Amelioration of Aspirin Induced Oxidative Impairment and Apoptotic Cell Death by a Novel Antioxidant Protein Molecule Isolated from the Herb Phyllanthus niruri

Aspirin has been used for a long time as an analgesic and anti-pyretic drug. Limitations of its use, however, remain for the gastro-intestinal side effects and erosions. Although the role of aspirin on gastro-intestinal injury has been extensively studied, the molecular mechanisms underlying aspirin-induced liver and spleen pathophysiology are poorly defined. The present study has been conducted to investigate whether phyllanthus niruri protein (PNP) possesses any protective role against aspirin mediated liver and spleen tissue toxicity, and if so, what signaling pathways it utilizes to convey its protective action. Aspirin administration in mice enhanced serum marker (ALP) levels, reactive oxygen species (ROS) generation, reduced antioxidant power and altered oxidative stress related biochemical parameters in liver and spleen tissues. Moreover, we observed that aspirin intoxication activated both the extrinsic and intrinsic apoptotic pathways, as well as down regulated NF-κB activation and the phosphorylation of p38 and JNK MAPKs. Histological assessments and TUNEL assay also supported that aspirin induced tissue damages are apoptotic in nature. PNP treatment after aspirin exposure effectively neutralizes all these abnormalities via the activation of survival PI3k/Akt pathways. Combining all results suggest that PNP could be a potential protective agent to protect liver and spleen from the detrimental effects of aspirin.


Introduction
Literature provides a consensus that conventional (Non Steroidal Anti Inflammatory Drugs) NSAIDs mediated ROS formation may suppress the risk of gastrointestinal related cancers [1,2]. Moreover, epidemiological and interventional studies have established that increased Reactive Oxygen Species (ROS) generation is one of the key mechanisms for NSAIDs-mediated anticancer effects on various cancer cells [3]. Thus modulation of redox biochemistry represents a fruitful approach to cancer prevention. NSAIDs like aspirin (a name coined by Bayer's chief pharmacologist, Heinrich Dreser) [4] inhibit cell cycle progression and induce apoptosis in cancer cells [5]. In the apoptotic action of aspirin (ASA) both COX-dependent and COX-independent mechanisms are involved [6]. Literature suggests that a regular dose of ASA reduces the risk of many diseases associated with aging [7]. ASA has pivotal role in modulating mast cell degranulation, COX-2 expression and release of pro-inflammatory cytokines [8]. At hepatotoxic dose ASA induces apoptotic death in hepatocellular carcinoma cells [9]. It is well known that ASAinduced gastric injury is caused by oxidative stress [10]. Liver and spleen are the gastrointestinal organs which are very much susceptible to ASA mediated diverse apoptotic damage. A common interest, therefore, lies in search of a safe antidote which could combat ASA mediated COX-2 independent apoptotic complications.
Since earlier times, many species of Phyllanthus family are employed in ayurvedic formulation for the treatment of various diseases like urolithiasis [24], gastric lesion [25], diuretics [26], etc. Different parts, particularly its leaf extracts are used as human consumable component in aqueous medium to maintain liver function properly. Besides, no side effect or toxicity has been reported so far in any of the clinical studies using this herb [27]. Phyllanthin [28] and corilagin [29] are the two bioactive compounds that have been isolated from organic extracts of P. niruri. It has been already reported that the aqueous extract [30], protein isolate [23,31,32] and a purified protein from P. niruri (PNP) possess the protective effects against various drugs and toxins mediated oxidative insults and pathophysiological complications [21,33]. However, the molecular signaling associated with its prophylactic action needs further studies. A couple of very recent reports described the possible pathways for the protective mechanism of PNP [33,34] against oxidative insults. We, therefore, designed our present study to explore the signal transduction pathways that are utilized by PNP to prevent aspirin induced hepatic and spleenic pathophysiology without interfering with its gastro-intestinal cancer preventive applications. Since apoptotic death is the ultimate fate of the cells in aspirin-induced pathophysiology, in vivo studies have been conducted to investi-gate whether PNP could effectively neutralize aspirin-induced abnormalities in the liver and spleen tissue.
The adverse effect of ASA administration and the protective action of PNP has been evaluated by measuring liver specific serum marker enzyme (ALP) leakage; lipid peroxidation, protein carbonylation; levels of cellular metabolites (GSH and GSSG) and activities of antioxidant enzymes (CAT, SOD, GST, GPX, GR etc). The molecular mechanism was determined by investigating the antiapoptotic Bcl-2 and pro-apoptotic Bax protein expressions, release of cytochrome c into the cytosol, caspase 3 as well as caspase 8 protein levels. Role of mitogen-activated protein kinase (MAPKs) and NF-kB under this pathophysiological situation were also investigated in this study. The mode of cell death in ASA induced spleen and hepatotoxicity and the protective role of PNP has been investigated by histology, TUNEL assay and FACS analysis. The consequences of the present study are anticipated to draw a clear picture of the protective mechanism of PNP against ASA induced hepatic and spleen injury as well as it may also shed light on an achievable solution to the devastating complications of aspirin administration.

Animals
Healthy Swiss strain male albino mice weighing approximately 24-25 g were purchased from CNCRI, Kolkata, India. The animals were accustomed under laboratory conditions for a fortnight prior to experiments. They were maintained on a standard diet and water ad libitum; exposed to 10-12 hours of daylight under standard conditions of temperature (25uC) and humidity (30%). All the studies with the experimental animals were performed following the standard ethical protocols of IAEC, Bose Institute, Kolkata. Full details of the study were approved by both IAEC and CPCSEA (Committee for the purpose of control and supervision on experiments on animals), Ministry of Environment and Forests, New Delhi, India (the permit number is: 95/99/ CPCSEA). Effect of Aspirin and PNP on Glutathione (GSH & GSSG) activities in liver tissue. GSH to GSSG ratio. Panel D. Effect of Aspirin and PNP on Glutathione (GSH & GSSG) activities in spleen tissue. GSH to GSSG ratio. Cont: normal mice, PNP: mice treated with only PNP, ASA: mice intoxicated with aspirin, ASA+PNP: mice treated with PNP after aspirin intoxication. ASA+VitC: mice treated with vitamin C after aspirin intoxication. ''a'' indicates the significant difference between the normal control and ASA intoxicated groups, and ''b'' indicates the significant difference between ASA intoxicated (toxin) and PNP post-treated groups. Each column represents mean 6 SD, n = 6; (P a ,0.05, P b ,0.05). doi:10.1371/journal.pone.0089026.g002 Plant Phyllanthus niruri is a shrub belonging to the family Euphorbiaceae. Fresh young leaves were collected from Bose Institute experimental farm.

Isolation and Purification of Protein from Phyllanthus niriri
The protein from Phyllanthus niruri (PNP) was isolated and purified following the method of Sarkar et al [21]. Briefly, all the fresh young leaves of the plant were homogenized in 50 mM phosphate buffer, pH 7.4. After centrifugation at 15,000 g, the soup was brought to 60% ammonium sulphate saturation. The pellet after centrifugation was reconstituted and dialysed against 50 mM phosphate buffer. It was applied to a DEAE cellulose column and the column was eluted in the same buffer using a linear gradient of 0-1 M NaCl. Two major peaks were obtained. The protein fractions from the first peak showed maximum biological activity. The materials of those fractions were collected, concentrated, dialyzed, in 50 mM phosphate buffer and subjected to gel filtration chromatography and re-chromatography using a gel filtration column (BIOSEP-SEC-S200, 60067.8 mm) attached to HPLC. Biological activity of each fraction was checked and the material of the active peak was subjected to rechromatography under identical conditions and the protein of the active fractions was used for experiments.

Test of Homogeneity of Purified PNP
The homogeneity and the molecular weight of the protein was confirmed by SDS-PAGE with known molecular weight marker proteins (25-225 kDa) by following the method of Sarkar et al. [21].

Effect of Heat Treatment and Protease on the Biological Activity of PNP
To check the biological activity of the purified PNP, the protein specific evidence-based experiments like effect of heat treatment and the effect of trypsin digestion have been carried out on PNP by following the techniques as described elsewhere [34].

Determination of Dose for ASA Induced Hepatic Dysfunctions in vivo
To establish the dose of ASA necessary for hepatic damage, mice were randomly allocated into six groups each consisting of six animals and they were treated as follows: First group served as normal control (received only water as vehicle). Remaining five groups were treated with five different doses of ASA orally (25 mg, 50 mg, 100 mg, 150 mg and 200 mg/kg body weight in distilled water for 6 weeks).
Twenty-four hours after the final dose of ASA intoxication, all mice were sacrificed and ALP levels were measured using serum of all experimental mice.

Determination of Dose and Time Dependent Activity of PNP by ALP Assay
For the dose-dependent study, mice were randomly distributed into six groups each consisting of six animals. First two groups were served as normal control (received only water as vehicle) and toxin control (received ASA 100 mg/kg body weight for 6 weeks, orally) respectively. Remaining four groups of animals were administrated with ASA (received 100 mg/kg body weight for 6 weeks, orally) followed by four different doses of PNP (2 mg, 5 mg, 10 mg and 15 mg/kg body weight for 2 weeks, intraperitoneally injected in distilled water). Previously it was found that at this level of dose the P. niruri protein fraction protected liver against oxidative stress [35,36].
To determine the time dependent effects of PNP treatment in ASA-dependent hepatic disorder, mice were divided into seven groups each consisting of six animals. First two groups were served as normal control (received only water as vehicle) and toxin control (received 100 mg/kg body weight for 6 weeks, orally) respectively. Other five groups of animals were treated with PNP intraperitoneally at a dose of 10 mg/kg body weight, once daily for 1, 1.5, 2, 2.5 and 3 weeks after ASA intoxication (received ASA at a dose 100 mg/kg body weight for 6 weeks, orally).
At selected times after ASA and PNP treatment, all mice were sacrificed. ALP levels were measured using serum of all experimental mice.

In vivo Experimental Set-up
The animals were divided into five groups, consisted of six mice in each and they were treated as follows.
Group 1: ''Normal control'': animals received only water as vehicle. Group 2: ''Toxin control (ASA)'': animals received ASA at a dose of 100 mg/kg body weight once daily for 6 weeks, orally. Group 3: ''PNP post-treated group (ASA+PNP)'': animals were intraperitoneally injected with PNP (10 mg/kg body weight in distilled water, once daily) for 2 weeks after ASA intoxication for 6 weeks. Group 4: ''Vitamin C post-treated group (ASA+VitC)'': animals were intraperitoneally injected with Vitamin C for 2 weeks after ASA intoxication for 6 weeks. Group 5: ''PNP alone treated group (PNP)'': no treatment for first 6 weeks, later animals were treated with PNP (intraperitoneally injected, 10 mg/kg body weight in distilled water, once daily) for 2 weeks.
The animals were sacrificed under light ether anesthesia and after that livers and spleens were collected.

Determination of Liver Weight to Body Weight Ratio
After scarification, the livers and spleens from experimental animals were quickly excised and weighed. Then the ratio of liver weight to body weight was measured for each.

Assessment of Serum Specific Markers Related to Hepatic Dysfunction
For assessment of serum specific marker (ALP levels) related to hepatic dysfunction, blood samples were collected by puncturing mice hearts of all experimental animals, kept overnight for clotting and then centrifuged at 3,000 g for 10 minutes. ALP level in the serum of experimented animals was measured by using standard kits according to the method of Kind and King [37] respectively.

Preparation of Liver Homogenate
Liver samples were homogenized using glass homogenizer in 100 mM potassium phosphate buffer containing 1 mM EDTA, pH 7.4 supplemented with protease and phosphatase inhibitors and centrifuged at 12,000 g for 30 minutes at 4uC. The supernatant was collected and used for the experiments.

Spleen Tissue Collection and Preparation of Homogenates
Spleen samples were also homogenized using glass homogenizer in 100 mM potassium phosphate buffer pH 7.4, containing 1 mM EDTA, 1 mM PMSF (proteinase inhibitor) and phosphatase inhibitor cocktail. The homogenized mixture was centrifuged at 120006g for 30 minutes at 4uC. The supernatant was collected and used for the experiments.

Determination of Protein Content
The protein content of experimental sets was measured following the method of Bradford (1976) using crystalline Bovine Serum Albumin (BSA) as standard.

Assay of Antioxidant Enzymes
The activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione  . Effect of PNP on aspirin induced ROS production. Panel A. Impact on ROS production in liver tissue. The intracellular ROS production was detected by FACS using DCF-DA. Normal: normal mice, PNP: mice treated with only PNP , ASA: mice intoxicated with aspirin, ASA+ PNP: mice treated with PNP after aspirin intoxication. Panel B. Impact on ROS production in spleen tissue. The intracellular ROS production was measured by spectroflurimeter. Normal: normal mice, PNP: mice treated with only PNP , ASA: mice administrated with aspirin, ASA+PNP: mice treated with PNP after aspirin intoxication. Panel C. Impact on ROS production in hepatocytes. The intracellular ROS production was detected by FACS using DCF-DA. Normal: normal mice, PNP: mice treated with only PNP , ASA: mice intoxicated with aspirin, ASA+PNP: mice treated with PNP after aspirin intoxication. ''a'' indicates the significant difference between the normal control and ASA intoxicated groups, and ''b'' indicates the significant difference between ASA intoxicated (toxin) and PNP treated groups. Each column represents mean 6 SD, n = 6; (P a ,0.05, P b ,0.05). doi:10.1371/journal.pone.0089026.g003 peroxidase (GP x ) and glutathione-S-transferase (GST) have been measured in liver and spleen homogenates of all experimental animals. Briefly, CAT activity was measured [20] by monitoring the decomposition of H 2 O 2 at 240 nm for 10 min spectrophotometrically. One unit of catalase activity is defined as the amount of enzyme, which reduces 1 mmol of H 2 O 2 per min.
SOD activity was measured [20] from the reaction mixture containing protein sample, phenazine methosulfate, NBT and NADH. The reaction mixture was incubated at 37uC and reaction was stopped by addition of glacial acetic acid. The color intensity was monitored at 560 nm. One unit of enzyme activity is defined as the amount of enzyme required for the inhibition of chromogen production by 50% in 1 min under assay condition.
GST activity was assayed [20] based on the conjugation reaction with glutathione in the first step of mercapturic acid synthesis. The reaction is carried out in presence of CDNB and GSH, at 37uC and monitored spectrophotometrically at 340 nm for 5 min. One unit of GST activity is 1 mmol product formation per min.
GR activity was determined [20] spectrophotometrically by monitoring the absorbance at 412 nm for 3 min at 24uC in presence of DTNB, NADPH and GSSG. The enzyme activity was calculated using molar extinction coefficient of 13,600 M 21 cm 21 .
One unit of enzyme activity is defined as the amount of enzyme, which catalyzes the oxidation of 1 mmol NADPH per min.
GPx activity was measured [20] by using H 2 O 2 and NADPH as substrates. The conversion of NADPH to NADP + was measured at 340 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 mmol NADPH per min.

Estimation of Lipid Peroxidation End Products and Protein Carbonyl Content
The lipid peroxidation in terms of malondialdehyde (MDA) formation and protein carbonylation were determined in liver and spleen homogenates of all experimental animals according to the method as described by Rashid et al. [38].

Assay of Cellular Metabolites
Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were estimated in liver and spleen homogenates of all experimental animals following the method of Ghosh et al. [39] using Ellman's reagent [40]. Briefly, tissue homogenates were harvested with metaphosphoric acid (5%) buffer and the reaction mixture containing EDTA, glutathione reductase, NADPH and DTNB. 5-thio-2-nitrobenzoic acid (TNB) formation was monitored at 412 nm. The oxidized glutathione (GSSG) level can also be estimated using 1-methyl-2-vinylpyridinium trifluoromethane- Figure 5. Western blot analysis of different mitogen activated protein kinases (MAPKs) in liver tissue homogenates. phosphorylated p38 (phospho-p38) and total p38 MAPK, phosphorylated JNK MAPK (phospho-JNK MAPK) and total JNK MAPK. b actin was used as an internal control. Data represent the average 6 SD, n = 6; (P a ,0.05, P b ,0.05). Cont: normal mice, PNP: mice treated with only PNP, ASA: mice intoxicated with aspirin, ASA+PNP: mice treated with PNP after aspirin intoxication. ASA+VitC: mice treated with vitamin C after aspirin intoxication. ''a'' indicates the significant difference between the normal control and ASA intoxicated groups, and ''b'' indicates the significant difference between ASA intoxicated (toxin) and PNP post-treated groups. Each column represents mean 6 SD, n = 6; (P a ,0.05, P b ,0.05). doi:10.1371/journal.pone.0089026.g005 sulfonate (M2VP) in order to eliminate GSH. The levels of GSH were measured from the difference between concentrations of total glutathione (GSH+GSSG) and GSSG. The intracellular levels of GSH and GSSG were calculated on the basis of cellular protein concentration.

Assay of Antioxidant Power of Hepatocytes: Ferric Reducing/Antioxidant Power (FRAP) Assay
Ferric Reducing/Antioxidant Power (FRAP) assay was performed in order to determine the antioxidant power of liver as well as spleen tissues following the method of Benzie and Strain [41].

Measurement of ASA Induced ROS Production in Hepatocytes
At first, the hepatocytes were isolated from the liver tissue of the experimental animals by following the method of Sarkar and Sil [18]. After that the intracellular ROS production was estimated by using 2,7-dichlorofluorescein diacetate (DCFDA) as a probe following the method of Ghosh et al. [39]. Briefly, hepatocytes were incubated with DCF-DA, washed and resuspended in phosphate buffered saline. The cells were analyzed by flow cytometry in order to investigate the ASA induced ROS production at cellular level.

Measurement of ASA Induced ROS Production using Tissue Homogenates
The ROS production from spleen tissue and liver tissue homogenates were estimated separately by following the method of Rashid et al. [38], using 2,7-dichlorofluorescein diacetate (DCFDA) as a probe as described elsewhere [42]. Briefly, tissue homogenates were incubated in the assay medium (20 mM Tris-HCl, 130 mM KCl, 5 mM MgCl 2 , 20 mM NaH 2 PO 4 , 30 mM glucose and 5 mM DCFDA) at 37uC for 15 minutes. The formation of DCF was measured at the excitation wavelength of 488 nm and emission wavelength of 610 nm for 10 minutes by using fluorescence spectrometer equipped with a FITC filter. Figure 6. Western blot analysis of Bcl-2 family proteins and survival pathway related proteins. Panel A. Western blot analysis of Bax, Bcl-2, Phospho-Akt, PI3k proteins in liver tissue homogenates. b actin was used as an internal control. Cont: normal mice, PNP: mice treated with only PNP, ASA: mice intoxicated with aspirin, ASA+PNP: mice treated with PNP after aspirin intoxication. ASA+VitC: mice treated with vitamin C after aspirin intoxication. ''a'' indicates the significant difference between the normal control and ASA intoxicated groups, and ''b'' indicates the significant difference between ASA intoxicated (toxin) and PNP post-treated groups. Each column represents mean 6 SD, n = 6; (P a ,0.05, P b ,0.05). Panel B. Western blot analysis of Bax, Bcl-2, Phospho-Akt, PI3k proteins in spleen tissue homogenates. b actin was used as an internal control. Cont: normal mice, PNP: mice treated with only PNP, ASA: mice intoxicated with aspirin, ASA+PNP: mice treated with PNP after aspirin intoxication. ''a'' indicates the significant difference between the normal control and ASA intoxicated groups, and ''b'' indicates the significant difference between ASA intoxicated (toxin) and PNP post-treated groups. Each column represents mean 6 SD, n = 6; (P a ,0.05, P b ,0.05). doi:10.1371/journal.pone.0089026.g006 Immunoblotting Proteins (50 mg) from each sample were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked using BSA and incubated separately with primary antibodies such as anti-caspase3, anti-caspase8, anti PARP and anti NF-kB (1:1000 dilution), anti Akt (1:1000 dilution), anti cytochrome c (1:1000 dilution), anti Bad (1:1000 dilution), anti Bax (1:1000 dilution), anti Bcl-2 (1:1000 dilution), anti p-38 (1:1000 dilution) and anti JNK (1:1000 dilution) at 4uC for overnight. The membranes were washed in TBST (50 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20) for 30 min and incubated with appropriate HRP conjugated secondary antibody (1:2000 dilution ) for 2 h at room temperature and developed by the HRP substrate 3,39-diaminobenzidine tetrahydrochloride (DAB) system (Bangalore, India).

Determination of Mitochondrial Membrane Potential
Fresh mitochondria were isolated from the liver tissue [42] and spleen tissue [38]. These mitochondria exhibit high respiratory control ratios using malate glutamate as substrate. The evaluation of the mitochondrial membrane potential was done on the basis of cell preservation of the fluorescent probe JC-1. The membrane Figure 7. Impact of aspirin and PNP on the mitochondrial membrane potential and apoptotic pathways. Panel A. Measurement of the mitochondrial membrane potential by flow cytometry analysis (using JC-1) from liver tissue homogenates, Panel B. Measurement of the mitochondrial membrane potential by flow cytometry analysis (using JC-1) from spleen tissue homogenates, Panel C. Release of cytochrome c, Total Caspase-3 and cleaved Caspase-3, Total PARP and cleaved PARP, Caspase-8, Bid. b actin was used as an internal control. Data represent the average 6 SD, n = 6; (P a ,0.05, P b ,0.05). Cont: normal hepatocytes, PNP: mice treated with only PNP, ASA: mice administrated with aspirin, ASA+PNP: mice treated with PNP after aspirin intoxication. ''a'' indicates the significant difference between the normal control and ASA intoxicated groups, and ''b'' indicates the significant difference between ASA intoxicated (toxin) and PNP treated groups. Each column represents mean 6 SD, n = 6; (P a ,0.05, P b ,0.05). doi:10.1371/journal.pone.0089026.g007 potential was measured using a FACS scan flow cytometer with an argon laser excitation at 488 nm and 525 nm band pass filter.

Histological Studies
Livers and spleens from the normal and experimental mice were fixed in 10% buffered formalin and were processed for paraffin sectioning. Sections of about 5 mm thickness were stained with haematoxylin and eosin to evaluate the histology under light microscope.

TUNEL Assay
The experimental paraffin embedded spleen tissue sections (5 mm) were warmed 30 minutes (64uC), deparaffinised and rehydrated. Terminal transferase mediated dUTP nick endlabelling of nuclei was performed by using APO-BrdU TUNEL Assay kit (A-23210; Molecular Probes, Eugene, OR) following the manufacturer's protocol.

Detection of Apoptosis by Flowcytometry
Normal and experimental hepatocytes were incubated with Annexin V and propidium iodide for 30 min at 37uC. Excess PI and Annexin V were washed off. After that, the cells were analyzed by flow cytometry using FACS Calibur (Becton Dickinson, Mountain View, CA) equipped with 488 nm argon laser light source; 515 nm band pass filter for FITC-fluorescence and 623 nm band pass filter for PI-fluorescence using Cell Quest software.

Statistical Analysis
All experimental values have been represented as mean 6 S.D. (n = 6). Data on biochemical investigation were analyzed using analysis of variance (ANOVA) and the group means were compared by Duncan's Multiple Range Test (DMRT). P values of 0.05 or less were considered significant.

Results and Discussion
At the beginning of our present study we have purified PNP and confirmed its biological activities via heat treatment and enzymatic digestion analysis. Later we evaluated its antioxidant activities against drug induced pathophysiology [15,[26][27]. Here, we should mention that very little information is available in the literature about the role of protein molecules against oxidative stress mediated organ pathophysiology. In this regard, a few articles published very recently, [43][44][45][46][47][48] revealed that some protein molecules from various plant sources possess antioxidant activities like our PNP, but all these reports lack the mechanism of protective actions of the active principles making the comparison a difficult task with the molecule of our interest. Moreover, the partial amino acid sequence of this novel protein (PNP) revealed only four peptide fragments of nominal mass 2128 Da, 2392 Da, 2533 Da and 2719 Da. The MS-MS analysis showed that none of these fragments possess similarity with any peptide sequences in the NCBI non-redundant databases indicating a unique nature of this molecule [21].
Aspirin (ASA) is safe and broadly utilized NSAID when used at the therapeutic level but higher doses or prolonged use of this drug may promote oxidative stress and result in gastrointestinal erosions and apoptotic lesions [6]. Thus ASA is responsible for the central action of anticancer effect [49]. The toxic effect of ASA is highly dependent on its dose. The present study demonstrated that ASA at higher dose induces hepatic and spleen toxicity by activating a cascade of apoptotic signaling pathways. However, PNP treatment could effectively neutralize those ASA induced apoptotic pathways.

Dose Dependent Activity of ASA
It is reported that aspirin causes hepatotoxicity and elevates the levels of serum marker enzymes (ALT, ALP, SGPT etc) [50][51][52][53][54]. We performed a dose-dependent study using ALP assay as an index of ASA mediated hepatic damage to determine the optimum dose of ASA. Transport function and membrane permeability are altered due to the damaged hepatocytes in the liver tissue, leading to the leakage of enzymes from these cells [55]. We used aspirin from lower dose to the higher dose (25 mg-200 mg/kg body weight) ranges in order to investigate the proper effect of aspirin on the liver as well as spleen tissue. These doses have pharmacological evidences. Previous reports suggested that the number of gastrointestinal (GI) complications induced by low-dose aspirin exposure may be greater than the number of cardiovascular (CV) events prevented [56]. Moreover, the effect of aspirin administration up to 300 mg dose is similar to aspirin administration at 75-100 mg/ day dose for the prevention of major vascular events and these doses increase the risk of bleeding [57,58]. The benefits of regular aspirin administration at these lower doses for a longer period may prevent CV disorder but increases the risks of gastrointestinal (GI) and intracerebral (IC) hemorrhage [59,60]. It is also to be mentioned that prolong treatment with aspirin at a lower dose damages the gastrointestinal mucosal barrier [61] and thereby releases serum marker enzymes from the organs. Figure 1A shows that in ASA exposed animals, maximum ALP level in plasma was reached at a dose of 100 mg/kg body weight for 6 weeks. Effect of ASA was not much beyond this concentration. The remarkable increased levels of serum ALP at this dose causes severe damage in hepatic tissue membranes. Therefore, this dose of ASA was selected as the optimum dose for the subsequent experiments.

Dose and Time Dependent Activity of PNP
Survival of cells under oxidative stress is an important parameter to evaluate the effectiveness of any prophylactic agent. The result of our study suggests that ASA at a dose of 100 mg/kg body weight up regulated the ALP level in plasma but that could be reversed with the treatment of PNP up to a dose of 10 mg/kg body weight daily up to 2 weeks (figure 1B). Time dependent study indicated those 2 weeks of treatment with PNP provided maximum beneficial effect against ASA intoxication ( figure 1C). Effect of PNP treatment was not much beyond this concentration. Therefore, after ASA administration 2 weeks was chosen as optimum period of time for the post treatment with PNP in this study.
After the fixation of the dose and treatment time, we designed our animal experimental protocol (figure S1) and performed all the subsequent experimental studies.

Effect on Liver Weight and Body Weight
The result of present study showed that ASA administration for 6 weeks reduced the liver weight to body weight ratio (figure 2A). Treatment with PNP after ASA administration was effective and inhibited this liver weight deficiency. The result suggests that PNP treatment could be beneficial against ASA mediated growth retarding effect.

Ameliorative Role of PNP against ASA Stimulated ROS Induced Oxidative Stress in Hepatic and Spleen Tissue
In mammalian tissue the complex endogenous antioxidant system and chemical sequesters help to prevent oxidative damage. In our study we explored the effect of the drug (ASA) on the activities of antioxidant enzymes in both the liver and spleen tissues. The results showed that ASA administration at the hepatotoxic dose reduced the activities of antioxidant enzymes (SOD, CAT, GST, GR, GPx) in both hepatic (Table 1) and spleen tissue ( Table 2) whereas, PNP treatment ameliorated the loss of antioxidant enzyme activities. We further compared the protective role of PNP with vitamin C.
As expected, ASA at hepatotoxic dose caused a significant reduction in FRAP value (figure 2B) but post treatment with PNP increased the antioxidant power (,90%) as compared to respective toxin control (,57%). This kind of alteration is encountered whenever any imbalance occurs between the productions of reactive oxygen species (ROS) and ability of detoxification of the reactive intermediates through the biological system. Reactive oxygen species (ROS) causes oxidative stress and that can attack lipid membranes, proteins and ultimately disrupt cellular integrity. Lipid peroxidation indicates cellular damage mediated by reactive oxygen intermediates with resultant destruction of membrane lipids and production of lipid peroxides. Peroxyl radicals can be rearranged via a cyclization reaction to endoperoxides, and produce malondialdehyde (MDA), the final product. In the present study we observed that ASA administration significantly enhanced lipid peroxidation in both the liver (Table 3) and spleen tissue (Table 4). PNP administration after ASA intoxication, however, almost normalized the pathophysiological condition.
The protein carbonyl group is generated by ROS through many different mechanisms and its concentration is a good measure of protein oxidation via oxidative stress. The side chains of all amino acid residues of proteins are susceptible to oxidation by the action of ROS. Literature suggests that enhanced protein carbonylation may be responsible for the decrease in antioxidant enzyme activity [62]. In the present study ASA administration stimulated protein oxidation but PNP treatment altered this pathophysiological condition in liver (Table 3) as well as spleen tissue (Table 4).
Reduced glutathione is present at high concentrations in all mammalian cells, especially in the renal cells, hepatocytes, and erythrocytes. GSH is the main non-protein thiol intracellular antioxidant that scavenges free radicals. A considerable amount of GSH is consumed to scavenge ROS [55]. Whenever the GSH level is reduced below the threshold level, the concentration of reactive radicals get elevated and cause oxidative stress. GSH itself is oxidized to GSSG in this process [34]. Therefore, the levels of both of these non-protein thiols were altered in the system during oxidative stress. Similar results were exhibited in our study in which ASA administration drastically down regulated the GSH level and thereby up regulated the GSSG level. Thus drug administration interrupts the redox status inside the system. PNP treatment, on the other hand, moderated the alteration of GSH/ GSSG level and maintained the normal redox status in both the liver (figure 2C) and spleen (figure 2D) tissue.
Early report suggested that ROS generation is one of the key mechanisms in NSAIDs mediated anti carcinogenic effect [49]. ROS formation depleted the intracellular glutathione level and activated NADPH oxidase enzyme activity. NADPH oxidase is the key enzyme for the activation of lipid peroxidation [63]. Moreover, this type of drug administration produces ROS and thereby decreases cellular ATP level [2]. In our study, we observed that ASA administration caused a right-shift of the fluorescence intensity of DCF signal of the respective H 2 O 2 content thereby enhanced ROS formation in the liver. Treatment with PNP reversed the phenomenon of the ASA administrated fluorescence intensity of DCF signal in the liver ( Figure 3A), spleen (figure 3B) as well as prevented the intracellular ROS elevation in the hepatocytes (figure 3C). The consequence was expected because PNP itself has free radical scavenging property as per evidenced from our previous reports [32,34]. Therefore, our result justifies that PNP possesses ROS scavenging activity.

ASA Mediated NF-kB Down Regulation: Beneficial Role of PNP
NF-kB is a ubiquitous transcription factor that regulates the transcription of many genes involved in immune and inflammatory responses as well as cell survival pathways [64]. NF-kB activation is also considered to be one of the major crucial factors for chemo resistant effect in many cancer cells [65]. Our result is also consistent with the earlier report as ASA inhibited NF-kB activation [66], however, PNP could ameliorated this incident (figure 4) and might help in transcription of the cell survival genes. ASA induced down regulation of NF-kB activity is mediated by preventing the phosphorylation and degradation of the inhibitory subunit IkB [67]. In this respect, it is important to shed light on the effects of PNP on the NF-kB activity. The result showed that prolong utilization with ASA, the NF-kB activity has been inhibited. ASA administration up regulated IkBa levels and there by blocked the release of NF-kB. Our study exhibited that PNP treatment might degraded IkBa. As a result nuclear translocation of p65 subunit of NF-kB occurred. However, PNP may stimulated the phosphorylation of IkBa at serine 32 and 36 positions before degradation by 26s proteosomal complex. Moreover, our result indicated that PNP somehow enhanced the phosphorylation of p65 subunit of the transcription factor at ser 536 position. This phosphorylation is required for many NF-kB target genes to combat ASA induced oxidative impairment in the liver and spleen. Although many critical queries still remained to be answered in order to understand the signaling mechanism of ASA induced NF-kB inactivation.
ASA Stimulated Pro-apoptotic Signaling via the Activation of MAPKs: Protective Role of PNP Any stimulus that causes oxidative stress might activate MAPKs family proteins. Literature suggests that NSAID drugs activated JNK and p38 MAPKs signaling pathways [49]. These proteins phosphorylate Bax and promote its translocation to mitochondria [68]. So, we performed western blotting analyses to elucidate whether p38 and JNK MAPK have any role in ASA-induced hepatic disorder as well as whether PNP treatment could ameliorate this phenomenon. We observed that ASA up regulated JNKs (,2 folds) and p38 (,1.8 folds) phosphorylation in liver tissue (figure 5) whereas, PNP treatment modulated this detrimental phenomenon. It is to be mentioned that JNK follows different signaling pathways to accomplish apoptotic cell death [69]. JNK also plays key role in both intrinsic and extrinsic apoptotic pathways [69]. ASA activated JNK might promote apoptotic signals by directly inhibiting Bcl-2 protein or translocated to mitochondria and thereby releasing cytochrome c [69] to accomplish the apoptotic task. On other side, ASA inhibited NF-kB activation is one of the key mechanisms for the inactivation of several important anti-apoptotic cell survival signals e.g. Bcl-X L , Flip as well as Bcl-2 [70]. Furthermore, ASA exposure might up regulate Bax translocation [66]. Our observation supported the previous report because in this study ASA administration activated Bax translocation and block Bcl-2 protein in both the liver (figure 6A) and spleen ( figure 6B). Therefore, Bax/Bcl-2 ratio status was disrupted by ASA administration and leading to the alteration of mitochondrial membrane potential. However, treatment with PNP maintained the balance of Bax/Bcl-2 and ameliorated the ASA mediated loss of mitochondrial membrane potential in the liver (figure 7A) as well as spleen (figure 7B).

Activation of both Mitochondria Dependent and Independent Apoptotic Signaling Pathways: Modulation by PNP
Apoptosis is one type of complex interplay of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) mitochondrial membrane proteins as well as the activation of caspase cascades [71]. Apoptosis occurs through the up regulation of pro-apoptotic proteins and down regulation of anti-apoptotic proteins. Gradual loss of the mitochondrial membrane potential creates mitochondrial permeability transition pores or MPTP. It is the key step in the mitochondria dependent apoptotic cell death pathway [34]. ASA administration induces apoptotic signals through both mitochondria dependent and independent pathways. Mitochondria dependent apoptotic pathways occur via the release of cytochrome c from mitochondria and the independent pathways occur via the activation of caspase 8 [72,73]. The extrinsic apoptotic signaling pathways (mitochondria independent pathways) involve cleavage of Bid, so that Bid could translocate to mitochondria and there by releases cytochrome c [6]. Our observation was similar to the earlier report as ASA administration up regulated cytosolic cytochrome c, activated caspase 3, PARP cleavage and also sensitized the cells to TRAIL mediated mitochondria independent apoptotic pathways such as caspase 8 activation and translocation of Bid (figure 8C). TRAIL binds to cell surface receptors and activates the extrinsic apoptotic pathways [66]. All of those apoptotic complications were ameliorated by PNP treatment as evidenced from flow cytometry analysis (figure 8) and TUNEL assay (figure 9). Besides, the histological studies of liver (figure 10A) and spleen tissue (figure 10B) could be evidenced for the alleviation effect of PNP treatment against ASA mediated apoptotic death.

Protective Mechanism of PNP: Activation of Survival Signaling Pathway
Another essential serine/threonine protein kinase that plays a vital role in cell survival pathways by inhibiting apoptosis is Akt. Activation of Akt requires the activation of PI3K and might promote cellular survivality [74]. Activation of PI3K occurs via the activation of the receptor Tyrosine Kinase. PNP, somehow stimulates the Tyrosine Kinase receptor and results in the oligomerization of the receptor. As a consequence self phosphorylation of the Tyrosine residues occurs. This might create a docking site for the binding of the p85 subunit of PI3K. After that, SH2-containing adaptor proteins bound to p85 subunit. Once activated, PI3K phosphorylates phosphoinositides and its major lipid product is phosphatidylinositol 3,4,5-triphosphate. Phosphatidylinositol 3,4,5-triphosphate facilitates the recruitment of Akt to the plasma membrane through binding with the pleckstrin homology domain of Akt [75]. Akt is activated by phosphoinositide-dependent kinase-1 (PDK1). AKT was fully activated when both residues, Thr308 and Ser473, were phosphorylated. A number of pro-apoptotic proteins have been identified as direct Akt substrates, including BAD, caspase-9, etc [66]. The proapoptotic activities of these proteins were suppressed upon phosphorylation through Akt [66]. This is consistent with our findings that phospho-Akt level was markedly reduced in ASA administrated liver (figure 6A) and spleen tissue ( figure 6B). Moreover, PNP supplementation up regulated PI3k and phospho-Akt level. Through the above mentioned probable mechanism PNP might increase the anti apoptotic potency of the cells against ASA mediated organ pathophysiology.

Conclusion
Overall, the present study demonstrated that like other NSAIDs, ASA administration at hepatotoxic dose induces ROS formation. Once formed, ROS altered the normal GSH/GSSG balance and at the same time caused lipid peroxidation of the cellular membrane. Most importantly, the cellular endogenous antioxidant defense mechanisms are also disrupted and thereby shifting the physiological redox status. Besides, ROS mediated activation of JNKs and p38 MAPKs altered the balance between the pro-apoptotic and anti-apoptotic proteins. Under these circumstances, the overall system might follow both intrinsic and extrinsic apoptotic signaling pathways. However, PNP treatment after ASA intoxication might be able to protect the liver and spleen via 1) scavenging free radicals thereby inhibiting ROS formation, 2) enhancing the antioxidant enzyme activities and maintaining the proper GSH/GSSG ratio, 3) ameliorating ASA mediated inhibition of NF-kB and thereby activation of antiapoptotic Bcl-2 proteins as well as inhibition of MAPKs activation and 4) by activating Akt/PI3k mediated cell survival signaling pathways. A possible mechanism of PNP induced survival signaling pathways against ASA induced hepatic and spleen toxicity has been depicted in figure 11. Finally, we would like to mention that without any adverse effects (as suggest by experimental results) PNP could be a safe antidote against aspirin induced detrimental complications. Figure S1 Schematic representation of group division for mice treatment in the study. (TIF)