Progressive hepatic fibrosis is one of the important public health problems worldwide, causing serious clinical consequences such as portal hypertension, hepatic dysfunction, and hepatocellular carcinoma.1,2 Liver fibrosis is a type of irregular tissue-repairing response to chronic hepatic injuries induced mainly by a virus infection, autoimmune disorders, and exposure to drugs or toxic compounds.3,4 Liver fibrosis is characterized by excessive accumulation of extracellular matrix (ECM) components primarily consisting of fibrillar collagens, resulting in the subsequent destruction of the normal hepatic architecture.5 Activated hepatic stellate cells (HSCs) are postulated as the main source of ECM deposition in the fibrotic liver and play a crucial role in the progression of fibrosis.6 During the fibrogenesis, enhanced pro-fibrotic cytokine responses occur upon HSC activation, and transforming growth factor β1 (TGFβ1) has been recognized as one of the key profibrogenic cytokines, which increases major ECM synthesis and changes the balance between tissue inhibitors of metalloproteinases and matrix metalloproteinases.7,8 Although the knowledge of liver fibrogenesis has been increasingly elucidated recently, the development of practical anti-fibrotic therapies is still challenging.9 Therefore, better identification of the mechanisms that regress fibrosis is required to develop safe and effective treatments for patients with hepatic fibrosis.

Calcitriol (1,25(OH)2D3), the primary biologically active metabolite of vitamin D, has been shown to exert marked effects on calcium homeostasis, cellular proliferation and differentiation, and immune regulation.10,11 In recent studies, calcitriol and vitamin D analogs have been demonstrated to play an important role in suppressing hepatic fibrogenic signals in vitro and in vivo.12, 13, 14, 15 The biological effects of calcitriol are mediated by the vitamin D receptor (VDR), an evolutionarily ancient member of the nuclear hormone receptor superfamily.16,17 The binding of calcitriol to VDR and subsequent interaction of the ligand-receptor complex with the vitamin D response elements (VDREs) in the promoter region of target genes lead to the suppression or activation of gene transcription.18,19 However, the mechanism of calcitriol in inhibiting liver fibrosis remains unclear.

Autophagy is a catabolic process in which damaged organelles, aggregate-prone proteins, and invasive pathogens are encapsulated in double-membrane compartments known as autophagosomes and fused with lysosomes for degradation and recycling. The above degradation sequence is called autophagic flux.20, 21, 22 Autophagy is known to impact multiple physiological and pathological processes and has emerged as a proposed therapeutic target in several human disorders including neurodegeneration, myopathy, diabetes, inflammatory bowel disease (IBD), and liver disease.23 A recent study showed that autophagy dysfunction could result in inflammation in the liver, causing activation of hepatic stellate cells (HSCs), which accelerate liver fibrosis.24 Furthermore, accumulating evidence has described that calcitriol and vitamin D analogs induce autophagy in a variety of disease models, such as spinal cord injury, preeclampsia, and human immunodeficiency virus infection.25, 26, 27 Autophagy-related gene 16-like 1 (ATG16L1), an important component of the autophagy machinery, directs the site of autophagosome formation and promotes the conjugation of phosphatidylethanolamine to LC3, subsequently producing a membrane-bound activated form of LC3, referred to as LC3-Ⅱ.28,29 Several studies pointed out that a close interaction between ATG16L1 and vitamin D existed in inflammatory bowel disease.30, 31, 32, 33 Therefore, we assumed that calcitriol may involve in the inhibition of liver fibrosis via ATG16L1-mediated autophagy.

In the present study, we aimed to explore the role of autophagy in anti-hepatic fibrosis afforded by calcitriol, as well as its potential molecular mechanism, using a carbon tetrachloride (Ccl4)-induced liver fibrosis mouse and TGFβ1-stimulated LX-2 and HSC cell models.