Jintiange Capsule Alleviates Rheumatoid Arthritis and Reverses Changes of Serum Metabolic Profile in Collagen-Induced Arthritic Rats

Figures & data

Figure 1 Schematic of the animal experiment design and process.

Table 1 Bioactive Peptides Identified from JTG

No.	Peptide	Mass	Length	m/z	RT	Accession
1	PSGPQTP	682.3286	7	683.3365	0.049	P21692|MMP1_PIG
2	GPAGDARVP	838.4297	9	420.2223	1.0986	Q29400|IBP2_SHEEP
3	HGCLLLSPGN	1009.5015	10	505.7546	2.0037	Q5MNU5|SCAP_PIG
4	AGPAGDARVP	909.4668	10	455.7408	4.2283	Q29400|IBP2_SHEEP
5	VKAHGQ(+0.98)KVADAL	1236.6826	12	619.3491	6.0053	P01965|HBA_PIG
6	GEKVAN(+0.98)AL	801.4232	8	401.7187	12.5431	P21379|HBA_RANTA; P01971|HBA_ALCAA
7	VGN(+0.98)HSAGSAVGPEAAGAPVPMQ(+0.98)GEAPQ	2487.1335	27	830.0527	16.886	Q9XT50|ATP7B_SHEEP
8	PASTPPAAPA	878.4498	10	440.2324	23.7267	Q9TU17|CXA3_SHEEP
9	AAALRKKH	893.5559	8	447.7883	29.1562	Q9TV63|MYH2_PIG; P79293|MYH7_PIG; Q9TV62|MYH4_PIG

Figure 2 The total ions chromatography of JTG. (A) Peptide fraction; (B) protein fraction.

Figure 3 Bioinformatic analysis of the constituents in JTG: Molecular functions of peptides (A) and proteins (B); Biological process of peptides (C) and proteins (D).

Figure 4 Effects of JTG on body weight, spleen and thymus in CIA rats. (A) Body weight; (B) Spleen index; (C) Thymus index. Data were expressed as the mean ± SD (n=10), ^#P < 0.05, ^##P < 0.01 vs normal group; *P < 0.05, **P < 0.01 vs CIA model group.

Figure 5 Effects of JTG on rheumatoid arthritis in CIA rats. (A) Pathological phenotype changes in CIA rats; (B) Paw swelling; (C) Arthritis score; (D) Serum IL-6 level; (E) Serum IL-1β level; (F) Histopathological changes of joints in CIA rats treated with JTG. The red arrow indicates cartilage hyperplasia and erosion; the black arrow indicates inflammatory cell infiltration; the blue arrow indicates the synovial hyperplasia; the green arrow indicates pannus formation. Data were expressed as the mean ± SD (n=10), ^##P < 0.01 vs normal group; *P < 0.05, **P < 0.01 vs CIA model group.

Figure 6 Effects of JTG on bone metabolism-related biochemical factors, bone tissue injury, bone morphology and bone micro-architecture in CIA rats. (A) Serum TRAP; (B) Serum RANKL; (C) Serum CTX-I; (D) Serum OCN; (E) Serum OPG; (F) Histopathological analysis of the bone tissue sections with Masson staining in CIA rats. The black arrow indicates articular cartilage, collagen and ossein; the purple arrow indicates muscle fiber and cellulose. Data were expressed as mean ± SD (n=10), ^#P < 0.05 vs normal group; *P < 0.05 vs CIA model group.

Figure 7 Effects of JTG on bone morphology and bone micro-architecture in CIA rats. (A) BMD; (B) BVF; (C) BS/BV; (D) Tb.Th; (E) Tb.N; (F) Tb.Sp; (G) 2D scanning images of the rat femur (can observe the density of bone trabecular); (H) 3D scanning images of the rat femur (can more intuitively observe the structure, density and morphology of bone trabecular.) Data were expressed as the mean ± SD (n=10), ^#P < 0.05, ^##P < 0.01 vs normal group; *P < 0.05, **P < 0.01 vs CIA model group.

Figure 8 PCA score plot shows preliminary separation between serum samples and QC. (A) Positive ion mode; (B) negative ion mode.

Figure 9 The analysis of UHPLC-Q/TOF-MS of serum samples of CIA rats. Statistical validation of the orthogonal partial least square discriminant analysis (OPLS-DA) model in 200 random permutation tests. Green dots are the R² values and blue blocks are the Q² values. Between normal and CIA model groups: (A) validation of OPLS-DA model in positive ion mode; (B) OPLS-DA score plot in positive ion mode; (C) an S-plot of the normal and CIA model groups. Between CIA model and JTG-360 mg/kg groups: (D) validation of OPLS-DA CIA model in positive ion mode; (E) OPLS-DA score plot in positive ion mode; (F) an S-plot of the CIA model and JTG-360 mg/kg groups.

Table 2 Potential Biomarkers Identified in Rat Serum

No.	Metabolite Identification	m/z	ESI	Normal vs CIA Model		CIA Model vs JTG-360mg/kg
				FC	Trend	FC	Trend
1	4,7,10,13,16,19-Docosahexaenoic acid	327.2320	[M-H]-	0.69	↑*	225.06	↓**
2	Gamma-L-Glutamyl-L-phenylalanine	295.1334	[M+H] +	2.69	↓**	0.37	↑*
3	L-Tyrosine	182.0808	[M+H] +	1.81	↓**	0.60	↑**
4	Lys-Thr	230.1496	[M+H] +	1.77	↓**	0.64	↑*
5	Arg-Met	305.1452	[M+H] +	1.75	↓*	0.60	↑**
6	Arg-Thr	339.1767	[M+H] +	1.99	↓**	0.47	↑**
7	Ile-Tyr	294.1566	[M+H] +	1.93	↓**	0.33	↑**
8	Leu-Thr	296.1603	[M+H] +	2.07	↓**	0.32	↑**
9	Val-Pro	278.1500	[M+H] +	1.82	↓**	0.35	↑**
10	N-Acetylaspartylglutamate	305.1041	[M+H] +	2.10	↓**	0.36	↑**
11	Glutaraldehyde	123.0435	[M+H] +	1.41	↓*	0.80	↑*
12	Dopamine	136.0753	[M+H] +	1.44	↓*	0.81	↑**
13	Cytidine	244.0940	[M-H]-	0.72	↑**	1.25	↓*
14	D-Ribose	149.0446	[M-H]-	1.57	↓**	35.52	↓**
15	2-hydroxy-butanoic acid	103.0394	[M-H]-	0.74	↑**	0.01	↑**
16	L-Citrulline	174.0915	[M-H]-	2.00	↓*	0.16	↑**
17	L-Galactono-1,4-lactone	237.1483	[M-H]-	0.73	↑*	0.01	↑**
18	Dihydroxyacetone	71.0136	[M-H]-	1.73	↓**	1.95	↓**
19	D-Quinovose	223.0815	[M-H]-	0.34	↑**	6.09	↓**
20	3-Methylhistidine	170.0922	[M+H] +	0.62	↑**	0.74	↑**
21	Val-Tyr	280.1410	[M+H] +	1.63	↓**	0.46	↑**
22	Phe-Tyr	328.1397	[M+H] +	1.71	↓*	0.36	↑**
23	16-Hydroxypalmitic acid	271.2266	[M-H]-	0.51	↑*	0.14	↑**
24	Ribothymidine	259.0931	[M+H] +	1.24	↓**	0.72	↑**
25	N-Acetylglutamine	233.0510	[M+H] +	1.36	↓**	0.72	↑**
26	Taurine	126.0223	[M+H] +	0.64	↑**	1.71	↓**
27	Nicotinate	124.0384	[M+H] +	2.65	↓*	0.35	↑*
28	Acetylglycine	118.0493	[M+H] +	1.39	↓**	0.57	↑**
29	Alpha-Tocopherol	430.3881	[M+H] +	0.53	↑*	0.52	↑*
30	Dimethylglycine	104.0704	[M+H] +	0.60	↑**	1.40	↓*
31	sn-Glycerol 3-phosphoethanolamine	214.0476	[M-H]-	0.61	↑*	0.71	↑*
32	Stearoylcarnitine	428.3663	[M+H] +	0.56	↑*	0.54	↑*
33	Tyramine	120.0813	[M+H] +	0.74	↑*	1.12	↓
34	Ile-Ala	203.1389	[M+H] +	1.93	↓**	0.85	↑
35	L-Asparagine	131.0455	[M-H]-	0.79	↑**	0.93	↑
36	Arachidonic Acid	305.2435	[M+H] +	0.69	↑**	1.12	↓
37	N4-Acetylcytidine	286.1036	[M+H] +	0.65	↑*	1.04	↓
38	Histamine	112.0863	[M+H] +	0.52	↑*	0.91	↑

Notes: FC was calculated based on mean ratios for Normal vs CIA model, or CIA model vs JTG-360mg/kg. The icons↑,↓ of trend represent up-regulation and down-regulation between the two groups; meanwhile * and ** represent change significance P < 0.05 and P < 0.01.

Figure 10 The pathway impact prediction of MetaboAnalyst 5.0 online database between CIA model and JTG-360 mg/kg groups: (A) bubble diagram. Bubble color represents the P-value: deeper colors represent smaller P -values, indicating larger differences. The size of the bubble represents the impact of the pathway during topological analysis. Larger size represents a higher impact; (B) heat map of the differential metabolites in CIA model and JTG-360 mg/kg groups. Each cell in the heat map represents the fold change between the two groups: blue color represents the metabolite expression down-regulation, and red color represents the metabolite expression up-regulation.