Role of palmitoyltransferase modified NOD2 in brain injury after cardiopulmonary resuscitation in mice

doi:10.11958/20231874

Abstract

Abstract:

Objective To investigate the role of nucleotide-binding oligomerization domain 2 (NOD2) modified by palmitoyltransferase (ZDHHC5) in brain injury after cardiopulmonary resuscitation (CPR) in mice. Methods Twenty-four male C57BL/6J mice were divided into the blank group, the control group, the ZDHHC5-si group and the NOD2-si group, with 6 mice in each group. Except for the blank group without any treatment, CPR modeling was performed in the other three groups. At 24 h before CPR, mice in the ZDHHC5-si group and the NOD2-si group were injected with ZDHHC5 siRNA and NOD2 siRNA via tail vein, respectively. The modified neurological deficit scale (mNSS) was used to evaluate the neurological function at 24 h, 48 h and 72 h in each group. Blood samples and brain tissue were collected 72 h after modeling. Real-time fluorescent quantitative PCR (qPCR) was used to detect ZDHHC5 and NOD2 in brain tissue. The protein expression levels of IL-1β, TNF-α and IL-6 in plasma were detected by enzyme-linked immunosorbent assay (ELISA). Colorimetric method and thiobarbituric acid (TBA) method were used to detect protein expression levels of MDA and MPO in brain tissue, respectively. Western blot assay was used to detect expression levels of Cleaved Caspase-3, ZDHHC5 and NOD2 in brain tissue. HE pathological sections of brain tissue were observed under light microscope. The pathological sections of brain tissue were observed by TUNEL under fluorescence microscope. Results Compared with the blank group, the mNSS score, the expression levels of IL-1β, TNF-α, IL-6, MDA and MPO, and the protein expression levels of Cleaved Caspase-3, ZDHHC5 and NOD2 were significantly increased (P<0.05), and brain tissue damage and cell apoptosis were aggravated in the other three groups. Compared with the control group, the above indicators were significantly decreased in the ZDHHC5-si group and the NOD2-si group (P<0.05), and brain tissue damage and cell apoptosis were significantly attenuated. Compared with the NOD2-si group, the above parameters were significantly decreased (P<0.05), and brain tissue damage and cell apoptosis were further attenuated in the ZDHHC5-si group. Conclusion In the mouse CPR model, NOD2 can produce palmitoylated NOD2 after regulated by ZDHHC5, which further promotes the release of inflammatory factors and causes neuronal apoptosis, ultimately damaging brain tissue and affecting neurological function.

Key words: cardiopulmonary resuscitation, brain injuries, lipoylation, neuroprotection, nucleotide-binding oligomerization domain containing 2, zinc finger DHHC-type palmitoyltransferase 5

CLC Number:

R459.7，R651.15

Figures/Tables 8

References 25

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基因名称	引物序列（5'→3'）	产物大小/bp
β-actin	上游：GGCTGTATTCCCCTCCATCG	154
	下游：CCAGTTGGTAACAATGCCATGT	154
ZDHHC5	上游：AAACCCAGCAAGTATGTACCG	132
	下游：AATTGCATTGTAAATGGGCACTG	132
NOD2	上游：CAGGTCTCCGAGAGGGTACTG	156
	下游：GCTACGGATGAGCCAAATGAAG	156

基因名称	引物序列（5'→3'）	产物大小/bp
β-actin	上游：GGCTGTATTCCCCTCCATCG	154
	下游：CCAGTTGGTAACAATGCCATGT	154
ZDHHC5	上游：AAACCCAGCAAGTATGTACCG	132
	下游：AATTGCATTGTAAATGGGCACTG	132
NOD2	上游：CAGGTCTCCGAGAGGGTACTG	156
	下游：GCTACGGATGAGCCAAATGAAG	156

组别	24 h	48 h	72 h
空白组	0.00±0.00	0.00±0.00	0.00±0.00
对照组	15.33±0.82^a	11.83±0.75^a	10.00±0.89^a
ZDHHC5-si组	7.83±0.75^ab	6.00±0.63^ab	4.17±0.75^ab
NOD2-si组	10.83±0.75^abc	7.33±1.03^abc	5.50±0.55^abc
F	554.815^＊＊	281.175^＊＊	244.400^＊＊

组别	24 h	48 h	72 h
空白组	0.00±0.00	0.00±0.00	0.00±0.00
对照组	15.33±0.82^a	11.83±0.75^a	10.00±0.89^a
ZDHHC5-si组	7.83±0.75^ab	6.00±0.63^ab	4.17±0.75^ab
NOD2-si组	10.83±0.75^abc	7.33±1.03^abc	5.50±0.55^abc
F	554.815^＊＊	281.175^＊＊	244.400^＊＊

组别	ZDHHC5	NOD2
空白组	1.00±0.00	1.00±0.00
对照组	1.84±0.06^a	1.97±0.06^a
ZDHHC5-si组	1.38±0.07^ab	1.40±0.06^ab
NOD2-si组	1.62±0.05^abc	1.55±0.05^abc
F	294.434^＊＊	380.478^＊＊