Rheumatoid arthritis (RA) is a chronic autoimmune disease with pathological features characterized by excessive hyperplasia of synovial tissue, joint swelling, vascular formation, and cartilage erosion 1. The high disability rate, poor prognosis, recurrent and long treatment cycle which brings a heavy economic burden on patients' families and society. The incidence of RA worldwide amounts to 0.18–1.07%, and it is increasing year by year 2. Depending on the U.S. administrative health insurance claims database, the prevalence of RA from 2004 to 2014 was from 0.41% to 0.54% 3. The prevalence of Chinese patients is about 0.4%. For patients who have not been diagnosed and treated promptly, the disability rate is 50% after 2 years, and 70% of patients after 3 years have varying degrees of disability, and their quality of life is severely affected 4. Current research shows that the pathogenesis of RA is related to multiple mechanisms such as infection, genetics, immune system disorders, and metabolic abnormalities 5, 6, but it has not yet been conclusively determined, and effective preventive and therapeutic drugs are continue to be lacking.

Western medicines currently available include non-steroidal anti-inflammatory drugs, glucocorticoids, anti-rheumatic drugs (DMARD) to relieve disease, and biological agents. However, its potential side effects may affect patient compliance with treatment 7. In addition, pathophysiological and genetic differences between patients also limit the therapeutic effectiveness of such drugs in RA treatment. Therefore, as an adjuvant treatment for patients with chronic diseases, complementary therapies have become more and more common 8. The most prevalent of these complementary therapies is traditional Chinese medicine (TCM). In China, a nationwide study of the incidence of TCM use in RA and non-RA populations has demonstrated that RA patients have a higher TCM use tendency compared to the general population, with a range of 30% 9.

Studies have shown 10 that compound Xuanju have significant anti-inflammatory and immune-regulating effects. Compound Xuanjuare mainly composed of Chinese herbal ingredients such as Xuanju (Polyrhachis vicine Roger), Epimrdii Herba, Cnidii Fructus, and Lycii Fructus. Xuanju is salty, sour, and mild in nature, which can make stronger, nourish blood, strengthen pain, and relieve pain and Quyu Tongluo. Lycii Fructus is sweet and flat, achieves strong bones, and nourishes liver and kidney; Epimrdii Herba can remove wind and dehumidification, kidney and aphrodisiac. Cnidii Fructus tastes bitter, mild, has warm kidney and impotence Cold effect. The compatibility of these drugs can achieve the effects of removing wind and dispel cold and warming kidney, and it can treat the RA from the two aspects of righting and eliminating evil. Although some studies have shown that compound Xuanjucan effectively treat RA 11, 12, there is still an urgent need to further clarify and describe its underlying material basis and mechanism of action. Therefore, this study uses network pharmacology to conduct a comprehensive network analysis of the effects of compound Xuanju for RA targets, and to construct a RA target network with hub genes, in order to explain the key active ingredients of compound Xuanju and their mechanism of action. The workflow is shown in Figure 1.

Figure 1

1. RA disease target acquisition and collection

Using "Rheumatoid arthritis" as a key topic word in Drugbank disease target screening database (https://www.drugbank.ca/), GeneCards database (https://www. genecards.org/), Therapeutic Target Database (TTD) database (http://db.idrblab. net/ttd/) and DisGeNET (http://www.disgenet.org/) database to search RA related targets. The Drugbank database contains detailed drug, drug target, drug action, and drug interaction information on FDA-approved drugs and experimental drugs that have undergone FDA approval procedures 13. The TTD database provides information on known therapeutic protein and nucleic acid targets, targeted disease conditions, pathway information, and target drug/ligand counterparts 14 . DisGeNET (http://www.disgenet.org/) is used to retrieve the molecular basis of specific human diseases, analysis of disease gene characteristics, generation of hypothesis for drug treatment effects and adverse drug reactions, verification of predicted disease genes and evaluation of text mining the performance of the method etc 15 . The chemical composition and target data obtained from the three databases are combined, duplicate and invalid targets are deleted, and the full names of the selected targets are entered into DrugBank to obtain the gene abbreviation and Uniprot ID.

2. Active ingredients and action targets of compound Xuanju

Through the Traditional Chinese Medicine System Pharmacology database (TCMSP) (http://tcmspw.com/tcmsp.php), Traditional Chinese Medicine Integrated Database (TCMID) (http://119.3.41.228:8000/tcmid/search/) and Pubmed, CNKI, Wanfang and VIP databases, the active ingredients and active targets of the four main components of compound Xuanju, namely, Xuanju, Epimrdii Herba, Cnidii Fructus, and Lycii Fructus were searched and retrieved. The TCMSP database is a unique traditional Chinese medicine system pharmacological data analysis platform that can analyze chemical components, targets, and disease networks, including oral bioavailability (OB), drug similarity (DL) and other natural compounds. 16, the screening conditions in this study were OB≥30%, DL≥0.18, and HL≥4. It has priority over TCMSP database retrieval. If TCMSP is not included, the TCMID database is used. The TCMID database is a comprehensive database that provides information for TCM and modern life sciences and bridge the gap. It collects information including prescriptions, herbs and herbal ingredients, and modern pharmacological information 17. If the two databases are not included, the literature query is conducted through the Pubmed, CNKI, Wanfang, and VIP databases to obtain the active ingredients and the target of traditional Chinese medicine.

3. Target screening and protein interaction network construction

Map the matching target of the active ingredient of compound Xuanju and the target of RA in OmicShare (https://www.omicshare.com/tools/index. php/) , and construct a Venn diagram. The common target with the intersection module may be the key target for active ingredients of compound Xuanjuto treat RA. The common targets were used to build a protein-protein interaction (PPI) network model on the String database online platform (https://string-db.org/).

4. Construction and analysis of the active ingredient-target network

OmicShare were used to construct and visualize the collected traditional Chinese medicine ingredients and their target information. The nodes represent the active ingredient and the target, and the edges represent the interaction between them, and the connectivity analysis is performed. The more connected knots are, the more important the compound or target is. The key active components and central genes of compound Xuanju against RA were determined by analyzing the strength of network connectivity.

5. Analysis of key target functions annotation and pathway enrichment

Through the DAVID database (https://david.ncifcrf.gov/), P <0.05 was used as the screening condition to obtain key target proteins for gene ontology (GO) functional enrichment analysis and Kyoto Gene and Genome Encyclopedia (KEGG) pathway enrichment analysis. Three modules including biological process, cellular component and molecular function were selected for mapping. Use GraphPad Prism 5.0 to visualize the top 20 entries of biological processes, cell composition, and molecular function entries in GO function enrichment analysis as a visual bar chart. Use the OmicShare to present the channel enrichment results in the form of a bubble chart for KEGG pathway analysis on key targets.

1. Active ingredients of compound Xuanju and targets

Three components of compound Xuanju(Epimrdii Herba, Cnidii Fructus, and Lycii Fructus) were searched in the TCMSP and TCMID databases. Xuanju was searched through Pubmed, CNKI, Wanfang and VIP databases. 42 active ingredients of Lycii Fructus, 14 of Cnidii Fructus, 18 of Epimrdii Herba, and 4 of Xuanju were obtained. After removing 5 repetitive ingredients, a total of 73 single active ingredients were obtained from the compound Xuanju Table 1 . A total of 842 targets were acted on by the active ingredients, and 229 targets were obtained after de-duplication.

Table 1

Molecule Name	MW	AlogP	OB (%)	Caco-2		DL	HL
Sitosterol alpha1	426.8	8.15	43.28	1.41	0.97	0.78	5.64
Cycloartenol	426.8	7.55	38.69	1.53	1.33	0.78	5
Mandenol	308.56	6.99	42	1.46	1.14	0.19	5.39
Ethyl linolenate	306.54	6.55	46.1	1.54	1.12	0.2	6.2
LAN	426.8	8.12	42.12	1.52	1.18	0.75	5.84
Stigmasterol	412.77	7.64	43.83	1.44	1	0.76	5.57
beta-sitosterol	414.79	8.08	36.91	1.32	0.99	0.75	5.36
campesterol	400.76	7.63	37.58	1.34	0.95	0.71	4.83
24-methylidenelophenol	412.77	7.75	44.19	1.41	1	0.75	5.1
daucosterol_qt	414.79	8.08	36.91	1.33	0.85	0.75	5.6
glycitein	284.28	2.32	50.48	0.56	-0.29	0.24	16.32
CLR	386.73	7.38	37.87	1.43	1.13	0.68	4.52
14b-pregnane	288.57	6.54	34.78	1.8	1.95	0.34	4.57
(24R)-4alpha-Methyl-24-ethylcholesta-7,25-dien-3beta-ylacetate	482.87	8.79	46.36	1.39	1	0.84	8.25
24-Methylenecycloartan-3beta,21-diol	456.83	6.7	37.32	0.95	0.3	0.8	5.67
24-ethylcholest-22-enol	414.79	7.89	37.09	1.33	0.99	0.75	5.32
24-ethylcholesta-5,22-dienol	412.77	7.64	43.83	1.31	0.84	0.76	5.76
24-methyl-31-norlanost-9(11)-enol	428.82	8.15	38	1.33	0.93	0.75	5.49
24-methylenelanost-8-enol	440.83	8.43	42.37	1.44	1.18	0.77	5.43
Fucosterol	412.77	7.83	43.78	1.34	1.01	0.76	5.44
31-Norcyclolaudenol	440.83	8.04	38.68	1.35	0.94	0.81	4.88
31-norlanost-9(11)-enol	414.79	7.9	38.35	1.34	1.03	0.72	5.37
31-norlanosterol	412.77	7.85	42.2	1.4	1.03	0.73	5.27
4,24-methyllophenol	414.79	7.95	37.83	1.43	1.1	0.75	4.9
Lophenol	400.76	7.7	38.13	1.36	1.1	0.71	4.76
4alpha,14alpha,24-trimethylcholesta-8,24-dienol	426.8	8.29	38.91	1.4	1.06	0.76	6.67
4alpha,24-dimethylcholesta-7,24-dienol	412.77	7.89	42.65	1.43	0.98	0.75	5.44
4alpha-methyl-24-ethylcholesta-7,24-dienol	426.8	8.35	42.3	1.45	1.08	0.78	5.83
6-Fluoroindole-7-Dehydrocholesterol	402.7	7.04	43.73	1.03	0.54	0.72	5.1
7-O-Methylluteolin-6-C-beta-glucoside_qt	318.3	2.01	40.77	0.33	-0.47	0.3	14.1
Atropine	289.41	1.72	42.16	0.57	0.39	0.19	5.27
Cryptoxanthin monoepoxide	568.96	9.5	46.95	1.49	-0.03	0.56	4.4
Cycloeucalenol	426.8	7.59	39.73	1.42	1.04	0.79	5.01
(E,E)-1-ethyl octadeca-3,13-dienoate	308.56	6.99	42	1.46	1.11	0.19	5.47
methyl(1R,4aS,7R,7aS)-4a,7-dihydroxy-7-methyl-1-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-1,5,6,7a-tetrahydrocyclopenta[d]pyran-4-carboxylate	406.43	-3.18	39.43	-1.43	-2.06	0.47	7.64
Lantadene A	552.87	7.21	38.68	0.32	-0.3	0.57	5.44
Physalin A	526.58	0.5	91.71	-0.78	-1.11	0.27	13.21
Physcion-8-O-beta-D-gentiobioside	608.6	-0.91	43.9	-2.68	-3.52	0.62	28.46
lanost-8-en-3beta-ol	428.82	8.37	34.23	1.48	1.25	0.74	5.48
lanost-8-enol	428.82	8.37	34.23	1.37	1.12	0.74	6.41
Obtusifoliol	426.8	8.15	42.55	1.47	1.25	0.76	5.91
quercetin	302.25	1.5	46.43	0.05	-0.77	0.28	14.4
Methyl oleate	296.55	7.09	31.9	1.42	1.13	0.17	5.37
cholesterol	386.73	7.38	37.87	1.43	1.13	0.68	4.52
cholest-4-en-3-one	412.77	8.18	36.08	1.46	1.22	0.76	5.49
β-sitosterol	546.57	1.55	33.94	-0.44	-1.37	0.7	13.56
24-epicampesterol	400.76	7.63	37.58	1.43	1.15	0.71	4.5
poriferast-5-en-3beta-ol	414.79	8.08	36.91	1.45	1.14	0.75	5.07
Diosmetin	300.28	2.32	31.14	0.46	-0.66	0.27	16.34
Ethyl oleate (NF)	310.58	7.44	32.4	1.4	1.1	0.19	4.85
ar-curcumene	426.5	5.05	52.34	0.59	0.17	0.65	9.88
cnidimol B	292.31	0.88	68.66	-0.06	-0.84	0.26	16.79
cnidimol F	290.29	0.65	54.43	-0.32	-1.39	0.28	14.68
(E)-2,3-bis(2-keto-7-methoxy-chromen-8-yl)acrolein	404.39	3.54	56.38	0.26	-0.22	0.71	6.98
cniforin A	374.42	3.09	55.89	0.37	-0.13	0.47	6.91
cniforin B	414.49	4.63	36.7	0.6	-0.05	0.6	6.65
o-Isovalerylcolum bianetin	330.41	3.78	64.03	0.73	0.38	0.36	5.79
Ostruthin	298.41	5.32	30.65	1.2	0.79	0.23	4.09
Linoleyl acetate	308.56	6.85	42.1	1.36	1.08	0.2	7.48
DFV	256.27	2.57	32.76	0.51	-0.29	0.18	17.89
Chryseriol	300.28	2.32	35.85	0.39	-0.53	0.27	16.31
8-Isopentenyl-kaempferol	354.38	3.63	38.04	0.53	-0.49	0.39	15.37
sitosterol	414.79	8.08	36.91	1.32	0.87	0.75	5.37
kaempferol	286.25	1.77	41.88	0.26	-0.55	0.24	14.74
Anhydroicaritin	368.41	3.88	45.41	0.72	0.01	0.44	15.01
C-Homoerythrinan, 1,6-didehydro-3,15,16-trimethoxy-, (3.beta.)-	329.48	2.89	39.14	1.02	0.68	0.49	6.58
Yinyanghuo A	420.49	4.2	56.96	0.38	-0.49	0.77	14.44
Yinyanghuo C	336.36	3.39	45.67	0.75	-0.11	0.5	15.74
Yinyanghuo E	352.36	3.12	51.63	0.51	-0.54	0.55	15.47
8-(3-methylbut-2-enyl)-2-phenyl-chromone	290.38	4.99	48.54	1.53	0.99	0.25	18.73
Anhydroicaritin-3-O-alpha-L-rhamnoside	676.73	0.77	41.58	-1.59	-2.89	0.61	16.23
Icariin	676.73	0.77	41.58	-1.82	-3	0.61	19.93
luteolin	286.25	2.07	36.16	0.19	-0.84	0.25	15.94

2. Screening of interaction targets of compound Xuanju and RA in addition construction of protein interaction network

A total of 2707 RA-related targets were collected through Drugbank, GeneCards, DisGeNET and TTD databases, 370 duplicate targets were deleted to obtain 2337 targets for RA. The RA targets were mapped and matched with 229 sole targets of compound Xuanju. The Venn diagram (Figure 2A) showed that the potential active ingredients of compound Xuanju intersect with RA. The module predicts a total of 155 targets Table 2. A protein-protein interaction (PPI) network model was constructed with 155 common targets in the String database (Figure 2B). These 155 common targets may be the key targets for the active ingredients of compound Xuanju to treat RA.

Table 2

NO.	Targets	Uniprot ID	NO.	Targets	Uniprot ID	NO.	Targets	Uniprot ID
1	MMP2	P08253	42	GABRA1	P14867	83	CHRNA7	P36544
2	KCNMA1	Q12791	43	PPARD	Q03181	84	ADRA2C	P18825
3	XDH	P47989	44	HMOX1	P09601	85	PLAU	P00749
4	NOS2	P35228	45	HTR1A	P08908	86	PTEN	P60484
5	CHRM5	P08912	46	MMP3	P08254	87	MMP13	P45452
6	MET	P08581	47	MMP8	P22894	88	ALOX5	P09917
7	BIRC5	O15392	48	GSTM1	P09488	89	HTR2C	P28335
8	PPP3CA	Q08209	49	IL10	P22301	90	DPP4	P27487
9	BCL2	P10415	50	MAPK1	P28482	91	PON1	P27169
10	ADRA2A	P08913	51	PLAT	P00750	92	KCNH2	Q12809
11	F7	P08709	52	MDM2	Q00987	93	E2F1	Q01094
12	HTR3A	P46098	53	EGFR	P00533	94	CASP8	Q14790
13	CHRM1	P11229	54	IKBKB	O14920	95	PPARG	P37231
14	CRP	P02741	55	SOD1	P00441	96	SELE	P16581
15	GSTP1	P09211	56	ERBB2	P04626	97	NR3C1	P04150
16	ADRA1A	P35348	57	IL4	P05112	98	THBD	P07204
17	AHR	P35869	58	MAPK8	P45983	99	MAPK14	Q16539
18	CHRNA2	Q15822	59	AKR1B1	P15121	100	RAF1	P04049
19	NR1I3	Q14994	60	CHEK2	O96017	101	MPO	P05164
20	CHRM4	P08173	61	RB1	P06400	102	ADRA1B	P35368
21	ADRB1	P08588	62	CDK2	P24941	103	PCNA	P12004
22	TNF	P01375	63	RUNX2	Q13950	104	CHUK	O15111
23	EGF	P01133	64	PGR	P06401	105	GABRA2	P47869
24	IL1A	P01583	65	ODC1	P11926	106	CASP7	P55210
25	IL8	P10145	66	E2F2	Q14209	107	ADRB2	P07550
26	PTGS2	P35354	67	CXCL10	P02778	108	CTSD	P07339
27	NCF1	P14598	68	TYR	P14679	109	MCL1	Q07820
28	RUNX1T1	Q06455	69	TP53	P04637	110	CCL2	P13500
29	CCND1	P24385	70	CDKN1A	P38936	111	IL6	P05231
30	ESR1	P03372	71	DIO1	P49895	112	CASP3	P42574
31	GABRG3	Q99928	72	GABRA5	P31644	113	PARP1	P09874
32	CDK4	P11802	73	IL1B	P01584	114	MGAM	O43451
33	PDE3A	Q14432	74	CHRM3	P20309	115	KDR	P35968
34	VEGFA	P15692	75	NFKBIA	P25963	116	ABCG2	Q9UNQ0
35	PIK3CD	O00329	76	IGFBP3	P17936	117	PPARA	Q07869
36	TGFB1	P01137	77	HTR2A	P28223	118	CYP1A2	P05177
37	MYC	P01106	78	GSTM2	P28161	119	AHSA1	O95433
38	ADRA1D	P25100	79	CTRB1	P17538	120	IGF2	P01344
39	CCNA2	P20248	80	CALM1	P0DP23	121	HTR1B	P28222
40	MMP1	P03956	81	ESR2	Q92731	122	ADRA2B	P18089
41	STAT1	P40763	82	LTA4H	P09960	123	IL2	P60568
124	FOS	P01100	135	CHRM2	P08172	146	JUN	P05412
125	IFNG	P01579	136	SERPINE1	P05121	147	AR	P10275
126	HSPA5	P11021	137	VCAM1	P19320	148	GABRA3	P34903
127	OPRD1	P41143	138	IFNGR1	P15260	149	BAX	Q07812
128	ICAM1	P05362	139	INSR	P06213	150	PRKCA	P17252
129	CxCL11	O14625	140	AKT1	P31749	151	CD40LG	P29965
130	BCL2L1	Q07817	141	AKR1C3	P42330	152	APP	P05067
131	CA2	P00918	142	PTGS1	P23219	153	PRSS1	P07477
132	RELA	Q04206	143	F3	P13726	154	MMP9	P14780
133	HIF1A	Q16665	144	NR1I2	O75469	155	RXRB	P28702
134	NOS3	P29474	145	CDKN2A	Q8N726

Figure 2

3. Analysis of key active ingredients and targets of compound Xuanju against RA

In order to further understand the mechanism of action of key genes, we identified 155 overlapping genes based on the predicted results and reversely searched for compounds that interacted with them. Finally, 55 compounds were identified from Xuanju, Epimrdii Herba, Cnidii Fructus, and Lycii Fructus. The compounds are shown in Table 3 . And visualize the traditional Chinese medicine-chemical composition-target network Figure 3. The results showed that the three components with the strongest connectivity were: Quercetin (96), Kaempferol (64), and Luteolin (46); the four targets with the strongest connectivity were: PGR (30), PTGS2 (28), PTGS1 (19) and DDP4 (17). It is suggested that compound Xuanju mainly treat targets such as PGR, PTGS2, PTGS1 and DDP through active ingredients such as quercetin, kaempferol and luteolin to treat RA.

Table 3

NO.	Compound	NO.	Compound
1	24-epicampesterol	29	Ethyl linolenate
2	Linoleyl acetate	30	LAN
3	poriferast-5-en-3beta-ol	31	campesterol
4	DFV	32	24-methylidenelophenol
5	Chryseriol	33	daucosterol_qt
6	8-Isopentenyl-kaempferol	34	glycitein
7	sitosterol	35	CLR
8	kaempferol	36	14b-pregnane
9	Anhydroicaritin	37	24-ethylcholest-22-enol
10	C-Homoerythrinan, 1,6-didehydro-3,15,16-trimethoxy-, (3.beta.)-	38	24-ethylcholesta-5,22-dienol
11	Yinyanghuo A	39	24-methyl-31-norlanost-9(11)-enol
12	Yinyanghuo C	40	24-methylenelanost-8-enol
13	Yinyanghuo E	41	Fucosterol
14	8-(3-methylbut-2-enyl)-2-phenyl-chromone	42	31-norlanost-9(11)-enol
15	luteolin	43	31-norlanosterol
16	quercetin	44	4,24-methyllophenol
17	Diosmetin	45	Lophenol
18	beta-sitosterol	46	4alpha,14alpha,24-trimethylcholesta-8,24-dienol
19	ar-curcumene	47	6-Fluoroindole-7-Dehydrocholesterol
20	cnidimol B	48	7-O-Methylluteolin-6-C-beta-glucoside_qt
21	cnidimol F	49	atropine
22	(E)-2,3-bis(2-keto-7-methoxy-chromen-8-yl)acrolein	50	lanost-8-en-3beta-ol
23	cniforin A	51	lanost-8-enol
24	cniforin B	52	Obtusifoliol
25	o-Isovalerylcolum bianetin	53	cholesterol
26	Stigmasterol	54	cholest-4-en-3-one
27	Sitosterol alpha1	55	β-sitosterol
28	Mandenol

Figure 3

4. Analysis of key target functions annotation and pathway enrichment

To further understand the function and enrichment of key targets, GO and KEGG analyses were performed. The results showed that the highest scores in the biological process, cellular component, and molecular function of GO functional enrichment analysis were positive regulation of transcription from RNA polymerase II promoter (GO: 0045944), extracellular space (GO: 000561), and protein homodimerization activity (GO : 0042803). KEGG pathway enrichment analysis showed that compound Xuanju mainly passed Toll-like receptor signaling pathway (ssc04620), PI3K-Akt signaling pathway (ssc04151), NF-κB signaling pathway (ssc04064), HIF-1 signaling pathway (ssc04066), TNF Signaling pathways (ssc04668) and other signaling pathways act on RA. Figure 4

Figure 4

The ingredient of traditional Chinese medicine is a complex chemical system, and the biological body is also a complex system. The interaction between the chemical substance entity of the traditional Chinese medicine product and the biological activity of the body has become an important entry point for modern Chinese medicine research. Network pharmacology researches the pharmacology of relating drugs through big data analysis. It takes herbs, chemical components, targets, diseases and other perspectives as the starting point to achieve a comprehensive and systematic comprehensive network analysis of traditional Chinese medicine research. It has the unique advantage and great potential to predicts and identifies active ingredients of traditional Chinese medicine, groups and targets, clarify the mechanism of action, reveal the formulas, discover new indications and new active compounds. Moreover, network pharmacology emphasizing on "one target, one drug" to "network target, multiple groups paradigm shift. This gives us a better understanding of the mechanism of action of drugs, then it is not difficult to understand that network pharmacology is widely used in drug research 18 In the treatment of RA,compound Xuanju have received more attention due to its obvious effectiveness and fewer side effects. Therefore, in this study, we utilized network pharmacology to study the potential targets of compound Xuanju for the treatment of RA. Construct and analyze target networks, perform enrichment analysis on hub genes, and uncover their potential mechanism.

In this study, we first excavated the targets of RA, the active ingredients and targets of compound Xuanju capsules. By mapping and matching the targets, we obtained the key targets of compound Xuanju for RA; Point the corresponding active ingredients; construct the protein-protein interaction network of key targets, and then perform GO analysis and KEGG analysis to clarify the functional annotation and enrichment of key targets.

The results of this study show that the four most active ingredients are: quercetin in Lycii Fructus and Epimrdii Herba, kaempferol in Epimrdii Herba, luteolin in Lycii Fructus and Epimrdii Herba, β-sitosterol in Xuanju and Cnidii Fructus. This component group may play a therapeutic role in RA through highly connected targets such as PGR, PTGS2, PTGS1, and DDP4. Among them, quercetin has the effects of anti-oxidation and scavenging of free radicals, and also has anti-inflammatory, immunomodulatory, anti-cancer, antibacterial, antiviral, hypoglycemic and antihypertensive effects, and cardiovascular protection 19, 20 Jayanti et al 21 reported that quercetin gel preparation can effectively treat rheumatoid arthritis. Kaempferol has anti-inflammatory, antibacterial, anti-cancer and other physiological activities 22, 23. β-sitosterol (beta-sitosterol) inhibit tumor cell proliferation and inhibit blood angiogenesis 24. PTGS1 is thought to play a key role in the pathophysiology of inflammation, arthritic disease, and cancer 25 Clinical studies have shown that as a specific inhibitor of PTGS, low-dose aspirin can effectively exert antipyretic, analgesic and anti-inflammatory effects 26. Importantly, PTGS1 inhibitors or silencing of PTGS1 expression through negative regulation greatly reduces the inflammatory response caused by LPS 27. Wang et al 28 showed that knockout of PTGS1 promotes osteogenic differentiation of ASCs in vitro and in vivo by targeting p65 cytoplasm/nuclear translocation, and that during bone remodeling, PTGS1 silencing may provide regulation of the inflammatory microenvironment a potential means. Wei et al 29 reported that PTGS2 plays an important role in inflammation and fibroblast-like synoviocytes in RA. MiR-101-3p reduced joint swelling and arthritis index in RA rats by down-regulating PTGS2. The negative expression of miR-101-3p and PTGS2 was found in the synovial tissues of RA patients and RA rats, overexpressed MiR-101-3p and silenced PTGS2. RA-FLS was observed to have reduced cell proliferation, migration and invasion, promote cells apoptosis.

In GO analysis, the biological process mainly involves positive regulation of transcription from RNA polymerase II promoter, adenylate cyclase-activating adrenergic receptor signaling pathway, and lipopolysaccharide-mediated signaling pathway; Molecular function mainly involves protein homodimerization activity, G-protein coupled acetylcholine receptor activity, and steroid hormone receptor activity. The KEGG analysis mainly involved TNF signaling pathway, HIF-1 signaling pathway, T cell receptor signaling pathway, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, NF-κB signaling pathway and p53 signaling pathway. The NF-κB signaling pathway plays an important role in the inflammatory process of rheumatoid arthritis. Its transcription factor NF-κB is involved in regulating the expression of multiple cytokines and cell adhesion factors during the inflammation process 30. Activation can lead to T cell activation and thereby mediate the inflammatory response. It can also induce abnormal proliferation of RA-FLS and stimulate the proliferation and activation of osteoclasts. As a result, joint deformities and bone erosion can be caused, which can aggravate the condition of RA. In addition, after activation of NF-κB, cytokines and acute response proteins are produced to activate the immune response and increase the expression of adhesion molecules. These immune molecules exacerbate the inflammation level of RA 31 Toll-like receptors (TLRs) in the toll-like receptor signaling pathway are type I transmembrane protein receptors, which are widely distributed on the surface of immune cells and epithelial cells. TLRs/NF-κB in signal transduction pathways that mediate immunity signaling pathways play an extremely important role in immune regulation 32, 33 TNF-α in the TNF signaling pathway can induce the expression of multiple inflammatory cytokines (such as IL-6、IL-8) and matrix metal protein (MMP) in synovial cells, which is a persistent and prolonged progression of rheumatoid arthritis key factors 34, 35. Study found that P53 is mutated in synovial tissue of RA patients. Mutated P53 can cause cells to over express IL-6. It can be seen that abnormal proliferation T cell differentiation, and various inflammations of RA synovial tissue is regulated by the P53 signal pathway Factor production 36.

In summary, we utilized network pharmacological methods to discover 73 ingredients and 229 potential targets in compound Xuanju, and 2337 targets related to RA disease, of which compound Xuanju-RA common targets 155 points, and through the GO biological process and KEGG signal pathway enrichment analysis of these targets, it is predicted that compound Xuanju may regulate the TNF signal pathway, toll-like receptors signal pathway, NF-κB signal pathway, P53 signal pathway to act on PTGS2, PTGS1, DDP4, and PGR targets. Thereby inhibiting inflammatory response, regulating immune function and regulating apoptosis to treat RA. But it's worth noting that network pharmacology develops rapidly. Our research present certain limitations. First of all, this study is based on a network pharmacological approach to study the potential targets of compound Xuanju for RA, but the specific mechanism of action remains to be confirmed by cell and animal experiments. Second, due to the difficulty of obtaining data, the database may not include all known or unknown active ingredients, targets, and protein-protein interactions. However, it is undeniable that the results of this study can provide ideas and reference for experimental research, and provide a theoretical basis for the wide application of compound Xuanju in clinical practice.

Conflicts of Interests : The authors have no conflicts of interests.

Acknowledgments: This work was supported by the Science and Technology Fund Project of Health and Family Planning Commission of Guizhou Province (gzwjkj2019-2-006) and the Science and Technology Plan Project of Qiannan Prefecture(Qiannan Keheshezi (2018) No. 36).

Hunter T M Boytsov N N Zhang X Schroeder K Michaud K Araujo A B Prevalence of rheumatoid arthritis in the United States adult population in healthcare claimsdatabases Rheumatol Int 2004 37 1551 1557 Jin X R Zhu M M Gao R X Research progress on the relationship between TOLL-like receptor-mediated signaling pathway and rheumatoid arthritis Hunan J Traditional Chinese Med 2017 33 193 195 Wang C L Fufang Xuanju Capsule for the treatment of 30 patients with RA rheumatoid arthritis TCM Res 2009 22 38 40 Wei Q Lv F Zhang H Wang X Geng Q Zhang X Li T Wang S Wang Y Cui Y MicroRNA-101-3p inhibits fibroblast-like synoviocyte proliferation and inflammation in rheumatoid arthritis by targeting PTGS2 Biosci Rep 2020 40 20191136 20191136 Boezio Baptiste Audouze Karine Ducrot Pierre Taboureau Olivier Network-based Approaches in Pharmacology Molecular Informatics 2017 36 10 1700048 1700048 1868-1743 10.1002/minf.201700048 Wiley https://dx.doi.org/10.1002/minf.201700048 McInnes Iain B Schett Georg Pathogenetic insights from the treatment of rheumatoid arthritis The Lancet 2017 389 10086 2328 2337 0140-6736 10.1016/s0140-6736(17)31472-1 Elsevier BV https://dx.doi.org/10.1016/s0140-6736(17)31472-1 Kargman S L O&apos;neil G P Vickers P J Evans J F Mancini J A Jothy S Expression of prostaglandin G/H synthase-1 and-2 protein in human colon cancer Cancer Res 1995 55 2556 2559 Feng Y Wang X Research progress on chemical constituents of different medicinal parts of Impatiens Shandong J Traditional Chinese Med 2016 35 749 753 Sun X R Chen P P Lin Y Research progress on antitumor targets of natural flavonoids China J Experimental Traditional Med Formulae 2017 23 218 228 Zhang Q Li Q Zhu J Guo H Zhai Q Li B Jin Y He X Jin F Comparison of therapeutic effects of different mesenchymal stem cells on rheumatoid arthritis in mice PeerJ 2019 7 Piñero Janet Ramírez-Anguita Juan Manuel Saüch-Pitarch Josep Ronzano Francesco Centeno Emilio Sanz Ferran Furlong Laura I The DisGeNET knowledge platform for disease genomics: 2019 update Nucleic Acids Research 2019 48 845 855 0305-1048, 1362-4962 10.1093/nar/gkz1021 Oxford University Press (OUP) https://dx.doi.org/10.1093/nar/gkz1021 Wang Y Liu Y Zhang M Lv L Zhang X Zhang P Zhou Y Inhibition of PTGS1 promotes osteogenic differentiation of adipose-derived stemcells by suppressing NF-kB signaling Stem Cell Res Ther 2019 10 57 57 Wishart David S Feunang Yannick D Guo An C Lo Elvis J Marcu Ana Grant Jason R Sajed Tanvir Johnson Daniel Li Carin Sayeeda Zinat Assempour Nazanin Iynkkaran Ithayavani Liu Yifeng Maciejewski Adam Gale Nicola Wilson Alex Chin Lucy Cummings Ryan Le Diana Pon Allison Knox Craig Wilson Michael DrugBank 5.0: a major update to the DrugBank database for 2018 Nucleic Acids Research 2018 46 D1 D1074 D1082 0305-1048, 1362-4962 10.1093/nar/gkx1037 Oxford University Press (OUP) https://dx.doi.org/10.1093/nar/gkx1037 Calvello Rosa Lofrumento Dario Domenico Perrone Maria Grazia Cianciulli Antonia Salvatore Rosaria Vitale Paola De Nuccio Francesco Giannotti Laura Nicolardi Giuseppe Panaro Maria Antonietta Scilimati Antonio Highly Selective Cyclooxygenase-1 Inhibitors P6 and Mofezolac Counteract Inflammatory State both In Vitro and In Vivo Models of Neuroinflammation Frontiers in Neurology 2017 8 251 251 1664-2295 10.3389/fneur.2017.00251 Frontiers Media SA https://dx.doi.org/10.3389/fneur.2017.00251 Bazzazi H Aghaei M Memarian A Asgarian-Omran H Behnampour N Yazdani Y Th1-Th17 Ratio as a New Insight in Rheumatoid Arthritis Disease Iran J Allergy Asthma Immunol 2018 17 68 77 Fuster V Sweeny J M Aspirin: a hitorical and contemporary therapeutic overview Circulation 2011 123 768 778 Luo X J Mo X R Zhou L L TNF-α-induced rheumatoid arthritis synovial cells. Discussion on activation of NF-κB signaling pathway J Immu 2012 28 321 323 Gokhale Jayanti P. Mahajan Hitendra S. Surana Sanjay J. Quercetin loaded nanoemulsion-based gel for rheumatoid arthritis: In vivo and in vitro studies Biomedicine & Pharmacotherapy 2019 112 108622 108622 0753-3322 10.1016/j.biopha.2019.108622 Elsevier BV https://dx.doi.org/10.1016/j.biopha.2019.108622 Jia J Li J Yao X Zhang Y Yang X Wang P Xia Q Hakonarson H Li J Genetic architecture study of rheumatoid arthritis and juvenile idiopathic arthritis PeerJ 2020 8 8234 8234 Ye P Li J H Xu J H Myeloid cell-induced receptor 2 expression in synovial tissue of uncle in rheumatoid arthritis model Chinese J of Tissue Engineering Res 2015 4 2807 2813 Shi H Wang D T Wu R G Role of TNF-α-mediated NF-κB signaling pathway in angiogenesis of rheumatoid arthritis Med Review 2012 18 2397 2400 Wang C Y Wang X Y Zhang Y Wang L P Effects of Fufang Xuanjuon Th17 cells/ interleukin 17 in patients with rheumatoid arthritis Pharmacology and Clinics of Chinese Medicine 2016 32 198 200 Ru J Li P Wang J Zhou W Li B Huang C Li P Guo Z Tao W Yang Y Xu X Li Y Wang Y Yang L TCMSP: a database of systems pharmacology for drug discovery from herbal medicines J Cheminform 2014 6 Huang Lin Xie Duoli Yu Yiran Liu Huanlong Shi Yan Shi Tieliu Wen Chengping TCMID 2.0: a comprehensive resource for TCM Nucleic Acids Research 2018 46 D1 D1117 D1120 0305-1048, 1362-4962 10.1093/nar/gkx1028 Oxford University Press (OUP) https://dx.doi.org/10.1093/nar/gkx1028 Sun J Yu S C Research Progress of Quercetin, Res Practice Chinese Med 2011 25 85 88 Wang Y Zhang S Li F Zhou Y Zhang Y Wang Z Zhang R Zhu J Ren Y Tan Y Qin C Li Y Li X Chen Y Zhu F Therapeutic target database 2020: enriched resource for facilitating research and early development of targeted therapeutics 2020 48 1031 1041 Lu S W Wang Q S Zhao S Research progress on the regulation of nuclear factor κB signaling pathway of rheumatoid arthritis by Chinese medicine J Cell Mol Immu 2015 11 1580 1583 Chen K H Yeh M H Livneh H Chen B C Lin I H Lu M C Tsai T Y Yeh C C Association of traditional Chinese medicine therapy and the risk of dementia in patients with hypertension: a nationwide population-based cohort study BMC Complement Altern Med 2017 17 178 178 Shu Y Tan T Zhang S Y Progress in pharmacological research of quercetin West China Pharmaceutical J 2008 23 689 691 Wong Siu Ling Wagner Denisa D. Peptidylarginine deiminase 4: a nuclear button triggering neutrophil extracellular traps in inflammatory diseases and aging The FASEB Journal 2018 32 12 6258 6370 0892-6638, 1530-6860 10.1096/fj.201800691r Wiley https://dx.doi.org/10.1096/fj.201800691r Xie X Q Wang Y Luo S S Research progress in the pathogenesis of rheumatoid arthritis World Abstracts in Med Inf 2019 19 109 111 Lin M C Lu M C Livneh H Lai N S Guo H R Tsai T Y Factors associated with sexual dysfunction in Taiwanese females with rheumatoid arthris BMC Womens Health 2017 17 12 12 Fan H Wu Y Huang X Wang W Ang B Cao X Wan T Toll-like receptor 4 (TLR4) is essential for Hsp70-like protein 1 (HSP70L1) to activate dendritic cells and induce Th1 response J Biol Chem 2011 286 30393 30400 Bin Sayeed Muhammad Shahdaat Ameen Syeda Sadia Beta-Sitosterol: A Promising but Orphan Nutraceutical to Fight Against Cancer Nutrition and Cancer 2015 67 8 1216 1222 0163-5581, 1532-7914 10.1080/01635581.2015.1087042 Informa UK Limited https://dx.doi.org/10.1080/01635581.2015.1087042 Smolen Josef S. Aletaha Daniel Barton Anne Burmester Gerd R. Emery Paul Firestein Gary S. Kavanaugh Arthur McInnes Iain B. Solomon Daniel H. Strand Vibeke Yamamoto Kazuhiko Rheumatoid arthritis Nature Reviews Disease Primers 2018 4 1 18001 18006 2056-676X 10.1038/nrdp.2018.1 Springer Science and Business Media LLC https://dx.doi.org/10.1038/nrdp.2018.1 Takatori Hiroaki Kawashima Hirotoshi Suzuki Kotaro Nakajima Hiroshi Role of p53 in Systemic Autoimmune Diseases Critical Reviews in Immunology 2014 34 6 509 516 1040-8401 10.1615/critrevimmunol.2014012193 Begell House https://dx.doi.org/10.1615/critrevimmunol.2014012193