Therapeutic mechanism of Cynanchum wilfordii for ulcerative colitis: an analysis using UPLC-QE-MS, network pharmacology and metabolomics

doi:10.12122/j.issn.1673-4254.2024.08.07

Abstract

Abstract:

Objective To explore the targets and pathways of Cynanchum wilfordii for treatment of ulcerative colitis (UC). Methods UPLC-QE-MS was used to identify the components of Cynanchum wilfordii ethanol extract, and their targets were screened using public databases for construction of the core protein-protein interaction (PPI) network and GO and KEGG enrichment analyses. Forty male C57 mice were randomized into normal control group, model group, mesalazine group and Cynanchum wilfordii group (n=10), and in the latter 3 groups, mouse UC models were established by treatment with 2.5% DSS and the latter 2 groups drug interventions by gavage. The therapeutic effect was evaluated by recording body weight changes and DAI score. Pathological changes of the colon tissue were observed with HE and AB-PAS staining, and JAK2 and STAT3 protein expressions were detected with Western blotting. The metabolites and metabolic pathways were identified by metabonomics analysis. Results We identified 240 chemical components in Cynanchum wilfordii alcoholic extracts, including 19 steroids. A total of 177 Cynanchum wilfordii targets, 5406 UC genes, and 117 intersection genes were obtained. JAK2 and STAT3 were the core targets and significantly enriched in lipid and atherosclerosis pathways. Cynanchum wilfordii treatment significantly increased the body weight and decreased DAI score of UC mice (P<0.05), alleviated intestinal pathologies, and decreased JAK2 and STAT3 protein expressions in the colon tissues. Most of the 83 intersecting differential metabolites between the control, model and Cynanchum wilfordii groups were identified as glycerophospholipids, arachidonic acid, and amino acids involving glycerophospholipid metabolism and other pathways. Correlation analysis suggested that the core targets of Cynanchum wilfordii for UC participated in regulation of the metabolites. Conclusion Cynanchum wilfordii alleviates lipid and amino acid metabolism disorders to lessen UC in mice by regulating the core targets including JAK2 and STAT3 and the levels of endogenous metabolites.

Key words: Cynanchum wilfordii (Maxim.) Hook. F, ulcerative colitis, metabolomics, UPLC-QE-MS, steroids

Guanzheng YU, Weiqiang CHENG, Xing TU, Man ZHANG, Hong LI, Juan NIE. Therapeutic mechanism of Cynanchum wilfordii for ulcerative colitis: an analysis using UPLC-QE-MS, network pharmacology and metabolomics[J]. Journal of Southern Medical University, 2024, 44(8): 1485-1496.

Figures/Tables 13

Tab.1 Results of identification of the steroids in Cynanchum wilfordii

NO.	t_R/min	Chemical compound	Chemical formula	Ionization mode	m/z	Theoretical m/z
1	11.54	Qingyangshengenin B	C₄₉H₇₈O₁₆	M-H, M+FA-H	967.5273	967.5272
2	9.72	Qingyangshengenin A	C₄₉H₇₂O₁₇	M+NH₄, M+Na	955.4642	955.4662
3	5.53	Convallatoxin	C₂₉H₄₂O₁₀	M+FA-H	595.2765	595.2760
4	9.7	Caudatin	C₂₈H₄₂O₇	M-H, M+FA-H	535.2915	535.2912
5	10.22	Cynatratoside A	C₂₈H₄₀O₈	M+H-H₂O	487.2679	487.2690
6	6.45	Qingyangshengenin	C₂₈H₃₆O₈	M+Na	523.2292	523.2303
7	7.33	5beta-Dihydrocortisol	C₂₁H₃₂O₅	M+H	365.2315	365.2323
8	9.13	Acetylarenobufagin	C₂₆H₃₄O₇	M+Na	481.2214	481.2196
9	9.36	Polyphyllin VI	C₃₉H₆₂O₁₃	M+K	777.3818	777.3822
10	9.2	Oleandrin	C₃₂H₄₈O₉	M+Na	599.3215	599.3190
11	10.47	Gracillin	C₄₅H₇₂O₁₇	M-H₂O-H	865.4589	865.4591
12	7.57	19-Oxocinobufagin	C₂₆H₃₂O₇	2M-H	911.4252	911.4223
13	10.28	Dioscin	C₄₅H₇₂O₁₆	M+FA-H	913.4814	913.4802
14	11.52	(25R)-Spirost-4-en-3,12-dion	C₂₇H₃₈O₄	M+NH₄	444.3099	444.3109
15	10.14	3-O-Acetylbufotalin	C₂₈H₃₈O₇	M+Na	509.2527	509.2509
16	11.57	Timosaponin A1	C₃₃H₅₄O₈	M+Na	601.3699	601.3711
17	5.83	Bufarenogin	C₂₄H₃₂O₆	2M+Na	855.4328	855.4289
18	5.38	Digoxigenin	C₂₃H₃₄O₅	M+H-H₂O	373.236	373.2373
19	4.82	Rubrosterone	C₁₉H₂₆O₅	M+NH₄	352.2113	352.2119

Fig.1 Venn diagrams, network regulation charts and core PPI. A: Venn diagram. B: Network regulation charts. C: PPI network. D: Core PPI. CW: Cynanchum wilfordii.

Fig.2 GO enrichment analysis. A: Biological process. B: Cellular components. C: Molecular function.

Fig.3 KEGG enrichment analysis.

Fig.4 Weight change rate and DAI score of the mouse models of UC. A: Weight change rate. B: DAI score. N: Normal group. M: Model group. Y: Mesalazine group. G: Cynanchum wilfordii Group. #P<0.05 vs normal group; *P<0.05 vs model group.

Fig.5 HE staining and AB-PAS staining of the colon tissue of the mice. A: Normal group. B: Model group. C: mesalazine group. D: Cynanchum wilfordii Group.

Fig.6 Expression of JAK2 and STAT3 proteins in the colon tissue of the mice detected by Western blotting. CW: Cynanchumwilfordii Group. #P<0.05 vs normal group; *P<0.05 vs model group;△P<0.05 vs mesalazine group.

Fig.7 Base peak chromatogram. A: Normal group. B: Model group. C: mesalazine group. D: Cynanchum wilfordii Group.

Fig.8 Metabolomics multivariate statistical analysis. A: PCA. B, C: OPLS-DA.

Fig.9 Volcano map based on different metabolites. A:M vs N; B: M vs G.

Fig.10 Heatmaps based on different metabolites.

Fig.11 Differential metabolite-enriched metabolic pathways.

Fig.12 Metabolite-target gene network. Squares represent differential genes, circles represent the core target. The darker the color, the stronger the correlation.

References 31

1	Ordás I, Eckmann L, Talamini M, et al. Ulcerative colitis[J]. Lancet, 2012, 380(9853): 1606-19.
2	Ukil A, Maity S, Das PK. Protection from experimental colitis by theaflavin-3, 3'-digallate correlates with inhibition of IKK and NF-kappaB activation[J]. Br J Pharmacol, 2006, 149(1): 121-31.
3	Gao X, Li J, Pang XP, et al. Animal models and pathogenesis of ulcerative colitis[J]. Comput Math Methods Med, 2022, 2022: 5927384.
4	Du L, Ha C. Epidemiology and pathogenesis of ulcerative colitis[J]. Gastroenterol Clin North Am, 2020, 49(4): 643-54.
5	周长琳, 郑学宝, 黄晓其, 等. 黄芩汤通过调节ILC3s-Th细胞反应减轻小鼠的溃疡性结肠炎[J]. 南方医科大学学报, 2021, 41(2): 256-63.
6	Sun Y, Zhang Z, Zheng CQ, et al. Mucosal lesions of the upper gastrointestinal tract in patients with ulcerative colitis: a review[J]. World J Gastroenterol, 2021, 27(22): 2963-78.
7	王康, 缪志伟, 董筠, 等. 基于STAT3/NF-kB/IL-6通路研究加味黄芩汤治疗溃疡性结肠炎的作用机制[J]. 南方医科大学学报, 2020, 40(2): 196-202.
8	张照, 杨菊, 王加伟, 等.左金丸对DSS诱导的溃疡性结肠炎的作用及其机制[J].中国实验方剂学杂志, 2023, 29(16): 1-11.
9	沈洪, 唐志鹏, 唐旭东, 等.消化系统常见病溃疡性结肠炎中医诊疗指南（基层医生版）[J].中华中医药杂志, 2019, 34(9): 4155-60.
10	孙佳, 刘利琴, 勾健, 等. 基于 “双态” 在体肠循环灌流模型研究隔山消提取物肠吸收特性差异[J]. 中国中药杂志, 2022, 47(23): 6340-7.
11	勾健, 吴宗芹, 孟欣, 等. 基于UHPLC Q-Exactive Plus Orbitrap HRMS高分辨质谱技术的隔山消提取物化学成分分析[J]. 中国新药杂志, 2024, 33(5): 489-98.
12	Ru JL, Li P, Wang JN, et al. TCMSP: a database of systems pharmacology for drug discovery from herbal medicines[J]. J Cheminform, 2014, 6: 13.
13	谢泽宇, 许一笑, 郑梦圆, 等. 基于网络药理学探究合欢皮-白蒺藜药对抑制肝星状细胞系LX2的抗焦亡作用[J]. 中国中药杂志, 2023, 48(2): 481-91.
14	胡雪黎, 周昌园, 徐睿, 等. 基于UPLC-Q-TOF-MS技术的土家族药三颗针干预溃疡性结肠炎的代谢组学研究[J]. 中国中药杂志, 2023, 48(9): 2490-9.
15	Cooper HS, Murthy SN, Shah RS, et al. Clinicopathologic study of dextran sulfate sodium experimental murine colitis[J]. Lab Invest, 1993, 69(2): 238-49.
16	Saber S, El-Kader EMA. Novel complementary coloprotective effects of metformin and MCC950 by modulating HSP90/NLRP3 interaction and inducing autophagy in rats[J]. Inflammo-pharmacology, 2021, 29(1): 237-51.
17	席进, 张敏, 张永玉, 等. 上调KLF11可改善结肠炎模型小鼠的肠道炎症: 基于抑制JAK2/STAT3信号通路[J]. 南方医科大学学报, 2024, 44(4): 765-72.
18	Lu Z, Xiong WN, Xiao SM, et al. Huanglian Jiedu Decoction ameliorates DSS-induced colitis in mice via the JAK2/STAT3 signalling pathway[J]. Chin Med, 2020, 15: 45.
19	刘星赐, 吴东升, 曹晖, 等. 基于LC-MS研究芍药汤对溃疡性结肠炎大鼠粪便代谢产物的影响[J]. 中国实验方剂学杂志, 2023, 29(9): 61-70.
20	张婷, 陈烨, 王中秋, 等. 炎症性肠病患者肠道菌群结构的变化及其与炎性指标的关系[J]. 南方医科大学学报, 2013, 33(10): 1474-7, 1498.
21	龚顺航, 杨杰, 张金涛, 等. 基于HMGCR及PPARα信号通路探究雪莲果浸膏对高脂血症大鼠脂质代谢的影响[J]. 南方医科大学学报, 2023, 43(11): 1977-83.
22	张甜甜, 项楚涵, 夏勇, 等. M1型巨噬细胞非靶向脂质组学分析[J]. 中国病理生理杂志, 2021, 37(4): 711-7.
23	杨慧, 蒋且英, 刘漩, 等. 基于UPLC-Q-TOF-MS技术白术多糖干预溃疡性结肠炎的代谢组学研究[J]. 中草药, 2023, 54(15): 4895-904.
24	Cho WH, Yeo HJ, Yoon SH, et al. Lysophosphatidylcholine as a prognostic marker in community-acquired pneumonia requiring hospitalization: a pilot study[J]. Eur J Clin Microbiol Infect Dis, 2015, 34(2): 309-15.
25	戎晓娟, 孔令霏, 康雨彤, 等. 基于脂质代谢组学的卵白蛋白诱导小鼠过敏性哮喘机制研究[J]. 中国药理学通报, 2023, 39(3): 477-83.
26	朱洁, 侯宝龙, 程雯, 等. 基于血清代谢组学探究色胺酮抗小鼠溃疡性结肠炎的作用机制[J]. 中国中药杂志, 2023, 48(8): 2193-202.
27	Nakanishi M, Menoret A, Tanaka T, et al. Selective PGE(2) suppression inhibits colon carcinogenesis and modifies local mucosal immunity[J]. Cancer Prev Res, 2011, 4(8): 1198-208.
28	Li MH, Zeng YX, Ge LL, et al. Evaluation of the influences of low dose polybrominated diphenyl ethers exposure on human early retinal development[J]. Environ Int, 2022, 163: 107187.
29	Diederen K, Li JV, Donachie GE, et al. Exclusive enteral nutrition mediates gut microbial and metabolic changes that are associated with remission in children with Crohn’s disease[J]. Sci Rep, 2020, 10(1): 18879.
30	Mammedova JT, Sokolov AV, Freidlin IS, et al. The mechanisms of L-arginine metabolism disorder in endothelial cells[J]. Biochemistry, 2021, 86(2): 146-55.
31	Seim GL, Britt EC, Fan J. Analysis of arginine metabolism using LC-MS and isotopic labeling[J]. Methods Mol Biol, 2019, 1978: 199-217.

[1]	Na ZHAO, Mengdi SHEN, Rui ZHAO, Di AO, Zetan LUO, Yinliang ZHANG, Zhidong XU, Fangtian FAN, Hailun ZHENG. column:Sanguinarine alleviates ulcerative colitis in mice by regulating the Nrf2/NF-κB pathway [J]. Journal of Southern Medical University, 2024, 44(8): 1467-1475.
[2]	HU Sigan, CHENG Zengwei, LI Min, GAO Shiyi, GAO Dasheng, KANG Pinfang. Correlation between insulin resistance and coronary collateral circulation in patients with chronic total coronary occlusion [J]. Journal of Southern Medical University, 2024, 44(4): 780-786.
[3]	SONG Zejun, DONG Haibin, MA Na, REN Yutang, JIANG Bo. Value of Improved Mayo Endoscopic Score for evaluating treatment efficacy for active ulcerative colitis [J]. Journal of Southern Medical University, 2023, 43(7): 1204-1213.
[4]	WANG Xuancheng, ZHU Yifan, ZHOU Hailin, HUANG Zongsheng, CHEN Hongwei, ZHANG Jiahao, YANG Shanyi, CHEN Guanghui, ZHANG Qisong. Integrated analysis of serum untargeted metabolomics and targeted bile acid metabolomics for identification of diagnostic biomarkers for colorectal cancer [J]. Journal of Southern Medical University, 2023, 43(3): 443-453.
[5]	SONG Zejun, ZHANG Mingjun, REN Yutang, JIANG Bo. Improved Mayo Endoscopic Score has a higher value for evaluating clinical severity of ulcerative colitis [J]. Journal of Southern Medical University, 2022, 42(7): 997-1005.
[6]	KUERBANNAIMU Kaheman, ZHAO Jianfeng, MUKAIDAISI Aihemaiti, WANG Hanming, ZHU Jiwei, PAN Wentao, KASIMUJIANG Aximujiang. E.faecium QH06 alleviates TNBS-induced colonic mucosal injury in rats [J]. Journal of Southern Medical University, 2022, 42(7): 976-987.
[7]	LIU Wenhu, TANG Jiancai, CHANG Jinxia. RUNX3 regulates trastuzumab resistance of gastric cancer cells: a metabolomic analysis based on UPLC-Q Exactive Focus Orbitrap mass spectrometry [J]. Journal of Southern Medical University, 2022, 42(4): 498-508.
[8]	WANG Jiayuan, YUAN Yiyi, ZHANG Kun, SUN Xiang, BU Xin, DONG Jian, WU Yousheng, TIAN Hongying, SHEN Lan. NDRG2 inhibits tumorigenesis of hepatocellular carcinoma by regulating metabolism of phospholipids and triglyceride: a metabonomic analysis [J]. Journal of Southern Medical University, 2022, 42(12): 1765-1773.
[9]	. rCsHscB derived from Clonorchis sinensis has therapeutic effect on dextran sodium sulfate-induced chronic ulcerative colitis in mice [J]. Journal of Southern Medical University, 2021, 41(5): 664-670.
[10]	. Huangqin decoction alleviates ulcerative colitis by regulating ILC3s-TH cell response [J]. Journal of Southern Medical University, 2021, 41(2): 256-263.
[11]	. Mechanism of Jiawei Huangqin decoction for treating ulcerative colitis in mice: the role of STAT3/NF-kB/IL-6 pathway [J]. Journal of Southern Medical University, 2020, 40(02): 196-202.
[12]	. Kirenol relieves dextran sulfate sodium-induced ulcerative colitis in mice by inhibiting inflammatory cytokines and inducing CD4+ T lymphocyte apoptosis [J]. Journal of Southern Medical University, 2019, 39(12): 1387-1392.
[13]	. Changes of cerebral cortical metabolomics in rats following benzo[a]pyrene exposure [J]. Journal of Southern Medical University, 2018, 38(02): 162-.
[14]	. Expressions of inflammatory cytokines in intestinal mucosa and their prognostic value in patients with ulcerative colitis [J]. Journal of Southern Medical University, 2016, 36(12): 1712-.
[15]	. Effect of chronic arsenic exposure on mouse brain tissue and serum metabolomics [J]. Journal of Southern Medical University, 2016, 36(09): 1192-.