Pig Serum-induced Liver Fibrosis Research Articles

Extracts of Periplaneta americana alleviate hepatic fibrosis by affecting hepatic TGF-β and NF-κB expression in rats with pig serum-induced liver fibrosis.

Liver fibrosis is caused by continuous wound healing responses to various harmful stimuli, including viral infection, drugs, alcohol, and autoimmune liver disease. The purpose of this study was to examine the effects of extracts of Periplaneta americana (EPA) in rats with pig serum-induced liver fibrosis to preliminarily assess the antifibrotic effect of EPA. Seventy rats were randomly divided into 7 groups (10 rats in each group): HC, the healthy control group; FC, the fibrotic control group; TL, low-dose EPA treatment group group; TM, medium-dose EPA group; TH, high-dose EPA treatment group; TC1, Panax notoginseng/Salvia mitiorrhiza treatment control group 1; TC2, colchicine treatment control group 2. TC1 and TC2 were used as the positive control to demonstrate the difference between EPA and the effects of other compounds. The liver fibrosis model was induced by intraperitoneal injection of 0.5 mL pig serum twice a week for 13 weeks in all groups except for the HC group. The hepatic fibrosis model was established at the 7th week, and followingly, the corresponding compounds were administered once a day in all groups for 6 weeks. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity was determined in rat blood serum. We also measured liver fibrosis-related serum markers, including hyaluronic acid (HA), mucin layer (LN), type III pre-collagen (PC-III) and type IV collagen (IV-C). Hematoxylin and eosin (H&E) and Masson stainings were used to assess liver morphology and determine the stage of fibrosis. Immunohistochemistry was used to detect the protein expression of NF-κB, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in rat liver tissue. Compared with that of the HC group, the liver tissue of the FC group presented obvious liver damage and collagen deposition. The serum levels of ALT, AST, HA, LN, PC-III and IV-C and the expression of NF-κB, α-SMA, TGF-β1 and TIMP-1 in the FC group were significantly higher than those in the HC group, the EPA treatment groups, the TC1 group and the TC2 group (P < 0.01). The levels of serum ALT, AST, HA, LN, PC-III and IV-C and the expression of α-SMA, NF-κB, TGF-β1 and TIMP-1 in the TL, TC1 and TC2 groups were significantly higher than those TM and TH groups (P < 0.05). EPA treatment significantly improved liver function, decreased collagen deposition and reversed the pathological changes related to liver fibrosis. We found that EPA could reduce liver inflammation, suppress liver cell degeneration and necrosis, and reduce the formation of liver fibrous tissue. Its mechanism might be associated with inhibiting the expression of TGF-β1, TIMP-1, NF-κB and α-SMA to block signal transduction pathways in the hepatic fibrosis process. Therefore, EPA, as a traditional Chinese medicine, might be potentially used to prevent and treat hepatic fibrosis in the future. However, further more experiments are necessary to verify its effectiveness and possible signaling pathways.

Inactivated Orf virus (Parapoxvirus ovis) elicits antifibrotic activity in models of liver fibrosis

Inactivated Orf virus (ORFV, Parapoxvirus ovis) demonstrates strong antiviral activity in animal models including a human hepatitis B virus (HBV)-transgenic mouse. In addition, expression of interferon (IFN)-γ and interleukin-10 (IL-10) was induced after administration of inactivated ORFV in these mice. IFN-γ and IL-10 are known to elicit antifibrotic activity. We therefore aimed to study antifibrotic activity of inactivated ORFV in models of liver fibrosis. We characterized ORFV-induced hepatic cytokine expression in rats. We then studied ORFV in two models of liver fibrosis in rats, pig serum-induced liver fibrosis and carbon tetrachloride (CCL4 )-induced liver fibrosis. ORFV induced hepatic expression of IFN-γ and IL-10 in rats. ORFV mediated antifibrotic activity when administrated concomitantly with the fibrosis-inducing agents in both models of liver fibrosis. Importantly, when CCL4 -induced liver fibrosis was already established, ORFV application still showed significant antifibrotic activity. In addition, we were able to demonstrate a direct antifibrotic effect of ORFV on stellate cells. These results establish a potential novel antifibrotic therapeutic approach that not only prevents but also resolves established liver fibrosis. Further studies are required to unravel the details of the mechanisms involved.

Pentoxifylline prevents pig serum‐induced rat liver fibrosis by inhibiting interleukin‐6 production

Pig serum-induced rat liver fibrosis is a model of liver fibrosis in the absence of obvious hepatocyte injury. Penoxifylline (PTX), a xanthine derivative, which is a well-known suppressor of tumor necrosis factor-alpha (TNF-alpha) production from inflammatory cells, has also been shown to inhibit the growth of hepatic stellate cells and to inhibit collagen synthesis in these cells in vitro. We investigated the effect of PTX on pig serum-induced liver fibrosis in vivo, and assessed the mechanisms of prevention of fibrogenesis by this drug. Male Wistar rats were given intraperitoneal injections of 0.5 ml normal pig serum twice a week for 10 weeks with or without concomitant oral administration of PTX (20 mg/kg). Rats that received pig serum showed significant liver fibrosis, and their serum interleukin-6 (IL-6) and hyaluronic acid levels were significantly increased. The serum levels of IL-6 were well correlated with the serum levels of hyaluronic acid, and increased as the liver fibrosis progressed. Penoxifylline prevented the development of fibrosis in this animal model and reduced the serum levels of IL-6 in a dose-dependent manner. In vitro, by the addition of PTX to the culture medium of the rat hepatic stellate cells (HSCs), the proliferation of the HSCs was significantly inhibited and IL-6 in the culture supernatant was also reduced significantly. Exogenous addition of IL-6 partially restored the proliferation. Penoxifylline prevents pig serum-induced rat liver fibrosis by inhibiting the proliferation of HSCs and by inhibiting the production of IL-6 from HSCs.

Efficacy of Chinese medicine Yi-gan-kang granule in prophylaxis and treatment of liver fibrosis in rats.

To investigate the efficacy of a Chinese medicine, Yi-gan-kang granule (granules for benefiting the liver), in prophylaxis and treatment of liver fibrosis in rats and its possible mechanism. One hundred and forty Sprague-Dawley rats were randomly divided into seven groups (20 each): group 1, blank control group without any interference during the study; group 2, CCl4-induced liver fibrosis group; group 3, pig serum-induced liver fibrosis group; group 4, prophylaxis group of CCl4-induced liver fibrosis by Yi-gan-kang; group 5, prophylaxis group of pig serum-induced liver fibrosis by Yi-gan-kang; group 6, treatment group of CCl4-induced liver fibrosis by Yi-gan-kang; group 7, treatment group of CCl4-induced liver fibrosis by Yi-gan-kang. At wk 6, 10, 14 and 20 (baseline for CCl4 or big serum induction), five rats in each group were anesthetized and their livers were removed for pathological studies including immunohistochemical studies for alpha-SMA, type I collagen and in situ hybridization of tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA of hepatic stellate cells (HSCs). Anti-lipid peroxidation in isolated mitochondria and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay for proliferation and terminal deoxynucleotidyl transferase-medicated dUTP-biotin nick end-labeling (TUNEL), flow cytometry and electron microscopy for apoptosis in isolated HSCs were also studied. The mean number of pseudolobuli at wk 10, 14 and 20 in the prophylaxis group was significantly less than that in the control group (P<0.05 or 0.01). The effect of prophylaxis at wk 14 in CCl4 rats and at wk 10 in pig serum-induced rats was much better than that of treatment group (P<0.01). The thickness (in microm) of fibers both in pig serum-induced prophylaxis and in treatment groups at wk 14 and 20 was significantly less than that in control group (P<0.05). The number of fibers both in prophylaxis and in treatment groups from wk 10 or 14 to 20 was significantly less than that in control group (P<0.05 or P<0.01). The tissue HSC positive rates of type I collagen, alpha-SMA and TIMP-1 mRNA, which represented the active phenotype of HSCs in tissues, remained very high from wk 6 to the end of model making in control group. While in prophylaxis group, they were at a relatively low level. In treatment group, there was a gradual decreasing trend. Time- and dose-dependent effects of anti-lipid peroxidation on isolated mitochondria, cell proliferation and apoptosis in cultured HSCs were also observed during the study. Yi-gan-kang can effectively inhibit or inverse the course of liver fibrogenesis in CCl4- and pig serum-induced rat models.

Treatment of pig serum-induced rat liver fibrosis withBoschniakia rossica, oxymatrine and interferon-α

To investigate the effect of Boschniakia rossica (BR), oxymatrine (OM) and interferon-alpha (IFN-alpha) 1b on the therapy of rat liver fibrosis and its mechanism. By establishing a rat model of pig serum-induced liver fibrosis, liver/weight index and serum alanine transaminase (ALT) were observed to investigate the therapeutic effect of BR,OM and IFN-alpha. Radioimmunoassay was utilized to measure procollagen type III (PCIII) and collagen type IV (CIV). RT-PCR was used to assay the expression of liver transforming growth factor-beta 1 (TGF-beta1) mRNA. Immunohistochemistry of alpha-smooth muscle actin (alpha-SMA) and pathologic changes of liver tissues were also under investigation. Serum PCIII and CIV in BR, OM and IFN-alpha groups were significantly declined compared with those in model group, and their RT-PCR revealed that TGF-beta1 mRNA expression was also reduced more than that in model group. Immunohistochemistry demonstrated that alpha-SMA also declined more than that in model group. Serum ALT in IFN-alpha, control and model groups was within normal level. Serum ALT in BR group had no significant difference from those of IFN-alpha, control and model groups. Serum ALT in OM group was significantly higher than those in BR, IFN-alpha, model, and control groups. BR, OM and IFN-alpha can prevent pig serum-induced liver rat fibrosis by inhibiting the activation of hepatic stellate cells and synthesizing collagen. OM has hepatotoxicity to rat liver fibrosis induced by pig serum.

Blockage of transforming growth factor beta receptors prevents progression of pig serum-induced rat liver fibrosis.

To test the hypothesis that introduction of antisense T beta R I and T beta R II eukaryotic expressing plasmids into a rat model of immunologically induced liver fibrosis might block the action of TGF-beta (1) and halt the progression of liver fibrosis. RT-Nest-PCR and gene recombination techniques were used to construct rat antisense T beta R I and T beta R II recombinant plasmids which could be expressed in eukaryotic cells. The recombinant plasmids and empty vector (pcDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model. Expression of exogenously transfected gene was assessed by Northern blot, and hepatic expressions of T beta R I and T beta R II were evaluated by RT-PCR and Western blot. We also performed ELISA for serum TGF-beta(1), hydroxyproline of hepatic tissues, immunohistochemistry for collagen types I and III, and VG staining for pathological study of the liver tissues. The exogenous antisense T beta R I and T beta R II plasmids could be well expressed in vivo, and block mRNA and protein expression of T beta R I and T beta R II in the fibrotic liver at the level of mRNA respectively. These exogenous plasmid expressions reduced the level of TGF-beta(1) (antisense T beta R I group 23.998+/-3.045 ng/mL, antisense T beta R II group 23.156+/-3.131 ng/mL, disease control group 32.960+/-3.789 ng/mL; F=38.19, 36.73, P<0.01). Compared with disease control group, the contents of hepatic hydroxyproline (antisense T beta R I group 0.169+/-0.015 mg/g liver, antisense T beta R II group 0.167+/-0.009 mg/g liver, disease control group 0.296+/-0.026 mg/g liver; F=14.39, 15.48, P<0.01) and the deposition of collagen types I and III decreased in the two antisense treatment groups (antisense T beta R I group, collagen type I 669.90+/-50.67, collagen type III 657.29+/-49.48; antisense T beta R II group, collagen type I 650.26+/-51.51, collagen type III 661.58+/-55.28; disease control group, collagen type I 1209.44+/-116.60, collagen type III 1175.14+/-121.44; F=15.48 to 74.89, P<0.01). Their expression also improved the pathologic classification of liver fibrosis models (compared with disease control group, chi(2)=17.14, 17.24, P<0.01). No difference was found in the level of TGF-beta(1), the contents of hepatic hydroxyproline and collagen types I and III and pathologic grade between pcDNA3 control group and disease control group or between the two antisense treatment groups (F=0.11 to 1.06, chi(2)=0.13 to 0.16, P>0.05). Antisense T beta R I and T beta R II recombinant plasmids have certain reverse effects on liver fibrosis and can be used as possible candidates for gene therapy.

Leptin receptor-deficient Zucker (fa/fa) rat retards the development of pig serum-induced liver fibrosis with Kupffer cell dysfunction

The aim of this study was to investigate the role of leptin in the development of liver fibrosis with Kupffer cell function using leptin receptor deficient rats. Male Zucker (fa/fa) and control (fa/-) rats received pig serum for 8 weeks. Animals were sacrificed to estimate the degree of liver fibrosis and stellate cell activation with the expression of α smooth muscle actin (αSMA). Microarray analysis was performed. Isolated Kuppfer cells of Zucker and control rats were treated with LPS. LPS uptake and TNF-α production were examined. Stellate cells were also isolated from Zucker and control rats. The expression of procollagen type I mRNAs was examined. Control rats developed liver fibrosis 8 weeks after injection of pig serum and showed an increased liver hydroxyproline content of 348 ± 34 μg/g (n = 10) compared with Zucker rats (225 ± 13, n = 10, P < 0.01). The procollagen type I mRNA level and αSMA expression of Zucker rats were also significantly reduced. Microarray analysis indicated significantly reduced expression of TNF-α, LPS-binding protein, urokinase-type plasminogen activator (uPA), IGF, IGF-binding protein (IGFBP)-3,5, and increased expression of apolipoprotein IV. Isolated Kupffer cells of Zucker rats showed significantly reduced LPS uptake as well as TNF-α production compared with control rats. However, no significant change was observed in procollagen type I mRNA levels of isolated stellate cells after 4 days of culture on plastic dishes. These results suggest that leptin receptor deficiency retards the development of liver fibrosis due to the dysfunction of Kuppfer cells.

73 Leptin receptor-deficient zucker (FA/FA) rat retards the development of pig serum-induced liver fibrosis with Kupffer cell dysfunction

73 Leptin receptor-deficient zucker (FA/FA) rat retards the development of pig serum-induced liver fibrosis with Kupffer cell dysfunction

Angiotensin-II type 1 receptor interaction is a major regulator for liver fibrosis development in rats

The renin-angiotensin system (RAS) is frequently activated in patients with chronic liver diseases. Angiotensin-II (AT-II) has been suggested to play an important role in liver fibrogenesis. It induces hepatic stellate cell (HSC) proliferation and up-regulates the transforming growth factor beta(1) (TGF-beta(1)) expression via AT-II type 1 receptor (AT(1)-R) in vitro. The aim of the present study was to examine the in vivo effect of candesartan (CA), a clinically used AT(1)-R blocker (ARB), and perindopril (PE), an angiotensin-converting enzyme (ACE) inhibitor (ACE-I), on pig serum-induced liver fibrosis development in rats. The clinically available comparable doses of CA and PE significantly attenuated the fibrosis development. These inhibitory effects of PE and CA were also found in the on-going liver fibrosis model. The hepatic hydroxyproline and serum fibrosis markers were significantly suppressed by CA and PE treatment. Furthermore, the alpha smooth muscle actin (alpha-SMA) positive cells in number were markedly suppressed by CA and PE treatment. Similarly, the hepatic TGF-beta(1) protein and messenger RNA (mRNA) levels were significantly suppressed. Our in vitro study showed that AT-II increased the TGF-beta(1) mRNA expression in the activated HSCs, and this effect was totally blocked by CA. These results suggested that the RAS, especially AT-II and AT(1)-R interaction plays a pivotal role in liver fibrosis development through HSC activation. Because both CA and PE are widely used in clinical practice without serious side effects, these drugs may provide an effective new strategy for anti-liver fibrosis therapy.

Angiotensin-II type 1 receptor interaction is a major regulator for liver fibrosis development in rats

The renin-angiotensin system (RAS) is frequently activated in patients with chronic liver diseases. Angiotensin-II (AT-II) has been suggested to play an important role in liver fibrogenesis. It induces hepatic stellate cell (HSC) proliferation and up-regulates the transforming growth factor β1 (TGF-β1) expression via AT-II type 1 receptor (AT1-R) in vitro. The aim of the present study was to examine the in vivo effect of candesartan (CA), a clinically used AT1-R blocker (ARB), and perindopril (PE), an angiotensin-converting enzyme (ACE) inhibitor (ACE-I), on pig serum-induced liver fibrosis development in rats. The clinically available comparable doses of CA and PE significantly attenuated the fibrosis development. These inhibitory effects of PE and CA were also found in the on-going liver fibrosis model. The hepatic hydroxyproline and serum fibrosis markers were significantly suppressed by CA and PE treatment. Furthermore, the α smooth muscle actin (α-SMA) positive cells in number were markedly suppressed by CA and PE treatment. Similarly, the hepatic TGF-β1 protein and messenger RNA (mRNA) levels were significantly suppressed. Our in vitro study showed that AT-II increased the TGF-β1 mRNA expression in the activated HSCs, and this effect was totally blocked by CA. These results suggested that the RAS, especially AT-II and AT1-R interaction plays a pivotal role in liver fibrosis development through HSC activation. Because both CA and PE are widely used in clinical practice without serious side effects, these drugs may provide an effective new strategy for anti–liver fibrosis therapy.

Enhanced Interstitial Collagenase (Matrix Metalloproteinase-13) Production of Kupffer Cell by Gadolinium Chloride Prevents Pig Serum-Induced Rat Liver Fibrosis

Hepatic fibrosis results from an imbalance between fibrogenesis and fibrolysis in the liver. It remains uninvestigated whether Kupffer cells produce matrix metalloproteinase-13 (MMP-13), which mainly hydrolyzes extracellular matrix (ECM). We sought to determine the role of Kupffer cells in fibrogenesis/fibrolysis. In vivo, we used the rat model of pig serum-induced liver fibrosis. A subset was treated with gadolinium chloride (GdCl3), which specifically acts on Kupffer cells. Administration of GdCl3 remarkably decreased the hydroxyproline content of the liver and increased the expression of MMP-13 mRNA in the liver without a difference in procollagen type I and tissue inhibitors of metalloproteinase-1 (TIMP-1) mRNA expression on Northern blot analysis with the elimination of ED2-positive cells. In vitro, addition of GdCl3 to isolated Kupffer cells showed increased type I collagen-degrading activity in a dose-dependent manner as well as MMP-13 mRNA expression on Northern blot analysis. It is concluded that Kupffer cells are a major source of MMP-13 and modulation of Kupffer cells by GdCl3 prevents liver fibrosis with increased expression of MMP-13 mRNA and protein, whereas procollagen type I and TIMP-1 mRNA, which encode two major effectors of fibrogenesis, were unchanged. This is the first report showing that Kupffer cells produce interstitial collagenase (MMP-13) resulting in the reduction of ECM. This discovery may provide new insights into therapy for hepatic fibrosis.

Prolyl 4-hydroxylase inhibitor (HOE 077) inhibits pig serum-induced rat liver fibrosis by preventing stellate cell activation

Background/Aims: The aim of this study was to investigate the effect and mechanism of fibrosuppression by a newly synthesized prolyl 4-hydroxylase inhibitor {HOE 077, 2, 4-pyridine dicarboxylic acids bis [(2-methoxyethyl) amide]} on pig serum-induced liver fibrosis in the rat.Methods: Male Wistar rats received 0.5 ml of pig serum twice a week for 10 weeks with 0, 100 or 200 ppm of HOE 077. At the end of the experiment, the hydroxyproline content of the liver, and alanine aminostransferase were measured. Histological stains used were HE, azan and a stain for α-smooth muscle actin (α-SMA). Electron microscopy was also performed. Messenger RNA expressions of type I and III procollagen were examined by Northern blot analysis. α-SMA positive cells and fibers with azan staining were assessed as percent area of the tissue specimen, using an image analysis system.Results: Rats that received pig serum for 10 weeks showed an increased liver hydroxyproline content of 318±39 μg/g we weight (n=15). HOE 077 at doses up to 200 ppm significantly (p<0.01) reduced this increase of liver hydroxyproline content (181±39 μg/g wet weight, n=15) in accordance with imporved histological findings. 200 ppm of HOE 077 significantly reduced mRNA expressions of α2(I) (486±102 vs 151±36, p<0.01) and α1(III) (276±127 vs 160±67, p<0.05) procollagen and percent area of α-SMA positive cells (2.94±2.14 vs 1.17±0.88%). Electron microscopy revealed that 200 ppm of HOE 077 prevented the loss of fat droplets.Conclusions: A prolyl 4-hydroxylase inhibitor (HOE 077) prevented pig serum-induced rat liver fibrosis by inhibiting stellate cell activation.

New prolyl 4-hydroxylase inhibitor inhibits pig serum-induced liver fibrosis by preventing ITO cell activation

New prolyl 4-hydroxylase inhibitor inhibits pig serum-induced liver fibrosis by preventing ITO cell activation