OBJECTIVETo investigate the effect of Tauroursodeoxycholic acid (TUDCA) on Taurodeoxycholic acid (TDCA)-induced HepG2 cell apoptosis and to clarify the molecular mechanism of its anti-apoptosis effect of TUDCA.

METHODSMorphologic evaluation of apoptotic cells was performed by Hoechst 33258 staining and electron microscope. DNA fragment was detected by electrophoresis on 1.5% agarose gels. Apoptosis rate was measured by flow cytometry using PI dye. Following incubation of HepG2 cells either with TDCA alone, or coincubation with TUDCA and TDCA, the releasing level of cytochrome c from mitochondria into cytosol was determined by western blot, also the activity of caspase-3, 8, 9.

RESULTSIncubating the cells with 400 micromol/L TDCA for 12 h induced the cells apoptosis significantly. The apoptotic rate decreased from 50.35% +/- 2.20% to 13.78% +/- 0.84% after coincubation with TUDCA, and this anti-apoptotic effect of TUDCA was confirmed by morphological and DNA ladder detection. TUDCA significantly inhibited the release of cytochrome C from mitochondria into cytosol, and the activity of caspase-9, 3 (t > or = 13.00, P < 0.01), especially at 12 h, caspase-3 activity decreased by 54.9% (t = 16.88, P < 0.01) and 52.5%, however it had no obvious effect on the activity of caspase-8 (t = 1.94, P > 0.05).

CONCLUSIONSTUDCA prevents HepG2 cells apoptosis induced by TDCA through modulating mitochondrial membrane stability, inhibiting the release of cytochrome c and the activation of procaspase-9 and 3. Anti-apoptotic mechanism of TUDCA may be considered to be one of the most important reasons that TUDCA exerts significant efficacy in the treatment of cholestatic liver diseases.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; Caspase 9 ; Caspases ; metabolism ; Cytochromes c ; pharmacology ; Humans ; Liver Neoplasms ; pathology ; Taurochenodeoxycholic Acid ; pharmacology ; Taurodeoxycholic Acid ; analogs & derivatives ; pharmacology ; Tumor Cells, Cultured

OBJECTIVE: This study was conducted to investigate the regulation of endoplasmic reticulum stress on Nrf2 signaling pathway in the kidneys of rats. METHODS: Rats were divided into twelve groups of six animals each. Some groups were pre-administered with bacitracin or tauroursodeoxycholic acid (TUDCA), and all of them were treated with 5-20 μmol/kg cadmium (Cd) for 48 h. The oxidative stress levels were analyzed using kits. The mRNA and protein expression levels of endoplasmic reticulum stress-related factors and Nrf2 signaling pathway-related factors were determined using RT-PCR and western blot. RESULTS: Cd exposure resulted in oxidative stress in the kidneys of rats and upregulated the expression of endoplasmic reticulum stress (ERS)-related factors and Nrf2 signaling pathway-related factors, especially at doses of 10 and 20 μmol/kg Cd, and the expression changes were particularly obvious. Moreover, after pretreatment with bacitracin, Cd upregulated the expression of ERS-related factors to a certain extent and, at higher doses, increased the mRNA expression of Nrf2. After pretreatment with TUDCA, Cd reduced the level of ERS to a certain extent; however, at these doses, there were no significant changes in the expression of Nrf2. CONCLUSION Cadmium can result in ERS and oxidative stress in the kidneys of rats, activate Nrf2, and upregulate the transcriptional expression of phase II detoxification enzymes under these experimental conditions. ERS has a positive regulation effect on Nrf2 signaling pathway but has little effect on the negative regulation of Nrf2 signaling pathway in cadmium toxicity.
Animals ; Cadmium ; toxicity ; Endoplasmic Reticulum Stress ; drug effects ; Environmental Pollutants ; toxicity ; Female ; Kidney ; drug effects ; metabolism ; Male ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Taurochenodeoxycholic Acid ; pharmacology

Taurochenodeoxycholic acid (TCDCA) is one of the main effective components of bile acid, playing critical roles in apoptosis and immune responses through the TGR5 receptor. In this study, we reveal the interaction between TCDCA and TGR5 receptor in TGR5-knockdown H1299 cells and the regulation of inflammation via the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding (CREB) signal pathway in NR8383 macrophages. In TGR5-knockdown H1299 cells, TCDCA significantly activated cAMP level via TGR5 receptor, indicating TCDCA can bind to TGR5; in NR8383 macrophages TCDCA increased cAMP content compared to treatment with the adenylate cyclase (AC) inhibitor SQ22536. Moreover, activated cAMP can significantly enhance gene expression and protein levels of its downstream proteins PKA and CREB compared with groups of inhibitors. Additionally, TCDCA decreased tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-12 through nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activity. PKA and CREB are primary regulators of anti-inflammatory and immune response. Our results thus demonstrate TCDCA plays an essential anti-inflammatory role via the signaling pathway of cAMP-PKA-CREB induced by TGR5 receptor.
Animals ; Cell Line ; Cyclic AMP/metabolism* ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Cytokines/metabolism* ; Humans ; Inflammation ; Macrophages ; Rats ; Receptors, G-Protein-Coupled/metabolism* ; Signal Transduction/drug effects* ; Taurochenodeoxycholic Acid/pharmacology*

BACKGROUND/AIMS: Deoxycholic acid (DCA) has been appeared to be an endogenous colon tumor promoter. In this study, we investigated whether DCA induces nuclear factor-kappa B (NF-kappa B) activation and IL-8 expression, and tauroursodeoxycholic acid (TUDC) inhibits this signaling in HT-29 cells. METHODS: After DCA treatments, time courses of NF-kappa B binding activity were determined by electrophoretic mobility shift assay (EMSA). Also, we performed Western blotting of I kappa B alpha to confirm NF-kappa B activation. Time and concentration courses of DCA-induced secretion of IL-8 were measured with ELISA in supernatants of cultured media from the cells. To evaluate the role of NF-kappa B, IL-8 levels were assessed after pretreatment with using phosphorothioate-modified anti-sense oligonucleotides (ODN). Moreover, DCA-induced secretions of IL-8 were measured after pretreatment with TUDC. RESULTS: DCA dose-dependently induced prominent NF-kappa B binding complexes from 30 min to 8 hr and degradation of I kappa B alpha. The secretions of IL-8 were increased with DCA (50~200 micro M) treatment in a time and dose-dependent manner. Pre-incubation of the cells with TUDC (0.1~10 micro M) for 2 hours caused significant decreases in DCA induced IL-8 secretion. However, transient transfection using p50 or p65 AS-ODN showed no effect on IL-8 secretion. CONCLUSIONS: DCA may play as a colonic tumor promoter through anti-apoptotic effect of NF-kappa B activation and IL-8 expression, and DCA-induced NF-kappa B independent IL-8 expression is inhibited by TUDC.
Blotting, Western ; Colonic Neoplasms ; Deoxycholic Acid/*pharmacology ; Dose-Response Relationship, Drug ; Electrophoretic Mobility Shift Assay ; English Abstract ; HT29 Cells ; Humans ; Interleukin-8/*metabolism ; NF-kappa B/*metabolism ; Oligonucleotides, Antisense/pharmacology ; Signal Transduction/*drug effects ; Taurochenodeoxycholic Acid/*pharmacology ; Trans-Activation (Genetics)/drug effects

OBJECTIVE: To explore the mechanism of tauroursodeoxycholic acid (TUDCA) in suppressing apoptosis in pulmonary tissues of intermittent hypoxia (IH) mice model.
 METHODS: A total of 32 C57 mice were randomly divided into a control group, a TUDCA group, an IH group and an IH+TUDCA group (8 mice per group). The mice were put in specially designed chambers and exposed to IH treatment for 4 weeks. In the chambers, oxygen levels repeatedly decreased from 21% to 10% and recovered from 10% to 21%, lasting for 8 hours in every day. After 4 weeks of IH exposure, the expression levels of caspase-12 and cleaved caspase-3 in pulmonary tissues were detected by Western blot. Meanwhile, the expression levels of glucose regulated protein-78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) were quantified by Western blot, immunochemistry and real-time PCR.
 RESULTS: Compared with the control group, the expression levels of caspase-12, cleaved caspase-3, GRP78 and CHOP were increased in the IH group (all P<0.01). TUDCA treatment could reduce these proteins expression (all P<0.05).
 CONCLUSION Endoplasmic reticulum stress-mediated apoptosis can be activated in pulmonary tissues after chronic IH exposure, and TUDCA can reduce the cellular apoptosis via suppressing endoplasmic reticulum stress.
Animals ; Apoptosis ; drug effects ; Caspase 12 ; metabolism ; Caspase 3 ; metabolism ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Hypoxia ; physiopathology ; Lung ; drug effects ; Mice ; Mice, Inbred C57BL ; Real-Time Polymerase Chain Reaction ; Taurochenodeoxycholic Acid ; pharmacology ; Transcription Factor CHOP ; metabolism