Physical and chemical characteristics of new Ibuprofen derivatives.

<p>Physical and chemical characteristics of new Ibuprofen derivatives.</p

Effect of compound 4f on A23187 induced (A) Translocation of cytosolic cytochrome C and activation of caspase-3 (B) Caspase-9 and (C) Caspase-3 activities and (D) PS externalization in PRP and washed platelets.

<p>Values are presented as mean ± SEM (n = 5), expressed as percentage increase in (B & C) caspase activity and (D) Annexin V-FITC fluorescence and expressed as percentage increase in apoptotic platelets expressing PS relative to control. ***<i>p</i><0.001; significant compared to control. ^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001; significant compared to A23187.</p

Efficacy of Ibuprofen and its derivatives measured in terms of (A) Reduction potential (B) DPPH radical scavenging activity in comparison with Quercetin.

<p>Values are presented as mean ± SEM (n = 5).</p

Effect of compound 4f on (A) Collagen induced protein phosphorylation (B) A23187 induced γ-glutamyltransferase activity (C) MTT cell viability assay (D) LDH release in platelets.

<p>(A) Lane I- resting platelets (untreated). Lane II- platelets treated with Collagen (1 µg/mL). Lanes III, IV and V- pre-loaded platelets with collagen and incubated with 4f in increasing concentration of 25, 50 and 100 µM respectively. Values are presented as mean ± SEM (n = 5). ***<i>p</i><0.001; significant compared to control. ^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001; significant compared to agonist.</p

Effect of compound 4f on A23187 induced (A) Endogenous generation of ROS (B) Intracellular calcium levels (C) Mitochondrial membrane depolarization and (D) Peroxidation of cardiolipin in PRP and washed platelets.

<p>Values are presented as mean ± SEM (n = 5), expressed as percentage increase in DCF fluorescence (for ROS), percentage increase in Fura-2/AM fluorescence (intracellular calcium), Rhodamine 123 fluorescence (ΔΨ<i>m</i>) and NAO fluorescence (cardiolipin), relative to control. ***<i>p</i><0.001; significant compared to control. ^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001; significant compared to A23187.</p

Reaction sequence for the synthesis of new Ibuprofen derivatives.

<p>Reaction sequence for the synthesis of new Ibuprofen derivatives.</p

MTX altered ER stress and mitochondrial apoptotic markers and its inhibition by NAC/NACA.

<p>Effect of MTX in presence or absence of NAC/NACA on (<b>A</b>) changes in intracellular calcium levels, (<b>B</b>) phospho-eIF2-α expression and (<b>C</b>) peroxidation of cardiolipin, (<b>D</b>) FACS analysis of ΔΨ<i>m</i> depolarization in washed platelets treated with MTX in presence or absence of NAC/NACA. Values are presented as mean ± SEM (n = 5), expressed as percentage increase in (<b>A</b>) fura-2/AM and (<b>C</b>) NAO fluorescence. p*/#< 0.05, p**/##< 0.01, p***/###< 0.001; *: significant compared to control. #: significant compared to MTX. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest were presented.</p

MTX induced platelet apoptosis and its inhibition by NAC/NACA.

<p>Effect of MTX in presence or absence of NAC/NACA/Dicumarol on (<b>A</b>) cyt. c release from mitochondria to cytosol, activation of caspase-9 and caspase-3, (<b>B</b>) protein tyrosine phosphorylation, (<b>C</b>) PS externalization, (<b>D</b>) LDH release and (<b>E</b>) MTT cell viability assay. <b>B,</b> Lane I: resting platelets (untreated), Lane II: platelets treated with A23187 (10 μM), Lane III: platelets treated with MTX (50 μM), Lane IV: pre-loaded platelets with MTX (50 μM) and incubated with NAC (500 μM), Lane V: pre-loaded platelets with MTX (50 μM) and incubated with NACA (50 μM). Values are presented as mean ± SEM (n = 5), expressed as percentage increase in (<b>C</b>) Annexin V-FITC fluorescence. COX IV and β-Tubulin were used as loading control. p*/#< 0.05, p**/##< 0.01, p***/###< 0.001; *: significant compared to control. #: significant compared to MTX. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest were presented.</p

MTX altered ROS levels in platelets and its inhibition by NAC/NACA/Mito-TEMPO.

<p><b>A,</b> FACS analysis of ROS generation in washed platelets treated with MTX in presence or absence of NAC/NACA. <b>B,</b> GSH/GSSG ratio and (<b>C</b>) γ‑Glutamyltransferase activity. <b>D,</b> Effect of Mito-TEMPO on MTX induced ROS generation, expressed as percentage increase in DCF fluorescence. Effect of MTX in presence or absence of NAC/NACA on components of mitochondrial electron transport chain, (<b>E</b>) Complex I-NADH: ubiquinone oxidoreductase activity (<b>F</b>) Complex II-succinate: ubiquinone oxidoreductase activity (<b>G</b>) Complex III-coenzyme Q: cytochrome c-oxidoreductase activity and (<b>H</b>) Complex IV-cytochrome c oxidase activity. Values are presented as mean ± SEM (n = 5). p*/#< 0.05, p**/##< 0.01, p***/###< 0.001; *: significant compared to control. #: significant compared to MTX.</p

JNK activation by mitochondrial ROS in MTX-treated platelets and its reversal by NAC/NACA.

<p><b>A,</b> Immunoblot showing the expression of phospho-JNK in time- and concentration-dependent manner in MTX-treated platelets. <b>B</b>, Immunoblots showing the effect of Dicumarol on the expression levels of phospho-JNK, Bcl-2, Bad, Bax, cyt. c, Cas-3 in MTX treated platelets. <b>C,</b> Immunoblots showing the effect of z-DEVD-fmk on the expression level of Cas-3 in MTX-treated platelets. <b>D,</b> Immunoblots showing the effect of Mito-TEMPO on the expression levels of phospho-JNK in MTX-treated platelets. <b>E,</b> Effect of NAC/NACA on the expression levels of phospho-JNK, Bcl-2, Bad, Bax, tBid and Cas-8 in MTX-treated platelets. <b>F,</b> Representative densitogram of immunoblots present in panel <b>A, B, C, D,</b> and <b>E</b>. JNK and β-Tubulin were used as loading control. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest were presented.</p