Transcriptome sequencing-based study on the mechanism of action of Jintiange capsules (金天格胶囊) in regulating synovial mesenchymal stem cells exosomal miRNA and articular chondrocytes mRNA for the treatment of osteoarthritis

doi:10.19852/j.cnki.jtcm.20240927.004

Abstract

Abstract:

OBJECTIVE: To corroborate the efficacy of Jintiange capsules (JTGs) (金天格胶囊) in the treatment of osteoarthritis (OA) by exploring the potential mechanism of action of synovial mesenchymal stem cell exosomes (SMSC-Exos) and articular chondrocytes (ACs) through transcriptome sequencing (RNA-seq).

METHODS: Type II collagenase was used to induce OA in rats. The efficacy of JTGs was confirmed by macroscopic observation of articular cartilage, micro-CT observation, and safranin fast green staining. After SMSC-Exos and ACs were qualified, RNA-seq was used to screen differentially expressed miRNAs and mRNAs. The target genes of differentially expressed miRNAs in Synovial mesenchymal stem cells (SMSCs) were predicted based on the multiMiR R package. The co-differentially expressed genes of SMSC-Exos and ACs were obtained by venny 2.1.0. The miRNA-mRNA regulatory network was constructed by Cytoscape software. Based on the OmicShare platform, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed on the mRNA regulated by key miRNAs. Expression trend analysis was performed for co-differentially expressed genes. Correlation analysis was performed on micro-CT efficacy indicators, co-differentially expressed genes mRNA and miRNA.

RESULTS: The efficacy of each administration group of JTGs was significant compared with the model group. SMSC-Exos and ACs were identified by their characteristics. The expression of rno-miR-23a-3p, rno-miR-342-3p, rno-miR-146b-5p, rno-miR-501-3p, rno-miR-214-3p was down-regulated in OA pathological state, and the expression of rno-miR-222-3p, rno-miR-30e-3p, rno-miR-676, and rno-miR-192-5p expression was up-regulated, and the expression of all these miRNAs was reversed after the intervention with JTGs containing serum. The co-differentially expressed genes were enriched in the interleukin 17 signaling pathway, tumor necrosis factor signaling pathway, transforming growth factor-β signaling pathway, etc. The expression trends of Ccl7, Akap12, Grem2, Egln3, Arhgdib, Ccl20, Mmp12, Pla2g2a, and Nr4a1 were significant. There was a correlation between micro-CT pharmacodynamic index, mRNA, and miRNA.

CONCLUSION: JTGs can improve the degeneration of joint cartilage and achieve the purpose of cartilage protection, which can be used for the treatment of OA. SMSCs-related miRNA expression profiles were significantly altered after the intervention with JTGs containing serum. The 9 co-differentially expressed genes may be the key targets for the efficacy of JTGs in the treatment of OA rats, which can be used for subsequent validation.

Key words: transcriptome sequencing technology, osteoarthritis, Jintiange capsules, synovial mesenchymal stem cells, articular chondrocytes

CHEN Zhongying, ZHANG Xue, ZHANG Xiaofei, ZOU Junbo, YUAN Puwei, SHI Yajun. Transcriptome sequencing-based study on the mechanism of action of Jintiange capsules (金天格胶囊) in regulating synovial mesenchymal stem cells exosomal miRNA and articular chondrocytes mRNA for the treatment of osteoarthritis[J]. Journal of Traditional Chinese Medicine, 2024, 44(6): 1153-1167.

Figures/Tables 8

Figure 1 Pharmacodynamic investigation of SMSCs regulating ACs through exosomal miRNAs A: gross observation of articular cartilage in rats; A1: Blank group; A2: Model group; A3: Diacerein Capsules group; A4: 0.324 g/kg JTGs group; A5: 0.648 g/kg JTGs group; A6: 1.296 g/kg JTGs group; B: micro-CT subchondral bone imaging of rat knee specimens; B1: Blank group; B2: Model group; B3: Diacerein Capsules group; B4: 0.324 g/kg JTGs group; B5: 0.648 g/kg JTGs group; B6: 1.296 g/kg JTGs group; C: Staining diagram of the Safranin fast green; C1: Blank group (×5); C2: Blank group (×20); C3: Model group (×5); C4: Model group (×20); C5: Diacerein Capsules group (×5); C6: Diacerein Capsules group (×20); C7: 0.324 g/kg JTGs group (×5); C8: 0.324 g/kg JTGs group (×20); C9: 0.648 g/kg JTGs group (×5); C10: 0.648 g/kg JTGs group (×20); C11: 1.296 g/kg JTGs group (×5); C12: 1.296 g/kg JTGs group (×20). Blank and model groups: 0.5% CMC-Na at a daily dose of 5 mL/kg for 30 d; diacerein capsules group: diacerein at a daily dose of 9 mg/kg for 30 d; 0.324 g/kg JTGs group: the JTGs at a daily dose of 0.324 g/kg for 30 d; 0.648 g/kg JTGs group: the JTGs at a daily dose of 0.648 g/kg for 30 d; 1.296 g/kg JTGs group: the JTGs at a daily dose of 1.296 g/kg for 30 d. SMSCs: synovial mesenchymal stem cells; ACs: articular chondrocytes; JTGs: Jintiange capsules. Dyeing method of all pictures are the Safranin fast green staining method.

Table 1 Results of micro-CT analysis of morphological parameters of subchondral bone microstructure ($\bar{x}±s$)

Bone microstructure index	Blank group (n = 6)	Model group (n = 6)	Diacerein Capsules group (n = 6)	JTGs (0.324 g/kg) (n = 6)	JTGs (0.648 g/kg) (n = 6)	JTGs (1.296 g/kg) (n = 6)
BV/TV	66.28±6.53	47.90±4.87^a	62.70±12.68^c	53.82±4.88	60.65±10.66^b	59.68±6.21^b
BS/TV	19.08±0.35	15.18±2.24^a	18.48±1.66^c	18.05±3.15^b	18.45±1.73^c	18.27±1.76^b
BS/BV	24.95±1.87	39.17±6.12^a	26.31±5.39^c	33.98±6.09	28.04±4.00^c	30.27±2.37^c
Tb.Sp	75.66±31.69	137.79±20.97^a	80.83±30.09^c	121.84±39.54	87.92±36.77^b	105.88±36.33
Tb.Th	159.20±10.60	109.36±16.40^a	147.27±23.38^c	128.52±5.75^b	140.12±19.32^c	136.30±11.47^c
Tb.N	4.68± 0.16	3.93±0.32^a	4.52±0.20^c	4.22±0.30	4.45±0.16^c	4.34±0.35^b

Table 2 Screening results of the drug-containing serum concentrations of JTGs ($\bar{x}±s$)

Survival rate	Control group (n = 3)	IL-1β group (n = 3)	5 % JTGs medicated serum group (n = 3)	10 % JTGs medicated serum group (n = 3)	15 % JTGs medicated serum group (n = 3)
SMSCs	1.000±0.000	0.146±0.063^a	0.844±0.052^b	0.972±0.067^b	0.672±0.209^b
ACs	1.000±0.000	0.136±0.017^a	0.742±0.132^b	0.930±0.078^b	0.714±0.134^b

Figure 2 Identification of SMSC-Exos and their differentially expressed gene screening results A: SMSC-Exos transmission electron microscope; B: Exosome particle size analysis; C: Western blots to detect the expression of calnexin and actin protein in SMSC; D: Nanoflow method to detect CD9 (D1), CD81 (D2) and blank control (D3) protein expression; E: Volcano map of SMSC-Exos miRNA differentially expressed genes in normal group compared with model group; F: Volcano map of SMSC-Exos miRNA differentially expressed genes in the model group compared with the JTGs group. M: Marker; control: 293T cell; 30 μg/lane; CD9 and CD81: exosome marker proteins; Control: 10% fetal bovine serum; Model: 10% fetal bovine serum + 10 ng/ml of IL-1β; JTG: 10% drug-containing serum + 10 ng/mL of IL-1β. SMSC: synovial mesenchymal stem cells; SMSC-Exos: synovial mesenchymal stem cell exosomes; JTG: Jintiange capsules; IL-1β: Interleukin-1beta. The miRNAs with significant differences were identified using the criteria of |log2FC| ≥ 1 and P < 0.05.

Table 3 Significantly differentially expressed miRNAs

miRNA	Up / down	log₂FC	P value
rno-miR-342-3p	down	-3.263143907	0.000652152
rno-miR-146b-5p	down	-1.688859604	0.003207717
rno-miR-501-3p	down	-4.154136556	0.019277237
rno-miR-23a-3p	down	-1.275428183	0.024102849
rno-miR-214-3p	down	-1.432467001	0.047196955
rno-miR-222-3p	Up	-1.150188066	0.001046597
rno-miR-30e-3p	Up	-4.438090482	0.004065579
rno-miR-676	Up	-1.859923545	0.030041356
rno-miR-192-5p	Up	-1.302132946	0.042020132

Figure 3 Identification of ACs and their differentially expressed gene screening results A: result of toluidine blue ACs staining (×50); B: result of type Ⅱ collagen immunofluorescence staining; C: volcano plot of ACs differentially expressed genes in the normal group compared with the model group; D: Volcano map of ACs differentially expressed genes in the model group compared with the JTGs group. ACs: articular chondrocytes. The identification of ACs was conducted through toluidine blue staining and immunofluorescence staining for type II collagen. The mRNA of ACs exhibiting significant differences was identified based on the criteria of |log2FC| ≥ 0.5 and P < 0.05.

Figure 4 Screening of differentially expressed genes and their GO and KEGG enrichment analysis A: miRNA and mRNA co-intersection differentially expressed genes; B: result of GO enrichment analysis of co-intersecting differentially expressed genes; C: result of KEGG enrichment analysis of co-intersecting differentially expressed genes. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes. The miRNA-mRNA regulatory network was constructed by Cytoscape software.

Table 4 Expression and trend of differential genes in common intersection

Group	Ccl7 (n = 3)	Akap12 (n = 3)	Egln3 (n = 3)	Arhgdib (n = 3)	Ccl20 (n = 3)	Mmp12 (n = 3)	Pla2g2a (n = 3)	Grem2 (n = 3)	Nr4a1 (n = 3)
Control	1.000±0.036	1.000±0.028	1.000±0.012	1.000±0.131	1.000±0.070	1.000± 0.137	1.000±0.047	1.000±0.119	1.000±0.128
Model	1.699±0.055^a	0.633±0.034^c	1.727± 0.050^a	1.995±0.117^a	2.722±0.241^a	1.555±0.184^a	1.889±0.044^a	1.404±0.046^a	1.683±0.218^a
JTG	0.948±0.059^b	1.364±0.236^b	0.923± 0.024^b	0.445±0.045^b	0.643±0.023^b	0.809±0.009^b	0.220±0.014^b	0.461±0.032^b	0.650±0.031^b

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