Assessment of Toxicity, Pro-Inflammatory Cytokines and Cytoprotective Potential of the Phytosaponin in Isolated Rat Pancreatic Islets

INTRODUCTION

Herbs and herb-derived drugs have played a crucial role in health and disease management. The global demand for herbal medicinal products has increased significantly in recent years. It is estimated that, the world’s population will be more than 7.5 billion in the next 10 to 15 years, where approximately 80% of the population still relies on a traditional system of medicine based on herbal drugs for primary healthcare.1 Herbal medicine toxicities which may arise from herbs administered alone or concomitantly with other herbs and/or drugs. However, challenges still exist for testing herbal medicines in this exciting field.

The mammalian In vivo micronucleus test is especially relevant for assessing genotoxicity because, although they may vary among species,factors of In vivo metabolism, pharmacokinetics and DNA repair processes are active and contribute to the responses. The mammalian In vivo micronucleus test is used for the detection of damage induced by the test chemical to the chromosomes or the mitotic apparatus of erythroblasts.2 The In vitro micronucleus test is a also genotoxicity for the detection of micronuclei (MNvit) in the cytoplasm of interphase cells. MN may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. Therefore the MNvit test is an In vitro method that provides a comprehensive basis for investigating chromosome damaging potential in vitro because both aneugens and clastogens can be detected in cells that have undergone cell division during or after exposure to the test chemical. In addition to using the MNvit test to identify substances that induce micronuclei, the use of immunochemical labelling of kinetochores, or hybridisation with centromeric/telomeric probes (Fluorescence In situ hybridisation (FISH)).3 The In vitro chromosomal aberration test is to identify substances that cause structural chromosomal aberrations in cultured mammalian cells. Structural aberrations may be of two types, chromosome or chromatid. Polyploidy (including endoreduplication) could arise in chromosome aberration assays in vitro. While aneugens can induce polyploidy, polyploidy alone does not indicate aneugenic potential and can simply indicate cell cycle perturbation or cytotoxicity.4 The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. This bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.5

Cytokines such as IL-1β, IL-6 and TNF- α are secreted by immune cells and directly harm beta cells. Furthermore, cytokines and chemokines modulate, activate, regulate and attract other immune cells and thereby play a crucial role in cellular immune reactivity.6 So far, complex up and down regulation of systemic cytokines in the patients with type I diabetes and subjects to increased diabetes risk and in relation to beta cell function and clinical remissions in type I diabetes patient. Chronic exposure to free fatty acids alone or with hyperglycemic conditions lead to pancreatic cell death possibly employing oxidative stress as the mechanism for cell destruction. Hence, blockage of multiple pathways, rather than a single pathway, leading to β-cell death may be necessary to fully protect β-cells from destruction in vitro. Cytokines inducing IL-1B, INF-Y, TNF-α have also been reported to inhibit insulin secretion and induces apoptosis of Beta-cells via INOS independent pathway. The inducible form of nitric oxide synthase (iNOS) has been also shown to contribute to DNA damage. Chronic exposure of pancreatic beta cell to IL-1B activates the expression of inducible nitric oxide synthase and results in excessive production of NO- Interferes with electron transfer – inhibits ATP synthase in the mitochondria and induces the expression of pro-inflammatory gene. IL-1B, INF-Y decrease the SR pump Ca2+ATPase2b protein expression and deplete ERCa2+ stores by NO Synthase, which subsequently activates the ER stress pathways-triggers beta cell apoptosis.7

 Therefore, we had already screened SMD for in vivo acute to chronic toxicities in animal model and reported in our previous paper. In light of these findings, have concluded SMD was not producing any toxic signs or evident symptoms on the doses of 250, 100 and 50mg/kg b.w.8 Here our aim was to investigate the mutagenicity and any protective effect of the phytochemical in cytokines induced high glucose and determined by ELISA along with a highly selective iNOS against STZ induced beta cell destruction in pancreatic isolated islets of rat, Expression analysis was performed by Western blotting.

METHODOLOGY

Extraction and isolation of saponin:

Extraction and Isolation of saponin from fruits of Momordica dioica was already done and reported in our previous paper.⁸

Animals:

Wistar male rats weighing 120-150g and mice 20-25gm was used for the experiment. Entire study was conducted in accordance with the CPCSEA guidelines. All the experiments conducted on the animals were in accordance with the standards set for the use of the laboratory animal use and the experimental protocols were duly approved by the IAEC of Karnataka College of pharmacy, Bangalore (Ref. no: KCP-IAEC2/17-18/15-04-07).

Cell lines – NB41A3 Neuroblastoma cells of human species cell line was brought from NCCS, Pune, India.

Pancreatic islet isolation and culture:

Rat was sacrificed and the pancreas isolated and pancreas was mincing and Collagenase Type XI digestion followed by cell straining (500μm), Ficoll gradient centrifugation and cell straining (70μm). Produced highest yield (800 to 900 per rat pancreas) of purified islets with long term viability and the procedure of isolation already reported in our previous paper.10

EXPERIMENTAL DESIGN

In vivo micronucleus test:

All the procedure was done by as per the OECD 474 guidelines. 

Requirements:

Animal: Mice (20-25gm) 

Chemicals:

Cyclophosphamide, Potassium dihydrogen phosphate, Disodium hydrogen phosphate, Bovine albumin, Sodium chloride, Sodium azide, May-grunwald’s stain, Giemsa stain. Test compound: Saponin isolated from fruits of Momordica dioica (SMD). Equipment’s: Centrifuge, Microsope

Preparation of the reagents:

• Preparation of phosphate buffer solution
• Preparation of suspending medium
• Preparation of May-Grunwald’s stain & phosphate buffer mixture (1:1)
• Preparation of giemsa’s stain & phosphate buffer mixture (1:5)

Procedure: 

• One animal was treated with vehicle (normal saline 1ml/100g body weight) which serves as control
• Other animal was treated with cyclophosphamide (200mg/kg) which serves as Positive control and left for 24hrs and one for test compound
• Bone marrow extraction
• A fine pellet of the suspension was formed by centrifuging the marrow content at 1000 rpm for 8 minutes.
• Smear made, air dried and staining procedure (methanol fixing-May- Grunwald’s stain (15min)-wash-giemsa stain (10min)-wash) done.
• The slides were air dried and observed under oil immersion objective to determine the frequency of MN.

In vitro micronucleus test:

In vitro micronucleus test was done as per OECD guidelines 487.

Procedure:

• Micronuclei is either the whole chromosome or a chromosomal fragment as a small body outside nucleus
• Principle: detection of the frequency of micronuclei:
• Cell cultures of human (Neuroblastoma cells) are exposed to the test chemical, formation of micronuclei in interphase cells.
• Harvested and stained interphase cells are analyzed for the presence of micronuclei.
•  Treated with a cytokinesis blocker, this is easily achieved by scoring only binucleate cells. Assay detects the activity of clastogenic and aneugenic chemicals

In vitro chromosomal aberration test:

In vitro chromosomal aberration test was done as per OECD guidelines 473. 

Procedure:

Human Neuroblastoma cells (NB41A3) were cultured in alpha MEM and Cells were exposed to test substance (SMD) for 3 to 6 hours. Sampled at a time equivalent to about 1.5 normal cell cycle lengths after the beginning of treatment. Chromosome preparation: culture treated with Colchicine (0.1µg/ml) for 3hr prior to harvesting, process involves hypotonic treatment of the cells, fixation and staining. Cells were analyzed microscopically for abnormalities in structural organization.

Slides were scanned and at least 100 well-spread metaphase cells were screened. Only those cells that were well spread, where nuclei were free of cytoplasm and chromosomes did not overlap were selected of aberrations. Mitotic index (MI) was determined from the ratio of dividing to non-dividing cells. The MI was determined by counting the number of metaphases in 100 cells.

MI = No. of dividing cells/No. of total counted cells x 100

Chromosomal aberrations (%) = No. of aberrations cells/No. of total counted cells x 100

Scoring Criteria

Different types of aberrations (breaks, exchanges, rings and minute) were recorded separately.

• Breaks: Chromosomal breaks are true discontinuities with clearly dislocated fragments and also include fragments without obvious origin.
• Exchanges: Also called as translocations. Here one portion of chromosome is transferred to another chromosome.
• Rings: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material.
• Minute: Chromosomes are broken into small particles like structure

 Ames test: Ames test was performed according to OECD guidelines 471 using the Muta-ChromoPlateTM basic kit with S9 activation version 2.0.

Procedure:

Overnight Growth of the Bacteria (It was incubated overnight at 37ºC for 16 to 18 hours). Once the reagents were mixed and the bacteria and samples for analysis were added the suspension was then dispensed into the 96 well plate. It was noticed that all the wells in the plates will be a purple color. The plates were placed into the Ziploc bags and incubated for 5 days at 37o C. Later the plates were removed and scored.

Principle:

• Each well of the 96 well plates is considered a colony.
• If the colony reverts back to the natural state, a mutation has occurred.
• If a reverse mutation has occurred, the bacteria in the colony have the ability to synthesize histidine and will continue to grow turning the color in the well from purple to yellow.
• The Muta-ChromoPlateTM kit (as with the traditional ‘Ames Test’) compares the natural background rate of reverse mutation to a rate of reverse mutation within a sample assay. Components:
• Bacteria- Salmonella typhimurium 
• Strain- TA100
• Standard mutagen- Sodium azide (5µg/ml)
•  Test compound- Saponin of fruits of Momordica dioica
• Test dose- 8, 40, 200, 1000, 5000µg/ml

Assessment of SMD on production of proinflammatory cytokines IL-1β, IL-6 and TNFα in isolated pancreatic islet of rat:

Pancreatic islets were cultured in a 12-well plate seeded at a 50 islets each plate and cultured at 370C in a humidified atmosphere of 5% CO2 in air in RPMI1640 medium.

The grouping of cells was done as follows:

Group 1: Normal control-Islet in growth medium (Normal glucose)

Group 2: High glucose control- Islet in high glucose (56mM) medium

Group 3: Treated group-High glucose control + 50µg/ml Saponin Momordica dioica (SMD)Standard

Group 4: High glucose control + 1µM Epalrestat

After incubation, the culture media were aspirated from each well without disturbing the cell monolayer and centrifuged at 10,000 rpm for 5 mins at 40C to sediment particulate matter, if any. The supernatants were used for the estimation of the cytokines viz, IL-1β, IL-6 and TNF-α was done using antigen capture was assayed by sandwich ELISA using a commercially available kit (Cat. No: 10-1250-01, Mercodia, Sweden).

Wells of ELISA plate were coated with 100 µl of IL-6, IL-1β and TNF-α primary antibodies (2.5µg/ ml) in carbonate buffer (Na2HPO4 and NaH2PO4, pH 9.6). Primary antibodies of IL-6, IL-1β, and TNF-α were used. The plates were incubated overnight at 40C to facilitate proper adsorption of antibodies on to the substrate. After 12-14hr of incubation, the plates were washed thrice with wash buffer (NaCl and Tween 20 in phosphate buffer, pH 7.4) and blocked with 250µl of blocking buffer (BSA in phosphate buffer, pH 7.4) per well followed by incubation for 1hr at 370C. After incubation, standard cytokines were added for the construction of calibration curve. Remaining wells were coated with 100µl of appropriately diluted cell lysate. The concentration range used for the standards was – 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.39 and 0.195ng/ml. The standard cytokines were used, Incubated for 1hr and washed thrice. Anti-cytokine antibodies (anti IL-6, anti IL-1β and anti TNF-α monoclonal antibodies were diluted 1:1000 and added 100µl per well to the strips containing the respective antigens and incubated at 370C for 1hr. After incubation, 100µl of HRP-conjugate was added. Plate incubated at 370C for 1hr. Then 100µl of freshly prepared substrate (TMB in DMSO containing H2O2) was added to all wells, and incubated in dark 370C for 15 mins for colour development. After incubation, the reaction was terminated by adding 50µl of 2.5N H2SO4 per well and the A450nm was measured using ELISA reader. A standard calibration graph was plotted (Abs vs Conc) and the concentrations of unknown samples were determined from the graph.

Cytoprotective activity against STZ-induced beta cell destruction in pancreatic isolated islets of rat:

Pancreatic islets were isolated from rat pancreas and cultured in a 12-well plate seeded at a 50 islets each plate and cultured at 370C in a humidified atmosphere of 5% CO2 in air in RPMI1640 medium.

Islet cells were stimulated with STZ 5mM (Sigma, USA) in PBS, pH 7.4 with or without the SMD. The cells were incubated for 6 h. After incubation, cells were lysed using ice-cold cell lysis buffer containing 1x Triton-X-100 (1%), Tris-HCl (50mM, pH 7.4), EDTA (5mM), NaCl (150mM) and a protease inhibitor cocktail. The lysates were spun at 10,000×g for 5 minutes at 4˚C to sediment particulate matter, if any. The supernatant was stored at -200C for the estimation of iNOS by western blot analysis, according to the manufacturer’s protocol.

Groups of 50 islets each in triplicate (n=3) were treated in the following manner:

Group 1: Normal control – Only islet cells

Group 2: STZ control- islet cells treated with 5mM STZ

Group 3: SMD treated- Islet cells treated with 5mM STZ + 50μg SMD

Group 4: Positive control - 5mM STZ + 50μM salicylic acid

Western blot analysis of iNOS:

The protein separation was done by SDS-PAGE (8% v/v gel) and blotted on to nitrocellulose membranes (0.22µm, Himedia, India). The membranes were blocked with 1% BSA in PBS (pH 7.4) for 90 mins and incubated overnight at 4 0C with continuous stirring with anti iNOS antibodies (1:2000; Abcam, USA) and anti β-actin antibodies (1:5000) followed by anti-mouse IgG-HRP conjugate. Substrate buffer was added containing 1mg/mL TMB (3’,3’,5’,5’-Tetramethylbenzidine) and 0.01% H2O2 and stop solution (2.5N H2SO4). The bands developed were scanned densitometrically and expression of iNOS was calculated with respect to the β-actin bands (loading control).

Statistical Analysis

The results are expressed as Mean ± SEM in each group. The significance of difference among the groups was assessed using one-way ANOVA followed by Tukey’s multiple comparison tests (Graph pad Prism v5.0). P-value < 0.05 was considered to indicate a statistically significant difference.

Results and Discussion

In vivo micronucleus test: In vivo micronucleus test was performed and it was observed that the number of poly-nucleated cells were more in the positive control than the test drug grouping i.e. saponin Momordica dioica. The slides were scanned under oil immersion (100X) for the presence of MN and PN was determined by counting a total of erythrocytes per animal (Fig. 1).

In vitro micronucleus test was performed and human Neuroblastoma cells (NB41A3) exposed to the test drug and it was observed that the number of poly- nucleated cells were more in the positive control than the test drug grouping i.e. saponin Momordica dioica. The slides were scanned under oil immersion (40X) for the presence of MN and PN was determined (Fig. 2).

In vitro chromosomal aberration test: In vitro chromosomal aberration test was performed and it was found that the test compound showed far less aberrations in comparison to the positive control. The slides were scanned under oil immersion (100X) for the different types of aberrations were recorded separately (Fig. 3).

Ames test: The number of revertant (positive) wells obtained on the 5th day of incubation was counted. The negative control plate did not show any change colour (all wells were purple) indicating the sterility of the test conditions. The numbers of revertant wells obtained are mentioned below (Table 4):

Interpretation of results: The number of revertant (positive) wells in each treatment dose plate of the saponin Momordica dioica was compared with those of the background plate. The number of positive wells in the background plate was found to be 12.

The background plate does not have the standard mutagen and test drug and is used to analyze the natural mutagenic changes which occur. The statistical significance of different doses was derived from the fluctuation by comparing the treatment plate with the background plate. It was concluded from the significance values that the saponin does not possess significant mutagenicity.

Assessment of SMD on production of pro-inflammatory cytokines IL-1β, IL-6 and TNF- α in isolated pancreatic islet of rat:

Assessment of the anti-inflammatory activity of SMD (50µg/ml) was done on pancreatic islet cells maintained in a high glucose (56mM) environment. It was found that the production of all three pro-inflammatory cytokines viz., IL-1β, IL-6 and TNF-α was increased considerably by the cells in high glucose state. However, treatment with SMD has significantly suppressed the production of the cytokines in comparison to the high glucose control (p<0.05) and the standard, Epalrestat (1µM) (Graph 2).

Cytoprotective activity against STZ-induced beta cell destruction in pancreatic isolated islets of rat.

The expression of inflammatory mediator viz., iNOS was found to be increased in the cells challenged with STZ in comparison to the normal control (***p<0.05). However, upon treatment with SMD (50µg) and salicylic acid (50µM) the production of the mediators was found to be reduced in comparison to the STZ control (##p<0.05). The anti-inflammatory activity of SMD was found to be comparable to the positive control salicylic acid. 

CONCLUSION

We can safely conclude from our study that, the saponin Momordica dioica (SMD) does not possess significant mutagenicity. Isolated pancreatic islet cells treated with high glucose produced pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) which were reduced upon treatment with SMD. Mediator of inflammation like iNOS was inhibited by SMD moderately in STZ stimulated pancreatic islet cells. The results concluded, that saponin of Momordica dioica possesses moderate to good cytoprotective nature and proves itself to be an auto-inflammatory disorders, pro-inflammatory disorders, and neurological disorders involving inflammation. 

ACKNOWLEDGEMENT

I would like to express heart full thanks to my beloved Parents, guide for their support and blessings.

CONFLICT OF INTEREST

None