LI Jinzhong, CHEN Ning, WANG Dang, GONG Xiaobing

(Department of Gastroenterology, the First Affiliated Hospital of Jinan University,Guangzhou 510632, China)

[Abstract] Objective: To study the efficacy and safety of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers(ACEIs/ARBs) in the treatment of non-alcoholic fatty liver disease(NAFLD) . Methods: The review studied the mechanisms of NAFLD and the therapeutic effect and mechanisms of ACEIs/ARBs from aspects of new and old RAS system, insulin resistance, oxidative stress, and adipocytokines,respectively. Results:ACEIs/ARBs improve the insulin resistance, lipid deposition, liver inflammatory and fibrosis of patients by regulating adipokines, inhibiting the release of inflammatory cytokines and activation of hepatic stellate cells． Conclusion:The review provides a new direction and theoretical evidence for the treatment of NAFLD by studying the pathogenesis of NAFLD and the possible action pathways of ACEIs/ARBs.

[Key words] nonalcoholic fatty liver disease; angiotensin-converting enzyme inhibitors; angiotensin receptor blocker; mechanism of action

In addition to the performance of the triglyceride over-accumulation in liver and thus causing inflammation and fibrosis,non-alcoholic fatty liver disease (NAFLD) also has some extrahepatic manifestations, such as chronic kidney disease, cardiovascular disease and Malignant tumor related[1].The global prevalence of NAFLD was 25.24% and metabolic comorbidities associated with NAFLD included obesity (51.34%), type 2 diabetes (22.51%), hyperlipidemia (69.16%), hypertension (39.34%) and metabolic syndrome (42.54%)[2].The pathophysiological changes of NAFLD can be explained by “two-hit theory” in which the first hit is represented by the synthesis and metabolism imbalance of hepatic lipid, resulting in the abnormal lipid deposition in hepatocytes.The second hit is a chronic inflammatory reaction in the fat-accumulating liver,which leads to microsomal, mitochondrial dysfunction, induces inflammation and fibrosis, resulting in hepatocyte apoptosis and stellate cell activation.ACEIs / ARBs is a new direction for the treatment of NAFLD, whose mechanism may be related to its regulation of adipocytokines, the improvement of oxidative stress, the inhibition of the release of inflammatory cytokines and the activation of HSCs.The pathogenesis of non-alcoholic fatty liver disease and the therapeutic effect and mechanism of ACEIs / ARBs are reviewed.

1 Old and new RAS system

Ang- (1～7), a new member of RAS system,is an extension of the traditional ACE / AngⅡ / AT1 axis, as Fig1. And ACEIs / ARBs mainly play an important role by increasing the expression of Angiotensin converting enzyme 2 (ACE2) and Ang- (1～7).High levels of serum Ang- (1～7) can antagonize AngⅡ / AT1, which can improve liver histology, as well as improve insulin resistance, lipid deposition and reduce portal hypertension[3-4].By inhibiting the mRNA transcription level of ACE gene,ACEIs / ARBs can indirectly induce the increase of precursor substance (AngI) of Angl (1～7) while reducing the production of Ang II.It can also up-regulate the mRNA expression of ACE2 and Mas gene and thus increase the production of Ang- (1～7).In addition, due to the inhibition of ACE activity,the amount of Ang- (1～5) produced by the cleavage of Ang (1-7) decrease, thereby reducing the metabolic pathway of Ang (1～7，F(xiàn)ig.1)[5].

Fig.1 Old and new RAS system

2 NAFLD and RAS system

2.1 Insulin resistance

Insulin resistance (IR) is the critical point of the pathogenesis of NAFLD and may be the result of various biochemical deficiencies in the cascade of insulin action[6]. Other factors (such as oxidative stress, adipocytokines, etc.) result in NAFLD by directly or indirectly affecting IR, or further exacerbate NAFLD on the basis of IR.IR is the result of impaired insulin signaling that may result from mutations or post-translation modification of the IR itself or any of its downstream effector molecules[7]. Multiple levels of postreceptor defects have been regarded as mechanisms of IR:1)the phosphorylation of serine (Ser) / threonine (Thr) of insulin receptor substrates (IRS) increase and thus make it degrade[8-9].2) increased activity of Tyr-phosphatases including SHIP2 (Srchomology 2 domain containing inositol 5’phosphatase 2)、PTEN (phosphatase and tension homolog deleted on chromosome ten) and protein tyrosine phosphatase-1B (PTP-1B),and decreased activation of IR downstream signaling molecules including serine threonine kinase (AKT) and protein kinase C (PKC)[10-11].Ang II enhances negative feedback of insulin by promoting the expression of signaling molecules downstream of IRS proteins (such as Phosphatidylinositol 3-kinase (PI3K), Akt and mTOR) in liver, muscle and adipose tissue:Ser / Thr phosphorylation of IRS (especially IRS-1) increases, whereas phosphorylation of Ser / Thr in specific residues induces IRS protein dissociation from IR, blocking the tyrosine phosphorylation site of the IRS protein and releasing it from the intracellular complex and thus in close contact with the receptor, thereby inducing IRS protein degradation or converting it into an inhibitor of insulin receptor kinase[7].Stimulating phosphorylation of serine residues in the insulin receptor beta-subunit and the p85 regulatory subunit of PI3-kinase,AngⅡ can also induce generation of ROS mainly by activating the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and regulates the production of pro-inflammatory mediators, including tumor necrosis factor α, interleukin-6 (IL-6) and PAI-1, leading to impairment of insulin signaling[12].In addition, Ang II attenuates the delivery of glucose and insulin to insulin-sensitive tissues by promoting the contraction of the pancreas and muscles, reducing blood flow in islets and muscles[13].In conclusion, AngⅡ can weaken the signal transduction of insulin and accelerate the degradation of IRS protein at the same time, thereby causing IR.Ang- (1-7) antagonizes Ang II in lowering insulin and systolic blood pressure levels as well as restoring insulin signaling in major target tissues (liver, adipose tissue and skeletal muscle) via the IR / IRS-1 / PI3K / Akt pathway Role[14].

2.2 Oxidative stress

Oxidation of fatty acids produces excess superoxide anion and ROS in NAFLD patients, resulting in oxidative overload of mitochondria and activating c-Jun N-terminal kinase (JUK). JUK and Receptor interacting protein 3 (RIP3) are mutually positive feedback, which cause hepatic inflammation and hepatic fibrosis through caspase-8 pathway;meanwhile RIP3-dependent JNK activation promotes the release of a proinflammatory mediator such as monocyte chemotactic protein-1 (MCP-1), attracting macrophages to the injured liver and further increasing RIP3-dependent signaling[15].The experiment found that the phosphorylation of JUK increased in ACE2 knockout mice, which also stimulate the phosphorylation of Ser in IRS-1.In addition, phosphorylation of JUK and Ser307 residues in IRS-1 was inhibited in control and ACE2 overexpressed hepatocytes, suggesting that ACE2 inhibits the ROS-activated JUK pathway[16].Fatty acids increase the expression of TNF-αby activating the activity of nuclear factor-kappa B (NF-κB) in skeletal muscle cells and 3T3-L1 cells, which inducing the expression of inflammatory mediators[17].Ang II activates membrane-bound NADH / NADPH oxidase to induce the production of cytokines and ROS, which leads to the inflammatory reaction on the vascular wall, enhancing the oxidative stress and increasing the level of NF-κB[18].ACEIs / ARBs inhibit the ROS-activated JUK pathway by up-regulating the expression of ACE2 gene mRNA, and it can also reduce the Ang II-mediated vasoconstriction, thus improving local tissue perfusion and oxygenation and reducing the increase of ROS caused by fatty acid accumulation.In addition, it inhibits the activation of the NF-κB pathway and further improves the development of inflammatory-related metabolic diseases by blocking the activation of related inflammatory cytokines.

2.3 Adipocyte cytokines

Leptin,adiponectin, resistin, TNF, interleukin-6 (IL-6), and other adipocytokines are secreted by adipose tissue to make the body adapt to the massive and complicated communication network In the body’s internal environment[19].Infiltration of immune cells in adipose tissue is more severe in the lipid-deficient internal environment,and the complex feedback regulation between adipocytes and immune cells plays an important role in the development of NAFLD.In addition to adipose tissue and immune system,peroxisome proliferator activated receptor (PPAR), a ligand-activated transcription factor, also expresses little in fat-accumulating cells like liver and kidney.PPARa is associated with lipid metabolism in the body,while PPARγplays a role in promoting adipocyte differentiation, regulating glucose and lipid metabolism and immune function[20].The expression of SREBP1c in the liver is directly regulated by PPAR-α[21], and ARBs reduce the expression of lipogenic genes by up-regulating the expression of PPAR-αin the liver, thus improving hyperlipidemia and hepatic steatosis.At the same time, ARBs can increase the activity of PPAR-γ and induce adipocyte differentiation and decrease the size of adipocytes by activating PPAR-α/γ[22-24].

2.3.1Leptin

While regulating appetite, eating behavior and energy metabolism (inhibiting fat synthesis and promoting fatty acid breakdown),leptin is directly responsible for autonomic and cardiovascular functions, so it is closely related to the occurrence, development and prognosis of NAFLD[25]. The normal “fat-insulin” axis is disrupted in NAFLD patients. On one hand, hyperlipidemia leads to leptin resistance while stimulating an increase in leptin levels, thereby limiting its ability to inhibit insulin synthesis and secretion.On the other hand, high concentration of leptin down-regulates the gene expression of glucose transporter 4 and affects the translocation of glucose transporter 4, thus reducing the uptake and sensitivity of peripheral tissues to glucose[26].In addition, due to the overlap of leptin and insulin signal transduction pathways,elevated leptin over-activate PI3-K (p85α),resulting in the imbalance between the two subunits of p85 and p110 in PI3-K.Overexpression of p85αcompetitively inhibits the binding of p85-p110 dimer to IRS, thereby interfering with insulin signaling[27-29].Though preventing hepatic steatosis in the initial stages of the disease,leptin,a double-edged sword for NAFLD, contributes to the disease’s persistence or progress as an inflammatory and fibrotic factor.Leptin improves insulin sensitivity and the function of anti-steatosis by inhibiting the production of glucose and de novo fat production and stimulating the oxidation of fatty acid in liver[30].However, excessive leptin may promote liver inflammation and fibrosis:it up-regulates TGF-β1 and other matrix remodeling enzymes by acting on sinus endothelial cells and Kupffer cells[31].Leptin can also up-regulate CD14 in Kupffer cells, making it susceptible to other stimuli such as lipopolysaccharides, thus increasing oxidative stress.In addition, leptin can further mediate and induce the activation of hepatic stellate cells (HSC) through the Janus protein kinase-STAT signal pathway (JAK-STAT), which produces TGF-β1, angiopoietin-1, Vascular endothelial growth factor and collagen-I, promoting the development of inflammation and fibrosis in liver. The activated HSC itself can produce leptin, which stimulates the proliferation of HSC and prevent its apoptosis, resulting in a vicious circle[32].Telmisartan, a selective Partial PPAR-γ agonist,play a bidirectional regulatory role:on one hand it enhances the liver’s ability to oxidize fatty acids,on the other hand it binds to leptin secreted by the omentum and down-regulates the expression of leptin,thus improving IR through the “fat-insulin” axis[33-35].

2.3.2Adiponectin

In addition to mature adipocytes, other cells, including hepatocytes, may also produce adiponectin upon attack (eg, hepatocytes as a response to liver injury).Adiponectin functions primarily through two transmembrane receptors (AdipoR1, AdipoR2) that are mainly found in skeletal muscle and moderately expressed in the liver. When combined with adiponectin, both receptors activate PPARα to stimulate the oxidation of fatty acid and increase the sensitivity of insulin[36].It has been experimentally found that fimasartan increases the oxidation of fatty acid and reduces the accumulation of hepatic fat by activating the oxidative metabolism through the representative PPARδ-Adenylate-activated protein kinase (AMPK) -peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1α) pathway,and thus improves NAFLD[37].It is also possible to increase the expression of adiponectin by activating AMPK-PGC-1αpathway through the bypass.In addition, adiponectin works by acting on HSC, Kupffer cells and sinusoids:on one hand, it exerts the effects of anti-inflammatory by inhibiting proinflammatory cytokines such as TNF-α and IL-6 and inducing anti-inflammatory cytokines such as IL-10; on the other hand, through reducing the activation and proliferation of HSC and inducing its apoptosis,adiponectin plays a role in anti-fibrosis[38]. Adiponectin also antagonizes leptin-induced liver fibrosis by the following pathways:1) Promoting the binding of suppressor of cytokine signaling (SOCS-3) to long-acting leptin receptor (Ob-Rb);2) Stimulating PTP1B expression and enhancing its activity.Both mechanisms inhibit JAK2/STAT3 at multiple sites, thereby preventing extracellular matrix deposition[39].In NAFLD patients, the expression of AdipoRs in adipose tissue and skeletal muscle was down-regulated by the PI-3K pathway in IR and hyperinsulinemia[40], whereas Ang- (1-7) could up-regulate adiponectin level and increase the expression of hepatic AdipoR2 to improve NAFLD[41].ACEIs / ARBs can play a hepatoprotective role as selective PPAR-c modulators and further improve lipid metabolism by activating and enhancing the effects of adiponectin.

2.3.3Resistin

Produced by macrophages infiltrating in human adipose tissue,resistin is also expressed in the liver and appears to increase as the increases in liver damage.

Resistin reduces the transport function of Glucose transporter protein 4 (GLUT4) by interfering with the IRS-1 / Akt pathway, which in turn affects the body’s ability to uptake glucose[42];It also affects the metabolism of carbohydrate and lipidactivity by inhibiting the activity of AMPK.Some scholars[43]proposed that resistin might up-regulate the expression of SOCS-3 by inhibiting tyrosine kinase-like orphan receptor 1, thus preventing the phosphorylation of insulin receptor.Recently, it has been demonstrated experimentally that resistin activates PKG via PKC, which activates p65 by phosphorylating Thr on NF-κB and promotes the interaction between p65 and PGC-1α,thus inactivating PGC-1α, which reducing mitochondrial content and inducing hepatic fat accumulation[44].It means that resistin reduces mitochondria and induces hepatic steatosis via the PKC/PKG /p65/PGC-1αpathway.So the reduction of mitochondria may occur before the changes in fat accumulation and development in patients with IR, which means that resistin-induced reduction of mitochondria may be one of the initiators of NAFLD onset. In addition, resistin activates HSC and promotes the expression of proinflammatory cytokines and chemokines (IL-6 and MCP-1) through the p38-NFKB pathway, resulting in enhancing proliferation and migration of HSCs while attenuating its apoptosis[45];at the same time,resistin promotes the expression of TGFβ1 through the JNK-p38 pathway,which subsequently activates HSCs by upregulating collagen I[46].Therefore, resistin may be one of the pro-fibrotic adipocytokines.While improving IR by reducing the expression of protein and mRNA of resistin in serum and liver[47], ACEIs / ARBs may also directly bind to resistin to impair their biological function.

2.3.4SOCS-3

SOCS is a class of proteins that have the effect on inhibiting JAK-STAT signal transduction.Normally, insulin or leptin binds to the receptor through the JAK-STAT pathway to regulate metabolism and meanwhile activate SOCS-3 that has a negative feedback on the JAK-STAT pathway.For NAFLD patients, inflammatory factors such as IL-6, TNF-α and AngII, leptin and insulin can induce the expression of SOCS-3 in the early stage of chronic liver disease[48].Highly expressed SOCS-3 leads to IR mainly through two pathways:1) SOCS-3 and STAT5b are competitive combined with the insulin receptor, inhibiting the tyrosine phosphorylation of STAT5b.It can also bind to IRS to ubiquitinate and make it easily recognized and degraded by the proteasome[49-50];2) SOCS-3 can act negatively on the JAK2 / STAT3 signal transduction pathway, inhibiting this signaling pathway and leading to leptin and insulin resistance[51].At the same time, it can upregulate the synthesis of fatty acid in hepatocytes by stimulating the expression of Sterol regulatory element binding protein-1 (SREBP-1c).But high levels of SOCS-3 can improve liver fibrosis as a stress response[52]:on one hand, STAT3 down-regulates the expression of TGF-β1 and has a negative feedback to the signal transduction of leptin, thereby blocking or ameliorating the status of hepatic fibrosis induced by TGF-β1 and leptin[53-54].In a recent study,ACE2 prevented the activation of the Ang II-mediated JAK2 / STAT3 / SOCS3 signaling pathway, thereby improving IR and steady-state glucose infusion rates[55].

2.3.5TNF-a

In NAFLD, a large amount of accumulated ROS stimulates Kupffer cells to synthesize TNF-α through the NF-κB and Binding protein-1 pathways.The activation of RAS in obese patients can up-regulate the expression of INF-a:In adipose tissue, TNF-α activates transcription factors Carbohydrate Response Element Binding Protein (Ch REBP) and SREBP that are transcriptionally expressed by lipogenic lipases and promote fat mobilization[56]; while activating the lipid synthesis gene SREBP-1 in the liver[57], TNF-α can also inhibit the fatty acid oxidase gene PPARa.Therefore,while increasing free fatty acids through fat mobilization,INF-a also increases liver fatty acid synthesis and inhibits its oxidation,which,finally, causing a large number of lipid accumulation in the liver and resulting in fatty liver.Overexpression of TNF-a leads to IR through the following pathways:1) activates NF-κB or JNK signal transduction pathway, inhibiting the activity of tyrosine kinase of insulin receptor and phosphorylation of hepatocyte IRS, thus attenuating insulin receptor signaling[58];2) down-regulates expression of insulin-dependent GLUT4, reducing glucose uptake and leading to IR in surrounding tissues, especially adipose tissue[59];3)directly damage the mitochondrial respiratory chain, activate the apoptotic enzyme 3 and promote the apoptosis of hepatocytes[60],4) miR-338-3p mediates TNF-α-induced hepatic insulin resistance by targeting PP4R1 to regulate PP4 expression,which promotes hepatic lipogenesis through the dephosphorylating acetyl-CoA carboxylase 1 on serine[61-62].In addition, INF-a activates MCP-1 through the Akt / PKB pathway,which plays a role in the chemotaxis of macrophages in the liver.Meanwhile,TNF-a stimulates the damaged hepatocytes to secrete tissue inhibitor of metalloproteinase-1 directly or through the TGF-βpathway, which may cause hepatic fibrosis by activating the expression of collagen type I α1 (Col1a1) gene[57].It has been found that TNF-αupregulates hepatic RAS component m RNA, including ACE, angiotensinogen and AT1R, in THLE-5b cells, a type of hepatocyte cell line.Therefore, inhibition of RAS system can reduce the expression of TNF-α from the source and the chain reaction caused by it.

In conclusion, adipocytokines play a bidirectional regulatory role in NAFLD.Most of the changes in adipokines during adipose tissue expansion are compensatory and they are often intended to provide a positive effect or neutralize the negative effects caused by another factor.However, side effects may be triggered at the same time during these changes:as fat mass increases, various adipokines increase compensatoryly. Leptin, as a compensatory mechanism for maintaining insulin sensitivity, has an anti-steatotic effect on the liver, and resistin may be targeted to reduce adipogenesis.However, if adipose tissue continues to expand, leptin may overexpress SOCS3 through the JAK2-STAT pathway or directly,then SOCS3 may become inflammatory and fibrotic adipokines and causes leptin and insulin resistance through negative feedback.While stimulating other inflammatory cytokines and hepatic fibrosis,resistin can also cause IR by inhibit the JAK2-STAT pathway and leads to the deposition of liver fat by blocking the AMPK pathway.Adiponectin and its downstream AMPK pathway are inhibited,which weakens its function to improve lipid metabolism(Fig.2).

Fig.2 Network diagram of the pathogenesis of NAFLD

Image：NF-κB:nuclear factor-κB;；IKK-β: NF-κβ kinase inhibitor；IR：insulin resistance；ROS：reactive oxygen species；RIP3：receptor-interacting protein 3；JUK：c-Jun N-terminal kinase；MCP-1：monocyte chemotactic protein-1；,PGC-1α：peroxisome proliferator activated receptor gamma coactivator 1 alpha；SOCS-3：suppressor of cytokine signaling-3；JAK-STAT：Janus kinase/signal transducer and activator of tran-scriptions；PI3K：Phosphatidylinositol 3-kinase；TNF-α：tumor necrosis factor-alpha；IL-6：interleukin 6；AdipoR1：Adiponectin receptor 1；AdipoR2：Adiponectin receptor 2；AMPK：Adenylate-activated protein kinase；PPAR：peroxisome proliferator activated receptor.

3 Summary

IR is the origin and key point of NAFLD and other factors such as oxidative stress and adipocytokines affect the signal transduction of insulin at all levels. The factors are not independent but interact with each other and change dynamically,as Fig 2.IR, lipid toxicity and inflammation are involved in the disease process[63].Lipotoxicity promotes the development of IR and inflammation, and the mutual promotion of inflammation and IR forms a vicious circle，which in turn, increase adipocyte lipolysis and exacerbates lipotoxicity.As an integrator of inflammatory pathway networks, NF-κβ plays a role in the regulation of pro-inflammatory cytokines such as IL-6, TNF-α and anti-inflammatory cytokines such as adiponectin[6].In NAFLD, the activation of the RAS system can be used as a pro-factor or cascade of amplification factors that causes NAFLD from the source and participates in all aspects of its progress and deterioration.ACEIs or ARBs has been shown to have protective effects on heart, brain and kidneys,but its protective effect on the liver remains unclear.Recent studies have shown that it can improve insulin sensitivity and secretion by inhibiting RAS system(especially AngII), enhancing insulin signaling, reducing sympathetic activity and improving ion balance.It can also reduce lipid deposition and improve liver inflammation and fibrosis.Telmisartan and irbesartan, with PPAR-γpartial agonist activity, has the characteristics of high selective activation of PPARγ, high lipophilicity and a large volume of plasma distribution, which can improve adipocyte dysfunction and decrease TNF-αand free fatty acids, thus making it stand out[22,33-34].

NAFLD has a tendency to coexist with cardiovascular and cerebrovascular diseases such as hypertension, hyperglycemia, and coronary heart disease[64].The clinical burden of NAFLD,therefore, is not only confined to liver-related morbidity and mortality,but also affecting extra-hepatic organs and regulatory pathways.However, no drugs currently specifically recommended in clinical practice or guidelines are used for NAFLD.The review provides a new direction and theoretical basis for the treatment of NAFLD by studying the pathogenesis of NAFLD and the possible pathways of action of ACEI or ARB.At the same time, some new molecular sites of action (SOCS, NF-κB, TNF-α) may be used as a new research direction, and Ang- (1-7) oral preparations may also be used as an endogenous RAS inhibition instead of relying on ACEIs / ARBs to indirectly increase Ang- (1-7) to achieve therapeutic effect.