Neotuberostemonine inhibits the differentiation of lung fibroblasts into myofibroblasts in mice by regulating HIF-1α signaling.

Pulmonary fibrosis may be partially the result of deregulated tissue repair in response to chronic hypoxia. In this study we explored the effects of hypoxia on lung fibroblasts and the effects of neotuberostemonine (NTS), a natural alkaloid isolated from Stemona tuberosa, on activation of fibroblasts in vitro and in vivo. PLFs (primary mouse lung fibroblasts) were activated and differentiated after exposure to 1% O2 or treatment with CoCl2 (100 µmol/L), evidenced by markedly increased protein or mRNA expression of HIF-1α, TGF-ß, FGF2, α-SMA and Col-1α/3α, which was blocked after silencing HIF-1α, suggesting that the activation of fibroblasts was HIF-1α-dependent. NTS (0.1-10 µmol/L) dose-dependently suppressed hypoxia-induced activation and differentiation of PLFs, whereas the inhibitory effect of NTS was abolished by co-treatment with MG132, a proteasome inhibitor. Since prolyl hydroxylation is a critical step in initiation of HIF-1α degradation, we further showed that NTS treatment reversed hypoxia- or CoCl2-induced reduction in expression of prolyl hydroxylated-HIF-1α. With hypoxyprobe immunofiuorescence staining, we showed that NTS treatment directly reversed the lower oxygen tension in hypoxia-exposed PLFs. In a mouse model of lung fibrosis, oral administration of NTS (30 mg·kg-1·d-1, for 1 or 2 weeks) effectively attenuated bleomycin-induced pulmonary fibrosis by inhibiting the levels of HIF-1α and its downstream profibrotic factors (TGF-ß, FGF2 and α-SMA). Taken together, these results demonstrate that NTS inhibits the protein expression of HIF-1α and its downstream factors TGF-ß, FGF2 and α-SMA both in hypoxia-exposed fibroblasts and in lung tissues of BLM-treated mice. NTS with anti-HIF-1α activity may be a promising pharmacological agent for the treatment of pulmonary fibrosis.

Astin B, a cyclic pentapeptide from Aster tataricus, induces apoptosis and autophagy in human hepatic L-02 cells.

Astins (including astin B) are a class of halogenated cyclic pentapeptides isolated from the medicinal herb of Aster tataricus. However, our previous works showed that the herbal medicine was hepatotoxic in vivo, and a toxicity-guided isolation method led to the identification of a cyclopeptide astin B. Astin B is structurally similar to cyclochlorotine, a well-known hepatotoxic mycotoxin. Thus, the aim of this study was to determine the potential cytotoxic effects and the underlying mechanism of astin B on human normal liver L-02 cells. We found that astin B has hepatotoxic effects in vitro and in vivo and that hepatic injury was primarily mediated by apoptosis in a mitochondria/caspase-dependent manner. Astin B provoked oxidative stress-associated inflammation in hepatocytes as evidenced by increased levels of reactive oxygen species (ROS), reduced contents of intracellular glutathione (GSH), and enhanced phosphorylation of c-Jun N-terminal kinase (JNK). Furthermore, the mitochondria-dependent apoptosis was evidenced by the depolarization of the mitochondrial membrane potential, the release of cytochrome c into cytosol, the increased ratio of Bax/Bcl-2, and the increased activities of caspases-9 and -3. Interestingly, astin B treatment also induces autophagy in L-02 cells, characterized by acidic-vesicle fluorescence, increased LC3-II and decreased p62 expression. Autophagy is a protective mechanism that is used to protect cells from apoptosis. The presence of autophagy is further supported by the increased cytotoxicity and the enhanced cleaved caspase-3 after co-treatment of cells with an autophagy inhibitor, also by increased LC3-II and decreased p62 after co-treatment with a caspase inhibitor. Taken together, astin B, most likely together with other members of astins, is the substance that is primarily responsible for the hepatotoxicity of A.tataricus.