Effect of bitter melon on liver fibrosis induced by carbon tetrachloride

AIM: To investigate the effects of bitter melon (BM) on liver fibrosis induced by CCl₄ in Wistar rats. METHODS: Healthy male Wistar rats were randomly divided into 4 groups (with 8 each): olive oil control group (group C), olive oil CCl₄ model group (group M), CCl₄+BM at low concentration (BM 100 g/kg, group BM-L), CCl₄+ BM at high concentration (BM 200 g/kg, group BM-H). All rats except those in group C were subcutaneously injected with CCl₄ twice a week for 8 weeks to induce liver fibrosis. After injection of CCl₄ for 8 weeks, all rats were sacrificed and the samples of blood and livers were collected. The weight ratio of liver to body was measured. The serum level of MDA and the activity of SOD were tested. The contents of total protein and albumin, the activity of GSH-Px, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were determined. Hepatic inflammation and collagen deposition were observed under microscope with Masson staining. RESULTS: In the rats treated with BM, the weight ratio of liver to body, the serum level of MDA, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were lower than those in group M (P<0.01). The serum activity of SOD, the contents of total protein and albumin, and the activity of GSH-Px in the liver homogenate were enhanced (P<0.01). The livers of the model rats had remarkable inflammatory necrosis, collagen accumulation and fibrosis. The rats in BM-treated group showed slighter hepatic injury and collagen deposition, and the liver functions were much better than those in the model group. High dose of BM showed more obvious liver-protective effects. CONCLUSION: BM attenuates liver fibrosis by its antioxidant effect and the mechanisms of reducing hydroxyproline content and monoamine oxidase activity.     

Hepatoprotective effect of Paecilomyces tenuipes polysaccharides on carbon tetrachloride -induced liver injury

AIM:To investigate the protective effect of crude Paecilomyces tenuipes polysaccharides (cPtPs) and Paecilomyces tenuipes polysaccharides (PtPs) in a rat model of acute hepatic necrosis induced by carbon tetrachloride (CCl4). METHODS:Wistar rats were divided into four groups (control group, CCl4 group, cPtPs+CCl4 group and PtPs +CCl4 group), the four groups were given intragastrically with normal saline, cPtPs and PtPs for 15 d, respectively. In the last two days, these groups except control group were injected peritoneally with CCl4. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL) and indirect bilirubin (IBIL) were detected by automatic biochemistry analyzer. Pathological changes of hepatic tissue were assessed by hematoxylin-eosine (HE) staining. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenate were analyzed using xanthinoxidase and thio-barbituric acid, respectively. The concentration of Ca2+ in hepatocyte mitochondria was determined by colorimetric method. The expression of α-smooth muscle actin (α-SMA) was examined in hepatic tissue by immunohistochemical method. RESULTS:Compared with control group, serum levels of ALT, AST, TBIL, DBIL and IBIL in CCl4 group increased significantly, denaturation and necrosis implicated to the whole hepatic lobules. Compared with CCl4 group, serum levels of ALT, AST, TBIL, DBIL and IBIL in PtPs +CCl4 group decreased significantly, denaturation and necrosis located in the third region of hepatic lobules, the level of SOD increased and MDA decreased (P<0.05) in endochylema. Concentration of Ca2+ in mitochondria decreased in PtPs +CCl4 group and cPtPs +CCl4 group (P<0.05, P<0.01, respectively). Expression of α-SMA was found little in PtPs+CCl4 group. CONCLUSION:PtPs, the effect is better than cPtPs, lessens CCl4-induced hepatonecrosis significantly. The role may be related with anti-lipid peroxidation.     

Changes of portal hemodynamics in the development of experimental liver cirrhosis

AIM: To understand the formation of portal hypertension through the change of portal hemodynamics on experiment cirrhosis. METHODS: Carbon tetrachloride was subcutaneously injected in the rat. The changes of the portal hemodynamics in the pathological process of liver tissue were observed. RESULTS: The liver underwent degeneration, necrosis of hepatocytes, and the normal architecture of the liver lobules was replaced by pseudolobule, which consist of regenerative hepatocytes and fibrous septa. The diameter, the blood flow velocity and the blood flow quantity of portal were significantly higher than that in former group (P＜0.05 or P＜0.01) two weeks after the injection of carbon tetrachloride. In the fifteenth week, these parameters were lower than that before owing to the forming of portacaval collateral circulation (P＜0.01). The congest index of the portal in second week, fifth week and fifteenth week were statistically higher than its predecessor (P＜0.05 or P＜0.01), except that in tenth week, which had no statistical significance (P>0.05). CONCLUSION: The changes in hemodynamics of the portal are in accordance with the changes in pathology of liver in the formation of liver cirrhosis.     

Expression of peroxisome proliferator-activated receptor α in rat fatty liver

AIM:To investigate the role of expression of peroxisome proliferator-activated receptor α(PPAR α) in pathogenesis of rat fatty liver.METHODS:The rats were treated with a low dose of carbon terachloride (CCl₄) and fed a high fat diet to produce fatty liver. We determined the concentrations of triglyceride (TG), total cholesterol (TC), free fatty acid (FFA) in liver and the alanine aminotransferase (ALT) activity, tumor necrosis factor-α (TNF-α), FFA in serum and the degree of hepatocytic steatosis. Total RNA of liver was extracted, and the expression of PPAR α were analyzed by semi-quantitative RT-PCR method.RESULTS:In model group, the hepatocytic PPAR α mRNA expression decreased to 0.41±0.28, compared to 1.41±0.29 in the control group (P＜0.01). The contents of TG, TC, FFA in model rat liver were (1.88±0.20) mmol·L^-1, (11.03±1.12) mmol·L^-1 and (1 260.38±151.27) μmol·L^-1, respectively, compared to (0.53±0.10) mmol·L^-1, (1.25±0.25) mmol·L^-1 and (334.30±27.09) μmol·L^-1 in the control group (P＜0.01). The activity of ALT, concentrations of TNF-α and FFA in serum were also increased remarkably in model group.CONCLUSION:Oxidation of fatty acid and utilization of lipids in liver are affected by reducing the expression of PPAR α, which result lipid accumulation in liver.     

Effect of intestinal endotoxemia on hepatic energy metabolism in acute liver failure

AIM:To study the effect of intestinal endotoxemia(IETM) on hepatic energy metabolism in acute liver failure. METHODS:Intoxication by thioacetamide (TAA) was used to establish rat model of acute liver injury.Ketone body(acetoacetate and β-hydroxybutyrate) in arterial blood and ATP content of hepatocellular mitochondria were determined by using enzymatic fluorimetric micromethod.Colectomy was adopted in observing the changes in plasma endotoxin content and serum alanine aminotransferase (ALT) activity. RESULTS:In the TAA group,plasma endotoxin content and serum ALT activity were all significantly higher than those in the control group(P＜0.01),arterial ketone body ratio of acetoacetate to β-hydroxybutyrate (AKBR) decreased below 0.4,total ketone body in arterial blood was significantly lower than that in the control group(P＜0.01).In the TAA+colectomy group,there was no endotoxemia to be found,ATP content of hepatocellular mitochondria was significantly higher than that in the TAA group(P＜0.01), though serum ALT activity was higher than that in the control group(P＜0.05),but significantly lower than that in the TAA group(P＜0.01). CONCLUSION:IETM played a key role in the occurrence of acute liver failure,hepatic dysfunction might be caused by IETM through damaging hepatic energy metabolism.     

A novel cDNA clone related with rat liver regeneration

AIM:To study a novel gene that probably related with liver regeneration, which was found by representational difference analysis(RDA). METHODS:cDNA sequence, tissue distribution and functions of the novel gene were studied by slot blot, Northern blot, RT-PCR, cDNA library screening and sequence analyzing. RESULTS:Two full-length clones were isolated from cDNA library of rat fetal livers and the sequence analysis identified that the positive cDNA encoded 76 amino acids only; Using the cDNA as a probe, the novel gene showed a specific liver distribution, a moment increasing expression in one hour after partial hepatectomy (PH) and high expression in fetal liver or liver tumor by Northern blot; EGF quickly induced its high expression in primary culture rat hepatocytes(FCS free).CONCLUSION:These results show that the novel gene is an early phase response gene that is closely related to a liver regeneration adjustment. It may encode peptide or has longer sequence at N tip.     

The reversal effects of recombinant human augmenter of liver regeneration in immunocomplex induced liver fibrosis

AIM: To observe the anti-fibrosis activity of recombinant human augmenter of liver regeneration (hALR). METHODS: Albumin induced rat model of liver fibrosis was established and hALR was given peritoneally after the model production. Serum concentration of alanine aminotransferase(ALT),aspartic aminotransferase(AST),lactate dehydrogenase(LDH), hepatic collagen contents and pathological examination were selected as observing parameter. RESULTS: Recombinant human augmenter of liver regeneration (hALR) could decrease ALT, AST, LDH concentration of fibrotic rats. The measurements of hepatic collagen contents showed that hepatic collagen contents in hALR treatment group was much lower than those of model group and negative control group. Pathological examination also indicated that the degree of liver fibrosis in hALR treatment group was attenuated in comparison with those of model group and negative control group. CONCLUSION: Recombinant human augmenter of liver regeneration (hALR) had reversal effects on immunocomplex induced rat liver fibrosis.control group. CONCLUSION: Recombinant human augmenter of liver regeneration (hALR) had reversal effects on immunocomplex induced rat liver fibrosis.     

Changes of histone modification levels in rats with liver fibrosis

AIM: To observe the changes of histone modifications in the liver of rats with hepatic fibrosis and its possible role in the development of hepatic fibrosis. METHODS: Male Wistar rats (n=20) were randomly divided into liver fibrosis group and normal control group. The liver fibrosis model was established by hypodermic injection of CCl₄, and the rats in normal control group were injected with vegetable oils. At the end of the 8th week, all rats were killed. Liver function indexes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and liver fibrosis indexes including haluronic acid (HA), laminin (LN), collagen type IV (Col Ⅳ) and procollagen type Ⅲ (PCⅢ) were determined by biochemical and RIA methods. The liver index was analyzed, and the liver fibrosis degree and the morphological change of the liver were detected by HE and Masson staining. The levels of α-smooth muscle actin (α-SMA), collagen type Ⅰ (ColⅠ), H3K4me2, H3K9me2, acH3K9 and acH4K12 were detected by Western blot. RESULTS: After hypodermic injection of CCl₄ for 8 weeks, the liver index, ALT, AST, HA, LN, Col Ⅳ and PCⅢ of the rats in liver fibrosis group were higher than those in control group (P<0.05). Compared with control group, the level of acH4K12 was decreased (P<0.05), while acH3K9, H3K9me2, α-SMA and ColⅠ were increased (P<0.05), but H3K4me2 had no significant change.CONCLUSION: The levels of acH4K12, acH3K9 and H3K9me2 may play essential roles in the pathogenesis of liver fibrosis, and these histone modifications may regulate gene expression associated with extracellular matrix metabolism.     

Functional analysis of differential microRNA expression profile in mouse fibrotic liver tissues induced by CCl4

AIM:To discover the expression profile of microRNAs (miRNAs) in mouse fibrotic liver tissues induced by carbon tetrachloride (CCl4), and to investigate the functions of these differential miRNAs based on the gene ontology (GO) analysis and KEGG Pathway analysis. METHODS:The mice were randomly divided into normal group and model group. Liver fibrosis was induced by subcutaneous injection of CCl4. miRNA expression profile of the liver tissues was assayed by a mouse miRNA microarray (Agilent 12.0). The differential expression of miRNAs between the normal and model mice was screened, and GO analysis and KEGG Pathway analysis were performed to determine the functions of these differential miRNAs. RESULTS:Thirty-nine miRNAs with differential expression were discovered in the model mice compared with the normal mice, among which 23 were up-regulated and 16 were down-regulated. GO analysis and KEGG Pathway analysis indicated that most pathological processes of liver fibrosis regulated by miRNAs included cell proliferation and activation, cell apoptosis, cell cycle, cell adhesion, inflammatory reaction, cell migration, transforming growth factor β (TGF-β) signaling pathway, Wnt signaling pathway and proteometabolism process. GO analysis revealed that the key up-regulated miRNAs were mmu-miR-322, mmu-miR-15b, mmu-miR-195, mmu-miR-200b and mmu-miR-214, and the key down-regulated miRNAs were mmu-miR-16, mmu-miR-130a, mmu-miR-101b, mmu-miR-30a and mmu-miR-30e. Analyzing the target genes screened out by GO analysis and Pathway analysis simultaneously, we found that the key up-regulated miRNAs included mmu-miR-200b, mmu-miR-322, mmu-miR-106b, mmu-miR-23a and mmu-miR-15b, and the key down-regulated miRNAs included mmu-miR-16, mmu-miR-30e, mmu-miR-30c, mmu-miR-30a and mmu-miR-130a. CONCLUSION: Differential expression of miRNAs is discovered in mouse fibrotic liver tissues induced by CCl4 compared with the normal liver tissues. Most of the pathological processes involved in liver fibrosis may be regulated by miRNA, such as cell proliferation and activation, cell adhesion and apoptosis, cell migration and differentiation, metabolism, TGF-β receptor signaling pathway and so on.     

Changes of liver sinusoidal endothelial cells and basement membrane in early stage of liver fibrosis induced by carbon tetrachloride in rats

AIM：To explore the development of hepatic sinusoidal capillarization in the early stage of liver fibrosis induced by carbon tetrachloride (CCl4) in rats. METHODS：Clean SD rats were randomly divided into normal control group (group N, n=6) and liver fibrotic model group (group M, n=32). The rats in group N were intraperitoneal injected with saline and the rats in group M were intraperitoneal injected with CCl4 (2 mL/kg, twice a week for 4 weeks). At the end of the 3rd day and the 1st, 2nd and 4th weeks, all rats were killed and then the samples were collected. The pathological changes in the livers were observed by HE staining and Masson straining. The development of hepatic sinusoidal capillarization was observed by transmission electron microscopy (TEM) and immunohistochemical staining. The cell surface expression of vascular endothelium-associated marker CD31, collagen type Ⅳ (Col IV) and laminin (LN) was determined. RESULTS：HE and Masson staining showed the formation of liver fibrosis after treatment with CCl4 for 4 weeks. TEM showed that the fenestrate diameter of liver sinusoidal endothelial cells (LSECs) grew down, the fenestrate numbers of LSECs were decreased along with the development of liver fibrosis, and the consecutive basement membrane was formed at the end of the experiment. The expression of CD31 was significantly increased along with the development of defenestration, and the expression of Col IV and LN was significantly increased after the treatment with CCl4 for 2 weeks and 4 weeks, respectively. CONCLUSION：The typical hepatic sinusoidal capillarization was detected in the early stage of liver fibrosis, and the deposition of LN in the liver sinusoidal walls was the mainly factor of formation of the consecutive basement membrane.     

Role of caspase-3 dependent hepatocyte apoptosis in liver ischemia-reperfusion injury in cirrhotic rats

AIM:To investigate whether hepatocyte apoptosis is contributed to liver ischemia-reperfusion (I/R) injury and the relationship between liver caspase-3 activity and hepatocyte apoptosis in cirrhotic rats. METHODS:Liver ischemia-reperfusion is induced by Pringle maneuver. The cirrhotic rats were randomized into two groups: Group A: simple hepatic blood inflow occlusion (HBIO); Group B: HBIO + inhibitor, before HBIO, ZVAD-fmk 15 mg/kg was injected via dorsal penis vein; Group C: healthy rat, simple HBIO. The ischemia time was 30 min in these groups. Serum aspartate aminotransferase(AST), liver caspase-3 activity, and apoptotic hepatocytes were examined in the three groups. RESULTS: After 6 h of reperfusion, the liver caspase-3 activity was markedly elevated and reached its peak, which was statistically higher than that of before I/R . The same change occurred in hepatocyte apoptosis between 6 h of reperfusion and before I/R (20.9%±4.9% vs 0.5%±0.3%, P＜0.01). As the reperfusion prolonged, the caspase-3 activity and apoptotic hepatocyte decreased gradually. The 7th-day survival rate was 62.5% in group A. The serum AST, liver caspase-3 activity and apoptotic hepatocytes were significantly higher in group A than those in group B and C, representing the most severe liver injury among the three groups. CONCLUSION:Hepatocyte apoptosis is the major form of cell death in liver ischemia-reperfusion injury in cirrhotic rats. Hepatoctye apoptosis induced by I/R is caspase-3 dependent, and inhibiting caspase-3 can alleviate liver injury. The caspase-3 dependent hepatocyte apoptosis is highly contributed to the pathological phenomenon that the ischemic sensitivity of cirrhotic liver is higher than normal liver.     

Expression of Hedgehog signaling molecules in rat livers after partial hepatectomy

AIM: To detect the expression of Hedgehog signaling molecules in rat livers after partial hepatectomy. METHODS: The model of rat partial hepatectomy was established by resecting the middle and left lobes of the liver. Eighteen male Sprague-Dawley rats were divided into 3 groups: sham operation group (group A), partial hepatectomy group 1 (group B) and partial hepatectomy group 2 (group C). Hepatic tissues were collected 24 h after the operation in group A and group B, and 48 h after the operation in group C. The expression of Ki-67,Sonic Hedgehog(Shh),Indian Hedgehog(Ihh) and Glioblastoma-2(Gli-2) in the hepatic tissues were detected by immunohistochemical staining and Western blotting. RESULTS: The edema and spotty necrosis in the hepatic tissues were observed in group B and group C by HE staining. The cells of different dividing stages were found in the hepatic tissues of group C. Compared with group A, the expression of Ki-67, Shh, Ihh and Gli-2 in group B (P<0.01) and group C (P<0.01) was significantly elevated, and the expression levels in group C were higher than those in group B (P<0.01). CONCLUSION: Hedgehog signaling in rat livers may be activated after partial hepatectomy and stimulate liver regeneration.     

Effect of sorafenib on liver regeneration after partial hepatoectomy in liver cirrhotic rats

AIM: To study the effect of sorafenib on the liver regeneration after partial hepatectomy (PH) in cirrhotic rats. METHODS: Thirty Wistar rats with liver cirrhosis induced successfully with diethylnitrosamine (DEN) underwent 30% PH and then were randomly divided into 2 groups (n=15). The rats in experimental group were fed with sorafenib at dose of 30 mg·kg^-1·d^-1 from the 1st day to the 10th day after PH, while those in control group were fed with vehicle by gavage. The blood and liver tissues of the rats were collected after PH and at the end of the experiment. Liver regeneration rate (LRR) and proliferating cell nuclear antigen (PCNA) expression were assessed for determining the hepatocyte proliferation. The content of alanine transaminase (ALT), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), angiogenesis related factors including vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β) and micro-vessel density (MVD) were measured in both groups. RESULTS: LRRs on day 10 after PH were 45.43%±3.36% and 44.21%±2.77% in experimental group and control group, respectively (P>0.05), and the expression of PCNA in hepatic tissues of the rats was not found by the method of immunohistochemistry in both groups. Liver function index had no significant difference between the 2 groups (P>0.05). However, other than VEGF, sorafenib resulted in inhibition of VEGFR-2 and PDGFR-β expression and reduction of MVD in experiment group, and significant difference between the 2 groups was observed (P<0.01). CONCLUSION: Sorafenib does not influence live regeneration after PH in liver cirrhotic rats.     

Expression of IL-6 mRNA in endometrium with endometriosis

AIM: The purpose of the present study was to explore the relationship between interleukin-6 mRNA expression and endometriosis. METHODS: Using the rat model, IL-6 mRNA expression in the endometrium was examined by RT-PCR. RESULTS: The expression of IL-6 mRNA in control rats did not change at 2, 4, 6 and 8 weeks after sham operation (P>0.05), but in model rats it gradually increased at 2, 4, 6 and 8 weeks after endometriosis (P<0.01). The expression of IL-6 mRNA in uterine endometrium with endometriosis was lower than in endometriotic tissue, but higher than in endometrium from healthy controls. CONCLUSION: The IL-6 mRNA expression may contribute to the development of endometriosis . The increase in IL-6 mRNA expression may promote the implantation and growth of endometriotic tissue.     

Morphology of endoplasmic reticulum and expression of GRP78 in rat fibrotic liver

AIM: To observe the changes of endoplasmic reticulum and the biomarker of endoplasmic retidum stress (ERS), glucose-regulated protein 78(GRP78),in the process of liver fibrosis induced by carbon tetrachloride (CCl₄). METHODS: Male Wistar rats weighing 180 g to 220 g were divided into control group and liver fibrosis group. The rats in liver fibrosis group were induced by hypodermic injection of CCl₄ for 4 weeks or 8 weeks. The liver index and the serumactivity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. The liver fibrosis and the morphological changes of endoplasmic reticulum were observed under light and electronic microscopes, respectively. Additionally, the expression of GRP78 at mRNA and protein levels was determined by real-time PCR and the method of immunohistochemistry, respectively. RESULTS: The liver index, serum ALT and AST activity in liver fibrosis group were obviously higher than those in control group. Swelling and reduced number of endoplasmic reticulum were observed in the hepatocytes of fibrotic rats compare to the controls. The levels of GRP78 protein and GRP78 mRNA in the liver of hepatic fibrotic rats were obviously higher than those in the control rats. CONCLUSION: In the process of liver fibrosis induced by CCl₄, the obvious morphological changes of endoplasmic reticulum and increased expression of ERS protein indicate that ERS plays an important role in the liver fibrosis.     

Effects of nitric oxide and endothelin on relaxation and contraction of isolated splanchnic vascular strips in cirrhotic rats

AIM: To investigate the different vasoactive effects of nitric oxide (NO) and endothelin (ET) on splanchnic arterial and venous vessels in cirrhotic rats. METHODS: Cirrhosis was induced in Wistar rats by subcutaneously administration of carbon tetrachloride. Maximal relaxation (Rmax) and contraction (Cmax) to NO and ET were determined in vitro using isolated vascular strips prepared from portal vein (PV) and mesenteric artery (MA) of both cirrhotic and normal rats, and EC50 was calculated for effects of NO and ET, respectively. RESULTS: Rmax of PV and MA to sodium nitroprusside (SNP) (releasing NO) were significantly higher in cirrhotic rats (n=8) than those in normal rats (n=7), and EC50 of NO were dramatically lower in cirrhotic rats than those in control (P<0.05,P<0.01). Cmax of PV and MA to ET were significantly decreased in cirrhotics compared with control, and EC50 of ET were obviously increased in cirrhotic rats compared with normal rats (P<0.05,P<0.01). Furthermore, there were significant differences in Rmax, Cmax and EC50 to NO and ET between PV and MA in both of cirrhotic and normal rats, but these differences in cirrhotics were greater than those in control (P<0.05 or P<0.01). CONCLUSION: There are significant different vasoactive effects of NO and ET on splanchnic arterial and venous vessels in cirrhotic rats, and it may play a crucial role in the pathogenesis of portal hypertension.