Screening of Differentially Expressed Genes in Rat Synovitis by Transcriptome Sequencing and in Vitro Verification of Therapeutic Target of Fraxetin

doi:10.12300/j.issn.1674-5817.2022.100

Abstract

Abstract:

Objective Using transcriptome sequencing to screen the differentially expressed genes between the synovial tissue of rats with knee osteoarthritis (KOA) induced by monosodium iodoacetate (MIA) and that of normal rats, and then screen the target of fraxetin in the treatment of synovitis. Methods SD rats were divided into KOA group and the negative control (NC) group. Rat right knee KOA model was prepared by MIA knee joint injection in KOA group and none treatments in NC group. Four weeks after modeling, the right knee synovial tissue of rats in each group was taken for transcriptome sequencing. Then the differential gene expression analysis, GO enrichment analysis, KEGG function enrichment analysis and PPI protein network interaction analysis were performed. The synovial macrophage Raw264.7 cells were divided into the control group, lipopolysaccharide (LPS) intervention group and LPS+60 μmol/L fraxetin intervention group, then RNA-sequencing results were verified by qRT-PCR in the three groups. Results The results of differential gene-expression analysis showed that there were 1 730 up-regulated genes and 1 546 down-regulated genes in the KOA group compared with the control group, among which the significantly up-regulated genes were mmp12, Acod1, Acan, Col2a1, Atp6v0d2 (|log2(FoldChange)|≥1, adjusted P<0.01). KEGG cluster analysis and GO cluster analysis showed that differential genes were mainly involved in the regulation of inflammation and immune metabolism, such as tricarboxylic acid cycle and mitochondrial function. The expressions of Acod1 and Atp6v0d2 in Raw264.7 cells after LPS intervention were significantly higher. Compared with the LPS intervention group, the expression level of Atp6v0d2 in Raw264.7 cells after LPS+fraxetin combined intervention was significantly lower. Conclusion After modeling KOA induced by MIA, macrophage-related genes mmp12, Acod1 and Atp6v0D2, which mediate inflammation and immune metabolism, were highly expressed in the synovial tissue of rats, suggesting that there might be immune metabolism changes mediated by synovial macrophages during the occurrence and development of KOA. The increased expression of Acod1 and Atp6v0d2 in macrophages Raw264.7 after LPS intervention can preliminarily confirm this result. Among them, Atp6v0d2 may be a potential target of fraxetin in the treatment of synovitis, which provides a new idea for KOA treatment.

Key words: Osteoarthritis, Transcriptome sequencing, Immunometabolism, Synovial macrophage, Fraxetin

CLC Number:

Q95-33

Ling YANG, Di ZHUANG, Lilun JIN. Screening of Differentially Expressed Genes in Rat Synovitis by Transcriptome Sequencing and in Vitro Verification of Therapeutic Target of Fraxetin[J]. Laboratory Animal and Comparative Medicine, 2023, 43(1): 11-20.

Figures/Tables 6

Figure 1 Gene expression correlation analysis （A） and principal components analysis （B） between the samplesNote： A, showed the correlation test, the closer the correlation coefficient is to 1, the higher the sample similarity is. In the negative control group (NC) and knee osteoarthritis model group (OA), the intra-group samples are more similar than inter-group samples. B, performed a principal component (PC) analysis, samples from the same group packed together in the figure, which indicates that the sequencing data are of reliable quality.

Table 1 Genes up-regulated and down-regulated in synovial tissues of knee osteoarthritis model rats iduced by monosodium iodoacetate

差异Difference	基因Gene	log2FC	P值P value	校正P值P_adj value
上调Up-regulated	Mmp12	9.933 625 864	9.132 91×10^-11	3.537 83×10^-9
	Acod1	9.272 106 280	0.000 127 507	0.000 802 842
	Col2a1	9.153 847 197	7.690 69×10^-9	1.807 62×10^-7
	Atp6v0d2	8.282 894 807	5.603 80×10^-6	5.607 99×10^-5
	Fbn2	7.973 227 345	2.921 38×10^-37	3.387 43×10^-34
	Acan	7.718 643 189	4.203 11×10^-20	1.015 34×10^-17
	Ocstamp	7.591 893 578	0.001 876 497	0.007 726 778
	LOC103690326	7.546 217 455	7.903 40×10^-5	0.000 529 726
	Cxcl2	7.422 908 965	1.713 86×10^-5	0.000 145 198
	Mmp9	7.188 248 072	2.374 04×10^-11	1.027 150×10^-9
下调Down-regulated	Plekha2	-1.000 555 825	1.696 39×10^-5	0.000 143 999
	Nudt7	-1.001 123 965	5.602 09×10^-5	0.000 395 268
	Cdc42bpg	-1.002 206 901	0.000 106 010	0.000 684 033
	Mtus1	-1.002 219 197	0.000 139 053	0.000 863 768
	Ppp1r37	-1.002 289 468	4.948 870×10^-20	1.179 120×10^-17
	Oard1	-1.003 517 453	0.000 375 535	0.002 019 066

Figure 2 Volcano plots （A） and heat map （B） of differentially expressed genesNote：A， the abscissa is log2FC， the ordinate is -log10（adjusted P）. Red or green dots indicate significantly up-regulated and down-regulated genes， respectively. While， genes with no significantly differentially expressed are indicated with blue dots. B， Gene heat map， the abscissa is sample names， the ordinate is gene names， red indicates high gene expression， while green indicates low expression. NC， negative control group； OA， knee osteoarthritis model group.

Figure 3 Gene ontology （GO） enrichment analysis （A-C） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis （D） of differentially expressed genesNote：A-C，GO enrichment analysis showed three ontologies “molecular function”（A）， “cellular component”（B） and “biological process”（C）. The abscissa is Fold Enrichment， the ordinate is term， the size of the bubbles indicates the number of term-enriched genes. The bigger the bubble is， the more genes enriched under the term. The colors of different bubbles correspond to P-value， the increasingly reddish colors means the more significant of P-value.

Figure 4 The protein-protein interaction （PPI） networks of differentially expressed genesNote：The size of the points means the degree of the node. The more protein connects with the node, the bigger the node will become. The red notes mean the up-regulated genes, the green ones mean down-regulated genes.

Figure 5 Atp6v0d2 and Acod1 gene expression in synovial macrophage RAW264.7 cells treated with lipopolysaccharide （LPS） and Fraxetin detected by real-time fluorescence quantitative PCRNote：Compared with the control group，****P<0.000 1； Compared with the LPS group， ###P<0.001. ns indicated none statistically significant differences. n=3。

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