Myricetin attenuates renal fibrosis by activating Nrf2/HO-1 pathway to inhibit oxidative stress / 药学学报

This paper investigates the effect of myricetin (MYR) on renal fibrosis induced by unilateral ureteral obstruction (UUO) and common bile duct ligation (CBDL) in mice and its mechanism. The animal experiment has been approved by the Ethics Committee of China Pharmaceutical University (NO: 2022-10-020). Thirty-five ICR mice were divided into control, UUO, UUO+MYR, CBDL and CBDL+MYR groups. H&E and Masson staining were used to detect pathological changes in kidney tissues. Western blot (WB) was used to detect the expression of fibrosis-related proteins in renal tissue, and total superoxide dismutase (SOD) activity detection kit (WST-8) was used to detect the changes of total SOD in renal tissue of CBDL mice. In vitro, HK-2 cells and transforming growth factor beta 1 (TGF-β1, 10 ng·mL-1) were used to induce fibrotic model, and high glucose (30 mmol·L-1) was used to induce oxidative stress model, and then treated with different concentrations of MYR, WB was used to detect the expression of fibrosis and oxidative stress-related proteins, while NIH/3T3 cells were treated with different concentrations of MYR, and their effects on cell proliferation were detected by 5-bromo-2′-deoxyuridine (Brdu). The results showed that the renal lesions in UUO group and CBDL group were severe, collagen deposition was obvious, the expression of collagen-Ⅰ (COL-Ⅰ), α-smooth muscle actin (α-SMA), fibronectin (FN), vimentin and plasminogen activator inhibitor-1 (PAI-1) protein was up-regulated, and the activity of SOD enzyme in CBDL group was significantly decreased. MYR partly reversed the above changes after treatment. MYR inhibited the proliferation of NIH/3T3 cells but had no effect on the proliferation of HK-2 cells, and decreased the upregulation of PAI-1, FN and vimentin in HK-2 cells stimulated by TGF-β1. MYR can also up-regulate the down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in HK-2 cells stimulated by high glucose. To sum up, MYR can improve renal fibrosis in vivo and in vitro, probably by inhibiting the proliferation of fibroblasts and activating Nrf2/HO-1 signal pathway to inhibit oxidative stress.

Bile duct ligation differently regulates protein expressions of organic cation transporters in intestine, liver and kidney of rats through activation of farnesoid X receptor by cholate and bilirubin

Body is equipped with organic cation transporters (OCTs). These OCTs mediate drug transport and are also involved in some disease process. We aimed to investigate whether liver failure alters intestinal, hepatic and renal Oct expressions using bile duct ligation (BDL) rats. Pharmacokinetic analysis demonstrates that BDL decreases plasma metformin exposure, associated with decreased intestinal absorption and increased urinary excretion. Western blot shows that BDL significantly downregulates intestinal Oct2 and hepatic Oct1 but upregulates renal and hepatic Oct2. In vitro cell experiments show that chenodeoxycholic acid (CDCA), bilirubin and farnesoid X receptor (FXR) agonist GW4064 increase OCT2/Oct2 but decrease OCT1/Oct1, which are remarkably attenuated by glycine-β-muricholic acid and silencing FXR. Significantly lowered intestinal CDCA and increased plasma bilirubin levels contribute to different Octs regulation by BDL, which are confirmed using CDCA-treated and bilirubin-treated rats. A disease-based physiologically based pharmacokinetic model characterizing intestinal, hepatic and renal Octs was successfully developed to predict metformin pharmacokinetics in rats. In conclusion, BDL remarkably downregulates expressions of intestinal Oct2 and hepatic Oct1 protein while upregulates expressions of renal and hepatic Oct2 protein in rats, finally, decreasing plasma exposure and impairing hypoglycemic effects of metformin. BDL differently regulates Oct expressions via Fxr activation by CDCA and bilirubin.

Effect of vitamin C inhibiting liver fibrosis and lipid peroxidation in biliary obstruction of Wistar rats

Background: The aim of this study was to evaluate the potential inhibiting effects of vitamin C as an antioxidant against liver fibrosis and lipid peroxidation in the bile duct ligation- induced biliary obstruction of Wistar rats.Methods: A total of 25 male Wistar albino rats were divided into five groups: sham operated, control (bile duct ligation/BDL) without given vitamin C, BDL with vitamin C 75 mg, BDL with vitamin C 150 mg, and BDL with vitamin C 225 mg. Each group contained 5 animals. Vitamin C was given orally on day 3 after operation and after 14 days following vitamin C administration, all animals were performed laparotomy to obtain liver tissue samples for histopathological investigation of liver fibrosis and blood samples for malondialdehyde (MDA) as lipid peroxidation measurement.Results: The changes demonstrating hepatic fibrosis including moderate to markedly thickened wall of central veins, localized to diffuse perisinusoidal fibrosis, enlarged portal track, increased number of septa, and thickened width of septa were observed in BDL groups. MDA measurement were also observed in all groups. Treatment of biliary obstruction in BDL groups with vitamin C given orally attenuated liver damage. Both the MDA measurement and histopathologic investigation of hepatic fibrosis were observed to be reduced with the vitamin C treatment.Conclusions: Our data indicate that vitamin C inhibited liver fibrosis and lipid peroxidation in bile duct ligation-induced biliary obstruction of Wistar rats.

CD24 regulates Ly6Chi macrophages and BDL-induced liver fibrosis / 中华微生物学和免疫学杂志

Objective To investigate the regulatory effects of CD24 on Ly6Chi macrophages in liv-er and its influences on bile duct ligation ( BDL)-induced hepatic fibrosis in mice. Methods Liver fibrosis was induced in wild-type ( WT) and CD24-/- mice by surgical ligation of the biliary duct. Levels of alanine amino transferase ( ALT) in serum were detected and liver sections were stained with haematoxylin and eosin ( H&E) staining to assess the severity of liver injury. Sirius Red staining was used to observe the degree of liver fibrosis. Real-time PCR was performed for detecting the expression of hepatic fibrosis-related markers and TGF-β1 at mRNA level. The percentage of macrophages and the number of TGF-β1-producing macro-phages were measured by flow cytometry. Results BDL-induced liver fibrosis was exacerbated in CD24-/-mice than in WT mice as demonstrated by more serious hyperplasia in bile duct, more inflammatory infiltra-tion at the portal area and higher levels of ALT in serum. Results of Sirius red staining also showed that the liver fibrosis was more severe in CD24-/- mice than in WT mice. Moreover, α-SMA and collagen typeⅠalpha 1 (Col1a1) were significantly upregulated in CD24-/- mice. Flow cytometry analysis revealed that CD24 was highly expressed by hepatic macrophages in BDL-induced WT mice, and the percentages of hepat-ic macrophages were significantly elevated in CD24-/-mice compared with those in WT mice. Further analy-sis revealed that the percentages of Ly6Chi hepatic macrophages in CD24-/- mice were higher than those in WT mice, but there was no significant difference in the percentages of Ly6Clo macrophages. The expression of TGF-β1 at mRNA level was increased in CD24-/-mice as compared with that in WT mice after BDL. Mo-reover, intracellular staining showed that Ly6Chi hepatic macrophages in CD24-/- mice secreted more TGF-β1 than the macrophages in WT mice. Conclusions CD24 might attenuate the BDL-induced liver fibrosis in mice via regulating the percentage of hepatic Ly6Chi macrophages and the secretion of TGF-β1.

The establishment of portal hypertension model in mice / 介入放射学杂志

Objective To establish two types of portal hypertension (PHT) models in mice by using bile duct ligation (BDL) method and carbon tetrachloride (CCl4) induction technique respectively. Methods A total of 24 C57BL/6 mice were randomly and equally divided into the following four groups with 6 mice in each group: group BDL, control group of BDL, group CCl4, and control group of CCI4. After the establishment of PHT, the main portal vein was punctured in all experimental mice to measure the portal vein pressure, and blood sampling was collected to test serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Using hematoxylin eosin (HE) and sirius red staining the liver tissues were pathologically examined. Immunohistochemical study of alpha smooth muscle actin (SMA) was adopted to evaluate the liver function, hepatic fibrosis and hepatic stellate cell activation status. Results Both modeling methods could make the portal vein pressure increased in experimental mice. The increasing of portal vein pressure in group CCl4 was more obvious. Compared with their corresponding control groups, the degree of liver damage, hepatic fibrosis and hepatic stellate cell activation in group BDL and group CCl4 were more serious. Conclusion Both BDL method and CCl4 induction technique can successfully establish the mouse model of PHT. All the portal venous pressure, the serum biochemical indices and the changes of liver pathology of the mouse model are well in line with the characteristics of PHT in human. (J Intervent Radiol, 2018, 27:242-246)

Mechanism of Astragaloside prevents cholestatic liver fibrosis through inhibition of Notch signaling activation / 中华肝脏病杂志

Objective@#The Notch signaling pathway is closely related to biliary fibrosis. Previous studies have shown that Astragaloside (AS) can prevent the progression of cholestatic liver fibrosis. The purpose of this study is to observe the effect of AS on the regulation of Notch signaling pathway in biliary fibrosis.@*Methods@#Cholestatic liver fibrosis was established by common bile duct ligation (BDL) in rats. Two weeks after BDL, the rats were randomly divided into a model group (i.e., BDL), an Astragalosides group (AS), and a sorafenib (SORA) positive control group and treated for 3 weeks. Bile duct proliferation and liver fibrosis were determined by tissue staining. Protein and gene expression were determined by immunostaining, immunoblotting and RT-PCR, respectively. Activation of the Notch signaling pathway was evaluated by analyzing expressions of Notch-1, -2, -3, -4, Jagged (JAG)1, Delta like (DLL)-1, -3, -4, Hes1, Numb and RBP-Jκ. Statistical analysis of variance analysis, q test, P < 0.05 showed that the difference was statistically significant.@*Results@#(1) AS significantly reduced the deposition of collagen and the Hyp content of liver tissue (500.15 ± 86.10 vs. 625.72 ± 105.62, P = 0.031), and inhibited the activation of hepatic stellate cells. (2) AS significantly decreased the protein and mRNA expressions of transforming growth factor (TGF)-β1 (1.02±0.15 vs. 1.89±0.36, P = 0.007; 1.17±0.18 vs. 1.68±0.29, P = 0.013, respectively) and α-smooth muscle actin (α-SMA, 0.41±0.11 vs. 0.72±0.16, P = 0.003; 1.71±0.57 vs. 2.68±0.46, P = 0.008, respectively) compared with BDL group. In contrast, AS significantly enhanced expression of the Smad 7 protein compared with the BDL group (0.72±0.008 vs. 0.33±0.001, P = 0.005). AS also reduced biliary epithelial cell proliferation. AS reduced the mRNA levels of CK7, CK8 and CK18 (1.31±0.39 vs. 2.63±0.82, P = 0.009; 0.71±0.09 vs. 0.87±0.08, P = 0.031; 2.56±0.32 vs. 3.41±0.39, P = 0.010, respectively) and reduced the positive areas of CK19 and OV6 (62 337.17±21 873.38 vs. 22 5472.67±26 933.63, P = 0.000; 92 237.43±15 894.11 vs. 171 298.13±61 761.37, P = 0.000, respectively). (3) The mRNA expression of Notch-2, -3, -4 and JAG1 were significantly reduced in the AS group compared to the BDL group (1.07±0.19 vs. 1.51±0.28, P = 0.044; 0.99±0.24 vs. 1.18±0.10, P = 0.043; 1.36±0.42 vs. 3.40±0.44, P = 0.048; 2.62±0.43 vs. 3.73±0.83, P = 0.046, respectively). In contrast, the mRNA level of Numb was clearly enhanced after AS treatment (0.90±0.05 vs. 0.75±0.11, P = 0.019). In addition, consistent with the mRNA levels, the protein expressions of Notch-2, -3, -4 and JAG1 were reduced significantly (1.27±0.18 vs. 1.71±0.26, P = 0.004; 0.99±0.11 vs. 4.38±0.60, P = 0.001; 1.76±0.32 vs. 4.01±0.74, P = 0.002; 1.62±0.33 vs. 2.74±0.63, P = 0.002) and the Numb protein level was increased significantly (1.50±0.15 vs. 0.85±0.11, P = 0.001) in AS group compared with BDL group.@*Conclusion@#AS may prevent cholestatic liver fibrosis via inhibition of the Notch signaling pathway, thereby inhibiting the abnormal proliferation of biliary epithelial cells. Results indicate that AS may be a potential treatment for cholestatic liver disease.

Long-term effects of human amniotic membrane in a rat model of biliary fibrosis

Liver fibrosis is the most common outcome of chronic liver diseases, and its progression to cirrhosis can only be effectively treated with liver transplantation. The amniotic membrane (AM) has been studied as an alternative therapy for fibrosis diseases mainly for its favorable properties, including anti-inflammatory, anti-scaring and immunomodulatory properties. It was recently demonstrated that the AM reduces the progression of biliary fibrosis to its advanced stage, cirrhosis, when applied on the liver for 6 weeks after fibrosis induction. Here, we investigated the effects of AM on rat fibrotic liver, during a prolonged period of time. Fibrosis was induced by bile duct ligation (BDL), and at the same time, a fragment of AM was applied around the liver. After 1, 3, 6, and 9 weeks, the degree of fibrosis was assessed by qualitative Knodell scoring, and by quantitative image analysis to quantify the area of collagen deposition in hepatic tissue. While fibrosis progressed rapidly in untreated BDL animals, leading to cirrhosis within 6 weeks, AM-treated livers showed confined fibrosis at the periportal area with few and thin fibrotic septa, but without cirrhosis. In addition, collagen deposition was reduced to about 36 and 55% of levels observed in BDL at 6 and 9 weeks after BDL, respectively, which shows that the longer the period of AM application, the lower the collagen deposition. These results suggested that AM applied as a patch onto the liver surface for longer periods attenuated the severity of biliary fibrosis and protected against liver degeneration caused by excessive collagen deposition.

Effect of Rifaximin on Hepatic Fibrosis in Bile Duct-ligated Rat Model / 대한소화기학회지

BACKGROUND/AIMS: The translocation of bacteria and their lipopolysaccharides from the gut can promote fibrosis in cirrhotic patients. The aim of this study was to investigate the effects of rifaximin on hepatic fibrosis in a bile duct-ligated rat model. METHODS: The bile duct ligation (BDL) was carried out for eight days (acute injury model: sham-operated rats [G1], BDL rats [G2], and BDL rats treated with rifaximin [G3]) or 22 days (chronic injury model: sham-operated rats [G4], BDL rats [G5], and BDL rats treated with rifaximin [G6]). Rifaximin (50 mg/kg/day) was administered daily via gavage after BDL. Liver function, serum tumor necrosis factor-alpha (TNF-α), and hepatic hydroxyproline levels were measured. Moreover, a histological analysis of fibrosis contents was performed using sirius red stain. RESULTS: In the acute injury model, the liver function and TNF-α level were not improved after the rifaximin treatment. The hydroxyproline levels (µg/g liver tissue) in G1, G2, and G3 were 236.4±103.1, 444.8±114.4, and 312.5±131.6, respectively; and fibrosis contents (%) were 0.22±0.04, 1.64±0.53, and 1.66±0.44, respectively. The rifaximin treatment did not ameliorate acute BDL-induced fibrosis. In the chronic injury model, the hydroxyproline levels in G4, G5, and G6 were 311.5±72.9, 1,110.3±357.9, and 944.3±209.3, respectively; and fibrosis contents (%) were 0.19±0.03, 5.04±0.18, and 4.42±0.68, respectively (G5 vs. G6, p=0.059). The rifaximin treatment marginally ameliorated chronic BDL-induced fibrosis. CONCLUSIONS: Rifaximin did not reduce inflammation and fibrosis in bile duct-ligated rat model.

Protective effects of a polymethoxy flavonoids-rich Citrus aurantium peel extract on liver fibrosis induced by bile duct ligation in mice

Objective To evaluate the possible protective effect of Citrus aurantium peel extract (CAE) against apoptosis in cholestatic liver fibrosis induced by bile duct ligation in mice. Methods Male ICR mice were divided to 5 groups: 1) Control group (Sham-operated mice), 2) Cholestatic liver injury group induced by bile duct ligation (BDL), 3) BDL mice treated with silymarin (200 mg/kg) for 4 weeks, 4) BDL mice treated with 50 mg/kg CAE for 4 weeks, 5) BDL mice treated with 200 mg/kg CAE for 4 weeks. Mice were sacrificed and liver fibrosis was evaluated by serum and hepatic tissue biochemistry tests and liver histopathological examination. Effects of CAE on inflammation and apoptosis gene regulation were investigated through real-time PCR. CAE effect on lipid metabolism related signaling was determined by western blot analysis. Results In BDL mice, administration of CAE for 4 weeks markedly attenuated liver fibrosis based on histopathological alteration. Serum and hepatic tissue biochemistry results revealed that CAE (50 and 200 mg/kg) decreased the levels of alanine transaminase, aspartate transaminase, gamma-glutamyl transferase, total bilirubin, nitric oxide, and thiobarbituric acid reactive substances. Real-time PCR and western blot analysis showed that CAE regulated inflammation, apoptosis, and lipid metabolism factors increased by BDL. Interleukin family, tumor necrosis factor α, and related apoptosis factors mRNA levels were increased by BDL treatment. However, these increases were suppressed by CAE administration. In addition, CAE effectively increased phosphorylation of AMP-activated protein kinase, nuclear factor E2-related factor 2, and related cytoprotective proteins. Conclusions CAE can efficiently regulate BDL-induced liver injury with antioxidant, anti-inflammatory, and anti-apoptotic activities.

Protective effects of a polymethoxy flavonoids-rich Citrus aurantium peel extract on liver fibrosis induced by bile duct ligation in mice

OBJECTIVE@#To evaluate the possible protective effect of Citrus aurantium peel extract (CAE) against apoptosis in cholestatic liver fibrosis induced by bile duct ligation in mice.@*METHODS@#Male ICR mice were divided to 5 groups: 1) Control group (Sham-operated mice), 2) Cholestatic liver injury group induced by bile duct ligation (BDL), 3) BDL mice treated with silymarin (200 mg/kg) for 4 weeks, 4) BDL mice treated with 50 mg/kg CAE for 4 weeks, 5) BDL mice treated with 200 mg/kg CAE for 4 weeks. Mice were sacrificed and liver fibrosis was evaluated by serum and hepatic tissue biochemistry tests and liver histopathological examination. Effects of CAE on inflammation and apoptosis gene regulation were investigated through real-time PCR. CAE effect on lipid metabolism related signaling was determined by western blot analysis.@*RESULTS@#In BDL mice, administration of CAE for 4 weeks markedly attenuated liver fibrosis based on histopathological alteration. Serum and hepatic tissue biochemistry results revealed that CAE (50 and 200 mg/kg) decreased the levels of alanine transaminase, aspartate transaminase, gamma-glutamyl transferase, total bilirubin, nitric oxide, and thiobarbituric acid reactive substances. Real-time PCR and western blot analysis showed that CAE regulated inflammation, apoptosis, and lipid metabolism factors increased by BDL. Interleukin family, tumor necrosis factor α, and related apoptosis factors mRNA levels were increased by BDL treatment. However, these increases were suppressed by CAE administration. In addition, CAE effectively increased phosphorylation of AMP-activated protein kinase, nuclear factor E2-related factor 2, and related cytoprotective proteins.@*CONCLUSIONS@#CAE can efficiently regulate BDL-induced liver injury with antioxidant, anti-inflammatory, and anti-apoptotic activities.

Decreased C-reactive protein induces abnormal vascular structure in a rat model of liver dysfunction induced by bile duct ligation

BACKGROUND/AIMS: Chronic liver disease leads to liver fibrosis, and although the liver does have a certain regenerative capacity, this disease is associated with dysfunction of the liver vessels. C-reactive protein (CRP) is produced in the liver and circulated from there for metabolism. CRP was recently shown to inhibit angiogenesis by inducing endothelial cell dysfunction. The objective of this study was to determine the effect of CRP levels on angiogenesis in a rat model of liver dysfunction induced by bile duct ligation (BDL). METHODS: The diameter of the hepatic vein was analyzed in rat liver tissues using hematoxylin and eosin (H&E) staining. The expression levels of angiogenic factors, albumin, and CRP were analyzed by real-time PCR and Western blotting. A tube formation assay was performed to confirm the effect of CRP on angiogenesis in human umbilical vein endothelial cells (HUVECs) treated with lithocholic acid (LCA) and siRNA-CRP. RESULTS: The diameter of the hepatic portal vein increased significantly with the progression of cirrhosis. The expression levels of angiogenic factors were increased in the cirrhotic liver. In contrast, the expression levels of albumin and CRP were significantly lower in the liver tissue obtained from the BDL rat model than in the normal liver. The CRP level was correlated with the expression of albumin in hepatocytes treated with LCA and siRNA-CRP. Tube formation was significantly decreased in HUVECs when they were treated with LCA or a combination of LCA and siRNA-CRP. CONCLUSION: CRP seems to be involved in the abnormal formation of vessels in hepatic disease, and so it could be a useful diagnostic marker for hepatic disease.

Rhus verniciflua Stokes attenuates cholestatic liver cirrhosis-induced interstitial fibrosis via Smad3 down-regulation and Smad7 up-regulation / 대한해부학회지

Cholestatic liver cirrhosis (CLC) eventually proceeds to end-stage liver failure by mediating overwhelming deposition of collagen, which is produced by activated interstitial myofibroblasts. Although the beneficial effects of Rhus verniciflua Stokes (RVS) on various diseases are well-known, its therapeutic effect and possible underlying mechanism on interstitial fibrosis associated with CLC are not elucidated. This study was designed to assess the protective effects of RVS and its possible underlying mechanisms in rat models of CLC established by bile duct ligation (BDL). We demonstrated that BDL markedly elevated the serological parameters such as aspartate aminotransferase, alanine transaminase, total bilirubin, and direct bilirubin, all of which were significantly attenuated by the daily uptake of RVS (2 mg/kg/day) for 28 days (14 days before and after operation) via intragastric route. We observed that BDL drastically induced the deterioration of liver histoarchitecture and excessive deposition of extracellular matrix (ECM), both of which were significantly attenuated by RVS. In addition, we revealed that RVS inhibited BDL-induced proliferation and activation of interstitial myofibroblasts, a highly suggestive cell type for ECM production, as shown by immunohistochemical and semi-quantitative detection of α-smooth muscle actin and vimentin. Finally, we demonstrated that the anti-fibrotic effect of RVS was associated with the inactivation of Smad3, the key downstream target of a major fibrogenic cytokine, i.e., transforming growth factor β (TGF-β). Simultaneously, we also found that RVS reciprocally increased the expression of Smad7, a negative regulatory protein of the TGF-β/Smad3 pathway. Taken together, these results suggested that RVS has a therapeutic effect on CLC, and these effects are, at least partly, due to the inhibition of liver fibrosis by the downregulation of Smad3 and upregulation of Smad7.

Antioxidant Effect of Sepia Ink Extract on Extrahepatic Cholestasis Induced by Bile Duct Ligation in Rats / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The aim of our study was to assess the complications of hepatic fibrosis associated with bile duct ligation and the potential curative role of sepia ink extract in hepatic damage induced by bile duct ligation.</p><p><b>METHODS</b>Rattus norvegicus rats were divided into 3 groups: Sham-operated group, model rats that underwent common bile duct ligation (BDL), and BDL rats treated orally with sepia ink extract (200 mg/kg body weight) for 7, 14, and 28 d after BDL.</p><p><b>RESULTS</b>There was a significant reduction in hepatic enzymes, ALP, GGT, bilirubin levels, and oxidative stress in the BDL group after treatment with sepia ink extract. Collagen deposition reduced after sepia ink extract treatment as compared to BDL groups, suggesting that the liver was repaired. Histopathological examination of liver treated with sepia ink extract showed moderate degeneration in the hepatic architecture and mild degeneration in hepatocytes as compared to BDL groups.</p><p><b>CONCLUSION</b>Sepia ink extract provides a curative effect and an antioxidant capacity on BDL rats and could ameliorate the complications of liver cholestasis.</p>

Dynamic expression of desmin, α-SMA and TGF-β1 during hepatic fibrogenesis induced by selective bile duct ligation in young rats

We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-β1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT‐PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-β1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT‐PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-β1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT‐PCR technique was more sensitive to expression changes than the semiquantitative method.

Protective effects of Baicalin on intestinal mucosa injury by inhibiting nuclear factor-κB expression in rats with partial common bile duct ligation / 中华实用儿科临床杂志

Objective To investigate the effects of Baicalin on intestinal mucosal injury in rats with partial common bile duct ligation (PCBDL).Methods Forty male Wistar rats were randomly divided into 4 groups equally:sham operation,PCBDL,PCBDL1 and PCBDL2.Rats in PCBDL,PCBDL1 and PCBDL2 groups were subjected to partial common bile duct ligation.Baicalin [80 mg/(kg · d)] was fed in PCBDL1 group (for 2 weeks) and PCBDL2 group (for 3 weeks),while for other groups,9 g/L saline in the same volume was fed.Ileum mucosa were prepared for microscopic examination.The intestinal mucosal injury in rats was observed and scored.The level of NF-κB mRNA expression by Fluorescent in Situ Hybridization,and the level of NF-κB protein were determined by immunohistochemistry.Results 1.Compared with PCBDL group (3.2 ± 0.5),the pathological severity scores of intestinal mucosa significantly declined (F =21.120,P ＜ 0.01) in PCBDL1 group (1.9 ± 0.2) and PCBDL2 group (1.5 ± 0.3).2.Compared with sham operation group(0.066 ± 0.006),PCBDL1 group (0.107 ± 0.011),and PCBDL2 group (0.098 ± 0.010),NF-κB expression in PCBDL group (0.155 ± 0.012) presented significantly up-regulation (F =76.8,P ＜ 0.01).3.Compared with sham operation,PCBDL1 group,and PCBDL2 group,the positive expression rates of NF-κB mRNA of intestinal mucosal epithelium in PCBDL group significantly increased.Conclusions It is suggested that Baicalin exert protective effects on the intestinal mucosal injury in rats with PCBDL,partially by inhibiting NF-κB mRNA,down-regulating NF-κB protein expression of intestinal mucosal epithelial cells.

Serum hyaluronic acid level: Correlation with quantitative measurement of hepatic fibrosis in a cirrhotic rat model / 대한간학회지

BACKGROUNDS/AIMS: The serum level of hyaluronic acid (HA) has been suggested as a useful serologic marker for hepatic fibrosis. However, the relationship between serum HA levels and quantitative markers of fibrosis from liver tissue has not been reported. The aim of this study was to determine the correlation between serum HA level and quantitative measurement of hepatic fibrosis in a cirrhotic rat model. METHODS: Cirrhosis was produced by common bile duct ligation (BDL) in adult Sprague-Dawley rats. The animals were classified into four groups: (1) G1, sham operated (n=5); (2) G2, BDL for 2 weeks (n=6); (3) G3, BDL for 3 weeks (n=6); and (4) G4, BDL for 4 weeks (n=6). Hepatic fibrosis was analyzed histomorphologically using the Batts and Ludwig scoring system. Serum HA level and hepatic hydroxyproline content were quantified. The gene expressions in the liver of procollagen, collagen, and transforming growth factor-beta (TGF-beta) were measured by reverse transcriptase-polymerase chain reaction. RESULTS: In groups G1, G2, G3, and G4, the Batts and Ludwig scores (mean+/-SD) were 0, 1.3+/-0.5, 2.6+/-0.5, and 3.4+/-0.5, respectively (P<0.05), serum HA levels were 12.5+/-3.2, 30.0+/-4.3, 228.6+/-157.7, and 391.3+/-207.7 ng/mL (P<0.05), and the concentration of hydroxyproline was 12.4+/-2.8, 17.6+/-3.8, 17.9+/-2.4, and 33.4+/-3.4 microgram/g liver tissue, and it was significantly higher in group G4 than in the other groups (P<0.05). The gene expressions of collagen, procollagen, and TGF-beta1 in the liver were also significantly higher in group G4 compared with the other groups (P<0.05). Direct linear correlations were observed between serum HA level and hepatic hydroxyproline content, hepatic gene expressions of collagen, procollagen, TGF-beta1, and histomorphological grade of hepatic fibrosis (P<0.001). CONCLUSIONS: These results indicate that serum HA is a useful and noninvasive serologic marker for the evaluation of advanced hepatic fibrosis.

Study on Improvement of Zea mays Extract on Cholestatic Liver Diseases / 中国药房

OBJECTIVE:To study the improvement of Zea mays extract on cholestatic liver diseases.METHODS: Acute cholestatic liver injury model of rat was induced by bile duct ligation and cholestatic liver model of rats was established by ANIT.Model rats were given 6.2 g?kg-1 and 3.1 g?kg-1 dose of Z.mays extract for 5 days.The content of TBIL,DBIL,ALT,AST and TBA in serum and the secretion of bile were determined.RESULTS: The corn stigma extract decreased the content of DBIL,ALT and TBA in serum of ANIT-inducing cholestatic liver model,and increased the flow and speed of bile (P

Expression of Inducible Nitric Oxide Synthase (iNOS) in the Epithelial Cells of the Distended Bile Duct by the Bile Ductal Ligation

PURPOSE: Recent studies in an obstructive jaundice rat model showed that the bile duct epithelium is also very important in the bile duct dilatation besides the increased luminal pressure. This study evaluated the role of iNOS in the bile duct epithelium in a rat obstructive jaundice model. METHODS: Bile duct ligations were performed in male Sprague-Dawley rats. The bile ducts were harvested on seven consecutive days. Immunohistochemical staining in the bile duct was performed using anti-iNOS polyclonal antibodies. Aminoguanidine (an iNOS antagonist) was injected intraperitoneally after bile duct ligation (0, 100, and 200 mg/kg/day, n=6 in each group). One week after surgery, the diameter of bile duct was measured and bile was collected for NO analysis by 280NOA (Silvers). RESULTS: The iNOS expression level was increased in the dilated ductal epithelium after the bile duct ligation but not in the normal epithelium. Aminoguanidine decreased the mean diameter of the bile duct after the bile duct ligation: 11/-2.3 mm in the duct ligation only group; 7.5+/-0.75 mm in the 100 mg/kg/day aminoguanidine; 6+/-0.82 mm in the 200 mg/kg/day of aminoguanidine group (mean+/-SE, P<0.05). The NO concentration in the bile was decreased by aminoguanidine: 16+/-4.2 mM in the sham operation group; 40+/-4.5 mM in duct ligation only group; 34+/-6.4 mM in the 100 mg/kg/day of aminoguanidine group; 11+/-1.2 mM in the 200 mg/kg/day of aminoguanidine group (mean+/-SE). CONCLUSION: Bile duct ligation induced iNOS expression in the dilated bile duct epithelium and the iNOS antagonist partially inhibited bile duct dilatation. iNOS induction in the epithelium is partly responsible for the dilatation of the bile duct after duct ligation.

Liver Fibrosis by Bile Duct Sclerosis with Ethanol: a New Experimental Model for Liver Fibrosis / 한국간담췌외과학회지

BACKGROUND/AIMS: Common bile duct ligation will induce cholestatic fibrosis or cirrhosis in the rat. However, the degree of histologic changes following ligation of the common bile duct appears to vary widely due to recanalization of the bile duct in the distensible extrahepatic system. The purpose of this study is to compare liver damages induced by traditional simple ligation of bile duct with those induced by ethanolic sclerosis of bile duct and ligation. METHODS: Twenty rats of 7 weeks-aged Spraugue-Dawley (body weight 200~250 gm) were divided into 2 groups, common bile duct ligation (BDL group n=10), and 60% ethanol infusion into the common bile duct before bile duct ligation (ETL group n=10). After 7 weeks, all animals were sacrificed. Gross findings, histologic findings (Ki-67 and Masson- trichrome staining), and liver function test were compared. RESULTS: During the experiment, 5 rats of BDL group and 4 rats of ETL group died. In the operative findings, diameter of bile duct in BDL group was dilatated 8~15 mm, but there was no dilatation in ETL group. In the histologic findings of extrahepatic bile duct, epithelial cells were maintained well in BDL group, but they were all cast off or destroyed in ETL group. Liver fibrosis and cell proliferation are more prominent in the ETL group. Also, ETL group showed worse liver function test than that of BDL group. CONCLUSION: Liver fibrosis or cirrhosis by prolonged bile duct ligation is a well-known experimental model. In this study, we demonstrated that a new method of bile duct ligation (ethanol infusion before bile duct ligation) which abolishes bile duct distention accompanied with simple bile duct ligation is more effective to produce liver fibrosis or cirrhosis.

Change of the Lymphocyte Stimulation Index and the Serum Immunoglobulin in Experimental Obstructive Jaundiced Rats

BACKGROUND: The high incidence of operative complications in patients with jaundice is associated with an impaired immune system, an increased systemic and portal endotoxemia due to the decreased hepatic reticuloendothelial function, and increased immune inhibitory factors such as bilirubin and bile acids. This study was designed to evaluate the immune response and the histological changes in rats with obstructive jaundice induced by common bile-duct (CBD) ligation. METHODS: Thirty-two male Wister rats weighing 250-320 gm were enrolled in this study and divided into 2 groups: a control group and a CBD ligation group. Under general anesthesia, the CBD was ligated with silk and resected for the CBD ligation group, but it was only isolated for the control group. The rats were sacrificed at the lst, the 2nd, and the 3rd weeks after ligation, and we evaluated the chemistries of the liver function, the lymphocytes stimulation index, the serum immunoglobulin levels, and the histological changes in the bile-duct-ligated livers. RESULTS: 1. There was no statistical difference in the serum creatinine levels between the control and the CBD ligation groups. At the lst week in the CBD ligation group, the serum AST, ALT, and bilirubin levels were statistically higher than those of the control group (p<0.05), which were slightly increased thereafter. 2. The lymphocyte stimulation index (LSI) of peripheral lymphocytes in the CBD ligation group was significantly decreased with the passing of time, but that of the control group was not. The LSI of splenocytes was statistically higher than that of peripheral lymphocytes in the control group, and the LSI of splenocytes was significantly decreased at the lst and the 2nd weeks, but was only slightly decreased, without statistical significance, at the 3rd week, in the CBD ligation group. 3. The serum immunoglobulin G (Ig G) level was significantly decreased at the lst week in the CBD ligation group compared with that of the control group and was slightly decreased, without statistical significance, at the 2nd and the 3rd weeks in the CBD ligation group. The serum immunoglobulin A (Ig A) level was extremely low in all groups, but this result had no statistical significance. 4. After CBD ligation, livertissues showed progressive bile ductular proliferation in the portal tract and an infarct with inflammatory infiltration into the central vein area and cholestatic change. CONCLUSION: These results suggest that derangement of the liver functions, suppresed lymphocytes function, and decreased immunoglobulin production might be associated with impairment of the immune response in bile-duct-ligated rats.