Effects of Hypoxic Stress and Reoxygenation on Hypoxic Response Genes and Physiological and Biochemical Indexes in the Gill Tissue of Leiocassis longirostris

doi:10.11924/j.issn.1000-6850.casb2022-0076

Abstract

Abstract:

In order to study the effects of hypoxic stress [(0.8±0.1) mg/L] and reoxygenation [(7.3±0.5 mg/L] on oxygen sensing, respiratory metabolism, oxidative stress, tissue structure, and apoptosis in the gill of L. longirostris, by using qRT-PCR, enzyme activity measurement, H&E staining and TUNEL slice detection, the experiments of hypoxic stress (0, 2, 4, 6 h) and reoxygenation (2, 4, 6 h) on L. longirostris were conducted and the changes of hypoxic response genes, and physiological and biochemical indexes in the gill of L. longirostris were analyzed. The results showed that: the expressions of genes related to oxygen sensors (HIF-1α, HIF-2α, PHDs and Vhl) were upregulated in the gill of L. longirostris during hypoxia, after reoxygenation, there was still a significant difference compared with the control (P<0.05). Expression activities and contents of glycolysis related enzymes (PFK, HK and PK), anaerobic respiration enzymes (LDH), antioxidant enzymes (GSH-Px, CAT and SOD) and oxidative stress indicators (MDA and LPO) significantly increased. The activities of TCA cycle-related enzymes (SDH and MDH) decreased significantly during hypoxia, but most physiological indexes gradually recovered to the level of the control upon reoxygenation. H&E staining showed that under hypoxic stress, the epithelium of the gill was uplifted, and some blood cells gathered in large numbers to make the gill small pieces have rod-like tips, and tissue changes such as swelling and proliferation of mitochondria-rich cells occurred, but these changes in tissue morphology caused by hypoxia were not improved after the recovery of dissolved oxygen. TUNEL sections showed that the number of apoptotic cells in the gill of L. longirostris increased with the prolongation of hypoxia time, the mRNA levels of apoptosis-related genes (Bax, Caspase 3, p53 and Apaf-1) increased significantly and Bcl-2 decreased significantly during hypoxia, and the apoptosis still existed compared with control upon reoxygenation. Hypoxic stress and reoxygenation can significantly affect the gill oxygen sensors, respiratory metabolism, oxidative stress, tissue structure, and apoptosis of L. longirostris. The results lay a theoretical foundation for exploring the molecular regulation mechanism of fish under hypoxic stress, and provide basic data for future development of new hypoxia tolerant varieties of L. longirostris.

Key words: Leiocassis longirostris, hypoxia, gill tissue, hypoxic response, physiological and biochemical

LI Yao, YANG Zhiru, CHENG Jinghao, LI Jie, WANG Tao, ZHANG Kai, ZHANG Guosong, YIN Shaowu. Effects of Hypoxic Stress and Reoxygenation on Hypoxic Response Genes and Physiological and Biochemical Indexes in the Gill Tissue of Leiocassis longirostris[J]. Chinese Agricultural Science Bulletin, 2023, 39(2): 107-116.

Figures/Tables 7

References 43

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基因	引物	引物序列(5’-3’)	长度/bp
IF-1α	HIF-1α-F	CTGGAAAGAGGGCTAAGGTG	20
IF-1α	HIF-1α-R	AGTGACGGTCCTGAATAGGG	20
HIF-2α	HIF-2α-F	AAAAGCCCAGTGGATGAC	18
HIF-2α	HIF-2α-R	TGGCGATGTTGCTGAAGT	18
PHD1	PHD1-F	AGCAATGGTGGCTTGTTAC	19
PHD1	PHD1-R	TCAAATAAGGGCTCAATGTC	20
PHD2	PHD2-F	AGCCTGGATGTGAGAAGATAGC	22
PHD2	PHD2-R	CCGTCTCCGTTAGGGTTGT	19
PHD3	PHD3-F	CTGTTTGATCGACTCCTGCTT	21
PHD3	PHD3-R	TGTGGTGCTGCTTTGTGAA	19
Vhl	Vhl-F	AGACGGATGACCCGATGTTG	20
Vhl	Vhl-R	GCACACTAGCTTCCTCACCA	20
Bax	Bax-F	TGACCGTGGCAAAGAGCA	18
Bax	Bax-R	GATCCAGGCGGAAATGTG	18
Caspase 3	Caspase 3-F	GGGAAACTGGGCTACAAA	18
Caspase 3	Caspase 3-R	CTCAGCAACACGCAAACA	18
Bcl-2	Bcl-2-F	CGTAGCCTCGCTTCAAAA	18
Bcl-2	Bcl-2-R	CGCGTCAGATCAATTCACA	19
p53	p53-F	CGTCGTAGCAGTGTCTAAAGT	21
p53	p53-R	GACCCTCTTGTCCATTGTG	19
Apaf-1	Apaf-1-F Apaf-1-R	TCGCCTCTGAACCCTGCTC CTGATGGAGTCCACTGGCTGTC	19 22
β-actin	β-actin-F	TGCTGCCTCTTCCTCCTCTC	20
β-actin	β-actin-R	GGACACCTGAACCTCTCATTGC	22

基因	引物	引物序列(5’-3’)	长度/bp
IF-1α	HIF-1α-F	CTGGAAAGAGGGCTAAGGTG	20
IF-1α	HIF-1α-R	AGTGACGGTCCTGAATAGGG	20
HIF-2α	HIF-2α-F	AAAAGCCCAGTGGATGAC	18
HIF-2α	HIF-2α-R	TGGCGATGTTGCTGAAGT	18
PHD1	PHD1-F	AGCAATGGTGGCTTGTTAC	19
PHD1	PHD1-R	TCAAATAAGGGCTCAATGTC	20
PHD2	PHD2-F	AGCCTGGATGTGAGAAGATAGC	22
PHD2	PHD2-R	CCGTCTCCGTTAGGGTTGT	19
PHD3	PHD3-F	CTGTTTGATCGACTCCTGCTT	21
PHD3	PHD3-R	TGTGGTGCTGCTTTGTGAA	19
Vhl	Vhl-F	AGACGGATGACCCGATGTTG	20
Vhl	Vhl-R	GCACACTAGCTTCCTCACCA	20
Bax	Bax-F	TGACCGTGGCAAAGAGCA	18
Bax	Bax-R	GATCCAGGCGGAAATGTG	18
Caspase 3	Caspase 3-F	GGGAAACTGGGCTACAAA	18
Caspase 3	Caspase 3-R	CTCAGCAACACGCAAACA	18
Bcl-2	Bcl-2-F	CGTAGCCTCGCTTCAAAA	18
Bcl-2	Bcl-2-R	CGCGTCAGATCAATTCACA	19
p53	p53-F	CGTCGTAGCAGTGTCTAAAGT	21
p53	p53-R	GACCCTCTTGTCCATTGTG	19
Apaf-1	Apaf-1-F Apaf-1-R	TCGCCTCTGAACCCTGCTC CTGATGGAGTCCACTGGCTGTC	19 22
β-actin	β-actin-F	TGCTGCCTCTTCCTCCTCTC	20
β-actin	β-actin-R	GGACACCTGAACCTCTCATTGC	22