTION H pylori has infected more than half the population of the world. Most people are unaware that they are infected because they remain asymptomatic throughout life and survive without any harmful infection-related clinical sequelae. However, so…     

Platelet aggregation is affected by nitrosothiols in patients with chronic hepatitis： In vivo and in vitro studies

AIM： To investigate the relationship among the number of platelets and plasma levels of S-nitrosothiols （S-NO）, nitrite, total non-protein SH （NPSH）, glutathione （GSH）, cysteine （CYS）, malondialdehyde （MDA）, 4-hydroxininenal （4HNE）, tumor necrosis factor-alpha （TNFα） and interleukin （IL）-6 in patients with chronic hepatitis C （CH）.
METHODS： In vitro the aggregation of platelets derived from controls and CH patients was evaluated before and after the addition of adenosine diphosphate （ADP） and collagen, both in basal conditions and after incubation with nitrosoglutathione （GSNO）.
RESULTS： In vivo, S-NO plasma levels increased significantly in CH patients and they were significantly directly correlated with platelet numbers. Patients with platelet counts 〈 150000/μL, had a smaller increase in S-NO, lower levels of GSH, CYS, NPSH, TNFα, and IL-6, and higher levels of nitrite, MDA, and 4-HNE relative to those of patients with platelet counts 〉 150000/μL. In vitro, the ADP and collagen aggregation time was increased in platelets from patients and not from controls; in addition, platelets from CH patients but not from controls also showed a latency time after exposure to collagen.
CONCLUSION： The incubation of platelets with GSNO improved the percentage aggregation and abolished the latency time.     

In vitro and in vivo effects of ferulic acid on gastrointestinal motility： Inhibition of cisplatin-induced delay in gastric emptying in rats

INTRODUCTIONPhenolic acids are plant components ubiquitously present in many fruits, vegetables and grains. They constitute a major portion of the human daily intake of non-nutrients[1]. There is increasing evidence of positive health benefits of the high…     

Ex-vivo evaluation of gene therapy vectors in human pancreatic （cancer） tissue slices

AIM： To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions.
METHODS： Human pancreatic tissue samples （malignant and normal） were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% 02. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection.
RESULTS： With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants.
CONCLUSION： The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.     

Hepatoprotective and antioxidant activity of pentagamavunon-0 against carbon tetrachloride-induced hepatic injury in rats

ObjectiveTo investigate the hepatoprotective and antioxidant activity of pentagamavunon-0(PGV-0) against CCl₄-induced hepatic injury in rats.MethodsThe groups of animals were administered with PGV-0 at the doses 2.5, 5, 10, and 20 mg/kgb.w., p.o. once in a day for 6 days and at day 7 the animals were administrated with carbon tetrachloride (CCl) (20%, 2 mL/kgb.w. in liquid paraffin (i.p.). The effect of PGV-0 on serum transaminase (SGPT), alkaline phosphates (ALP) and total bilirubin were determined in CCl₄-induced hepatotoxicity in rats. Further, the effects of PGV-0 on glutathione (GSH) content, catalase (CAT) and NO free radical scavenging activity also were investigated.ResultsThe results demonstrated that PGV-0 significantly reduced the activity of SGPT, serum ALP and total bilirubin in CCl₄induced rat hepatotoxicity. PGV-0 has effect on the antioxidant and free radical defense system. It prevented the depletion level of GSH and decrease activity of CAT in CCl₄-induced liver injury in rats. PGV-0 also demonstrated the free radical scavenger effects on NO free radical scavenging activity with ES value of 32.32μM.ConculsionAll of our findings suggests that PGV-0 could protect the liver cells from CCl₄-induced liver damages and the mechanism may through the antioxidative effect of PGV-0 to prevent the accumulation of free radicals and protect the liver damage.     

In vitro and in vivo protective effects of proteoglycan isolated from mycelia of Ganoderma lucidum on carbon tetrachloride-induced liver injury

AIM: To investigate the possible mechanism of the protective effects of a bioactive fraction,Ganoderma lucidum proteoglycan (GLPG) isolated from Ganoderma lucidum mycelia, against carbon tetrachloride-induced liver injury. METHODS: A liver injury model was induced by carbon tetrachloride. Cytotoxicity was measured by MTT assay.The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined with an automatic multifunction-biochemical analyzer and the levels of superoxide dismutase (SOD)and TNF-alpha were determined following the instructions of SOD kit and TNF radioimmunoassay kit. Liver sections were stained with hematoxylin and eosin (H and E) for histological evaluation and examined under light microscope. RESULTS: We found that GLPG can alleviate the L-02 liver cells injury induced by carbon tetrachloride (CCl4) through the measurements of ALT and AST activities and the administration of GLPG to L-02 cells did not display any toxicity. Furthermore, histological analysis of mice liver injury induced by CCl4 with or without GLPG pretreatment indicated that GLPG can significantly suppress the toxicity induced by CCl4 in mice liver. We also found that GLPG reduced TNF-alpha level induced by CCl4 in the plasma of mice, whereas increased SOD activity in the rat serum. CONCLUSION: GLPG has hepatic protective activity against CCl4-induced injury both in vitro and in vivo. The possible anti-hepatotoxic mechanisms may be related to the suppression of TNF-alpha level and the free radical scavenging activity.     

In vitro study of lovastatin interactions with amiodarone and with carbon tetrachloride in isolated rat hepatocytes

AIM： To investigate the interactions at a metabolic level between Iovastatin, amiodarone and carbon tetrachloride in isolated rat hepatocytes.
METHODS： For cell isolation two-step collagenase liver perfusion was performed. Lovastatin was administered alone in increasing concentrations （1μmol/L, 3μmol/L, 5μmol/L and 10μmol/L） and in combination with CCh （86μmol/L）. The cells were also pretreated with 14μmol/L amiodarone and then the other two compounds were added.
RESULTS： Lovastatin promoted concentration-dependent significant toxicity estimated by decrease in cell viability and GSH level by 45% and 840, respectively, LDH- activity increased by 114% and TBARS content by 90%, CCl4 induced the expected severe damage on the examined parameters, CCh induced toxicity was attenuated after Iovastatin pretreatment, which was expressed in less increased values of LDH activity and TBARS levels, as well as in less decreased cell viability and GSH concentrations, However, the pretreatment of hepatocytes with amiodarone abolished the protective effect of Iovastatin.
CONCLUSION： We suggest that the observed cytoprotective effect was due to interactions between Iovastatin, CCh and amiodarone at a metabolic level.     

Hepatoprotective effects of baicalein against CCl4-induced acute liver injury in mice

AIM: To investigate the hepatoprotective effect of baicalein against carbon tetrachloride (CCl₄)-induced liver damage in mice.METHODS: Mice were orally administered with baicalein after CCl₄ injection, and therapeutic baicalein was given twice a day for 4 d. The anti-inflammation effects of baicalein were assessed directly by hepatic histology and serum alanine aminotranferease and aspartate aminotransferase measurement. Proliferating cell nuclear antigen was used to evaluate the effect of baicalein in promoting hepatocyte proliferation. Serum interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assay and liver IL-6, TNF-α, transforming growth factor-α (TGF-α), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) genes expression were determined by quantitative real-time polymerase chain reaction.RESULTS: CCl₄-induced acute liver failure model offers a survival benefit in baicalein-treated mice. The data indicated that the mRNA levels of IL-6 and TNF-α significantly increased within 12 h after CCl₄ treatment in baicalein administration groups, but at 24, 48 and 72 h, the expression of IL-6 and TNF-α was kept at lower levels compared with the control. The expression of TGF-α, HGF and EGF was enhanced dramatically in baicalein administration group at 12, 24, 48 and 72 h. Furthermore, we found that baicalein significantly elevated the serum level of TNF-α and IL-6 at the early phase, which indicated that baicalein could facilitate the initiating events in liver regeneration.CONCLUSION: Baicalein may be a therapeutic candidate for acute liver injury. Baicalein accelerates liver regeneration by regulating TNF-α and IL-6 mediated pathways.     

Hepatoprotective and antioxidant capacity of Melochia corchorifolia extracts

ObjectiveTo evaluate hepato protective and antioxidant capacity of Melochia corchorifolia (M. corchorifolia) aerial part extracts.MethodsAntioxidant activity was evaluated by using three free radicals (Superoxide, Hydroxyl and DPPH) and hepatoprotective activity was assessed against CCl₄ induced liver intoxication in rats.ResultsThe extracts produced concentration dependent percentage protection in decrease of serum enzymes and percentage inhibition on free radicals. Among all extracts methanol extract showed better activity with percentage protection of SGOT (78.98%), SGPT (79.65%), ALP (82.48%) and total bilirubin (80.0%) levels against CCl₄ liver intoxication and also methanolic extract showed better activity with IC₅₀ values on superoxide, hydroxyl and DPPH radicals were 127 μ g, 240 μ g and 179 μ g.ConclusionsFrom the results obtained during the study it could be concluded that M. corchorifolia aerial part extracts have antioxidant and hepatoprotective components. Further study is necessary for isolation and characterization of bioactive molecules which are responsible for hepatoprotective and antioxidant activity.     

Heparin-antithrombin III binding. In vitro and in vivo studies

Heparin antithrombin III binding was studied by crossed immunoelectrophoresis. In plasma and purified antithrombin III standard, multicomponent patterns were obtained with low concentrations of mucosal heparin. There is evidence that antithrombin III may bind more than one heparin molecule. At high heparin concentration (greater than 16 U/ml), single symmetrical peaks were obtained. Serum samples showed two antithrombin III peaks due to a decreased heparin binding of the slower peak (2.1-3.9 times), which was probably antithrombin III-activated procoagulant complexes. Heparin analogue (A 73025) also bound antithrombin III in vitro but the mobility of the peak was slower than with mucosal heparin and only a single peak was obtained in serum samples. Radioimmunoassay showed a decreased binding of antithrombin III antibody to heparin-antithrombin III complex. Venous occlusion to the forearm resulted in a slow second peak in the plasma. Heparin therapy gave rise to a double peak in the plasma antithrombin III profile and with continuous infusion, quantitative decreases were noted in all subjects studied, two of whom rethrombosed at the end of 7 days therapy.     

Human lymphoblastoid interferon: In vitro and in vivo studies in hepatocellular carcinoma

We have examined the growth inhibitory effects of human lymphoblastoid interferon (IFN) on the human hepatocellular carcinoma (HCC) cell line PLC/PRF/5. In vitro, PLC/PRF/5 cells were sensitive to the antiproliferative effects of IFN, growth inhibition being noted at concentrations as low as 1.25 i.u. ml-1. Athymic mice with xenografted tumours derived from the PLC/PRF/5 cell line were treated daily with IFN or a saline control. An IFN dose of 2 X 10(5) i.u./day was found capable of significantly slowing tumour growth rate and prolonging mouse survival. Further studies to examine the mechanisms involved in growth inhibition in vivo demonstrated that IFN was capable of inducing the activity of the enzyme 2,5-oligoadenylic acid (2,5 A) synthetase, a potent inhibitor of protein synthesis, in tumour xenografts but not in mouse tissue, and that IFN significantly enhanced the membrane display of HLA class I glycoproteins on tumour cells, though histology did not reveal any increase in tumour infiltration by host lymphocytes. We conclude that IFN exerts potent growth inhibitory effects on the HCC cell line PLC/PRF/5 both in vitro and in vivo and its mode of action in this animal model system appears to be predominantly mediated by a direct antiproliferative effect on tumour cells.     

Modulation of rat hepatocyte proliferation by bile salts: In vitro and In vivo studies

In this study, the stimulatory effect of bile salts (BS) was evaluated both In vitro, using hepatocyte primary cultures, and In vivo, in normal and 40% partially hepatectomized rats previously fed on BS- enriched diets for 4 weeks. In vitro results show that conjugated cholate (CA) and chenodeoxycholate (CDCA) augmented proliferative activity in rat hepatocytes cultured in absence of mitogens, whereas conjugated deoxycholate (DCA), and ursodeoxycholate (UDCA) did not have any significant effect. None of these BSs increased significantly the replicative response induced by submaximal concentrations of epidermal growth factor (EGF). In vivo, at the end of dietary treatment all animals fed on CA or DCA but not those fed on either CDCA, or UDCA, or tauroursodeoxycholate (TUDCA) developed cholestatic hepatitis and a burst of damage-induced hepatocyte proliferation. After 40% partial hepatectomy (PH), CA- and DCA-treated groups underwent a deterioration of cholestatic hepatitis. On the other hand, in CDCA-, and UDCA-, and TUDCA-treated groups liver histology, serum glutamic pyruvic transaminase (SGPT) and cholestasis indices did not change significantly compared with controls. As far as the proliferative activity, a significant increase was observed not only in CA and DCA but also in UDCA- and TUDCA-fed groups compared with controls, whereas a slight decrease was observed in CDCA-treated animals. In conclusion, our data indicate that conjugated BSs had only a modest stimulatory effect on hepatocyte proliferation In vitro. However, In vivo, in PH rats, UDCA or TUDCA treatment determined a further increase of hepatocellular proliferation not attributable to hepatotoxic effects. Our result suggest that modifications of bile acid pool could modulate hepatocellular proliferation. (Hepatology 1996 May;23(5):1159-66)