Effects of lipopolysaccharides stimulated Kupffer cells on activation of rat hepatic stellate cells

AIM:To study the effects of Kupffer cell-conditioned medium (KCCM) derived from lipopolysaccharide (LPS) treatment on proliferation of rat hepatic stellate cells (HSC).METHODS:HSC and Kupffer cells were isolated from the liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz and cultured. KCCM was prepared and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to detect HSC proliferation. The content of type Ⅳ collagen and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay.TGF-β1 production in the KCCM was detected by enzymelinked immunosorbent assay (ELISA).RESULTS:HSC and Kupffer cells isolated had high purity.One microgram per mililiter LPS-activated KCCM and unstimulated KCCM could significantly promote HSC proliferation [0.132±0.005 and 0.123±0.008 vscontrol group (0.100±0.003), P<0.01], and there was a difference between them (P<0.05). Ten microgram per mililiter LPS-activated KCCM (0.106±0.010) was unable to promote HSC proliferation (P>0.05). Adding anti-TGF-β1 antibodies could suppress the proliferation promoted by unstimulated KCCM and LPS(1μg/ml)-activated KCCM (0.109±0.009 vs 0.123±0.008,0.115±0.008 vs 0.132±0.005, P<0.01). LPS (1μg/ml or 10μg/ml) could not promote HSC proliferation immediately (0.096±0.003 and 0.101±0.004 vs 0.100±0.003, P>0.05). There was a parallel behavior between HSC proliferation and increased ECM level. One microgram per mililiter LPS-activated KCCM contained a larger amount of TGF-β1 than unstimulated KCCM.CONCLUSION:The technique for isolation of HSC and Kupffer cells described here is simple and reliable. KCCM stimulated by LPS may promote HSC proliferation and collagen accumulation, which are associated with hepatic fibrogenesis.

c-Jun N-terminal kinase is required for vitamin E succinate-induced apoptosis in human gastric cancer cells

AIM:To investigate the roles of c-Jun N-terminal kinase (JNK) signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells. METHODS:Human gastric cancer cell lines (SGC-7901) were treated with vitamin E succinate (VES) at 5,10,20 mg/L. Succinic acid and vitamin E were used as vehicle controls and condition medium only as an untreated (UT) control. Apoptosis was observed by 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining for morphological changes and by DNA fragmentation for biochemical alterations. Western blot analysis was applied to measure the expression of JNK and phosphorylated JNK.After the cells were transiently transfected with dominant negative mutant of JNK (DN- JNK) followed by treatment of VES,the expression of JNK and c-Jun protein was determined. RESULTS:The apoptotic changes were observed after VES treatment by DNA fragmentation.DNA ladder in the 20 mg/L VES group was more clearly seen than that in 10 mg/L VES group and was not detected following treatment of UT control,succinate and vitamin E.VES at 5,10 and 20 mg/L increased the expression of p-JNK by 2.5-,2.8- and 4.2- fold,respectively.VES induced the phosphorylation of JNK beginning at 1.5 h and produced a sustained increase for 24 h with the peak level at 12 h.Transient transfection of DN-JNK blocked VES-triggered apoptosis by 52%.DN-JNK significantly increased the level of JNK,while decreasing the expression of VES-induced c-Jun protein. CONCLUSION:VES-induced apoptosis in human gastric cancer SGC-7901 cells involves JNK signaling pathway via c-Jun and its downstream transcription factor.