Anatomical Characterization, HPLC Analysis, and Biological Activities of Ilex dipyrena

Ilex dipyrena Wall (Aquifoliaceae), is a traditional medicinal plant abundantly found in India and Pakistan. In the current research work, initially, the anatomical characteristics were recorded through microscopic examination of selected plant parts, such as leaf, petiole, and midrib. Then, the quantitative phytochemical screening was performed using standard tests reported in literature. The whole-plant powdered sample was then soaked in methanol to obtain crude extract, which was then fractionated into solvents of different polarities to obtain ethyl acetate, chloroform, butanol, hexane, and aqueous extracts. The phytochemical composition of the crude ethyl acetate and chloroform extracts (being the most active fractions) was then confirmed through HPLC analyses, where the possible phytochemical present were predicted through comparison of retention time of a given compound peak with the available standards. The extracts were also evaluated for their in vitro antioxidant and ani-lipoxygenase potentials using standard methods. The microscopic examination revealed the presence of anomocytic type stomata on the abaxial side of the leaf as well as unicellular trichrome and calcium oxalate druses crystals in the midrib and petiole, with a single, centered U-shaped collateral arterial bundle, which was directed toward the adaxial and the phloem toward the abaxial sides of the selected plant parts, respectively. Almost all tested representative groups of phytochemicals and essential minerals were detected in the selected plant, whereas five possible phytochemicals were confirmed in crude and chloroform extract and seven in ethyl acetate fraction. As antioxidant, chloroform fraction was more potent, which exhibited an IC50 value of 64.99, 69.15, and 268.52 µg/mL, determined through DPPH, ABTS, and FRAP assays. Ethyl acetate extract was also equally potent against the tested free radicals. Chloroform and ethyl acetate extracts were also potent against lipoxygenase, with IC50 value of 75.99 and 106.11 µg/mL, respectively. Based on the results of biological studies, Ilex dipyrena was found to good inhibitor of free radicals and lipoxygenase that could be further investigated to isolate compounds of medicinal importance

A Novel MicroRNA-132-Surtuin-1 Axis Underlies Aberrant B-cell Cytokine Regulation in Patients with Relapsing-Remitting Multiple Sclerosis

<div><p>Clinical trial results demonstrating that B-cell depletion substantially reduces new relapses in patients with multiple sclerosis (MS) have established that B cells play a role in the pathophysiology of MS relapses. The same treatment appears not to impact antibodies directed against the central nervous system, which underscores the contribution of antibody-independent functions of B cells to disease activity. One mechanism by which B cells are now thought to contribute to MS activity is by over-activating T cells, including through aberrant expression of B cell pro-inflammatory cytokines. However, the mechanisms underlying the observed B cell cytokine dysregulation in MS remain unknown. We hypothesized that aberrant expression of particular microRNAs might be involved in the dysregulated pro-inflammatory cytokine responses of B cells of patients with MS. Through screening candidate microRNAs in activated B cells of MS patients and matched healthy subjects, we discovered that abnormally increased secretion of lymphotoxin and tumor necrosis factor α by MS B cells is associated with abnormally increased expression of miR-132. Over-expression of miR-132 in normal B cells significantly enhanced their production of lymphotoxin and tumor necrosis factor α. The over-expression of miR-132 also suppressed the miR-132 target, sirtuin-1. We confirmed that pharmacological inhibition of sirtuin-1 in normal B cells induces exaggerated lymphotoxin and tumor necrosis factor α production, while the abnormal production of these cytokines by MS B cells can be normalized by resveratrol, a sirtuin-1 activator. These results define a novel miR-132-sirtuin-1 axis that controls pro-inflammatory cytokine secretion by human B cells, and demonstrate that a dysregulation of this axis underlies abnormal pro-inflammatory B cell cytokine responses in patients with MS.</p></div

SIRT1 regulates LT and TNFα production from B cells.

<p>A: Levels of lymphotoxin (LT), tumor necrosis factor (TNF)α, and interleukin (IL)-10 produced by B cells from healthy subjects (HS: n = 6) treated with the selective pharmacological inhibitor of sirtuin (SIRT)-1, EX-527 (10 µM), or vehicle (0.1% DMSO), and stimulated through the B-cell antigen receptor and CD40 (Wilcoxon test). B: Levels of LT, TNFα, and IL-10 produced by B cells from MS patients (n = 10) treated with the small molecule activator of SIRT1, resveratrol (10 µM), or vehicle (0.1% DMSO), and stimulated through the B-cell antigen receptor and CD40 (Wilcoxon test). C: Levels of LT and TNFα produced by B cells from MS patients (n = 10) treated with either vehicle control (0.1% DMSO) or resveratrol (10 µM) and those from HS (n = 6) treated with vehicle control are shown. NS: not significant (unpaired t-test).</p

Activated B cells of MS patients express lower levels of SIRT1.

<p>A: Expression levels of SIRT1 mRNA in B cells immediately after isolation (Fresh), or when kept in culture for 48 hours either unstimulated (US), stimulated through CD40 (CD40), or stimulated through the BCR and CD40 (BCR+CD40) (Mann-Whitney U-test). B: Frequency of SIRT1⁺ B cells (CD20⁺) within whole PBMC that were kept unstimulated (US: left panel) or stimulated through the BCR and CD40 (BCR+CD40: right panel) (Mann-Whitney U-test).</p

MS B cells express increased levels of miR-132, in association with an abnormal cytokine profile.

<p>A: Levels of lymphotoxin (LT), tumor necrosis factor (TNF)α, and interleukin (IL)-10 (CD40: following CD40 stimulation alone; BCR+CD40: following dual B-cell antigen receptor and CD40 stimulation) in the culture supernatants of B cells from healthy control subjects (HS, n = 13) and untreated MS patients (n = 14) (unpaired t-test). The proportion of memory cells among total B cells, and expression levels of CD40 were not different between B cells from MS patients and HS (data not shown). B: Expression level of miR-132 in B cells either immediately after isolation (Fresh), or following 48 hours in culture when left unstimulated (US), stimulated through CD40 alone (CD40), or stimulated through both the B-cell antigen receptor and CD40 (BCR+CD40). HS n = 13, MS n = 14; (Mann-Whitney U-test).</p

miR-132 enhances LT and TNFα production in association with SIRT1 suppression in B cells.

<p>A: Levels of lymphotoxin (LT), tumor necrosis factor (TNF)α, and interleukin (IL)-10 in B cells from healthy subjects (HS: n = 7) transfected with miR-132 mimic or negative control (NC), and stimulated through the B-cell antigen receptor (BCR) and CD40 (Wilcoxon test). B: Protein level of sirtuin (SIRT)-1 in B cells from HS transfected with miR-132 mimic or NC. A representative result of 2 experiments is shown. The arrow and the arrowhead indicate the bands corresponding to the molecular weight of SIRT1 and β-actin, respectively. C: Level of SIRT1 mRNA in B cells from HS transfected with miR-132 mimic or NC (n = 5) (Paired t-test).</p