Kangfuxin alleviates ulcerative colitis in rats by inhibiting NF-κB p65 activation and regulating T lymphocyte subsets

Document Type : Original Article


1 Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Dali, China

2 Yunnan Provincial 2011 Collaborative Innovation Center for Entomoceutics, Dali, Yunnan, China

3 School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China

4 Good Doctor Pharmaceutical Group, Chengduo, Sichuang, China

5 Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Dali, China

6 Cancer and Anticancer Drug Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China


Objective(s): Ulcerative colitis (UC) remains an enduring, idiopathic inflammatory bowel disease marked by persistent mucosal inflammation initiating from the rectum and extending in a proximal direction. An ethanol extract of Periplaneta americana L., namely Kangfuxin (KFX), has a significant historical presence in Traditional Chinese Medicine and has been broadly utilized in clinical practice for the treatment of injury. Here, we aimed to determine the effect of KFX on 2,4,6-trinitro’benzene sulfonic acid (TNBS)-induced UC in Sprague-Dawley rats.
Materials and Methods: We established the UC model by TNBS/ethanol method. Then, the rats were subject to KFX (50, 100, 200 mg/kg/day) for 2 weeks by intragastric gavage. The body weight, disease activity index (DAI), colonic mucosal injury index (CMDI), and histopathological score were evaluated. The colonic tissue interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), IL-10, transforming growth factor-1 (TGF-β1), and epidermal growth factor (EGF) were determined by Elisa. To study T-lymphocyte subsets, flow cytometry was performed. In addition, the expression level of NF-κB p65 was evaluated by immunohistochemistry and western blot analysis.
Results: Compared with the TNBS-triggered colitis rats, the treatment of rats with KFX significantly increased the body weight, and decreased DAI, CMDI, and histopathological score. Also, KFX elicited a reduction in the secretion of colonic pro-inflammatory cytokines, namely IL-1β, IL-6, and TNF-α, concomitant with up-regulation of IL-10, TGF-β1, and EGF levels. Upon KFX treatment, the CD3+CD4+/CD3+CD8+ ratio in the spleen decreased, while the CD3+CD8+ subset and the CD3+CD4+CD25+/CD3+CD4+ ratio demonstrated an increase. In addition, the expression of NF-κB p65 in the colon was decreased.
Conclusion: KFX effectively suppresses TNBS-induced colitis by inhibiting the activation of NF-κB p65 and regulating the ratio of CD4+/CD8+.


Main Subjects

1. Ordás I, Eckmann L, Talamini M, Baumgart DC, Sandborn WJ. Ulcerative colitis. Lancet 2012; 380: 1606-1619.
2. Eftychi C, Schwarzer R, Vlantis K, Wachsmuth L, Basic M, Wagle P, et al. Temporally distinct functions of the cytokines IL-12 and IL-23 drive chronic colon inflammation in response to intestinal barrier impairment. Immunity 2019; 51:367-380.
3. Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature 2011; 474:298-306.
4. Neurath MF. Cytokines in inflammatory bowel disease. Nat Rev Immunol 2014; 14:329-342.
5. Acovic A, Simovic Markovic B, Gazdic M, Arsenijevic A, Jovicic N, Gajovic N, et al. Indoleamine 2,3-dioxygenase-dependent expansion of T-regulatory cells maintains mucosal healing in ulcerative colitis. Therap Adv Gastroenterol 2018; 11:1756-1759.
6. Fukaura K, Iboshi Y, Ogino H, Ihara E, Nakamura K, Nishihara Y, et al. Mucosal profiles of immune molecules related to T helper and regulatory T cells predict future relapse in patients with quiescent ulcerative colitis. Inflamm Bowel Dis 2019; 25:1019-1027.
7.  Golubovskaya V, wu L. Different subsets of T cells, memory, effector functions, and CAR-T immunotherapy. Cancers (Basel) 2016; 8:36-48.
8. Konjar S, Ferreira C, Blankenhaus B, Veldhoen M. Intestinal barrier interactions with specialized CD8 T cells. Front Immunol 2017; 8:1-15.
9. Kappeler A, Mueller C. The role of activated cytotoxic T cells in inflammatory bowel disease. Histol Histopathol 2000; 15:167-172.
10. Funderburg NT, Stubblefield Park SR, Sung HC, Hardy G, Clagett B, Ignatz-Hoover J, et al. Circulating CD4(+) and CD8(+) T cells are activated in inflammatory bowel disease and are associated with plasma markers of inflammation. Immunology 2013; 140:87-97.
11. Muller S, Lory J, Corazza N, Griffiths GM, Z’Graggen K, Mazzucchelli L, et al. Activated CD4+ and CD8+ cytotoxic cells are present in increased numbers in the intestinal mucosa from patients with active inflammatory bowel disease. Am J Pathol 1998; 152:261-268.
12.Lin S, Y Li, L Shen, R Zhang, L Yang, M Li, et al. The anti-inflammatory effect and intestinal barrier protection of HU210 differentially depend on TLR4 signaling in dextran sulfate sodium-induced murine colitis. Dig Dis Sci 2017; 62:372-386.
13. Zhang MS, Du Y. Advances in studies on probiotics for treatment of ulcerative colitis based on TLR4-NF-κB signaling pathway. Chin J Microecol 2019; 31:104-107.
14. Li LJ, Xu XH, Yuan TJ, Hou J, Yu CL, Peng LH. Periplaneta americana L. as a novel therapeutics accelerates wound repair and regeneration. Biomed Pharmacother 2019; 114:1-9. 
15. Lin HL, Li GJ, Wu JZ. Effect of aluminum phosphate gel and kangfuxin on esophageal pathology and IL-8 and PGE2 expressions in a rat model of reflux esophagitis. Nan Fang Yi Ke Da Xue Xue Bao 2015; 35:573-577.
16. Chen P, Shen Y, Shi H, Ma X, Lin B, Xiao T, et al. Gastroprotective effects of kangfuxin-against ethanol-induced gastric ulcer via attenuating oxidative stress and ER stress in mice. Chem Biol Interact 2016; 260:75-83.
17. Shen Y, Sun J, Niu C, Yu D, Chen Z, Cong W, et al. Mechanistic evaluation of gastroprotective effects of Kangfuxin on ethanol-induced gastric ulcer in mice. Chem Biol Interact 2017; 273:115-124.
18. Lu S, Wu D, Sun G, Geng F, Shen Y, Tan J, et al. Gastroprotective effects of kangfuxin against water-immersion and restraint stress-induced gastric ulcer in rats: roles of antioxidation, anti-inflammation, and pro-survival. Pharm Biol 2019; 57:770-777.
19. Li HB, Chen MY, Qiu ZW, Cai QQ, Li DT, Tang HM, et al. Efficacy and safety of kangfuxin liquid combined with aminosalicylic acid for the treatment of ulcerative colitis: A systematic review and meta-analysis. Medicine (Baltimore) 2018; 97:1-11.
20. Arunachalam K, Damazo AS, Macho A, Lima J, Pavan E, Figueiredo FF, et al. Piper umbellatum L. (piperaceae): phytochemical profiles of the hydroethanolic leaf extract and intestinal anti-inflammatory mechanisms on 2,4,6 trinitrobenzene sulfonic acid induced ulcerative colitis in rats. J Ethnopharmacol 2020; 254:112707-112720.
21. Zheng L, Zhang YL, Dai YC, Chen X, Chen DL, Dai YT, et al. Jianpi qingchang decoction alleviates ulcerative colitis by inhibiting nuclear factor-kappaB activation. World J Gastroenterol 2017; 23:1180-1188.
22. Yu WG, Lu B, Zhang HW, Zhang YX, Yan J. Effects of the sijunzi decoction on the immunological function in rats with dextran sulfate-induced ulcerative colitis. Biomed Rep 2016; 5:83-86.
23. Millar AD, Rampton DS, Chander CL, Claxson AW, Blades S, Coumbe A, et al. Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis. Gut 1996; 39:407-415.
24. Zhao H, Wang M, Gao Y, Wu X, Xiao H, Yang D, et al. Vespakinin-M, a natural peptide from vespa magnifica, promotes functional recovery in stroke mice. Commun Biol 2022; 5:1-14.
25. He J, Liang J, Zhu S, Zhao W, Zhang Y, Sun W. Protective effect of taurohyodeoxycholic acid from pulvis fellis suis on trinitrobenzene sulfonic acid induced ulcerative colitis in mice. Eur J Pharmacol 2011; 670:229-235.
26. Obermeier F, Dunger N, Strauch UG, Grunwald N, Herfarth H, Scholmerich J, et al. Contrasting activity of cytosin-guanosin dinucleotide oligonucleotides in mice with experimental colitis. Clin Exp Immunol 2003; 134:217-224.
27. Tang Z, Xiong D, Song J, Ye M, Liu J, Wang Z, et al. Antitumor drug combretastatin-A4 phosphate aggravates the symptoms of dextran sulfate sodium-induced ulcerative colitis in mice. Front Pharmacol 2020; 11:339-349.
28. Dou B, Hu W, Song M, Lee RJ, Zhang X, Wang D. Anti-inflammation of erianin in dextran sulphate sodium-induced ulcerative colitis mice model via collaborative regulation of TLR4 and STAT3. Chem Biol Interact 2020; 324:1-11.
29. Zhu XM, Shi YZ, Cheng M, Wang DF, Fan JF. Serum IL-6, IL-23 profile and Treg/Th17 peripheral cell populations in pediatric patients with inflammatory bowel disease. Pharmazie 2017; 72:283-287.
30. Garrett WS, Lord GM, Punit S, Lugo-Villarino G, Mazmanian SK, Ito S, et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 2007; 131:33-45.
31. Zhang SZ, Zhao XH, Zhang DC. Cellular and molecular immunopathogenesis of ulcerative colitis. Cell Mol Immunol 2006; 3:35-40.
32. Ordas I, Eckmann L, Talamini M, Baumgart DC, Sandborn WJ. Ulcerative colitis. Lancet 2012; 380:1606-1619.
33. Li S, Li L, Guo C, Qin H, Yu X. A promising wound dressing material with excellent cytocompatibility and proangiogenesis action for wound healing: strontium loaded silk fibroin/sodium alginate (SF/SA) blend films. Int J Biol Macromol 2017; 104: 969-978.
34. Ou HL. Study on the anti-tumor effects and immunomodulatory function of periplaneta americana peptide. 2017, Dali University.
35. Du WW, Liu H, Zhang HC, Li GK, Zhang CG, Geng FN, et al. Therapeutic effect and mechanism of kangfuxin liquid on oxazolone-induced ulcerative colitis in rats. Chin J Exp Trad Med Formul 2017; 23:126-131.
36. Zhang JN, Sun MZ, Liu H, Zhang HC, Xiao H, Zhao Y, et al. The ethanol extract of Periplaneta americana L. improves ulcerative colitis induced by a combination of chronic stress and TNBS in rats. Acta Cir Bras 2022; 37:370505-370519.
37. Zhu L, Gu P, Shen H. Protective effects of berberine hydrochloride on DSS-induced ulcerative colitis in rats. Int Immunopharmacol 2019; 68:242-251.
38. Zhang HC, Wang PC, Liu H, Geng FN, Ma XY, He M, et al. Effect of kangfuxin on ulcerative colitis induced by trinitrobenzene sulfonic acid in rats. Chin Pharmacol Bull 2018; 34:496-501.
39. Fan SJ, Pan ZY, Geng Q, Li X, Wang YF, An Y, et al. Layered signaling regulatory networks analysis of gene expression involved in malignant tumorigenesis of non-resolving ulcerative colitis via integration of cross-study microarray profiles. PLoS One 2013; 8:1-13.
40. Cao SG, Wang H, Lin LM, Xia XP, Wu H. Cryptotanshinone inhibits prostaglandin E2 production and COX-2 expression via suppression of TLR4/NF-κB signaling pathway in LPS-stimulated Caco-2 cells. Microb Pathogen 2018; 116:313-317.
41. Rezayat SM, Dehpour AR, Motamed SM, Yazdanparast M, Chamanara M, Sahebgharani M, et al. Foeniculum vulgare essential oil ameliorates acetic acid-induced colitis in rats through the inhibition of NF-kB pathway. Inflammopharmacology 2018; 26:851-859.
42. Luo S, Deng XL, Liu Q, Pan ZF, Zhao ZX, Zhou L, et al. Emodin ameliorates ulcerative colitis by the flagellin-TLR5 dependent pathway in mice. Int Immunopharmacol 2018; 59:269-275.
43. Antoniou E, Margonis GA, Angelou A, Pikouli A, Argiri P, Karavokyros I, et al. The TNBS-induced colitis animal model: an overview. Ann Med Surg (Lond) 2016; 11:9-15.
44. El-Salhy M, Hatlebakk JG. Changes in enteroendocrine and immune cells following colitis induction by TNBS in rats. Mol Med Rep 2016; 14:4967-4974.
45. Motavallian-Naeini A, Andalib S, Rabbani M, Mahzouni P, Afsharipour M, Minaiyan M. Validation and optimization of experimental colitis induction in rats using 2, 4, 6-trinitrobenzene sulfonic acid. Res Pharm Sci 2012; 7:159-169.
46. Mehto S, Jena KK, Nath P, Chauhan S, Kolapalli SP, Das SK, et al. Mol Cell 2019; 73:429-445.
47. Wang L, Liu WZ, Pei LY, Ke YS, Cui J, Li SC. Garidisan: improving the quality of ulcer healing in rats with ulcerative colitis. Evid Based Complement Alternat Med 2017; 2017:1-12.
48. Long Y, Li SX, Qin JC, Xie LW, Gan LP, Jie FM, et al. Kuijieling regulates the differentiation of treg and Th17 cells to ameliorate experimental colitis in rats. Biomed Pharmacother 2018; 105:781-788.
49. Postovalova EA, Khochansky DN, Zolotova NA, Gao Y, Makarova OV, Dobrynina. Morphological changes in mesenteric lymph nodes and lymphocyte subpopulation composition in experimental ulcerative colitis. Bull Exp Biol Med 2016; 160:835-839.