, Volume 27, Issue 6, pp 1297–1307 | Cite as

Total flavonoids from Tetrastigma hemsleyanum ameliorates inflammatory stress in concanavalin A-induced autoimmune hepatitis mice by regulating Treg/Th17 immune homeostasis

  • Weiwei JiEmail author
  • Xin PengEmail author
  • Tianling Lou
  • Juan Wang
  • Wenyu Qiu
Original Article



Tetrastigma hemsleyanum, a rare and endangered medicinal plant, has attracted much attention due to its immunoregulatory and hepatoprotective activities. This study aimed to evaluate the anti-inflammatory effects and underlying mechanisms of total flavonoids from T. hemsleyanum(TFT)on Con A-induced hepatitis in mice.


TFT (1, 2 and 4 g/kg) and a positive control drug bifendate (200 mg/kg) were administered intragastrically to mice once daily for 10 consecutive days. On the 10th day, the model autoimmune of hepatitis was established by intravenous injection of Con A (20 mg/kg) 1 h after drug administration. Liver injury was assessed by serum levels of alanine amino transferase (ALT and AST) and histopathology 8 h after Con A injection. The levels of pro-inflammatory Th17 cytokines (IL-17, IL-6) and anti-inflammatory Treg cytokines (IL-10, TGF-β1) in serum were evaluated by ELISA, the levels of Th17 and Treg cells infiltrated into spleen were investigated by flow cytometry methods, and hepatic tissue transcription factor Foxp3 and RORγt mRNA were determined using quantitative real-time PCR.


Pretreatment with TFT and bifendate significantly reduced the serum levels of ALT and AST, and attenuated histopathological alterations in Con A-induced liver injury. With respect to samples treated with Con A alone, TFT and bifendate pretreatments differentially attenuated the increase of serum inflammatory factors interleukin (IL)-17 and IL-6 levels, the proportions of Th17 cells in spleen and the expression of RORγt in hepatic tissues. Meanwhile, TFT and bifendate pretreatments could enhance the percentage of Treg cells in spleen and the expression of Foxp3 in hepatic tissues, as well as the levels of transforming growth factor (TGF)-β1, IL-10 in serum.


The anti-inflammatory effects of TFT was mediated by regulating Treg/Th17 immune homeostasis, which, therefore, suppressed the inflammatory immune response. This study provided scientific basis for the further researches and clinical applications of Tetrastigma hemsleyanum.


Tetrastigma hemsleyanum Concanavalin A-induced hepatitis Th17 cells Treg cells Inflammatory immune response 



Alanine aminotransferase


Aspartate aminotransferase

Con A

Concanavalin A


Enzyme-linked immunosorbent assay


Forkhead box protein3


Haematoxylin and eosin






Retinoid-related orphan nuclear receptor-γt


Total flavonoids from Tetrastigma hemsleyanum


Transforming growth factor-β


T helper type


Tumor necrosis factor-α


T regulatory



This work was supported by the Natural Science Foundation of Ningbo City (Grant no. 2018A610430), the New Shoot Talents Program of Zhejiang Province (Grant no. 2017R435001), the Science Foundation of Zhejiang education department (Grant no. Y201534579), the Science and technology projects of Zhejiang Food and Drug Administration (YP201603), the Leading and top talent training project of Ningbo City (Grant no. NBLJ201801011), Key Laboratory of Ningbo, China (no. 2016b22002).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abe M, Hiasa Y, Onji M (2013) T helper 17 cells in autoimmune liver diseases. Clin Dev Immunol 2013:607073PubMedPubMedCentralGoogle Scholar
  2. Beringer A, Miossec P (2018) IL-17 and IL-17-producing cells and liver diseases, with focus on autoimmune liver diseases. Autoimmun Rev 17(12):1176–1185PubMedGoogle Scholar
  3. Casal Moura M, Liberal R, Cardoso H, Vale Horta E, Macedo AMG (2014) Management of autoimmune hepatitis: focus on pharmacologic treatments beyond corticosteroids. World J Hepatol 6(6):410–418PubMedPubMedCentralGoogle Scholar
  4. Chen LG, Ding L, Yu AM, Yang RL, Wang XP, Li JT, Jin HY, Zhang HQ (2007) Continuous determination of total flavonoids in Platycladus orientalis (L.) Franco by dynamic microwave-assisted extraction coupled with on-line derivatization and ultraviolet-visible detection. Anal Chim Acta 596(1): 164-170PubMedGoogle Scholar
  5. Dehghan P, Tolouie S, Baradaran B, Nami S, Morovati H (2018) TLR-2, IL-10 and IL-17-mediated immunity in experimental chemotherapy murine model of systemic candidiasis; cyclophosphamides’ impact and roles. Microb Pathog 119:183–192PubMedGoogle Scholar
  6. El-Agamy DS (2016) Pirfenidone ameliorates concanavalin A-induced hepatitis in mice via modulation of reactive oxygen species/nuclear factor kappa B signalling pathways. J Pharm Pharmacol 68(12):1559–1566PubMedGoogle Scholar
  7. Feng TT, Zou T, Wang X, Zhao WF, Qin AL (2007) Clinical significance of changes in the Th17/Treg ratio in autoimmune liver disease. World J Gastroentero 23(21):3832–3838Google Scholar
  8. Feng ZQ, Hao WQ, Lin XY, Fan DP, Zhou JH (2014) Antitumor activity of total flavonoids from Tetrastigma hemsleyanum Diels et gilg is associated with the inhibition of regulatory T cells in mice. Oncotargets Ther 7:947–956Google Scholar
  9. Fyfe B, Zaldana F, Liu C (2018) The pathology of acute liver failure. Clin Liver Dis 22(2):257–268PubMedGoogle Scholar
  10. Hirota K, Hashimoto M, Ito Y, Matsuura M, Ito H, Tanaka M, Watanabe H, Kondoh G, Tanaka A, Yasuda K, Yasuda M, Potocnik AJ, Stockinger B, Sakaguchi N, Sakaguchi S (2018) Autoimmune Th17 cells induced synovial stromal and innate lymphoid cell secretion of the cytokine GM-CSF to initiate and augment autoimmune arthritis. Immunity 48(6):1220–1232PubMedPubMedCentralGoogle Scholar
  11. Hong J, Kim BS (2018) Regulatory T cell-mediated tissue repair. Adv Exp Med Biol 1064:221–233PubMedGoogle Scholar
  12. Hou L, Jie Z, Desai M, Liang Y, Soong L, Wang T, Sun J (2013) Early IL-17 production by intrahepatic T cells is important for adaptive immune responses in viral hepatitis. J Immunol 190(2):621–629PubMedGoogle Scholar
  13. Hu BJ, Zou Y, Liu SS, Wang J, Zhu JL, Li JB, Bo LL, Deng XM (2014) Salidroside attenuates concanavalin A-Induced hepatitis via modulating cytokines secretion and lymphocyte migration in mice. Med Inflamm 1:314081Google Scholar
  14. Kuwabara T, Ishikawa F, Kondo M, Kakiuchi T (2017) The Role of IL-17 and Related Cytokines in Inflammatory Autoimmune Diseases. Mediators Inflamm 12:3908061Google Scholar
  15. Lee WY, Lee GR (2018) Transcriptional regulation and development of regulatory T cells. Exp Mol Med 50:e456PubMedPubMedCentralGoogle Scholar
  16. Li K, Liu H, Guo T (2017) Th17/Treg imbalance is an indicator of liver cirrhosis process and a risk factor for HCC occurrence in HBV patients. Clin Res Hepatol Gas 41(4):399–407Google Scholar
  17. Liao SB, Cai WW, Chen D, Xie P, Huang J, Xm Zhu, Ma GP (2017) Anti-inflammatory and Analgesic Effects of the Extracts of Tetrastigmatis Hemsleyanum’s Aerial Parts from Fujian in Vivo. Chin J Mod Appl Pharm 34(3):319–324Google Scholar
  18. Liu B, Yang J, Ma Y, Yuan E, Chen C (2010) Antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of ethanol extract and pure flavonoids from Adinandra nitida leaves. Pharm Biol 48(12):1432–1438PubMedGoogle Scholar
  19. Liu T, Zhao J, Ma L, Ding YS, Su DQ (2012) Hepatoprotective Effects of Total Triterpenoids and Total Flavonoids from Vitis vinifera L against Immunological Liver Injury in Mice. Evid Based Complement Alternat Med 2012:969386PubMedPubMedCentralGoogle Scholar
  20. Liu DD, Cao G, Han LK, Ye YL, Zhang Q, Sima YH, Ge WH (2016) Flavonoids from Radix Tetrastigmae improve LPS-induced acute lung injury via the TLR4/MD-2-mediated pathway. Mol Med Rep 14(2):1733–1741PubMedGoogle Scholar
  21. Liu YL, Yan WM, Yuan W, Wang P, Huang D, Luo XP, Ning Q (2018) Treg/Th17 imbalance is associated with poor autoimmune hepatitis prognosis. Clin Immunol PubMedGoogle Scholar
  22. Ma DD, Li WP, Ma ZL, He LY, Jiang FS, Ding ZS (2012) Anti-liver Damage Activity Analysis of Polysaccharide in Radix Tetrastigmatis Hemsleyani. J Med Res 41(1):33–36 (in Chinese)Google Scholar
  23. Malemud CJ (2018) Defective T-Cell Apoptosis and T-Regulatory Cell Dysfunction in Rheumatoid Arthritis. Cell 7(12): pii: E223, PubMedCentralGoogle Scholar
  24. MiX Wang, Zhang H (2018) The pathogenesis of autoimmune hepatitis. Front Lab Med 2:36–39Google Scholar
  25. Noack M, Miossec P (2014) Th17 and regulatory T cell balance in autoimmune and inflammatory diseases. Autoimun Rev 13(6):668–677Google Scholar
  26. Oh IS, Park SH (2015) Immune-mediated Liver Injury in Hepatitis B Virus Infection. Immune Netw 15(4):191–198PubMedPubMedCentralGoogle Scholar
  27. Owczarczyk-Saczonek A, Czerwińska J, Placek W (2018) The role of regulatory T cells and anti-inflammatory cytokines in psoriasis. Acta Dermatovenerol Alp Pannonica Adriat 27(1):17–23PubMedGoogle Scholar
  28. Pandiyan P, Zhu J (2015) Origin and functions of pro-inflammatory cytokine producing Foxp3 + regulatory T cells. Cytokine 76(1):13–24PubMedPubMedCentralGoogle Scholar
  29. Paquissi FC (2007) Immunity and Fibrogenesis: the Role of Th17/IL-17 Axis in HBV and HCV-induced Chronic Hepatitis and Progression to Cirrhosis. Front Immunol 8:1–16Google Scholar
  30. Peng X, Zhang YY, Wang J, Ji QY (2016) Ethylacetate extract from Tetrastigma hemsleyanum induces apoptosis via the mitochondrial caspase-dependent intrinsic pathway in HepG2 cells. Tumor Biol 37:865–876Google Scholar
  31. Qu N, Xu M, Mizoguchi I, Furusawa J, Kaneko K, Watanabe K, Mizuguchi J, Itoh M, Kawakami Y, Yoshimoto T (2013) Pivotal roles of T-helper 17-related cytokines, IL-17, IL-22, and IL-23, in inflammatory diseases. Clin Dev Immunol 2013:968549PubMedPubMedCentralGoogle Scholar
  32. Roeleveld DM, Koenders MI (2015) The role of the Th17 cytokines IL-17 and IL-22 in Rheumatoid Arthritis pathogenesis and developments in cytokine immunotherapy. Cytokine 74(1):101–107PubMedGoogle Scholar
  33. Ru Y, Chen X, Wang J, Guo L, Lin Z, Peng X, Qiu B (2018) Polysaccharides from Tetrastigma hemsleyanum Diels et Gilg: extraction optimization, structural characterizations, antioxidant and antihyperlipidemic activities in hyperlipidemic mice. Int J Biol Macromol 123:775–783PubMedGoogle Scholar
  34. Sun DM, Liang DC, Kaplan HJ, Shao H (2015) The role of Th17-associated cytokines in the pathogenesis of experimental autoimmune uveitis (EAU). Cytokine 74(1):76–80PubMedPubMedCentralGoogle Scholar
  35. Taubert R, Hupa-Breier KL, Jaeckel E, Manns MP (2018) Novel therapeutic targets in autoimmune hepatitis. J Autoimmun 95:34–46PubMedGoogle Scholar
  36. Tian MX, Liu WL, You HJ, Zhao QZ, Ouyang LD, Gao BE, Zhang X, Che NC (2016) Protective effect of Yiguanjian decoction against DNA damage on concanavalin A-induced liver injury micemodel. J Tradit Chin Med 36(4):471–478PubMedGoogle Scholar
  37. Tiegs G (2007) Cellular and cytokine-mediated mechanisms of inflammation and its modulation in immune-mediated liver injury. Z GastroenterolZeitschrift Fur Gastroenterologie 45(1):63–70Google Scholar
  38. Vicente O, Boscaiu B (2018) Flavonoids: Antioxidant Compounds for Plant Defence…and for a Healthy Human Diet. Not Bot Horti Agrobo 46(1):14-21Google Scholar
  39. Wang CJ, Liu XY, Li ZJ, Liu SK (2012a) Systematic review of randomized control trials on treatment with bifendatatum for chronic hepatitis B. Int J Pathol Clin Med 32(2): 118–123. (In Chinese) Google Scholar
  40. Wang HX, Liu M, Weng SY, Li JJ, Xie C, He HL, Guan W, Yuan YS, Gao J (2012b) Immune mechanisms of Concanavalin A model of autoimmune hepatitis. World J Gastroenterol 18(2):119–125PubMedPubMedCentralGoogle Scholar
  41. Wang F, Xue Y, Yang JY, Lin F, Sun Y, Li T, Wu CF (2016a) Hepatoprotective effect of apple polyphenols against concanavalin A-induced immunological liver injury in mice. Chem Biol Interact 258:159–165PubMedGoogle Scholar
  42. Wang WH, Wu ZZ, Niu GJ, Zhu S, Li SZ, Liang YC (2016b) Studies on the Supersonic Extraction Process Total Flavonoids from Tetrastigma Hemsleyanum. Strait Pharmaceutical Journal 28(3):45–48 (in Chinese) Google Scholar
  43. Wang XL, Zhou SQ, Ma XZ, Zhang LP (2016c) Tetrastigma hemsleyanum (Sanyeqing) extracts reduce inflammation and oxidative stress in a chronic obstructive pulmonary disease rat model. Int J Clin Exp Med 9:19447–19453Google Scholar
  44. Wang LY, Tu L, Zhang JP, Xu KS, Qian W (2017) Stellate cell activation and imbalanced expression of TGF-beta 1/TGF-beta 3 in acute autoimmune liver lesions induced by conA in mice. Biomed Res Int 2017:2540540PubMedPubMedCentralGoogle Scholar
  45. Wang CY, Jang HJ, Han YK, Su XD, Lee SW, Rho MC, Wang HS, Yang SY, Kim YH (2018a) Alkaloids from Tetrastigma hemsleyanum and Their Anti-Inflammatory Effects on LPS-Induced RAW264.7 Cells. Mol 23(6): pii: E1445PubMedCentralGoogle Scholar
  46. Xia GT, Wu SS, Wang X, Fu M (2018) Inhibition of microRNA-155 attenuates concanavalin- A-induced autoimmune hepatitis by regulating Treg/Th17 cell differentiation. Can J Physiol Pharmacol 96(12):1293–1300PubMedGoogle Scholar
  47. Xie GX, Zhou J, Zhang CZ, Yang JH, Chen JX, Jin HQ (1981) Synthesis of schisandrin C analogs. Acta Pharm Sin 16(4):306–309 (In Chinese) Google Scholar
  48. Xu CJ, Ding GQ, Fu JY, Meng J, Zhang RH, Lou XM (2008) Immunoregulatory effects of ethylacetate fraction of extracts from Tetrastigma Hemsleyanum Diels et. Gilg on immune functions of ICR mice. Biomed Environ Sci 21:325–331PubMedGoogle Scholar
  49. Yan S, Wang L, Liu N, Wang Y, Chu Y (2012) Critical role of interleukin-17/interleukin-17 receptor axis in mediating Con A-induced hepatitis. Immunol Cell Biol 90(4):421–428PubMedGoogle Scholar
  50. Yu H, Cao NJ, Pan WK, Su L, Zhao YY, Tian DH, Xu WY, Gao Y, Zheng BJ (2018) Correlation of spatio-temporal characteristics of intestinal inflammation with IL-17 in a rat model of hypoganglionosis. Biochem Bioph Co 506(4):956–961Google Scholar
  51. Yüksel M, Laukens D, Heindryckx F, Van Vlierberghe H, Geerts A, Wong FS, Wen L, Colle I (2014) Hepatitis mouse models: from acute-to-chronic autoimmune hepatitis. Int J Exp Path 95:309–320Google Scholar
  52. Zhang Y, Shan L, Hua YP, Wang D, Zeng HW, Liu RH, Zhang WD, Hu ZL (2013) Baicalein selectively induces apoptosis in activated lymphocytes and ameliorates concanavalin A-induced hepatitis in mice. PLoS One 8(7):e69592PubMedPubMedCentralGoogle Scholar
  53. Zhang ZM, Xue ZY, Liu Y, Liu HK, Guo XD, Li Y, Yang HW, Zhang LJ, Da YR, Yao Z, Zhang RX (2018) MicroRNA-181c promotes Th17 cell differentiation and mediates experimental autoimmune encephalomyelitis. Brain Behav Immun 70:305–314PubMedGoogle Scholar
  54. Zhao X, Liu MJ, Li JG, Yin S, Wu Y, Wang A (2017) Antimalarial agent artesunate protects Concanavalin A-induced autoimmune hepatitis in mice by inhibiting inflammatory responses. Chem-Biol Interact 274:116–123PubMedGoogle Scholar
  55. Zhou YQ, Weng XF, Dou R, Tan XS, Zhang TT, Fang JB, Wu XW (2017) Betulin from Hedyotis hedyotidea ameliorates concanavalin A-induced and T cell-mediated autoimmune hepatitis in mice. Acta Pharmacol Sin 38(2):201–210PubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Institute of Biopharmaceutical, Zhejiang Pharmaceutical CollegeNingboPeople’s Republic of China

Personalised recommendations