Blocking ATP-P1Rs axis attenuate alcohol-related liver fibrosis.

AIMS: The aim of this study was to explore the fibrogenic effects of ATP-P1Rs axis and ATP-P2Rs axis on alcohol-related liver fibrosis (ALF). MATERIALS AND METHODS: C57BL/6J CD73 knock out (KO) mice were used in our study. 8-12 weeks male mice were used as an ALF model in vivo. In conclusion, after one week of adaptive feeding, 5 % alcohol liquid diet was given for 8 weeks. High-concentration alcohol (31.5 %, 5 g/kg) was administered by gavage twice weekly, and 10 % CCl4 intraperitoneal injections (1 ml/kg) were administered twice weekly for the last two weeks. The mice in the control group were injected intraperitoneally with an equivalent volume of normal saline. Fasting for 9 h after the last injection, blood samples were collected, and related indicators were tested. In vitro, rat hepatic stellate cells (HSCs) were treated with 200 µM acetaldehyde to establish an alcoholic liver fibrosis for 48 h, then tested related indicators. KEY FINDINGS: We found that both adenosine receptors including adenosine A1, A2A, A2B, A3 receptors (A1R, A2AR, A2BR, A3R) and ATP receptors including P2X7, P2Y2 receptors (P2X7R, P2Y2R) were expressed increased in ALF. After CD73 was knocked out, we found that adenosine receptors expression decreased, ATP expression increased, and fibrosis degree decreased. SIGNIFICANCE: Based on the research, we discovered that adenosine plays a more important role in ALF. Therefore, blocking the ATP-P1Rs axis represented a potential treatment for ALF, and CD73 will become a potential therapeutic target.

CD73 aggravates alcohol-related liver fibrosis by promoting autophagy mediated activation of hepatic stellate cells through AMPK/AKT/mTOR signaling pathway.

CD73 is a membrane-bound glycoprotein that can dephosphorylate AMP to adenosine. Increasing evidence has shown that CD73 is involved in the occurrence and development of liver fibrosis. However, the potential mechanism by which CD73 affects the progression of alcohol-related liver fibrosis (ALF) remains unknown. This study aimed to examine the role and mechanism of CD73 in autophagy in HSC-T6 cells and its role in ALF in mice that treated with alcohol plus CCl4. We found that CD73 knockout reduced serum alanine aminotransferase and aspartate aminotransferase levels and decreased liver injury and collagen deposition. Furthermore, autophagy-related indicators were downregulated in the liver fibrosis tissues of CD73-/- (EtOH + CCl4) mice. In vitro, the expression of CD73 and autophagy increased in activated HSC-T6 cells. Autophagy inhibitor, 3-methyladenine, reduced autophagy and activation of acetaldehyde-induced HSC-T6 cells. When using CD73-siRNA, autophagy in HSC-T6 cells was found to be downregulated. However, the CD73 plasmid increased the activation and autophagy of hepatic stellate cells (HSCs). In addition, CD73 induced autophagy through the AMPK/AKT/mTOR pathway, which is characterized by an increase in the ratio of P-AMPKα/AMPKα and a decrease in the ratio of P-AKT/AKT and P-mTOR/mTOR. Our study found that CD73 promotes HSCs activation by regulating autophagy through the AMPK/AKT/mTOR signaling pathway.

Blockade of the P2Y2 Receptor Attenuates Alcoholic Liver Inflammation by Targeting the EGFR-ERK1/2 Signaling Pathway.

Purpose: It is well known that inflammation plays a key role in complex pathological progressions of alcohol-associated liver disease (ALD). To date, effective therapy for ALD is lacking. P2Y2 receptor (P2Y2R), a G protein-coupled P2Y purinergic receptor, represents a novel pharmacological target in many inflammations. Methods: The alcohol-associated liver injury and inflammation mouse model was established. The effect of P2Y2R on alcohol-induced liver injury and inflammation was evaluated using quantitative real-time PCR, Western blot and immunohistochemical assay. An alcohol-stimulated (100 mmol/L, for 24 h) AML-12 cell model was established. Different agonists, antagonists and P2Y2R siRNA were used to explore the possible mechanisms of P2Y2R. Results: In vivo, results showed that the hepatoprotective effect of P2Y2R blockade by significantly suppressed liver structural abnormalities and lipid infiltration, and decreased levels of ALT/AST and TNF-α/IL-1ß in the high dosage group of suramin (20 mg/kg) compared to control diet (CD)-fed mice. At the same time, we found that alcohol feeding promoted the phosphorylation of EGFR and ERK1/2, both of which were effectively inhibited by suramin (20 mg/kg). In vitro, suramin or P2Y2R silencing effectively inhibited the phosphorylation of EGFR and ERK1/2, similar to the down-regulated effects of their corresponding inhibitors (EGFR inhibitor AG1478 and ERK1/2 inhibitor U0126) accompanied by reduced levels of TNF-α and IL-1ß compared to alcohol-induced AML-12 cell. In addition, we found that silencing P2Y2R attenuated the apoptosis of hepatocyte. Conclusion: Our findings suggest that P2Y2R regulates alcoholic liver inflammation by targeting the EGFR-ERK1/2 signaling pathway and plays an important role in hepatocyte apoptosis, which may provide new ideas for the development of methods to treat ALD.

CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway.

BACKGROUND: Alcoholic liver disease (ALD) is liver damage caused by long-term drinking. Inflammation plays a central role in the progression of ALD. CD73 is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that is a key enzyme that converts ATP into adenosine. Evidence has shown that CD73 plays an important role in many diseases, but the role and mechanism of CD73 in alcohol-induced liver injury and inflammation is still unclear. METHODS: The alcohol-induced liver injury and inflammation mouse model was established. The rAAV9-CD73 was used to overexpress CD73. Isolation of primary macrophages (MΦ) from the liver was conducted. The effects of CD73 on alcohol-induced liver injury and inflammation were evaluated by quantitative real­time PCR, Western blotting, ELISA, and immunohistochemical assay. Flow cytometry was used to detect the cell cycle and apoptosis. RESULTS: Our results showed that overexpression of CD73 can reduce alcohol-induced liver damage, lipid accumulation, and the secretion of inflammatory cytokines. pEX3-CD73 can promote RAW264.7 cells proliferation and inhibit apoptosis via suppressing the activation of TLR4/MyD88/NF-κB signaling pathway. Inhibition of TLR4 further enhanced the anti-inflammatory effect of overexpression of CD73. CONCLUSION: Overexpression of CD73 can reduce alcohol-induced liver injury and inflammation. CD73 may serve as a potential therapeutic target for ALD.

Construction of a CRISPR-Biolayer Interferometry Platform for Real-Time, Sensitive, and Specific DNA Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been widely applied for nucleic acid detection because of its high specificity. By using the highly specific and irreversible bond between HaloTag and its alkane chlorine ligand, we modified dCas9 (deactivated CRISPR/Cas9) with biotin as a biosensor to detect nucleic acids. The CRISPR biosensor was facilely prepared to adequately maintain its DNA-recognition capability. Furthermore, by coupling biolayer interferometry (BLI) with the CRISPR biosensor, a real-time, sensitive, and rapid digital system called CRISPR-BLI was established for the detection of double-stranded DNA. The CRISPR biosensor immobilised on the biolayer could recruit the target DNA onto the biosensor surface and change its optical thickness, resulting in a shift in the interference pattern and responding signal of the BLI. The CRISPR-BLI system was further applied to detect the ALP gene of Escherichia coli DH5α combined with a polymerase chain reaction, which demonstrated a linear range from 20 to 20 000 pg and a low detection limit (1.34 pg). The CRISPR-BLI system is a promising approach for rapid and sensitive detection of target DNA analytes.

CD73 regulates hepatic stellate cells activation and proliferation through Wnt/ß-catenin signaling pathway.

Alcoholic liver fibrosis (ALF) is commonly associated with long-term alcohol consumption and the activation of hepatic stellate cells (HSCs). Inhibiting the activation and proliferation of HSCs is a critical step to alleviate liver fibrosis. Increasing evidence indicates that ecto-5'-nucleotidase (CD73) plays a vital role in liver disease as a critical component of extracellular adenosine pathway. However, the regulatory role of CD73 in ALF has not been elucidated. In this study, both ethanol plus CCl4-induced liver fibrosis mice model and acetaldehyde-activated HSC-T6 cell model were employed and the expression of CD73 was consistently elevated in vivo and in vitro. C57BL/6 J mice were intraperitoneally injected with CD73 inhibitor Adenosine 5'-(α, ß-methylene) diphosphate sodium salt (APCP) from 5th week to the 8th week in the development of ALF. The results showed APCP could inhibit the activation of HSCs, reduce fibrogenesis marker expression and thus alleviate ALF. Silencing of CD73 inhibited the activation of HSC-T6 cells and promoted apoptosis of activated HSC-T6 cells. What's more, the proliferation of HSC-T6 cells was inhibited, which was characterized by decreased cell viability and cycle arrest. Mechanistically, Wnt/ß-catenin pathway was activated in acetaldehyde-activated HSC-T6 cells and CD73 silencing or overexpression could regulate Wnt/ß-catenin signaling pathway. Collectively, our study unveils the role of CD73 in HSCs activation, and Wnt/ß-catenin signaling pathway might be involved in this progression.

Regulation of CD39 expression in ATP-P2Y2R-mediated alcoholic liver steatosis and inflammation.

Inflammation plays a central role in the progression of alcoholic liver disease. ATP-P2Y2R signaling and CD39 play an important role in various diseases, but little is known about their role in alcoholic liver steatosis and inflammation. As a transmembrane hydrolase, CD39 hydrolyzes ATP, while the mutual regulation of CD39 and ATP-P2Y2R in alcoholic steatohepatitis is poorly understood. Here, we found that the expression of ATP, P2Y2R, and CD39 is increased significantly both in the liver of alcohol-fed mice and alcohol-induced RAW264.7 cell lines. In this study, C57BL/6 mice were intrapretationally injected with P2Y2R inhibitor suramin from day 4 until day 10 during the induction of a chronic/binge drinking model. Pharmacological blockade of P2Y2R largely prevents liver damage, lipid accumulation, and inflammation, with concomitant down-expression of CD39 in liver. We found that the inhibition of P2Y2R in vitro reduces inflammation via down-expression of interleukin 6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α), and the expression of CD39 was reduced, whereas the activation of P2Y2R showed an opposite effect. Silencing of CD39 promoted the expression of ATP and P2Y2R. These results indicate that CD39 attenuates alcohol-induced steatohepatitis by scavenging extracellular ATP to indirectly regulate the expression of P2Y2R. Interestingly, P2Y2R paradoxically boosts CD39 activity. Thus, blockade of the extracellular ATP-P2Y2R signalling represents a potential therapeutic approach against alcoholic liver disease, and CD39 is a potential therapeutic target.

Purinergic P2X7 receptor blockade mitigates alcohol-induced steatohepatitis and intestinal injury by regulating MEK1/2-ERK1/2 signaling and egr-1 activity.

The P2X7 receptor is an ATP-binding cation channel involved in a broad range of inflammatory diseases. However, little is known about the potential role of P2X7R in alcohol-induced steatohepatitis and intestinal injury. In our study, C57BL/6 mice were intraperitoneally injected with P2X7R antagonists Brilliant Blue G and A438079 from the 4th day to the 10th day during the induction of chronic plus binge alcohol feeding model. Our results showed that alcohol feeding induced significant steatohepatitis and liver injury, which were mitigated by P2X7R blockade as evidenced by decreased serum levels of ALT, AST, T-CHO and TG, reduced lipid accumulation, and less inflammation. The increased intestinal inflammatory cytokines production and the prominent intestinal barrier disruption caused by alcohol were also modulated by P2X7R antagonism. Interestingly, alcohol feeding increased the relative abundance of phylum Bacteroidetes while decreased the number of phylum Verrucomicrobia and genus Akkermansia in the cecal content, which were reversed by P2X7R antagonist. Importantly, the improvement of intestinal barrier function and the restoration of partial taxonomic alterations in the gut microbiota might contribute to protect the liver from gut microbiota dysbiosis-induced second hit. Furthermore, P2X7R blockade inhibited MEK1/2-ERK1/2 phosphorylation and egr-1 expression in both liver and intestine from alcohol-fed mice. Collectively, P2X7R blockade mitigates alcohol-induced steatohepatitis and intestinal injury by inhibiting MEK1/2-ERK1/2 signaling and egr-1 expression. These studies strongly suggest that P2X7R blockade may be a promising therapeutic approach for treating alcoholic liver disease.