[Expressions of inflammatory cytokines in mouse peritoneal macrophages induced by MRP8/MRP14 in vitro].

OBJECTIVE: To investigate MRP8/MRP14-induced expression of inflammatory cytokines in mouse peritoneal macrophages and explore the mechanism. METHODS: Tumor necrosis factor-α (TNF-α), interferon-γ inducible protein 10 (IP-10), interleukin-2 (IL-2), IL-6, IL-5 and interferon-γ (IFN-γ) proteins in the culture supernatants of mouse peritoneal macrophages treated with recombinant MRP8/MRP14 were quantified using Luminex xMAP system. TNF-α, IP-10 and IL-6 levels were detected in the culture supernatants of the peritoneal macrophages after treatment with different domains of MRP14. Western blotting was used to detect the phosphorylation of p38 MAPK, JNK and ERK in the cells after MRP8/MRP14 treatment. The effects of p38 MAPK, JNK, ERK inhibitors, TLR4 or RAGE receptor antagonists on MRP8/MRP14-induced expressions of TNF-α, IP-10 and IL-6 were tested. RESULTS: MRP8/MRP14 significantly increased TNF-α, IP-10 and IL-6 levels in mouse peritoneal macrophages by 98.2, 378.6 and 6.3 folds (P<0.01), respectively, but did not obviously affect IL-2, IL-5 and IFN-α levels. MRP14 protein and its calcium binding motifs such as EF hand-1, EF hand-2, EF hand-1+2, but not CT terminal domain, all induced TNF-α, IP-10 and IL-6 expressions (P<0.01). Phosphorylation of p38 MAPK, JNK and ERK were detected by Western blotting in the cells at 1 h after MRP8/MRP14 stimulation and sustained to 2 h. Compared with MRP8/MRP14, SB203580 (p38 MAPK inhibitor) significantly inhibited TNF-α, IP-10 and IL-6 expression (P<0.05), and SP600125 (JNK inhibitor) inhibited the expression of TNF-α and IP-10 (P<0.05) but not IL-6, PD98059 and U0126 (ERK and MEK1/2 inhibitor) reduced IL-6 expression (P<0.05). TNF-α, IP-10 and IL-6 levels were inhibited by TAK242 (P<0.05); IL-6 level in the cells was also partially inhibited by RAGE neutralizing antibody (P<0.05). CONCLUSION: MRP8/MRP14 can induce the expression of TNF-α, IP-10 and IL-6 in mouse peritoneal macrophages. MRP14 protein, which contain calcium binding motifs, has the biological activity of inducing cytokine expression. TNF-α and IP-10 expressions are related with TLR4 and its downstream p38 MAPKs and JNK; IL-6 is regulated by both TLR4 and RAGE and their downstream p38 MAPKs and ERK.

Kelch-like Protein 21 (KLHL21) Targets IκB Kinase-ß to Regulate Nuclear Factor κ-Light Chain Enhancer of Activated B Cells (NF-κB) Signaling Negatively.

Activation of IKKß is the key step in canonical activation of NF-κB signaling. Extensive work has provided insight into the mechanisms underlying IKKß activation through the identification of context-specific regulators. However, the molecular processes responsible for its negative regulation are not completely understood. Here, we identified KLHL21, a member of the Kelch-like gene family, as a novel negative regulator of IKKß. The expression of KLHL21 was rapidly down-regulated in macrophages upon treatment with proinflammatory stimuli. Overexpression of KLHL21 inhibited the activation of IKKß and degradation of IκBα, whereas KLHL21 depletion via siRNA showed the opposite results. Coimmunoprecipitation assays revealed that KLHL21 specifically bound to the kinase domain of IKKß via its Kelch domains and that this interaction was gradually attenuated upon TNFα treatment. Furthermore, KLHL21 did not disrupt the interaction between IKKß and TAK1, TRAF2, or IκBα. Also, KLHL21 did not require its E3 ubiquitin ligase activity for IKKß inhibition. Our findings suggest that KLHL21 may exert its inhibitory function by binding to the kinase domain and sequestering the region from potential IKKß inducers. Taken together, our data clearly demonstrate that KLHL21 negatively regulates TNFα-activated NF-κB signaling via targeting IKKß, providing new insight into the mechanisms underlying NF-κB regulation in cells.

AGE/RAGE/Akt pathway contributes to prostate cancer cell proliferation by promoting Rb phosphorylation and degradation.

Metabolomic research has revealed that metabolites play an important role in prostate cancer development and progression. Previous studies have suggested that prostate cancer cell proliferation is induced by advanced glycation end products (AGEs) exposure, but the mechanism of this induction remains unknown. This study investigated the molecular mechanisms underlying the proliferative response of prostate cancer cell to the interaction of AGEs and the receptor for advanced glycation end products (RAGE). To investigate this mechanism, we used Western blotting to evaluate the responses of the retinoblastoma (Rb), p-Rb and PI3K/Akt pathway to AGEs stimulation. We also examined the effect of knocking down Rb and blocking the PI3K/Akt pathway on AGEs induced PC-3 cell proliferation. Our results indicated that AGE-RAGE interaction enhanced Rb phosphorylation and subsequently decreased total Rb levels. Bioinformatics analysis further indicated a negative correlation between RAGE and RB1 expression in prostate cancer tissue. Furthermore, we observed that AGEs stimulation activated the PI3K/Akt signaling pathway and that blocking PI3K/Akt signaling abrogated AGEs-induced cell proliferation. We report, for the first time, that AGE-RAGE interaction enhances prostate cancer cell proliferation by phosphorylation of Rb via the PI3K/Akt signaling pathway.

Potential protective effects of Clostridium butyricum on experimental gastric ulcers in mice.

AIM: To investigate the effects of Clostridium butyricum (C. butyricum) on experimental gastric ulcers (GUs) induced by alcohol, restraint cold stress, or pyloric ligation in mice, respectively. METHODS: One hundred and twenty mice were randomly allocated into three types of gastric ulcer models (n = 40 each), induced by alcohol, restraint cold stress, or pyloric ligation. In each GU model, 40 mice were allocated into four groups (n = 10 each): the sham control group; model group (GU induction without pretreatment); C. butyricum group (GU induction with C. butyricum pretreatment); and Omeprazole group (GU induction with Omeprazole pretreatment). The effects of C. butyricum were evaluated by examining the histological changes in the gastric mucosal erosion area, the activities of superoxide dismutase (SOD) and catalase (CAT), the level of malondialdehyde (MDA), and the contents of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, leukotriene B4 (LTB4) and 6-keto-PGF-1α (degradation product of PGI2) in the gastric tissue. RESULTS: Our data showed that C. butyricum significantly reduced the gastric mucosal injury area and ameliorated the pathological conditions of the gastric mucosa. C. butyricum not only minimized the decreases in activity of SOD and CAT, but also reduced the level of MDA in all three GU models used in this study. The accumulation of IL1-ß, TNF-α and LBT4 decreased, while 6-keto-PGF-1α increased with pretreatment by C. butyricum in all three GU models. CONCLUSION: Our data demonstrated the protective effects of pretreatment with C. butyricum on anti-oxidation and anti-inflammation in different types of GU models in mice. Further studies are needed to explore its potential clinical benefits.

Angiotensin II facilitates fibrogenic effect of TGF-ß1 through enhancing the down-regulation of BAMBI caused by LPS: a new pro-fibrotic mechanism of angiotensin II.

Angiotensin II has progressively been considered to play an important role in the development of liver fibrosis, although the mechanism isn't fully understood. The aim of this study was to investigate a possible pro-fibrotic mechanism, by which angiotensin II would enhance the pro-fibrotic effect of transforming growth factor beta 1 (TGF-ß1) through up-regulation of toll-like receptor 4 (TLR4) and enhancing down-regulation of TGF-ß1 inhibitory pseudo-receptor-BAMBI caused by LPS in hepatic stellate cells (HSCs). Firstly, the synergistic effects of angiotensin II, TGF-ß1 and LPS on collagen 1α production were confirmed in vitro by ELISA, in which angiotensin II, LPS and TGF-ß1 were treated sequentially, and in vivo by immunofluorescence, in the experiments single or multiple intra-peritoneally implanted osmotic mini-pumps administrating angiotensin II or LPS combined with intra-peritoneal injections of TGF-ß1 were used. We also found that only LPS and TGF-ß1 weren't enough to induce obvious fibrogenesis without angiotensin II. Secondly, to identify the reason of why angiotensin II is so important, the minute level of TLR4 in activated HSCs - T6 and primary quiescent HSCs of rat, up-regulation of TLR4 by angiotensin II and blockage by different angiotensin II receptor type 1 (AT1) blockers in HSCs were assayed by western blotting in vitro and immunofluorescence in vivo. Finally, BAMBI expression level, which is regulated by LPS-TLR4 pathway, was detected by qRT-PCR and results showed angiotensin II enhanced the down-regulation of BAMBI mRNA caused by LPS in vitro and in vivo, and TLR4 neutralization antibody blocked this interactive effect. These data demonstrated that angiotensin II enhances LPS-TLR4 pathway signaling and further down-regulates expression of BAMBI through up-regulation of TLR4, which results in facilitation of pro-fibrotic activity of TGF-ß1. Angiotensin II, LPS and TGF-ß1 act synergistically during hepatic fibrogenesis, showing crosstalks between angiotensin II-AT1, LPS-TLR4 and TGF-ß1-BAMBI signal pathways in rat HSCs.