Effects of the Calcium-Activated Chloride Channel Inhibitors T16Ainh-A01 and CaCCinh-A01 on Cardiac Fibroblast Function.

BACKGROUND/AIMS: Calcium-activated chloride channels (CaCCs) regulate numerous physiological processes including cell proliferation, migration, and extracellular matrix secretion. T16Ainh-A01 and CaCCinh-A01 are selective inhibitors of CaCCs. But it is unknown whether these two compounds have functional effects on cardiac fibroblasts (CFs). METHODS: Primary CFs were obtained by enzymatic dissociation of cardiomyocytes from neonatal rat hearts. Intracellular Ca2+ ([Ca2+]i) and Cl- ([Cl-]i) were measured using the fluorescent calcium indicators (Fluo-4 AM) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide respectively. The expression of anoctamin-1 (ANO1) and α-smooth muscle actin (α-SMA) was detected by quantitative RT-PCR, immunofluorescence, and western blotting. A hydroxyproline assay was used to examine collagen secretion. Cell proliferation, cell cycle distribution, and cell migration were assessed by Cell Counting Kit-8, flow cytometry, and Transwell assays, respectively. RESULTS: ANO1 was preferentially expressed on the nuclear membrane and partially within intracellular compartments around the nucleus. T16Ainh-A01 and CaCCinh-A01 displayed different inhibitory effects on [Cl-]i in CFs. T16Ainh-A01 considerably decreased [Cl-]i in the nucleus, whereas CaCCinh-A01 reduced [Cl-]i in intracellular compartments around the nucleus, and both inhibitors exhibited a minimal effect on [Ca2+]i in CFs. ANO1 and α-SMA expression levels were significantly repressed by CaCCinh-A01. T16Ainh-A01 showed a marked inhibitory effect on the mRNA levels of ANO1 and α-SMA, but had a negligible effect on ANO1 at the protein level. T16Ainh-A01 and CaCCinh-A01 led to the significant repression of cell proliferation, cell migration, and collagen secretion in CFs. CONCLUSION: Our findings indicate that T16Ainh-A01 and CaCCinh-A01 have the potential to inhibit the proliferation and collagen secretion of CFs and may serve as novel anti-fibrotic therapeutic drugs in the future.

Isolation and genetic analysis of a variant porcine epidemic diarrhea virus in China.

Porcine epidemic diarrhea virus (PEDV) is having a severe effect on the pig breeding industry in central China. The mucosa and the content of the small intestine from newborn pre-weaned piglets with diarrhea were tested for the presence of PEDV by molecular and morphologic methods, and found to be positive. Negative-staining electron microscopy (EM) revealed the presence of coronavirus- like particles in the samples. The result of molecular detection by nested RT-PCR based on the amplification of the M gene was positive. Using a novel alternative method we successfully propagated the PEDV strain (CH/QX-2) in Vero cells, confirmed by ultrathin sections of the cells and Immunofluorescence assay (IFA). Phylogenetic analysis based on the partial S gene showed that the CH/QX-2 isolate was genetically closer to strains more commonly found in China, but differed genetically from two domestic strains (CH/S, 1986 and LZC, 2007), Korean strains (DR13, 2007), and the vaccine strain (CV777 vs) currently being used in China. CH/QX-2 formed a unique clade in the derived phylogenetic tree indicating that the CH/QX-2 strain currently circulating in central China is a new variant of PEDV. This study extends current knowledge on the diversity and epidemiology of PEDV.

[Expression of ANO1 during cardiac fibroblasts differentiation and its electrophysiological characteristics as calcium-activated chloride channel protein].

OBJECTIVE: To investigate the expression and electrophysiological characteristics of calcium-activated chlorine channel anoctamin-1 (ANO1) protein during the differentiation of cardiac fibroblasts (CFs) into myofibroblasts (MFs), and to elucidate the role of ANO1 in myocardial fibrosis. METHODS: The primary CFs from neonatal rats were isolated and the cells differentiated into MFs by subculture. The Ca2+-activated Cl- current (ICl(Ca)) in CFs and MFs were measured by whole-cell patch clamp, and the expressions of ANO1, α-smooth muscle actin(α-SMA)and vimentin in CFs and MFs were detected by immunofluorescence assay and Western blot, respectively. RESULTS: The current density in the early adherent CFs was stronger than that in MFs. ANO1 was expressed preferentially within and around the nuclei, and a small amount of ANO1 was expressed on the cell membrane. Moreover, ANO1 expression was weak in the early adherent CFs and displayed stronger expression in the MFs with proliferation tendency. CONCLUSION: The expression of ANO1 is closely related to the differentiation of MFs and it may be involved in modulation myocardial fibrosis.

[Recent advances in the study of natural homoisoflavonoids].

Homoisoflavonoid is a special type in flavonoids. There are more than 110 homoisoflavonoid compounds isolated from natural materials. Homoisoflavonoid compounds show many bioactivities on anti-inflammatory, estrogenicy, antiestrogenic, anticancer and angioprotective etc. This paper summarized the plant sources, structural types spectrocopy features and the biology activities of homoisoflavonoids.