Treatment schedule and division of groups.

<p>Treatment schedule and division of groups.</p

Possible role of CG administration in nimesulide-induced hepatotoxicity.

<p>In our study, nimesulide was found to enhance ROS/RNS generation and compromise antioxidant defenses in mitochondria leading to oxidative stress. As a consequence of oxidative stress mitochondrial electron flow and NAD(P)H decreased along with significant mitochondrial depolarization. Oxidative stress along with mitochondrial dysfunction facilitated membrane permeability transition (MPT). Cell death proteins like AIF, EndoG, Cyt <i>c</i> released from mitochondria to cytosol due to MPT. In such a condition, where significant oxidative stress ensued and antioxidant defenses were compromised at the transcriptional level, significant macromolecular damage occurred. Apoptotic protein Cyt <i>c</i> along with other factors like dATP, ApoAF-1 and caspase-9 activated effector caspase-3 leading to DNA damage and cell death. A combination of terpenes, camphene and geraniol (1∶1), showed their potency to prevent nimesulide-induced imbalance in oxidant-antioxidant homeostasis and its downstream effects in mitochondrial dysfunction during hepatotoxicity.</p

GSH content and Antioxidant enzyme activities.

<p>Mitochondrial thiol (majorly GSH) was assessed using CellTracker^™ Green CMFDA fluorescent probe on flow cytometer. Data represent mean fluorescent intensity (MFI) of chloromethylfluorescein (CMF). Superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase activity of mitochondrial and cytosolic fractions were demonstrated as unit activity/min/mg protein. Level of significance is denoted as */# <i>P</i><0.05, **/## <i>P</i><0.01 and ***/### <i>P</i><0.001. *, compared to vehicle control and #, compared to nimesulide stress.</p

Oxidative stress status. A.

<p>superoxide (O₂^·-) and, <b>B</b>. secondary ROS/RNS generation were assessed using DHE and DCFH-DA dye on flow cytometer. t-BHP (oxidative stress generator) was used as positive control for oxidative stress in mitochondria.</p

ROS/RNS induced damage to proteins and lipids.

<p>This was observed in both cytosolic and mitochondrial fractions. <b>A.</b> Protein oxidative damage is demonstrated as protein carbonyl formation (nM/mg protein), while, <b>B.</b> protein nitrosative damage as nitrotyrosine formation, and, <b>C.</b> oxidative lipid damage as MDA formation (nM/mg protein). Carbonyl and MDA formation was estimated using biochemical assays. Tyrosine formation was observed by western blot and demonstrated as fold change of densitometric values with respect to vehicle control. Level of significance is denoted as */# <i>P</i><0.05, **/## <i>P</i><0.01 and ***/### <i>P</i><0.001. *, compared to vehicle control and #, compared to nimesulide stress.</p

Mitochondrial membrane permeability transition (MPT). A.

<p>Immunoblot analysis of released proteins: AIF, EndoG and Cyt <i>c</i> from mitochondria to cytosol were assessed and dansitometric analysis was done. Cyto-Ox-IV and β-Actin were used as internal loading controls for mitochondrial and cytosolic proteins respectively. All the densitometric values are normalized with respective controls and values are represented as compared to vehicle control in fold change. Densitometry of bands was done using ImageJ software (V1.41o, NIH, USA). <b>B.</b> Mitochondrial swelling as a function of MPT change was also observed as decrease in absorbance at 540 nm. Cyclosporine A (CsA, MPT inhibitor) + Ca²⁺ and <i>Pi</i> (KH₂PO₄, MPT inducer) were used as controls of MPT. During <i>in vivo</i> treatment nimesulide showed some (25%) initial swelling after that in <i>ex vivo</i> condition during the experiment remaining swelling was observed. Dotted line in bar graph showed 100% swelling induced by <i>Pi.</i> Level of significance is denoted as */# <i>P</i><0.05, **/## <i>P</i><0.01 and ***/### <i>P</i><0.001. *, compared to vehicle control and #, compared to nimesulide stress.</p

Activation of Caspase-9/caspase-3, and DNA damage.

<p>Cleavage of procaspase into cleaved low molecular weight protein as activation of caspases, was observed in cytosolic fraction and OGG formation, marker for oxidative DNA damage were assessed by immunoblotting (<b>A</b>). β-Actin was used as internal loading control and the densitometric values in bar graph (<b>B</b>) are normalized with it. Densitometry of bands was done using ImageJ software (V1.41o, NIH, USA). Values are represented as compared to vehicle control in fold change. Level of significance is denoted as */# <i>P</i><0.05, **/## <i>P</i><0.01 and ***/### <i>P</i><0.001. *, compared to vehicle control and #, compared to nimesulide stress.</p

Structure of the drug - nimesulide, and camphene and geraniol.

<p>Nimesulide is a non-steroidal anti-inflammatory drug (NSAID). Camphene is a bicyclic mono-terpenoid whereas geraniol is acyclic monoterpene-alcohol.</p

Determination of nimesulide-induced hepatotoxicity. A.

<p>Clinical biochemistry (levels of SGPT, SGOT and bilirubin) of blood serum. <b>B.</b> Histopathology of H&E stained liver tissue of vehicle control (<b>i</b>), nimesulide administered rats (<b>ii</b>) and CG pre-administered rats (<b>iii</b>). Pictures were taken at 125X (i, ii, and iii) and 500X (iv, v and vi) magnification. In figures yellow arrow heads represent normal hepatocytes; red arrowhead represents edema, green arrowhead represents hyperplastic bile ductule, and white arrowheads represent degenerating hepatocytes with infiltration of inflammatory cells. Level of significance is denoted as */# <i>P</i><0.05, **/## <i>P</i><0.01 and ***/### <i>P</i><0.001. *, compared to vehicle control and #, compared to nimesulide stress.</p

Mitochondrial Activity. A

<p>and <b>B.</b> %MTT activity as mitochondrial electron flow and UV auto-fluorescence of mitochondria as reduced pyridine nucleotides [NAD(P)H] respectively. <b>C.</b> Mitochondrial membrane potential was assessed using JC-1dye on flow cytometer. In dot plot, red fluorescence of J-aggregates represents polarized mitochondria (in right panel, red colored) whereas green fluorescence of JC-1 monomers represents depolarized mitochondria (left panel, green colored). CCCP (mitochondrial uncoupler) was used as negative control for mitochondrial membrane potential. Level of significance is denoted as **/## <i>P</i><0.01 and ***/### <i>P</i><0.001. *, compared to vehicle control and #, compared to nimesulide stress.</p