Molecular mechanism and research progress on pharmacology of traditional Chinese medicine in liver injury

Abstract Context: Liver disease is a common threat to human health, caused by a variety of factors that damage the liver. Recent studies have shown that active ingredients (for example: flavonoids, saponins, acids, phenols, and alkaloids) from Traditional Chinese Medicine (TCM) can have hepatoprotective benefits, which represents an attractive source of drug discovery for treating liver injury. Objective: We reviewed recent contributions on the chemically induced liver injury, immunological liver damage, alcoholic liver injury, and drug-induced liver injury, in order to summarize the research progress in molecular mechanism and pharmacology of TCM, and provides a comprehensive overview of new TCM treatment strategies for liver disease. Materials and methods: Relevant literature was obtained from scientific databases such as Pubmed, Web of Science. and CNKI databases on ethnobotany and ethnomedicines (from January 1980 to the end of May 2018). The experimental studies involving the antihepatic injury role of the active agents from TCM and the underlying mechanisms were identified. The search terms included ‘liver injury’ or ‘hepatic injury’, and ‘traditional Chinese medicine’, or ‘herb’. Results: A number of studies revealed that the active ingredients of TCM exhibit potential therapeutic benefits against liver injury, while the underlying mechanisms appear to contribute to the regulation of inflammation, oxidant stress, and pro-apoptosis signaling pathways. Discussion and conclusions: The insights provided in this review will help further exploration of botanical drugs in the development of liver injury therapy via study on the effective components of TCM.


Introduction
The liver is the largest parenchymal organ in the abdominal cavity, it plays an important role in regulating physiological functions, such as digestive and excretory functions, storage of nutrients, metabolic homeostasis, synthesis of new substances, and detoxification of harmful chemicals. Therefore, liver injuries and diseases are serious health problems that threaten human health. Liver diseases have a wide range of liver pathologies, including hepatic steatosis, fatty liver, hepatitis, fibrosis, cirrhosis, and hepatocarcinoma. However, only a limited number of studies have been performed up to now on the treatment options for liver injuries and diseases. Therefore, discovering a new treatment that could safely and effectively block or reverse liver injuries and diseases remains a top priority. Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk. Immunological liver injury is caused by immune response, which is characterized by infiltrate of inflammatory cell, formate of inflammatory granuloma, and damage of hepatic cell cable structure. Chronic or heavy drinking can lead to alcoholic liver injury, resulting in the damage of liver function. Idiosyncratic drug reactions can be extremely severe and are not accounted for by the regular pharmacology of a drug (Tailor et al. 2015). This review briefly discusses the molecular mechanism of liver damage, in the aspects of chemical liver injury, immunological liver injury, alcoholic liver injury, and drug-induced liver injury . The roles of Traditional Chinese Medicine (TCM) as the agents for antihepatic injury, the pharmacological effects of all the monomers separated from Chinese medicine, and Chinese herbs on liver injury from January 1980 to May 2018 are summarized.

Chemical liver injury
Carbon tetrachloride (CCl 4 ) CCl 4 , a hepatotoxin, is the oldest and most frequently used toxin for inducing hepatic injury in experimental animal studies. The underlying mechanism is that various cellular enzymes are massively released into the bloodstream during liver injury (Balogun and Ashafa 2016). Cyclooxygenase-2 (COX-2) catalyzes the synthesis of prostaglandins (PGs) to promote inflammatory response as well as increase the activation and proliferation of hepatic stellate cells (HSC), which then results in hepatic fibrosis. Cytochrome P450 (CYP450) transforms CCl 4 into toxic metabolite, affect DNA and cell membrane, which result in the oxidative degradation of lipids and change the gene expression of liver cells. In addition, hepatic cells secrete large amount of inflammatory factors such as tumor necrosis factor a (TNF-a) to stimulate immunity-associated cells and generate large amount of cytokines. The resulting mediators then induce local inflammatory response, activate cysteine aspirate protease and lead to cellular apoptosis.
The interleukin (IL) is an important inflammatory mediator and immunomodulatory factor that can up-regulate the expression of nuclear factor jB (NF-jB), heterodimer compound activated protein transcription factor 1 (AP-1) and COX-2, stimulate the induction of phosphatase A2 (PLA2) and promote the activation of B and T lymphocyte that infiltrate endothelial cells. The transforming growth factor b (TGF-b) can activate HSC, generate large amount of extracellular matrix and result in hepatic fibrosis. NF-jB can regulate the expression of leukocyte adhesion molecules and soluble inflammatory factors. In addition, CCl 4 generates Cl and CCl 3 ions through phase I CYP450 metabolism, which leads to the loss of membrane integrity oxidative stress, cell membrane lipid peroxidation and ultimately degeneration and necrosis of liver cells. CCl 4 -induced liver injury may also be related to Bcl-2 family and mitochondrial pathway, which can integrate with the lipid bilayer to destroy the bilayer structure of mitochondria, activate caspase-8 and modulate mitochondrial membrane permeability. Cytochrome c (Cyt-C) is often released from mitochondria to the cytoplasm during mitochondrial injury, leading to apoptotic death. Nonetheless, the autophagy level is up-regulated in CCl 4 -induced liver injury model, which can activate the stellate cells and result in hepatic fibrosis (Wunjuntuk et al. 2016).

D-Galactosamine (D-GalN)
D-GalN is a hepatotoxic and liver-damaging drug that has been extensively used as an animal model for the induction of experimental liver failure. The induced mechanism is similar to that of CCl 4 -induced liver injury. D-GalN causes glucosamine accumulation, combines with the uridine diphosphate (UDP), induces deficiency of uridine triphosphate (UTP), and suppress the activation and amount of UDP glucose pyrophosphorylase (UDPase), which result in the metabolic inhibition of carbohydrate and phospholipids. D-GalN also suppresses the synthesis of RNA and proteins in liver cells, while increasing the loss of membrane and further lead to necrocytosis. Meanwhile, Kupffer cells and liver sinusoidal endothelial cells release cytokines that enhance the expression of various enzymes and inflammatory factors. D-GalN can also combine with specific parenchymal hepatic cells and affect its integrity. A large amount of calcium ions can damage calcium homeostasis, which subsequently leads to metabolic disorders. Apart from that, it also causes the depletion of glutathione (GSH) that prohibits carbohydrate and lipid metabolisms, induces free radicals and disturbs redox balance, releases TNF-a and induces apoptosis. Therefore, D-GalN is a Figure 1. CCl 4 -induced liver injury. CCl 4 trigger the continuous development of hepatocyte injury by inducing cell stress via inflammation, macrophages activation, mitochondrion path, and oxidative stress, resulting in apoptosis, hepatic fibrosis, and liver injury. COX-2: epoxide hydrolase; PGs: prostaglandins; HSC: hepatic stellate cells; CYP450: cytochrome P450; TNF-a: tumor necrosis factor a; IL: interleukin; NF-jB: nuclear factor jB; AP-1: activated protein transcription factor 1; PLA 2 : phosphatase A2; TGF-b: transforming growth factor b; Cyt-C: cytochrome C.
hepatotoxic agent that causes panlobular focal hepatocyte necrosis, polymorphonuclear cell infiltration and macrophages enlargement, which closely resembles human viral hepatitis infection. Due to its unique propensity, D-GalN has been widely used in inflammatory liver injury models to screen for potential hepatoprotective agents .
Chinese herb medicine Panax ginseng C. A. Mey (Araliaceae) (Almajwal and Elsadek 2014), Abrus mollis Hance (Leguminosae) , wild ginseng , and Nymphaea candida C. Presl (Nymphaeaceae)  can attenuate D-GalN-induced liver injury, and was shown to possess hepatoprotective activity against D-GalN-induced liver injury, whereas its bioactive constituent nicotiflorin can decrease the high levels of serum enzymatic and cytokines. In addition, compounds such as limonin (Mahmoud et al. 2014), genistein (Ganai and Husain 2016), and schisandrin A (Lu et al. 2014) can increase the activation of autophagy flux, inhibit apoptosis and enhance immunity towards liver injury.

a-Naphthyl isothiocyanate(ANIT)
ANIT is a hepatotoxicant that been used in rodents as a model for human intrahepatic cholestasis. ANIT-induced liver damage is mediated by an ANIT-GSH conjugate, which can dissociate upon crossing the canalicular membrane, yielding free GSH and ANIT in the bile. The accumulated ANIT and GSH damage the biliary epithelial cells, and thus result in intrahepatic bile duct proliferation and inflammation of interlobular bile duct. Bile regurgitation or expansion to form surrounding inflammation and liver cells injury can decrease the secretion of cholestatic jaundice, hyperbilirubinemia, and bilifaction, while increasing the levels of chemotactic factors and adhesion molecules, mediating inflammatory cells and neutrophil granulocytes as well as aggravating the inflammatory reaction. ANIT induces hepatocellular apoptosis, upregulates caspase-9 and cytochrome c expression levels, and inhibits the proliferating cell nuclear antigen (PCNA) mRNA and protein expression. In the mice model, the expression levels of endoplasmic reticulum stress-related gene markers, including glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2 (eIF2), inositol-requiring enzyme-1 (IRE-1) and activating transcription factor 6 (ATF6) are increased by ANIT . ANIT upregulates GRP78nprotein expression and activates the phosphorylation of IRE1. Additionally, ANIT increases NF-jB/IL-6/ STAT signaling, triggers NF-jB activation in a concentrationdependent manner, activates PPARa, and induces liver injury by increasing ALT and AST levels. ANIT also enhances the elevation of macrophage inflammatory protein-2 (MIP-2) through the binding to the IL8Rb receptor on neutrophils (Fang et al. 2017). Furthermore, ANIT generates a large amount of the reactive oxygen through nicotinamide adenine dinucleotide phosphate (NADPH), oxidizes the superoxide dismutase (SOD) and subsequently blocks their activation, which triggers the inflammatory response and aggravates the cholestasis and liver necrosis (Fang et al. 2017).
Chinese herb medicine Artemisia capillaries Thunb. (Composite) , Calculus Bovis Bezoar. (Wu et al. 2013), and rhubarb (Zhao et al. 2009) can significantly improve the liver injury caused by ANIT. For example, both emodin and rhein compounds containing in rhubarb can markedly increase neutrophil infiltration and sinus congestion in the liver cells, increase bile flow, remove jaundice and gallbladder, as well as alleviate liver damage and necrosis. The inhibitory effect of chlorogenic acid , sweroside , dioscin , gentiopicroside , and geniposide  can counteract the liver injury caused by ANIT. Geniposide prevents ANIT-induced liver injury in a dose-dependent manner and reduces basolateral bile acids uptake via repression of organic anion transporting polypeptide 2 (OATP2).

Dimethyl nitrosamine(DMN)
Dimethyl nitrosamine is a potent hepatotoxic, carcinogenic and mutagenic agent, which has been used to induce hepatic fibrosis model. DMN can generate formaldehyde and methyl alcohol after combined with CYP2EI, which results in macromolecule damages, promotes Sinusoid capillarization, and upregulates the expression of TNF-a and IL-lb. The combined substance also stimulates inflammatory response, increases the serum procollagen type III, collagen type IV and relative antigen and induces the expressions of a smooth muscle actin (a-SMA), TGF-b1, connective tissue growth factor (CTGF), TIMP-1 and its mRNA (Liang and Tan 2013). The TLR4/MyD88 signaling pathway that suppresses TLR4 and myeloid differentiation factor 88 (MyD88) can inhibit the translocation of NF-jB and affect the levels of iNOS, NO, IL-1, IL-6, and TNF-a. Sirt1/Nrf2 signaling pathway is critical for regulating oxidative stress. Sirtuin 1 (Sirt1) adjusts oxidative stress and regulates Nrf2. Additionally, some biological molecules including HO-1, glutathione cysteine ligase catalytic subunit (GCLC), glutathione cysteine ligase modifier subunit (GCLM), and glutathione-S-transferase (GST) are representing Nrf2 target genes. HO-1 catalyzes heme metabolism to scavenge free radicals, while GCLC and GCLM regulate the cellular redox state to eliminate ROS. Among the interferon regulatory factors, IRF-9 induces hepatocyte apoptosis after suffering from liver injury, by decreasing Sirt 1 level and increasing p53 level. p53 plays a key role in promoting apoptosis through the upregulation of some apoptotic proteins such as caspase-9 and caspase-3 ). In addition, p53 significantly increases the protein levels of tropomyosin 1, TGF-b1, drosophila mothers against decapentaplegic protein (Smad) 3, a-SMA, and their  mRNA expression. p53 also suppresses the mRNA expression of Smad7, in order to increase the signal transmission of TGF-b1/ Smad3, enhance the activation of HSC, increase the sedimentation of extracellular matrix (ECM), accelerate the course of hepatic fibrosis and ultimately lead to the cellular apoptosis of liver cells. Following the damage to hepatic sinusoidal endothelial cells (SECs), the apoptosis of HSCs and denaturation of SECs may trigger liver cell death .

Thioacetamide (TAA)
Thioacetamide is commonly used to induce hepatotoxicity in hepatic model. The toxic effect of TAA is attributed to its biological activity exerted through oxidase systems, particularly CYP450 2E1 and FAD monooxygenase. The bioactivation of TAA further lead to the formation of reactive metabolites (thioacetamide-S-dioxide), followed by thioacetamide sulphene and sulfone that covalently bind to cellular macromolecules or cellular membrane. Upon binding, TAA enhances oxidative stresses, and subsequently destroys the cellular integrity and initiates necrosis. TAA can be transformed into sulfone, and then to sulfoxide through the conversion of TAA-oxysulfide by CYP450 metabolizing enzymes. These metabolites suppress the phagocytosis of the monocyte to red blood cells, eliminate the capability of the endotoxin of Kupffer cells, promote endotoxemia formation, and lead to enterogenous toxicemia. Depletion of macrophages extends coagulation necrosis with elevated levels of hepatic enzymes at the early stage, which results in unsuccessful reparative fibrosis and dystrophic calcification at the end stage.
After the formation of intestinal endotoxemia (IETM), the endotoxin can inhibit hepatic microcirculation, down-regulate the expression of endothelial nitric oxide synthase (eNOS) and up-regulate the expression of iNOS, in order to increase the NO content of the liver homogenate, cause large-scale induction of hepatocyte necrosis and promote thrombosis formation. On the other hand, the endotoxin can affect the hepatic microcirculation via Kupffer-induced thromboxane A2 (TXA2) and leukotriene formation. The generated free radicals and reactive oxygen can irreversibly bind with liver macromolecules, and thereby contribute to oxidative stress. TAA also enhances the production of ROS and leads to lipid peroxidation, depletion of glutathione, and SH-Thiol group reduction. TAA induces the mobilization of calcium from intracellular stores. Both calcium and ROS can promote cell damage or proliferation by activating multiple mechanisms. In addition, TAA can interfere with the transfer of RNA from nucleus to cytoplasm, which linked to the membrane injury. Consequently, the elevated liver enzymes (ALT, AST) are the indicators for cellular liver necrosis (Khattab et al. 2017). The oxidative stress generated by TAA may also decrease the level of endogenous antioxidant such as a-tocopherol. Besides, TAA can affect the synthesis of proteins and enzyme activity, induce DNA damage and cause liver apoptosis (Zhou and Zhao 2013).

Concanavalin A (ConA)
Concanavalin A is a plant lectin, mainly mediated by CD4 þ T lymphocyte and natural killer T cells. ConA-induced liver injury model demonstrated better simulation on the pathogenesis of human immune disease and immunological liver injury mechanism. Autoimmune liver injury induced by ConA displays a timebased feature, in which the most severe liver lesions occurred at 8 h after ConA treatment . ConA is absorbed into the splenic organ in order to activate the proliferation of T lymphocytes (e.g Th1 and Th2) and secrete Thl and Th2 cytokines. Large amount of cytokines including TNF-a, IFN-c, IL-2, IL-1b, etc. enter the liver through portal vein and combine with the macrophage to activate CD4 þ T, mitogen of T lymphocytes, and Kupffer cells. Subsequently, NF-jB and IRF-3 are activated to induce the expression of proinflammatory cytokine and adhesion molecules, generate various inflammatory cytokines, injure liver cells, and cause damage to vascular endothelial cells, lymphocytes, monocytes, and Kupffer cells . Upon the activation and upregulation of NKT cells, a variety of cytokines such as IFN-c and IL-4 are rapidly secreted. NKT cells can directly cause liver injury through Fas/Fas ligand mechanism that secretes various cytokines to recruit and activate other innate immune cells for exacerbating inflammatory responses in the liver (Wei et al. 2016). ConA expresses major histocompatibility complex (MHC) class II structures and stimulates Kupffer cells to secrete cytokines, TNF-a and IL-1b, which subsequently activating CD4 þ T cells that initiate an inflammatory reaction. After ConA administration, autophagy is initiated to decompose damages on SECs. Autophagy contributes to the transport of proteins from the cytoplasm into the lumen of antigen-processing compartments in Kupffer cells, which enhances MHC class II-mediated antigen presentation and prolongs CD4 þ T cell activation. The MHCII-dependent activation of CD4þ helper T cells secrete IFN-g and further activate Kupffer cells to form a positive feedback loop. The IFN-c can enhance ConA-induced endothelial cell autophagy and accelerate cell death .
Chinese herb medicine such as Hypericum perforatum L. (Oleaceae) , Angelica sinensis (Oliv.) Diels (Umbelliferae) , and Dracocephalum heterophyllum Benth. (Labiatae) ) may attenuate the liver injury caused by ConA. Pretreatment with Dracocephalum heterophyllum extract ameliorates liver injury and suppresses the production of inflammatory cytokines (e.g TNF-a and IFN-c) in ConA-induced hepatitis model. This herbal extract recruites CD11b þ Gr1þ myeloid-derived suppressor cells to the liver and causes suppression of macrophage infiltration. Other TCM compounds such as quercetin , shikonin , fucoidan , and astaxanthin  can be used to treat liver injury induced by ConA. Astaxanthin can attenuate serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It also exhibited anti-apoptotic effects via decreasing the phosphorylation of b-cell lymphoma-2 (Bcl-2) downregulated by JNK/p-JNK pathway.

Bacillus calmette guerin (BCG) combined with lipopolysaccharide (LPS)
The combination of Bacillus calmette guerin and lipopolysaccharide may result in acute liver injury, which is widely used as a model to study the pathogenesis of viral hepatitis and select liver-protective drugs through immunization route. The pathological changes and liver injury mechanisms are similar to that of viral hepatitis type B. Tissue damage is caused by the liver necrosis and large amount of monocyte infiltration, which is characterized by the presence of granuloma in the liver cells. This is an allergic reaction caused by cell-mediated immunity. BCG attenuates the activated TB-PCR vaccine, by triggering and sensitizing T lymphocytes and increasing the number of Kupffer cells, macrophages, and neutrophil granulocyte in the liver, in order to remain the allergic state (Liu 1991). After treatment with LPS, the allergic Kupffer cells and macrophages become activated to release a large number of cytotoxic factors such as TNF-a, NO, IL, free radicals, and leukotrienes, which ultimately resulted in hepatic necrosis. NO is a major intercellular and intracellular signaling molecule, which is essential in maintaining homeostasis and acts as a cytoprotective mediator or apoptosis inducer. NO induces vasodilatation, inhibits platelet aggregation and reduces hepatic damage. The BCG/LPS-induced immunological liver injury is an important model for pathophysiological course in sepsis and systemic inflammatory syndrome with a massive release of both NO and inducible nitric oxide synthase (iNOS). Excess production of NO from iNOS-mediated hepatotoxicity has the potential to induce various cytokines. The reaction between NO and superoxide anions may produce peroxynitrite, which the latter is a potent cytotoxic oxidative agent that elicits cellular damage (Gound et al. 2015).

D-GalN combined with LPS
The combination of D-GalN with LPS-induced liver injury model is commonly used to study endotoxic liver injury. D-GalN combines with UDP of liver cells, depletes UTP, suppresses the synthesis of RNA and proteins, and results in the metabolic blockage and cell death. LPS can directly activate Toll-like receptor (TLR) 4 on the surface of HSC as well as NF-JB and ERK  pathways, suppress the expression of receptors and induce the expression of inflammatory cytokines and adhesive factors ). In addition, it can also activate the expression of TNF-a, IL-6, and NO, and damage the structural integrity of the endothelial cells in response to inflammatory mediators. LPS activates Kupffer and inflammatory cells to secrete various inflammatory cytokines, including TNF-a, IL-1b, COX-2, etc. After confronted with LPS/TNF-a stimulation, Sirt1 may deacetylate p65 and compromise NF-jB activity in hepatocytes, leading to the increased susceptibility to endotoxemic injury. Among them, TNF-a is the most important pleiotropic cytokine, which can trigger an inflammatory cascade to induce cytokines, such as IL-1b, IL-6, NO, and cell adhesion molecules ). The binding of TNF to its receptor on the hepatocyte membrane may activate caspase-3, stimulate the expressions of endothelial cells, promote the formation of thrombus and induce hepatocyte apoptosis or necrosis in acute liver failure. In addition, the combination of D-GalN and LPS can cause damage to intracellular DNA of the liver cells, by breaking into 80-200 bp or other oligonucleotide probe sections. It also activates the proteins and pathways related to the apoptosis of endoplasmic reticulum stress, induces CHOP, caspase-12, and C-Jun/JUK, as well as increasing the serum and hepatic tissue levels of TNF-a, the liver caspase-3 activity and mitochondrial pathway, in order to induce cell death (Yan et al. 2016).

Alcoholic liver injury
The alcoholic liver injury is defined as the hepatocytic damage due to excessive intake of alcohol for long-term. In human, alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase-2 (ALDH2) are the two important enzymes that can eliminate the alcohol found primarily in the liver. ADH2 can catalyze the conversion of ethyl alcohol into acetaldehyde, while ALDH2 is involved in the oxidation of acetaldehyde to acetic acid. The acetaldehyde can induce oxidative stress and injure the mitochondria and microtubule system. The ethyl alcohol can generate ROS through CYP 2E1 metabolism, trigger mitochondria dysfunction, induce mitochondrial permeability transition pore and result in the release of mitochondria apoptotic factors such as cytochrome c and Smac. The release of cytochrome c promotes the activation of caspase-9 and caspase-3, whereas Smac inhibits X-linked inhibitor of apoptosis protein (XIAP) to abolish its inhibition on caspases, which then leads to hepatocyte apoptosis. The long-term ethyl alcohol exposure may alter the mitochondrial structure and function, suppress the oxidative phosphorylation and tricarboxylic acid cycle, decrease the amount of ATP and GSH as well as increase ROS . Therefore, mitochondrial DNA damage and the impaired synthesis of mitochondrial proteins may trigger liver injuries. In addition, longterm overconsumption of alcohol activates Kupffer cells to generate large amount of NF-jB, which promotes the release of TNF-a, IL-1, and other inflammatory cytokines. TNF-a binds to its tumor necrosis factor receptor 1 (TNFR1) and recruits tumor necrosis factor receptor-related domain death protein (TRADD), fas-associating protein with a novel death domain (FADD), caspase-8, and FLIPL. As a result, caspase-8 is activated when receptor-interacting protein (RIP) 1 is de-ubiquitinated by cylindromatosis (CYLD). Activation of caspase-8 cleaves Bid to trigger the mitochondrial apoptotic pathway and cell death. Activated caspase-8 also cleaves RIP1 and RIP3 to prevent the unleashing of necroptosis mediated by RIP1 and RIP3. RIP1 and RIP3 interact with each other via RHIM domains to form the Figure 9. Alcoholic-induced liver injury. Alcohol triggers the continuous development of hepatocyte injury by inducing cell stress via inflammation, oxidative stress, protein synthesis decrease, and hepatocyte DNA fracture and degradation, resulting in hepatic necrosis, apoptosis, hepatic fibrosis, and liver injury. ADH: alcohol dehydrogenase; ALDH: aldehyde dehydrogenase; CYP2EI: cytochrome P2EI; ROS: reactive oxygen species; Cyt-C: cytochrome C; RIP: receptor-interacting protein; TRADD: tumor necrosis factor receptor-related domain death protein; FADD: fas-associating protein with a novel death domain; TNF-a: tumor necrosis factor a; IL: interleukin; NF-jB: nuclear factor jB.
amyloid-like necrosome, during the depletion of cIAPs and inhibition of caspase-8. The auto-and transphosphorylated RIP1 and RIP3 further recruit and phosphorylate downstream mixed lineage kinase domain-like pseudokinase (MLKL) to initiate necroptosis. Thus alcohol overconsumption may initiate a series of molecular events that can lead to the fatty degeneration, inflammation, and necrosis, stimulate the liver cell proliferation, suppress the activation of collagenase and promote the formation of liver fibrosis (Souli et al. 2015).
Chinese herb medicine of Ceratonia siliqua (Neyrinck et al. 2015), Lindera aggregata (Sims) Kosterm. (Lauraceae)  and Crude Rhubarb  have been used to improve the alcoholic liver injury. Rhubarb contained the highest amount of anthraquinones that can reduce the liver inflammation induced by alcohol, promote the growth of gut bacterium Akkermansia muciniphila and regulate colon antibacterial protein secretion. Several bioactive compounds such as salvianolic acid B ), dihydroartemisinin , and Panax notoginseng saponins (Zhou et al. 2015) can inhibit hepatic injury induced by alcohol. Panax notoginseng saponins inhibit acute ethanol-induced liver injury, by reducing lipolysis in white adipose tissue and decreases the expression of hormone-sensitive lipase total protein and its phosphorylation. Moreover, this herb inhibits alcohol-induced hepatic fatty acid uptake by elevating liver cluster of differentiation 36 (CD36) expressions, improves liver lipid accumulation, reduces CYP2E1-mediated oxidative stress, and prevents induction of CYP2E1 in response to chronic alcohol consumption.

Acetaminophen (APAP)
Acetaminophen-induced acute liver injury model is commonly used to test drugs through the depletion of hepatic GSH in liver injury. A large dose of APAP can be oxidized into N-acetylbenzoquinoneimine (NABQI) through the combination of CYP450 and GSH, to produce bromphenyl-acetyl-cysteine or cysteine. Meantime, the remaining N-acetyl-p-benzoquinonimine (NAPQI) can have complexation with perssads containing electrons and impaired mitochondrial functions, by generating superoxide anions and hydrogen peroxide, stimulating oxidative stress reaction, increasing iNOS, and NO synthesis. Consequently, these reactions may initiate lipid peroxidation, protein oxidation, and nitration, alter the calcium homeostasis, decrease the synthesis of ATP, release miR-122 from hepatocytes, and regulate Notch signaling Singh et al. 2017). APAP aggravates the Figure 10. APAP-induced liver injury. APAP trigger the continuous development of hepatocyte injury by inducing cell stress via inflammation, oxidative stress, and JNK pathway, resulting in hepatic necrosis, apoptosis, hepatic fibrosis, and liver injury. CYP450: cytochrome P450; NAPQI: N-acetyl-p-benzoquinonimine; GSH: glutathione; ROS: reactive oxygen species; iNOS: inducible nitric oxide synthase; NF-jB: nuclear factor jB; TNF-a: tumor necrosis factor a. oxidative damage to mitochondrial functions and changes in membrane permeability to increase the level of superoxide. However, mitophagy was shown to improve toxicity from APAP, by clearing the damaged mitochondria and reduced the production of free radicals and ROS. APAP can result in the formation of autophagosomes that engulfed mitochondria. SQSTM1/p62, an autophagy receptor protein, was recruited to APAP adducts, which may be removed by the induction of autophagy. Therefore, timely removal of adducts via autophagy may promote protection from APAP, by increasing the expression of TNF-a, activating NF-KB p65, inflammatory factors and JNK signaling pathways. In APAP experimental model, the JNK/Sab/Src/ROS pathway sustains JNK activation, amplifies mitochondrial ROS production and promotes MPT-mediated necrosis. For apoptosis, this pathway also sustains elevated levels of JNK activation and modulated multiple antiapoptotic Bcl2 family members that mediate mitochondrial outer membrane permeabilization. In fact, cell death in APAP model is a form of regulated necrosis, which is mediated by MPT and ultimately leads to liver cells death (Dara and Kaplowitz 2017).  (Xie et al. 2015) can improve the liver injury caused by APAP. For instance, A. venetum inhibited the activation of caspase-3, cleavage of DNA, released of cytochrome C and significantly reduced serum aminotransferase, MDA, and 3-nitrotyrosine (3-NT) levels, which exhibited a protective effect against liver injury. Furthermore, compounds such as hyperoside (Xie et al. 2016), baicalin , astaxanthin , and resveratrol  have therapeutic effect on liver injury induced by APAP. For example, resveratrol can inhibit the activation of APAP into toxic metabolites NAPQI, inhibit APAP-induced JNK activation, negatively regulate p53 signaling to induce cell proliferation-associated proteins including cyclin D1, cyclin-dependent kinase 4 (CDK4),  Lxeris chinensis Thunb. proliferating cell nuclear antigen (PCNA), and sirtuin type 1 (SIRT1); thus promote the proliferation of liver cells.

Isoniazid (INH) combined with rifampin (RIF)
The combined use of Isoniazid and rifampin hepatotoxic drugs is a common treatment for tuberculosis. INH is primarily metabolized in the liver through acetylation process, catalyzed by N-acetyl transferase 2 enzyme. INH is acetylated into acetyl-isoniazid (AcINH), which can be oxidized further into acethydrazide, and subsequently hydrolyzed into hydrazine by covalently binding with biomacromolecule. AcINH is then metabolized to form toxic monoacetyl hydrazine (MAH), lesser toxic diacetyl hydrazine (DAH) and other minor metabolites of INH. The CYP450 superfamily further metabolize INH intermediates through phase I pathways of drug metabolism. MAH and its reactive metabolites cause liver injury possibly through free radical generation. The production of reactive oxygen species enhances the extent of lipid peroxidation, thereby causing damage to the cellular membrane of hepatocytes. Depletion of GSH can result in lipid peroxidation of cytomembrane and mitochondrial membrane, change the permeability of mitochondrial membrane and lead to liver damage. RIF reacts through deacetylation in liver, in order to donate acetyl group for the acetylation of INH, induction of CYP450 and accelerate the metabolism of AcINH into acethydrazide. Hence, RIF is thought to potentiate the formation of toxic INH metabolites, by enhancing hepatic idiosyncratic drug reactions in the liver cells. This combination of drugs can downregulate the expressions of peroxisome proliferator-activated receptors (PPAR) a mRNA and Bsep mRNA. As a result, these drugs have been reported to cause necrosis/cell injury, inflammation, hepatic steatosis induce cholestasis by impairing bile secretion, increase the consumption of hepatic glycogen, affect the microcirculation of liver, and ultimately lead to cell death and destroy endoplasmic reticulum (Baskaran and Sabina 2015). Ficus religiosa L. (Moraceae) (Parameswari et al. 2013), Bacopa monnieri L. (Composite) (Evan et al. 2016), Tamarix gallica L. (Tamaricaceae) (Urfi et al. 2018), monoammonium glycyrrhizin ) and kaempferol (Shih et al. 2013) can reduce the depletion of glutathione and MDA levels, and thus inhibit hepatic injury induced by INF/RIF.

Conclusions
Necrosis and apoptosis induced by liver injury exist in almost all liver diseases, but the mechanism of triggering liver injury and apoptosis is specific. This is mainly due to the combined effect of the process of metabolism and detoxification associated with hepatotoxic drugs or components in the liver and the imbalance between different degrees of injury in liver cell and cell protection pathway (For example: apoptosis, innate immune response, repair, and regeneration, etc). Although Chinese medicine has a certain effect, its ingredients are numerous, and the effective components are still not clear. Therefore, the more definite pathogenesis and mechanism of liver protection is the key and difficult points of today's research. This process is very complex and miscellaneous and requires the cooperation of experts in various fields to further study and promote clinical treatment and drug development. Strengthen the synergy of TCM research TCM and its effective components can improve liver functions, reduce injury caused by oxidative stress, regulate immunologic functions, decrease inflammation, and apoptosis, as well as protect against liver damage. These studies lay the foundation for future development of hepatoprotective drugs. However, the basic concept of TCM is the effective compound groups, especially herbal medicine that has large group of chemical components. Therefore, the study on TCM drug action cannot be limited to one or several effective constituents but is relying on the exact mechanisms. Moreover, the use of the synergistic approach in TCM is needed for disease prevention, diagnosis, and treatment.
The synergistic effects of the same and different targets Because of the different pathogenic mechanisms in the development of liver injury, multiple targets are involved in different liver injury models. The current challenge is to identify the key target and index model. The target selection should be based on the conceptual foundation of pharmacology, by establishing theoretical models of TCM and thorough assessment of the target and related biology. Thus, the safety and efficiency of TCM can be improved. Although there are several studies on the multiple targets of TCM in the treatment of liver injury, further studies on the synergistic effects of the effective components of TCM with the same target are needed. It is proposed that different components of TCM with the same target and the synergistic effects of different targets may be the possible mechanisms of effective TCM compounds. Therefore, more fundamental studies should be conducted on the functions of different constituents for the key target as well as the synergistic effects of same and different targets, in order to identify the mechanisms underlying potential therapeutic role of TCM.

Highlight the combination of the basis with clinical applications
By understanding the molecular mechanisms underlying liver injury and pharmacological effects of TCM, the discovery and development of new TCM with potential clinical applications can be improved. However, there are numerous basic fundamental researches been carried out, but clinical studies are lacking. In future, the combined efforts of basic, translational, and clinical studies should be highlighted, in the context of treatment combination.
The beneficial values of the combined therapy between Chinese and Western medicines should be exploited, in order to develop an effective and inexpensive drug for the treatment of liver injuries.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The investigation was supported by The National Natural Science Foundation of China (Grant No. 81660702), Jiangxi University of Traditional Chinese Medicine.