Vol 61, No 2 (2023)
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Published online: 2023-06-20

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Cryptotanshinone ameliorates hemorrhagic shock-induced liver injury via activating the Nrf2 signaling pathway

Jiahui Han1, Di Jia1, Hao Yao1, Ting Lv2, Xi Xu2, Xin Ge13
Pubmed: 37435898
Folia Histochem Cytobiol 2023;61(2):109-122.


Introduction. Hemorrhagic shock (HS) is an important cause of high mortality in traumatized patients. Cryptotanshinone
(CTS) is a bioactive compound extracted from Salvia miltiorrhiza Bunge (Danshen). The current study aimed to explore the effect and underlying mechanism of CTS on the liver injury induced by HS.
Material and methods. Male Sprague-Dawley rats were used to establish the HS model by hemorrhaging and monitoring mean arterial pressure (MAP). CTS was intravenously administered at concentration of 3.5 mg/kg, 7 mg/kg, or 14 mg/kg 30 minutes before resuscitation. Twenty-four hours after resuscitation, the liver tissue and serum samples were collected for the following examinations. Hematoxylin and eosin (H&E) staining was used to evaluate hepatic morphology changes. The myeloperoxidase (MPO) activity in liver tissue and the serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were examined to reveal the extent of liver injury. The protein expression of Bax and Bcl-2 in liver tissue was detected by western blot. The TUNEL assay determined the apoptosis of hepatocytes. Oxidative stress of liver tissue was assessed by the examination of reactive oxygen species (ROS) generation. The content of malondialdehyde (MDA), glutathione (GSH), and adenosine triphosphate (ATP), the activity of superoxide dismutase (SOD) and oxidative chain complexes (complex I, II, III, IV), as well as cytochrome c expression in cytoplasm and mitochondria, were also used to determine the extent of oxidative injury in the liver. Immunofluorescence (IF) was employed to estimate nuclear factor E2-related factor 2 (Nrf2) expression. The mRNA and protein levels of heme oxygenase 1 (HO-1), NAD(P)H: quinone oxidoreductases 1 (NQO1), cyclooxygenase-2 (COX-2), and nitric oxide synthase (iNOS) were assessed by real-time qPCR, western blot to investigate the mechanism of CTS regulating HS-induced liver injury.
Results. H&E staining and a histological score of rat liver suggested that HS induced liver injury. The activity of ALT, AST, and MPO was significantly increased by HS treatment. After CTS administration the ALT, AST, and MPO activities were suppressed, which indicates the liver injury was alleviated by CTS. The HS-induced upregulation of the TUNEL-positive cell rate was suppressed by various doses of CTS. HS-induced ROS production was decreased and the protein expression of Bax and Bcl-2 in the HS-induced rat liver was reversed by CTS administration. In the liver of HS-induced rats, the upregulation of MDA content and the downregulation of GSH content and SOD activity were suppressed by CTS. Additionally, CTS increases ATP content and mitochondrial oxidative complexes activities and suppressed the release of cytochrome c from mitochondria to the cytoplasm. Moreover, IF and western blot demonstrated that the activation of Nrf2 blocked by HS was recovered by different doses of CTS in liver tissue. The expression of downstream enzymes of the Nrf2 pathway, including HO-1, NQO1, COX-2, and iNOS, was reversed by CTS in the HS rat model.
Conclusions. The current study for the first time revealed the protective effect of CTS in HS-induced liver injury. CTS
effectively recovered hepatocyte apoptosis, oxidative stress, and mitochondria damage induced by HS in the rat liver
partly via regulating the Nrf2 signaling pathway.

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  1. Eastridge BJ, Holcomb JB, Shackelford S, et al. Outcomes of traumatic hemorrhagic shock and the epidemiology of preventable death from injury. Transfusion. 2019; 59(S2): 1423–1428.
  2. Cannon JW, Cannon JW. Hemorrhagic Shock. N Engl J Med. 2018; 378(4): 370–379.
  3. Chen H, Huang RS, Yu XX, et al. Emodin protects against oxidative stress and apoptosis in HK-2 renal tubular epithelial cells after hypoxia/reoxygenation. Exp Ther Med. 2017; 14(1): 447–452.
  4. Liang W, Greven J, Qin K, et al. Sulforaphane exerts beneficial immunomodulatory effects on liver tissue via a Nrf2 pathway-related mechanism in a murine model of hemorrhagic shock and resuscitation. Front Immunol. 2022; 13: 822895.
  5. Liu H, Xiao X, Sun C, et al. Systemic inflammation and multiple organ injury in traumatic hemorrhagic shock. Front Biosci (Landmark Ed). 2015; 20(6): 927–933.
  6. Matot I, Katz M, Pappo O, et al. Resuscitation with aged blood exacerbates liver injury in a hemorrhagic rat model. Crit Care Med. 2013; 41(3): 842–849.
  7. Guan Z, Zhou L, Zhang Yu, et al. Sulforaphane ameliorates the liver injury of traumatic hemorrhagic shock rats. J Surg Res. 2021; 267: 293–301.
  8. Jin YC, Kim CW, Kim YM, et al. Cryptotanshinone, a lipophilic compound of Salvia miltiorrriza root, inhibits TNF-alpha-induced expression of adhesion molecules in HUVEC and attenuates rat myocardial ischemia/reperfusion injury in vivo. Eur J Pharmacol. 2009; 614(1-3): 91–97.
  9. Kim EJu, Jung SN, Son KHo, et al. Antidiabetes and antiobesity effect of cryptotanshinone via activation of AMP-activated protein kinase. Mol Pharmacol. 2007; 72(1): 62–72.
  10. Zhu R, Wang W, Yang S, et al. Cryptotanshinone inhibits hypoxia/reoxygenation-induced oxidative stress and apoptosis in renal tubular epithelial cells. J Cell Biochem. 2019; 120(8): 13354–13360.
  11. Wu YH, Wu YR, Li Bo, et al. Cryptotanshinone: A review of its pharmacology activities and molecular mechanisms. Fitoterapia. 2020; 145: 104633.
  12. Jin HJ, Xie XL, Ye JM, et al. TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells. PLoS One. 2013; 8(1): e51720.
  13. Wang Na, Dong X, Shi D, et al. Cryptotanshinone ameliorates placental oxidative stress and inflammation in mice with gestational diabetes mellitus. Arch Pharm Res. 2020; 43(7): 755–764.
  14. Liu H, Xie J, Fan L, et al. Cryptotanshinone protects against PCOS-induced damage of ovarian tissue via regulating oxidative stress, mitochondrial membrane potential, inflammation, and apoptosis via regulating ferroptosis. Oxid Med Cell Longev. 2022; 2022: 8011850.
  15. Shi D, Li H, Zhang Z, et al. Cryptotanshinone inhibits proliferation and induces apoptosis of breast cancer MCF-7 cells via GPER mediated PI3K/AKT signaling pathway. PLoS One. 2022; 17(1): e0262389.
  16. Vundavilli H, Datta A, Sima C, et al. Anti-tumor effects of cryptotanshinone (C(19)H(20)O(3)) in human osteosarcoma cell lines. Biomed Pharmacother. 2022; 150: 112993.
  17. Bahn G, Jo DG. Therapeutic approaches to alzheimer's disease through modulation of NRF2. Neuromolecular Med. 2019; 21(1): 1–11.
  18. Li C, Tang B, Feng Yu, et al. Pinostrobin exerts neuroprotective actions in neurotoxin-induced Parkinson's disease models through Nrf2 induction. J Agric Food Chem. 2018; 66(31): 8307–8318.
  19. Cui Y, Ma S, Zhang C, et al. Pharmacological activation of the Nrf2 pathway by 3H-1, 2-dithiole-3-thione is neuroprotective in a mouse model of Alzheimer disease. Behav Brain Res. 2018; 336: 219–226.
  20. Cinegaglia N, Acosta-Navarro J, Rainho C, et al. Association of omnivorous and vegetarian diets with antioxidant defense mechanisms in men. J Am Heart Assoc. 2020; 9(12): e015576.
  21. Zhang Q, Liu J, Duan H, et al. Activation of Nrf2/HO-1 signaling: an important molecular mechanism of herbal medicine in the treatment of atherosclerosis the protection of vascular endothelial cells from oxidative stress. J Adv Res. 2021; 34: 43–63.
  22. Bian Y, Chen Y, Wang X, et al. Oxyphylla A ameliorates cognitive deficits and alleviates neuropathology via the Akt-GSK3β and Nrf2-Keap1-HO-1 pathways in and in murine models of Alzheimer's disease. J Adv Res. 2021; 34: 1–12.
  23. Fragoulis A, Schenkel J, Herzog M, et al. Nrf2 ameliorates DDC-induced sclerosing cholangitis and biliary fibrosis and improves the regenerative capacity of the liver. Toxicol Sci. 2019; 169(2): 485–498.
  24. Taguchi K, Masui S, Itoh T, et al. Nrf2 activation ameliorates hepatotoxicity induced by a heme synthesis inhibitor. Toxicol Sci. 2019; 167(1): 227–238.
  25. Yamamoto M, Kensler TW, Motohashi H. The KEAP1-NRF2 system: a thiol-based sensor-effector apparatus for maintaining redox homeostasis. Physiol Rev. 2018; 98(3): 1169–1203.
  26. Liu FC, Chaudry IH, Yu HP. Hepatoprotective effects of corilagin following hemorrhagic shock are through Akt-dependent pathway. Shock. 2017; 47(3): 346–351.
  27. Liu FC, Liu FW, Yu HP. Ondansetron attenuates hepatic injury via p38 MAPK-dependent pathway in a rat haemorrhagic shock model. Resuscitation. 2011; 82(3): 335–340.
  28. El-Emam SZ, Soubh AA, Al-Mokaddem AK, et al. Geraniol activates Nrf-2/HO-1 signaling pathway mediating protection against oxidative stress-induced apoptosis in hepatic ischemia-reperfusion injury. Naunyn Schmiedebergs Arch Pharmacol. 2020; 393(10): 1849–1858.
  29. Lu WJ, Lin KH, Tseng MF, et al. New therapeutic strategy of hinokitiol in haemorrhagic shock-induced liver injury. J Cell Mol Med. 2019; 23(3): 1723–1734.
  30. Hsieh YC, Yu HP, Suzuki T, et al. Upregulation of mitochondrial respiratory complex IV by estrogen receptor-beta is critical for inhibiting mitochondrial apoptotic signaling and restoring cardiac functions following trauma-hemorrhage. J Mol Cell Cardiol. 2006; 41(3): 511–521.
  31. Poulose N, Raju R. Aging and injury: alterations in cellular energetics and organ function. Aging Dis. 2014; 5(2): 101–108.
  32. Wang H, Guan Y, Karamercan MA, et al. Resveratrol rescues kidney mitochondrial function following hemorrhagic shock. Shock. 2015; 44(2): 173–180.
  33. Mao Y, Qu Y, Wang Q. Cryptotanshinone reduces neurotoxicity induced by cerebral ischemia-reperfusion injury involving modulation of microglial polarization. Restor Neurol Neurosci. 2021; 39(3): 209–220.
  34. Wolf A, Thakral S, Mulier KE, et al. Evaluation of novel formulations of d-β-hydroxybutyrate and melatonin in a rat model of hemorrhagic shock. Int J Pharm. 2018; 548(1): 104–112.
  35. Nugent WH, Sheppard FR, Dubick MA, et al. Microvascular and systemic impact of resuscitation with pegylated carboxyhemoglobin-based oxygen carrier or hetastarch in a rat model of transient hemorrhagic shock. Shock. 2020; 53(4): 493–502.
  36. Wilfred BS, Madathil SK, Cardiff K, et al. Alterations in peripheral organs following combined hypoxemia and hemorrhagic shock in a rat model of penetrating ballistic-like brain injury. J Neurotrauma. 2020; 37(4): 656–664.
  37. Tomiyama K, Ikeda A, Ueki S, et al. Inhibition of Kupffer cell-mediated early proinflammatory response with carbon monoxide in transplant-induced hepatic ischemia/reperfusion injury in rats. Hepatology. 2008; 48(5): 1608–1620.
  38. Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 1997; 275(5303): 1132–1136.
  39. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997; 275(5303): 1129–1132.
  40. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008; 9(1): 47–59.
  41. Muñoz-Pinedo C, Guío-Carrión A, Goldstein JC, et al. Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration. Proc Natl Acad Sci U S A. 2006; 103(31): 11573–11578.
  42. Kinnally KW, Antonsson B. A tale of two mitochondrial channels, MAC and PTP, in apoptosis. Apoptosis. 2007; 12(5): 857–868.
  43. Sun F, Zhou Q, Pang X, et al. Revealing various coupling of electron transfer and proton pumping in mitochondrial respiratory chain. Curr Opin Struct Biol. 2013; 23(4): 526–538.
  44. Reisman SA, Buckley DB, Tanaka Y, et al. CDDO-Im protects from acetaminophen hepatotoxicity through induction of Nrf2-dependent genes. Toxicol Appl Pharmacol. 2009; 236(1): 109–114.
  45. Xu W, Hellerbrand C, Köhler UA, et al. The Nrf2 transcription factor protects from toxin-induced liver injury and fibrosis. Lab Invest. 2008; 88(10): 1068–1078.
  46. Battino M, Giampieri F, Pistollato F, et al. Nrf2 as regulator of innate immunity: A molecular Swiss army knife! Biotechnol Adv. 2018; 36(2): 358–370.
  47. Thimmulappa RK, Lee H, Rangasamy T, et al. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest. 2006; 116(4): 984–995.
  48. Kahroba H, Ramezani B, Maadi H, et al. The role of Nrf2 in neural stem/progenitors cells: From maintaining stemness and self-renewal to promoting differentiation capability and facilitating therapeutic application in neurodegenerative disease. Ageing Res Rev. 2021; 65: 101211.
  49. Saji N, Francis N, Blanchard CL, et al. Rice bran phenolic compounds regulate genes associated with antioxidant and anti-inflammatory activity in human umbilical vein endothelial cells with induced oxidative stress. Int J Mol Sci. 2019; 20(19).
  50. Kudoh K, Uchinami H, Yoshioka M, et al. Nrf2 activation protects the liver from ischemia/reperfusion injury in mice. Ann Surg. 2014; 260(1): 118–127.
  51. Zheng Yi, Tao S, Lian F, et al. Sulforaphane prevents pulmonary damage in response to inhaled arsenic by activating the Nrf2-defense response. Toxicol Appl Pharmacol. 2012; 265(3): 292–299.
  52. Lin W, Wu RT, Wu T, et al. Sulforaphane suppressed LPS-induced inflammation in mouse peritoneal macrophages through Nrf2 dependent pathway. Biochem Pharmacol. 2008; 76(8): 967–973.
  53. Jiang Xi, Hu Y, Zhou Y, et al. Irisin protects female mice with LPS-induced endometritis through the AMPK/NF-κB pathway. Iran J Basic Med Sci. 2021; 24(9): 1247–1253.
  54. Hsu JT, Kuo CJ, Chen TH, et al. Melatonin prevents hemorrhagic shock-induced liver injury in rats through an Akt-dependent HO-1 pathway. J Pineal Res. 2012; 53(4): 410–416.
  55. Matsiukevich D, Piraino G, Lahni P, et al. Metformin ameliorates gender-and age-dependent hemodynamic instability and myocardial injury in murine hemorrhagic shock. Biochim Biophys Acta Mol Basis Dis. 2017; 1863(10 Pt B): 2680–2691.
  56. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 2007; 47: 89–116.
  57. McGrath-Morrow S, Lauer T, Yee M, et al. Nrf2 increases survival and attenuates alveolar growth inhibition in neonatal mice exposed to hyperoxia. Am J Physiol Lung Cell Mol Physiol. 2009; 296(4): L565–L573.
  58. Zoja C, Benigni A, Remuzzi G. The Nrf2 pathway in the progression of renal disease. Nephrol Dial Transplant. 2014; 29 Suppl 1: i19–i24.