Influence of miR-139-5p on cognitive dysfunction in rats with chronic cerebral hypoperfusion by targeting RIPK1/RIPK3/MLKL signal pathway

doi:10.11958/20230110

Abstract

Abstract:

Objective To investigate the influence of miR-139-5p on cognitive dysfunction in chronic cerebral hypoperfusion (CCH) rats by targeting receptor-interacting protein kinase-1 (RIPK1)/receptor-interacting protein kinase-3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL) signal pathway. Methods Sixty SPF SD rats were randomly divided into the sham operation group, the model group, the miR-NC group, the miR-139-5p mimic group and the miR-139-5p mimic+RIPK1 inhibitor Nec-1 group (miR-139-5p mimic+Nec-1 group), with 12 rats in each group. Except the sham group, the other groups used permanent ligation of bilateral common carotid arteries to construct rat CCH model. New object recognition experiment and Morris water maze were used to evaluate the cognitive function of rats. Enzyme linked immunosorbent assay (ELISA) was used to detect levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), inflammatory factor tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) in rat hippocampus. Real-time quantitative PCR (qPCR) was used to detect the expression of miR-139-5p, RIPK1, RIPK3, MLKL mRNA in rat hippocampus. Western blot method was used to detect the protein expression levels of RIPK1, RIPK3, MLKL, synaptophysin (SYP), postsynaptic density protein 95 (PSD95) and α-synuclein (α-SYN). Double luciferase reporter gene experiment was used to verify the relationship between miR-139-5p and RIPK1. Results Compared with the sham group, the resolution coefficient, target quadrant residence time, levels of miR-139-5p, GSH-Px, SOD and the protein expression levels of SYP and PSD95 decreased obviously in hippocampus of the model group rats (P<0.05). The escape latency extended, and levels of MDA, TNF-α and IL-6 in hippocampus increased. The mRNA and protein expression of RIPK1, RIPK2, MLKL, the protein expression of α-SYN increased obviously (P<0.05). Compared with the model group and the miR-NC group, the expression trends of related indexes were opposite to the above in the miR-139-5p mimic group (P<0.05). Nec-1 further promoted the recovery of cognitive function in CCH rats by up-regulating the expression of miR-139-5p (P<0.05). MiR-139-5p negatively regulated the expression of RIPK1. Conclusion The up regulation of miR-139-5p may alleviate the cognitive dysfunction of CCH rats by targeting the inhibition of RIPK1/RIPK3/MLKL signaling pathway.

Key words: cognitive dysfunction, RIPK1/RIPK3/MLKL pathway, MicroRNA-139-5p, chronic cerebral hypoperfusion

CLC Number:

R743.3

Figures/Tables 7

References 24

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基因名称	引物序列（5'→3'）	产物大小/bp
miR-139- 5p	上游：CAGCAGTGGCAGAGTGCG	159
miR-139- 5p	下游：CGATCCAGCCTACCTAGAGTGCA	159
RIPK1	上游：TTCATGGTGCACAAACCCCT	121
RIPK1	下游：TGACACAAGCGCAACAAAGG	121
RIPK3	上游：CATCGATCTCTCTCGTGGGC	185
RIPK3	下游：CGTACACGTAGTCCCACTCG	185
MLKL	上游：GGGAACACTCCACGGAAACA	117
MLKL	下游：GATCTTGCAGTCTCTCGGGG	117
U6	上游：GGTCAGCCAGCTCAAGCGTC	136
U6	下游：TGGACACGCTCGCTCTGAATCG	136
β-actin	上游：CCCGCGAGTACAACCTTCTT	98
β-actin	下游：AGGGTCAGGATGCCTCTCTT	98

基因名称	引物序列（5'→3'）	产物大小/bp
miR-139- 5p	上游：CAGCAGTGGCAGAGTGCG	159
miR-139- 5p	下游：CGATCCAGCCTACCTAGAGTGCA	159
RIPK1	上游：TTCATGGTGCACAAACCCCT	121
RIPK1	下游：TGACACAAGCGCAACAAAGG	121
RIPK3	上游：CATCGATCTCTCTCGTGGGC	185
RIPK3	下游：CGTACACGTAGTCCCACTCG	185
MLKL	上游：GGGAACACTCCACGGAAACA	117
MLKL	下游：GATCTTGCAGTCTCTCGGGG	117
U6	上游：GGTCAGCCAGCTCAAGCGTC	136
U6	下游：TGGACACGCTCGCTCTGAATCG	136
β-actin	上游：CCCGCGAGTACAACCTTCTT	98
β-actin	下游：AGGGTCAGGATGCCTCTCTT	98

组别	1 d	2 d	3 d	4 d	5 d
Sham组	30.28±3.75	26.62±3.37	24.39±2.72	20.38±2.30	16.60±1.87
Model组	53.65±5.02^a	50.40±4.16^a	48.45±4.21^a	45.32±3.85^a	41.06±3.77^a
miR-NC组	53.23±4.86	50.06±4.22	48.64±4.26	45.53±4.08	41.65±3.51
miR-139-5p mimic组	42.36±3.25^bc	37.14±3.54^bc	33.71±2.92^bc	29.42±2.39^bc	25.37±2.14^bc
miR-139-5p mimic+Nec-1组	33.55±2.38^d	28.10±1.95^d	25.55±1.67^d	22.83±1.23^d	18.62±1.15^d
F	88.897^＊＊	125.718^＊＊	154.959^＊＊	199.106^＊＊	241.944^＊＊

组别	1 d	2 d	3 d	4 d	5 d
Sham组	30.28±3.75	26.62±3.37	24.39±2.72	20.38±2.30	16.60±1.87
Model组	53.65±5.02^a	50.40±4.16^a	48.45±4.21^a	45.32±3.85^a	41.06±3.77^a
miR-NC组	53.23±4.86	50.06±4.22	48.64±4.26	45.53±4.08	41.65±3.51
miR-139-5p mimic组	42.36±3.25^bc	37.14±3.54^bc	33.71±2.92^bc	29.42±2.39^bc	25.37±2.14^bc
miR-139-5p mimic+Nec-1组	33.55±2.38^d	28.10±1.95^d	25.55±1.67^d	22.83±1.23^d	18.62±1.15^d
F	88.897^＊＊	125.718^＊＊	154.959^＊＊	199.106^＊＊	241.944^＊＊

组别	GSH-Px/（kU/g）	SOD/（kU/g）	MDA/（mmol/g）	TNF-α/（ng/L）	IL-6/（ng/L）
Sham组	28.04±3.36	50.47±2.63	0.78±0.08	95.15±9.24	12.53±1.14
Model组	8.67±0.95^a	12.24±0.88^a	3.24±0.25^a	316.42±26.32^a	58.63±6.05^a
miR-NC组	8.96±0.78	11.82±0.95	3.12±0.21	309.48±27.26	57.03±5.76
miR-139-5p mimic组	16.83±1.22^bc	26.41±2.38^bc	1.18±0.09^bc	248.37±25.40^bc	30.28±2.51^bc
miR-139-5p mimic+Nec-1组	27.74±2.30^d	43.10±3.59^d	0.80±0.03^d	176.45±16.34^d	16.49±1.73^d
F	140.124^＊＊	343.236^＊＊	383.479^＊＊	108.404^＊＊	178.559^＊＊