Bird’s Saliva Soup
mainly comprises a secretion of the salivary gland of different Swiftlet
species has been used as a symbol of wealth and power as well as an crucial
ingredient in traditional Chinese Medicine. The MTT assay and flow cytometry
analysis were carried out to evaluate the effect of Bird’s Saliva Soup extract
in treated Influenza A virus infected MDCK cells. MTT assay was performed to
determine the cell viability and Ha assay was used to determine the virus titer
after 24 hour of post treatment with Bird’s Saliva Soup extract towards MDCK
cells challenged with influenza A virus. The results showed that there was a
significant increase in cell viability (p < 0.05) of Bird’s Saliva Soup
extract treatment compared to positive infected control. Annexin V-fitc double
staining method was carried out to identify the mode of cell death in MDCK
cells by calculating the means of each apoptosis stage. The results showed that
there was a significant reduced early apoptotic injury in infected cells
treated with Bird’s Saliva Soup extract. In summary, the current finding
suggests that Bird’s Saliva Soup may be have the potentiality to be as an
apoptotic inhibitor and ameliorate the infected cells caused by virus.
Introduction
Apoptosis is an important physiological necessary for development and
maintenance of tissues homeostasis including tissue atrophy, the immune system
development and biological tumor [1-2]. It also plays an important function in
the pathogenesis of many infectious diseases including those caused by viruses
[3-5]. Many virus infections result in apoptosis of host cells, and several
viruses have evolved mechanisms to inhibit apoptosis. Influenza viruses induce
apoptosis through mechanism then both cellular and viral factors depending on
the cell type. However, the precise mechanism still remain unclear.Bird’s Saliva Soup is natural food product made from Aerodramus genus Swiftlet’s saliva. Studies show its unique glycoprotein properties provide health benefits, cure many illnesses and rejuvenate cells. The nests contain almost all of the bessential ingredients for body maintenance. Amino acids, minerals, and others keep the body healthy and increase immunity against a host of illness. Another studies have shown that this animal saliva previously found to contain bioactive compounds that are powered with anti-apoptotic and antioxidant properties [6-7]. As apoptosis have been suggested as crucial events in Influenza virus infection, Bird’s Saliva Soup, the salivary secretion of swiftlets, may have anti-apoptosis relevance in the therapeutic context of viral infection. Hence, this study aimed to investigate the anti-apoptosis effect of Bird’s Saliva Soup.
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| The evaluation of Bird’s Saliva Soup Extract Reduced Influenza A virus Induced Apoptosis on Cultured Cells in vitro study |
Materials and Methods
Preparation of Bird’s Saliva Soup ExtractsRaw Bird’s Saliva Soup from the swiftlet of Aerodramus genus collected from Bird’s Saliva Soup house in Teluk Intan, Perak, Malaysia. The cleaning process was carried out by first soaking after the unprocessed Bird’s Saliva Soup in distilled water until softened. Debris and feathers were removed after which the Bird’s Saliva Soup was subjected to drying process at 70 oC for 16 hrs and followed by grounding and sifting through a mesh (60μM in pore size). The grounded samples were kept in distilled water at 3oC in about 16 hours and continued heated for 30-60 min at 60oC. The extract was then filtered and freeze-dried after 48 hours freezing at -800C, and stored for the following experiment.
Madin Darby Canine Kidney (Mdck) Cell Culture
MDCK cell line was purchased from the American Type Culture Collection (ATCC,CCL-34TM) and grown in Dulbecco’s Modified Eagle’s Medium (Gibco,UK) supplemented with 10% fetal bovine serum (Gibco, UK), antibiotic-antimycotic (Gibco,UK). The cells then were seeded into a sterile 96-well flat bottom plate (Nunc, USA) and maintained at 37oC humidified incubator with 5% CO2 (Galaxy, UK) for 2-3 days until 70%-80% confluency is achieved.
Virus Propagation
Influenza A virus, strain A/Puerto Rico/1934 (H1N1)was purchased from the American Type Culture Collection (ATCC,® VR-95TM) and propagated in MDCK cells, then the stock virus was titrated by TCID50. In obtained 100 TCID50, the resultant virus titer was further diluted with 100 μl taken from the amount ofTCID50, then it was to be used as a constant positive control in the following procedure
Cell Viability Based On Combination Treatment
This procedure was performed by using Maximal Non- Cytotoxic Concentration of Bird’s Saliva Soup and constant virus titer (100 TCID50). Briefly,the virus was first inoculated into the MDCK cells and later followed by the Bird’s Saliva Soup extract replacement for 1 hrs followed by change with media. All the samples were incubated at 37oC in 5% CO2 incubator.
After 24 hour of post-treatment incubation, the plates were exposed to the MTT assay. Treated cells were subjected to MTT reagent (Sigma Aldrich, USA) in which reacts to quantify the viable cells. Pure DMSO solution (Merck, Germany) was added to the cells after 2-3 hr exposure to the MTT reagent to solubilise the formazon precipitation. Absorbance of the solution was measured spectrophotomerically by using ELISA reader at 540nm wavelength (Bio Tek Instruments EL800,USA).
Apoptotic Cells Analysis
The protocol of Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, USA) by Rieger et al was performed in this procedure. Briefly, upon completion of treatments, cells were harvested and washed with binding buffer. Then, Annexin V and propidium iodide (PI) were added and incubated in dark for 15 min. Subsequently, washing was performed twice with binding buffer which then incubated for 15 min at 37oC. Finally, the samples were reads for analysis with Becton Dickson FACS Calibur Flow Cytometer (BD Biosciences, USA).
Statistical Analysis
Data was collected as triplicate for each experiments. The results were expressed as mean ± standard deviation. Statistical significance was assessed with Anova test and P value < 0.05 was considered significant.
Result And Discussion
Cytotoxic Profile Of Bird’s Saliva Soup ExtractToxicity study was first performed with addition of Bird’s Saliva Soup extract to MDCK cells to determine the concentration side effect as well as MNCCs of the extract on cultured cells. MNCC is maximal non-cytotoxic concentration of the Bird’s Saliva Soup extract.
Based on the [Figure 1], cytotoxicity percentage in MDCK cells showed an increasing trend of optimal density (OD) of cell viabilities along the concentration. As determined from the bar graph, MNCCs was 12.5 mg/ml.
The Bird’s Saliva Soup extract in water soluble form is likely less cytotoxic, thus same as previous report that pointed out the water soluble subtances possess less cytotoxic effect compared to the crude Bird’s Saliva Soup extract. This is due to compound solubility and stability are major factors that contribute to varied activities in different extract [8] such as the application of high temperature during the water extraction process caused affected the potency of proteins within the Bird’s Saliva Soup, possibility through denaturation [9].
Bird’s Saliva Soup extracts improves cell viability in H1N1 viruschalleged MDCK cells
The MTT was performed in order to quantify the cell viability based on the apoptotic reduction activity. Upon challeged with 100TCID50/ul HINI virus for 24 hour post treatment with Bird’s Saliva Soup extract, cell viability increased to about 20% of that of infected control [Figure 2]. Meawhile, the Bird’s Saliva Soup alone treatment generally did not affect the infected group. This could be due to the mitogenic property of Bird’s Saliva Soup that promoted cell growth, as made evident a study by Zainal Abidin et el. Which shows that Bird’s Saliva Soup promoted cell divison in rabbit corneal keratocytes [10].
Bird’s Saliva Soup Extracts Reduce Early Apoptotic Event in H1N1 Virus Challeged MDCK Cells
Apoptotic cells resulted due to phosphatidylserine (PS) translocation towards the outer leaflet of the plasma membrane caused the apoptotic or death cells and membrane integrity become loss. Therefore, the annexin V-FITC double stainig was performed in this study to calculate the percentage of cell death in different stages of apoptosis. In this assay, cells residing at different level of apoptosis upon exposure to the virus infection (A/Puerto Rico/8/1934 (H1N1)) by differential staining of DNA and also PS, whereby cells are grouped and revealed in dot plot. Annexin V-FITC-/PI- (lower left quadrant) are classified as healthy cells, the cells that are considered early apoptotic showed Annexin V-FITC+/PI- (lower right quadrant), whereas late
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