Heme oxygenase-1 prevents liver fibrosis in rats by regulating the expression of PPAR_γ and NF-_κB

AIM:To investigate the effects of heme oxygenase(HO)-1 on liver fibrosis and the expression of peroxisome proliferator-activated receptor gamma(PPARγ) and nuclear factor-kappa B(NF-κB) in rats.METHODS:Sixty Wistar rats were used to construct liver fibrosis models and were randomly divided into 5 groups:group A(normal,untreated),group B(model for 4 wk,untreated),group C(model for 6 wk,untreated),group D [model for 6 wk,treated with zinc protoporphyrin Ⅸ(ZnPP-Ⅸ) from week 4 to week 6],group E(model for 6 wk,treated with hemin from week 4 to week 6).Next,liver injury was assessed by measuring serum alanine aminotransferase(ALT),aspartate aminotransferase(AST) and albumin levels.The degree of hepatic fibrosis was evaluated by measuring serum hyaluronate acid(HA),type Ⅳ collagen(Ⅳ-C) and by histological examination.Hydroxyproline(Hyp) content in the liver homogenate was determined.The expres-sion levels of alpha-smooth muscle actin(α-SMA) in liver tissue were measured by real-time quantitative polymerase chain reaction(RT-PCR).The expression levels of PPARγ and NF-κB were determined by RT-PCR and Western blotting.RESULTS:The expression of HO-1 increased with the development of fibrosis.Induction of HO-1 by hemin significantly attenuated the severity of liver injury and the levels of liver fibrosis as compared with inhibition of HO-1 by ZnPP-Ⅸ.The concentrations of serum ALT,AST,HA and Ⅳ-C in group E decreased compared with group C and group D(P < 0.01).Amount of Hyp and α-SMA in the liver tissues in group E decreased compared with group C(0.62 ± 0.14 vs 0.84 ± 0.07,1.42 ± 0.17 vs 1.84 ± 0.17,respectively,P < 0.01) and group D(0.62 ± 0.14 vs 1.11 ± 0.16,1.42 ± 0.17 vs 2.56 ± 0.37,respectively,P < 0.01).The expression of PPARγ at levels of transcription and translation decreased with the development of fibrosis especially in group D;and it increased in group E compared with groups C and D(0.88 ± 0.15 vs 0.56 ± 0.19,0.88 ± 0.15 vs 0.41 ± 0.11,respectively,P < 0.01).The expression of NF-κB increased with the development of fibrosis especially in group D;and it decreased in group E compared with groups C and D(1.43 ± 0.31 vs 1.89 ± 0.29,1.43 ± 0.31 vs 2.53 ± 0.54,respectively,P < 0.01).CONCLUSION:Our data demonstrate a potential mechanism that HO-1 can prevent liver fibrosis by enhancing the expression of PPARγ and decreasing the expression of NF-κB in liver tissues.

Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

AIM： To investigate the effects of transforming growth factor β1 （TGF-β1） on the differentiation of colonic lamina propria fibroblasts （CLPF） into myofibroblasts in vitro.METHODS： Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of m-smooth muscle actin （α-SMA）, fibronectin （FN） and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase （FAK） in CLPF were analyzed after induction of migration.did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SIVlA production. Short term incubation （6 h） with TGF-β1 enhanced CLPF migration, while long term treatment （6 d） of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 （2 d） in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells.CONCLUSION： Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact （6 h） of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.

THE BI DIRECTIONAL REGULATION OF FILAMIN ON THE ATPase ACTIVITY OF SMOOTH MUSCLE MYOSIN

The aim of this study is to investigate the functional relationship between filamin, a known actin binding protein, and myosin and the effects of filamin on the interaction between myosin and actin. Methods.Ultra centrifugation method was used to investigate the binding of filamin to both phosphorylated and unphosphorylated myosins. Mg ATPase activities of both phosphorylated and unphosphorylated myosins in the presence and absence of actin were measured to observe the effects resulted from filamin actin and filamin myosin interactions. Results. It was found that filamin is also a myosin binding protein. Filamin inhibited the actin activated Mg ATPase activity of phosphorylated myosin and stimulated Mg ATPase of phosphorylated myosin in the absence of actin; in addition, filamin stimulated Mg ATPase activity of unphosphorylated myosin in both the presence or absence of actin. Conclusion. The results suggest that the effects of filamin on the myosin Mg ATPase activities are bi directional, i.e., stimulatory via binding to myosin and inhibitory via binding to actin.

Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase

Myosin light chain kinases （MLCK） phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase （S-MLCK） to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase （L-MLCK） has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules （2Ig） in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased ＂spike＂-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.

Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor-pi and a-smooth muscle actin in rats with hepatic fibrosis

AIM:To investigate the effect of interferon-a (IFN-α) on preventing or reversing hepatic fibrosis in rat experimental model induced by CCI4. METHODS: One hundred and ten Sprague-Dawley rats were divided into five groups: group A (normal controls, n = 18), group B (fibrotic model controls, n = 22), group C (IFN-α prevention, n = 22) initially treated with intramuscular injection of IFN-a in saline daily at the doses of 1× 105 U for 6 wk, group D (IFN-a treatment, n = 24) treated with intra-muscular injection of IFN-a in saline daily at the doses of 1×105 U for 6 wk after the first 6 wk, group E (0.9% sodium chloride treatment control, n = 24) treated with intra-muscular injection of 0.01 mL/kg daily for 6 wk after the first 6 wk. At the end of the experiment, all rats of each group were killed. Samples of the liver obtained by biopsy were subjected to histological, immunohistochemical and electron microscopic studies for the expressions of transforming growth factor-pi (TGF- μ41) and α-smooth muscle actin (α-SMA). RESULTS: The expressions of TGF-pl, the number of activated hepatic stellate cells and a-SMA in hepatic tissue of group C were significantly less than those of group B (P<0.01). The degree of fibrosis score in group B was also significantly less than that of group C under light microscope (P<0.01). CONCLUSION: IFN-a can inhibit the production of TGF-pl, decrease HSC activation and stimulate its apoptosis.

Gastrointestinal stromal tumor of duodenum:a cause of upper gastrointestinal hemorrhage

Objective:The aim of the study was to report an anemia patient with melena for five years caused by duodenal gastrointestinal stromal tumor (GIST), who required surgical treatment. Methods: A 44-year old man present with anemia appearance was admitted to our center (Department of Hepatobiliary Surgery, Union Hospital, Huazhong University of Science and Technology, China) due to sustaining melena for five years. Endoscopy found no special mucosal abnormalities in the duodenal lumen. Computed Tomography showed a well-demarcated mass, 7.4 cm in diameter, located between the C loop of duodenum and pancreatic head. Pylorus-preserving pancreaticoduodenectomy and right hemicolectomy were performed when the patient's general conditions were improved. He recuperated successfully and was discharged on the 21st postoperative day. No complications happened during the period of hospital stay. Results: Histological and immunohistochemical study revealed a high risk invasive duodenal GIST which was positive for CD117, CD34, α-smooth muscle actin and negative for S-100. Conclusion: Duodenal GIST can be a source of upper gastrointestinal hemorrhage; surgical treatment is still a reasonable choice for the patients with invasive duodenal GIST.

IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

AIM： To examine the pathway related to the IL-1β induced activation of mitogen-activated protein （MAP） kinases in cat esophageal smooth muscle cells. METHODS： Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-β3. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases. RESULTS： In the primary cultured cells, although IL-β3 failed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of 1344142 HAP kinase and p38 MAP kinase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively. The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 HAP kinase band to the basal level. Phosphokinase C （PKC） was found to play a mediating role in the IL-1β-induced p44/42 MAP kinase activity. In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds. CONCLUSION： Based on these results, IL-1β-induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C （PLC） and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL- 1β.

大黄素抑制IgG型抗双链DNA抗体诱导的系膜细胞表型转化的研究

目的探讨大黄素对IgG型抗双链DNA(dsDNA)抗体诱导的大鼠系膜细胞(MCs)表型转化的抑制作用。方法利用马疫锥虫动基体DNA诱导大鼠产生IgG型抗dsDNA抗体,小牛胸腺DNA纤维素层析法提纯抗体。体外培养正常MCs(空白组),或在培养上清中分别加入IgG型抗dsDNA抗体、25 mg/L大黄素(对照组),或同时加IgG型抗dsDNA抗体与大黄素(实验组),检测MCs合成转化生长因子-β_1(TGF-β_1)、α-平滑肌肌动蛋白(α-SMA)等水平,并在透视电镜下观察细胞显微结构的变化。结果与空白组比较,抗体对照组合成TGF-β_1等水平明显升高(P<0.05),并出现肌成纤维细胞样结构变化;而大黄素对照组的上述指标下降(P<0.05),且细胞显微结构基本正常。与抗体对照组比较,实验组合成TGF-β_1等水平降低,细胞显微结构大致正常。结论抑制IgG型抗dsDNA抗体诱导的系膜细胞表型转化可能是大黄素治疗狼疮肾炎的药理机制之一。

Inhibition mechanism of compound ethanol extracts from Wuweizi(Fructus Schisandrae Chinensis) on renal interstitial fibrosis in diabetic nephropathy model mice

OBJECTIVE：To evaluate inhibition effect and mech- anism of compound ethanol extracts from Wuweizi （Fructus Schisandrae Chinensis）, Chuanxiong （Rhi- zoma Chuanxiong） and Muli （Cocha Ostreae） （FRC） on glomerular and tubular interstitial fibrosis in streptozocin （STZ）-induced diabetic nephropathy （ND） model mice. METHODS： Twenty-seven male C57BL/6 mice were divided randomly into 3 groups： nondibetic （ND）, STZ-induced diabetic （D）, and STZ-induced diabetic that were treated with .5 g. kg1. daylof FRC by oral gavage （DFRc）, with 9 in each group. The protein ex- pressions of E-cadherin, a-smooth muscle actin （a-SMA）, Plasminogen Activator Inhibitor-1 （PAl-l） in renal tissues were investigated by Western blot- ting. The expressions of fibronectin （FN） and o-SMA were detected by immunohistochemical method. The morphological changes of renal tissues were observed under a microscope. RESULTS： Renal tissues in the DFRC group showed a lessened degree of fibrosis. Meanwhile, the expres- sions of FN, o-SMA and PAl-lwere significantly lower in the DrRc group than those in the D group （all P〈0.05）.CONCLUSION： FRC can ameliorate the DN in the C57BL/6 mice, and its mechanism may relate to in- hibition on the epithelial to mesenchymal transdif- ferentiation, endothelial-myofibroblast transition and PAl-1 expression.