Therapeutic use of Fisetin and Fisetin Loaded on Mesoporous Carbon Nanoparticle (MCN) in Thioglycollate-induced Peritonitis

Background: The pathophysiology of aseptic peritonitis involves inflammation of the serosal membrane that lines the abdominal cavity and the organs contained therein. The etiology of peritonitis is complicated and is involved in various processes, of which, the most important one is the inflammatory reaction. During the pathological process of peritonitis, NF-κB plays an activating role in the inflammatory reaction, which might be a potential therapeutic target in the therapy of certain inflammatory diseases. We studied the anti-inflammatory and pro-regenerative actions of Fisetin, a flavonol found in many plants, in a mouse model of thioglycollate-induced peritonitis, as well as the actions of fisetin administered with a nanoparticle such as mesoporous carbon nanoparticle (MCN). BALB/c mice were used in this study. Results: We found cell recruitment in the blood increased with the administration of thioglycollate (TG) after 24 h, 48 h, 72 h and 96 h, showing that it has induced inflammation. Cell recruitment was successfully inhibited by fisetin, and with MCN+fisetin. In the peritoneal fluid, total cell recruitment was increased, which was successfully inhibited with fisetin and MCN+fisetin treatment. TG treatment significantly reduced cell proliferation in the blood, PF and BM, within 24 h, till 96 h. Interestingly, cell proliferation increased with fisetin treatment, and with MCN+fisetin. The clonogenic potential of the tissues decreased significantly within 24 h, with administration of TG. Both fisetin treatment and MCN+fisetin treatment restored the clonogenic potential of the tissues. There was a decrease in Th2 cytokines with TG treatment, in blood after 48 h, and both fisetin and MCN+fisetin increased the cytokine content. Conclusion: In conclusion, we found that fisetin had a promising therapeutic effect on the peritonitis. *Corresponding author: Ena Ray Banerjee, Associate Professor, Department of Zoology, Immunology and Regenerative Medicine Research Laboratory, University of Calcutta, 35, Ballygunge Circular Road, Kolkata-700019, West Bengal, India, Tel: 9163739975; Fax: 91-33-24614849; E-mail: enarb1@gmail.com Received October 13, 2015; Accepted November 25, 2015; Published December 05, 2015 Citation: Mitra S, Biswas S, Sinha A, Jana NR, Banerjee ER (2015) Therapeutic use of Fisetin and Fisetin Loaded on Mesoporous Carbon Nanoparticle (MCN) in Thioglycollate-induced Peritonitis. J Nanomed Nanotechnol 6: 332. doi:10.4172/2157-7439.1000332 Copyright: © 2015 Mitra S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

effective on the treatment, the morbidity and mortality remain kept at a high level [9,10].
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants [11,12]. It is a protective mechanism by the organism to eliminate injurious stimuli and to initiate the healing process. Inflammation is a mechanism of innate immunity [13,14]. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes from the blood into the injured tissues. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. The process of acute inflammation is initiated by cells already present in all tissues, mainly resident macrophages, dendritic cells, histiocytes, Kupffer cells and mast cells. At the onset of an infection, these cells are activated and release inflammatory mediators, which are responsible for the clinical signs of inflammation [15]. Inflammation leads to increased production of reactive species like ROS (reactive oxygen species), NOS (nitric oxide synthase) and their product peroxynitrite (ONO 2 -) by activated macrophages [16]. This increase in oxidative stress leads to decrease in effectiveness of oxidant defenses, that is, reduction in antioxidants.
Due to the various side effects and other complications of modern medicine, the use of traditional medicines and natural products is gaining popularity. Phytochemicals from fruits are being exploited as possible sources of therapeutic agents. Different biological activities of these chemicals, including their anti-oxidant properties and their antiinflammatory properties have been tested in vitro, as well as in vivo [17][18][19]. Nanomaterials, either as nanodrugs or as nano-vehicles, have the advantages of being small devices that are less invasive than normal medicines, that can be targeted to reach a particular site and that can possibly be implanted inside the body; also biochemical reaction times are much shorter. These devices are faster and more sensitive than typical drug delivery [20][21][22].
Mesoporous Carbon Nanoparticle (MCN) is a type of porous nanomaterial with a size of 100-200 nm. It has several important properties such as high surface area, large pore volume, and a uniform pore size of 3 nm. It is chemically inert, biocompatible and dispersible in water. Pores of MCN can be loaded with a large amount of drug molecules and then the drugs can be released by slow diffusion or other methods. These properties of the MCN make them useful for biomedical application [23][24][25][26].
The aim of our study was to test the anti-inflammatory and pro-regenerative actions of Fisetin, a flavonol found in many plants, including strawberries and apples, in a mouse model of thioglycollateinduced peritonitis. It protects against oxidative stress-induced cell death, by up-regulating expression of heme oxygenase 1 (HO -1 ). We also aimed to test whether the anti-inflammatory and pro-regenerative actions of fisetin were enhanced when it was administered with a nanovehicle such as mesoporous carbon nanoparticle (MCN).

Reagents and materials
Sodium thioglycollate, Fetal Bovine Serum (FBS), RBC Lysis Buffer, Iscove's Modified Dulbecco's Media (IMDM), powdered Methyl Cellulose and Penicillin-Streptomycin were bought from Himedia, India. EDTA, methanol, Sulfanilamide and NED were bought from Sisco Research Laboratory (SRL), India. DMEM from Gibco, Murine Stem Cell Factor (SCF) from Biovision, and Bovine Serum Albumin (BSA) from Biosera were used. Ortho-phosphoric acid and NaNO 2 were purchased from Merck, India. 1X phosphate buffered saline (PBS) was prepared using 137 mM NaCl (Merck, India), 2.7 mM KCl (Himedia, India), 10 mM Na 2 HPO 4 (Qualigens, India), 2 mM KH 2 PO 4 (Himedia, India). 24 well plates and 96 well plates were obtained from Nest Biotech Co. Ltd., China. Dispovan syringes were used to obtain blood and peritoneal fluid. Smears for cell counting were prepared using Cytospin (Centurion Scientific C 2 Series) after centrifuging the sample in a cold centrifuge (Vision VS-15000CFN). Smears were observed under a light microscope (Debro DX-200). Absorbance readings were taken in a multiplate reader (Thermo Fisher Multiskan EX). Plates were incubated in a CO 2 incubator (Thermo Fisher), and colonies in CFU assay were observed using Floid Cell Imaging Station (Life Technologies, India). All cell-culture work was done inside the biosafety cabinet.

Statistical analysis
All data are presented as mean ± SEM, and only p values less than 0.05 have been considered as statistically significant. Statistical significance has been calculated using t-test in Graph Pad Prism 6. dissolved in 1X PBS and stored at 4°C.
Peritoneal Fluid (PF): 2 ml of 1X PBS was slowly injected into the peritoneal cavity, and the cavity was massaged well to wash the cavity extensively. A 5 ml syringe was inserted into the side of the mouse, and the plunger slowly pulled out to retrieve the maximal amount of peritoneal fluid. The PF was collected in DMEM medium.

Bone Marrow (BM):
The bone of the hind leg of the mouse was taken into the biosafety cabinet, and flushed with DMEM till the bone turned white.

Total and differential cell count (TC/DC)
Differential white blood cell count is an examination and enumeration of the distribution of leukocytes in a stained blood smear. Increases in any of the normal leukocyte types or the presence of immature leukocytes or erythrocytes in peripheral blood are important diagnostically in a wide variety of inflammatory disorders.
The total number of cells and their viabilities were determined using a hemocytometer. Differential count was taken using a smear prepared in a cytospin. 100 µl of each sample was loaded into the wells of a cytospin, and centrifuged at 2000 rpm for 3 mins. The slides were removed, air dried and fixed with methanol. The smears are then stained with hematoxylin, counter-stained with Eosin, and observed under a microscope. The TC and the DC were plotted against each sample.

Determination of nitric oxide content (NO Assay)
Activation of immune system is associated with increase in macrophage NO production. Transient nature of NO makes it unsuitable for detection, but it is oxidized to Nitrite (NO 2 -) and Nitrate (NO 3 -) by nitrate reductase. The concentrations of these anions are used as quantitative measure of NO production using the Griess reaction. In this reaction, acidified NO 2 produces a nitrosating agent, which reacts with sulfanillic acid to produce diazonium ion. This ion couples with NED (N-1-naphthyl ethylene diamine dihydrochloride) to form a coloured product that is measured spectrophotometrically at 540 nm.
The reaction was standardized using different concentrations of NaNO 2 (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM and 0 μM), using the method in Promega User Guide (Product G2930). 50 μl of cells from each sample (PB, PF and BM) from all the groups (Control, TG24, TG24F, TG24MF, TG48, TG48F, TG48MF,  TG72, TG72F, TG72MF, TG96, TG96F, TG96MF) were plated in the wells of a 96-well plate. The cells were incubated for 24 hours, in a CO 2 incubator at 5% CO 2 , 37°C. Sulfanilamide solution was prepared by dissolving 1% Sulfanilamide in 5% ortho-phosphoric acid. 0.1% NED solution was prepared in distilled water. 50 μl of sulfanilamide solution was added to each well, and incubated at room temperature for 5 minutes, in dark. 50 μl of NED solution was then added, and incubated at room temperature for 5 minutes, in dark. Absorbance was measured in a plate reader at 540 nm. Using the standard curve prepared, the absorbance values of the samples were plotted to get the concentrations of NO produced (in μM). The concentrations of NO were plotted against each sample.

Cell proliferation assay (MTS Assay)
The MTS assay is a colourimetric method for determining the number of viable cells in culture. It uses solutions of a novel tetrazolium compound MTS [3-(4, 5-dimethyl thiazol-2-yl)-5-(3-carboxy methoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] and an electron-coupling reagent PMS (Phenazine Methosulfate). MTS is bioreduced by cells into a formazan product that is soluble in tissue culture medium. Absorbance of formazan is measured at 492 nm. The conversion of MTS to aqueous soluble formazan is accomplished by dehydrogenase enzymes found in metabolically active cells. Quantity of formazan product, as measured by the absorbance at 492 nm, is directly proportional to the number of living cells in culture. The assay was performed using the Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay Kit. 100 μl of cells (PB, PF, and BM) from all the samples were added to the wells of a 96-well plate, and incubated for 1 hour in a CO 2 incubator at 5% CO 2 , 37°C. 20 μl of MTS/PMS solution was added to each well, and incubated in a CO 2 incubator for 1-4 hours. Absorbance was measured immediately in a plate reader at 492 nm.
The absorbance values for each sample were plotted against time.
Taking absorbance of the control as 100 % cell viability, the viabilities of the other samples were calculated. Fold changes in the viability of the samples were calculated.

Determination of clonogenic potential of cells (CFU-c Assay)
CFU-c (Colony Forming Units in culture) assay measures the clonogenic potential of cells. The assay is based on the ability of cells to proliferate and differentiate into colonies in a semi-solid medium, in response to cytokine stimulation. The colonies formed can be enumerated and characterised according to their unique morphology. Clonogenic potential is determined by dividing the number of colonies formed by the number of cells plated.
Number of cells per well taken was 1 × 10 6 . For PF, 10 5 cells were taken per well. CFU-c media was prepared using IMDM, supplemented with 30% FBS, 10% BSA, 1% Penicillin-Streptomycin and 5 ng/ml murine SCF. Lastly, 1.5% methylcellulose was added into the concoction. 1 ml CFU-c assay media and 500 μl cell suspension was plated in each 24 well cell culture plate. The plates were kept in CO 2 incubator at 5% CO 2 and 37°C. All Colony types were counted after 7 days using Floid Cell Imaging Station, and pooled to get total CFU-c. A graph of Clonogenic Potential-vs-samples was plotted for each tissue sample.

Cytokine analysis of peripheral blood
The BD CBA Mouse Th1/Th2 Cytokine Kit (Catalog No. 551287) was used to measure Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interferon-γ (IFN-γ) and Tumour Necrosis Factor (TNF) protein levels in peripheral blood samples. Bead array technology was used to simultaneously detect multiple cytokines in samples. Five bead populations with distinct fluorescent intensities are coated with capture antibodies, specific for the above-mentioned proteins. The beads are mixed to form the bead array, and resolved in a red channel of a flow cytometer. After addition of the samples to the sample assay tubes containing the capture beads, the Mouse Th1/Th2 PE Detection Reagent was added to each tube. The tubes were incubated for 1 hour at room temperature, in the dark, and then washed with 1 ml of wash buffer (centrifuge at 200 g for 5 mins). The supernatant is carefully discarded and 300 µl of wash buffer added to resuspend the bead pellet.  Figure 1).  Figure 2).

DC of neutrophils in PB:
The count of polymorphonuclear (PMN) cells in the blood-eosinophils, basophils and neutrophils, increased (data not given) with TG. The numbers of neutrophils in the blood were found to have increased. There is a 1.10 fold increase after 24 Figure 3).       Figure 4).         Figure 6).  Figure 7).

Cell proliferation (MTS) assay of PB
The MTS assay gives an idea about the proliferative potential of the cells. The cell viabilities decreased with TG treatment, as compared to untreated control. There is a 1.23 fold decrease in viability after 24 Figure 8).  fold after 24 h and by 1.15 fold after 72 h (Table 9 and Figure 9).  (Table 10 and Figure 10).

CFU-c assay of PB
CFU-c assay gives the clonogenic potential of cells, which is the ability of cells to form colonies on a semi-solid matrix. The clonogenic potential of cells in the peripheral blood decreased with TG treatment, as compared to control.     MCN+fisetin, the clonogenic potential increased 1.45 fold after 24 h and 1.65 fold after 72 h. MCN has enhanced the clonogenic potential by 1.15 fold after 24 h and by 1.75 fold after 72 h (Table 11 and Figure 11).

CFU-c assay of PF
The clonogenic potential of cells in PF decreased 1.33 fold after 24 h, 2.00 fold after 48 h, 2.29 fold after 72 h and 3.20 fold after 96h, with TG treatment. Compared to TG, with fisetin treatment, it increased 1.42 fold after 24h, 1.75 fold after 48 h, 1.71 fold after 72 h and 3.20 fold after 96 h. With MCN+fisetin, the clonogenic potential increased 1.33 fold after 24 h, 1.38 fold after 48 h, 2.14 fold after 72 h and 6.80 fold after 96 h. Compared to fisetin, MCN has improved the clonogenic potential by 1.25 fold after 72 h and by 2.13 fold after 96 h (Table 12 and Figures  12-14).

CFU-c assay of BM
The clonogenic potential of cells in the bone marrow decreases with TG treatment, as compared to control. There is a 1.73 fold decrease (p<0.05) after 24 h, a 2.52 fold decrease (p<0.05) after 48 h, a 3.39 fold decrease (p<0.05) after 72 h and a 4.59 fold (p<0.05) decrease after 96 h, with TG. It increases 1.20 fold after 24 h, 1.03 fold after 48 h and 1.53 fold after 96 h, with fisetin, compared to TG. With MCN+fisetin,

Discussion
The pathophysiology of peritonitis is complicated and is involved in various processes, of which, the most important one is the inflammatory reaction [27]. During the pathological process of the peritonitis, NF-κB plays an activating role in the inflammatory reaction [7].
Our study was designed to investigate the potential therapeutic effects of fisetin and fisetin loaded on mesoporous carbon nanoparticle (MCN) on the thioglycollate-induced peritonitis in rodent models. The thioglycollate-induced peritonitis in mice is used as a model to study the potential anti-inflammatory action of investigated test compounds [28]. In this present study, we have induced peritonitis in 6-8 weeks old BALB/c mice using thioglycollate, and then assessed the antiinflammatory effects of plant flavonoid, fisetin, when administered therapeutically. We have also assesses the anti-inflammatory effects of fisetin, when administered with a nanovehicle, mesoporous carbon nanoparticle. We found that fisetin had a positive therapeutic effect on the peritonitis. Acute peritonitis differs from other infections because of the broad variety of causes, severity of the infection [29]. Acute peritonitis is one of the most headachy postoperative complications, which was an important cause of death in surgical practice and intensive care units [9]. The most serious consequence of acute peritonitis is sepsis, often leading to an unacceptably high morbidity and mortality [30]. So the research of acute peritonitis is always the hotspot of surgery and critical care medicine. The animal model is one of the most important methods in the scientific research. It can not only provide convenience in deriving a better understanding of the pathophysiology of disease, but also provide important and indispensable tools to explore the therapy of disease. It is the bridge between the fundamental research and clinical application.
The process of peritonitis is mediated by the activation of inducible transcription factors, such as NF-κB, which play a pivotal role in the immune and inflammatory responses. Previous investigators have found that acute peritonitis and sepsis were associated with the activation of the transcription factor NF-κB in various organs and tissues [10,31,32] which can regulate the synthesis of TNF-α, IL-6, inducible nitric oxide synthase, cyclooxigenase-2 and many other molecules involved in the inflammatory reaction [18,33].
In this study, we found that, with administration of TG, cell recruitment in the blood increases progressively with time, with maximum recruitment after 96 h. This shows that it has induced inflammation, and the body is synthesizing more immune cells to counter the infection. Cell recruitment is successfully inhibited by fisetin, and with MCN+fisetin. In the peritoneal fluid, total cell recruitment was increased, which was successfully inhibited with fisetin and MCN+fisetin treatments.
Nitric acid is produced by macrophages as a defense against oxidative stress. In case of inflammation, NO content is expected to increase. Our assays have shown that the NO content of the tissues was increased with TG challenge. NO content decreased with fisetin and with MCN+fisetin. However, the addition of MCN to fisetin did not make a significant difference to the NO content.
We found that, TG treatment significantly reduced cell proliferation in the blood, PF and BM. Interestingly, cell proliferation was increased with fisetin treatment, and with MCN+fisetin. In another assay, the clonogenic potential of the tissues decreases significantly within 24 h, with administration of TG. Both fisetin treatment and MCN+fisetin treatment have restored the clonogenic potential of the tissues. Our study demonstrated that, there was a decrease in Th2 cytokines (IL-2, IL-5 and TNF-α) with TG treatment, in blood after 48 h and fisetin and MCN+fisetin was increased the cytokine content. However, in all the   cases, MCN has not had a significant effect. So, the anti-inflammatory and pro-regenerative effects of MCN+fisetin is mainly because of the fisetin.
One of the main results of an inflammatory reaction in a body is the over-production of pro-oxidative agents, like nitric oxide. In response to the increase in pro-oxidative radicals, the body attempts to maintain the oxidative balance, by producing more anti-oxidants, which can scavenge the harmful radicals. This leads to an increase in the proliferation of cells. This has been demonstrated clearly in our experiment, where treatment with TG has led to an increase in NO concentration. Due to the attack caused by TG, the body loses the battle to maintain the oxidative balance, and proliferation is reduced. The cells lose the ability to proliferate and form colonies on semi-solid medium. Fisetin is successful in restoring this balance to some extent. It successfully reduced the NO content, and increased the proliferation. It also increased the clonogenic potential of the cells, showing the cells have regained their ability to form colonies when given a matrix for growth. Fisetin, when loaded on MCN, has a similar effect to fisetin alone, but it does not have any additional effect, which we would want our nano-vehicle to have.

Conclusion
In conclusion, we demonstrated that fisetin and fisetin loaded on mesoporous carbon nanoparticle (MCN) may have anti-inflammatory effects on thioglycollate-induced peritonitis. To our knowledge, this is the first study to date to assess new therapeutic approaches using phytochemicals such as fisetin against peritonitis. Further studies are required to verify the clinical use of fisetin in the treatment of peritonitis, and also to determine the pathways by which it acts. The future research could focus on the combination of fisetin therapy and traditional antibiotics, which might be more efficient than using antibiotics alone. We can also investigate other nano-particles that can be used as vehicles, for better delivery of the drug.

Contribution of Authors
SM performed all experiments, analyzed data, SB gave valuable input to the manuscript, AS and NRJ have prepared the MCN, and ERB initiated the project with her idea, designed the experiments, analyzed all data and wrote the manuscript.