The evaluation of Bird Nest Extract Reduced Influenza A virus Induced Apoptosis on Cultured Cells in vitro study

Bird Nest mainly comprises a secretion of the salivary gland of different Bird Nest 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 Nest 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 Nest 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 Nest 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 Nest extract. In summary, the current finding suggests that Bird Nest 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 Nest is natural food product made from Aerodramus genus Bird Nest’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 Nest, the salivary secretion of Bird Nests, may have anti-apoptosis relevance in the therapeutic context of viral infection. Hence, this study aimed to investigate the anti-apoptosis effect of Bird Nest.

The evaluation of Bird Nest Extract Reduced Influenza A virus Induced Apoptosis on Cultured Cells in vitro study

Materials and Methods

Preparation of Bird Nest Extracts
Raw Bird Nest from the Bird Nest of Aerodramus genus collected from Bird Nest house in Teluk Intan, Perak, Malaysia. The cleaning process was carried out by first soaking after the unprocessed Bird Nest in distilled water until softened. Debris and feathers were removed after which the Bird Nest 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 Nest and constant virus titer (100 TCID50). Briefly,the virus was first inoculated into the MDCK cells and later followed by the Bird Nest 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 Nest Extract
Toxicity study was first performed with addition of Bird Nest 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 Nest 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 Nest 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 Nest 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 Nest, possibility through denaturation [9].
Bird Nest 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 Nest extract, cell viability increased to about 20% of that of infected control [Figure 2]. Meawhile, the Bird Nest alone treatment generally did not affect the infected group. This could be due to the mitogenic property of Bird Nest that promoted cell growth, as made evident a study by Zainal Abidin et el. Which shows that Bird Nest promoted cell divison in rabbit corneal keratocytes [10].
Bird Nest 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

Conclusion

It has been successfully demonstrated that Bird Nest extracts confer apoptotic protection in H1N1-challenged MDCK cells. Particularly, Bird Nest demonstrated apoptotic protection potential by improving cell viability. Thus, it can be concluded that Bird Nest extracts shown to inhibit apoptosis.

Acknowledgement

Our sincere gratitude to Dr Fadzillah A’ini, from Department of Veterinary Services, Putrajaya, Malaysia, who kindly provided Bird Nest sample. This project was supported by Coe Bird Nest grants no.6371400 from Ministry of Agriculture,Putrajaya,Malaysia.

About Bird Nest - Nutritional Value

Bird Nest has been regarded as the most precious natural nourishment and health product and it has been considered as a precious item by aristocrats of the royal household in the Tang Dynasty in China . It was not until the Ming Dynasty that it became a treasure used as tributes by minor South East Asian countries, thus its name “Gong Yan (Tribute Bird Nest) or “Guan Yan (Official Bird Nest). Today, the production rate of Bird Nest has become more stable and it has become a common commodity with nutritional values recommended by both Chinese physicians and Western doctors.

Chinese medicine:

From the Chinese medicine point of view, Bird Nest principally fortifies the lungs, stomach and kidneys. In addition, its mild and sweet taste which is not heaty or cold, benefits the fortification of qi, nourishes the lungs, beautifies skin, tranquillizes and promotes sleep, nourishes the spleen, upgrades appetite, dissolves phlegm and stops coughs.
It has been recorded in the “Ben Cao Gang Mu” that Bird Nest is an excellent medicine for nourishing the lungs, dissolving phlegm, stopping cough, replenishing nourishment, clearing the body and regulating physical weakness”. “Its ability to penetrate the lungs promotes the movement of qi and penetration of the kidneys promotes movement of water, while tranquillizing the stomach making it the most ideal food. The consumption of Bird Nest stewed with rock sugar is effective in the regulation of physical weakness, cough and phlegm containing strands of blood. However, this is only suitable for those in the initial stage of such problems. If body heat is in excess and the flow of blood has reversed, this method may not be useful anymore.”

Western medicine:

From the nutritional point of view based on Western medicine, Bird Nest is rich in proteins, amino acids, carbohydrates, phosphorous, iron, calcium and potassium which are able to strengthen resistance of the human body. Proteins effectively promote the growth, renewal and repair of cells while helping muscles to contract, aid in the transportation of nutrients and also oxidation. Proteins are made of more than 20 types of amino acids in which 18 of them are found in Bird Nest. Out of these, 7 are essential amino acids. As Bird Nest contain many different types of essential amino acids, and are rich in them, it has been nicknamed an excellent perfect protein.

Proteins found in Bird Nest consist of Glycoprotein and Epidermal Growth Factors.

Epidermal Growth Factors stimulates the growth of skin and they are able to stimulate cell fission, promote regenerative power of body tissues, accelerate body metabolism, promote healing of wounds and also strengthen oxygen affinity in cells and resist ageing. This will fortify the immunity of the body and increase resistance of the body towards damage caused by X-rays and other radiation, making it the best health and beauty product for modern living.