Chinese medicine in the treatment of autoimmune hepatitis: Progress and future opportunities

Abstract Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease occurring in individuals of all ages with a higher incidence in females and characterized by hypergammaglobulinemia, elevated serum autoantibodies and histological features of interface hepatitis. AIH pathogenesis remains obscure and still needs in‐depth study, which is likely associated with genetic susceptibility and the loss of immune homeostasis. Steroids alone and in combination with other immunosuppressant agents are the primary choices of AIH treatment in the clinic, whereas, in some cases, severe adverse effects and disease relapse may occur. Chinese medicine used for the treatment of AIH has proven its merits over many years and is well tolerated. To better understand the pathogenesis of AIH and to evaluate the efficacy of novel therapies, several animal models have been generated to recapitulate the immune microenvironment of patients with AIH. In the current review, we summarize recent advances in the study of animal models for AIH and their application in pharmacological research of Chinese medicine‐based therapies and also discuss current limitations. This review aims to provide novel insights into the discovery of Chinese medicine‐originated therapies for AIH using cutting‐edge animal models.

At present, AIH is recognized as two types: AIH-1, positive for anti-nuclear (ANA) and/or anti-smooth muscle (SMA) antibodies, and AIH-2, positive for anti-liver kidney microsomal antibody type 1 (anti-LKM1), anti-LKM3 and/or anti-liver cytosol antibody type 1 (anti-LC1). 9,10 Recent studies have revealed that the etiology and pathogenesis of AIH are correlated with genetic susceptibility, the loss of self-tolerance, molecular mimicry or non-specific activation of T lymphocytes. 11 Notably, the interaction between genetic susceptibility and the loss of self-tolerance caused by environmental factors is the core of pathogenesis. 12 In adults, genetic predisposition to AIH is strongly associated with the specific genes located within the humanleukocyte-antigen (HLA) region on the short arm of chromosome 6, especially with genes encoding HLA-DR4 and HLA-DR3 alleles. 13 In patients with HLA-DRB1 cross-reactions with liver autoantigens and rapid disease progression are more likely to occur. 14 In addition, several environmental factors including drugs, xenobiotics and viruses result in the loss of self-tolerance and initiate the immune response, and current studies aim to address this issue by developing animal models that mimic the liver damage caused by the immune system dysfunction in AIH patients. 15 T lymphocytes control the balance of intrinsic and extrinsic peripheral tolerance in healthy individuals, alteration of which may give rise to the dysregulation of immunoregulatory networks. Support for the role of loss of self-tolerance in AIH comes from studies showing that the deletion of medullary thymic epithelial cells that regulate T cell tolerance by eliminating autoreactive T cells and expressing self-antigens caused the enrichment of anti-ANA and initiated AIH in mice. 16,17 Moreover, antigen-presenting cells (APCs) are involved in the process and presentation of self-antigens to T cell receptor (TCR) on naive CD4 + T helper (T H 0) cells and trigger immunoregulation. Exposure to self-mimicking exogenous sequences results in the production of anti-LKM1 antibodies in patients who have α1-antitrypsin deficiency and acquired hepatitis C virus after liver transplantation. 18,19 Cytochrome P4502D6 (CYP2D6) is reported to mainly express in endoplasmic reticulum and hepatocyte membranes and become the target of anti-LKM1 antibodies, thus leading hepatocytes to be directly attacked by humoral immunity. 20 At present, first-line therapeutic drugs targeting AIH and complications are glucocorticoids and azathioprine, which can be used either alone or in combination to achieve biochemical remission, defined as normal serum transaminase and IgG levels. 21,22 However, patients with AIH generally relapse after cessation of these drugs or suffer side effects caused by their long-term use. 23 When AIH progresses into end-stage cirrhosis or fulminant liver failure, patients are no longer sensitive to pharmacotherapy and are forced to choose the final treatment option, liver transplantation. 24 Therefore, exploring novel medications with high efficiency and low toxicity is of both basic science and clinical relevance. Traditional Chinese medicine (TCM) consists of different medicinal formulas and has shown success in regulating immune responses, thus exhibiting therapeutic effects against acute or chronic liver diseases. 25 Considering the multi-component and multi-target characters of TCM, identifying various Chinese herbal prescriptions or natural Chinese medicine extracts is potentially an efficient approach to discovery of novel therapeutics against AIH. Recently, more AIH patients have prefered to take TCM to alleviate their severe symptoms and suppress steroidinduced side effects. 26 Many experimental studies have illustrated the promising therapeutic effects of several natural products from TCM, including phenols, glycosides, flavonoids, polysaccharides and alkaloids, in the treatment of AIH by activating T/B lymphocytes, inducing helper T (Th2) cell development, promoting T cell proliferation and ultimately regulating T cell-mediated immunity. 27,28 In the current review, we mainly focus on the hepatoprotective and immunoregulative effects of TCM and its natural products on several kinds of AIH-related animal models. This review also provides a comprehensive summary of recent studies, highlights findings that have been ignored, and indicates potential prospects of clinical application of TCM for the management of AIH and associated manifestations.

| MURINE INJ URY MODEL S FOR AIH
Given that AIH pathogenesis is not yet been understood, multifarious animal models have been generated to mimic the histopathological features of AIH patients and help to identify mechanisms responsible for immunological intolerance. A variety of drugs has been associated with the induction of AIH, including concanavalin A (Con A), lipopolysaccharide/D-galactosamine (LPS/D-GalN), complete Freund's adjuvant (CFA), α-galactosylceramide (α-GalCer), carbon tetrachloride (CCl 4 ) and alcohol ( Figure 1). However, owing to differences in the major histocompatibility complex (MHC) class I and II proteins involved in the T cell response between mouse and human, mouse models used to mimic human autoimmune diseases often don't share the same target autoantigens and have certain limitations. 29 Recently, Chinese herbal prescriptions, single Chinese herbs and active ingredients isolated from Chinese medicines have been shown to possess protective effects in these animal models of AIH and related diseases (Table 1).

| TCM and Con A-induced liver injury models
Con A, a lectin isolated from jack beans, is a T cell mitogen for inducing antigen non-specific T cell activation and severe acute hepatitis with cytokine storm. 82 Recent studies report that Con A can stimulate CD4 + T cells, natural killer T (NKT) cells, macrophages and APCs, and cause the secretion of proinflammatory cytokines including interleukins (IL-6, IL-12, IL-18), tumor necrosis factorα (TNFα), interferonγ (IFNγ) and several chemokines in mice. 83 Although Con A represents a model of non-specific and T cell-mediated acute liver injury rather than a typical AIH model, Con A-induced hepatitis has been extensively used to investigate the immune system-mediated liver injury and evaluate possible therapies for human AIH.
According to their different biological functions in the immune response, T cells are further divided into three subgroups: cytotoxic T cells (CTLs), namely CD8 + T cells, which release granzyme and perforin to induce the apoptosis of target cells; CD4 + helper T cells (Th cells), which secrete a variety of cytokines such as interleukins, TNFα and IFNγ and activate B cells to differentiate and produce antibodies; regulatory T cells (Tregs), which inhibit the immune response by secreting forkhead box p3 (Foxp3), transforming growth factorβ (TGFβ), IL-4, and IL-10. [84][85][86] It has been well-established that aberrant activation of CTLs, cytokines released by Th1 cells and macrophages, and adhesion of NKT cells to hepatocytes coated with autoantibodies contribute to hepatocyte destruction in Con A-induced animal models. 87 Wang et al. 30 obtained pomegranate peel extract (PoPx) from Punica granatumand and demonstrated that pretreatment in a Con A-induced AIH murine model with PoPx obviously reduced the level of cytokines (TNFα, IFNγ and IL-6), serum transaminases and the murine mortality rate, and played a protective role by decreasing the liver damage caused by ROS. It also remarkably reduced the hepatic infiltration of activated CD4 + /CD8 + T cells. Yunfang Cao isolated abietoquinones A from Prunella vulgarisis and found that it decreased the serum level of aminotransferases, inhibited the production of proinflammatory cytokines and alleviated the proliferation and infiltration of CD3 + T cells in a Con A-induced hepatitis mouse model. 31 NKT cells, a T cell subset expressing surface markers of both NKT cells and T lymphocytes, are crucial first responders against multiple infectious diseases. Once stimulated, NKT cells secrete a large number of cytokines and chemokines, especially IL-4 and IFNγ, and simultaneously inhibit the differentiation of Th1/Th2 cells. 88 It has also been reported that betulin, extracted from Hedyotis hedyotidea, inhibited the activation of NKT and conventional T cells by decreasing the enhanced expression of CD69, prevented the production of pro-inflammatory cytokines, particularly Th1-type cytokines, in these two cell populations, and subsequently ameliorated liver injury. 32 Additionally, betulin abolished NKT cell death in mice, and dose-dependently attenuated splenocyte proliferation stimulated by Con A in vitro. 32 Researchers from the same lab further extracted periplocoside A (PSA) from Periploca sepium Bge and investigated whether this ingredient also alleviated Con A-induced hepatitis in mice. As expected, PSA inhibited the proliferation of primary T cells and the production of NKT-derived inflammatory cytokines (IL-4 and IFNγ), reduced the necrosis and infiltration of leukocytes and protected mice from Con A-induced AIH damage. 33 Apoptosis is programmed cell death including internal and external apoptosis pathways and is of great importance in resisting pathogen invasion and regulating the immune response. In addition to its role in stimulating T cells, Con A can cause AIH injury by directly triggering hepatic parenchymal cell death. The intrinsic pathway includes endoplasmic reticulum (ER) stress or mitochondrial dysfunction.
After the destruction of Ca 2+ balance or the aberrant accumulation of ER protein, endoplasmic reticulum stress (ERS) triggers the apoptosis signals and promotes cell apoptosis by activating caspase 3/7/12. 89 Under the stimulation of various apoptotic signals, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) form a heteromer and migrate to the outer membrane of mitochondria, causing the release of cyto C and a cascade reaction of downstream caspases. 90 Yongdamsagan-tang (YST) extracts markedly inhibited the apoptosis of hepatocytes and prevented the Con A-induced AIH injury in mice via the suppression of caspase-3/8/9 and Bax. 34 In addition, YST also The transcription factor nuclear factor-kappa B (NF-κB) is considered a central mediator of the immune response and exists as an inhibitor of nuclear factor kappa B (IκB) kinase (IKK) complex with its inhibitory protein α (IκBα) in the cytoplasm in a quiescent condition.
Once activated, NF-κB is translocated from the cytoplasm to the nucleus, driving the expression of target genes involved in immune and inflammation responses. 94 It was reported that SIRT1 negatively regulated the transcriptional activity of NF-κB/P65 and thus controlled the immune response of macrophages. 38 Interestingly, the higher level of NF-κB/P65, lower expression of SIRT1, accumulated macrophages and hepatocyte apoptosis that appeared in the livers of aged mice following long-term administration of Con A were all completely reversed by resveratrol pretreatment. 95  by inhibiting NF-κB and activating Nrf2 signaling pathway. [49][50][51][52][53][54]102 Another study further suggested that SIRT1 was associated with the regulation of the inflammatory response and oxidative stress by

| TCM and alcohol-induced liver injury models
Considering its increased clinical incidence, alcohol is regarded as another potential trigger of liver autoimmunity. 109  , oxidative stress, LPO and hepatocyte apoptosis. 111 It has been reported that platycodon grandiflorum inhibited the activity of SOD and CAT, restrained the production of MDA, alleviated a high accumulation of microvesicular-type fat in the hepatocyte cytoplasm and prevented alcohol-induced oxidative stress and acute hepatitis. 73 It was also reported that Cichorium intybus root extract reduced lipid accumulation and alleviated alcohol-induced liver injury by upregulating alternative alcohol metabolism pathways mediated by ADH and ALDH and inhibiting the levels of GOT, GPT, TG and CYP2E1. 74 Under the stimulation of alcohol, excessive accumulation of fatty acids resulted in activation of lymphocytes, infiltration of macrophages and hepatic inflammation. 112 Penthorum chinense Pursh (PCP) reduced oxidative stress, improved inflammation and protected the liver from acute alcohol-induced hepatitis by activating the Nrf2/HO-1 pathway and downregulating the expression of CYP2E1 and hepatic TG levels. 75 A previous study showed that taraxasterol effectively improved hepatocyte steatosis and prevented the development of fatty liver by significantly reducing TG content, and inhibiting the secretion of pro-inflammatory cytokines (TNFα and IL-6) via CYP2E1/Nrf2/ HO-1 and NF-κB signaling pathways in alcohol-induced liver injury in mice. 76 Notably, pretreatment with PCP inhibited the upregulation of fatty acid translocation enzyme (CD36) expression, and ameliorated dysfunctional white adipose tissue derived-fatty acid influx to the liver, thereby protecting the liver from acute alcohol-induced hepatitis. 77 Inflammatory infiltration and hepatocyte apoptosis are widely observed in the alcohol-induced AIH mouse model. It is well established that alcohol and its metabolites promote the release of cytokines and experimental hepatitis, which are closely related to the activation of NF-κB signaling. Gastrodin ameliorated histopathological lesions of hepatocytes, alleviated liver inflammation and reduced the level of cytokines and chemokines, such as TNFα, IFNγ and IL-6, chemokine (C-X-C motif) ligand 1 (CXCL-1), vascular cell adhesion molecule 1 (VCAM-1) in a dose-dependent manner by regulating the NF-κB and AMPK/Nrf2 pathways in alcohol-induced AIH. 78 Akt acts as a critical upstream activator in the inflammatory reaction involving Nrf2 nuclear translocation, NF-κB activation and ROS generation. 113 Triticum aestivum sprout-derived polysaccharide protected against alcohol-induced hepatitis by inhibiting the phosphatidylinositol 3-kinase (PI3K)/phosphor-protein kinase B (Akt) signaling pathway and regulating Nrf2 nuclear translocation. 79 Another study reported that cinnamomea mycelia (AC) strongly reduced lipid droplet formation, hepatocyte apoptosis and inflammatory infiltration in livers by suppressing the increased levels of TNFα, inhibiting the phosphorylation of Akt and NF-κB and suppressing caspase 3/8/9 signaling in an alcohol-exposed rat model. 80 Under physiological conditions, excessive alcohol downregulates the production of retinol-binding protein 4 (RBP4), upregulated the expression of inflammatory factors including chitinase-3-like protein 1 (YKL 40) and plasminogen activator inhibitor type 1 (PAI-1) in the liver. 114 A recent study further reported that Morchella esculenta prevented inflammatory cell infiltration, inhibited the production of YKL-40, IL-7, PAI-1 and RBP4 and improved the alcohol-induced imbalance in prooxidative and anti-oxidative signaling by altering Nrf2 and NF-κB pathways in the liver. 81,115

| CON CLUS I ON AND PROS PEC T
AIH is a chronic inflammatory disorder characterized by interface hepatitis, increased serum autoantibodies and hypergammaglobulinemia, representing a major health burden without any available cure. Many animal models for human AIH have been generated to either provide an appropriate approach to investigate regulatory immune mechanisms or evaluate possible therapeutics. However, based on all the evidence summarized above, almost all these animal models of AIH have obvious limitations that raise the difficulty of exploring AIH pathogenesis.
Although the Con A-related hepatic injury is an extensively used model mimicking the histopathological changes of human AIH, it is more suitable for studying the underlying mechanisms of immune activation and/or regulation. Moreover, while chemicals such as CCl 4 are capable of triggering liver inflammation and disrupting immune tolerance, they are more likely to induce a non-specific immune response. In addition, there is controversy about whether a liver injury model induced by MAA with soluble proteins can be widely applied to reflect the AIH injury in humans, due to the loss of immunogenicity and lack of detailed mechanisms. Furthermore, the feasibility of several established AIH animal models is still questionable. Collectively, several phenotypic characteristics need to be fulfilled in generating any novel AIH models in the future: firstly, inducing the production of autoantibodies against liver antigens; secondly, regulating the serum levels of inflammatory factors such as cytokines and chemokines; finally, altering the levels of T-cell subtypes or triggering the disorder of the immune system.
Emerging evidence shows that Chinese medicine prescriptions and natural products isolated from TCM provide additional benefits in the prevention of chronic liver diseases, including AIH, and represent a critical source of drug screening and development. In the current review, we comprehensively reviewed these researches.
However, most of the above studies only investigated the regulatory effects of TCM on the typical inflammatory signaling pathways (especially those for NF-κB/MAPKs) and inflammatory factors (like IFNγ/TNF-α/IL-6), without detecting the changes of lgG/autoantibodies and T cell subsets in the respective AIH animal models.
Therefore, the precise mechanism of the protective effects of the above-reported TCM components against AIH still requires extensive research. Considering the existing deficiencies in the current AIH animal models, researchers should test combinations of different drugs or construct new animal models. For example, the NTxPD-1 −/− mouse model is an inducible AIH model that lacks the programmed cell death 1 (PD-1) gene and a sufficient number of regulatory T cells to maintain the immune T cell balance. 116 Another novel AIH model is the CYP2D6 mouse model, which is generated by the infection of an adenovirus encoding for human CYP2D6, the best-characterized autoantigen recognized by T cells and antibodies of AIH-2 patients. 117 These novel animal models have not been summarized in this review, because, to date, no research has yet investigated the hepatoprotective effects of TCM on these models.

Further comprehensive evaluations of the effects and mechanisms
of TCM on AIH-related animal models and identification of specific molecular targets are urgently needed.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest.