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Estrogen-related receptor α (ERRα) functions in the hypoxic injury of microglial cells

Chao-Yang Deng
Chao-Yang Deng
, 
Ting-Ting Zhu
Ting-Ting Zhu
, 
Shuai Lian
Shuai Lian
, 
Jian-Fa Wang
Jian-Fa Wang
, 
Rui Wu
Rui Wu
 e 
Jia-San Zheng
Jia-San Zheng
   | 10 mar 2022
Journal of Veterinary Research's Cover Image
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Pubblicato online: 10 mar 2022
Pagine: 131 - 140
Ricevuto: 17 lug 2021
Accettato: 15 feb 2022
DOI: https://doi.org/10.2478/jvetres-2022-0009
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© 2022 C.Y. Deng et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

The effects of CoCl2 on BV2 cell activity. Values represent the mean ± standard deviation for three independent experiments. **P < 0.01
The effects of CoCl2 on BV2 cell activity. Values represent the mean ± standard deviation for three independent experiments. **P < 0.01

Fig. 2

The effects of different CoCl2 treatment times on the expression of hypoxic inducible factors. The nucleus is indicated with blue fluorescence (4′,6-diamidino-2-phenylindole – DAPI) and the hypoxic-inducible factor 1-alpha (HIF-1α) protein is indicated with green fluorescence. Scale bar: 50 μm
The effects of different CoCl2 treatment times on the expression of hypoxic inducible factors. The nucleus is indicated with blue fluorescence (4′,6-diamidino-2-phenylindole – DAPI) and the hypoxic-inducible factor 1-alpha (HIF-1α) protein is indicated with green fluorescence. Scale bar: 50 μm

Fig. 3

Expression of ERRα in BV2 cells. A –Expression of ERRα in the control, and XCT790 groups; B –Expression of ERRα in the control and ERRα agonist groups. Values represent the mean ± standard deviation for three independent experiments. **P < 0.01
Expression of ERRα in BV2 cells. A –Expression of ERRα in the control, and XCT790 groups; B –Expression of ERRα in the control and ERRα agonist groups. Values represent the mean ± standard deviation for three independent experiments. **P < 0.01

Fig. 4

The changes in hypoxic-inducible factor 1-alpha (HIF-1α) mRNA levels in BV2 cells after corresponding treatments. Values represent the mean ± standard deviation for three independent experiments. *P < 0.05; **P < 0.01
The changes in hypoxic-inducible factor 1-alpha (HIF-1α) mRNA levels in BV2 cells after corresponding treatments. Values represent the mean ± standard deviation for three independent experiments. *P < 0.05; **P < 0.01

Fig. 5

Expression of Beclin1 and light chain 3 (LC3) in BV2 cells. A – Expression of Beclin1 in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; B – The light chain (LC)3-II/LC3-I ratio in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; C – Expression of Beclin1 in the control, oestrogen-related receptor alpha (ERRα) agonist, ERRα agonist+CoCl2 and CoCl2 groups; D – The LC3-II/LC3-I ratio in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups; E – The changes in p62 mRNA levels in BV2 cells after corresponding treatments. Values represent the mean ± standard deviation for three independent experiments. **P < 0.01
Expression of Beclin1 and light chain 3 (LC3) in BV2 cells. A – Expression of Beclin1 in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; B – The light chain (LC)3-II/LC3-I ratio in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; C – Expression of Beclin1 in the control, oestrogen-related receptor alpha (ERRα) agonist, ERRα agonist+CoCl2 and CoCl2 groups; D – The LC3-II/LC3-I ratio in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups; E – The changes in p62 mRNA levels in BV2 cells after corresponding treatments. Values represent the mean ± standard deviation for three independent experiments. **P < 0.01

Fig. 6

Expression of p65, p38 and inhibitor of nuclear factor of kappa light polypeptide gene enhancer in B-cells, alpha (IκB-α) in BV2 cells. A – The p-p65/p65 ratio in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; B – The p-p38/p38 ratio in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; C – Expression of IκB-α in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; D – The p-p65/p65 ratio in the control, oestrogen-related receptor alpha (ERRα) agonist, ERRα agonist+CoCl2 and CoCl2 groups; E – The p-p38/p38 ratio in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups; F – Expression of IκB-α in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups. Values are expressed as means ± standard deviation. *P < 0.05; **P < 0.01
Expression of p65, p38 and inhibitor of nuclear factor of kappa light polypeptide gene enhancer in B-cells, alpha (IκB-α) in BV2 cells. A – The p-p65/p65 ratio in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; B – The p-p38/p38 ratio in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; C – Expression of IκB-α in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; D – The p-p65/p65 ratio in the control, oestrogen-related receptor alpha (ERRα) agonist, ERRα agonist+CoCl2 and CoCl2 groups; E – The p-p38/p38 ratio in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups; F – Expression of IκB-α in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups. Values are expressed as means ± standard deviation. *P < 0.05; **P < 0.01

Fig. 7

The mRNA expression in BV2 cells after the corresponding treatments. A– Interleukin 6 (IL-6) mRNA; B – Tumour necrosis factor alpha (TNF-α) mRNA; C – p65 mRNA; D – Interleukin 4 (IL-4) mRNA; E – Interleukin 10 (IL-10) mRNA. Values represent the mean ± standard deviation for three independent experiments. *P < 0.05; **P < 0.01
The mRNA expression in BV2 cells after the corresponding treatments. A– Interleukin 6 (IL-6) mRNA; B – Tumour necrosis factor alpha (TNF-α) mRNA; C – p65 mRNA; D – Interleukin 4 (IL-4) mRNA; E – Interleukin 10 (IL-10) mRNA. Values represent the mean ± standard deviation for three independent experiments. *P < 0.05; **P < 0.01

Fig. 8

Expression of fibronectin type III domain containing protein 5 (FNDC5), brain-derived neurotrophic factor (BDNF) and extracellular signal-regulated kinase 1/2 (ERK1/2) in BV2 cells. A– Expression of FNDC5 in the control, XCT790, ERRα) agonist, XCT790+CoCl2, ERRα agonist+CoCl2 and CoCl2 groups; B– Expression of BDNF in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; C– The p-ERK/ERK ratio in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; D– Expression of BDNF in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups; E– The p-ERK/ERK ratio in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups. Values represent the mean ± standard deviation for three independent experiments. *P < 0.05; **P < 0.01
Expression of fibronectin type III domain containing protein 5 (FNDC5), brain-derived neurotrophic factor (BDNF) and extracellular signal-regulated kinase 1/2 (ERK1/2) in BV2 cells. A– Expression of FNDC5 in the control, XCT790, ERRα) agonist, XCT790+CoCl2, ERRα agonist+CoCl2 and CoCl2 groups; B– Expression of BDNF in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; C– The p-ERK/ERK ratio in the control, XCT790, XCT790+CoCl2 and CoCl2 groups; D– Expression of BDNF in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups; E– The p-ERK/ERK ratio in the control, ERRα agonist, ERRα agonist+CoCl2 and CoCl2 groups. Values represent the mean ± standard deviation for three independent experiments. *P < 0.05; **P < 0.01

Fig. 9

Wound-healing assay of BV2 cells (400×). Values represent the mean ± standard deviation for three independent experiments. ERRα – oestrogen-related receptor alpha; *P < 0.05; **P < 0.01
Wound-healing assay of BV2 cells (400×). Values represent the mean ± standard deviation for three independent experiments. ERRα – oestrogen-related receptor alpha; *P < 0.05; **P < 0.01

Primary and secondary antibodies used for Western blotting

Antibody Dilution Catalogue number
Beclin1 1:1,000 11306-1-AP
LC3 1:1,000 14600-1-AP
BDNF 1:1,000 28205-1-AP
FNDC5 1:1,000 23995-1-AP
p-p65 1:1,000 19771-1-AP
p65 1:2,000 10745-1-AP
Primary p-p38 1:1,000 19771-1-AP
p38 1:1,000 14064-1-AP
IκB-α 1:1,000 10268-1-AP
P-ERK1/2 1:1,000 9101
ERK1/2 1:1,000 4695S
ERRα 1:1,000 13826
Tubulin 1:15,000 11224-1-AP
β-actin 1:15,000 60008-1-lg
HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) 1:8,000 SA00001-1
Secondary HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) 1:8,000 SA00001-2

Primers used for protein amplification

Protein Forward primer Reverse primer
IL-4 5ʹ-GGTCTCAACCCCCAGCTAGT-3ʹ 5ʹ-GCCGATGATCTCTCTCAAGTGAT-3ʹ
IL-10 5ʹ-GCTCTTACTGACTGGCATGAG-3′ 5′-CGCAGCTCTAGGAGCAT GTG-3′
IL-6 5ʹ-CCGGAGAGGAGACTTCACAG-3′ 5′-GGAAAT TGGGGTAGGAAGGA-3′
TNF-α 5ʹ-TACTGAACTTCGGGGTGAT TGGTCC-3ʹ 5ʹ-CAGCCTTGTCCCTTGAAGAGAAC-3ʹ
P65 5ʹ-GTATTGCTG TGCCTACCCGAAAC-3ʹ 5ʹ-GTTTGAGATCTGCCCTGATGGTAA-3ʹ
P62 5'-GTTATGGCGTCGTTCACGGT-3′ 5′-TCACAATGGTGGAGGGTGC-3′
HIF-1 α 5ʹ-GATGGGTTATGAGCCGGAAGA-3ʹ 5ʹ-CTGTGGCTGGGAGTTCTTC G-3ʹ
β-actin 5ʹ-CGTTGACATCCGTAAAGACC-3′ 5′-AACAGTCCGC CTAGAAGCAC-3′
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