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. 2022 Jun 22:13:772680.
doi: 10.3389/fphar.2022.772680. eCollection 2022.

Bioinformatics Analysis of miRNAs and mRNAs Network-Xuefu Zhuyu Decoction Exerts Neuroprotection of Traumatic Brain Injury Mice in the Subacute Phase

Zhao-Yu Yang  1   2 , Yao Wu  1   2 , Xuexuan Li  1   2 , Tao Tang  1   2 , Yang Wang  1   2 , Ze-Bing Huang  2   3 , Rong Fan  1   2
Affiliations

Bioinformatics Analysis of miRNAs and mRNAs Network-Xuefu Zhuyu Decoction Exerts Neuroprotection of Traumatic Brain Injury Mice in the Subacute Phase

Zhao-Yu Yang et al. Front Pharmacol. .

Abstract

Xuefu Zhuyu decoction (XFZYD) is used to treat traumatic brain injury (TBI). XFZYD-based therapies have achieved good clinical outcomes in TBI. However, the underlying mechanisms of XFZYD in TBI remedy remains unclear. The study aimed to identify critical miRNAs and putative mechanisms associated with XFYZD through comprehensive bioinformatics analysis. We established a controlled cortical impact (CCI) mice model and treated the mice with XFZYD. The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) confirmed the quality of XFZYD. The modified neurological severity score (mNSS) and Morris water maze (MWM) tests indicated that XFZYD improved the neurological deficit (p < 0.05) and cognitive function (p < 0.01). Histological analysis validated the establishment of the CCI model and the treatment effect of XFZYD. HE staining displayed that the pathological degree in the XFZYD-treated group was prominently reduced. The transcriptomic data was generated using microRNA sequencing (miRNA-seq) of the hippocampus. According to cluster analysis, the TBI group clustered together was distinct from the XFZYD group. Sixteen differentially expressed (5 upregulated; 11 downregulated) miRNAs were detected between TBI and XFZYD. The reliability of the sequencing data was confirmed by qRT-PCR. Three miRNAs (mmu-miR-142a-5p, mmu-miR-183-5p, mmu-miR-96-5p) were distinctively expressed in the XFZYD compared with the TBI and consisted of the sequencing results. Bioinformatics analysis suggested that the MAPK signaling pathway contributes to TBI pathophysiology and XFZYD treatment. Subsequently, the functions of miR-96-5p, miR-183-5p, and miR-142a-5p were validated in vitro. TBI significantly induces the down-expression of miR-96-5p, and up-expression of inflammatory cytokines, which were all inhibited by miR-96-5p mimics. The present research provides an adequate fundament for further knowing the pathologic and prognostic process of TBI and supplies deep insights into the therapeutic effects of XFZYD.

Keywords: Bioinformatic analysis; MicroRNAs; neurological recovery; traumatic brain injury; xuefu zhuyu decoction.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Qualitative analysis. (A) Amygdalin, neohesperidin, rutin, and digaoxin of standard agents detected by LC chromatogram. (B) Amygdalin, neohesperidin, rutin, and digaoxin of XFZYD in plasma samples post-CCI.
FIGURE 2
FIGURE 2
Effects of XFZYD on TBI. (A) The mNSS of sham, TBI, and XFZYD groups on post-injury, 1st day, 3rd day, 7th day, and 14th day. (0 day presents post-injury) (B) Time in the target quadrant of sham, TBI, and XFZYD groups. (C) The swimming speed of sham, TBI, and XFZYD groups. (D) HE staining (400 x) in CA1 region on 14th day, scar bar = 20 μm. Data are displayed as mean ± SD, n = 6 mice per group, *p < 0.05, **p < 0.01.
FIGURE 3
FIGURE 3
(A) Volcano plot of distinctively expressed miRNA in XFZYD/TBI. The red dots represent upregulated miRNAs, and the blue dots represent downregulated miRNAs (log2 (fold change) > 0.3 and p-value < 0.05). (B) Hierarchical clustering of DEMs in XFZYD/TBI. Ten samples were obtained from the TBI group and XFZYD group. Hierarchical clustering was formed based on miRNAs’ expression levels. Green, downregulated; red, upregulated (n = 5 mice per group). TBI: traumatic brain injury; XFZYD: Xuefu Zhuyu decoction.
FIGURE 4
FIGURE 4
Validation of the RNA-sequencing data. Statistical difference analysis of the relative expression levels of miR-96-5p, miR-183-5p, and miR-142a-5p in TBI and XFZYD groups. Data are exhibited as mean ± SEM, n = 5 mice per group, *p < 0.05, **p < 0.01.
FIGURE 5
FIGURE 5
The miRNA-target genes network. Pink dots represent upregulated miRNAs (miR-96-5p and miR-183-5p); orange nodes represent downregulated miRNAs (miR-142a-5p); blue dots represent target genes, and solid gray lines illustrate correlations between miRNAs and target genes.
FIGURE 6
FIGURE 6
The biological functions analysis of differential expressed miRNAs target genes. The significantly enriched GO biological processes of upregulated miRNAs (A) and downregulated miRNAs (B). GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes.
FIGURE 7
FIGURE 7
The KEGG enrichment analysis of up-regulated genes. The y-axis and x-axis depict KEGG-enriched terms and the Gene Ratio, respectively. The size of the dot means the number of genes and the color of the dots represents the p-value. The left cluster plot shows a chord dendrogram of clustering the expression spectrum of significantly DE genes. DE, differentially expressed; KEGG, Kyoto Encyclopedia of Genes and Genomes.
FIGURE 8
FIGURE 8
:The KEGG enrichment analysis of down-regulated genes. The y-axis and x-axis depict KEGG-enriched terms and the Gene Ratio, respectively. The size of the dot means the number of genes and the color of the dots represents the p-value. The left cluster plot shows a chord dendrogram of clustering the expression spectrum of significantly DE genes. DE, differentially expressed; KEGG, Kyoto Encyclopedia of Genes and Genomes.
FIGURE 9
FIGURE 9
Expression of miR-96-5p (A), miR-183-5p (B), and miR-142a-5p (C) in BV2 cells with scratch injury (*p < 0.05, ***p < 0.01 presented TBI compared to control group). qRT-PCR was used to detect the expression of miR-96-5p (D), miR-183-5p (E), and miR-142a-5p (F) in the BV2 cells transfected with miR-96-5p mimic, miR-183-5p mimic, and miR-142a-5p inhibitor (*p < 0.05, ***p < 0.01 presented miRNAs compared to NC group). The data are exhibited as the mean ± SEM (n = 3).
FIGURE 10
FIGURE 10
Overexpression of miR-96-5p or miR-183-5p and inhibition of miR-142a-5p suppress the expression of inflammatory cytokines. ELISA was applied to detect the levels of IL-1β (A), IL-6 (B), and TNF-α (C) in the BV2 cells culture medium. (D) The binding region and seed sequence between miR-96-5p and Rasa1. (E) Validation of Rasa1’s levels by qRT-PCR. miRNAs could silence the mRNAs, Rasa1 was significantly upregulated in scratch injury and downregulated in miR-96-5p mimic. The data are exhibited as the mean ± SD (n = 3). ***p < 0.01, TBI group vs. control group; ## p < 0.01 and ### p < 0.001, miRNAs mimic or inhibitor groups vs. TBI group.
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