Antagonizing interleukin-5 receptor ameliorates dextran sulfate sodium-induced experimental colitis in mice through reducing NLRP3 inflammasome activation.

Inflammatory bowel disease (IBD) is a condition characterized by inflammation in the gastrointestinal tract. Reducing intestinal inflammation is a promising approach for treating IBD. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, a critical component of the innate immune system, is implicated in the pathogenesis of IBD. Therefore, inhibiting NLRP3 inflammasome activation is a potential therapeutic strategy for IBD. In this study, we investigated the effects of the interleukin-5 (IL-5) receptor antagonist YM-90709 on dextran sulfate sodium-induced experimental colitis in mice. We found that YM-90709 reduced the expressions of IL-1ß and caspase-1 p20 in the colon and ameliorated colitis. Furthermore, we identified YM-90709 as an effective agent for inhibiting NLRP3 inflammasome activation. Knockdown of IL-5 receptor or using an inhibitor of STAT5, a key transcription factor downstream of the IL-5/IL-5 receptor signal pathway, also reduced NLRP3 inflammasome-dependent IL-1ß release and ASC speck formation. Our study is the first to demonstrate that the NLRP3 inflammasome may be a downstream signal of IL-5/IL-5 receptor and that YM-90709 protects against IBD by inhibiting IL-5 receptor. These findings suggest a new strategy for regulating intestinal inflammation and managing IBD.

Discovery of a selective NLRP3-targeting compound with therapeutic activity in MSU-induced peritonitis and DSS-induced acute intestinal inflammation.

The aberrant activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is known to contribute to the pathogenesis of various human inflammation-related diseases. However, to date, no small-molecule NLRP3 inhibitor has been used in clinical settings. In this study, we have identified SB-222200 as a novel direct NLRP3 inhibitor through the use of drug affinity responsive target stability assay, cellular thermal shift assay, and surface plasmon resonance analysis. SB-222200 effectively inhibits the activation of the NLRP3 inflammasome in macrophages, while having no impact on the activation of NLRC4 or AIM2 inflammasome. Furthermore, SB-222200 directly binds to the NLRP3 protein, inhibiting NLRP3 inflammasome assembly by blocking the NEK7 - NLRP3 interaction and NLRP3 oligomerization. Importantly, treatment with SB-222200 demonstrates alleviation of NLRP3-dependent inflammatory diseases in mouse models, such as monosodium urate crystal-induced peritonitis and dextran sulfate sodium-induced acute intestinal inflammation. Therefore, SB-222200 holds promise as a lead compound for the development of NLRP3 inhibitors to combat NLRP3-driven disease and serves as a versatile tool for pharmacologically investigating NLRP3 biology.

Bortezomib inhibits NLRP3 inflammasome activation and NF-κB pathway to reduce psoriatic inflammation.

The abnormal activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the pathogenesis of psoriasis. Accordingly, the inhibition of NLRP3 inflammasome may be an effective strategy for psoriasis treatment. However, the NLRP3 inflammasome inhibitors are not available in the clinic. Repurposing FDA-approved drugs is a highly attractive way for identifying new drugs. Here, proteasome inhibitor bortezomib, a marketed drug for treating multiple myeloma, was found to specifically inhibit NLRP3 inflammasome activation at nanomolar concentrations. Mechanistically, bortezomib did not inhibit reactive oxygen species generation, ion efflux, NLRP3 oligomerization, and NLRP3-ASC interactions. Bortezomib reduced ASC oligomerization and ASC speck formation. In addition, bortezomib inhibited the activity of the core subunit ß5i in the immunoproteasome and reduced ß5i binding to NLRP3. Bortezomib reduced the production of interleukin-1ß and attenuated the severity of skin lesions in the imiquimod-induced psoriatic mouse model. Thus, bortezomib is a potential therapeutic drug for psoriasis. Our study also revealed that ß5i may be an indirect target for regulating NLRP3 inflammasome activation and treating psoriasis and other NLRP3 inflammasome-related diseases.

Artemisinin-derived artemisitene blocks ROS-mediated NLRP3 inflammasome and alleviates ulcerative colitis.

Artemisinins are well-known antimalarial drugs with clinical safety. In addition to antimalarial effects, their anti-inflammatory and immunoregulatory properties have recently attracted much attention in the treatment of inflammatory diseases. However, these artemisinins only have sub-millimolar anti-inflammatory activity in vitro, which may pose a high risk of toxicity in vivo with high doses of artemisinins. Here, we identified another derivative, artemisitene, which can increase the activity of inhibiting the NLRP3 pathway by more than 200-fold through introducing a covalent binding group while retaining the peroxide bridge structure. Mechanistically, artemisitene inhibits the production of ROS (especially mtROS) and prevents the assembly and activation of NLRP3 inflammasome, thereby inhibiting IL-1ß production. In addition, it can also block IL-1ß secretion mediated by NLRC4 and AIM2 inflammasome and IL-6 production. Furthermore, treatment with artemisitene significantly attenuated inflammatory response in DSS-induced ulcerative colitis. Our work provides a potential artemisinin derivative, which is worthy of further structural optimization based on pharmacokinetic properties as a drug candidate for inflammatory disorders.

Discovery of a Novel Oral Proteasome Inhibitor to Block NLRP3 Inflammasome Activation with Anti-inflammation Activity.

NLRP3 inflammasome activation plays a critical role in inflammation-related disorders. More small-molecule entities are needed to study the mechanism of NLRP3 inflammasome activation and to validate the efficacy and safety of the NLRP3 pathway. Herein, we report the discovery of an orally bioavailable proteasome inhibitor NIC-0102 (27) that specifically prevents NLRP3 inflammasome activation but has no effect on NLRC4 or AIM2 inflammasomes. In vitro studies revealed that NIC-0102 induced the polyubiquitination of NLRP3, interfered with the NLRP3-ASC interaction, and blocked ASC oligomerization, thereby resulting in the inhibition of NLRP3 inflammasome activation. In addition, NIC-0102 also inhibited the production of pro-IL-1ß. Importantly, NIC-0102 showed potent anti-inflammatory effects on DSS-induced ulcerative colitis model in vivo. As a result of these studies, a potential small molecule is identified to demonstrate the possible link between the proteasome and NLRP3 pathway, which supports further exploration of potentially druggable nodes to modulate NLRP3 inflammasome activation.

Seratrodast, a thromboxane A2 receptor antagonist, inhibits neuronal ferroptosis by promoting GPX4 expression and suppressing JNK phosphorylation.

More than 30 % of individuals with epilepsy are refractory to currently available drugs, highlighting the urgent need to develop novel candidate drugs. Accumulating evidence implicates the key role of ferroptosis in the pathophysiology of epileptic seizuresand its potential as a new drug target. Drug repurposing is a promising strategy for the rapid generation of new candidate drugs from the market drugs with new therapeutic indications, such as the best-selling drug thalidomide. Herein, we reported the discovery of Seratrodast, a market drug of thromboxane A2 receptor antagonist as a new ferroptosis inhibitor (IC50: 4.5 µmol·L-1). Seratrodast could reduce lipid ROS production, regulate the system xc-/glutathione (GSH)/glutathione peroxidase 4 (GPX4) axis, and inhibit JNK phosphorylation and p53 expression. In addition, Seratrodast elevated GPX4 expression and decreased JNK phosphorylation in pentylenetetrazole-induced seizures in mice. Seratrodast increased the latency of seizures and reduced seizure duration in pentylenetetrazole-induced seizures. Our results suggest Seratrodast might be either a ferroptosis inhibitor or a novel lead compound for further optimization of novel drug discovery.

Chlorquinaldol inhibits the activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 inflammasome and ameliorates imiquimod-induced psoriasis-like dermatitis in mice.

Psoriasis is a common chronic autoinflammatory/autoimmune skin disease associated with elevated pro-inflammatory cytokines. The pivotal role of interleukin (IL)-1ß and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome in the pathogenesis of psoriasis has been widely described. Accordingly, the suppression of NLRP3-dependent IL-1ß release is a potential therapy for psoriasis. Repurposing marketed drugs is a strategy for identifying new inhibitors of NLRP3 inflammasome activation. Herein, chlorquinaldol (CQD), a historic antimicrobial agent used as a topical treatment for skin and vaginal infections, was found to have a distinct effect by inhibiting NLRP3 inflammasome activation at concentrations ranging from 2 to 6 µM. CQD significantly suppressed apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) oligomerization, NLRP3-ASC interaction, and pyroptosis in macrophages. The levels of cleaved IL-1ß and caspase-1 were reduced by CQD in the cell lysates of macrophages, suggesting that CQD acted on upstream of pore formation in the cell membrane. Mechanistically, CQD reduced mitochondrial reactive oxygen species production but did not affect the nuclear factor-κB (NF-κB) pathway. Intraperitoneal administration of CQD (15 mg/kg) for 6 days was found to improve the skin lesions in the imiquimod-induced psoriatic mouse model (male C57BL/6 mice), while secretion of pro-inflammatory cytokines (IL-17 and IL-1ß) and keratinocyte proliferation were significantly suppressed by CQD. In conclusion, CQD exerted inhibitory effects on NLRP3 inflammasome activation in macrophages and decreased the severity of psoriatic response in vivo. Such findings indicate that the repurposing of the old drug, CQD, is a potential pharmacological approach for the treatment of psoriasis and other NLRP3-driven diseases.

Ciclopirox inhibits NLRP3 inflammasome activation via protecting mitochondria and ameliorates imiquimod-induced psoriatic inflammation in mice.

The maturation and secretion of interleukin-1ß (IL-1ß) mediated by NLRP3 inflammasome activation plays an important role in the progression of many inflammatory diseases. Inhibition of NLRP3 inflammasome activation may be a promising strategy to treat these inflammation-driven diseases, such as psoriasis. As a broad-spectrum antifungal agent, ciclopirox (CPX) is widely used in the treatment of dermatomycosis. Although CPX has been reported to have anti-inflammatory effects in many studies, there has been little research into its underlying mechanisms. In our study, CPX reduced lipopolysaccharide (LPS)/nigericin-induced NLRP3 inflammasome activation (IC50: 1.684 µM). Mechanistically, CPX upregulated peroxisome proliferator-activated receptor-γ coactivator-1α expression (by 82.7% at 5 µM and 87.5% at 10 µM) to protect mitochondria. Our studies showed that CPX reduced mitochondrial reactive oxygen species production, increased mitochondrial membrane potential, elevated mitochondrial biosynthesis, and up-regulated intracellular adenosine triphosphate level. Furthermore, treatment with CPX promoted the up-regulation of mRNA expression, which involved mitochondrial biosynthesis (NRF1, NRF2, TFAM) and antioxidation (SOD1 and CAT). In addition, CPX ameliorated inflammatory response in imiquimod-induced psoriasis mice. This study provides a potential pharmacological mechanism for CPX to treat psoriasis and other NLRP3-driven inflammatory diseases.

Tanshinones inhibit NLRP3 inflammasome activation by alleviating mitochondrial damage to protect against septic and gouty inflammation.

Tanshinones, the active ingredients derived from the roots of Salvia miltiorrhiza, have been widely used as traditional medicinal herbs for treating human diseases. Although tanshinones showed anti-inflammatory effects in many studies, large knowledge gaps remain regarding their underlying mechanisms. Here, we identified 15 tanshinones that suppressed the activation of NLRP3 inflammasome and studied their structure-activity relationships. Three tanshinones (tanshinone IIA, isocryptotanshinone, and dihydrotanshinone I) reduced mitochondrial reactive-oxygen species production in lipopolysaccharide (LPS)/nigericin-stimulated macrophages and correlated with altered mitochondrial membrane potentials, mitochondria complexes activities, and adenosine triphosphate and protonated-nicotinamide adenine dinucleotide production. The tanshinones may confer mitochondrial protection by promoting autophagy and the AMP-activated protein kinase pathway. Importantly, our findings demonstrate that dihydrotanshinone I improved the survival of mice with LPS shock and ameliorated inflammatory responses in septic and gouty animals. Our results suggest a potential pharmacological mechanism whereby tanshinones can effectively treat inflammatory diseases, such as septic and gouty inflammation.

Discovery of chalcone analogues as novel NLRP3 inflammasome inhibitors with potent anti-inflammation activities.

NLRP3 inflammasome activation plays a critical role in inflammation and its related disorders. Herein we report a hit-to-lead effort resulting in the discovery of a novel and potent class of NLRP3 inflammasome inhibitors. Among these, the most potent lead 40 exhibited improved inhibitory potency and almost no toxicity. Further mechanistic study indicated that compound 40 inhibited the NLRP3 inflammasome activation via suppressing ROS production. More importantly, treatment with 40 showed remarkable therapeutic effects on LPS-induced sepsis and DSS-induced colitis. This study encourages further development of more potent inhibitors based on this chemical scaffold and provides a chemical tool to identify its cellular binding target.

Compound AD16 Reduces Amyloid Plaque Deposition and Modifies Microglia in a Transgenic Mouse Model of Alzheimer's Disease.

Microglial dysfunction is involved in the pathological cascade of Alzheimer's disease (AD). The regulation of microglial function may be a novel strategy for AD therapy. We previously reported the discovery of AD16, an antineuroinflammatory molecule that could improve learning and memory in the AD model. Here, we studied its properties of microglial modification in the AD mice model. In this study, AD16 reduced interleukin-1ß (IL-1ß) expression in the lipopolysaccharide-induced IL-1ß-Luc transgenic mice model. Compared with mice receiving placebo, the group treated with AD16 manifested a significant reduction of microglial activation, plaque deposition, and peri-plaques microgliosis, but without alteration of the number of microglia surrounding the plaque. We also found that AD16 decreased senescent microglial cells marked with SA-ß-gal staining. Furthermore, altered lysosomal positioning, enhanced Lysosomal Associated Membrane Protein 1 (LAMP1) expression, and elevated adenosine triphosphate (ATP) concentration were found with AD16 treatment in lipopolysaccharide-stimulated BV2 microglial cells. The underlying mechanisms of AD16 might include regulating the microglial activation/senescence and recovery of its physiological function via the improvement of lysosomal function. Our findings provide new insights into the AD therapeutic approach through the regulation of microglial function and a promising lead compound for further study.

Synthesis and biological evaluation of parthenolide derivatives with reduced toxicity as potential inhibitors of the NLRP3 inflammasome.

Parthenolide (PTL) can target NLRP3 inflammasome to treat inflammation and its related disease, but its cytotoxicity limits further development as an anti-inflammatory drug. A series of PTL analogs and their Michael-type adducts were designed and synthesized, and most of them showed high activities against the NLRP3 inflammasome pathway. The most potent compound 8b inhibited the release of IL-1ß with IC50 values of 0.3 µM in J774A.1 cell and 1.0 µM in primary glial cells, respectively. Moreover, 8b showed low toxicity against J774A.1 cell (IC50 = 24.1 µM) and HEK-293T (IC50 = 69.8 µM) with a ~8 folds increase of therapeutic index compared to its parent PTL. The preliminary mechanism study revealed that 8b mediated anti-inflammation is associated with the NLRP3 inflammasome signal pathway. Based on these investigations, we propose that 8b might be a potential drug candidate for ultimate development of the anti-inflammation drug.

Disulfiram suppresses NLRP3 inflammasome activation to treat peritoneal and gouty inflammation.

The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome plays a vital role in mediating the innate immune system. Its aberrant activation contributes to the progression of several devastating diseases such as acute peritonitis, acute liver injury, sepsis, gout, and others. However, the medications targeting NLRP3 inflammasome are not available in the clinic. Reusing marketed drugs, which have been already proved to possess good pharmacokinetic profiles and safety, is a strategy to develop new NLRP3 inflammasome inhibitors for clinical trials. In this study, we identified disulfiram (DSF), an old marketed drug as a treatment for alcoholism, could effectively inhibit NLRP3 inflammasome activation and suppress pyroptotic cell death. DSF prevented lysosomal cathepsin B releasing into the cytoplasm, which in turn inactivated the NLRP3 inflammasome. DSF also reduced mitochondrial-independent ROS production. More importantly, treatment with DSF showed remarkable therapeutic effects on the LPS-induced peritoneal inflammation and MSU-induced gouty inflammation. This study provides a potential pharmacological approach to treating NLRP3-driven diseases and a tool to study NLRP3 biology.

Compound AD110 Acts as Therapeutic Management for Alzheimer's Disease and Stroke in Mouse and Rat Models.

Anti-inflammatory therapy may be an effective therapeutic intervention for neurological diseases, such as Alzheimer's disease (AD) and stroke. As an important anti-inflammatory cytokine, interleukin-10 (IL-10) inhibits proinflammatory responses of both innate and adaptive immune cells. We tested the hypothesis that drug-induced promotion of IL-10 expression is effective in improving cognitive abilities and neurologic outcomes of AD and stroke. An orally small molecule AD110 was synthesized and subjected to in vitro and in vivo analyses. We found that AD110 enhanced IL-10 release in lipopolysaccharide (LPS)-activated BV2 microglial cells. Y-Maze and Morris water maze tests showed improved cognitive abilities in AD mice treated with AD110. Moreover, AD110 attenuated cerebral ischemic injury in a transient middle cerebral artery occlusion (tMCAO) rat model. This study not only provides a promising lead compound with IL-10-promoting activity, but also supports the hypothesis that promoting IL-10 expression is a potential therapeutic strategy for AD and stroke.

Sphingosine kinase 1 promotes liver fibrosis by preventing miR-19b-3p-mediated inhibition of CCR2.

Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) leads to liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the sphingosine kinase (SphK)1-dependent effect on liver fibrosis. The levels of expression and activity of SphK1 were significantly increased in fibrotic livers compared with the normal livers in human. SphK1 was coexpressed with a range of HSC/KC markers including desmin, α-smooth muscle actin (α-SMA) and F4/80 in fibrotic liver. Deficiency of SphK1 (SphK1-/- ) resulted in a marked amelioration of hepatic injury, including transaminase activities, histology, collagen deposition, α-SMA and inflammation, in CCl4 or bile duct ligation (BDL)-induced mice. Likewise, treatment with a specific inhibitor of SphK1, 5C, also significantly prevented liver injury and fibrosis in mice induced by CCl4 or BDL. In cellular levels, inhibition of SphK1 significantly blocked the activation and migration of HSCs and KCs. Moreover, SphK1 knockout in KCs reduced the secretion of CCL2, and SphK1 knockout in HSCs reduced C-C motif chemokine receptor 2 ([CCR2] CCL2 receptor) expression in HSCs. CCL2 in SphK1-/- mice was lower whereas microRNA-19b-3p in SphK1-/- mice was higher compared with wild-type (WT) mice. Furthermore, microRNA-19b-3p downregulated CCR2 in HSCs. The functional effect of SphK1 in HSCs on liver fibrosis was further strengthened by the results of animal experiments using a bone marrow transplantation (BMT) method. CONCLUSION: SphK1 has distinct roles in the activation of KCs and HSCs in liver fibrosis. Mechanistically, SphK1 in KCs mediates CCL2 secretion, and SphK1 in HSCs upregulates CCR2 by downregulation of miR-19b-3p. (Hepatology 2018).

Osteopontin promotes collagen I synthesis in hepatic stellate cells by miRNA-129-5p inhibition.

Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. HSCs are believed to be the major source of collagen-producing myofibroblasts in fibrotic livers. A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen I (Col 1) deposition. Osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in HSCs, could drive fibrogenesis by modulating the HSC pro-fibrogenic phenotype and Col 1 expression. Here, we aimed to investigate the molecular mechanism of OPN regulating the activation of HSCs. Our results showed that hepatic expression of OPN was increased in patients with liver fibrosis. In addition, hepatic OPN was positively correlated with Col 1 and α-SMA. Recombinant OPN (rOPN) upregulated Col 1 and α-SMA expression in LX-2 cells. However, OPN knockdown downregulated Col 1 expression. The 3'-UTR of the collagen 1 (Col 1) was identified to bind miR-129-5p. Transfection of miR-129-5p mimic in HSC resulted in a marked reduction of Col 1 expression. Conversely, a decrease in miR-129-5p in HSCs transfected by anti-sense miR-129-5p (AS-miR-129-5p) caused Col 1 upregulation. Furthermore, luciferase reporter assay showed that miR-129-5p directly target the 3'-UTR of Col1α1 mRNA via repressing its post-transcriptional activities. Finally, miR-129-5p level was decreased in fibrotic liver of human, and reduced by rOPN treatment. In contrast, miR-129-5p was induced in HSCs transfected by OPN siRNA. These data suggested that OPN induces Col 1 expression via suppression of miR-129-5p in HSCs.

Andrographolide ameliorates diabetic nephropathy by attenuating hyperglycemia-mediated renal oxidative stress and inflammation via Akt/NF-κB pathway.

Diabetic nephropathy (DN) is characterized by proliferation of mesangial cells, mesangial hypertrophy and extracellular matrix (ECM) accumulation. Our recent study found that andrographolide inhibited high glucose-induced mesangial cell proliferation and fibronectin expression through inhibition of AP-1 pathway. However, whether andrographolide has reno-protective roles in DN has not been fully elucidated. Here, we studied the pharmacological effects of andrographolide against the progression of DN and high glucose-induced mesangial dysfunction. Diabetes was induced in C57BL/6 mice by intraperitoneal injection of streptozotocin (STZ). After 1 weeks after STZ injection, normal diet was substituted with a high-fat diet (HFD). Diabetic mice were intraperitoneal injected with andrographolide (2 mg/kg, twice a week). After 8 weeks, functional and histological analyses were carried out. Parallel experiments uncovering the molecular mechanism by which andrographolide prevents from DN was performed in mesangial cells. Andrographolide inhibited the increases in fasting blood glucose, triglyceride, kidney/body weight ratio, blood urea nitrogen, serum creatinine and 24-h albuminuria in diabetic mice. Andrographolide also prevented renal hypertrophy and ECM accumulation. Furthermore, andrographolide markedly attenuated NOX1 expression, ROS production and pro-inflammatory cytokines as well. Additionally, andrographolide inhibited Akt/NF-κB signaling pathway. These results demonstrate that andrographolide is protective against the progression of experimental DN by inhibiting renal oxidative stress, inflammation and fibrosis.

Activation of Slit2-Robo1 signaling promotes liver fibrosis.

BACKGROUND & AIMS: The secretory protein Slit2 and its receptor Robo1 are believed to regulate cell growth and migration. Here, we aimed to determine whether Slit2-Robo1 signaling mediates the pathogenesis of liver fibrosis. METHODS: Serum levels of Slit2 in patients with liver fibrosis were determined by ELISA. Liver fibrosis was induced in wild-type (WT), Slit2 transgenic (Slit2-Tg) and Robo1(+/-)Robo2(+/-) double heterozygotes (Robo1/2(+/-)) mice by carbon tetrachloride (CCl4). The functional contributions of Slit2-Robo1 signaling in liver fibrosis and activation of hepatic stellate cells (HSCs) were investigated using primary mouse HSCs and human HSC cell line LX-2. RESULTS: Significantly increased serum Slit2 levels and hepatic expression of Slit2 and Robo1 were observed in patients with liver fibrosis. Compared to WT mice, Slit2-Tg mice were much more vulnerable to CCl4-induced liver injury and more readily develop liver fibrosis. Development of hepatic fibrosis in Slit2-Tg mice was associated with a stronger hepatic expression of collagen I and α-smooth muscle actin (α-SMA). However, liver injury and hepatic expression of collagen I and α-SMA were attenuated in CCl4-treated Robo1/2(+/-) mice in response to CCl4 exposure. In vitro, Robo1 neutralizing antibody R5 and Robo1 siRNA downregulated phosphorylation of Smad2, Smad3, PI3K, and AKT in HSCs independent of TGF-ß1. R5 and Robo1 siRNA also inhibited the expression of α-SMA by HSCs. Finally, the protective effect of R5 on the CCl4-induced liver injury and fibrosis was further verified in mice. CONCLUSIONS: Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs.