Abstract—
Nonalcoholic steatohepatitis (NASH) is the inflammatory subtype of nonalcoholic fatty liver disease (NAFLD), which can lead to liver fibrosis and cirrhosis. Bile acid levels are correlated with markers of hepatic injury in NASH, suggesting a possible role for bile acids in the progression of NAFLD. Here, we examined the role of deoxycholic acid (DCA) in driving steatotic hepatocytes to pyroptosis, a pro-inflammatory form of programmed cell death. HepG2 cells were stimulated with odium oleate and sodium palmitate for modeling steatotic hepatocytes and then treated with DCA alone or in combination with a specific mitophagy agonist, carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Our results showed that DCA dose-dependently induced a pro-inflammatory response in steatotic hepatocytes but had no significant effect on lipid accumulation. Moreover, activation of the NLRP3 inflammasome and pyroptosis were triggered by DCA. Expression levels of the mitophagy markers PTEN-induced kinase 1 (PINK1) and E3 ubiquitin ligase Parkin were significantly diminished by DCA, whereas induction of mitophagy by CCCP prevented DCA-induced inflammatory response and restored the pyroptosis. Collectively, our data showed that DCA-induced pyroptosis involves the inhibition of PINK1-mediated mitophagy and the activation of the NLRP3 inflammasome. These findings provide insight into the association of DCA with mitophagy, pyroptosis, and inflammation in NASH.
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References
Younossi, Z., F. Tacke, M. Arrese, B. Chander Sharma, I. Mostafa, E. Bugianesi, V. Wai-Sun Wong, Y. Yilmaz, J. George, J. Fan, and M.B. Vos. 2019. Global perspectives on nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology 69: 2672–2682.
Jia, W., M. Wei, C. Rajani, and X. Zheng. 2020. Targeting the alternative bile acid synthetic pathway for metabolic diseases. Protein & Cell 12: 411–425.
Belgaumkar, A.P., R.P. Vincent, K.A. Carswell, R.D. Hughes, J. Alaghband-Zadeh, R.R. Mitry, C.W. Le Roux, and A.G. Patel. 2015. Changes in bile acid profile after laparoscopic sleeve gastrectomy are associated with improvements in metabolic profile and fatty liver disease. Obesity Surgery 26: 1195–1202.
Yara, S.I., T. Ikegami, T. Miyazaki, M. Murakami, J. Iwamoto, T. Hirayama, M. Kohjima, M. Nakamuta, and A. Honda. 2019. Circulating bile acid profiles in Japanese patients with NASH. Gastroenterology and Hepatology 1: 302–310.
Thomas, C., R. Pellicciari, M. Pruzanski, J. Auwerx, and K. Schoonjans. 2008. Targeting bile-acid signalling for metabolic diseases. Nature Reviews Drug Discovery 7: 678–693.
Aranha, M.M., H. Cortez-Pinto, A. Costa, I.B.M. Da Silva, M.E. Camilo, M.C. De Moura, and C.M.P. Rodrigues. 2008. Bile acid levels are increased in the liver of patients with steatohepatitis. European Journal of Gastroenterology and Hepatology 20: 519–525.
Kwan, S.Y., J. Jiao, J. Qi, Y. Wang, P. Wei, J.B. Mccormick, S.P. Fisher-Hoch, and L. Beretta. 2020. Bile acid changes associated with liver fibrosis and steatosis in the Mexican-American population of South Texas. Hepatology Communications 4: 555–568.
Puri, P., K. Daita, A. Joyce, F. Mirshahi, P.K. Santhekadur, S. Cazanave, V.A. Luketic, M.S. Siddiqui, S. Boyett, H.K. Min, D.P. Kumar, R. Kohli, H. Zhou, P.B. Hylemon, M.J. Contos, M. Idowu, and A.J. Sanyal. 2018. The presence and severity of nonalcoholic steatohepatitis is associated with specific changes in circulating bile acids. Hepatology 67: 534–548.
Nimer, N., I. Choucair, Z. Wang, I. Nemet, L. Li, J. Gukasyan, T. L. Weeks, N. Alkhouri, N. Zein, W. H. W. Tang, M. A. Fischbach, J. M. Brown, H. Allayee, S. Dasarathy, V. Gogonea and S. L. Hazen. 2021. Bile acids profile, histopathological indices and genetic variants for non-alcoholic fatty liver disease progression. Metabolism 116: 154457.
Bader, M., Y. Nakade, R. Kitano, K. Sakamoto, S. Kimoto, T. Yamauchi, T. Inoue, Y. Kobayashi, T. Ohashi, Y. Sumida, K. Ito and M. Yoneda. 2021. Characteristics of bile acid composition in high fat diet-induced nonalcoholic fatty liver disease in obese diabetic rats. Plos One 16: e0247303.
Suga, T., H. Yamaguchi, J. Ogura, S. Shoji, M. Maekawa and N. Mano. 2019. Altered bile acid composition and disposition in a mouse model of non-alcoholic steatohepatitis. Toxicology and Applied Pharmacology 379: 114664.
Janssen, A.W.F., T. Houben, S. Katiraei, W. Dijk, L. Boutens, N. Van Der Bolt, Z. Wang, J.M. Brown, S.L. Hazen, S. Mandard, R. Shiri-Sverdlov, F. Kuipers, K. Willems Van Dijk, J. Vervoort, R. Stienstra, G.J.E.J. Hooiveld, and S. Kersten. 2017. Modulation of the gut microbiota impacts nonalcoholic fatty liver disease: A potential role for bile acids. Journal of Lipid Research 58: 1399–1416.
Arrese, M., D. Cabrera, A.M. Kalergis, and A.E. Feldstein. 2016. Innate immunity and inflammation in NAFLD/NASH. Digestive Diseases and Sciences 61: 1294–1303.
Abderrazak, A., T. Syrovets, D. Couchie, K. El Hadri, B. Friguet, T. Simmet, and M. Rouis. 2015. NLRP3 inflammasome: From a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. Redox Biology 4: 296–307.
Csak, T., M. Ganz, J. Pespisa, K. Kodys, A. Dolganiuc, and G. Szabo. 2011. Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells. Hepatology 54: 133–144.
Wen, H., D. Gris, Y. Lei, S. Jha, L. Zhang, M.T.-H. Huang, W.J. Brickey, and J.P.Y. Ting. 2011. Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nature Immunology 12: 408–415.
Lamkanfi, M., and V.M. Dixit. 2010. Manipulation of host cell death pathways during microbial infections. Cell Host & Microbe 8: 44–54.
Wree, A., M.D. Mcgeough, C.A. Pena, M. Schlattjan, H. Li, M.E. Inzaugarat, K. Messer, A. Canbay, H.M. Hoffman, and A.E. Feldstein. 2014. NLRP3 inflammasome activation is required for fibrosis development in NAFLD. Journal of Molecular Medicine 92: 1069–1082.
Xu, B., M. Jiang, Y. Chu, W. Wang, D. Chen, X. Li, Z. Zhang, D. Zhang, D. Fan, Y. Nie, F. Shao, K. Wu, and J. Liang. 2018. Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice. Journal of Hepatology 68: 773–782.
Mridha, A.R., A. Wree, A.A.B. Robertson, M.M. Yeh, C.D. Johnson, D.M. Van Rooyen, F. Haczeyni, N.C. Teoh, C. Savard, G.N. Ioannou, S.L. Masters, K. Schroder, M.A. Cooper, A.E. Feldstein, and G.C. Farrell. 2017. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. Journal of Hepatology 66: 1037–1046.
Sheng, L., P.K. Jena, Y. Hu, H.-X. Liu, N. Nagar, K.M. Kalanetra, S.W. French, S.W. French, D.A. Mills, and Y.-J.Y. Wan. 2017. Hepatic inflammation caused by dysregulated bile acid synthesis is reversible by butyrate supplementation. The Journal of Pathology 243: 431–441.
Gaul, S., A. Leszczynska, F. Alegre, B. Kaufmann, C.D. Johnson, L.A. Adams, A. Wree, G. Damm, D. Seehofer, C.J. Calvente, D. Povero, T. Kisseleva, A. Eguchi, M.D. Mcgeough, H.M. Hoffman, P. Pelegrin, U. Laufs, and A.E. Feldstein. 2021. Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis. Journal of Hepatology 74: 156–167.
Zhou, R., A.S. Yazdi, P. Menu, and J. Tschopp. 2011. A role for mitochondria in NLRP3 inflammasome activation. Nature 469: 221–225.
Wu, J., X. Li, G. Zhu, Y. Zhang, M. He, and J. Zhang. 2016. The role of resveratrol-induced mitophagy/autophagy in peritoneal mesothelial cells inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS. Experimental Cell Research 341: 42–53.
Roberts, R.F., M.Y. Tang, E.A. Fon, and T.M. Durcan. 2016. Defending the mitochondria: The pathways of mitophagy and mitochondrial-derived vesicles. The International Journal of Biochemistry & Cell Biology 79: 427–436.
Ashrafi, G., and T.L. Schwarz. 2013. The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death and Differentiation 20: 31–42.
Zhang, N.-P., X.-J. Liu, L. Xie, X.-Z. Shen, and J. Wu. 2019. Impaired mitophagy triggers NLRP3 inflammasome activation during the progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis. Laboratory Investigation 99: 749–763.
Shi, D., X. Zhan, X. Yu, M. Jia, Y. Zhang, J. Yao, X. Hu, and Z. Bao. 2014. Inhibiting CB1 receptors improves lipogenesis in an in vitro non-alcoholic fatty liver disease model. Lipids in Health and Disease 13: 173.
Qiu, T., P. Pei, X. Yao, L. Jiang, S. Wei, Z. Wang, J. Bai, G. Yang, N. Gao, L. Yang, S. Qi, R. Yan, X. Liu, and X. Sun. 2018. Taurine attenuates arsenic-induced pyroptosis and nonalcoholic steatohepatitis by inhibiting the autophagic-inflammasomal pathway. Cell Death & Disease 9: 946.
Sheka, A.C., O. Adeyi, J. Thompson, B. Hameed, P.A. Crawford, and S. Ikramuddin. 2020. Nonalcoholic steatohepatitis: A review. The Journal of the American Medical Association 323: 1175–1183.
Manne, V., P. Handa, and K.V. Kowdley. 2018. Pathophysiology of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Clinics in Liver Disease 22: 23–37.
Jiao, N., S.S. Baker, A. Chapa-Rodriguez, W. Liu, C.A. Nugent, M. Tsompana, L. Mastrandrea, M.J. Buck, R.D. Baker, R.J. Genco, R. Zhu, and L. Zhu. 2018. Suppressed hepatic bile acid signalling despite elevated production of primary and secondary bile acids in NAFLD. Gut 67: 1881–1891.
Amaral, J.D., R.J.S. Viana, R.M. Ramalho, C.J. Steer, and C.M.P. Rodrigues. 2009. Bile acids: Regulation of apoptosis by ursodeoxycholic acid. Journal of Lipid Research 50: 1721–1734.
Péan, N., I. Doignon, and T. Tordjmann. 2013. Bile acids and liver carcinogenesis: TGR5 as a novel piece in the puzzle? Clinics and Research in Hepatology and Gastroenterology 37: 226–229.
Zhang, N.-P., X.-J. Liu, L. Xie, X.-Z. Shen and J. Wu. 2019. Impaired mitophagy triggers NLRP3 inflammasome activation during the progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis. Laboratory Investigation; a Journal of Technical Methods and Pathology 99: 749–763.
Kang, X., H. Wang, Y. Li, Y. Xiao, L. Zhao, T. Zhang, S. Zhou, X. Zhou, Y. Li, Z. Shou, C. Chen, and B. Li. 2019. Alantolactone induces apoptosis through ROS-mediated AKT pathway and inhibition of PINK1-mediated mitophagy in human HepG2 cells. Artificial Cells Nanomedicine and Biotechnology 47: 1961–1970.
Kubli, D.A., and Å.B. Gustafsson. 2012. Mitochondria and mitophagy: The yin and yang of cell death control. Circulation Research 111: 1208–1221.
Liu, P., H. Lin, Y. Xu, F. Zhou, J. Wang, J. Liu, X. Zhu, X. Guo, Y. Tang and P. Yao. 2018. Frataxin-mediated PINK1-Parkin-dependent mitophagy in hepatic steatosis: the protective effects of quercetin. Molecular Nutrition & Food Research 62: e1800164.
Chen, M.-Y., X.-J. Ye, X.-H. He, and D.-Y. Ouyang. 2021. The signaling pathways regulating NLRP3 inflammasome activation. Inflammation 44: 1229–1245.
Cassel, S.L., S. Joly, and F.S. Sutterwala. 2009. The NLRP3 inflammasome: A sensor of immune danger signals. Seminars in Immunology 21: 194–198.
Wree, A., A. Eguchi, M.D. Mcgeough, C.A. Pena, C.D. Johnson, A. Canbay, H.M. Hoffman, and A.E. Feldstein. 2014. NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology 59: 898–910.
Franchi, L., R. Muñoz-Planillo, and G. Núñez. 2012. Sensing and reacting to microbes through the inflammasomes. Nature Immunology 13: 325–332.
González-Rodríguez, A., R. Mayoral, N. Agra, M. P. Valdecantos, V. Pardo, M. E. Miquilena-Colina, J. Vargas-Castrillón, O. Lo Iacono, M. Corazzari, G. M. Fimia, M. Piacentini, J. Muntané, L. Boscá, C. García-Monzón, P. Martín-Sanz and Á. M. Valverde. 2014. Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD. Cell Death & Disease 5: e1179.
Yang, L., P. Li, S. Fu, E.S. Calay, and G.S. Hotamisligil. 2010. Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance. Cell Metabolism 11: 467–478.
Madrigal-Matute, J., and A.M. Cuervo. 2016. Regulation of liver metabolism by autophagy. Gastroenterology 150: 328–339.
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Discipline construction project of Guangdong Medical University (4SG21016G); Funds for PHD researchers of Guangdong Medical University in 2019 (B2019001).
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Xuebin Gao and Honghui Guo designed the study. Xuebin Gao, Xuan Zhu, Yan Xin, Xiaozhuan Lin, Xiang Li, and Meiqing Mai performed the experiments and analyzed the data. Xuebin Gao and Yongdui Ruan wrote the manuscript. Honghui Guo revised the manuscript.
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Gao, X., Ruan, Y., Zhu, X. et al. Deoxycholic Acid Promotes Pyroptosis in Free Fatty Acid-Induced Steatotic Hepatocytes by Inhibiting PINK1-Mediated Mitophagy. Inflammation 45, 639–650 (2022). https://doi.org/10.1007/s10753-021-01573-1
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DOI: https://doi.org/10.1007/s10753-021-01573-1