Ting-Hui Lin

The fungal metabolite, citrinin, inhibits lipopolysaccharide/interferon-γ-induced nitric oxide production in glomerular mesangial cells.

The mycotoxin, citrinin (CTN), is a secondary metabolite of the fermented products of Monascus. The mycotoxin can either suppress or stimulate immune responses. In the present study, the immunomodulatory role of CTN in nitric oxide (NO) production, a proinflammatory mediator in the process of inflammation, was investigated. NO is well known as a mediator of immune responses. Overproduction of NO catalyzed by inducible nitric oxide synthase (iNOS) protects host cells against microbial invasion, while aberrant iNOS induction is associated with the pathophysiology of inflammatory events. Herein, we report that CTN significantly suppressed lipopolysaccharide (LPS)/interferon (IFN)-γ-induced NO production in MES-13 cells, a glomerular mesangial cell line. The percentage of NO reduction caused by CTN was far greater than that of the decline in cell viability. CTN decreased iNOS gene and protein expressions in concentration-dependent manners. CTN caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation. Furthermore, LPS/IFN-γs induction of interferon response factor-1 (IRF-1) mRNA expression was inhibited by CTN. Moreover, CTN attenuated IκB-α phosphorylation and reduced NF-κBs translocation to the nuclear fraction. Taken together, our data indicated that CTN significantly suppressed NO and iNOS expressions in MES-13 cells via inhibition of the JAK/STAT-1α and NF-κB signaling pathways.

TGF‐β inhibits prolactin‐induced expression of β‐casein by a Smad3‐dependent mechanism

Transforming growth factor‐β (TGF‐β) is a multifunctional growth factor, affecting cell proliferation, apoptosis, and extracellular matrix homeostasis. It also plays critical roles in mammary gland development, one of which involves inhibition of the expression of milk proteins, such as β‐casein, during pregnancy. Here we further explore the underlying signaling mechanism for it. Our results show that TGF‐β suppresses prolactin‐induced expression of β‐casein mRNA and protein in primary mouse mammary epithelial cells, but its effect on protein expression is more evident. We also find out that this inhibition is not due to the effect of TGF‐β on cell apoptosis. Furthermore, inhibition of TGF‐β type I receptor kinase activity by a pharmacological inhibitor SB431542 or overexpression of dominant negative Smad3 substantially restores β‐casein expression. By contrast, inhibition of p38 and Erk that are known to be activated by TGF‐β does not alleviate the inhibitory effect of TGF‐β. These results are consistent with our other observation that Smad but not MAPK pathway is activated by TGF‐β in mammary epithelial cells. Lastly, we show that prolactin‐induced tyrosine phosphorylation of Jak2 and Stat5 as well as serine/threonine phosphorylation of p70S6K and S6 ribosomal protein are downregulated by TGF‐β, although the former event requires considerably long exposure to TGF‐β. We speculate that these events might be involved in repressing transcription and translation of β‐casein gene, respectively. Taken together, our results demonstrate that TGF‐β abrogates prolactin‐stimulated β‐casein gene expression in mammary epithelial cells through, at least in part, a Smad3‐dependent mechanism. J. Cell. Biochem. 104: 1647–1659, 2008.

Galangin ameliorates cisplatin-induced nephrotoxicity by attenuating oxidative stress, inflammation and cell death in mice through inhibition of ERK and NF-kappaB signaling

&NA; Cisplatin is a chemotherapeutic agent widely used in the treatment of various cancers. However, cisplatin can induce nephrotoxicity and neurotoxicity, limiting its dosage and usage. Galangin, a natural flavonol, has been found to exhibit anti‐oxidant and anti‐inflammatory effects in vivo. Here, we investigated the effects of galangin on cisplatin‐induced acute kidney injury (AKI) and its molecular mechanisms in mice. Galangin administration reduced the cisplatin‐induced oxidative stress by decreasing renal MDA and 3‐NT formations. Galangin administration also increased renal anti‐oxidative enzyme activities (SOD, GPx, and CAT) and GSH levels depleted by cisplatin. Furthermore, galangin administration inactivated stress‐induced Nrf2 protein and its downstream products, HO‐1 and GCLC. In terms of the inflammatory response, galangin administration reduced I&kgr;B&agr; phosphorylation, NF‐&kgr;B phosphorylation and nuclear translocation, and then inhibited cisplatin‐induced secretions of pro‐inflammatory TNF‐&agr;, IL‐1&bgr; and IL‐6. In addition, cisplatin‐induced ERK and p38 phosphorylations were inhibited by galangin administration. In terms of cell death, galangin administration reduced levels of p53, pro‐apoptotic Bax and activated caspase‐3 to inhibit the cisplatin‐induced apoptosis. Galangin administration also reduced the expression levels of RIP1 and RIP3 to inhibit cisplatin‐induced RIP1/RIP3‐dependent necroptosis. Therefore, galangin administration significantly ameliorates cisplatin‐induced nephrotoxicity by attenuating oxidative stress, inflammation, and cell death through inhibitions of ERK and NF‐&kgr;B signaling pathways. Galangin might be a potential adjuvant for clinical cisplatin therapy. HighlightsGalangin alleviates cisplatin‐induced acute kidney injury through ERK and NF‐kappaB signaling pathways.Galangin reduces cisplatin‐induced oxidative stress and inflammatory response.Galangin suppresses cisplatin‐induced cell death, apoptosis and necroptosis.

Aristolochic acid I suppressed iNOS gene expression and NF-κB activation in stimulated macrophage cells

Aristolochic acid I (AAI) is a phytotoxin that has been found in various herbal remedies and linked to the development of human carcinogenesis. To investigate the playing role of AAI in the function of macrophages, lipopolysaccharide (LPS)-stimulated macrophage cells RAW264.7 were employed as a model to examine the effect of AAI on the expression of the inducible nitric oxide synthase (iNOS) gene. AAI reduced the expression of iNOS mRNA and protein, as well as the production of NO in LPS-stimulated macrophages. Treatment of transfected macrophages with AAI effectively suppressed the luciferase activities of the iNOS promoter which is activated by LPS. The results of promoter deletion and electrophoretic gel mobility shift assay (EMSA) indicated that the NF-κB binding site at nucleotides -86 to -76 was the major site that was most responsible for the inhibitory effect of AAI. Moreover, the presence of AAI substantially reduced the phosphorylation of the inhibitory κBα (IκBα) protein in LPS-stimulated cultures. AAI also down-regulated the LPS-induction of TNF-α, a NF-κB regulated gene. On the other hand, AAI did not modulate the luciferase activities of reporter construct that contained iNOS mRNA 3-UTR. Taken together, the data herein suggest that in activated macrophages, AAI effectively down-regulated the expression of iNOS gene by interfering with the activation of NF-κB at the transcription level. The stability of iNOS mRNA was not the target of AAI inhibition.

Panax notoginseng Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Panax notoginseng (PN) is a traditional Chinese herb experimentally proven to have anti-inflammatory effects, and it is used clinically for the treatment of atherosclerosis, cerebral infarction, and cerebral ischemia. This study aimed to determine the anti-inflammatory effects of PN against bleomycin-induced pulmonary fibrosis in mice. First, in an in vitro study, culture media containing lipopolysaccharide (LPS) was used to stimulate macrophage cells (RAW 264.7 cell line). TNF-α and IL-6 levels were then determined before and after treatment with PN extract. In an animal model (C57BL/6 mice), a single dose of PN (0.5u2009mg/kg) was administered orally on Day 2 or Day 7 postbleomycin treatment. The results showed that TNF-α and IL-6 levels increased in the culture media of LPS-stimulated macrophage cells, and this effect was significantly inhibited in a concentration-dependent manner by PN extract. Histopathologic examination revealed that PN administered on Day 7 postbleomycin treatment significantly decreased inflammatory cell infiltrates, fibrosis scores, and TNF-α, TGF-β, IL-1β, and IL-6 levels in bronchoalveolar lavage fluid when compared with PN given on Day 2 postbleomycin treatment. These results suggest that PN administered in the early fibrotic stage can attenuate pulmonary fibrosis in an animal model of idiopathic pulmonary fibrosis.

Downregulation of connective tissue growth factor by LPS/IFN-γ-induced nitric oxide is reversed by aristolochic acid treatment in glomerular mesangial cells via STAT-1α and NF-κB signaling

Aristolochic acid (AA) is a common cause of Chinese herb nephropathy. The mechanisms involved in the pathogenesis of AA nephropathy (AAN) are intricate. One well-documented effect of AA in the kidney is its pro-fibrotic activity. Nitric oxide (NO), a messenger gas generated from l-arginine, is the product of nitric oxide synthase (NOS). NO is involved in renal hemodynamics and exerts cytoprotective effects against renal injury. In the present study, the role of NO in AAN was investigated in MES-13 cells, a glomerular mesangial cell line. NO endogenously generated by the induction of inducible nitric oxide synthase (iNOS) with lipopolysaccharide (LPS)/interferon-γ (IFN-γ) significantly downregulated connective tissue growth factor (CTGF) protein expression in MES-13 cells. AA significantly suppressed LPS/IFN-γ-induced NO production and reversed CTGF expression that was downregulated by LPS/IFN-γ. AA decreased iNOS gene and protein expressions in a concentration-dependent manner. AA caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation and interferon response factor-1 (IRF-1) mRNA expression. Furthermore, AA attenuated IκB phosphorylation and reduced NF-κB translocation to the nuclear fraction. Taken together, our data indicate that AA reversed the CTGF expression inhibited by LPS/IFN-γ treatment via suppression of NO and iNOS expressions in MES-13 cells through inhibition of the JAK/STAT-1α and NF-κB signaling pathways. NO potentially exerts antifibrotic activity by down regulation of CTGF in MES-13 cells and inhibition of the iNOS gene by AA might partially account for the fibrotic effects of AA in nephropathy.

Differential effects of LY294002 and wortmannin on inducible nitric oxide synthase expression in glomerular mesangial cells.

Nitric oxide (NO) that is produced by inducible nitric oxide synthase (iNOS) is associated with the pathophysiology of glomerulonephritis. Numerous studies have focused on the regulation of NO production by iNOS to reduce NO-mediated cytotoxicity. In the present study, we demonstrated the differential effects of two phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, on lipopolysaccharide- (LPS) and interferon (IFN)-γ-induced NO production in a glomerular mesangial cell line, MES-13 cells. At dosages without affecting cell viability of MES-13 cells, 5μM LY294002 showed a more-significant inhibitory effect on LPS/IFN-γ-induced NO production, and iNOS protein and gene expressions than did 1μM wortmannin. Akt phosphorylation in MES-13 cells declined upon the addition of wortmannin, but not upon treatment with LY294002. Suppression of PI3K expression by small interfering (si)RNA exhibited no effect on LPS/IFN-γ-stimulated NO production or iNOS protein expression in MES-13 cells. Neither LY294002 nor wortmannin reduced IFN-γ-induced STAT-1α phosphorylation. LY294002 exhibited a more-significant inhibitory effect on NF-κB luciferase activities than wortmannin in LPS/IFN-γ-stimulated MES-13 cells. Moreover, LY294002, but not wortmannin, accelerated iNOS protein degradation and reduced the iNOS dimer/monomer ratio in MES-13 cells. Although both LY294002 and wortmannin are known as PI3K inhibitors, their differential effects on iNOS expression in MES-13 cells indicate that the effects of LY294002 on inhibiting NF-κB activation and accelerating iNOS protein degradation are through a mechanism independent of PI3K.

Candida albicans DBF4 gene inducibly duplicated by the mini-Ura-blaster is involved in hypha-suppression.

The opportunistic human fungal pathogen Candida albicans is a natural diploid that does not have a complete sexual cycle. The ability to switch between diverse cellular forms is important to its virulence. Here, we describe the characterization of the C. albicans DBF4 gene, a Saccharomyces cerevisiae homolog that encodes a regulatory subunit of Cdc7 kinase that is known to initiate DNA replication. We made a C. albicans strain, with one DBF4 allele deleted by the mini-Ura-blaster and the other controlled by a repressible promoter. We also found a third CaDBF4 copy that was later verified to be inducibly duplicated by targeted recombination with the min-Ura-blaster. Surprisingly, the strain deleted with the third CaDBF4 copy exhibited hyphal growth under repressed conditions. We conclude that the CaDBF4 gene is prone to being duplicated by the mini-Ura-blaster and that it suppresses hyphal growth in C. albicans.

The protective roles of phosphorylated heat shock protein 27 in human cells harboring myoclonus epilepsy with ragged-red fibers A8344G mtDNA mutation

Mitochondrial DNA (mtDNA) mutations are associated with a large number of neuromuscular diseases. Myoclonus epilepsy with ragged‐red fibers (MERRF) syndrome is a mitochondrial disease inherited through the maternal lineage. The most common mutation in MERRF syndrome, the A8344G mutation of mtDNA, is associated with severe defects in mitochondrial protein synthesis, which impair the assembly and function of the respiratory chain. We have previously shown that there is a decreased level of heat shock protein 27 (HSP27) in lymphoblastoid cells derived from a MERRF patient and in cytoplasmic hybrids (cybrids) harboring the A8344G mutation of mtDNA. In the present study, we found a dramatic decrease in the level of phosphorylated HSP27 (p‐HSP27) in the mutant cybrids. Even though the steady‐state level of p‐HSP27 was reduced in the mutant cybrids, normal phosphorylation and dephosphorylation were observed upon exposure to stress, indicating normal kinase and phosphatase activities. To explore the roles that p‐HSP27 may play, transfection experiments with HSP27 mutants, in which three specific serines were replaced with alanine or aspartic acid, showed that the phosphomimicking HSP27 desensitized mutant cybrids to apoptotic stress induced by staurosporine (STS). After heat shock stress, p‐HSP27 was found to enter the nucleus immediately, and with a prolonged interval of recovery, p‐HSP27 returned to the cytoplasm in wild‐type cybrids but not in mutant cybrids. The translocation of p‐HSP27 was correlated with cell viability, as shown by the increased number of apoptotic cells after p‐HSP27 returned to the cytoplasm. In summary, our results demonstrate that p‐HSP27 provides significant protection when cells are exposed to different stresses in the cell model of MERRF syndrome. Therapeutic agents targeting anomalous HSP27 phosphorylation might represent a potential treatment for mitochondrial diseases.

Novel findings of secreted cyclophilin A in diabetic nephropathy and its association with renal protection of dipeptidyl peptidase 4 inhibitor

BACKGROUNDnOur previous clinical indicated that urinary cyclophilin A was a good marker for diabetic nephropathy.nnnMETHODSnWe used animal and cell models of diabetic nephropathy to examine the role of cyclophilin A in disease progression.nnnRESULTSnSignificantly increased urinary cyclophilin A could be detected in db/db at the 8th week. Linagliptin (3mg/kg/day and 15mg/kg/day) could suppress urinary 8-hydroxy-2-deoxyguanosine at the 8th and 16th week but only the high dose Linagliption could suppress cyclophilin A at the 8th week. Compared to 8-hydroxy-2-deoxyguanosine, cyclophilin A was a stronger, earlier, and more sensitive marker. Immunohistochemical staining for cyclophilin A was also positive for db/db. In cell studies, oxidative stress and hyperglycemia could stimulate MES-13 and HK-2 cells to secrete cyclophilin A. Hyperglycemia stimulated HK-2 cells to secrete TGFβ1, which caused secretion of cyclophilin A. The secreted cyclophilin A further stimulated CD 147 to move outward from cytosol onto cell membrane in confocal microscopy, which was associated with the p38 MAPK pathway in the downstream.nnnCONCLUSIONSnSecreted cyclophilin A may play an important role in diabetic nephropathy in the mouse model and is associated with TGFβ1, CD 147, and the p38 MAPK pathway.