Effect of Shenlian Extract on Macrophage Function and Inflammatory Resolution in Lipid Peroxidation Inflammatory Injury Models / 中国实验方剂学杂志

Objective:To study the effect of Shenlian extract (SL extract) on macrophage function and inflammatory resolution in lipid peroxidation inflammatory injury models. Method:The effects of different concentrations of SL extract (2.5, 5.0, 10.0, 20.0 mg·L-1) on the polarization type, foam formation and chemotactic function of macrophages were detected with RAW264.7 cells induced by oxidized low-density lipoprotein(ox-LDL). Western blot was used to detect pro-inflammatory resolution factor arachidonate 5-lipoxygenase(ALOX5) and inducible inducible nitric oxide synthase(iNOS), and phosphorylated p65 (p-p65) and phosphorylated IκK (p-IκK) in nuclear factor(NF)-κB related signaling pathways. Result:Compared with the control group, ox-LDL enhanced the expressions of M1 macrophage markers TNF-α, IL-1β, iNOS (PPPα, IL-1β, and iNOS (PPPPκK was inhibited significantly (PConclusion:In the inflammatory damage model of lipid peroxidation, SL extract can regulate the polarization of macrophage, inhibit the chemotaxis and foaming of ox-LDL, increase the inflammatory resolution molecular expression, and improve the state of lipid peroxidation, which may be related to the inhibition of NF-κB signaling pathway.

Inhibition of ATM Promotes Neuronal Apoptosis through Upregulating Bim Expression / 中山大学学报(医学科学版)

@#【Abstract】 【Objective】To investigate the mechanisms implicated in ataxia telangiectasia mutated(ATM)inhibitioncaused apoptosis in the cultured cerebellar granule neurons.【Methods】Primary cerebellar granule neurons(CGN)isolated from neonatal Sprague Dawley rats of 7-8 days were divided into the following groups:25 K group(survival group),5 K group(apoptosis group)and 25 K + KU-55933 treatment group(ATM inhibition group),25 K + KU-55933 + Mithramycin A treatment group(MMA group),25 K + KU-55933 + Chromonycin A3 treatment group(CMA3 group). The protein expression of p-ATM,ATM,Bim and Caspase 3 were detected by Western Blot. The apoptotic cells with nuclear pyknosis were detected by Hoechst-staining.【Results】Compared with 25 K group,the result of western blot showed that the protein expression of Bim and Caspase 3 were increased in the ATM inhibition group(P < 0.05). Compared with 25 K group,the nuclear pyknosis rate of 5 K group and ATM inhibition group were significantly increased(P < 0.05). Inhibition of Bim by Mithramycin A or Chromomycin A3 remarkably reversed ATM inhibition-caused apoptosis.【Conclusion】Inhibition of ATM induce Bim dependent apoptosis in cultured cerebellar granule neurons.

Identification and differential expression analysis of ABC transporter gene from medicinal plant Dendrobium officinale / 药学学报

ABC transporters (ATP-binding cassette transporters) are a family of trans-membrane transport proteins, which are ubiquitous in prokaryotes and eukaryotes. They are functionallyinvolved in the transport and accumulation of plant secondary metabolites, phytohormone transport, lipid metabolism, exogenous toxins detoxification, plant disease and other aspects. Based on the genome and transcriptome data of Dendrobium officinale, 88ABC transporters were preliminarily identified in D. officinale and their functions and subcellular localization were predicted. These proteins are divided into seven subfamilies, ABCA-ABCI, including 4 ABCA, 19 ABCB, 12 ABCC, 3ABCD, 9 ABCF, 37ABCG and 4 ABCI, which are mainly located in plasma membrane, vacuole and Golgi apparatus. Comparative transcriptomic analysis of ABC protein expression profile between asymbiotic and symbiotic germination of D. officinale show that some members of ABCB and ABCG proteins are highly expressed during seed germination inoculated fungi. qPCR validated that 2 ABCB11 and 2 ABCG-PDR are significantly up-regulated in symbiotic assay compared to asymbiotic germination (fold change ≥ 10.0). These proteins are mainly involved in abscisic acid and auxin transport, suggesting that these proteins play an important role in the germination of D. officinale seed and in the interaction with microorganisms.

Cloning and expression analysis of gibberellin 3-oxidase gene in Dendrobium officinale / 中草药

Objective: To clone the gibberellin 3-oxidase gene DoGA3ox from an important and endangered medicinal plant Dendrobium officinale (Orchidaceae), followed by bioinformatic and expression analysis. Methods: RACE and RT-PCR were used to isolate the full-length gene. The physicochemical properties, conserved domains, and subcellular localization of DoGA3ox protein were determined using a series of bioinformatic tools. The phylogenetic analyses were performed using DNASTAR 7.0 and MEGA 6.0 software. Real time quantitative PCR was employed for gene expression analysis. Results: The full-length cDNA of DoGA3ox (GenBank registration number KT597694) was 1 318 bp in size, and encoded a 353-amino acid peptide chain with a molecular weight of 39052.5 and an isoelectric point (pI) of 6.21; The deduced DoGA3ox protein without transmembrane or signal peptide residues, contained the gibberellin 3-oxidases conserved domains that DIOX_N (40-130) and 2OG-FeII_Oxy (197-299). DoGA3ox had 51%-56% similarity with GA3ox proteins from various plants, and was closely related to the monocot Allium fistulosum, Elaeis guineensis, and Phoenix dactylifera. The relative transcript levels of DoGA3ox were increased at stage 1, then decreased (stage 2), and increased again (stage 3) during D. officinale symbiotic/asymbiotic seed germination, the fold change to ungerminated seeds was 13.44 (symbiotic)/5.21 (asymbiotic), 7.28/2.32 and 9.40/6.21, respectively. Moreover, its transcript level was higher in symbiotic germination than that in the asymbiotic status. Conclusion: The full-length DoGA3ox gene is cloned for the first time, and its expression in different stages during seed germination indicates that DoGA3ox gene plays a crucial role in the regulation of seed germination in D. officinale.

Establishment of vulnerable plaque model by mast cell activation in adventitia and evaluation of effectiveness of intervention of Shenlian tablet / 药学学报

This study was designed to investigate the activity of Shenlian tablet in stabilization of the atherosclerosis (As) plaque in apoE-/- mice and explore the mechanisms. Rat peritoneal mast cells were randomly allocated and treated with Shenlian tablet (100, 50, 25, 12.5 mg ·L-1) or cromoglicate sodium (200 μg·L-1) for 2 h before exposure to substance P. Histamine, tryptase, IL-1β and NF-κB were measured in the cell culture supernatant by ELISA assay. The plaque formation was induced by common carotid artery cannula method combined with high-fat diet in apoE-/- mice, and the plaque instability was induced by substance P through local mast cell degranulation. Mice were divided into eight groups that included the model 1 (M1, sham-operated group), M2 (carotid artery cannula combined with high-fat diet), M3 (M2 combined with substance P 0.5 μg/mouse), Shenlian extract (95, 190 and 380 mg·kg-1·d-1), atorvastatin (2.6 mg·kg-1·d-1) and normal control group. Total cholesterol (TC), high-density lipoprotein (HDL-C), high-sensitivity C-reactive protein (hs CRP), matrix metalloproteinases 9 (MMP-9) and histamine were measured by ELISA. Thickness, plaque area, mast cell degranulation were observed by hematoxylin and eosin staining, toluidine blue staining. CD117 antigen expression were observed by confocal microscopy. Intracellular phosphorylation was detected using the Bio-Plex 6-plex phosphoprotein assay kit. The results show that the mast cell membrane was stabilized by Shenlian tablet. Histamine, tryptase, interleukin l-β and NF-κB exhibited a significantly reduction in the Shenlian tablet-treated group (PP-/- mice model group. The proliferation, degranulation and inflammation of mast cell were significantly inhibited by Shenlian tablet. On the other hand, the same treatment decreased hs-CRP, MMP-9 and histamine in serum. IκB, p38 MAPK phosphorylation, intraplaque hemorrhage and collagen degradation were reduced in the presence of Shenlian tablet, which increased the stability of the As plaque. The results show that the vulnerable plaque model induced by mast cell activation in adventitia was established. Shenlian tablet exhibited a protective effect in this model. Shenlian tablet may increase the plaque stability via inhibition of mast cell-mediated inflammatory response.

Molecular cloning and characterization of a novel DoSWEET1 gene from Dendrobium officinale / 药学学报

SWEET (sugars will be eventually exported transporters) constitute a large and conserved gene family of sugar transporters in eukaryotes, which are important in the cellular metabolisms, growth and development, and plant-microbe interaction in plants. In the present study, a full length cDNA of SWEET encoding gene, designed as DoSWEET1(GenBank accession No. KT957550), was identified in Dendrobium officinale using RT-PCR and RACE approaches. DoSWEET1 was 1150 bp in length and encoded a 262-aa protein with a molecular weight of 29.18 kD and an isoelectric point of 9.49. The deduced DoSWEET1 protein contained seven transmembrane regions and two conserved MtN3-slv domains (11-94, 130-212). Multiple sequence alignment revealed that DoSWEET1 had high identities (45%-54.6%) with SWEET proteins from various plants. A neighbor joining phylogenetic analysis suggests that DoSWEET1 belonged to the class II subgroup of the SWEET evolutionary tree, and was closely related to rice OsSWEET13, OsSWEET14, and OsSWEET15. qPCR analysis demonstrated that DoSWEET1 gene was differentially expressed in the three included organs of D. officinale, and the expression was most abundant in the roots at 9.88 fold over that of the stems, followed by that of the leaves with 2.85 fold higher. In the 3rd symbiotic germinating seeds infected by Tulasnella sp., the transcipts were dramatically induced by 1359.06 fold over that in the ungerniamted control seeds, suggesting a vital role of the gene in the D. officinale symbiotic germination process. Molecular cloning and characterization of the novel DoSWEET1 gene provides a foundation for the functional study of the gene in sugar translocation during the D. officinale symbiotic germination process.