Effects of traditional Chinese medicine Dan-shao-hua-xian capsule on expression of miR-200s in rat fibrotic livers

AIM: To investigate the effect of Dan-shao-hua-xian (DSHX) capsule on the expression of the family of microRNA-200 (miR-200s) in rat fibrotic livers. METHODS: Forty male Wistar rats weighing 180 g to 220 g were divided into 5 groups (control group, two model groups and two interference groups). The rats in model groups and interference groups were induced by hypodermic injection of CCl4 for 4 weeks and 8 weeks. The rats in interference groups were also treated with DSHX capsule (0.5 g/kg) once daily for 4 weeks and 8 weeks at the same time. The liver index and serum activity of ALT and AST were analyzed. The liver fibrosis was observed under microscope. Additionally, the expression of miR-200a, -200b, -200c, -141 and -429 was determined by quantitative real-time PCR. RESULTS: The liver index, and serum activity of ALT and AST in model groups and 4-week interference group were obviously higher than those in normal control group. The apparent liver fibrosis was observed in 8-week model group. The expression of miR-200a,-200b, -200c, -141 and -429 in the liver of 8-week model groups was obviously higher than that in control group. CONCLUSION: In the process of liver fibrosis induced by CCl4, the obvious changes of miR-200s may play an important role in the development of liver fibrosis. The miR-200s might be the potential target that DSHX capsule inhibits the process of liver fibrosis.     

Protective effects of somatostatin and octreotide on hepatocytes

AIM: To investigate the protective effect of somatostatin (SST) and octreotide (OCT) on rat hepatocytes. METHODS: The primary hepatocytes were pretreated with different concentrations of SST and OCT. The levels of alanine minotransferase (ALT) and aspartate aminotransferase (AST) in culture supernatant were analyzed by the model of ethanol/carbon tetrachloride (CCl4)-induced hepatocyte injury. Additionally, 75 Sprague-Dawley rats were divided into 5 groups at random, including normal control, model control, SST-treated model groups at high, medium and low doses (200 μg·kg-1·d-1, 100 μg·kg-1·d-1 and 50 μg·kg-1·d-1, respectively). Except for the normal controls, all rats were injected with 40% CCl4 subcutaneously for 8 weeks to establish hepatic fibrosis. Meanwhile, rats of SST-treated model groups were given at different doses of SST twice a day in the same way. Thereafter, the liver function and apoptosis index of hepatocytes were detected by standard enzyme method, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. RESULTS: Compared with those of injury model group, the hepatocytes pretreated with SST (10-8-10-6 mol/L) and OCT (10-7-10-5 mol/L) exhibited significantly decreased levels of ALT and AST in the culture supernatant. Furthermore, most indices of liver function including ALT, AST, alkaline phosphatase (ALP), total bilirubin (TBIL) and albumin (ALB) improved obviously in all SST-treated groups, especially in the group treated with low dose of SST. The apoptosis index of hepatocytes in the fibrotic liver was also reduced greatly by the treatment with low dose of SST. CONCLUSION: SST and OCT may protect hepatocytes against CCl4-induced injury, inhibit hepatocyte apoptosis, and improve the liver function. These findings suggest them a potential efficiency in the prevention of hepatic fibrosis.     

Effects of diammonium glycyrrhizinate on the lipid peroxidation and interstitial collagenase activity in fibrotic liver in rats

AIM: To investigate the effect of diammonium gycyrrhizinate (Ganlixin) against liver fibrosis through preventing lipid peroxidation and regulating interstitial collagenase activity. METHODS: The liver fibrotic model was induced through subcutaneous injection of CCl4 and the feeding with high fat and low protein in rats. Diammonium gycyrrhizinate was administered (70 mg/kg rat BW). Hepatic inflammation and collagen were observed with H-E and Sirius red staining. The liver function including serum ALT, AST activity, Alb and total bilirubin levels were determined. The hepatic lipid peroxidation including SOD and GSH-Px activities, MDA and GSH content were also measured. Hepatic hydroxyproline (Hyp) content was detected with Jamall's method. The activity of interstitial collagenase in liver was assayed by the reaction with ［3H］ labeled type I collagen, and the gene expression of α1(Ⅰ) pro-collagen was analyzed by RT-PCR. RESULTS: The model rats had remarkable inflammatory necrosis, collagen accumulation and fibrosis at liver, while the diammonium gycyrrhizinate treated group showed slighter hepatic injury and collagen deposition, and the much better liver function than the model. The diammonium gycyrrhizinate-treated group had lower levels of hepatic MDA, Hyp and α1 (Ⅰ) pro-collagen mRNA expression than those in the model group, but had higher levels of interstitial collagenase activity, GSH content, SOD and GSH-Px activity than those in the model group. CONCLUSION: Diammonium gycyrrhizinate has a good effect against liver fibrosis, which may be related to the prevention from lipid peroxidation and improvement of interstitial collagenase activity in fibrotic livers.     

Changes of endoplasmic reticulum stress-related molecules CHOP and TRB3 in rat fibrotic liver

AIM: To observe the changes of endoplasmic reticulum stress-related molecules CCAAT/enhancer-binding protein homologous protein(CHOP) and Tribbles homolog 3（TRB3） in the process of liver fibrosis induced by carbon tetrachloride (CCl4). METHODS: Male Wistar rats weighing 180 g to 200 g were divided into 4-week normal control group, 8-week normal control group, 4-week liver fibrosis group and 8-week liver fibrosis group. The rats in liver fibrosis groups were induced by subcutaneous injection of 40% CCl4 for 4 weeks or 8 weeks. The pathological changes of the liver were observed under light microscope. The protein level of ATF6 was determined by Western blotting. The protein and mRNA levels of CHOP and TRB3 in the liver were analyzed by immunohistochemistry, Western blotting and real-time PCR, respectively. The apoptosis of hepatocytes was measured by TUNEL assay. RESULTS: Pseudolobuli formed in the liver tissue of hepatic fibrotic rats. Compared with the control rats, the protein level of p90ATF6 was obviously decreased, the protein level of p50ATF6 was obviously increased, and the protein and mRNA levels of CHOP and TRB3 were obviously higher in the hepatocytes of hepatic fibrotic rats. The apoptosis of hepatocytes was also increased in 4-week and 8-week fibrosis groups. CONCLUSION: In the process of liver fibrosis induced by 40% CCl4, the obviously increased expression of endoplasmic reticulum stress-related molecules CHOP and TRB3 at protein and mRNA levels indicates that endoplasmic reticulum stress may play an important role in the liver fibrosis by promoting the apoptosis of hepatocytes.     

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.     

Effect of chelerythrine on TGF-β/Smads signaling pathway in mouse livers with hepatic fibrosis

AIM:To investigate the anti-hepatic fibrosis effect of chelerythrine on mice and the regulation of transforming growth factor-β (TGF-β)/Smads signaling pathway. METHODS:C57BL/6N mice (n=50) were randomly divided into control group, model group and chelerythrine groups (10 mg·kg^-1·d^-1, 20 mg·kg^-1·d^-1 and 40 mg·kg^-1·d^-1, ig). The mouse model of hepatic fibrosis was established by intraperitoneal injection of carbon tetrachloride (CCl₄) in combination with the olive oil for 8 weeks. At the 5th week, different doses of chelerythrine was used to treat hepatic fibrosis in the mice. At the 14th week, hepatic index was detected. Histopathological changes and the degree of hepatic fibrosis were observed by hematoxylin-eosin staining and Van Gieson staining. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA), and hepatic hydroxyproline (Hyp) content were assayed by spectrophotometry and ELISA. The mRNA expression of TGF-β₁, Smad3, Smad4 and Smad7 in the liver was detected by RT-qPCR, and the protein expression of TGF-β₁, Smad4 and Smad7 was determined by Western blot. RESULTS:The degree of hepatic fibrosis changed markedly in model group compared with control group. The hepatic index, the serum levels of ALT and AST, and the contents of HA and Hyp were significantly increased (P<0.05). The mRNA expression of TGF-β₁, Smad3 and Smad4 was significantly up-regulated, while the mRNA expression of Smad7 was significantly down-regulated (P<0.05). The protein expression of TGF-β₁ and Smad4 was significantly up-regulated, while the protein expression of Smad7 was significantly down-regulated (P<0.05). Compared with model group, the changes of the above indexes in chelerythrine groups were inhibited. CONCLUSION:Chelerythrine protects the mouse liver from CCl₄-induced fibrogenesis injury by regulating TGF-β/Smads signaling pathway.     

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 thalidomide on the expression of NF-κB and TNF-α in experimental hepatic fibrotic rats

AIM: To study the effects of thalidomide on the expressions of nuclear factor κB (NF-κB) and tumor necrosis factor-α (TNF-α) in rat liver fibrosis.METHODS: The fibrosis of rat liver was induced by intraperitoneal injection of carbon tetrachloride thrice weekly.Meanwhile thalidomide (10 mg·kg^-1·d^-1 or 100 mg·kg^-1·d^-1) was given daily by the intragastric route for 8 weeks.Serum alanine aminotransferase (ALT),aspartate aminotransferase (AST),prealbumin (PA),hyaluronic acid (HA) and laminin (LN),and hydroxyproline (HYP) contents in the liver,NF-κB p65 and α-smooth muscle actin (α-SMA) protein in the liver,IκBα and TNF-α protein in cytoplasm and NF-κB p65 protein in nucleus and TNF-α mRNA levels in the liver were studied.RESULTS: Compared with the model group,the Knodell score,serum ALT,AST,HA,LN levels and HYP contents in liver,NF-κB p65 protein in nucleus and α-SMA protein in the liver,and TNF-α mRNA and protein in the liver of rats given high dose of thalidomide were decreased significantly (P<0.01).Meanwhile PA level and IκBα protein in cytoplasm were elevated significantly (P<0.01).CONCLUSION: Thalidomide exerts its effect on the down-regulation of NF-κB-induced TNF-α via inhibiting dissociation and degradation of IκB and prevents liver fibrosis in rats.     

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 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.     

LPS preconditioning relieves chronic liver injury induced by carbon tetrachloride

AIM:To investigate the effect of lipopolysaccharides(LPS)preconditioning on CCl4-induced liver injury and the change of LPS signal transduction.METHODS:The male Wistar rats were divided randomly into liver-injury group, which were injected with CCl4 5 mL/kg first, three days later were injected 0.3 mL 40% CCl4 and 60% olive oil.Animals in LPS preconditioning group were injected with LPS 0.5 mg/kg before the day CCl4 was given.Rats received high fat diet were as liver injury group, and normal control group received normal diet.The lymphocytes infiltrated in the liver tissue were counted.The endotoxin and ALT level in rat plasma, TNF-α content and expressions of TLR4, p38, p-p38, IκΒ, NF-κΒ in the rat livers were also determined.RESULTS:The lymphocytes in liver slice and ALT level of the plasma in LPS preconditioning group were lower significantly than those in the liver injury group, and the expressions of TLR4, p-p38, NF-κΒ in the liver were the same.In contrast, the expression of IκΒ was higher.CONCLUSION:LPS preconditioning relieves obviously CCl4-induced chronic liver injury.The mechanism may be associated with change of signal transduction of LPS, which results in decrease of pre-inflammatory cytokines.     

mRNA expression of insulin receptor and leptin receptor in liver of nonalcoholic fatty liver disease from diabetic rats

AIM: To observe the pathologic changes of liver in diabetic rats and to investigate the role of mRNA expression of insulin receptor and leptin receptor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). METHODS: Twenty male Sprague-Dawley rats were divided randomly into two groups: normal control group and diabetic group. After fed with high-fat diet for 4 weeks, diabetic rats were injected with streptozotocin at a dosage of 30 mg/kg intraperitoneally to induce NAFLD model of type 2 diabetes mellitus. Then the diabetic animals were fed with high-fat diet continuously for 12 weeks. At the end of the experiment, the rats were sacrificed, the concentrations of blood glucose, serum lipid, ALT and AST were measured biochemically. The levels of serum leptin and serum insulin were detected by enzyme-linked immunosorbent assay (ELISA) and radio immunoassay (RIA), respectively. The pathologic changes of liver were observed under light microscopy (LM) stained with HE, Sudan Ⅲ and Masson trichrome staining, respectively. The ultra-structural changes of liver were observed under transmission electron microscopy (TEM). Additionally, the mRNA expressions of PEPCK, G6Pase, insulin R and leptin R from rat livers were assayed by semi-quantitative RT-PCR. RESULTS: The levels of blood glucose, serum insulin, serum TG, ALT and AST increased significantly (P<0.01), serum TC elevated (P<0.05), and the levels of serum leptin decreased (P<0.01) in diabetic group compared to those in normal control group. Obvious liver fatty degeneration, piecemeal necrosis with accompanying inflammatory infiltration and fibrosis were found under LM. Hepatocytes pyknosis, lots of lipid deposits in cytoplasm of hepatocytes, proliferation of collagen in space of Disse were observed under TEM in diabetic group. The expression of insulin R and leptin R mRNA in liver from diabetic rats increased significantly (P<0.01) while the expression of PEPCK and G6Pase mRNA remained unchanged. CONCLUSION: Insulin resistance plays an important role in the pathogenesis of NAFLD. Low level of serum leptin, up-regulation of mRNA expression of insulin R and leptin R in liver caused by insulin resistance may be involved in this process.     

Apoptosis and expression of apoptosis-related genes in kidneys of the rats with 5/6 nephrectomy

AIM: To establish a model of subtotal nephrectomy (SNx) and investigate the changes of apoptosis and apoptosis-related genes (Bax, bcl-2, caspase-3, caspase-8 and caspase-9) in the rat remnant kidney. METHODS: Remnant kidneys were produced in adult male SD rats by 5/6 nephrectomy. The renal function and histopathological changes were evaluated at 1, 2, 4, 8, 12, 16, 26 and 40 weeks after operation. The tissues of remnant kidneys were collected to detect apoptosis cells by in situ end-labeling of cleaved DNA (TUNEL) and proliferating cells by determining the proliferating cell nuclear antigen (PCNA). The expression of Bax, bcl-2, caspase-3, caspase-8 and caspase-9 was measured by RT-PCR and Western blotting. The proteins were detected by immunohistochemistry staining. The relation between apoptosis, proliferation, glomerulosclerosis and renal interstitial fibrosis was also observed. RESULTS: The results showed the renal pathological dynamic changes in 5/6 nephrectomy remnant kidneys were tubule-interstitial inflammation and fibrosis, as well as glomerulosclerosis. There were transient increases in both proliferating and apoptotic processes in glomerulus, tubules and interstitium. Apoptosis was increased and most of apoptotic cells were detected in tubular epithelial cells and interstitial area. The mRNA and protein expression of Bax, caspase-3, caspase-8 and caspase-9 were increased in all course, and peaked at week 4 and 40 in the SNX rats. The successive changes of these parameters were parallel to the level of focal inflammation in interstitium. Glomerulosclerosis index was related with focal inflammation cells and 24 hours urine protein (r=0.788, 0.822; P<0.01). The tubuler apoptosis index was related with Scr, BUN, 24 hours urine protein and focal inflammation cells (r=0.824, 0.794, 0.883, 0.948; P<0.01). The mRNA and protein expression of Bcl-2 was not significantly increased than those in control rats. CONCLUSION: Apoptosis may be involved in the development of chronic renal damaged in 5/6 nephrectomy and play an important role in chronic glomerulosclerosis, tubule atrophy and interstitial fibrosis. The renal interstitial inflammation may promote the apoptosis in the remnant kidney.     

Apoptosis in rat hepatocytes with ischemia/reperfusion injury

AIM: To investigate the distributive rules of apoptosis index (AI) in liver with ischemia/reperfusion (I/R) injury and evaluate the factors related to the hepatocyte apoptosis. METHODS: Sixty SD rats in specific pathogen free grade were randomly divided into three groups: control group (n=18), sham operation group (n=18) and I/R group (n=24). In I/R group, liver injury was induced by blocking blood inflow in rat liver for 20 min, then reperfusion for 22 h. Rats in the control group didnt receive any management. Rats in the sham operation group only subjected sham operation. All rat blood samples and livers were obtained for determination. Blood serum ALT, AST, TBIL, TNF-α, IFN-γ, IL-4, plasma endotoxin concentration, MDA level and SOD activity in liver were detected. Hepatic histological analysis was conduced through HE staining. Apoptosis was detected by TUNEL methods. RESULTS: Focal necrosis occurred in six rats livers in I/R group, in control group and sham group no necrosis cell was found in livers. The hepatic AI of I/R group was significantly increased compared with other groups. The AI in region under hepatic amicula was higher than that in central veins region and portal area. The necrotic regions contained apoptotic cells and AI was higher than that of other regions. Hepatic AI was significantly associated with ALT, AST, TNF-α, IFN-γ and SOD/MDA. CONCLUSION: In liver with I/R injury, the apoptotic cells in the region under hepatic amicula and the focus of necrosis are significantly higher than those in other regions, apoptotic cells and necrosis cells co-exist in the same zone. Hepatic AI may be significantly associated with ALT, AST, TNF-α and SOD/MDA.     

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.     

亮菌多糖ATPS-2对小鼠四氯化碳和酒精肝损伤的保护作用

分别采用四氯化碳（CCl4）和北京红星二锅头诱导小鼠肝损伤，比色法测定血清中丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）的含量；肝脏中超氧化物歧化酶（SOD）活力和丙二醛（MDA）浓度，并作肝组织切片病理观察。亮菌多糖ATPS-2（25mg／kg、50mg／kg、100mg／kg）给药明显降低小鼠血清中升高的ALT和AST水平，抑制肝脏中上升的MDA水平和提高过低的SOD活性。病理检查结果显示亮菌多糖ATPS-2有明显的保肝作用。亮菌多糖ATPS-2对小鼠四氯化碳肝损伤和酒精肝损伤均具有显著的保护作用。     

Effect of HOCs implantation on protein expression of TGF-β/Smad signaling pathway in liver tissue of hepatic fibrosis rats

AIM: To investigate the effect of hepatic oval cells (HOCs) on the protein expression of TGF-β/Smad signaling pathway in the liver tissue of hepatic fibrosis rats.METHODS: The SD rat models of liver fibrosis were made by treating with carbon tetrachloride and combined factors. The HOCs was isolated from the model rats. HOCs suspension (0.5 mL at a density of 1×1012cells/L) were transplanted via portal vein into the hepatic fibrosis rats at 8th week and observed continuously for 30 days. Meanwhile, WuLing capsules were used for positive control. The blood samples were collected through trail vein at 8th day, 15th day, 23th day and 30th day after transplantation of HOCs. The levels of aspartate aminotransferase (AST) and alamine aminotransferase (ALT) in serum were determined by enzyme method. The morphological changes of hepatic tissues were observed under microscope with HE and Musson staining. The protein levels of collagen type I (Col-Ⅰ), extracellular-signal regulated protein kinase (ERK), phosphory-lation extracellular regulatedprotein kinases (p-ERK), TGF-β receptor type Ⅰ (TβRⅠ), TGF-β receptor type Ⅱ (TβRⅡ), mothers against decapentaplegic homolog 2/3 (Smad 2/3) and mothers against decapentaplegic homolog (Smad 7) were assessed in liver tissues by Western blotting. RESULTS: In HOCs and WuLing capsules treated groups, the levels of ALT and AST decreased significantly at 15th day, 23th day and 30th day after the transplantation of HOC. The damage degree of hepatic fiber hyperplasia of the liver histological structure reduced notably. The expression levels of Col-Ⅰ, ERK, p-ERK, TβRⅠ and TβRⅡ in liver tissues of hepatic fibrosis rats were down-regulated obviously while the expression of Smad 7 increased significantly.CONCLUSION: The implantation of HOCs prevents the progress of liver fibrosis in rats. The mechanism of action is to inhibit the protein expression of p-ERK, TβRⅠ, TβR Ⅱ for TGF-β/Smad signaling pathway of liver tissue.     

Role of 78 kD glucose-regulated protein in the development of liver cirrhosis promoted by intestinal endotoxemia in rats

AIM: To explore the role of 78 kD glucose-regulated protein (GRP78) in the development of liver cirrhosis in rats promoted by intestinal endotoxemia (IETM). METHODS: Fifty-one male Wistar rats were randomly divided into liver cirrhosis groups of 4th-week, 6th-week and 8th-week, and normal control group at the corresponding time points. The rat model of hepatic cirrhosis was induced by employing multiple pathogenic factors to the animals. The liver injury and hepatic fibrosis were observed with the staining of HE and VG, respectively. The expression of GRP78 at the mRNA and protein levels was measured by the methods of RT-PCR and immnunohistochemistry, respectively. The concentrations of alanine aminotransferase(ALT), endotoxin, TNF-α and homocystine (HCY) in plasma, and the content of TNF-α, malondialdehyde(MDA) and PⅢP in liver tissues were detected. RESULTS: As liver cirrhosis developed, the levels of ALT, endotoxin, TNF-α and HCY in plasma, the expression of GRP78 at mRNA and protein, the content of TNF-α, MDA and PⅢP in liver tissues, and the index of liver fibrosis were gradually increased and were significantly higher than those in normal control group (P<0.05). Elevated endotoxin in plasma was correlated positively with the protein expression of GRP78, the content of MDA and HCY in plasma and the index of liver fibrosis (P<0.01). Elevated protein expression of GRP78 was correlated positively with the content of MDA and HCY in plasma and the index of liver fibrosis (P<0.01). CONCLUSION: GRP78 plays an important role in the development of liver cirrhosis. Endoplasmic reticulum stress is a possible mechanism in the development of liver cirrhosis promoted by IETM.