Inhibition of Pim-1 kinase ameliorates dextran sodium sulfate-induced colitis in mice.

BACKGROUND: Pim-1 kinase is involved in the control of cell growth, differentiation and apoptosis. Recent evidence suggests that Pim kinases play a role in immune regulation and inflammation. However, the role of Pim-1 kinase in inflammatory bowel diseases (IBD) remains unclear. AIMS: The aims of this study were to explore the role of Pim-1 kinase in the pathology of IBD and to assess whether inhibiting Pim-1 kinase may be of therapeutic benefit as a treatment regimen for IBD. METHODS: Colitic mouse model was established by the induction of dextran sodium sulfate. The expression of Pim-1 in the colonic samples of control and colitic mice was examined. Furthermore, the mice were treated with Pim-1inhibitor (PIM-Inh), then the body weight and colon inflammation were evaluated, and the production of cytokines including IFN-γ, IL-4, TGF-ß and IL-17 in colon tissues was determined by ELISA. The expression of T cell master transcription factors T-bet, ROR-γt, GATA-3 and Foxp3 and Nuclear factor κB (NF-κB) and inducible nitric oxide synthase in colon tissues was detected by real-time PCR and western blot. Finally, the effect of LPS on Pim-1 expression and the effects of PIM-Inh on LPS-induced upregualtion of p65 and TNF-α in RAW264.7 cells were examined by real-time PCR and western blot. RESULTS: Pim-1 expression was correlated with the degree of mucosal inflammation in vivo, and it was significantly induced by LPS in vitro. PIM-Inh had protective effects on acute colitis in vivo. Mechanistically, PIM-Inh reduced the proinflammatory immune response through the inhibition of the overactivation of macrophages and the down-regulation of excessive Th1- and Th17-type immune responses. Furthermore, PIM-Inh could skew T cell differentiation towards a Treg phenotype. CONCLUSIONS: Pim-1 kinase is involved in mucosal injury/inflammation and Pim-1 kinase inhibitor may provide a novel therapeutic approach for IBD.

The combined treatment of praziquantel with osteopontin immunoneutralization reduces liver damage in Schistosoma japonicum-infected mice.

The aim of this study was to evaluate the therapeutic effects of osteopontin neutralization treatment on schistosome-induced liver injury in BALB/C mice. We randomly divided 100 BALB/C mice into groups A, B, C, D and group E. Mice in all groups except group A were abdominally infected with schistosomal cercariae to induce a schistosomal hepatopathological model. Mice in group C, D and group E were respectively administered with praziquantel, praziquantel plus colchicine and praziquantel plus neutralizing osteopontin antibody. We extracted mouse liver tissues at 3 and 9 weeks after the 'stool-eggs-positive' day, observed liver histopathological changes by haematoxylin-eosin and Masson trichrome staining and detected the expression of osteopontin, alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-ß1) by immunohistochemistry, RT-PCR and Western blot. We found that praziquantel plus neutralizing osteopontin antibody treatment significantly decreased the granuloma dimension, the percentage of collagen and the expression of osteopontin, α-SMA and TGF-ß1 compared to praziquantel plus colchicine treatment in both the acute and chronic stage of schistosomal liver damage (P<0·05). So we believe that the combined regimen of osteopontin immunoneutralization and anti-helminthic treatment can reduce the granulomatous response and liver fibrosis during the schistosomal hepatopathologic course.

Osteopontin expression is associated with hepatopathologic changes in Schistosoma japonicum infected mice.

AIM: To investigate osteopontin expression and its association with hepatopathologic changes in BALB/C mice infected with Schistosoma japonicum. METHODS: The schistosomal hepatopathologic mouse model was established by abdominal infection with schistosomal cercaria. Liver samples were obtained from mice sacrificed at 6, 8, 10, 14, and 18 wk after infection. Liver histopathological changes were observed with hematoxylin-eosin and Masson trichrome staining. The expression of osteopontin was determined with immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blotting. The expression of α-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1) were determined by immunohistochemistry. Correlations of osteopontin expression with other variables (α-SMA, TGF-ß1, hepatopathologic features including granuloma formation and degree of liver fibrosis) were analyzed. RESULTS: Typical schistosomal hepatopathologic changes were induced in the animals. Dynamic changes in the expression of osteopontin were observed at week 6. The expression increased, peaked at week 10 (P < 0.01), and then gradually decreased. Positive correlations between osteopontin expression and α-SMA (r = 0.720, P < 0.01), TGF-ß1 (r = 0.905, P < 0.01), granuloma formation (r = 0.875, P < 0.01), and degree of liver fibrosis (r = 0.858, P < 0.01) were also observed. CONCLUSION: Osteopontin may play an important role in schistosomal hepatopathology and may promote granuloma formation and liver fibrosis through an unexplored mechanism.

[Effect of ursolic acid on proliferation and apoptosis of hepatic stellate cells in vitro].

OBJECTIVE: To investigate the effect of ursolic acid on proliferation and apoptosis of hepatic stellate cells (HSC) in vitro and explore the mechanisms of apoptosis of HSC induced by ursolic acid by studying the expressions of apoptosis-regulating proteins Bcl-2, Bax and Caspase 3 in HSC. METHODS: Hepatic stellate cells HSC-T6 and hepatocytes L02 were incubated with different concentrations of ursolic acid (25, 50, 75, 100, 125 and 150 micromol/L) for 24 h, 48 h and 72 h. The effect of ursolic acid on the cell proliferation was studied by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The rate of HSC-T6 apoptosis was identified by flow cytometry (FCM) and the morphological change of apoptosis was observed with light microscopy. The expressions of apoptosis-regulating protein Bcl-2, Bax and Caspase 3 in HSC-T6 after apoptosis induced by ursolic acid were examined by immunocytochemical staining assay. RESULTS: MTT analysis indicated administration of 25-150 micromol/L ursolic acid incubated with HSC-T6 for 24 h, 48 h and 72 h significantly inhibited HSC-T6 proliferation in a dose-dependent and time-dependent manner compared with the control group. Promotive effect of ursolic acid on proliferation of hepatocyte L02 was observed in the 25, 50, 75 micromol/L concentration groups. Ursolic acid inhibited L02 proliferation when its concentration was higher than 100 micromol/L and for 72 hours or longer. HE stained histological slides demonstrated morphologic changes of HSC-T6, including karyorrhexis and cytoplasm vacuolization, when they were treated with ursolic acid at 75 micromol/L concentrations for 48 h. FCM showed the apoptosis ratios of HSC-T6 were 10.30%+/-3.85%, 21.87%+/-4.46% and 31.33%+/-6.18% after treating HSC-T6 with ursolic acid at concentrations of 25, 50 and 75 micromol/L for 48 h. They were significantly higher than that of the control group 2.93%+/-1.60%. Immunocytochemistry also indicated the expressions of Bax and caspase 3 protein in HSC-T6 cells were up-regulated in a dose-dependent manner, but expressions of Bcl-2 protein were not significantly different from that of the blank control group (P more than 0.05). CONCLUSIONS: Ursolic acid could significantly inhibit HSC proliferation and induce apoptosis in a dose-dependent and time-dependent manner. Ursolic acid in low concentration promotes proliferation of L02 cells, but in high concentrations (more than 100 micromol/L) it inhibits the growth of hepatocytes. Expressions of Bax and Caspase 3 in apoptotic HSC were increased; expressions of Bcl-2 protein were not significantly different from that of the control group, while Bcl-2/Bax ratio was reduced. Our results suggest that HSC-T6 cell apoptosis induced by ursolic acid occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.