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Galectin‐3 (Gal‐3) plays a critical role in vascular inflammation and fibrosis. The role of TGF‐β1 in mediating pulmonary vascular fibrosis is well documented; thus, we suspected that Gal‐3 could be an important factor in TGF‐β1‐induced fibrosis in pulmonary adventitial fibroblasts (PAFs). We treated rats with monocrotaline (MCT) and cultured PAFs with TGF‐β1 to stimulate fibrosis. We found that MCT injection induced vessel thickening and extracellular matrix deposition in vivo. TGF‐β1 stimulated the production of collagen and fibronectin (Fn) protein in vitro. TGF‐β1 promoted the expression of Gal‐3 and its translocation, while silencing Gal‐3 reduced Col‐1a deposition. Blockage of STAT3 decreased the expression of Gal‐3 induced by TGF‐β1. Gal‐3 increased Col‐1a accumulation and downregulated matrix metallopeptidase 9 (MMP‐9) expression in PAFs, but it did not affect Fn expression. These findings demonstrate that Gal‐3 is required for TGF‐β1‐stimulated vascular fibrosis via a STAT3 signaling cascade and that MMP‐9 is also involved in TGF‐β1/Gal‐3‐induced vascular fibrosis.     

The IκB kinase inhibitor ACHP strongly attenuates TGFβ1‐induced myofibroblast formation and collagen synthesis

Excessive accumulation of a collagen‐rich extracellular matrix (ECM) by myofibroblasts is a characteristic feature of fibrosis, a pathological state leading to serious organ dysfunction. Transforming growth factor beta1 (TGFβ1) is a strong inducer of myofibroblast formation and subsequent collagen production. Currently, there are no remedies for the treatment of fibrosis. Activation of the nuclear factor kappa B (NF‐κB) pathway by phosphorylating IκB with the enzyme IκB kinase (IKK) plays a major role in the induction of fibrosis. ACHP {2‐Amino‐6‐[2‐(cyclopropylmethoxy)‐6‐hydroxyphenyl]‐4‐(4‐piperidinyl)‐3 pyridinecarbonitrile}, a selective inhibitor of IKK, prohibits the activation of the NF‐κB pathway. It is not known whether ACHP has potential anti‐fibrotic properties. Using adult human dermal and lung fibroblasts we have investigated whether ACHP has the ability to inhibit the TGFβ1‐induced transition of fibroblasts into myofibroblasts and its excessive synthesis of ECM. The presence of ACHP strongly suppressed the induction of the myofibroblast markers alpha‐smooth muscle actin (αSMA) and SM22α, as well as the deposition of the ECM components collagen type I and fibronectin. Furthermore, post‐treatment with ACHP partly reversed the expression of αSMA and collagen type I production. Finally, ACHP suppressed the expression of the three collagen‐modifying enzymes lysyl hydroxylase (PLOD1, PLOD2 and PLOD3) in dermal fibroblasts, but did not do so in lung fibroblasts. We conclude that the IKK inhibitor ACHP has potent antifibrotic properties, and that the NF‐κB pathway plays an important role in myofibroblast biology.     

Oxidative stress and NF-κB signaling are involved in LPS induced pulmonary dysplasia in chick embryos

Inflammation or dysbacteriosis-derived lipopolysaccharides (LPS) adversely influence the embryonic development of respiratory system. However, the precise pathological mechanisms still remain to be elucidated. In this study, we demonstrated that LPS exposure caused lung maldevelopment in chick embryos, including higher embryo mortality, increased thickness of alveolar gas exchange zone, and accumulation of PAS⁺ immature pulmonary cells, accompanied with reduced expression of alveolar epithelial cell markers and lamellar body count. Upon LPS exposure, pulmonary cell proliferation was significantly altered and cell apoptosis was inhibited as well, indicating a delayed progress of pulmonary development. LPS treatment also resulted in reduced CAV-1 expression and up-regulation of Collagen I, suggesting increased lung fibrosis, which was verified by Masson staining. Moreover, LPS induced enhanced Nrf2 expression in E18 lungs, and the increased reactive oxygen species (ROS) production was confirmed in MLE-12 cells in vitro. Antioxidant vitamin C restored the LPS induced down-regulation of ABCA3, SP-C and GATA-6 in MLE-12 cells. Furthermore, LPS induced activation of NF-κB signaling in MLE-12 cells, and the LPS-induced decrease in SP-C expression was partially abrogated by blocking NF-κB signaling with Bay-11–7082. Bay-11–7082 also inhibited LPS-induced increases of ROS and Nrf2 expression. Taken together, we have demonstrated that oxidative stress and NF-κB signaling are involved in LPS induced disruption of pulmonary cell development in chick embryos.     

Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

Bacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl₄ injection alone, CCl₄ plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.     

Mefunidone ameliorates renal inflammation and tubulointerstitial fibrosis via suppression of IKKβ phosphorylation

Mefunidone is a new pyridone agent that attenuates renal tubulointerstitial fibrosis. However, the signaling pathways involved in the effect of mefunidone on renal tubulointerstitial fibrosis have not been well explained. Inflammatory response initiates and promotes renal tubulointerstitial fibrosis, and the inhibitor of nuclear factor kappa-B kinase beta (IKKβ) is a master regulator of inflammation. This study is determined to clarify the influence of mefunidone on renal inflammation and the phosphorylation of IKKβ. Experimental renal tubulointerstitial fibrosis was induced by unilateral ureteral obstruction (UUO) for 3, 7 and 14 days in sprague dawley rat. Treatment with mefunidone was conducted simultaneously. Obstructed kidneys were harvested for the assessment. Our results showed that treatment with mefunidone ameliorated renal inflammatory injury, renal tubular lesions and interstitial fibrosis. Further studies indicated that treatment with mefunidone mitigated the expressions of tumor necrosis factorα (TNFα) and interleukin-1β (IL-1β) in the kidney. The phosphorylation of IKKβ and inhibitor of kappa-B (IκB) and the expression of NOD-like receptor family, pyrin domain containing 3 (NALP3) were also reduced in vivo after treatment with mefunidone. In vitro, peritoneal macrophages were incubated with necrotic cells or lipopolysaccharide in the presence or absence of mefunidone. Mefunidone markedly decreased necrotic cell or LPS induced IL-1β production and LPS induced TNFα production in primary peritoneal macrophages. Furthermore, mefunidone significantly inhibited the phosphorylation of IKKβ/IκB and nuclear transition of NF-κB p65 in peritoneal macrophages stimulated by necrotic cell or lipopolysaccharide. In conclusion, mefunidone serves as a novel anti-inflammatory agent that attenuates UUO-induced renal interstitial inflammation and fibrosis, possibly through suppressing IKKβ phosphorylation.     

CYP27A1 inhibits bladder cancer cells proliferation by regulating cholesterol homeostasis

CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis, converts cholesterol into 27-hydroxycholesterol (27-HC). The relationship between CYP27A1 and cell proliferation was studied to determine the role of CYP27A1 in bladder cancer. The expression of CYP27A1 in three bladder cancer cell lines (T24, UM-UC-3 and 5637) were assessed by qRT-PCR and Western blotting, and cells with stable CYP27A1 expression were generated by lentiviral infection. Cell proliferation was detected by MTT assays, colony formation assays and a tumor xenograft model in vitro and in vivo, and the intracellular 27-HC and cholesterol secretion levels were detected by enzyme-linked immunosorbent assays (ELISA). The results revealed that CYP27A1 expression was downregulated in androgen receptor (AR)-positive T24/UM-UC-3 cells compared with AR-negative 5637 cell. After CYP27A1 expression was restored, cell proliferation was inhibited in vitro and in vivo because much more intracellular 27-HC was produced in the CYP27A1-overexpressing cells than in the control cells. Both T24 and UM-UC-3 cells treated with 27-HC showed similar results. In addition, CYP27A1/27HC could reduce the cellular cholesterol level in both T24 and UM-UC-3 cells by upregulating ATP-binding cassette transporters G1 and A1 (ABCG1 and ABCA1) through Liver X receptors (LXRs) pathway and downregulating low-density lipoprotein receptor (LDLR) expression. These findings all suggest that CYP27A1 is a critical cholesterol sensor in bladder cancer cells that may contribute significantly to bladder cancer proliferation.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

Beneficial effect of quercetin on cholestatic liver injury

Bile duct obstruction and subsequent cholestasis are associated with hepatocellular injury, cholangiocyte proliferation, stellate cell activation, Kupffer cell activation, oxidative stress, inflammation and fibrosis. Flavonoids have been shown to confer beneficial health effects, including hepatoprotection. However, the molecular mechanism of flavonoid-mediated hepatoprotection is incompletely understood. In this study, we report the protective effect of quercetin on cholestatic liver injury. Cholestasis was produced by bile duct ligation (BDL) in male Sprague–Dawley rats for 3 weeks. Daily oral administration of quercetin was started 1 week before injury and lasted for 4 weeks. In comparison with the control group, the BDL group showed liver injury, as evidenced by histological changes, and elevation in serum biochemicals, ductular reaction, fibrosis, inflammation and oxidative stress. These pathophysiological changes were attenuated by daily quercetin supplementation. Quercetin alleviated BDL-induced transforming growth factor beta-1 (TGF-β1), interleukin-1 beta, connective tissue growth factor and collagen expression. The antifibrotic effect of quercetin was accompanied by reductions in α-smooth muscle actin-positive matrix-producing cells and Smad 2/3 activity critical to the fibrogenic potential of TGF-β1. Quercetin also attenuated BDL-induced oxidative stress, leukocyte accumulation, nuclear factor (NF)-κB activation and proinflammatory cytokine production. Further studies demonstrated an inhibitory effect of quercetin on MyD88 and TGF-β-activated kinase-1 critical for linking toll-like receptor (TLR) and NF-κB. Taken together, the hepatoprotective, anti-inflammatory and antifibrotic effects of quercetin seem to be multifactorial. The beneficial effects of daily quercetin supplementation are associated with antioxidative and anti-inflammatory potential as well as down-regulation of NF-κB and TGF-β/Smad signaling, probably via interference with TLR signaling.     

Activity of the selective IκB kinase inhibitor BMS-345541 against T-cell acute lymphoblastic leukemia

Several lines of evidence suggest that the IκB kinase (IKK)/nuclear factor-κB (NFκB) axis is required for viability of leukemic cells and is a predictor of relapse in T-cell acute lymphoblastic leukemia (T-ALL). Moreover, many anticancer agents induce NFκB nuclear translocation and activation of its target genes, which counteract cellular resistance to chemotherapeutic drugs. Therefore, the design and the study of IKK-specific drugs is crucial to inhibit tumor cell proliferation and to prevent cancer drug-resistance. Here, we report the anti-proliferative effects induced by BMS-345541 (a highly selective IKK inhibitor) in three Notch1-mutated T-ALL cell lines and in T-ALL primary cells from pediatric patients. BMS-345541 induced apoptosis and an accumulation of cells in the G₂/M phase of the cell cycle via inhibition of IKK/NFκB signaling. We also report that T-ALL cells treated with BMS-345541 displayed nuclear translocation of FOXO3a and restoration of its functions, including control of p21^Cip1 expression levels. We demonstrated that FOXO3a subcellular re-distribution is independent of AKT and ERK 1/2 signaling, speculating that in T-ALL the loss of FOXO3a tumor suppressor function could be due to deregulation of IKK, as has been previously demonstrated in other cancer types.

It is well known that, differently from p53, FOXO3a mutations have not yet been found in human tumors, which makes therapeutics activating FOXO3a more appealing than others. For these features, BMS-345541 could be used alone or in combination with traditional therapies in the treatment of T-ALL.     

An interaction between BRG1 and histone modifying enzymes mediates lipopolysaccharide-induced proinflammatory cytokines in vascular endothelial cells

Vascular inflammation is the culprit for a host of human diseases. The underlying mechanism, however, is not definitively elucidated. In the present study, we investigated the interplay between different epigenetic factors during lipopolysaccharide (LPS) induced synthesis of proinflammatory cytokines in cultured vascular endothelial cells. We report that in response to LPS treatment, NF-κB was deplored to its target promoters along with the chromatin remodeling protein BRG1. Paralleling these changes trimethylated H3K9 became erased from while trimethylated H3K4 started to accumulate on the NF-κB target promoters. Further analysis revealed that LPS stimulation resulted in sequential recruitment of the H3K9 tri-demethylase JMJD2A and the H3K4 trimethyltransferase SET1A to the NF-κB target promoters. JMJD2A mediated–H3K9 demethylation served as a prerequisite for SET1A to bind to the NF-κB target promoters. Both JMJD2A and SET1A were essential for LPS-induced transactivation of proinflammatory cytokines by sustaining the binding of NF-κB. Of key importance, BRG1 coordinated the sequential recruit of and the interplay between JMJD2A and SET1A. In conclusion, our data unveil a novel epigenetic mechanism that contributes to LPS-induced vascular inflammation.     

MiR‐590‐3p regulates proliferation,migration and collagen synthesis of cardiac fibroblast by targeting ZEB1

Previous studies have implicated the attractive and promising role of miR‐590‐3p to restore the cardiac function following myocardial infarction (MI). However, the molecular mechanisms for how miR‐590‐3p involves in cardiac fibrosis remain largely unexplored. Using human cardiac fibroblasts (HCFs) as the cellular model, luciferase report assay, mutation, EdU assay and transwell migration assay were applied to investigate the biological effects of miR‐590‐3p on the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts. We found that miR‐590‐3p significantly suppressed cell proliferation and migration of HCFs. The mRNA and protein expression levels of α‐SMA, Col1A1 and Col3A were significantly decreased by miR‐590‐3p. Moreover, miR‐590‐3p directly targeted at the 3’UTR of ZEB1 to repress the translation of ZEB1. Interfering with the expression of ZEB1 significantly decreased the cell proliferation, migration activity, mRNA and protein expressions of α‐SMA, Col1A1 and Col3A. Furthermore, the expressions of miR‐590‐3p and ZEB1 were identified in infarct area of MI model in pigs. Collectively, miR‐590‐3p suppresses the cell proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts by targeting ZEB1. These works will provide useful biological information for future studies on potential roles of miR‐590‐3p as the therapeutic target to recover cardiac function following MI.     

Hepatoprotective effect of Herpetospermum caudigerum Wall. on carbon tetrachloride‐induced hepatic fibrosis in rats

Tibetan medicine Herpetospermum caudigerum Wall. (HCW) has long been employed to treat hepatitis, inflammatory diseases and jaundice according to the records of “The Four Medical Tantras” in China. This study was investigated to explore the protective effects of HCW on hepatic fibrosis and the possible mechanism in a rat model. Hepatic fibrosis was established by intragastric administration of 3 ml/kg carbon tetrachloride (CCl₄) twice a week for 6 weeks. CCl₄‐treated rats were received HCW (1 and 3 g/kg/d) and silymarin (0.1 g/kg/d) from 3 to 6 weeks. The results showed that HCW could significantly decrease the levels of AST, ALT, HA, LN, PCIII, Col IV, TNF‐α, IL‐1β and IL‐6. Moreover, HCW could effectively inhibit collagen deposition and reduce the pathological damage. Analysis experiments finally exhibited that HCW was able to markedly inhibit hepatic fibrosis by modulating the expressions of NF‐κB p65, IκBα, Samd3 and TGF‐β1 proteins. Therefore, our results suggest that HCW has hepatoprotective activity against CCl₄‐induced hepatic fibrosis in rats by regulating the inflammatory responses.     

miR-627/HMGB1/NF-κB regulatory loop modulates TGF-β1-induced pulmonary fibrosis

Pulmonary fibrosis (PF) is a fibroproliferative disease that can eventually lead to fatal lung failure. It is characterized by abnormal proliferation of fibroblasts, dysregulated fibroblast differentiation to myofibroblast, and disorganized collagen and extracellular matrix production, deposition and degradation. There is still a lack of effective treatment strategies for PF. Extracellular high-mobility group box protein 1 (HMGB1) induces PF through NF-κB-mediated TGF-β1 release. Herein, we first validate the suppressive effect of HMGB1 knockdown on TGF-β1-induced α-smooth muscle actin (α-SMA) and collagen I protein expression. In PF, miRNAs exert different effects through targeting various downstream target messenger RNAs. We searched an online database for dysregulated miRNAs in PF tissues; among them, miR-627 was predicted by online tools to target HMGB1 to inhibit its expression. miR-627 overexpression could partially reverse TGF-β1-induced normal human lung fibroblast proliferation, as well as α-SMA and collagen I protein expression. miR-627 inhibition could partially reverse the suppressive effect of HMGB1 knockdown on TGF-β1-induced α-SMA and collagen I protein expression through direct binding to the 3′-untranslated region of HMGB1. Moreover, miR-627/HMGB1 affected TGF-β1 release through RAGE/NF-κB signaling; miR-627/HMGB1 and RAGE/NF-κB signaling formed a regulatory loop to modulate TGF-β1-induced PF in vitro. In conclusion, miR-627 may be a potential agent that targets HMGB1 to inhibit its expression, thereby improving TGF-β1-induced PF in vitro.     

Steatohepatitis in laboratory opossums exhibiting a high lipemic response to dietary cholesterol and fat

Plasma VLDL and LDL cholesterol were markedly elevated (>40-fold) in high-responding opossums, but moderately elevated (6-fold) in low-responding opossums after they had consumed a high-cholesterol and high-fat diet for 24 wk. In both high- and low-responding opossums, plasma triglycerides were slightly elevated, threefold and twofold, respectively. Dietary challenge also induced fatty livers in high responders, but not in low responders. We studied the lipid composition, histopathological features, and gene expression patterns of the fatty livers. Free cholesterol (2-fold), esterified cholesterol (11-fold), and triglycerides (2-fold) were higher in the livers of high responders than those in low responders, whereas free fatty acid levels were similar. The fatty livers of high responders showed extensive lobular disarray by histology. Inflammatory cells and ballooned hepatocytes were also present, as were perisinusoidal fibrosis and ductular proliferation. In contrast, liver histology was normal in low responders. Hepatic gene expression revealed differences associated with the development of steatohepatitis in high responders. The accumulation of hepatic cholesterol was concomitant with upregulation of the HMGCR gene and downregulation of the CYP27A1, ABCG8, and ABCB4 genes. Genes involved in inflammation (TNF, NFKB1, and COX2) and in oxidative stress (CYBA and NCF1) were upregulated. Upregulation of the growth factor genes (PDGF and TGFB1) and collagen genes (Col1A1, Col3A1, and Col4A1) was consistent with fibrosis. Some of the histological characteristics of the fatty livers of high-responding opossums imitate those in the livers of humans with nonalcoholic steatohepatitis.