Abstract

   graphic file with name ao2c04641_0012.jpg

   Yuxuebi tablet (YXB) is a Chinese patent medicine with the effect of
   activating blood circulation and dissipating blood stasis and has been
   used to treat “Bi” syndrome in China. The aim of this study was to
   reveal its anti-inflammatory efficacy and mechanism. A
   carrageenan-induced inflammation mouse model was established to
   demonstrate the anti-inflammatory efficacy of YXB by detecting the paw
   swelling degree and inflammatory cell infiltration in paws. The active
   chemical ingredients and anti-inflammatory targets of YXB were obtained
   through network pharmacology analysis. Finally, the core
   anti-inflammatory targets of YXB were determined by the ELISA method
   and western blot. YXB significantly reduced the paw swelling degree and
   inflammatory cell infiltration in paws. A total of 120 key active
   components included in YXB interacted with 56 core inflammatory targets
   (such as TNF, IL1B, IL6, PTGS2, RELA, MAPK1, MAPK8, and MAPK14), mainly
   involving in the TNF signaling pathway, Toll-like receptor signaling
   pathway, NF-kappaB signaling pathway, and NOD-like receptor signaling
   pathway. Further studies in vivo found that YXB reduced the levels of
   TNF-α, IL-1β, and IL-6 in serum and inhibited the expression of COX-2
   and the phosphorylation levels of NF-κB p65, JNK, and p38 protein in
   paws. Taken together, YXB had a good anti-inflammatory effect, which
   might be related to inhibiting the phosphorylation of NF-κB, JUN, and
   p38 and the decrease of COX-2 expression and the levels of inflammatory
   factors.

1. Introduction

   Inflammation is the body’s adaptive response to infection and tissue
   injury,^[38]1 with the occurrence and development of a variety of
   chronic diseases,^[39]2 such as the “Bi” syndrome characterized by pain
   and numbness in the limb joints and muscles or swelling and difficulty
   in flexion-extension in the joints. During the inflammation, different
   types of cellular mediators are released and activated, including
   chemokines, cytokines, vasoactive amines, and adhesion molecules.^[40]3
   The most commonly used anti-inflammatory agents are nonsteroidal
   anti-inflammatory drugs (NSAIDs) such as aspirin and celecoxib, which
   mainly inhibit cyclooxygenase-2 (COX-2) activity and anticytokine
   agents such as antitumor necrosis factor (TNF) α.^[41]3,[42]4 However,
   NSAIDs can cause severe gastrointestinal, cardiovascular, hepatic,
   kidney, and cerebral injury,^[43]5,[44]6 and anticytokine agents can
   lead to infection from more common bacterial pathogens.^[45]7 In recent
   years, more and more traditional Chinese medicines (TCMs) have been
   used in the treatment of inflammatory diseases because of their good
   anti-inflammatory effect and low adverse reaction.

   Shentong-Zhuyu decoction (STZY) was first recorded in the “Yilin
   Gaicuo” written by Wang Qingren during the Qing Dynasty and had been
   documented for the treatment of shoulder pain, arm pain, low back pain,
   leg pain, or body aches. STZY has been recognized by the Chinese
   National Administration of Traditional Chinese Medicine as a classic
   prescription of Chinese medicine. Yuxuebi tablet (YXB) is a Chinese
   patent medicine approved by the National Medical Products
   Administration and derived from the addition and reduction of
   Shentong-Zhuyu decoction. It is produced by China Resources Liaoning
   Benxi Third Pharmaceutical Co., Ltd. and composed of Boswellia
   carteriiBirdw (named “Ru xiang” in Chinese; RX), Clematis
   chinensisOsbeck (named “Wei ling xian” in Chinese; WLX), Carthamus
   tinctoriusL. (named “Hong hua” in Chinese; HH), Salvia
   miltiorrhizaBunge (named “Dan shen” in Chinese; DS), Commiphora myrrha
   (Nees) Engl. (named “Mo yao” in Chinese; MY), Cyathula officinalisKuan
   (named “Chuan niu xi” in Chinese; CNX), Conioselinum
   anthriscoides’Chuanxiong’ (named “Chuan xiong” in Chinese; CX),
   Angelica sinensis (Oliv.) Diels (named “Dang gui” in Chinese, DG),
   Curcuma longa L. (named “Jiang huang” in Chinese; JH), Cyperus rotundus
   L. (named “Xiang fu” in Chinese; XF), and Astragalus mongholicusBunge
   (named “Huang qi” in Chinese; HQ). YXB has the effect of activating
   blood circulation, dissipating blood stasis, and relieving pain without
   obvious adverse reactions and can be used to treat “Bi” syndrome caused
   by the invasion of evil “Qi” in the theory of Chinese medicine, such as
   rheumatoid arthritis and knee osteoarthritis.^[46]8,[47]9 In the YXB,
   the chemical composition of RX, such as boswellic acid type and
   nonterpenoid type, could affect the levels of cytokines and oxygen
   species and inhibit the synthesis of leukotriene, COX-1/2, and
   5-lipoxygenase (5-LOX).^[48]10 The extracts from WLX inhibited the
   levels of nitric oxide (NO), prostaglandin E2 (PGE2), TNF-α,
   interleukin-1beta (IL-1β), and interleukin-6 (IL-6) in RAW 264.7 cells
   induced by lipopolysaccharide and alleviated paw edema and inflammatory
   cell infiltration of carrageenan-induced mice.^[49]11,[50]12 The
   aqueous extract from HH could improve neutrophilic lung inflammation in
   mice by activating nuclear factor E2-related factor 2 (Nrf2).^[51]13 DS
   could downregulate TNF-α, COX-2, inducible nitric oxide synthase
   (iNOS), and ILs by activating Nrf2, thus inhibiting the inflammatory
   response.^[52]14 Other TCMs included in YXB also have good
   anti-inflammatory effects.^[53]15−[54]21 However, as a complex Chinese
   traditional patent medicine, the anti-inflammatory mechanisms of YXB
   are still poorly understood.

   Network pharmacology has been a popular research method in recent years
   and has been widely used in the study of the mechanism of action of
   TCMs.^[55]22 It is to build a network of drug, disease, and therapeutic
   targets by linking published drug data with the disease, gene
   expression information, and protein–protein interaction data, better
   revealing the characteristics of multicomponent drug synergy, in line
   with the complex characteristics of the human body and providing a
   prerequisite for the discovery of effective natural
   drugs.^[56]23,[57]24 The aim of this paper was to study the
   anti-inflammatory effect and mechanism of YXB based on network
   pharmacology and experimental verification of carrageenan-induced
   inflammation mice.

2. Results

2.1. YXB Inhibited the Paw Edema Induced by Carrageenan

   Changes in the thickness of the paw after inflammation were used to
   evaluate the swelling degree of the paw.^[58]25 Compared with the
   control group, the swelling degree of the right hind paw in the model
   group increased significantly (P < 0.01), suggesting that carrageenan
   induced inflammation successfully. YXB 3.0 g/kg and Cele 0.08 g/kg
   markedly suppressed the swelling degree of carrageenan-induced mice paw
   (P < 0.05) ([59]Figure [60]1
   ). These results indicated the potent anti-inflammatory effect of YXB.

Figure 1.

   [61]Figure 1
   [62]Open in a new tab

   Effect of YXB on paw swelling in mice. Data were represented as mean ±
   SD, n = 6. ^#P < 0.05, ^##P < 0.01 vs control. *P < 0.05, ^**P < 0.01
   vs model.

2.2. YXB Reduced Inflammatory Cell Infiltration in the Paw

   To further observe the anti-inflammatory effect of YXB on
   carrageenan-induced inflammation mice, the morphological changes of the
   paw were observed by the hematoxylin and eosin (H&E) staining method.
   Only a few inflammatory cells were found in the control group, but
   subcutaneous injection of carrageenan resulted in massive inflammatory
   cell infiltration in the model group. Cele and YXB could alleviate the
   inflammatory cell infiltration, especially YXB 1.50 g/kg and 3.00 g/kg
   and Cele 0.08 g/kg ([63]Figure [64]2
   ). These results suggested that YXB could reduce the infiltration of
   inflammatory cells in local tissues.

Figure 2.

   Figure 2
   [65]Open in a new tab

   Effect of YXB on inflammatory cell infiltration of mouse paw tissue
   (H&E).

2.3. Potential Active Ingredients and Targets of YXB for Anti-Inflammation

   A total of 352 potential active compounds of YXB were obtained by
   searching TCMSP and TCMID databases: 17 for RX, 37 for WLX, 38 for HH,
   61 for DS, 35 for MY, 4 for CNX, 54 for CX, 61 for DG, 52 for JH, 29
   for XF, and 19 for HQ. Some TCMs had the same chemical composition, and
   1215 targets were obtained from the SwissTargetPrediction.

   Five-hundred inflammation targets were collected from the Genecards
   database. The overlap targets of YXB and inflammation were screened
   using Venny 2.1, and 137 genes were obtained. The results suggested
   that these targets might be the anti-inflammatory targets of YXB
   ([66]Figure [67]3
   ).

Figure 3.

   Figure 3
   [68]Open in a new tab

   Overlap targets of YXB and inflammation.

2.4. Protein–Protein Interaction (PPI) Network Analysis

   One-hundred and thirty seven YXB-anti-inflammatory targets were
   imported into STRING 11.5, and the PPI network was visualized using
   Cytoscape 3.7.2. The network consisted of 114 nodes and 496 edges where
   each node represented a gene and each edge connected to two genes
   ([69]Figure [70]4
   ). According to the topological parameter analysis of the network, the
   median degree of nodes was 6, and there were 56 targets whose degree
   values were higher than the median, which indicated that these targets
   were in the center of the network and closely related to other targets.
   That was to say they might be the key anti-inflammatory targets of YXB
   ([71]Table [72]1).

Figure 4.

   [73]Figure 4
   [74]Open in a new tab

   YXB-anti-inflammatory target PPI network. Nodes represent proteins, and
   lines represent relationships between proteins and proteins. The larger
   the node, the closer the color was to red, indicating that its degree
   value was higher and the protein was more important. The thicker the
   line, the bluer the color was, indicating that the protein binding was
   stronger.

Table 1. 56 Key Targets of the Anti-Inflammatory Effect of YXB.

   uniprot ID  target degree uniprot ID   target  degree uniprot ID  target
   degree
   [75]Q04206  RELA   37     [76]P15692  VEGFA    15     [77]P51617  IRAK1  10
   [78]P40763  STAT3  35     [79]O15111  CHUK     14     [80]P03956  MMP1   10
   [81]P12931  SRC    31     [82]Q9Y6K9  IKBKG    14     [83]Q96EB6  SIRT1  10
   [84]P19838  NFKB1  31     [85]P45983  MAPK8    13     [86]P04150  NR3C1  10
   [87]P05412  JUN    27     [88]P42345  MTOR     13     [89]Q04759  PRKCQ  9
   [90]P28482  MAPK1  26     [91]O14920  IKBKB    13     [92]Q07817  BCL2L1 8
   [93]P01375  TNF    24     [94]Q05655  PRKCD    13     [95]Q07869  PPARA  8
   [96]P31749  AKT1   24     [97]P43405  SYK      13     [98]P08253  MMP2   8
   [99]P04637  TP53   23     [100]Q13546 RIPK1    13     [101]P37231 PPARG  8
   [102]Q16539 MAPK14 23     [103]P19438 TNFRSF1A 13     [104]Q02763 TEK    8
   [105]P05231 IL6    21     [106]P00734 F2       12     [107]P29474 NOS3   7
   [108]P42336 PIK3CA 20     [109]O00206 TLR4     12     [110]P98170 XIAP   7
   [111]P42224 STAT1  20     [112]P10145 CXCL8    12     [113]P35354 PTGS2  7
   [114]Q14790 CASP8  18     [115]P00747 PLG      12     [116]P29466 CASP1  7
   [117]P01584 IL1B   17     [118]P42574 CASP3    11     [119]O43353 RIPK2  7
   [120]O60674 JAK2   17     [121]P14780 MMP9     11     [122]P17706 PTPN2  7
   [123]P00533 EGFR   15     [124]P05107 ITGB2    11     [125]P19320 VCAM1  7
   [126]P60568 IL2    15     [127]P35228 NOS2     10     [128]P08254 MMP3   7
   [129]P23458 JAK1   15     [130]Q16665 HIF1A    10
   [131]Open in a new tab

2.5. Screening of Key Ingredients of YXB

   To further screen the key ingredients of YXB, we established the
   YXB-potential active ingredient-target network ([132]Figure [133]5
   ). The network consisted of 348 nodes (281 for potential active
   ingredients, 56 for core targets, and 11 for TCMs) and 1948 edges. The
   topological parameter analysis of the network showed that the median
   degree value of 281 active compounds was 6, and the number of compounds
   whose degree values were higher than the median was 120. These
   compounds might play a key role in the anti-inflammatory process of
   YXB. The top 10 compounds in the degree value are shown in [134]Table
   [135]2.

Figure 5.

   [136]Figure 5
   [137]Open in a new tab

   YXB-potential active ingredient-target network. Circles represent
   protein targets, diamonds and triangles represent active ingredients,
   and hexagons represent TCM. The larger the node, the closer the color
   was to red, indicating that the degree value was higher.

Table 2. Key Compounds of YXB for Anti-Inflammation.

   [138]2.5.
   [139]Open in a new tab

2.6. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
Enrichment

   The 56 core targets were imported into the DAVID database for
   enrichment analysis to obtain the biological process (BP), molecular
   function (MF), cellular component (CC), and KEGG. The top 20 terms are
   shown in [140]Figure [141]6
   . According to the results, YXB-anti-inflammatory core targets were
   involved in the BP, MF, and CC. The BP included positive regulation of
   transcription from RNA polymerase II promoter, positive regulation of
   NF-kappaB transcription factor activity, and positive regulation of the
   nitric oxide biosynthetic process and inflammatory response. In terms
   of MF, the targets were mainly related to protein kinase activity,
   transcription factor binding, protein serine/threonine kinase activity,
   and protein phosphatase binding. As for the CC, there were mainly
   cytosol, membrane raft, and nucleoplasm. In addition, 99 pathways were
   revealed from the KEGG pathway enrichment analysis, including the TNF
   signaling pathway, Toll-like receptor signaling pathway, NF-kappaB
   signaling pathway, NOD-like receptor signaling pathway, and so on. Four
   pathways closely related to inflammation were selected to construct the
   pathway-target network ([142]Figure [143]6).

Figure 6.

   [144]Figure 6
   [145]Open in a new tab

   GO and KEGG enrichment analysis of anti-inflammatory targets of YXB.

   Combined with the analysis of “YXB-potential active ingredient-target
   network” and “Anti-inflammatory pathway-target network of YXB”, PTGS2
   (protein name: Cyclooxygenase-2, COX-2), RELA (protein name: Nuclear
   factor NF-kappa-B p65 subunit, NF-κB p65), MAPK1 (protein name:
   Mitogen-activated protein kinase 1, ERK2), MAPK8 (protein name:
   Mitogen-activated protein kinase 8, JNK1), MAPK14 (protein name:
   Mitogen-activated protein kinase 14, p38), TNF (protein name: Tumor
   necrosis factorm, TNF-α), IL1B (protein name: Interleukin-1 beta,
   IL-1β), and IL6 (protein name: Interleukin-6, IL-6) were selected for
   further study of the anti-inflammatory mechanism of YXB because of
   their high degree value and close relation to inflammation ([146]Figure
   [147]7
   ).^[148]26,[149]27

Figure 7.

   [150]Figure 7
   [151]Open in a new tab

   Anti-inflammatory pathway-target network of YXB. Circles represent
   protein targets; the arrows represent pathways.

2.7. YXB Decreased the Levels of Serum Inflammatory Factors

   The levels of TNF-α, IL-1β, and IL-6 in the serum were significantly
   increased in the model group (P < 0.01), showing that the acute
   inflammation model was successful, compared with the control group.
   However, YXB 3.0 g/kg markedly decreased the levels of TNF-α and IL-1β
   (P < 0.05). Meanwhile, Cele 0.08 g/kg and each dose of YXB markedly
   inhibited the production of IL-6 (P < 0.05) ([152]Figure [153]8
   A–C).

Figure 8.

   [154]Figure 8
   [155]Open in a new tab

   Levels of inflammatory cytokines in the serum of mice. (A) Levels of
   TNF-α; (B) levels of IL-1β; (C) levels of IL-6. Data were represented
   as mean ± SD, n = 4. ^#P < 0.05, ^##P < 0.01 vs control. *P < 0.05,
   ^**P < 0.01 vs model.

2.8. YXB Inhibited the Expression of Target Proteins

   Western blot analysis revealed that the expression of COX-2 and
   Phospho-NF-kappaB p65 (p-NF-κB p65) in paw tissue of the model group
   were increased (P < 0.05). However, YXB and Cele decreased the
   expression of COX-2 and p-NF-κB p65 (P < 0.05) ([156]Figure [157]9
   A–C).

Figure 9.

   [158]Figure 9
   [159]Open in a new tab

   Inhibitory effect of YXB on the target proteins of carrageenan-induced
   inflammation mice. (A) Electrophoretic images of COX-2 and p-NF-κB p65;
   (B) levels of COX-2; (C) levels of p-NF-κB p65; (D) electrophoretic
   images of p-JNK, p-ERK1/2 and p-p38; (E) levels of p-JNK; (F) levels of
   p-ERK1/2; (G) levels of p-p38. Data were represented as mean ± SD, n =
   3. ^#P < 0.05, ^##P < 0.01 vs control. *P < 0.05, ^**P < 0.01 vs model.

   Meanwhile, compared with the control group, the expression of
   phospho-38 (p-p38) in the paw tissue of the model group was
   significantly increased (P < 0.01), and the expression of phospho-JNK
   (p-JNK) and phospho-ERK1/2 (p-ERK1/2) was also increased (P > 0.05). As
   expected, YXB and Cele inhibited the expression of p-JNK and p-p38 (P <
   0.05). However, there was no significant difference in the expression
   of p-ERK1/2 ([160]Figure [161]9
   D–G).

3. Discussion

   Inflammation is the first sign of infection and injury, divided into
   acute inflammation, which develops quickly and lasts for a short time,
   and chronic inflammation, which develops slowly and lasts for a long
   time. Uncontrolled inflammation is a major cause of the development of
   many chronic diseases.^[162]28 “Bi” syndrome is the symptom causing
   inflammation of limb joints and muscles under the stimulation of
   external factors such as wind, cold, dampness, and heat. YXB is a
   common Chinese patent medicine used to treat “Bi” syndrome. In this
   study, the carrageenan-induced inflammation mouse model was used to
   evaluate the anti-inflammatory effect and mechanism of YXB.
   Subcutaneous injection of carrageenan in mice is a common animal model
   for screening anti-inflammatory drugs, which can cause edema and
   inflammatory cell infiltration in paw.^[163]29 In this research, we
   found that a high dose of YXB could effectively reduce the swelling
   degree of paws caused by carrageenan and the infiltration of
   inflammatory cells in paws, indicating that it had a good
   anti-inflammatory effect.

   YXB is composed of 11 TCMs, which contains many kinds of compounds and
   has the characteristics of multiple targets and multiple pathways.
   Therefore, we analyzed the anti-inflammatory mechanism of YXB through
   network pharmacology. By establishing the network of “YXB-potential
   active ingredient-target network”, we found that 120 active compounds
   might have acted on 56 key targets related to inflammation. Among them,
   the top 10 compounds with anti-inflammatory effects were quercetin,
   beta-sitosterol, luteolin, kaempferol, stigmasterol, myristicin,
   isorhamnetin, caffeic acid, hederagenin, and methyl eugenol, which were
   sorted by the degree value and reported in the literature. It has been
   reported that quercetin, a flavonoid widely found in natural plants,
   could inhibit LPS-induced macrophage inflammation and oxidative
   stress,^[164]30 and it also reduced the expression of C-C motif
   chemokine ligand (CCL) 17, CCL22, IL-4, IL-6, interferon (IFN)-γ, and
   TNF-α in atopic dermatitis by local administration.^[165]31 For
   beta-sitosterol, it inhibited the NF-κB pathway and activated the heme
   oxygenase-1(HO1)/nuclear factor erythroid 2-related factor 2(Nrf2)
   pathway to exert an anti-inflammatory effect.^[166]32 Luteolin affected
   M1/M2 polarization of macrophages and played an anti-inflammatory role
   by downregulating phosphorylated signal transducer and activator of
   transcription 3(p-STAT3) and upregulating p-STAT6.^[167]33
   Stigmasterol^[168]34 and kaempferol^[169]35 significantly inhibited the
   NF-κB and MAPK pathways mediated by Toll-like receptor 4 (TLR4)/myeloid
   differentiation factor-88 adaptor protein (MyD88) and reduced the
   release of TNF-α, IL-1β, IL-6, and IL-18. Myristicin,^[170]36
   isorhamnetin,^[171]37 hederagenin,^[172]11 and methyl eugenol^[173]38
   could eliminate reactive oxygen species and inhibit the expression of
   inflammatory factors and had antioxidant and anti-inflammatory effects.
   Therefore, as a compound Chinese medicine preparation, the key
   anti-inflammatory compounds of YXB have synergistic effects.

   Through network pharmacology analysis, we found that the key
   anti-inflammatory targets of YXB included TNF-α, IL-1β, IL-6, COX-2,
   NF-κB p65, ERK1/2, JNK, and p38. TNF-α, IL-1β, and IL-6 were mainly
   produced by monocytes.^[174]30 TNF-α binding to the receptors TNFR1 and
   TNFR2 could activate downstream NF-κB and MAPK signaling pathways and
   participate in the inflammatory response and apoptosis.^[175]39,[176]40
   IL-1β could rapidly stimulate the transcription of hundreds of gene
   mRNA in macrophages, epithelial cells, and endothelial cells by
   activating NF-κB and MAPK signaling pathways.^[177]41 TNF-α and IL-1β
   played a synergistic role in the development of inflammation^[178]39
   and were the main activators of IL-6 expression.^[179]42 IL-6 could
   activate NF-κB and other signaling pathways and further enhance the
   transcription of mRNA for inflammatory cytokines, such as IL-6, TNF-α,
   and IL-1β.^[180]43,[181]44 In addition, IL-6 could also promote
   overproduction of VEGF, increasing angiogenesis and vascular
   permeability, which were the pathological features of inflammatory
   lesions.^[182]43 COX-2, the key enzyme for prostaglandin
   synthesis,^[183]45 was highly expressed in inflammatory diseases and
   the main target of anti-inflammatory agents. Activation of NF-κB could
   promote the transcription of COX-2.^[184]46 ERK1/2, JNK, and p38 were
   members of the highly conserved protein kinase family in the MAPK
   signaling pathway, which could promote the production of TNF-α, IL-1β,
   and IL-6.^[185]47 In animal experiments, we found that YXB could
   effectively reduce the levels of TNF-α, IL-1β, and IL-6 in the serum of
   carrageenan-induced inflammatory mice. Similarly, YXB significantly
   reduced the expression of COX-2, p-NF-KB p65, p-JNK, and p-p38, while
   it had little effect on expression of p-ERK.

4. Conclusions

   In summary, this study showed that YXB exerted an anti-inflammatory
   effect, which might be related to inhibiting the phosphorylation of
   NF-κB p65, JNK, and p38, downregulating expression of COX-2, and
   reducing the levels of TNF-α, IL-1β, and IL-6. It provides a
   theoretical basis for YXB to treat “Bi” syndrome and has a better
   understanding of the anti-inflammatory mechanism of YXB, providing
   reference for searching safe and effective anti-inflammatory agents.

5. Materials and Methods

5.1. Materials

   Male KM mice, weighing 16–18 g, were purchased from the Animal
   Laboratory Centre of Jinzhou Medical University (Animal license number:
   SCXK [Liao] 2019-0007). YXB (NO: 20210303) was supplied by China
   Resources in Liaoning Benxi Third Pharmaceutical Co., Ltd. Celecoxib
   Capsules (Cele) (NO: DK1011) were purchased from Pfizer. Carrageenan
   (NO: C804872) was purchased from Shanghai Macklin Biochemical Co., Ltd.
   Isoflurane (NO: R510-22) was purchased from RWD Life Science Co., Ltd.
   RIPA lysis buffer (NO: P0013B), phosphatase inhibitor cocktail (NO:
   P1081), and phenylmethylsulfonyl fluoride (PMSF) (NO: ST506) were
   purchased from Beyotime Biotechnology. A Pierce Rapid Gold BCA Protein
   Assay Kit (NO: [186]A53227) was purchased from Thermo Scientific.
   SAPK/JNK Antibody (NO: 9252), Phospho-SAPK/JNK Antibody (NO: 9251), and
   p38 MAPK Antibody (NO: 8690) were purchased from Cell Signaling
   Technology. p-p38 Antibody (NO: WLP1576), ERK1/2 Antibody (NO:
   WL02371), p-ERK1/2 Antibody (NO: WLP1512), NF-κB p65 Antibody (NO:
   WL01273b), p-NF-κB p65 Antibody (NO: WL02169), and COX-2 Antibody (NO:
   WL01750) were purchased from Wanleibio Co., Ltd. β-actin Antibody (NO:
   AC026) and HRP Goat Anti-Rabbit IgG (H + L) (NO: AS014) were purchased
   from ABclonal. An Ultra-High Sensitivity ECL Kit (NO: BL523A) was
   purchased from Biosharp. Mouse TNF-α, IL-1β, and IL-6 ELISA kits were
   purchased from R&D.

5.2. Screening the Active Ingredients and Targets of YXB

   The compound information of YXB was obtained from the TCMSP database
   ([187]https://old.tcmsp-e.com/tcmsp.php) and TCMID database
   ([188]http://119.3.41.228:8000/tcmid/). Bioavailability (OB) ≥ 30% and
   drug-like (DL) ≥ 0.18 were used as parameters to screen active
   ingredients in the TCMSP database. The compounds obtained from the
   TCMID database were imported into SwissADME
   ([189]http://www.swissadme.ch/) in the style of 2D structure files to
   screen active ingredients. The two conditions must be met at the same
   time. That was to say GI absorption was high and at least two of the
   Druglikeness (Lipinski, Ghose, Veber, Egan and Muegge) were “yes”. The
   targets of active ingredients were predicted by the
   SwissTargetPrediction database
   ([190]http://www.swisstargetprediction.ch/).

5.3. Collecting Inflammation Targets

   The keyword of “inflammation” was searched in the GeneCards database
   ([191]https://www.genecards.org/). The targets were ranked by
   “Relevance score”, and the first 500 targets were selected to improve
   their reliability. All targets were standardized by Uniprot
   ([192]http://beta.uniprot.org/).

5.4. PPI Analysis

   The intersection targets of action targets of YXB and inflammatory
   targets, the potential anti-inflammatory targets of YXB, were obtained
   using Venny 2.1.0
   ([193]https://bioinfogp.cnb.csic.es/tools/venny/index.html) and
   imported into STRING 11.5 ([194]https://www.string-db.org/). The
   organism was limited to homo sapiens, and the minimum required
   interaction score was set as highest confidence (0.900) to construct a
   PPI network model. The result of PPI was visualized using Cytoscape
   3.7.2. Topology parameter analysis was carried out, and degree,
   betweenness centrality, and closeness were the key indicators to
   measure the importance of nodes. Targets whose degree values were
   greater than the median, the key anti-inflammatory targets of YXB, were
   selected for further analysis.

5.5. Go and KEGG Enrichment Analysis

   To understand the enrichment of the collective targets in BPs, CCs,
   MFs, and pathways, DAVID Bioinformatics Resources 6.8
   ([195]https://david.ncifcrf.gov/) was used for the analysis of GO and
   KEGG of core targets of YXB anti-inflammatory effects, and then the
   results were visualized using R project.

5.6. Animal Experiment

   All procedures involved in animal experiments in this study were
   approved by the Ethics Committee of Jinzhou Medical University (SYXK
   [Liao] 2019-0007), and the guidelines for the Guide for the Care and
   Use of Laboratory Animals issued by the National Institutes of Health
   of the United States were strictly followed.

   Cele is a selective inhibitor of COX-2. In this study, low and high
   doses of Cele in clinical use were selected as the positive control
   group.^[196]48 Mice were fed with standard laboratory food and water
   for 1 week under a 12 h light/dark cycle. The humidity was set at
   60–70%, and the temperature was 23 ± 2 °C. The mice were randomly
   divided into seven groups: control, model, Cele 0.04 g/kg, Cele 0.08
   g/kg, YXB 0.75 g/kg, YXB 1.50 g/kg, and YXB 3.00 g/kg (n = 6). Cele and
   YXB were diluted in 0.5% carboxymethylcellulose sodium to form a
   suspension for gavage. The dose of Cele and YXB was converted according
   to the dose relationship between the adult and mouse in the third
   edition of Pharmacological Research Methodology of Traditional Chinese
   Medicine edited by Chen Qi.

   The treatment lasted for 7 days. To evaluate the effect of YXB on paw
   edema, 100 μL of 1% carrageenan suspension (W/V, containing 0.9%
   saline) was subcutaneously injected into the right hind paw of mice 30
   min after the last administration, except for the control group, which
   received the same amount of sterile saline.^[197]49 After the
   experiment, the mice were killed by cervical dislocation, and the right
   paw was immediately dissected. Part of the paw was prepared for
   histological analysis, and the other parts were stored at −80 °C for
   western blotting.

5.7. Paw Edema Evaluation

   The thickness was measured before inflammation (0 h) and at 2, 4, and 6
   h after the intraplantar injection of carrageenan by vernier calipers.
   The degree of swelling was calculated as the thickness of the paws
   after inflammation minus that of the paws before inflammation.

5.8. Hematoxylin and Eosin Staining

   The part of right paws of mice were collected and fixed in 10% neutral
   buffered formalin for 24 h and then decalcified in 10% EDTA for 15
   days. Thereafter, the right paws were embedded in paraffin and cut into
   five-micrometer section. Subsequently, H&E were used for staining, and
   morphological changes were observed by using a light microscope
   (OLYMPUS, NO: DP72).

5.9. Evaluation of the Serum Levels of Cytokines

   After six hours of inflammation, mice were subjected to light
   anesthesia induced by isoflurane, and blood samples were collected by
   retroorbital sinus puncture in the absence of anticoagulant and
   centrifuged at 35 00 r/min for 10 min at 4 °C. The supernatant was
   stored at −80 °C and used for measurement of TNF-α, IL-1β, and IL-6 by
   ELISA.

5.10. Western Blotting Analysis

   The soft tissue from paw was harvested and homogenized in lysis buffer
   containing 1% PMSF and 2% phosphatase inhibitor cocktail. The BCA
   protein detection kit was used to determine the protein level of the
   extract. Protein samples were separated by 10% SDS-PAGE and transferred
   to a polyvinylidene fluoride (PVDF) membrane. After blocking in
   Tris-buffered saline containing Tween 20 (TBST) and 5% bovine serum
   albumin at room temperature for 1 h, PVDF membranes were washed three
   times in TBST and incubated overnight at 4 °C with primary antibodies
   of COX-2 (1:500), NF-κB p65 (1:500), p-NF-κB p65 (1:500), JNK (1:1000),
   p-JNK (1:500), p-p38 (1:500), p38 (1:1000), ERK1/2 (1:500), p-ERK1/2
   (1:500), and β-actin (1:1000). After washed with TBST three times at
   room temperature, the PVDF membranes were incubated with HRP Goat
   Anti-Rabbit IgG (H + L) (1:10000) for 1 h and washed three times with
   TBST. Proteins were scanned by using an Ultra-High Sensitivity ECL Kit.
   The protein expression was analyzed using Image J software.

5.11. Statistical Analysis

   The data were recorded using the software SPSS 26.0 and reported as the
   mean ± standard deviation (mean ± SD). One-way analysis of variance
   analysis was used to evaluate the significance of the differences
   between groups with normal distribution; otherwise, the independent
   sample test was used for the nonparametric test. P < 0.05 was
   considered statistically significant.

Acknowledgments