Identification of UQCRB as an oxymatrine recognizing protein using a T7 phage display screen.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophora flavescens Aiton (Radix Sophorae Flavescentis, Kushen) is used in traditional Chinese medicine to treat chronic hepatitis B (CHB), and has the ability to clear heat and dampness from the body. Oxymatrine is one of the major bioactive compounds extracted from Sophora flavescens Aiton and constitutes more than 90% of the oxymatrine injection commonly used for CHB treatment in clinics in China. AIM OF THE STUDY: We aim to analyze the protein binding target of oxymatrine in treating CHB by screening a T7 phage display cDNA library of human CHB and examine the biochemistry of protein-ligand binding between oxymatrine and its ligands. MATERIALS AND METHODS: A T7 phage cDNA library of human CHB was biopanned by affinity selection using oxymatrine as bait. The interaction of oxymatrine with its candidate binding protein was investigated by affinity assay, molecular docking, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR). RESULTS: A library of potential oxymatrine binding peptides was generated. Ubiquinol-cytochrome c reductase binding protein (UQCRB) was one of the candidate binding proteins of oxymatrine. UQCRB-displaying T7 phage binding numbers in the oxymatrine group were significantly higher than that in the control group, biotin group, and matrine group (p<0.05 or p<0.01). Three-dimensional structure modeling of the UQCRB with oxymatrine showed that their binding interfaces matched and oxymatrine inserted into a deeper pocket of UQCRB, which mainly involved amino acid residues Tyr21, Arg33, Tyr83, Glu84, Asp86, Pro88, and Glu91. The binding affinity constant (Kb) from SPR was 4.2mM. The Kb from ITC experiment was 3.9mM and stoichiometry was fixed as 1, which fit very well with the result of SPR. The binding of oxymatrine to UQCRB was driven by strong enthalpy forces such as hydrogen bonds and polar interactions as the heat released was about 157kcal/mol and ΔG was less than zero. CONCLUSIONS: In this study, using the T7 phage display system, we have identified UQCRB as a direct binding protein of oxymatrine. Furthermore, the specificity and molecular interaction of oxymatrine with UQCRB were also determined. The binding of UQCRB to oxymatrine suggests that UQCRB is a potential target of oxymatrine in treating CHB. These results provide new understanding into the mechanism of oxymatrine and insights into the strategy on the treatment of CHB.

[Community structure and difference of endophtic bacteria in Aquilaria sinensis with and without agarwood].

Aquilaria sinensis can generate agarwood, which is closely related with endophyte. Up to now, studies mainly focused on the effects of endophytic fungi on agarwood formation, but studies about endophytic bacteria are rarely reported. In our research, the T-RFs and Shannon index of endophytic bacteria in samples of agarwood increase. The number of distinctive T-RFs fragments of corresponding samples in the same group accounted for more than 60% the number of total T-RFs fragments. In samples of no-agarwood, the dominant bacterial population are Anoxybacillus, Clostridium, Candidatus endobugula, Lysinibacillus. In samples of agarwood, the dominant bacterial population are Clostridium, Lysinibacillus, Luteimonas, phytoplasma. Besides, there are. specific T-RFs fragment in samples of agarwood and no-agarwood respectively. When we perform cluster analysis, we found samples of agarwood highly gather together and samples of no-agarwood highly gather together. This means community of endophytic bacteria emerge significant and regular changes during agarwood formation, which may be result of agarwood production, or maybe it is important reason of agarwood production. In this paper, we obtain more comprehensive and accurate community of endophytic bacteria in Aquilaria sinensis and it's variation during agarwood formation using T-RFLP, which is first study of effects of endophytic bacteria on agarwood formation, and will help to exploit resource of endophytic bacteria more reasonably.

[Thoughts on pathogenesis of Helicobacter pylori related gastrosis with splenogastric hygropyrexia syndrome].

Splenogastric hygropyrexia syndrome, a common syndome in clinical practice, responds well to the therapy of traditional Chinese medicine (TCM). In view of the complexity of TCM syndrome formation, proceeding from different aspects such as Helicobacter pylori (Hp) infection, expression of inflammatory factors, mucosal protection and change in micro-ecosystem, the authors proposed that Hp infection in gastric mucosa, nuclear factor-kappa B activation, over-expression of heat shock protein 70, and "excessive evil, dyssplenism and confliction between healthy energy and evils" due to disequilibrium between Hp and lactobacillus acidophilus in gastric mucosa and coated tongue may be the important links in the formation of Hp related gastrosis with splenogastric hygropyrexia syndrome.

[Preliminary study on intestinal flora in diarrhea type irritable bowel syndrome with pi-wei dampness-heat syndrome].

OBJECTIVE: To observe the changes of intestinal flora in diarrhea type irritable bowel syndrome with Pi-wei dampness-heat syndrome (IBS-PDS). METHODS: The seven kinds of common intestinal bacteria in feces, including enteri bacillus, enterococci, saccharomycete, bifid bacteria, lactobacillus, bacteroides and peptococcus were studied in 21 patients suffered from IBS-PDS, and compared with those in 22 patients with IBS with deficiency of Pi syndrome (DPS) and 25 healthy subjects as control. RESULTS: As compared with the healthy subjects, the levels of enteri bacillus and enterococci were significantly increased (P<0.01), the levels of bifid bacteria, Lactobacillus and Peptococcus were significantly decreased (P < 0.01), and saccharomycete and Bacteroides were insignificantly different in patients with PDS. As compared with patients with DPS, the levels of enteri bacillus, enterococci, bifid bacteria, Lactobacillus, Peptococcus and Bacteroidaceae were significantly increased except the level of saccharomycete. CONCLUSION: There may be alteration of intestinal flora in patients with IBS-PDS.