Short-Chain Acyl-CoA Dehydrogenase as a Therapeutic Target for Cardiac Fibrosis.

Cardiac fibrosis is considered as unbalanced extracellular matrix (ECM) production and degradation, contributing to heart failure. Short-chain acyl-CoA dehydrogenase (SCAD) negatively regulates pathological cardiac hypertrophy. The purpose of this study was to investigate the possible role of SCAD in cardiac fibrosis. In-vivo experiments were performed on spontaneously hypertensive rats (SHR) and SCAD knockout mice. The cardiac tissues of hypertensive patients with cardiac fibrosis were used for measurement of SCAD expression. In-vitro experiments, with angiotensin II (Ang II), SCAD siRNA and adenovirus-SCAD (Ad-SCAD) were performed using cardiac fibroblasts (CFs). SCAD expression was significantly decreased in the left ventricles of SHR. Notably, swim training ameliorated cardiac fibrosis in SHR in association with the elevation of SCAD. The decrease in SCAD protein and mRNA expression levels in SHR CFs were in accordance with those in the left ventricular myocardium of SHR. In addition, SCAD expression was downregulated in CFs treated with Ang II in vitro, and SCAD siRNA interference induced the same changes in cardiac fibrosis as Ang II-treated CFs, while Ad-SCAD treatment significantly reduced the Ang II-induced CFs proliferation, α-SMA and collagen expression. In SHR infected with Ad-SCAD, the cardiac fibrosis of the left ventricle was significantly decreased. On the other hand, cardiac fibrosis occurred in conventional SCAD knockout mice. SCAD immunofluorescence intensity of cardiac tissue in hypertensive patients with cardiac fibrosis was lower than that of healthy subjects. All together, the current experimental outcomes indicate that SCAD has a negative regulatory effect on cardiac fibrosis and support its potential therapeutic target for suppressing cardiac fibrosis.

Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor.

P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

Gold-Nanocluster-Mediated Delivery of siRNA to Intact Plant Cells for Efficient Gene Knockdown.

RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a "green" alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.

Surface plasmon resonance biosensor combined with lentiviral particle stabilization strategy for rapid and specific screening of P-Glycoprotein ligands.

A novel surface plasmon resonance-based P-gp ligand screening system (SPR-PLSS) combined with lentiviral particle (LVP) stabilization strategy was constructed to screen out potential P-gp inhibitors from natural products. Firstly, we constructed LVPs with high and low expression levels of P-gp. The LVPs can ensure the natural conformation of P-gp based on the principle that LVPs germinated from packaging cells will contain cell membrane fragments and P-gp they carry. Then the LVPs with high P-gp expression for active channel and LVPs with low P-gp expression for reference channel were immobilized on CM5 chip respectively. The affinity detection was thus carried out with the signal reduction on the two channels. The P-gp inhibitors, Valspodar (Val) and cyclosporin (CsA), as positive compounds, were detected to characterize the chip's activity, and the KD of Val and CsA were 14.09 µM and 16.41 µM, respectively. Forty compounds from natural product library were screened using the SPR CM5 chip, and magnolol (Mag), honokiol (Hon), and resveratrol (Res) were screened out as potential P-gp ligands, showing a significant response signal. This work presented a novel P-gp ligand screening system based on LVP-immobilized biosensor to rapidly screen out P-gp ligands from natural product library. Compared with traditional cell experiments which the screening time may take up to several days, our method only takes several hours. Furthermore, this study has also provided solid evidences to support that some complicated membrane proteins would apply to the lentivirus-based SPR screening system.

The Effects of Traditional Chinese Medicine on P-Glycoprotein-Mediated Multidrug Resistance and Approaches for Studying the Herb-P-Glycoprotein Interactions.

As a member of the ATP-dependent membrane transport proteins, P-Glycoprotein (P-gp) is known to pump substrates out of cells using an ATP-dependent mechanism. The overexpression of P-gp in tumor cells reduces the intracellular drug concentrations, which decreases the efficacy of extensive antitumor drugs and leads to multidrug resistance (MDR) clinically. The combination of anticancer drugs with P-gp inhibitor has been an attractive and promising strategy to reverse MDR in cancer treatment. However, nonspecific or nonselective distribution of P-gp inhibitors to nontarget organs is one of the most fatal shortcomings in clinical application. Thus, there is an urgent need for effective and nontoxic MDR reversal agents, particularly in P-gp-mediated MDR. Traditional Chinese medicine (TCM) natural products may prove less toxic for use in P-gp inhibition to promote MDR reversal. P-gp modulatory effects have been previously demonstrated using selected TCM, including the flavonoid, alkaloid, terpenoid, coumarin, and quinonoid compounds, and some Chinese medicine extracts. Moreover, the approaches for screening active components from TCM are necessary, and these approaches face challenges. At present, the approaches to study the interaction between TCM and P-gp are divided into in vitro, in vivo, and in silico methods. This review will provide an overview and update on the role of TCM in overcoming P-gp-mediated MDR and the approaches to study the interaction between TCM and P-gp. SIGNIFICANCE STATEMENT: This review summarized some traditional Chinese medicines identified to have a modulatory effect on P-gp, including flavonoids, alkaloids, terpenoids, coumarins, quinonoid compounds, and some Chinese medicine extracts, and it introduced possible mechanisms. The approaches to study the interaction between TCM and P-gp are divided into in vitro, in vivo, and in silico methods.

Ginsenoside Rd Is Efficacious Against Acute Ischemic Stroke by Suppressing Microglial Proteasome-Mediated Inflammation.

A great deal of attention has been paid to neuroprotective therapies for cerebral ischemic stroke. Our two recent clinical trials showed that ginsenoside Rd (Rd), a kind of monomeric compound extracted from Chinese herbs, Panax ginseng and Panax notoginseng, was safe and efficacious for the treatment of ischemic stroke. In this study, we conducted a pooled analysis of the data from 199 patients with acute ischemic stroke in the first trial and 390 in the second to reanalyze the efficacy and safety of Rd. Moreover, animal stroke models were carried out to explore the possible molecular mechanisms underlying Rd neuroprotection. The pooled analysis showed that compared with placebo group, Rd could improve patients' disability as assessed by modified Rankin Scale (mRS) score on day 90 post-stroke and reduce neurologic deficits on day 15 or day 90 post-stroke as assessed by NIH Stroke Scale (NIHSS) and Barthel Index (BI) scores. For neuroprotective mechanisms, administration of Rd 4 h after stroke could inhibit ischemia-induced microglial activation, decrease the expression levels of various proinflammatory cytokines, and suppress nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα) phosphorylation and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear translocation. An in vitro proteasome activity assay revealed a significant inhibitory effect of Rd on proteasome activity in microglia. Interestingly, Rd was showed to have less side effects than glucocorticoid. Therefore, our study demonstrated that Rd could safely improve the outcome of patients with ischemic stroke, and this therapeutic effect may result from its capability of suppressing microglial proteasome activity and sequential inflammation.

Phenotypic and functional switch of macrophages induced by regulatory CD4+CD25+ T cells in mice.

CD4(+)CD25(+) regulatory T cells (Treg cells) are important in maintenance of peripheral tolerance. The direct effect of CD4(+)CD25(+) Treg cells on macrophages was studied using a mouse model in which syngeneic CD4(+)CD25(+) Treg cells were adoptively transferred into the peritoneal cavity of SCID mice. Peritoneal macrophages in mice transferred with CD4(+)CD25(+) Treg cells expressed significantly higher levels of CD23, CD47 and CD206 and less CD80 and major histocompatibility complex class II molecules as compared with those mice that received either CD4(+)CD25(-) T cells or no cells. Macrophages of mice injected with CD4(+)CD25(+) Treg cells displayed a remarkably enhanced phagocytosis of chicken red blood cells, and arginase activity together with an increased interleukin-10 (IL-10) production, whereas they showed a decreased antigen-presenting ability and nitric oxide production. Furthermore, CD4(+)CD25(+) Treg cells and CD4(+)CD25(-) T cells showed strong antagonistic effects on macrophage polarizations in vivo. Blocking arginase, IL-10 and/or transforming growth factor-ß (TGF-ß) partially but significantly reversed the effects of CD4(+)CD25(+) Treg cells to induce M2 macrophages in vivo suggesting that CD4(+)CD25(+) Treg cells have the ability to induce M2 macrophages at least in part through arginase, IL-10 and TGF-ß pathways. Thus, we have provided the in vivo evidence to support the unknown pathways for CD4(+)CD25(+) Treg cells to regulate innate immunity by promoting the differentiation of M2 macrophages as well as by inhibiting M1 macrophage induction by CD4(+)CD25(-) T cells in mice. CD4(+)CD25(+) Treg cells efficiently induced M2 macrophage differentiation in mice, offering the in vivo evidence to support the role of CD4(+)CD25(+) Treg cells in regulating innate immunity.

[Synthesis and characterization of octa-carboxylic phthalocyanine aluminum and its interaction with bovine serum albumin].

Octa-carboxylic phthalocyanine aluminium (AlPc(COOH)8) was used as fluorescent probe of infrared region. The interaction of octa-carboxylic phthalocyanine aluminium (AlPc(COOH)s) and bovine serum albumin(BSA) was studied by UV/ Vis and fluorescence spectra methods. The binding constant KA and n of phthalocyanine aluminium with BSA were determined. The results were K=5. 74 X 10(5) , n= 5. 7 and K= 3. 51 X 10(5) , for these two methods respectively. The same results by using two different analytical methods were obtained. Besides, hemin chloride(HE), ibuprofen(IB) and L-tryptophan(TRP) were used as probes, and the effects of these probes on the spectra of AlPc(COOH)8 )-BSA were studied by competitive binding method. The result indicated that, by adding HE into the AlPc(COOH)8)-BSA system, obvious spectral change of the system was observed, while adding TRP and IB caused no spectral changes. The binding site of octa-carboxylic phthalocyanine aluminium on the BSA was found to be at the I site by competitive binding method.

[The study on reversing mechanism of multidrug resistance of K562/A02 cell line by curcumin and erythromycin].

OBJECTIVE: To investigate the effects of curcumin (Cur) and erythromycin (EM) on multidrug resistance (MDR) reversal of K562/A02 cell line and their mechanism. METHODS: MTT assay was employed to determine the sensitivity of Cur, EM-treated K562/A02 cells to adriamycin (ADM). Flow cytometry was used to measure intracellular mean fluorescence intensity (MFI) of daunorubicin (DNR). P-gp expression was determined by immunohistochemistry. RT-PCR technique was used to examine the mdr1 mRNA level. RESULTS: IC(50) of ADM in K562/A02 cells was decreased when treated with Cur or EM, and the reversal times (RvT) was 4.9, 3.7 respectively. The RvT reached to 11.3 when treated with Cur (2.5 microg/ml) combined with EM (120 microg/ml). The DNR MFI in K562/A02 cells was significantly lower than that in K562 cells (P < 0.01), and was increased significantly when treated with Cur (2.5 microg/ml) or EM (120 microg/ml) (P < 0.05). There was no significant difference between DNR MFI of K562/A02 cells treated with Cur (2.5 microg/ml) or EM (120 microg/ml). Immunohistochemistry showed that P-gp expression was significantly higher in K562/A02 cells than in K562 cells (P < 0.01), and was reduced in K562/A02 cells treated with each (P < 0.01), though being still higher than that in K562 cells (P < 0.01). P-gp expression of K562/A02 cells treated with each drug for 5 days were lower than that for 3 days (P < 0.01), and lowered further when treated with Cur and EM together (P < 0.01). Mdr1 mRNA level in K562/A02 cells was higher than in K562 cells (P < 0.01), and was decreased when treated with each of the drugs (P < 0.01). The mdr1 mRNA level of K562/A02 cells treated with Cur (2.5 microg/ml) plus EM (120 microg/ml) was decreased most significantly than that treated with other group of drugs. After 5 day treatment the mdr1 mRNA level of K562/A02 cells with Cur (2.5 microg/ml) was lower than that with EM 120 microg/ml (P < 0.01). CONCLUSIONS: Either Cur or EM can partly reverse the multidrug resistance of K562/A02 cells and decrease the expression and function of P-gp in a time-dependent way. MDR reversing effect of Cur combined with EM is stronger than that of Cur or EM alone.

[Genetic diversity of human Parvovirus B19 VP1 unique region].

OBJECTIVE: Human Parvovirus B19 (HPV B19) is a small (23 nm), non-enveloped DNA virus found in 1974. It has been proved that HPV B19 is associated with a variety of childhood diseases, such as erythema infectious, transient aplastic crisis, aplastic anemia, idiopathic thrombocytopenic purpura and arthropathy, etc. There have been no any effective vaccines to prevent HPV B19 infection so far. The HPV B19 genome is composed of 5.6 kb single strand DNA. This genome encodes a nonstructural protein NS1, two structural proteins VP1 and VP2. Most neutralizing linear epitopes of HPV B19 cluster in the VP1 unique and VP1-VP2 junction regions. Only proteins encoded by genes of the VP1 unique and VP1-VP2 junction regions can stimulate bodies to produce protective antibodies. Aim of the present study was to get the VP1 unique region gene of HPV B19 and to analyze the genetic diversity so as to further study its function and application. METHODS: The VP1 unique region gene of HPV B19 was amplified from the serum of a child with idiopathic thrombocytopenic purpura by PCR. The purified PCR product was cloned into pGEM-T easy vector and transfected into the host strain E. coli (DH5 alpha). Positive clones were chosen and then the target gene was sequenced. RESULTS: The target gene sequence of HPV B19 VP1 unique region was amplified and cloned successfully. It had 705 nucleotides. Compared with the relevant sequences published in Genbank, the sequencing results were revealed with two nucleotides changes in the HPV B19 VP1 unique region, but their coding amino acid were not changed. CONCLUSION: It is suggested that genetic diversity exists in the VP1 unique region of HPV B19. Construction of the recombinant plasmid of HPV B19 VP1 unique region gene might benefit to further study.