2020.05.03.13

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SHORT RESEARCH REPORT / REPORTE DE INVESTIGACIÓN CORTO

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Inflammatory response of stem cell secreting conditioned media in SH-SY5Y cell line

Faizan Ahmad

Available from: http://dx.doi.org/10.21931/RB/2020.05.03.13

ABSTRACT

Mesenchymal stem cells (MSCs) are reported to secrete anti-inflammatory cytokines and growth factors, which makes MSCs a promising candidate in the treatment of various neurodegenerative diseases. SH-SY5Y show extreme inflammatory response under LPS and an inadequate inflammatory response when treated with Wharton's jelly, conditioned media. This study mainly focuses on the inflammatory (pro and anti-inflammatory) response of SH-SY5Y by gene expression study. SH-SY5Y cell line used for cell culture and RT- q PCR was done with 5 different primers. In this article, lipopolysaccharides (LPS) show a significant result in pro-inflammatory and pro-apoptotic. In this article, we focus on the therapeutic approach of stem cells, which reduce inflammation by secreting stem cell factors to cure various neurodegenerative diseases.

keywords- Inflammation, MSCs, SH-SY5Y, Gene expression, quantitative PCR

INTRODUCTION

SH-SY5Y is neuroblastoma human cell lines. SH-SY5Y secrete growth factors, cytokines, micro-RNAs, proteasomes, and exosomes, which play a vital role in inflammation¹. Inflammation includes two forms of signals, the signal that intimate and retain inflammation and signal that bar down the process Inequality between the two signals frond inflammation unhampered, which leads to cellular and tissue damage². In macrophages, inflammation has three significant roles, i.e., antigen presentation, phagocytosis, and immunomodulation, through the production of various cytokines and growth factors³. Macrophages have a choleric role in the initiation, maintenance, and resolution of inflammation ^4,11. Macrophages get activated and get deactivated during the inflammatory process^4,11. Activation signals include cytokines, lipopolysaccharides, and extracellular matrix proteins^2,3. LPS increases inflammation in which there is speedy cell proliferation with fast cell death⁵. Activated macrophages get deactivated by anti-inflammatory cytokines and cytokines rival, which mostly produced by macrophages^5,11. Macrophages have both beneficial and detrimental outcomes in inflammation and are promising therapeutic interference for inflammation^5,11. Conditioned media from MSCs contain various cytokines; growth factors and micro RNAs play an essential role in attuning inflammatory process⁸. Cytokines are called inflammatory mediators^2,5. IL-β is pro-inflammatory mediators, and IL10 and TGF-β are anti-inflammatory mediators². When there are neuroinflammation chemokines, regulate cell migration. Chemokines act as a neuro-modulators that increase inflammation and development^4,10.

When the expression of Bcl-2 is high, it protects neurons from neuronal death (in vivo and in vitro)¹³. Bcl2 is a protein that maintains cellular homeostasis by regulating apoptosis and has both anti-apoptotic and anti- necrotic effect^15,16. BAX is linked with Bcl-2, which is protein-encoding gene^15,16. Our study focuses on the inflammatory response of five different genes. BAX, IL-10 and TGF- β1 has anti- an inflammatory response which protects from neural cell death by reducing inflammation. We are moving towards therapeutics, so instead of stem cells, we focus on stem cell factors^6,7,14. Stem cell factors are considered as a promising therapeutic approach that helps in Parkinson's disease or Alzheimer's disease^6,7. In the future, it is predicted that stem cell factors can activate death neurons and can be a novel therapeutic approach^6,7,14.

METHODS

CELL CULTURE – DMEM media is used with 10% FBS, and 1% penicillin as an antibiotic which used for SHSY5Y cell culture, and the cells were incubated in T25 flask at 37°C with 5% CO2.

EXTRACTION and RT-q PCR – RNA was isolated by using Isoplus reagent. cDNA was synthesized using the Primer Script III First- Strand cDNA Synthesis kit (TAKARA BIO). Real-time RT-PCR analysis was performed using the Quanti Tect SYBR Green PCR kit (QIAGEN). All the above methods were executed according to the manufacturer's instructions. Real-time analysis of like BAX, Bcl-2, IL-1β, TGF- β1, and IL-10 was done in duplicates on a CFX384 Real-time PCR system (BIO-RAD). CFX Manager software was used to analyze the results.

Table 1.

Table 1. - List of primer sequences used for real-time RT-PCR analysis

RESULTS

IL-1β is pro-inflammatory, IL -10, and TGF –β1 are anti-inflammatory genes. When we see our result under three different concentrations i.e. LPS, Wharton's jelly conditioned media, and cord line conditioned media, we find that LPS under IL-1β has a fold change of 1.4. IL-10 and TGF-β have a fold change of 0.4 and 0.05, respectively. Cord line conditioned media in IL-10 and TGF-β1 has a fold change of 3.0 and 0.44, respectively. LPS is least in both IL-10 and TGF-β1 with 0.25and 0.005fold change, respectively. Wharton's jelly in all the three genes IL-1β, IL-10, and TGF-β1, show moderate fold change with 0.8, 1.7, and 0.34, respectively. Figure 1

BAX is pro-apoptotic, and Bcl2 is anti-apoptotic. In our result in BAX and Bcl2, there is a fold change of 7.2and 1.15 under LPS. BAX and Bcl2, when treated with Wharton's jelly conditioned media and cord line conditioned media, there is a fold change of 0.5,1.5,0.9 and 1.2, respectively.

Figure 1. Pro and anti-inflammatory, pro and anti-apoptotic patterns, under three different concentrations i.e. LPS, Wharton's jelly conditioned media, and cord line conditioned media

DISCUSSION

Cheng Chen et al., 2018 observed an anti-inflammatory effect under lipopolysaccharide¹². Cheng Chen et. Al., 2018 explains that when SH-SY5Yis treated with SG-Tang, then SH-SY5Y inflammation is reduced under retinoic acid added conditioned media¹². When we compare our result, we observe that our results are entirely contradictory in which LPS show inflammation and CM reduces inflammation. Hong Na Yang et al., 2018 result explains that NSC-CM improves the expression of Bcl-2 to inhibit cell apoptosis¹³. Our result also gives similar result in which Bcl2 under conditioned media inhibit apoptosis. Zhongyang Xu et al. .2019 paper explains that miR-124 suppresses cell apoptosis by inhibiting Bax in H₂O₂-treated BV-2 cells⁹. When we observe our result in BAX treated with LPS and LPS is very high, so speedy cell proliferation and so fast neuronal cell death.

CONCLUSIONS

Our main aim of this research study is to check the inflammatory response. Due to inflammation, there is a death of neuronal cells, and cell proliferation occurs. In our research instead of stem cells, we are using stem cell factors which act as therapeutic agent produced by genes likes IL-10, TGF-β1, and Bcl2 which can protect the neurons from death like in case of Parkinson's disease, Alzheimer disease, etc. In the future, stem cell factors can become a promising therapeutic approach for various neurodegenerative diseases.

Abbreviation: -

LPS- Lipopolysaccharide

WJ-CM- Wharton' Jelly Conditioned Media

CL-CM- Cord Line Conditioned Media

Acknowledgments

I would like to thanks Dr. Durai for technical support during this project.

Competing interests and Funding

The authors declare that there are no conflicts of interest, and the entire project was performed in CBCMT, VIT UNIVERSITY, VELLORE, INDIA.

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Received: 28 June 2020

Accepted: 15 July 2020

Faizan Ahmad*, Himalayan University, India

*Corresponding Author

Faizan Ahmad

BSc Biotechnology

HIMALAYAN UNIVERSITY, Itanagar

Arunachal Pradesh, India

Tel -91-9523395056

Email- [email protected]