Abstract Background In traditional Chinese medicine, Fuzi-Lizhong pill (FLZP) has been used for millennia as a treatment for the Spleen-Kidney-Yang-deficiency (SKYD) diseases. FLZP has increasingly been employed in the clinic as a therapeutic option for ulcerative colitis with SKYD syndrome (SKYD-UC). In the present study, we revealed the kernel material basis and underlying mechanisms of the FLZP for treating SKYD-UC. Methods and results The therapeutic effects of FLZP were assessed in SKYD-UC rats. In total, 55 absorbed components of FLZP were identified, thus forming the main material basis for the use of FLZP for treating SKYD-UC. Network pharmacology analyses revealed that the ability of FLZP to exert multi-target synergistic activity was found to be related to both antioxidant and anti-inflammatory activity. More specifically, FLZP was suggested to alleviate SKYD-UC through the regulation of targets associated with inflammation such as interleukin-6 (IL-6), myeloperoxidase (MPO), and tumor necrosis factor-α (TNF-α), while also regulating the mitogen-activated protein kinase (MAPK), TNF, and phosphoinositol-3 kinase-RAC-alpha serine/threonine-protein kinase (PI3K-Akt) pathways. Ultimately, the integration of network analyses, molecular docking studies, and Pearson correlation analyses enabled the identification of 9 core compounds (including linolenic acid, liquirtigenin, 7-hydroxycoumarin, glycyrrhizic acid, 6-shogaol, dehydro-10-gingerdione, caffeic acid, 6-gingerol, liquiritin), which can serve as kernel material basis for FLZP in the treatment of SKYD-UC. Conclusion Together, these findings offer a valuable foundation for additional research focused on the mechanistic effects and broader clinical application of FLZP as a treatment option for SKYD-UC. Keywords: Ulcerative colitis, Fuzi-Lizhong pill, Serum pharmacochemistry, Network pharmacology, Anti-inflammatory Graphical abstract Image 1 [39]Open in a new tab 1. Introduction Ulcerative colitis (UC) is a form of chronic inflammatory bowel disease that causes severe inflammation of the mucosa in the colon in millions of people throughout the world.[40]^1 Affected patients experience a relapsing and remitting disease course that can have an extremely negative effect on quality of life. A range of therapies for UC has been developed to date, including aminosalicylates, immunomodulatory agents, steroids, and particular biologic agents, all of which are associated with adverse side effects that limit their utility. Therefore, there remains a constant need for the design of safe and effective alternative treatments for this devastating disease.[41]^2 Traditional Chinese medicine (TCM) provides great promise to effectively treat UC and alleviate associated symptoms, providing patients with reliable benefits and very low rates of adverse side effects.[42]^3 TCM has been practiced for over 2000 years since first being documented in the Inner Canon of Huangdi, enabling the prevention and treatment of UC.[43]^4 According to TCM theory, the pathogenesis of UC is spleen-kidney-Yang deficiency (SKYD).[44]^5 Therefore, the number of UC patients with SKYD (SKYD-UC) syndrome is the largest in the clinic. Fuzi Lizhong pill (FLZP) is a first-line TCM prescription that is often administered to SKYD-UC patients as recommended by The Expert Consensus on Traditional Chinese Medicine Diagnosis and Treatment of Ulcerative Colitis (2017).[45]^5 First originating in Taiping Huimin Heji Ju Fang during the Song Dynasty, FLZP consists of several herbal medicines including Aconitum carmichaeli Debx. (Fuzi), Zingiber officinale Rosc. (Ganjiang), Glycyrrhiza uralensis Fisch. (Gancao), Codonopsis pilosula (Franch.) Nannf. (Dangshen), and Atractylodes macrocephala Koidz. (Baizhu). Research has demonstrated that FLZP exhibits a range of pharmacological activities, including analgesic, anti-inflammatory, spasmolytic, and increasing adaptive thermogenesis.[46]^6 Given its advantageous properties, FLZP has been established as a national essential drug in China that is available over the counter (OTC) and widely used to treat diseases of the digestive system such as diarrhea, functional dyspepsia, chronic gastritis, chronic enteritis, and UC.[47]^7^,[48]^8 The complex composition of FLZP, however, has hampered efforts to fully understand the material basis for its therapeutic efficacy in SKYD-UC and the associated molecular mechanisms, in large part owing to an absence of any systematic research focused on this topic. Serum pharmacochemistry studies can offer insight into the absorption, metabolic processing, and interactions of a given drug in the body while avoiding any artificial effects that may arise from the identification and analysis of specific chemical components in an in vitro setting, although they do not offer insight into the functional mechanisms through which drugs function.[49]^9^,[50]^10 Network pharmacology studies, however, can address this latter point by leveraging robust databases and other tools to generate complex “disease-gene-target-drug” interaction networks that predict the functional effects of particular drugs of interest.[51]^10^,[52]^11 In several recent reports, researchers have conducted integrated serum pharmacochemistry and network pharmacology analyses as a means of better understanding the material basis of TCM prescriptions and their mechanistic effects when administered to diseases.[53]12, [54]13, [55]14 Therefore, in this study, a similar approach was herein employed to better understand the material and mechanistic basis for the beneficial effects of FLZP on SKYD-UC. 2. Materials and methods 2.1. Reagents and materials Chromatographic grade methanol and formic acid were from Fisher (USA). Watson distilled water was used as a mobile phase. Carboxymethyl Cellulose-Na (CMC-Na, No. 2020110401) was from Chengdu Kelong Chemical Co., Ltd. A qualitative fecal occult blood test kit (o-tolidine method, No. 0118A21) was from HeFei BoMei Biotechnology Co., Ltd. A hematoxylin-eosin (H&E) Staining Kit (No. G1005) was from Wuhan Google Biotechnology Co., Ltd. Dextran sulfate sodium (DSS, 40 kDa, No. OUO403A, Shanghai, China) was from Seebio Biotech (Shanghai) Co., Ltd. Interleukin-6 (IL-6, No. 20210603LA2C), tumor necrosis factor-α (TNF-α, No.20210607M3C), myeloperoxidase (MPO, No.20210607M3C), glutathione peroxidase (GSH-Px, No.20210607M3C), gastrin (GAS, No.20210607M3C), and motilin (MTL, No.20210607M3C) ELISA kits were from HeFei BoMei Biotechnology Co., Ltd. (Hefei, China). All 12 reference materials were >98 % pure. Of these materials, the following were from Chengdu Kloma Biotechnology Co.: glycyrrhetinic acid (CHB180607), glycyrrhizic acid (CHB 180610), 7-hydroxycoumarin (CHB190103), atractylode Ⅱ (CHB 180223), atractylode Ⅰ ([56]CHB80222), and 6-gingerol (CHB180306). The following reference materials were from Chengdu Manster Biotechnology Co., Ltd.: 6-gingerol (must-20042101), benzoylaconitine (dst170210-055), benzoylneoaconitine (must-20022710), benzoylhypoaconitine (must-20033111), mesaconine, and hypaconine. 2.2. FLZP and senna water extract preparation Aconitum carmichaelii Debx. (Fuzi, No. 2002110), Codonopsis pilosula (Franch.) Nannf. (Dangshen, No. 2011066), Atractylodes macrocephala Koidz. (Baizhu, No. 2101026), Zingiber officinale Rosc. (Ganjiang, No. 2101055) and Glycyrrhiza uralensis Fisch. (Gancao, No. 2102059) were obtained from Sichuan New Lotus Traditional Chinese Medicine Slices Co., Ltd. (Chengdu, China) and authenticated by Prof. Jin Pei (Department of Pharmacognosy of the Chengdu University of Chinese Medicine). Fuzi, Dangshen, Baizhu, Ganjiang, and Gancao were each ground into a fine powder and combined at a 1: 2: 1.5: 1: 1 ratio as per the instructions of the Chinese Pharmacopoeia (2020 edition). After mixing thoroughly with water, FLZP was produced. To prepare senna water extract, Cassia angustifolia Vahl was placed in a 70 °C deionized water bath overnight. The solution was then filtered such that 1 g of crude drug was present per 1 mL of aqueous extract. The filtrate was stored at 4 °C for subsequent use. 2.3. Experimental animals Sprague-Dawley (SD) rats (males, 220 ± 20 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) (Certificate No. SCXK (Jing) 2019-0010), as was their food. These rats were housed in a controlled setting (20–25 °C, 65–69 % relative humidity, 12 h light/dark cycle) and allowed to acclimatize for 1 week with access to standard food and water. All animal research was performed as per the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China (2017), and the Committee on the Ethics of Animal Experiments of Chengdu University of Traditional Chinese Medicine approved these research protocol (CDUTCM, permit SYXK(Chuan)2020-124). 2.4. Animal model establishment and treatment (Ⅰ) To establish an SKYD model, from days 1–6, rats were fed with 10 g of cabbage on odd days and 10 g of high-fat mixed feed on even days. Every afternoon, rats were placed in a pool filled with 38 °C water and allowed to swim until fatigued, as determined by their inability to swim any further with their neck being entirely submerged. From days 7–21, these same procedures were maintained and rats were also administered with senna water extract (10 mL/kg, i.g.). The following characteristics were considered indicative of successful SKYD modeling: loose stool, significant weight loss, loss of appetite, gathering for warmth, withered and dull hair, significantly reduced swimming time, and mucous around the anus.[57]^15^,[58]^16 (Ⅱ) SKYD-UC model: From 22 to 28 days, the operation of the previous 21 days was maintained with the addition of ad libitum drinking 3 % DSS.[59]^7^,[60]^8 Following a one-week adaptive feeding period, 30 total SD rats were randomized into the following groups: (1) control, (2) SKYD-UC, (3) SKYD-UC + FLZP (low), (4) SKYD-UC + FLZP (medium), and (5) SKYD-UC + FLZP (high) groups. The SKYD-UC model was established from days 1–28 in groups 2–5. From days 29–49, FLZP suspended in 0.5 % CMC-Na was administered (i.g.) to rats in group 3 (60 mg crude drug/mL), group 4 (120 mg crude drug/mL), and group 5 (240 mg crude drug/mL). Over this treatment interval, rats in groups 2–5 were maintained as per the SKYD (I) modeling procedures detailed above. For a full overview of the study approach, see [61]Fig. 1. Fig. 1. [62]Fig. 1 [63]Open in a new tab Animal model establishment and drug administration. 2.5. DAI scores Disease activity index (DAI) scores were measured for all rats based on body weight loss, bloody stool production, and stool viscidity ([64]Table S1). 2.6. Biochemical and histopathological analyses Serum IL-6, TNF-α, MPO, GSH-P, GAS, and MTL levels were analyzed with ELISA kits based on provided directions. Sichuan SCIENTIST Biotechnology Co., Ltd. performed all colon sample pathological analyses. Briefly, 0.5 cm colon segments from each rat were fixed for 24 h with 10 % formalin, embedded in paraffin, cut to produce 5 μm sections, and subjected to H&E staining. Histological scoring was performed with a scale published previously.[65]^17 2.7. Serum sample processing According to the previous study, blood samples of control, SKYD-UC, and SKYD-UC + FLZP (medium) groups were collected from the abdominal aorta 45 min after oral administration and were placed at room temperature for 1 h until solidification.[66]^18 The samples were then spun in a centrifuge at 7992 g for 10 min at 4 °C. The samples were kept at a temperature of −80 °C until they were examined. After adding methanol to the 2 mL serum samples three times, they were vortexed and then centrifuged at 7992 g for 20 min. The nitrogen gas was used to dry the supernatant. The remaining substance was dissolved again in 50 μL methanol, spun in a vortex, and then spun at 7992 g for 20 min. The UHPLC-MS sample was derived from the filtrate. A 2 μL aliquot was administered for UHPLC-MS analysis. 2.8. UHPLC-MS analysis condition and data analysis The serum sample (results in Section [67]2.7) was injected into the UHPLC-MS instrument to identify absorbed components of FLZP. Specific parameters are as follows: chromatographic column: Thermo Scientific accucoretm C18 column (3 mm × 100 mm, 2.6 μm). Mobile phase A is 0.1 % formic acid aqueous solution, mobile phase B is methanol, gradient elution procedure: 0–15 min, 95-70 % A; 15–30 min, 70-48 % A; 30–40 min, 48-25 % A; 40–45 min, 25-15 % A; 45–50 min, 15-2% A; 50–55 min, 2-2% A. Column equilibrium for 10 min, column temperature: 30 °C, injection volume: 2 μL, flow rate: 0.3 mL/min. The electrospray spray ion source (ESI), positive ion, and negative ion scanning modes were used for detection. The spray voltage is 3.5 kV (+)/3.0 kV (−), the auxiliary gas heating temperature is 350 °C, the sheath gas flow rate is 35 arb, the auxiliary gas flow rate is 10 arb, and the ion transfer tube temperature is 320 °C. The scanning mode is full scanning of primary mass spectrometry combined with automatic triggering secondary mass spectrometry scanning mode (Full MS/dd-MS[68]^2). The primary resolution is 35000, the second resolution is 17500, the collision energy gradient is 20, 40, and 60 eV, and the data acquisition range is m/z 100–1200 Da. 2.9. Network pharmacology Serum pharmacochemistry theory posits that the main active components of a given drug preparation are likely to be those that are absorbed in the serum.[69]^19 These absorbed compounds were thus used to predict the mechanistic basis for the ability of FLZP to treat SKYD-UC. The TCMSP and SwissTarget Prediction databases were used to screen for putative target genes of identified compounds, and these targets were normalized with the UniProt database. Target genes associated with SKYD-UC were identified with the GeneCards and OMIM databases, after which the overlap between these two sets of target genes was assessed. These putative therapeutic target genes were then imported into the STRING and Cytoscape tools to generate a protein-protein interaction (PPI) network with a high confidence threshold (0.7). The Cytoscape CytoNCA plugin was used to identify core targets in this PPI network, and these core targets were used for KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses with DAVID 6.8 (FDR<0.01). 2.10. Molecular docking Typically studies in network pharmacology often use molecular docking to aid in validation. Molecular docking analyses performed using AutoDock Vina were employed to validate predicted interactions between particular compounds and target proteins. Crystal structures for core targets were obtained from the RCSB Protein Data Bank and enhanced with AutoDock Tools by the removal of ligands and water molecules, together with the addition of hydrogen. Binding free energy (kcal/mol) was calculated with the Lamarckian Genetic Algorithm (LGA), and the most favorable ligand-protein binding interactions were identified as those with the lowest free energy conformation values. 2.11. Statistics Data were analyzed with SPSS 26.0. Results were reported as means ± SD and compared via one-way ANOVAs with LSD or Dunnett's T3 post hoc test. P < 0.05 served as the cut-off to define statistical significance. 3. Results 3.1. Assessment of the therapeutic effects of FLZP on SKYD-UC On the last day of administration, rats in the SKYD-UC group exhibited symptoms of UC including a 56 % weight loss, elevated DAI scores, a 32 % decreased in spleen index and a decreased in 50 % thymus index, and a 22 % colon shortening relative to the control group (P < 0.05, P < 0.01, P < 0.001) ([70]Fig. 2A–I), while FLZP administration reversed these changes. H&E staining similarly confirmed that DSS induced marked damage to colonic crypts and epithelial cells, whereas FLZP administration alleviated such damage ([71]Fig. 2J). Fig. 2. [72]Fig. 2 [73]Open in a new tab Effects of FLZP on SKYD-UC symptoms and colonic lesions. (A) Body weight, n = 6; (B) Body weight (day 49), n = 6; (C) DAI score, n = 6; (D) DAI score (day 49), n = 6; (E) Spleen index, n = 6; (F) Kidney index, n = 6; (G) Thymus index, n = 6; (H) Colon length, n = 6; (I) Images of rat colon; (J) Histological scores of the colon, n = 3; (K–M) Changes in the serum levels of GAS, MTL, and GSH-Px, n = 6. *P < 0.05, **P < 0.01, ***P < 0.001. The results showed that decreased GAS and GSH-Px levels were evident in the SKYD-UC group relative to control rats ([74]Fig. 2K-M), together with increased MTL expression (P < 0.01, P < 0.001). FLZP administration, however, reversed all of these UC-related pathological changes (P < 0.05, P < 0.01, P < 0.001). Together, these data highlight the ability of FLZP to prevent and treat SKYD-UC rat models. 3.2. Analysis of material basis of the FLZP in treating SKYD-UC [75]Table S2 provides a summary of the reference standards, while [76]Fig. 3 and [77]Figs. S1–S2 presents their proposed fragmentation pathway. As an illustration, the positive ion mode detected the reference standards (RS) 8 (6-gingerol) in [78]Table S2 at the retention time (Rt) in 37.57 min, with a m/z value of 295.1901 [M+H]^+. The MS/MS data exhibited m/z values of 195.1750 [M + H–C[6]H[12]O]^+, 180.1068 [M + H–C[7]H[15]O]^+, and 137.0596 [M + H–C[6]H[12]O–CH[3]–C[2]H[3]O]^+. Component 38 in [79]Table 1 was detected in the positive ion mode at the Rt in 37.59 min with the m/z of 295.1905 (C[17]H[26]O[4]), 195.1747 [M + H–C[6]H[12]O]^+, 180.1068 [M + H–C[7]H[15]O]^+, and 137.0930 [M + H–C[6]H[12]O–CH[3]–C[2]H[3]O]^+. The characterization of Component 38 indicated that it contained 6-gingerol. The remaining elements were recognized employing a comparable procedure to the one elucidated earlier. Fig. 3. [80]Fig. 3 [81]Open in a new tab Mass fragments and fragmentation pathways of the reference standards. (A) Mesaconine; (B) Hypaconine; (C) 7-Hydroxycoumarin; (D) Benzoylmesaconine Table 1. Characterization of chemical constituents in vivo of FLZP in the SKYD-UC model by UHPLC-MS. No. Rt (min) Systematic name Molecular formula Molecular weight Measured value (Da) PPM Fragmentations (m/z) Source C1 0.63 Cinnamaldehyde C[9]H[8]O 133.0647 [M+H]^+ 133.0649 1.5030 105.0703 Dang Shen C2 0.62 5-Hydroxymethylfurfural C[6]H[6]O[3] 127.0390 [M+H]^+ 127.0393 2.3615 109.0288, 115.3209 Bai Zhu C3 1.34 Arginine C[6]H[14]N[4]O[2] 175.1190 [M+H]^+ 175.1195 3.1282 158.0923, 130.0977, 116.0709 Gan Cao C4 1.53 Citric acid C[6]H[8]O[7] 191.0197 [M − H]^- 191.0193 −2.0940 173.0085, 111.0078, 87.0078 Dang Shen C5 1.86 Nicotinamide C[6]H[6]N[2]O 123.0553 [M+H]^+ 123.0556 2.4379 106.0291, 80.0501, 53.0394 Dang Shen C6 1.99 Tyrosine C[9]H[11]NO[3] 182.0811 [M+H]^+ 182.0813 1.0984 155.9674 Bai Zhu C7 2.06 Caffeic acid C[9]H[8]O[4] 179.0556 [M − H]^- 179.0552 −2.2339 89.0234 Dang Shen C8 2.59 Leucine C[6]H[13]NO[2] 132.1019 [M+H]^+ 132.1021 1.5140 86.097 Dang Shen C9 3.75 Phenprobamate C[9]H[11]NO[2] 166.0862 [M+H]^+ 166.0865 1.8063 149.0599 Dang Shen C10 5.47 3-Indoleacrylicacid C[11]H[9]NO[2] 188.0706 [M+H]^+ 188.0706 0 170.0601, 146.0601, 118.0654, 91.0546 Dang Shen C11 6.01 Elemenone C[15]H[22]O 219.1743 [M+H]^+ 219.1748 2.2813 189.0437 Bai Zhu C12 6.17 Tryptophan C[11]H[12]N[2]O[2] 205.0976 [M+H]^+ 205.0976 0 188.0706, 170.0600, 159.0916 Dang Shen C13 11.59 Songorine C[22]H[31]NO[3] 358.2376 [M+H]+ 358.2365 −3.0706 358.2365, 340.2592 Fu Zi C14 12.76 LG-O-GluA-O-Sul C[21]H[20]O[13]S 511.0552 [M − H]^- 511.0534 −3.4918 431.0985, 335.0237, 255.0658 Gan Cao C15 14.57 Linolenic acid C[18]H[30]O[2] 279.2319 [M+H]^+ 279.2317 −0.7163 95.0862, 81.0704, 67.0550 Dang Shen C16 15.56 7-Hydroxycoumarin C[9]H[6]O[3] 163.0390 [M+H]^+ 163.039 0 143.9949 Bai Zhu C17 16.88 Liquirtigenin C[15]H[12]O[4] 257.0808 [M+H]^+ 257.0807 −0.3890 119.0494, 135.0445 Gan Cao C18 17.54 Liguiritigenin-7-O-d-apiosyl-4′-O-D-glucoside C[26]H[30]O[13] 549.1614 [M − H]^- 549.1615 0.1821 255.0661, 135.0079 Gan Cao C19 17.97 Abscisic acid C[15]H[20]O[4] 265.1434 [M+H]^+ 265.1437 1.1315 219.1383 Dang Shen C20 19.43 Liquiritin C[21]H[22]O[9] 417.1195 [M − H]^- 417.1185 −2.3974 135.0443 Gan Cao C21 20.69 Lepenine C[22]H[33]NO[3] 360.2533 [M+H]^+ 360.2551 4.9965 342.1446 Fu Zi C22 23.59 Azelaic acid C[9]H[16]O[4] 187.0976 [M − H]^- 187.0973 −1.6034 125.0963, 97.0648 Dang Shen C23 24.20 Sulfurein C[21]H[20]O[10] 433.1129 [M+H]^+ 433.1126 −0.6927 257.0807 Gan Cao C24 27.86 Syringaldehyde C[9]H[10]O[4] 183.0651 [M+H]^+ 183.0657 3.2775 183.0657 Dang Shen C25 30.88 Cyclopenta (isoleucine) C[30]H[55]N[5]O[5] 566.4289 [M+H]^+ 566.4279 −1.7654 548.4173, 453.3437, 435.3320 Bai Zhu C26 32.44 Dehydro-10-gingerdione C[21]H[30]O[4] 347.2218 [M+H]^+ 347.2219 0.2880 177.1276, 142.0779 Gan Jiang C27 32.65 Eudesma-4(15),7(11)-dien-8-one C[15]H[22]O 219.1743 [M+H]^+ 219.1744 0.4563 203.1432, 141.0009 Bai Zhu C28 33.90 3β-Hydroxyatractylon C[15]H[20]O[2] 233.1536 [M+H]^+ 233.1534 −0.8578 95.086, 159.1169, 187.1481 Bai Zhu C29 34.38 Costunolide C[15]H[20]O[2] 233.1536 [M+H]^+ 233.1536 −0.0274 105.0702, 131.0856, 187.148 1, 215.1431 Bai Zhu C30 34.65 14-Acetyltalatisamine C[26]H[41]NO[6] 464.3007 [M+H]^+ 464.3022 3.3071 464.3022 Fu Zi C31 35.71 10-Epi-atractyloside A C[15]H[18]O[2] 231.1380 [M+H]^+ 231.1379 −0.4326 128.0623 Bai Zhu C32 35.74 Vanillylacetone C[11]H[14]O[3] 195.1015 [M+H]^+ 195.1014 −0.5126 137.0958 Gan Jiang C33 36.46 9,10,13-Trihydroxy-10-Octadecenoic Acid C[18]H[34]O[5] 329.2333 [M − H]^- 329.2334 0.3037 293. 2133, 229. 1443, 211. 1335, 171. 1020 Dang Shen C34 36.79 8-Shogaol C[19]H[28]O[3] 305.2111 [M+H]^+ 305.2103 −2.6211 137.0598 Gan Jiang C35 37.02 Dehydrocostus lactone C[15]H[18]O[2] 231.1379 [M+H]^+ 231.1381 0.8653 213.1275, 185.1326, 157.1014 Dang Shen C36 37.03 Atractylenolide I C[15]H[18]O[2] 231.1380 [M+H]^+ 231.1379 −0.4326 128.0620, 173.0965 Bai Zhu C37 37.50 Atractylenolide II C[15]H[20]O[2] 233.1536 [M+H]^+ 233.1537 0.4289 105.0703, 115.0759, 133.1013, 159, 1169, 215.1434 Bai zhu C38 37.58 6-Gingerol C[17]H[26]O[4] 317.1723 [M+Na]^+ 317.1726 0.9459 145.1013 Gan Jiang C39 37.65 Atractylenolide III C[15]H[18]O[2] 231.1361 [M+H]^+ 231.1358 −1.2979 133.1013 Bai Zhu C40 37.8 Licoflavonol C[20]H[18]O[6] 353.1030 [M − H]^- 353.1027 −1.1328 285.1126 Gan Cao C41 39.26 Uralsaponin B C[42]H[62]O[16] 821.3975 [M − H]^- 821.3964 −1.3392 351.0576, 193.0349 Gan Cao C42 39.27 Licorice saponin H2 C[42]H[62]O[16] 821.3965 [M − H]^- 821.3971 0.7305 645.3682, 469.3364 Gan Cao C43 39.85 Methyl-6-gingerol C[18]H[28]O[4] 309.2060 [M+H]^+ 309.2062 0.6468 291.1954, 151.1121 Gan Jiang C44 40.35 Glycyrrhizic acid C[42]H[62]0[16] 821.3965 [M − H]^- 821.3971 0.7305 351.0569, 193.0348, 113.0235 Gan Cao C46 40.93 6-Shogaol C[17]H[24]O[3] 277.1797 [M+H]^+ 277.1797 0 259.1697, 177.1273, 163.0753, 137.0753, 131.0857 Gang Jiang C47 41.63 Nootkatone C[15]H[22]O 219.1743 [M+H]^+ 219.1745 0.9125 81.0704 Dang Shen C48 43.11 Bullatine B C[24]H[39]NO[6] 438.2850 [M+H]^+ 438.2851 0.2282 438.2851, 420. 2742 Fu Zi C49 43.74 10-Shogaol C[21]H[32]O[3] 333.2043 [M+H]^+ 333.2037 −1.8007 137.06 Gang Jiang C50 44.34 Columbianine C[21]H[33]NO[4] 364.2482 [M+H]^+ 364.2482 0 346.2379 Fu Zi C51 44.51 9-Hydroxy-10,12-octadecadienoic acid C[18]H[32]O[3] 295.2281 [M − H]^- 295.2278 −1.0162 277.2173, 195.1386, 171.1021 Dang Shen C52 45.00 methoxy-6-gingerol C[18]H[28]O[4] 309.2415 [M+H]^+ 309.2423 2.5870 291.2323, 277.2162, 259.2057, 179.1429, 137.0962, 83.0861, 55.0550 Gang Jiang C53 46.33 Aconicarchamine A C[22]H[35]NO[4] 378.2638 [M+H]^+ 378.2638 0 378.2638, 332.2014 Fu Zi C54 46.37 KaraKoline C[22]H[35]NO[4] 378.2638 [M+H]^+ 378.2638 0 332.2594, 328.2301, 310.1999 Fu Zi C55 47.77 Glycyrrhetinic Acid C[30]H[46]O[4] 471.3468 [M − H]^- 471.3478 2.1216 453.3359, 407.3319 Gan Cao [82]Open in a new tab The absorbed components of FLZP were thus identified. [83]Fig. 4 displays the total ion chromatograms for both positive and negative ion modes. As shown in [84]Table 1, 55 absorbed components were detected in the serum of SKYD-UC model rats based on these MS data. These included 15 alkaloids, 16 terpenoids, 7 flavonoids, 7 gingerols, 3 phenylpropanoids, and 7 others ([85]Fig. 5). Fig. 4. [86]Fig. 4 [87]Open in a new tab The total ion chromatogram of FLZP in the SKYD-UC model. (A) positive and (B) negative ion modes. Fig. 5. [88]Fig. 5 [89]Open in a new tab Chemical structures of FLZP in the SKYD-UC models. (A) Alkaloids; (B) Terpenoids; (C) Flavonoids; (D) Gingerols; (E) Phenylpropanoids; (F) Others. 3.3. Prediction of mechanisms of FLZP in SKYD-UC 3.3.1. Predictive identification of SKYD-UC-related therapeutic targets When analyzing the 55 absorbed FLZP-derived compounds in SKYD-UC model rats, 1012 FLZP-related target genes were identified. In addition, 4324 SKYD-UC-associated target genes were identified from extant databases. Based on the overlap between these two gene sets, 408 potential target genes associated with the FLZP-mediated treatment of SKYD-UC were identified. 3.3.2. Core target identification The Cytoscape and the STRING database were next employed to construct a PPI network containing these target genes ([90]Fig. 6A). This network included 318 nodes (target proteins) and 4184 edges (protein-protein interactions). Additionally, 141 targets with degree values greater than the average (75.96) are compiled in [91]Table S3, including TNF, IL-6, VEGFA, and MPO. These targets may represent core targets associated with the FLZP-mediated treatment of SKYD-UC. Accordingly, ELISAs were used to validate a subset of these core target proteins ([92]Fig. 6B), revealing significant increases in IL-6, TNF-α, and MPO levels in SKYD-UC rat models relative to control rats (P < 0.001), while FLZP suppressed the upregulation of these three factors in a dose-dependent fashion. These results thus offer evidence in support of the ability of FLZP to regulate these core targets and to thereby effectively treat SKYD-UC. Fig. 6. [93]Fig. 6 [94]Open in a new tab The PPI network of the therapeutic targets (A) and changes in the levels of IL-6, TNF-α, and MPO (B). *P < 0⋅05, **P < 0.01, ***P < 0.001. n = 6 rats per group. 3.3.3. KEGG pathway analyses of core targets A KEGG pathway enrichment analysis identified 67 enriched pathways, the top 20 of which are presented in [95]Fig. S3. These pathways primarily included inflammation-, cancer-, and disease-related pathways. Identified inflammation-related pathways included the PI3K-Akt, MAPK, VEGF, and TNF pathways, while cancer-related pathways included pathways in cancer and proteoglycans in cancer, and disease-related pathways mainly include the human papillomavirus infection, hepatitis B virus pathways. Functional analyses of these core targets suggested that they are associated with the regulation of signal transduction, the immune system, metabolism, and circadian rhythms ([96]Fig. S4). These enrichment analysis results suggest that the absorbed components of FLZP may alleviate SKYD-UC at least in part via these different signaling pathways. 3.4. Identification and analysis of core bioactive FLZP-derived compounds Based on the results of part 3.2 and 3.3, a Components-Targets-Pathways (C-T-P) network was established ([97]Fig. 7A), consisting of 196 nodes (35 absorbed components, 141 core targets, 20 pathways) and 795 edges. Nine of these components exhibited degree values greater than the average (4.02), including linolenic acid, liquiritigenin, 7-hydroxycoumarin, glycyrrhizic acid, 6-shogaol, dehydro-10-gingerdione, caffeic acid, 6-gingerol, and liquiritin, suggesting that they may represent the core bioactive components of FLZP. Fig. 7. [98]Fig. 7 [99]Open in a new tab Prediction and analysis of core components. (A) Components-Targets-Pathways; (B) Molecular docking. Docking binding energies less than 5.0 kJ/mol suggest that the chemicals and their target proteins show good binding; (C) Schematic (3D) and (2D) representation that molecular model of the glycyrrhizic acid was in the protein EGFR; (D) Clustering heatmap based on Pearson correlations. The line color represents the direction of correlation: blue (positive), red (negative); the deeper the color, the stronger the correlation. *p < 0.05, and **p < 0.01. The ability of the 9 core compounds to interact with core target proteins was explored using the AutoDock Vina program. The resultant docking scores are presented in [100]Fig. 7B, highlighting good binding energy for the analyzed interactions. These molecular docking results suggest that these 9 core compounds can bind to and regulate a range of targets including IL-6, TNF-α, and MPO, with such binding being potentially relevant to the alleviation of UC. Among them, glycyrrhizic acid are equipped with the best binding ability to combine with EGFR. As shown in [101]Fig. 7C, the interaction between glycyrrhizic acid and EGFR is not only hydrogen bond, but also van der Waals, Alkyl1, and Pi-Alkyl1. The hydroxyl group of glycyrrhizic acid can form hydrogen bond with amino acid residues (such as Asp292, and Asn210), have van der Waals with Leu240, and form Pi-Alkyl1 with Pro241. These forces promote the binding of the compound to the protein. To explore the correlation intensions between 9 core compounds, biochemical parameters, and core targets, a series of Pearson correlation analyses were next conducted. Levels of 6-shogaol were negatively correlated with TNF-α, IL-6, MTL, and MPO levels but positively correlated with GAS and GSH-Px levels in the SKYD-UC model ([102]Fig. 7D). Liquiritin was negatively correlated with TNF-α, IL-6, MTL, and MPO levels, but positively correlated with GAS and GSH-Px levels in the SKYD-UC model. As such, liquiritin and 6-shogaol may help reduce the severity of SKYD-UC at least in part by influencing these targets and associated biological pathways. 4. Discussion In terms of the principles of TCM theory in The Expert Consensus on Traditional Chinese Medicine Diagnosis and Treatment of Ulcerative Colitis (2017), SKYD is a root cause of UC. The spleen, in this context, is associated with a range of functions including digestive, endocrine, immunologic, and hematopoietic functions. Appropriate spleen function is dependent on appropriate “spleen qi” and “spleen yang”, and the disruption of such function manifests in the form of “not warming” based on disordered spleen metabolic function, “not resisting” based on impaired spleen-related immune regulation, and “not transporting” based on the dysregulation of spleen-related regulation of gastrointestinal motility.[103]^5^,[104]6, [105]20 These changes, in turn, can contribute to symptoms including diarrhea, tenesmus, and abdominal pain consistent with the clinical characteristics of UC.[106]^21 Kidney Yang can also warm the spleen and ensure that it functions properly. Accordingly, in TCM, SKYD is widely recognized as the primary cause of UC and the explanation for why treating this disease is so challenging.[107]^9^,[108]^22 FLZP has been established for millennia as a potent TCM treatment for SKYD. TCM theory posits that FLZP offers the advantages of warming the middle-Jiao and tonifying the spleen and kidney yang, thereby relieving symptoms of UC such as blood in the stool and irregular stools. However, TCM prescriptions are complex and composed of a range of bioactive components that affect target diseases through myriad mechanisms. This has complicated efforts to fully elucidate the material basis and mechanisms underlying the TCM-mediated treatment of UC. Here, 55 absorbed FLZP-derived components were identified in SKYD-UC model rats, with these compounds comprising the material basis for the FLZP-mediated treatment of SKYD-UC. Network pharmacology analyses were then used to explore the mechanisms through which FLZP can treat SKYD-UC based on these identified absorbed compounds, establishing antioxidant and anti-inflammatory mechanisms as the basis for the benefits of such treatment. Lastly, a C-T-P network was established to predict 9 core FLZP-derived compounds including liquiritin and 6-shogaol, with these targets being validated through molecular docking and Pearson correlation analyses. 4.1. FLZP exerts anti-inflammatory activity to alleviate pathological changes in SKYD-UC model rats In light of the absorbed compounds identified above, core target genes of FLZP were established and used to conduct KEGG pathway enrichment analyses. These core targets included IL-6, TNF, and MPO, all of which are closely related to the inflammatory response. Inflammation is integral to UC incidence and progression. UC is characterized by pathological changes associated with the destruction of the intestinal epithelium as a result of inflammation.[109]^23 SKYD-UC rat models tested in this study exhibited severe intestinal barrier damage, while FLZP administration effectively reversed these pathological changes. Mechanistically, FLZP was predicted to alleviate SKYD-UC rats at least in part by reducing the serum levels of IL-6, TNF-α, and MPO. In addition, inflammation is often accompanied by oxidative stress in the development of UC. Therefore, we will subsequently evaluate the antioxidant role of FLZP by measuring activities such as SOD, GAT, and albumin thiol groups. KEGG enrichment analyses revealed a close association between FLZP-mediated treatment and the PI3K-Akt, MAPK, VEGF, and TNF pathways. The PI3K-Akt pathway is central to the regulation of UC-associated inflammatory signaling.[110]^24 Dysregulated PI3K-Akt pathway activity can markedly alter pro-inflammatory cytokine expression and secretion, with these cytokines, in turn, driving or accelerating UC development.[111]^24 Inhibiting PI3K-Akt and MAPK pathway signaling has repeatedly been shown to alleviate diarrhea, mucous production, and hematochezia symptoms in animal models of UC.[112]^25^,[113]^26 In some reports, significantly elevated serum VEGF concentrations have been reported in UC patients relative to healthy controls, indicating that UC may be associated with the activation of VEGF signaling activity.[114]^27 TNF is a cytokine with multiple functions associated with inflammation, immunity, apoptotic death, and cell survival. TNF is also closely tied to intestinal barrier defects in UC, with TNF pathway activation occurring following TNF binding to cell membrane receptors.[115]^28^,[116]^29 These data indicate that FLZP may be capable of inhibiting the PI3K-Akt, MAPK, VEGF, and TNF pathways and suppressing the release of inflammatory factors including IL-6 and TNF-α, thereby alleviating the symptoms of SKYD-UC ([117]Fig. 8). Fig. 8. [118]Fig. 8 [119]Open in a new tab Predicted mechanisms of FLZP in the mitigation of SKYD-UC. 4.2. Core FLZP-derived compounds associated with UC treatment In a previous study, our group uncovered 67 components in FLZP extracts, such as triterpenes, gingerols, phenylpropanoids, alkaloids, flavonoids, and volatile oils. According to the theory of serum pharmacochemistry, the primary bioactive constituents of TCM prescriptions are probably those that are assimilated within the serum.[120]^18^,[121]^19 Here, we got absorbed components. Given the absorbed components and mechanistic analyses conducted above, a C-T-P network was used to ultimately identify 9 core components of FLZP associated with the treatment of SKYD-UC. Molecular docking and Pearson correlation analyses were then conducted for these compounds, which included 3 gingerols (6-gingerol, 6-shogaol, dehydro-10-gingerdione), 2 flavonoids (liquiritin, liquirtigenin), 2 phenylpropanoids (caffeic acid, 7-hydroxycoumarin), 1 terpenoid (glycyrrhizic acid), and 1 unsaturated fatty acid (linolenic acid). Core gingerols identified in this study included 6-shogaol, 6-gingerol, and dehydro-10-gingerdione. Gingerols reportedly exhibit antioxidant and anti-inflammatory activity, thereby exerting their pharmacological effects.[122]^30 Here, a negative correlation was noted between 6-shogaol and TNF-α and IL-6 levels in the established SKYD-UC rats, suggesting that this gingerol may provide therapeutic benefits by suppressing the production of these inflammatory cytokines. Previously, 6-shogaol has similarly been reported to inhibit TNF-α and IL-6 expression in the colon of DSS-induced UC model mice.[123]^31^,[124]^32 Zhang et al. also employed a single-step surface-functionalization approach to generate nanoparticles loaded with 6-shogaol, and they found that orally administering these nanoparticles in DSS-treated mice was sufficient to reduce colitis symptoms and to enhance the repair of colitis-related tissue damage through the regulation of TNF-α and IL-6 production.[125]^33 There is also prior evidence for the ability of 6-gingerol to modulate the Th17/Treg cell balance and NF-κB signaling activity in UC model mice, thereby reducing the severity of inflammation.[126]^34 In addition, dehydro-10-gingerdione is reportedly able to suppress LPS-induced nitric oxide production and iNOS expression, thus reducing the severity of inflammatory activity.[127]^35 The flavonoids liquiritin and liquirtigenin derived from Gancao are also important sources of antioxidant and anti-inflammatory activity.[128]^36^,[129]^37 Here, liquiritin levels were noted to be negatively correlated with TNF-α, IL-6, GSH-Px, and MPO levels in SKYD-UC rats, indicating that it may suppress the expression and activity of these targets following FLZP administration, thereby reducing UC symptom severity. This is consistent with prior evidence that liquiritin can inhibit colonic TNF-α expression in a DSS-induced UC mouse model system.[130]^38 Similarly, liquirtigenin is reportedly able to inhibit NF-κB pathway activity and TNF-α, IL-1β, and IL-6 expression in a TNBS-induced mouse model of UC, thereby alleviating disease-related symptoms. The phenylpropanoid caffeic acid is frequently employed in medicinal contexts owing to its purported antimicrobial, antitumor, anti-inflammatory, and antioxidant properties.[131]^39 In one recent report, caffeic acid was found to reduce the severity of DSS-induced UC in mice via the suppression of macrophage activation.[132]^40 The phenylpropanoid 7-hydroxycoumarin is often detected in Chinese herbs (e.g. Gan cao or Eucommiae Cortex) and vegetables such as carrots and coriander. Given its reported anti-inflammatory, antioxidant, and antitumor activity, 7-hydroxycoumarin has been a recent target of interest for the production of medicines, nutritional supplements, and functional foods.[133]^41 Glycyrrhizic acid is both a pentacyclic triterpene and a saponin, and it is the key bioactive ingredient derived from dried Gancao roots. This terpenoid exhibits a range of pharmacological activities, including antiviral, anti-inflammatory, and antitumor properties.[134]^42 In one study, glycyrrhizic acid was found to regulate tight junction proteins and thereby repair damage to the intestinal mucosal barrier in UC.[135]^43 Linolenic acid is an essential fatty acid in humans and a key bioactive compound derived from Dangshen. In prior pharmacological analyses, linoleic acid was demonstrated to possess antioxidant, antitumor, and anti-inflammatory activity.[136]^44 Oral α-linolenic acid administration can reportedly suppress inflammation and thereby mitigate acute DSS-induced UC symptoms.[137]^45 Given the above results, 6-gingerol, 6-shogaol, dehydro-10-gingerdione, liquiritin, liquirtigenin, caffeic acid, 7-hydroxycoumarin glycyrrhizic acid, and linolenic acid represent the most promising core bioactive compounds of FLZP for treating SKYD-UC. As such, these targets can serve as a foundation for efforts to develop novel drugs to treat SKYD-UC and related diseases. However, it is important to note that these results are subject to some limitations. For one, the absorbed compounds of FLZP identified in the blood of experimental model rats were regarded as prototypic compounds with no consideration for the in vivo metabolic pathways associated with their processing. Additional studies of metabolites of FLZP will be vital to more fully explore the mechanisms through which this TCM prescription can treat SKYD-UC. Second, while linolenic acid, liquiritigenin, 7-hydroxycoumarin, glycyrrhizic acid, 6-shogaol, dehydro-10-gingerdione, caffeic acid, 6-gingerol, and liquiritin were identified as core compounds of FLZP, their effects alone may not be sufficient to fully recapitulate the efficacy of FLZP. Additional experimental in vivo and in vitro validation of these results will thus be necessary. 5. Conclusions In summary, an integrated serum pharmacochemistry and network pharmacology-based approach was employed to gain novel insights into the material basis for the ability of FLZP to treat SKYD-UC and the associated molecular mechanisms. In total, 55 absorbed components were identified following oral FLZP administration to SKYD-UC model rats, including 15 alkaloids, 16 terpenoids, 7 flavonoids, 7 gingerols, 3 phenylpropanoids, and 7 others. Of these, 9 core compounds associated with multiple core targets and pathways were identified. Mechanically, FLZP was suggested to alleviate SKYD-UC through the regulation of targets associated with inflammation such as IL-6, MPO, and TNF-α, while may also regulate the MAPK, TNF, VEGF, and PI3K-Akt pathways. 6. Credit authorship contribution statement You Huang &Xia Lin: performed experiments, analyzed, Writing–original draft. Qiuhong Wu & XunJian Wu: performed experiments. Shasha Yang & Yidian Dong: Formal analysis. Zhen Zhang & Chaomei Fu & Wei Lin: Conceptualization, Writing–review & editing, Funding acquisition. All authors read and approved the final manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments