MiR-9-5p reduces neuroinflammation and apoptosis in rats with traumatic brain injury by down-regulating CXCR4

doi:10.3877/cma.j.issn.2095-1221.2024.02.001

Abstract

Abstract:

Objective

To explore the mechanism of miR-9-5p in reducing neuroinflammation and apoptosis in rats with traumatic brain injury (TBI) by regulating CXC chemokine receptor 4 (CXCR4) .

Methods

The rats with TBI model were constructed by a hydraulic controllable injury device. They were randomly divided into the model group, miR-9-5p agomir group, miR- 9-5p negative control group, CXCR4 agonist NUCC-390 (2.2 mg/kg) group, and miR-9-5p agomir+NUCC-390 group, with 12 rats per group, another 12 rats were selected as the sham group. After intervention with drugs, the neurological deficit scores of rats were evaluated; The Morris water maze experiment was used to test the cognitive ability of rats, and the average escape latency and the times of crossing platform of each group were compared; TUNEL staining was used to detect the apoptosis rate of hippocampal neurons in rats; kits were used to detect the levels of inflammatory mediator interleukin-18 (IL-18) , inducible nitric oxide synthase (iNOS) , and tumor necrosis factor-α (TNF-α) in serum and brain tissue of rats; qRT-PCR experiment was used to detect the expression level of miR-9-5p, CXCR4 mRNA in rat brain tissue; Western blot was used to detect the expression levels of CXCR4, apoptosis protein Bcl- 2, Bcl-2 associated X protein (Bax) , cleaved caspase-3, and caspase-3 in rat brain tissue. One- way analysis of variance was used for comparison among multiple groups, and SNK-q test was used for further pairwise comparison.

Results

Compared with the sham group, the times of crossing platform, brain tissue miR-9-5p, Bcl- 2 expression level of rats in the model group was reduced, the neurological impairment score, average escape latency, hippocampal neuron apoptosis rate [ (43.92 ± 4.75) % vs (1.31 ± 0.36) %], serum and brain tissue IL-18, iNOS and TNF-α levels [serum: (156.45 ± 20.62) vs (64.59 ± 12.04) ng/L, (67.81 ± 9.56) vs (12.03 ± 2.68) U/mL, (78.69 ± 8.56) vs (31.52 ± 5.03) ng/L; brain tissue: (169.47 ± 15.61) vs (75.58 ± 11.33) ng/g pro, (177.86 ± 19.54) vs (82.04 ± 9.67) U/g pro, (151.67 ± 12.57) vs (61.58 ± 6.53) ng/g pro], brain tissue CXCR4 mRNA and protein expression levels [ (1.54 ± 0.32) vs (0.97 ± 0.17) , (1.03 ± 0.12) vs (0.41 ± 0.10) ], Bax, cleaved caspase-3, and caspase-3 protein expression levels were increased (P < 0.05) . Compared with the model group, the times of crossing platform, brain tissue miR-9-5p, Bcl-2 expression level of rats in the miR-9-5p agomir group was increased, the neurological impairment score, average escape latency, hippocampal neuron apoptosis rate [ (2.10 ± 0.68) % vs (43.92 ± 4.75) %], serum and brain tissue IL-18, iNOS and TNF-α levels [serum: (68.03 ± 13.15) vs (156.45 ± 20.62) ng/L, (14.12 ± 3.71) vs (67.81 ± 9.56) U/ mL, (33.41 ± 6.40) vs (78.69 ± 8.56) ng/L; brain tissue: (82.01 ± 14.16) vs (169.47 ± 15.61) ng/g pro, (91.89 ± 11.72) vs (177.86 ± 19.54) U/g pro, (70.01 ± 7.42) vs (151.67 ± 12.57) ng/g pro], brain tissue CXCR4 mRNA and protein expression levels [ (1.08 ± 0.21) vs (1.54 ± 0.32, (0.45 ± 0.09) vs (1.03 ± 0.12) ], Bax, cleaved caspase-3, and caspase-3 protein expression levels were decreased (P < 0.05) ; NUCC-390 could attenuate the improving effect of miR-9-5p agomir on nerve function in TBI rats.

Conclusion

MiR-9-5p could down-regulate the expression of CXCR4 to reduce neuroinflammation, inhibit hippocampal neuronal cell apoptosis, improve the nerve function of TBI rats, and enhance their cognitive ability.

Key words: miR-9-5p, CXCR4, Traumatic brain injury, Neuroinflammation, Apoptosis

Ying Wang, Deping Wu, Yu Liu, Guodong Liu. MiR-9-5p reduces neuroinflammation and apoptosis in rats with traumatic brain injury by down-regulating CXCR4[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(02): 65-72.

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References 20

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基因	序列（5'→3'）	预期扩增长度（bp）
miR-9-5p	上游GCCGCTCTTTGGTTATCTAGCT	189
	下游GTGCAGGGTCCGAGGTAT
U6	上游CTCGCTTCGGCAGCACA	114
	下游AACGCTTCACGAATTTGCGT
CXCR4	上游CTCTGAGGCGTTTGGTGCT	325
	下游TGCCCACTATGCCAGTCAAG
GADPH	上游CTGGAGAAACCTGCCAAGTATG	81
	下游GGTGGAAGAATGGGAGTTGCT

基因	序列（5'→3'）	预期扩增长度（bp）
miR-9-5p	上游GCCGCTCTTTGGTTATCTAGCT	189
	下游GTGCAGGGTCCGAGGTAT
U6	上游CTCGCTTCGGCAGCACA	114
	下游AACGCTTCACGAATTTGCGT
CXCR4	上游CTCTGAGGCGTTTGGTGCT	325
	下游TGCCCACTATGCCAGTCAAG
GADPH	上游CTGGAGAAACCTGCCAAGTATG	81
	下游GGTGGAAGAATGGGAGTTGCT

分组	神经功能缺损评分（分）	凋亡率（%）	平均逃避潜伏期（s）	穿越平台所在位置次数（次）
假手术组	0.41 ± 0.13	1.31 ± 0.36	9.14 ± 2.35	7.92 ± 1.07
模型组	2.74 ± 0.52^a	43.92 ± 4.75^a	76.85 ± 10.52^a	3.48 ± 0.23^a
miR-9-5p阴性对照组	2.83 ± 0.57	43.18 ± 5.61	75.98 ± 11.16	3.46 ± 0.32
miR-9-5p agomir组	0.82 ± 0.27^b	2.10 ± 0.68^b	11.31 ± 3.08^b	7.01 ± 1.85^b
NUCC-390组	3.63 ± 0.64^b	65.86 ± 7.12^b	110.63 ± 15.64^b	1.03 ± 0.27^b
miR-9-5p agomir+NUCC-390组	2.50 ± 0.71^c	42.37 ± 5.12^c	75.27 ± 13.01^c	3.26 ± 0.85^c
F值	71.331	366.683	179.827	87.629
P值	< 0.001	< 0.001	< 0.001	< 0.001

分组	神经功能缺损评分（分）	凋亡率（%）	平均逃避潜伏期（s）	穿越平台所在位置次数（次）
假手术组	0.41 ± 0.13	1.31 ± 0.36	9.14 ± 2.35	7.92 ± 1.07
模型组	2.74 ± 0.52^a	43.92 ± 4.75^a	76.85 ± 10.52^a	3.48 ± 0.23^a
miR-9-5p阴性对照组	2.83 ± 0.57	43.18 ± 5.61	75.98 ± 11.16	3.46 ± 0.32
miR-9-5p agomir组	0.82 ± 0.27^b	2.10 ± 0.68^b	11.31 ± 3.08^b	7.01 ± 1.85^b
NUCC-390组	3.63 ± 0.64^b	65.86 ± 7.12^b	110.63 ± 15.64^b	1.03 ± 0.27^b
miR-9-5p agomir+NUCC-390组	2.50 ± 0.71^c	42.37 ± 5.12^c	75.27 ± 13.01^c	3.26 ± 0.85^c
F值	71.331	366.683	179.827	87.629
P值	< 0.001	< 0.001	< 0.001	< 0.001

分组	血清			脑组织
分组	IL-18 （ng/L）	iNOS （U/mL）	TNF-α （ng/L）	IL-18 （ng/g pro）	iNOS （U/g pro）	TNF-α （ng/g pro）
假手术组	64.59 ± 12.04	12.03 ± 2.68	31.52 ± 5.03	75.58 ± 11.33	82.04 ± 9.67	61.58 ± 6.53
模型组	156.45 ± 20.62^a	67.81 ± 9.56^a	78.69 ± 8.56^a	169.47 ± 15.61^a	177.86 ± 19.54^a	151.67 ± 12.57^a
miR-9-5p阴性对照组	154.87 ± 23.48	67.04 ± 10.15	80.49 ± 9.82	164.89 ± 20.42	173.84 ± 20.13	62.41 ± 8.84
miR-9-5p agomir组	68.03 ± 13.15^b	14.12 ± 3.71^b	33.41 ± 6.40^b	82.01 ± 14.16^b	91.89 ± 11.72^b	70.01 ± 7.42^b
NUCC-390组	238.56 ± 27.03^b	128.56 ± 12.37^b	115.78 ± 10.18^bc	247.58 ± 26.06^b	268.57 ± 26.33^bc	243.48 ± 23.85^b
miR-9-5p agomir+NUCC-390组	152.42 ± 18.47^c	65.12 ± 11.04^c	74.93 ± 8.29^c	156.48 ± 16.45	169.14 ± 21.01^c	140.21 ± 11.94^c
F值	129.155	271.358	180.589	150.909	154.506	356.443
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

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