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TAF and TDF attenuate liver fibrosis through NS5ATP9, TGFβ1/Smad3, and NF-κB/NLRP3 inflammasome signaling pathways

  • Jing Zhao
  • Ming Han
  • Li Zhou
  • Pu Liang
  • Yun Wang
  • Shenghu Feng
  • Hongping Lu
  • Xiaoxue Yuan
  • Kai Han
  • Xiaofan Chen
  • Shunai Liu
  • Jun ChengEmail author
Original Article

Abstract

Background

This study aimed to investigate the roles and mechanisms of tenofovir alafenamide fumarate (TAF)/tenofovir disoproxil fumarate (TDF) in treating liver fibrosis.

Methods

The effects of TAF/TDF on carbon tetrachloride (CCl4)-induced liver fibrosis in C57BL/6 wild-type or nonstructural protein 5A transactivated protein 9 (NS5ATP9) knockout mice were studied. The differentiation, activation, and proliferation of LX-2 cells after TAF/TDF treatment were tested in vitro. The expression of NS5ATP9 and activities of transforming growth factor-β1 (TGFβ1)/Sekelsky mothers against decapentaplegic homolog 3 (Smad3) and NF-κB/NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome signaling pathways were detected in TAF/TDF-treated mice and LX-2 cells. The genes related to extracellular matrix accumulation were detected in vivo and in vitro after NS5ATP9 silencing or knockout.

Results

TAF/TDF significantly inhibited CCl4-induced liver fibrosis in mice, and regulated the differentiation, activation, and proliferation of hepatic stellate cells (HSCs). Furthermore, TAF/TDF suppressed the activities of TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways in vivo and in vitro. NS5ATP9 inhibited liver fibrosis through TGFβ1/Smad3 and NF-κB signaling pathways. TAF/TDF upregulated the expression of NS5ATP9 in vivo and in vitro. Finally, TAF/TDF could only show marginal therapeutic effects when NS5ATP9 was silenced and knocked out in vivo and in vitro.

Conclusions

TAF/TDF prevented progression and promoted reversion of liver fibrosis through assembling TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways via upregulating the expression of NS5ATP9. TAF/TDF also regulated the differentiation, activation, and proliferation of HSCs. The findings provided strong evidence for the role of TAF/TDF as a new promising therapeutic strategy in liver fibrosis.

Keywords

TAF TDF Liver fibrosis NS5ATP9 

Notes

Author contributions

JC designed the experiments. JZ performed the experiments and wrote the manuscript. MH and LZ performed most of the experiments involving cells. PL performed molecular, biochemical assays, and participated in the revision of the manuscript. YW, SF, and HL provided NS5ATP9-KO mice. XY and KH analyzed and interpreted the data. XC performed molecular and biochemical assays.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81470863 and No. 81670547), the National Key Research and Development Program of China (No. 2017YFC0908100 / 2017YFC0908104), the Beijing Municipal Administration of Hospitals (XMLX201711 to JC), the Beijing Municipal Administration of Hospitals’ Ascent Plan (DFL20151701), and the National Science and Technology Major Project (No. 2017ZX10302201-005-004 and No. 2017ZX10202202-005-008). Support was also provided by the Program of Beijing Advanced Innovation Center for Big Data-Based Precision Medicine and the Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China.

Compliance with ethical standards

Conflict of interest

Jing Zhao, Ming Han, Li Zhou, Pu Liang, Yun Wang, Shenghu Feng, Hongping Lu, Xiaoxue Yuan, Kai Han, Xiaofan Chen, Shunai Liu, and Jun Cheng declare that they have no competing interests.

Statement of human and animal rights

a) Statement of human rights: this chapter does not contain any studies with human participants performed by any of the authors. b) Statement on welfare of animals: all procedures performed in studies involving animals were in accordance with the ethical standards of Institute Research Ethics Committee of Beijing Ditan Hospital. The entire study was approved by the Institute Research Ethics Committee of Beijing Ditan Hospital.

Supplementary material

12072_2019_9997_MOESM1_ESM.pdf (4.2 mb)
Supplementary Fig. 1. TAF and TDF inhibited liver fibrosis. TAF-L, TAF-M, TAF-H, TDF-M, and TDF-H represented 0.45 mg/(kg · d), 4.5 mg/(kg · d), 45 mg/(kg · d), 5 mg/(kg · d), and 50 mg/(kg · d), respectively. (A) Expression of α-SMA and collagen I mRNAs in mice. The CCl4 group was compared with the corn oil group, while the TAF or TDF group was compared with the CCl4 group (n = 3). (B) Plasma ALT and AST levels (n = 7). (C) Survival curve is shown. (D) Expression of TGFβ1 mRNAs (n = 8). The results shown are mean ± standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001. Supplementary Fig. 2. TAF and TDF attenuated liver fibrosis via TGFβ1/Smad3 and NF-κB signaling pathways. (A and C) Western blot of protein extracted from LX2 cells treated with TAF or TDF either at various concentrations for 48 h (B, D) or at a concentration of 250 μM for different times. (E) Expression of molecules of TGFβ1/Smad3 and NF-κB signaling pathways at the protein level in liver tissue significantly increased (F) and significantly decreased after treatment with TAF and TDF, respectively. Western blot analysis of proteins of the two signaling pathways. (G) LX2 cells were stimulated with TGFβ1 (2.5 or 5 ng/mL) for 24 h. (H) The LX-2 cells were treated with different concentrations of SD-208 (a TGF-βRI inhibitor), and PDTC (an NF-κB inhibitor) for 24 h to explore the optimal dose of inhibitors. (H) The total protein was extracted from LX-2 cells treated with SD-208 (1 μM), and PDTC (2 μM) for 24 h. Supplementary Fig. 3. NS5ATP9 -KO mice. (A) DEG and (B) GO of fibrosis-related genes for WT and NS5ATP9-KO mice. (C) Plasma ALT and AST levels (n = 6). (D) Western blot analysis of protein levels of NS5ATP9 in liver tissues. (E) Immunohistochemistry for NS5ATP9 of liver tissues (200 ×). (F) Gene sequencing for WT and NS5ATP9-KO mice was performed. Mice lacking only five bases were considered to be homozygous for NS5ATP9-KO mice. Supplementary Fig. 4. TAF and TDF upregulated the expression of NS5ATP9. (A and B) Expression of NS5ATP9 was detected using Western blot analysis and RT-qPCR. (C) The HepG2 cells were transfected transiently with 250 ng NS5ATP9 promoter plasmids with or without different concentrations of TAF or TDF. NS5ATP9 promoter activity was detected using dual-luciferase reporter assay. The results shown are mean ± standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001. (PDF 4284 kb)
12072_2019_9997_MOESM2_ESM.doc (134 kb)
Supplementary Table 1. Optimized primers used in the study. Supplementary Table 2. Primary antibodies used in the study. Supplementary Table 3. Comparison between TAF and TDF. The molecular formula, relative molecular mass, absorption, transformation, half-life, and clinical dosage were compared between TAF and TDF (DOC 133 kb)

References

  1. 1.
    Del Campo JA, Gallego P, Grande L. Role of inflammatory response in liver diseases: therapeutic strategies. World J Hepatol 2018;10(1):1–7.PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Roeb E. Matrix metalloproteinases and liver fibrosis (translational aspects). Matrix Biol 2018;68–69:463–473.PubMedCrossRefGoogle Scholar
  3. 3.
    European Association for the Study of the Liver. Electronic address EEE, European Association for the Study of the L. EASL Clinical practice guidelines for the management of patients with decompensated cirrhosis. J Hepatol 2018;69(2):406–460.CrossRefGoogle Scholar
  4. 4.
    Li K, Ma Q, Shi L, Dang C, Hong Y, Wang Q, et al. NS5ATP9 gene regulated by NF-kappaB signal pathway. Arch Biochem Biophys 2008;479(1):15–19.PubMedCrossRefGoogle Scholar
  5. 5.
    Wu DM, Han XR, Wen X, Wang S, Fan SH, Zhuang J, et al. Salidroside protection against oxidative stress injury through the Wnt/beta-catenin signaling pathway in rats with Parkinson’s disease. Cell Physiol Biochem 2018;46(5):1793–1806.PubMedCrossRefGoogle Scholar
  6. 6.
    Marchetti P, Trinh A, Khamari R, Kluza J. Melanoma metabolism contributes to the cellular responses to MAPK/ERK pathway inhibitors. Biochim Biophys Acta 2018;1862(4):999–1005.CrossRefGoogle Scholar
  7. 7.
    Shi L, Zhang SL, Li K, Hong Y, Wang Q, Li Y, et al. NS5ATP9, a gene up-regulated by HCV NS5A protein. Cancer Lett 2008;259(2):192–197.PubMedCrossRefGoogle Scholar
  8. 8.
    Karg E, Smets M, Ryan J, Forne I, Qin W, Mulholland CB, et al. Ubiquitome analysis reveals PCNA-associated factor 15 (PAF15) as a specific ubiquitination target of UHRF1 in embryonic stem cells. J Mol Biol 2017;429(24):3814–3824.PubMedCrossRefGoogle Scholar
  9. 9.
    Li G, Luna C, Gonzalez P. miR-183 inhibits UV-induced DNA damage repair in human trabecular meshwork cells by targeting of KIAA0101. Invest Ophthalmol 2016;57(4):2178–2186.CrossRefGoogle Scholar
  10. 10.
    Quan M, Liu S, Li G, Wang Q, Zhang J, Zhang M, et al. A functional role for NS5ATP9 in the induction of HCV NS5A-mediated autophagy. J Viral Hepat 2014;21(6):405–415.PubMedCrossRefGoogle Scholar
  11. 11.
    Sang Y, Zang W, Yan Y, Liu Y, Fu Q, Wang K, et al. Study of differential effects of TGF-beta3/BMP2 on chondrogenesis in MSC cells by gene microarray data analysis. Mol Cell Biochem 2014;385(1–2):191–198.PubMedCrossRefGoogle Scholar
  12. 12.
    Wang X, Jung YS, Jun S, Lee S, Wang W, Schneider A, et al. PAF-Wnt signaling-induced cell plasticity is required for maintenance of breast cancer cell stemness. Nat Commun 2016;7:10633.PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Zhang M, Zhang J, Liu S, Wang Q, Lin G, Qiu R, et al. NS5ATP9 suppresses activation of human hepatic stellate cells, possibly via inhibition of Smad3/phosphorylated-Smad3 expression. Inflammation 2015;38(1):278–289.PubMedCrossRefGoogle Scholar
  14. 14.
    Hladik F, Burgener A, Ballweber L, Gottardo R, Vojtech L, Fourati S, et al. Mucosal effects of tenofovir 1% gel. eLife 2015.  https://doi.org/10.7554/elife.04525.PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Kuo MT, Hu TH, Hung CH, Wang JH, Lu SN, Tsai KL, et al. Hepatitis B virus relapse rates in chronic hepatitis B patients who discontinue either entecavir or tenofovir. Aliment Pharmacol Ther 2019;49:218–228.PubMedCrossRefGoogle Scholar
  16. 16.
    Agarwal K, Brunetto M, Seto WK, Lim YS, Fung S, Marcellin P, et al. 96 weeks treatment of tenofovir alafenamide vs. tenofovir disoproxil fumarate for hepatitis B virus infection. J Hepatol 2018;68(4):672–681.PubMedCrossRefGoogle Scholar
  17. 17.
    Papatheodoridis GV, Idilman R, Dalekos GN, Buti M, Chi H, van Boemmel F, et al. The risk of hepatocellular carcinoma decreases after the first 5 years of entecavir or tenofovir in Caucasians with chronic hepatitis B. Hepatology 2017;66(5):1444–1453.PubMedCrossRefGoogle Scholar
  18. 18.
    Chang TT, Liaw YF, Wu SS, Schiff E, Han KH, Lai CL, et al. Long-term entecavir therapy results in the reversal of fibrosis/cirrhosis and continued histological improvement in patients with chronic hepatitis B. Hepatology 2010;52(3):886–893.PubMedCrossRefGoogle Scholar
  19. 19.
    Choi J, Kim HJ, Lee J, Cho S, Ko MJ, Lim YS. Risk of hepatocellular carcinoma in patients treated with entecavir vs. tenofovir for chronic hepatitis B: a Korean Nationwide Cohort Study. JAMA Oncol 2019;5(1):30–36.PubMedCrossRefGoogle Scholar
  20. 20.
    Bangen JM, Hammerich L, Sonntag R, Baues M, Haas U, Lambertz D, et al. Targeting CCl4-induced liver fibrosis by RNA interference-mediated inhibition of cyclin E1 in mice. Hepatology 2017;66(4):1242–1257.PubMedCrossRefGoogle Scholar
  21. 21.
    Sun YW, Zhang YY, Ke XJ, Wu XJ, Chen ZF, Chi P, et al. Pirfenidone prevents radiation-induced intestinal fibrosis in rats by inhibiting fibroblast proliferation and differentiation and suppressing the TGF-beta1/Smad/CTGF signaling pathway. Eur J Pharmacol 2018;822:199–206.PubMedCrossRefGoogle Scholar
  22. 22.
    Chen L, Li J, Zhang J, Dai C, Liu X, Wang J, et al. S100A4 promotes liver fibrosis via activation of hepatic stellate cells. J Hepatol 2015;62(1):156–164.PubMedCrossRefGoogle Scholar
  23. 23.
    Yoshida K, Matsuzaki K, Murata M, Yamaguchi T, Suwa K, Okazaki K. Clinico-pathological importance of TGF-beta/Phospho-Smad signaling during human hepatic fibrocarcinogenesis. Cancers 2018;10(6):183.PubMedCentralCrossRefGoogle Scholar
  24. 24.
    Peng J. The pharmacological targets and clinical evidence of natural products with anti-hepatic inflammatory properties. Front Pharmacol 2018;9:455.PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Yu X, Lan P, Hou X, Han Q, Lu N, Li T, et al. HBV inhibits LPS-induced NLRP3 inflammasome activation and IL-1beta production via suppressing the NF-kappaB pathway and ROS production. J Hepatol 2017;66(4):693–702.PubMedCrossRefGoogle Scholar
  26. 26.
    Xu F, Ji Q, Zhang J, Huang W, Cao Z, Li Y. AlCl3 inhibits LPS-induced NLRP3 inflammasome activation and IL-1beta production through suppressing NF-kappaB signaling pathway in murine peritoneal macrophages. Chemosphere 2018;209:972–980.PubMedCrossRefGoogle Scholar
  27. 27.
    Wree A, Eguchi A, McGeough MD, Pena CA, Johnson CD, Canbay A, et al. NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology 2014;59(3):898–910.PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Weiskirchen R, Weiskirchen S, Tacke F. Organ and tissue fibrosis: molecular signals, cellular mechanisms and translational implications. Mol Aspects Med 2019;65:2–15.PubMedCrossRefGoogle Scholar
  29. 29.
    Li X, Wang Y, Wang H, Huang C, Huang Y, Li J. Endoplasmic reticulum stress is the crossroads of autophagy, inflammation, and apoptosis signaling pathways and participates in liver fibrosis. Inflamm Res 2015;64(1):1–7.PubMedCrossRefGoogle Scholar
  30. 30.
    Li L, Zhao J, Zhou L, Chen J, Ma Y, Yu Y, et al. Tenofovir alafenamide fumarate attenuates bleomycin-induced pulmonary fibrosis by upregulating the NS5ATP9 and TGF-beta1/Smad3 signaling pathway. Respir Res 2019;20(1):163.PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Ma PF, Gao CC, Yi J, Zhao JL, Liang SQ, Zhao Y, et al. Cytotherapy with M1-polarized macrophages ameliorates liver fibrosis by modulating immune microenvironment in mice. J Hepatol 2017;67(4):770–779.PubMedCrossRefGoogle Scholar
  32. 32.
    Chen A, Tang Y, Davis V, Hsu FF, Kennedy SM, Song H, et al. Liver fatty acid binding protein (L-Fabp) modulates murine stellate cell activation and diet-induced nonalcoholic fatty liver disease. Hepatology 2013;57(6):2202–2212.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Asian Pacific Association for the Study of the Liver 2019

Authors and Affiliations

  1. 1.Peking University Ditan Teaching HospitalBeijingChina
  2. 2.Institiute of Infectious Diseases, Beijing Ditan HospitalCapital Medical University, Beijing Key Laboratory of Emerging Infectious DiseasesBeijingChina
  3. 3.Department of Infectious DiseaseChina–Japan Friendship HospitalBeijingChina
  4. 4.Department of Infectious Diseases and Clinical Microbiology, Beijing Chaoyang HospitalCapital Medical UniversityBeijingChina
  5. 5.Department of Infectious Diseases, Center for Liver DiseasesPeking University First HospitalBeijingChina
  6. 6.Beijing Advanced Innovation Center for Big Data-Based Precision MedicineBeihang University & Capital Medical UniversityBeijingChina

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