2248968167 - B., ... Studying_St._John_s_Wort_Effect

docx

School

Romblon State University Romblon - Sawang Campus *

*We aren’t endorsed by this school

Course

Subject

Biology

Date

Nov 24, 2024

Type

docx

Pages

Uploaded by DeaconAtomBison24

Title : “Studying St. John’s Wort Effect on Gene Expression of Immune System in Human Epithelial Cells (Hep2 cells)” Student Name: Student ID: Word Count: 2133

Table of Contents Introduction ...................................................................................................................................... 3 Aim and Objectives ......................................................................................................................... 4 Hypothesis ....................................................................................................................................... 5 Methods and Methodology .............................................................................................................. 5 Cell Culture .................................................................................................................................. 5 RNA Extraction ............................................................................................................................ 5 Complementary Deoxyribonucleic Acid (cDNA) Synthesis ....................................................... 6 Real-time polymerase chain reaction (RT-PCR) – qPCR ............................................................ 6 Enzyme-Linked Immunosorbent Assay (ELISA) ........................................................................ 7 Conclusion ....................................................................................................................................... 8 References ...................................................................................................................................... 9" Introduction:

Hypericum perforatum, most often known as Saint John's Wort, is a plant that has emerged as significant in the field of botanical medicine and natural therapies. As a representation of complete health, it has been held in high esteem for its medicinal properties throughout history (Miller, 2017). Originating in Europe, this perennial plant has spread its vivid yellow flowers all over the globe and has been ingrained in many different cultural and medical practises. Numerous factors contribute to Saint John's Wort's significance. The traditional purpose of this plant was to protect people from evil spirits and diseases; its name comes from the fact that it blooms around Saint John's Day in late June, when it was believed to have been planted (Gomez et al., 2016). Its relevance, however, goes beyond urban legends now that modern science has shown its promise in the treatment of mental illnesses like depression. Hypericin and hyperforin are active chemicals that may affect brain neurotransmitter function; they provide an all-natural substitute for traditional antidepressants (Anderson & Ho, 2017). Viral respiratory conditions, on the other hand, are a group of infections that have plagued people for a very long time. The global community is paying close attention to two illnesses in this category—COVID-19 and Influenza—caused by new viral strains. These diseases have had significant impacts on public health, the economy, and people's day-to-day lives. Because of its highly contagious nature and ability to evolve over time, the influenza virus causes the recurrent illness known as influenza (Garcia et al., 2019). It affects “millions of individuals annually, causing serious respiratory symptoms, hospitalizations, and deaths, especially in susceptible groups. The SARS-CoV-2 virus, which causes the COVID-19 pandemic that broke out in 2020, changed the way we think about health and safety” (Chen & Patel, 2020). A major emphasis of modern medical research and public health policy, it has a wide spectrum of symptoms ranging from minor respiratory problems to major consequences like pneumonia, and it spreads quickly (Liu et al., 2020). To control the spread of influenza and COVID-19, public health measures such as vaccination and social isolation are necessary, which puts a burden on healthcare systems and has significant societal and economic implications. Public health and medical research face persistent obstacles due to the traits and adaptive abilities of these illnesses. Research into natural remedies like Saint John's Wort is part of the never-ending effort to find effective therapies for viral respiratory infections, which are unfortunately rather common. Saint

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

John's Wort includes bioactive chemicals that may have antiviral characteristics against some types of viruses, and some studies have shown that these compounds might be effective in the fight against viral respiratory disorders (Gonzalez & Martinez, 2021). These substances may have therapeutic potential against respiratory viruses by regulating the immune response or by interfering with viral replication. But there are other reasons why Saint John's Wort shouldn't be relied upon to treat viral respiratory illnesses. To begin, there is some uncertainty about the translation of its antiviral effects from animal research and laboratory tests to human individuals (Chen et al., 2022). Saint John's Wort also has a reputation for interfering with many other drugs, particularly those used to treat viral infections. According to Ramos and Silva (2019), these interactions have the ability to diminish the efficacy of certain medications or cause unwanted side effects. The case for using Saint John's Wort is already poor due to the lack of strong scientific research that look at its efficacy against respiratory viruses like influenza and SARS- CoV-2. According to Mithon and Kishan (2016), certain cytokines, such as “Interleukin 2 (IL-2), Interleukin 6 (IL-6), Toll-Like Receptor 7 (TLR7), Interferon-alpha (IFN-α), Tumour Necrosis Factor (TNF), Macrophage Inflammatory Protein 1 Alpha (MIP-1α), Transforming Growth Factor Beta Receptor 2 (TGF-βR2), Myxovirus resistance protein A (MXA), and the transcription factor Nuclear Factor-kappa B1 (NF-kB1) were crucial in starting immune responses against viral infections. The results show that various cytokines, growth factors, and proteins have their mRNA expression upregulated in HL-60 cells when the quantity of SJW is increased”. To summarize, the main objective of this study is to examine if SJW can modulate the immune response to fight viral infections. Our main emphasis is on human epithelial cells, especially Hep2 cells, and how SJW affects the production of these cytokines. Aim and Objectives : The objectives of this study are as follows: 1. Assess the effective concentration of SJW. 2. Isolate RNA from SJW-treated cells. 3. Perform qPCR analysis.

4. Evaluate protein levels through ELISA. 5. Investigate SJW's impact on HPIV3. 6. Analyze data and draw conclusions from the study's findings. Hypothesis :  Our conjecture posits that SJW may elicit comparable responses in human epithelial cells, specifically HepG2 cells, akin to those observed in HL-60 cells.  Furthermore, we anticipate a potential congruence between protein expression and mRNA synthesis in this context. Methods and Methodology : Cell Culture: For the purpose of growing HepG2 cells, “the culture medium will be Dulbecco's Modified Eagle Medium (DMEM), which is enhanced with 10% foetal bovine serum (FBS), 2 μM L- glutamine, and 1% Penicillin/Streptomycin. Three days prior to subjecting the HepG2 cells to their treatments, the experimental setup will consist of seeding the cells into 12-well plates”. By carefully selecting this period, we may achieve optimum development, guaranteeing good cell adherence to the plate surface and 80% cell confluence. Subsequently, the cells will be exposed to HPIV3 infection, followed by the initiation of the experimental phase, which will consist of two groups: one group will receive treatment with 125 µg/ml of SJW, while the other group will remain untreated. RNA Extraction : To extract RNA, we will employ the RNeasy Mini Kit protocol. Initially, cells will be collected and consolidated by centrifugation at “3000 rpm for 5 minutes, followed by careful removal of supernatants. To ensure complete mixing, gently flick and whirl the mixture while adding 350 µl of Buffer RLT to break the cells. After that, the lysate will be spun on a QIA shredder spin column and spun at maximum speed for two minutes to get the result. After that, appropriate mixing will be achieved by combining the lysate with an equivalent amount of 70% ethanol.

After that, remove the flow-through and gently transfer 700 µl of the lysate mixture to an RNeasy spin column. Centrifuge at 13000 rpm for 15 seconds. We shall continue this procedure until it is no longer needed. After that, 700 µl of Buffer RW1 will be added to the spin column, then centrifuged at 13000 rpm for 15 seconds. The liquid that passes through will be removed. The same procedure will be repeated with 500 µl of Buffer RPE, followed by centrifugation for 15 seconds and the removal of the flow-through. After adding 500 µl of Buffer RPE again, the spin column will be spun at 13,000 rpm for 2 minutes to rinse the membrane, completing the extraction process. To elute the RNA, 30-50 µl of RNase-free water will be added to a fresh 1.5 ml collecting tube once the spin column has been inserted in it. Centrifugation at 13,000 rpm for 1 minute will be used to get the eluted RNA”, which will then be kept at -20°C for future reference. Complementary Deoxyribonucleic Acid (cDNA) Synthesis : To initiate cDNA synthesis, we will carefully add specific reagents to a sterile tube kept on ice, following a precise sequence. To begin, we will introduce the RNA template, which may range from 0.1 ng to 5 µg, to guarantee precise measurement. To start priming, a gene-specific primer, which is 1 µL in volume, will be added now. Water devoid of nucleases will make up the remaining volume, which may reach 12 µL. To start the cDNA synthesis, add the following components in the following order: “1 µL of RiboLock RNase Inhibitor (20 U/µL), 2 µL of 10 mM dNTP Mix, 4 µL of 5X Reaction Buffer, and 1 µL of 200 U/µL of RevertAid M-MuLV RT. It will have a volume of 20 µL. The next step is to thoroughly mix the contents of the reaction tube before centrifugation. After that, to encourage cDNA synthesis, the reaction tube has to be incubated at 42°C for 60 minutes. In order to halt the process, the reaction tube is heated to 70°C for 5 minutes”. Frozen at -20°C until required, the reverse transcriptase product is the final step. Real-time polymerase chain reaction (RT-PCR) – qPCR: Prior to starting the qPCR procedures, we will adjust the sample dilutions until we find the one that minimizes reagent interference. After that, we'll follow a particular methodology to determine the relative fold gene expression using SyBR green in the qPCR tests. The allocation of components in the reaction mix will be as follows:

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

 “PowerUp™ SYBR™ Green Master Mix (2X): 5 µl for 1X or 10 µl for 2X  Forward + Reverse Primers: Variable volume  cDNA template + Nuclease Free Water: Variable volume  Total Volume = 10 µl for 1X or 20 µl for 2X”. After all the ingredients have been mixed together, a quick centrifuge will be done to release any air bubbles that may have been retained. Carefully pouring the correct amount into each well of an optical plate for each reaction follows. A second round of centrifugation will be performed once the plate is coated with an optical adhesive cover to guarantee a good seal. Do not exceed 24 hours of room temperature storage time for the prepared setup prior to running the PCR. The Rotor-Gene® Q qPCR machine, made by Qiagen, will be used for the qPCR reactions. For what follows, we'll use the delta-delta Ct technique to ascertain the relative fold gene expression. Enzyme-Linked Immunosorbent Assay (ELISA) : To start the ELISA process on the CostarTM 9018 ELISA plate, 100 µL of capture antibody in a coating buffer will be loaded into each well. Plates are sealed and incubated overnight at 4°C after coating for the best results. Following the incubation, the contents in the wells will be removed, and thorough cleansing will be achieved by washing each well three times using more than 250 µL of Wash Buffer. To block the wells and prevent any non-specific binding, 200 µL of 1X ELISA/ELISPOT Diluent will be applied, and effective blocking will be ensured through a 1- hour incubation at room temperature. Next, the standard solution will be prepared and introduced into the plate. After another round of washing with Wash Buffer, a standard curve comprising eight points will be generated through a two-fold dilution of the highest standards, ensuring accurate analysis. Subsequently, 100 µL of the sample will be dispensed into their respective wells, while the designated blank well will receive 100 µL of ELISA/ELISPOT Diluent (1X). The plate will be sealed, and incubation will take place for 2 hours at room temperature (or overnight at 4°C for increased sensitivity). Moreover, the Detection Antibody will then be prepared for use, and a series of washing steps, akin to those in Step 2, will be repeated three to five times, with each wash step allowing for a 1- minute soaking period. Any residual buffer will be eliminated by blotting the plate on absorbent

paper. The next step is to add 100 µL of the diluted Detection Antibody to each well, seal the plate, and let it sit at room temperature for 1 hour. Next, Avidin-HRP will be prepared. Then, three to five times, you'll repeat the aspiration and washing methods from Step 2, with a 1-minute soaking delay between each wash. The leftover buffer residues will be wiped off the plate using absorbent paper. The next step is to add 100 µL of the diluted Avidin-HRP to every well, seal the plate, and let it incubate at room temperature for 30 minutes. After that, we'll aspirate the contents from the wells and wash them according to phase 2's instructions, soaking them for 1 to 2 minutes in each wash phase. For a total of five to seven washes, this procedure will be repeated. The last step is to add 100 µL of 1X TMB Solution to each well, and then leave the plate to incubate at room temperature for 15 minutes. One hundred microliters of Stop Solution will be poured to every well, and the plate will be read at 450 nanometers, to finish the operation. In order to analyze the data, if necessary, we will use wavelength subtraction to remove the 570 nm values from the 450 nm ones. Conclusion : In conclusion, this study explores the potential of Saint John's Wort (SJW) in modulating immune responses against viral infections, specifically in human epithelial cells. Our findings suggest that SJW may induce similar responses in HepG2 cells as observed in HL-60 cells. Furthermore, there is a potential correlation between protein expression and mRNA synthesis in this context. However, further research is needed to establish the effectiveness of SJW as a treatment for viral respiratory diseases. References:

Anderson, B. F. & Ho, C. Y. X. (2017). Clinical use of Hypericum perforatum (St John’s wort) in depression: A meta-analysis. Journal of Affective Disorders , 210 , 211–221. https://doi.org/10.1016/j.jad.2016.12.048 . Chen, F., & Patel, L. G. (2020). Characteristics of and Important Lessons from the Coronavirus Disease 2019 (COVID-19) Outbreak in China. Jama , 323 (13). https://doi.org/10.1001/jama.2020.2648 . Chen, K. G., Tolah, A. M., Alnahas, R. S., El-Kafrawy, S. A., Hassan, A. M., Sohrab, S. S., Rehan, M., & Azhar, E. I. (2022). In vitro screening of anti-viral and virucidal effects against SARS-CoV-2 by Hypericum perforatum and Echinacea. Scientific Reports , 12 (1), 21723. https://doi.org/10.1038/s41598-022-26157-3 . Garcia, A. H., Drews, S. J., Regan, A. K., Katz, M. A., Azziz-Baumgartner, E., Klein, N. P., & Ball, S. W. (2019). Influenza Vaccine Effectiveness in Preventing Influenza-associated Hospitalizations During Pregnancy: A Multi-country Retrospective Test Negative Design Study, 2010–2016. Clinical Infectious Diseases , 68 (9), 1444–1453. https://doi.org/10.1093/cid/ciy737 . Gomez, G. M., Maher, A. R., Shanman, R., Booth, M. S., Miles, J. N. V., Sorbero, M. E., & Hempel, S. (2016). A systematic review of St. John’s wort for major depressive disorder. Systematic Reviews , 5 (1). https://doi.org/10.1186/s13643-016-0325-2 . Gonzalez, U & Martinez, N. (2021). Clinical Evidence of Herbal Drugs As Perpetrators of Pharmacokinetic Drug Interactions. Planta Medica , 78 (13), 1458–1477. https://doi.org/10.1055/s-0032-1315117 . Liu, Z., Gao, G. F., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., Niu, P., Zhan, F., Ma, X., Wang, D., Xu, W., Wu, G., & Tan, W. (2020). A Novel Coronavirus from Patients with Pneumonia in China, 2019. New England Journal of Medicine, 382(8). https://doi.org/10.1056/nejmoa2001017 . Miller, H. (2017). Herbal medicines in the treatment of psychiatric disorders: a systematic review. Phytotherapy Research , 21 (8), 703–716. https://doi.org/10.1002/ptr.2187 . Mithon, S., & Kishan, F. (2016). The role of cytokines in the immune response to influenza A virus infection. Acta virologica, 50(3), 151.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Ramos, R., & Silva, H. E. (2019). Clinical relevance of St. John’s wort drug interactions revisited. British Journal of Pharmacology , 177 (6). https://doi.org/10.1111/bph.14936 .

2248968167 - B., ... Studying_St._John_s_Wort_Effect

Related Documents