The Sensitivity of Different Neural Cells to Hypoxia

doi:10.13523/j.cb.2202052

China Biotechnology

2022, Vol. 42

Issue (7): 1-11 DOI: 10.13523/j.cb.2202052

The Sensitivity of Different Neural Cells to Hypoxia

Wen-yu HU^1,^**,Shuo-shuo LI^2,^**,Jin-bo CHENG^2,^***(

),Zeng-qiang YUAN^2,^***(

)

1. School of Medicine, University of South China, Hengyang 421001, China
2. Institute of Military Cognition and Brain Science, Academy of Military Medical Sciences, Beijing 100850, China

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Abstract

Objective: In order to illustrate the hypoxia-induced changes of neural cells in inflammatory response, oxidative stress, and energy metabolism process and to compare the sensitivity of neural cells’ responses to hypoxia. Methods: Different types of neural cells (BV2, N9, Gl261, HT22) were treated with hypoxia (0.1% O₂, 5% CO₂) for 0-24 hours. Cell proliferation was detected by Cell Counting Kit-8 method and cell viability was assayed by CellTiter-Glo Luminescent Cell Viability Assay. Total RNA was extracted by Trizol reagent, and the inflammation, oxidative stress, and energy metabolism-related genes expression were measured by quantitative real-time PCR and Western blot. The ROS production was detected by flow cytometer with fluorescence probe. Results: Hypoxia stimulation decreased cell proliferation and cell viability. The hypoxia-induced changes of microglial cells (BV2 and N9) were mainly involved in inflammatory response and glucose metabolism process. The changes of astrocytes Gl261 and neural cell HT22 were mainly involved in glucose metabolism process. Hypoxia stimulation significantly increased oxidative stress in microglia and astrocytes. Conclusion: Different types of neural cells have different degrees of sensitivity in response to hypoxic stimulation. In terms of energy metabolism and inflammatory response, microglia are more sensitive to hypoxia treatment, which is manifested as a significant up-regulation of glycolytic enzymes and inflammation genes, whereas microglia and astrocytes are more sensitive to hypoxia treatment in terms of oxidative stress, which is indicated by their quick response and significant increase of ROS production.

Key words： Hypoxia Neural cells Inflammation Oxidative stress Energy metabolism

Received: 28 February 2022 Published: 03 August 2022

ZTFLH:

R34

Corresponding Authors: Wen-yu HU,Shuo-shuo LI,Jin-bo CHENG,Zeng-qiang YUAN E-mail: cheng_jinbo@126.com;zyuan620@yahoo.com

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Cite this article:

Wen-yu HU,Shuo-shuo LI,Jin-bo CHENG,Zeng-qiang YUAN. The Sensitivity of Different Neural Cells to Hypoxia. China Biotechnology, 2022, 42(7): 1-11.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2202052 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I7/1

Table 1 qRT-PCR primer sequence

Fig.1 Hypoxia stimulation decreased cell proliferation Neural cell proliferation curve [(BV2(a),N9(b),Gl261(c),HT22(d)] under hypoxia/normoxia treatment for 0-36 h. n=5;Student t-test; ^* P<0.05, ^** P<0.01, ^*** P<0.001; ns: No significant. Data are presented as mean±SEM

Fig.2 Hypoxia treatment decreased cell viability Intracellular ATP content across 4 neural cell lines at different timepoint of hypoxia [(BV2(a),N9(b),Gl261(c),HT22(d)] under hypoxia/normoxia treatment for 0-36 h. n=3; Student t-test; ^* P<0.05, ^** P<0.01, ^*** P<0.001; ns: No significant. Data are presented as mean ± SEM

Fig.3 Hypoxia treatment altered glucose metabolism in varies neural cells Dynamic alteration of quantitative real-time PCR was utilized to evaluate the mRNA level of glucose metabolism-related genes (HK1,HK2,LDHA,PDK1,PGK1,PKM2) in mRNA level in BV2 (a),N9 (b),Gl261 (c),HT22 (d) under hypoxia treatment. n=3; ND: Not determined

Fig.4 Changes in protein level after hypoxia Representative images of immunoblotting of glucose metabolism-related genes (HK1,HK2,PKM2,LDHA),oxidative stress-related genes (iNOS,SOD) in different neural cells [BV2 (a),N9 (b),Gl261 (c),HT22 (d)] under hypoxia treatment

Fig.5 Hypoxia treatment induced ROS generation in different neural cells ROS levels of neural cells [BV2 (a),N9 (b),Gl261 (c),HT22 (d)], treated with hypoxia, were determined by flow cytometry. NC: Negative control; PC: Positive control

Fig.6 Hypoxia treatment increased neural cells oxidative stress mRNA level of oxidative stress-related genes (iNOS,SOD,CAT) in different neural cells [BV2 (a),N9 (b),Gl261 (c),HT22 (d)] under hypoxia treatment. n=3; ND: Not determined

Fig.7 Inflammatory response in different neural cells after hypoxia Quantitative real-time PCR was utilized to evaluate the mRNA level of inflammatory factor (anti-inflammatory: IL-10,Arg1,CD206; pro-inflammatory: TNF-α,IL-6,IL-1β) in different neural cells [BV2 (a),N9 (b),Gl261 (c),HT22 (d)] under hypoxia treatment. n=3; ND: Not determined


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