Research progress on the fibrinolytic enzymes produced from traditional fermented foods.

Cardiovascular diseases (CVDs) are a global health problem and leading cause of death worldwide. Thrombus formation, one of the CVDs, is essentially the formation of fibrin clots. The existing thrombolytic agents have the disadvantages of high price, short half-life, and high bleeding risk; hence, there is an urgent need to find the alternative thrombolytic agents. In recent years, traditional fermented foods have been widely investigated for their outstanding effects in the prevention and treatment of thrombus formation. In this review, we have focused on fibrinolytic enzymes produced by microorganisms during the fermentation of traditional fermented foods and their potential use for treating CVDs. First, we discussed about the sources of fibrinolytic enzymes and microbial strains that produce those enzymes followed by the optimization of fermentation process, purification, and physicochemical properties of fibrinolytic enzymes. Finally, we have summarized the thrombolytic effects of fibrinolytic enzymes in humans and mice. Fibrinolytic enzymes produced by microorganisms during the fermentation of traditional fermented foods not only lyse thrombi but also acts as anti-atherosclerotic, anti-hyperlipidemia, and neuroprotection agents. Therefore, fibrinolytic enzymes from traditional fermented foods have great potential for the prevention and treatment of CVDs.

Galangin alleviates rheumatoid arthritis in rats by downregulating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that greatly affect patients' quality of life. Galangin extract is renowned for its anti-proliferative and anti-oxidative characteristics. However, galangin cytotoxicity studies are presently inadequate. We aimed to investigate the therapeutic potential of galangin on RA by investigating the PI3K/AKT signaling pathway.Fibroblast-like synovial cells (FLSs) were exposed to lipopolysaccharide (LPS) to establish an RA model in vitro. An ELISA assay was used to detect the levels of IL-1ß, TNF-α, and IL-6. Cell viability and apoptosis were determined by CCK8/EdU and flow cytometry assays. A western blot assay was used to analyze the protein expression levels. An RA rat model was established to evaluate the function of galangin through histopathological examination. Our results found that galangin induced apoptosis, inhibited cell proliferation, and increased cell invasion of rheumatoid arthritis fibroblast-like synovial cells (RAFLSs). Galangin inactivated the PI3K/AKT signaling pathway and the inflammatory response. An agonist of PI3K signaling, 740Y-P, restored the cellular functions of RAFLSs. Moreover, galangin suppressed the development of RA in vivo. Galangin effected its anti-arthritic influence through the PI3K/AKT signaling pathway. Galangin has potential as an alternative treatment for RA.

[Screening and identification of GABA-producing microbes in fermentation process of Sojae Semen Praeparatum].

A high-content GABA was found in Sojae Semen Praeparatum(SSP), which is a famous traditional Chinese medicine and officially listed in Chinese Pharmacopoeia. To screen out and identify GABA-producing microbes from samples at different time points during the fermenting process of SSP, traditional microbiological methods combined with molecular biological methods were used to study the predominant GABA-producing microorganisms existing in the fermenting process of SSP. This study would lay a foundation for further studying the processing mechanism of SSP. The fermenting process of SSP was based on Chinese Pharmacopoeia(2010 edition), and samples were taken at different time points during the fermenting process of SSP. The bacteria and fungi from samples at different time points in the fermenting process of SSP were cultured, isolated and purified by selective medium, and dominant strains were selected. The dominant bacteria were cultured in the designated liquid medium to prepare the fermentation broths, and GABA in the fermentation broth was qualitatively screened out by thin-layer chromatography. The microbial fermentation broth with GABA spots in the primary screening was quantitatively detected by online pre-column derivatization and high performance liquid chromatography established in our laboratory. GABA-producing microorganisms were screened out from predominant strains, and their GABA contents in fermentation broth were determined. The DNA sequences of GABA-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA sequences by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was made by phylogenetic tree constructed by MEGA 7.0 software. Through the homology comparison of NCBI and the construction of phylogenetic tree by MEGA 7.0 software, nine GABA-producing microorganisms were screened out and identified in this study. They were Bacillus subtilis, Enterococcus faecium, E. avium, Aspergillus tamarii, A. flavus, A. niger, Cladosporium tenuissimum, Penicillium citrinum and Phanerochaete sordida respectively. For the first time, nine GABA-producing microorganisms were screened out and identified in the samples at different time points during the fermenting process of SSP in this study. The results indicated that multiple predominant GABA-producing microorganisms exist in the fermenting process of SSP and may play an important role in the formation of GABA.

Liuwei Dihuang pill suppresses metastasis by regulating the wnt pathway and disrupting -catenin/T cell factor interactions in a murine model of triple-negative breast cancer.

OBJECTIVE: To investigate if the Liuwei Dihuang pill (LWDHP) can inhibit metastasis to the liver and lungs in mice bearing triple-negative breast cancer (TNBC), and the molecular mechanism underpinning this action. METHODS: Ninety-nine TNBC bearing-mice were distributed randomly to five groups: control (Con), paclitaxel (PTX), low-dose LWDHP (LLP, 2.3 g·kg－1·d－1), middle-dose LWDHP (MLP, 4.6 g·kg－1·d－1) and high-dose LWDHP (HLP, 9.2 g·kg－1·d－1). The LWDHP were administered (p.o.) to the agonal stage. The morphology of BC cells was observed by hematoxylin & eosin staining. Expression of axin-2, ß-catenin, T cell factor (TCF), cyclin- D1 and vascular endothelial growth factor (VEGF) was detected by western blotting or immunofluorescence. ß-catenin/TCF-1 interaction was measured using a co-immunoprecipitation assay. RESULTS: After LWDHP treatment, metastasis of BC cells to the lungs and liver was inhibited, expression of axin-2 was increased, expression of TCF-1, ß-catenin, cyclin-D1 and VEGF was decreased, and ß-catenin/TCF-1 interaction was disrupted. CONCLUSION: The LWDHP could inhibit metastasis of BC cells to the liver and lungs. The molecular mechanism underlying this action may be regulation of protein expression and ß-catenin/TCF-1 interactions in the Wnt pathway.

Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.

NF-κB, JNK and p53 pathways are involved in tubeimoside-1-induced apoptosis in HepG2 cells with oxidative stress and G2/M cell cycle arrest.

Tubeimoside-1 is a triterpenoid saponin extracted from the traditional Chinese herb Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae). We investigated the cytotoxic effect and apoptosis mechanism of tubeimoside-1. Tubeimoside-1 was cytotoxic in seven human cancer cell lines, with HepG2 the most sensitive. Tubeimoside-1 induced apoptosis of HepG2 cells dose and time dependently. Both the extrinsic and intrinsic pathways were triggered by tubeimoside-1. Caspase-3, -8 and -9 were activated and the expression of Fas, Fas ligand, Bcl-2, Bak and Bax was regulated. Moreover, tubeimoside-1 induced accumulation of reactive oxygen species and arrested cell cycle at the G(2)/M phase, thus contributing to apoptosis, through signaling regulation by tumor necrosis factor α, nuclear factor κB (NF-κB), Jun N-terminal kinase (JNK) and p53. We provide further insight into the tubeimoside-1 cytotoxic effect for antitumor chemotherapeutic treatment.

Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase.

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to ß-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.

Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.

Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.