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Purine signaling pathway plays an important role in inflammation and tissue damage. To investigate the role of purine signaling pathway in acute alcoholic liver injury and chronic alcoholic liver fibrosis, we replicated two animal models and two cellular models. We found that body weights, liver indexes, serum biochemical parameters, serum fibrosis indexes, and pathological and immunohistochemical results had significant changes in two treatment groups compared with two control groups. In addition, gene expressions of purine receptors, inflammatory cytokines, fibrogenic cytokines, and inflammasomes increased obviously in two animal models and two cellular models. Furthermore, purine receptor inhibitors could significantly inhibit protein expressions of purine receptors and reduce protein expressions of inflammatory cytokines, fibrogenic cytokines, and inflammasomes. Besides, P2X7R small interfering ribonucleic acid (siRNA) had the same effects. Meanwhile, we detected protein expressions of inflammatory cytokines secreted by inflammasomes, and we found that purine receptor-mediated inflammasomes activation was a key event in the process of chronic alcoholic liver fibrosis. In summary, this study shows that inhibition of purine receptors can alleviate acute alcoholic liver injury and chronic alcoholic liver fibrosis in mice. Therefore, purine receptor is a potential new target for the treatment of acute alcoholic liver injury and chronic alcoholic fibrosis.

Alcoholic liver disease is one of the most common chronic liver diseases in the world. It is a liver disease caused by prolonged heavy drinking and its main clinical features are nausea, vomiting, enlargement of the liver, and jaundice. Recent studies suggest that Kupffer cell-mediated inflammatory response is a core driver in the development of alcoholic steatohepatitis and alcoholic liver fibrosis. As a danger signal, extracellular ATP activates the assembly of NLPR3 inflammasome by acting on purine P2X7 receptor, the activated NLRP3 inflammasome prompts ASC to cleave pro-cCaspase-1 into active caspase-1in KCs. Active caspase-1 promotes the conversion of pro-IL-1β to IL-1β, which further enhances the inflammatory response. Here, we briefly review the role of the P2X7R-NLRP3 inflammasome axis in the pathogenesis of alcoholic liver disease and the evolution of alcoholic steatohepatitis and alcoholic liver fibrosis. Regulation of the inflammasome axis of P2X7R-NLRP3 may be a new approach for the treatment of alcoholic liver disease.

Zhen-Ni Liu,1– 3 Xue Wu,1– 3 Qian Fang,1– 3 Zi-Xuan Li,1– 3 Guo-Qing Xia,1– 3 Jun-Nan Cai,1– 3 Xiong-Wen Lv1– 3 1Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, People’s Republic of China; 2The Key Laboratory of Anti-Inflammatory and Immune Medicines, Ministry of Education, Hefei, People’s Republic of China; 3Institute for Liver Diseases of Anhui Medical University, Hefei, People’s Republic of ChinaCorrespondence: Xiong-Wen LvSchool of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, Anhui Province, 230032, People’s Republic of ChinaEmail lyuxw@ahmu.edu.cnBackground: 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 real‑time 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.Keywords: CD73, alcohol-induced liver injury and inflammation, TLR4/MyD88/NF-κB signaling pathway, apoptosis, RAW264.7 cells

Alcoholic liver fibrosis（ALF）, as a liver disease caused by long-term alcoholism, attracts international attention. Activation of hepatic stellate cells is a key step in the development of alcoholic-associated liver fibrosis. Increasing studies have shown that P2X4 receptor, as a component of purinoceptor family in adenosine pathway, plays an important role in numerous liver diseases. In this study, it was found that the expression of P2X4 receptor was significantly increased in the mouse liver fibrosis model fed with ethanol plus CCL4 and in the HSC-T6 cell model stimulated by acetaldehyde. In vivo, C57BL/6J mice were used to establish ALF models, and 5-BDBD, a specific inhibitor of P2X4 receptor, was injected intraperitoneally at 6-8 weeks of ALF development. The results indicated that 5-BDBD could reduce the expression of fibrotic markers and attenuate the pathological features of fibrosis, thus demonstrating the alleviation of ALF.In vitro, PI3K/AKT pathway was activated in HSC-T6 cells stimulated by acetaldehyde. Silencing P2X4 receptor or administration of 5-BDBD could inhibit the phosphorylation of PI3K and AKT, thereby inhibiting the activation of HSC-T6 cells. In addition, 5-BDBD was administered to RAW264.7 cells activated by acetaldehyde, and then part of the supernatant was added to HSC-T6 cells culture medium. The results showed that 5-BDBD could reduce the expression of classical inflammatory pathways such as TGF-β pathway in RAW267.4 cells, thus inhibiting the activation of HSC-T6 cells. Taken together, these results suggest that P2X4 receptors may influence the progression of alcohol-related liver fibrosis by directly mediating the PI3K/AKT pathway, or indirectly by influencing RAW264.7 cells to regulate hepatic stellate cell activation.

Zhen-Ni Liu,1– 3,* Qian-Qian Su,4,* Yu-Hui Wang,1– 3,* Xue Wu,1– 3 Xiong-Wen Lv1– 3 1Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, People’s Republic of China; 2The Key Laboratory of Anti-Inflammatory and Immune Medicines, Ministry of Education, Hefei, People’s Republic of China; 3Institute for Liver Diseases of Anhui Medical University, Hefei, People’s Republic of China; 4Department of Pharmacy, the First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiong-Wen Lv, School of Pharmacy, Anhui Medical University, 81 Mei Shan Road, Hefei, Anhui Province, 230032, People’s Republic of China, Email xiongwen_lv2019@163.comPurpose: 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-1β 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-1β 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.Keywords: P2Y2 receptor, alcoholic liver inflammation, EGFR, ERK1/2

Alcoholic hepatitis is a serious form of liver damage. Inflammation is a key factor in alcoholic hepatitis and plays a key role in the progression of alcoholic liver disease. Adenosine receptor A2B (A2BAR) is a member of the adenosine receptor family and generally considered to be a negative regulator of the inflammatory response. We found that A2BAR was the most highly expressed adenosine receptor in ETOH-fed mouse liver tissue and was also highly expressed in primary Kupffer cells and ETOH-induced RAW264.7 cells. In addition, injection of BAY 60-6583 stimulated A2BAR, induced upregulation of the expression levels of cAMP, and reduced ETOH-induced steatosis and inflammation in mice. At the same time, knockdown of A2BAR in vitro increased the inflammatory response in RAW264.7 cells triggered by ETOH. After knockdown of A2BAR in vitro, the release of the inflammatory cytokines IL-6, IL-1β and TNF-α was increased. After overexpression of A2BAR in vitro, the cAMP level was significantly increased, PKA expression was increased, the expression of phosphorylated proteins in the NF-kB signal transduction pathway was significantly affected, and the expression of the key phosphorylated protein p-P65 was decreased. However, after the simultaneous overexpression of A2BAR and inhibition of PKA, the expression of the key phosphorylated protein p-P65 was still significantly decreased. In addition, after the expression of A2BAR increased or decreased in RAW264.7 cells, AML-12 cells were cultured in the supernatant of RAW264.7 cells stimulated by ETOH, and the apoptosis rate was significantly changed by flow cytometry. These results suggest that A2BAR can reduce alcoholic steatohepatitis by upregulating cAMP levels and negatively regulating the NF-kB pathway. Overall, these findings suggest the significance of A2BAR-mediated inflammation in alcoholic liver disease.

Alcoholic liver disease caused by chronic excessive drinking has become one of the most common types of liver disease. Alcohol-induced inflammatory immune responses play a central role in the development of alcohol-associated steatohepatitis. The content and expression of ATP and P2X4 in the livers of alcoholic steatohepatitis mice are significantly increased. The content of ATP increased by 20 percent and the expression of P2X4 receptor protein was 1.3 times higher than that in the livers of normal mice. Treatment with 5-BDBD, a P2X4 receptor-specific inhibitor, significantly reduced alcohol-induced liver inflammation and lipid deposition. In RAW264.7 cell experiments, 5-BDBD inhibited the expression of P2X4 and alleviated alcohol-induced inflammation, while the CD39-specific inhibitor POM-1 reduced extracellular ATP degradation and promoted the expression of P2X4, thereby exacerbating inflammation. After treatment with 5-BDBD, P2X4 receptor protein expression decreased by 0.2 times and after treatment with POM-1, P2X4 receptor protein expression increased by 0.1 times compared to the alcohol-stimulated group. In addition, inhibition of P2X4 expression in RAW264.7 cells reduced calcium influx in RAW264.7 cells. P2X4 may induce the activation of NLRP3 inflammasomes by mediating calcium influx, thus exacerbating the inflammatory response, and inhibition of P2X4 expression can effectively block this process. Conclusion: These results suggest that the ATP-P2X4 signaling pathway promotes the inflammatory response in alcoholic steatohepatitis and that CD39 may play a protective role in regulating P2X4 expression by hydrolyzing ATP. In conclusion, the CD39 and ATP-P2X4 signaling pathways may be potential therapeutic targets for alcoholic steatohepatitis.

Introduction This study aimed to determine the prices, availability, and affordability of national essential medicines in public primary hospitals in poverty-stricken areas of Anhui province, China. Methods A cross-sectional study was conducted in 143 public primary hospitals in Anhui province, eastern China. Data on access to 44 essential medicines was evaluated using the standardized methodology available in the World Health Organization and Health Action International manual. Results Median price rates show that 46.51% (21 of 44) of the lowest price generics and 100% of the originator brands were more expensive than the international reference price. The median availability of the 44 medicines was 31.47%, and 65.91% (29 of 44) of the medicines had less than 50% availability. The majority of the medicines were affordable as they would cost less than a day's income in sample areas. Suppliers could respond to 88.27% of the procuring orders raised by the 143 hospitals in the study, but this ranged from 43.96% to 99.86%. Conclusions There is poor availability and non-ideal response rate of medicine delivery in public primary hospitals in poverty-stricken areas in eastern China. Further implementation of national essential medicine policy needs to focus on improving both availability and distribution efficiency in these areas.

The incidence and mortality of malignant tumors are on the rise, which has become the second leading cause of death in the world. At present, anti-tumor drugs are one of the most common methods for treating cancer. In recent years, with the in-depth study of tumor biology and related disciplines, it has been gradually discovered that the essence of cell carcinogenesis is the infinite proliferation of cells caused by the disorder of cell signal transduction pathways, followed by a major shift in the concept of anti-tumor drugs research and development. The focus of research and development is shifting from traditional cytotoxic drugs to a new generation of anti-tumor drugs targeted at abnormal signaling system targets in tumor cells. In this review, we summarize the targets of anti-tumor drugs and analyse the molecular mechanisms of their effects, which lay a foundation for subsequent treatment, research and development.

AIMS In acute alcoholic liver injury, alcohol can directly or indirectly induce endoplasmic reticulum stress (ERS) to participate in liver injury, and it is found that the expression of serum exosomal miR-122 is significantly affected. Therefore, the present study investigated the effects of endoplasmic reticulum stress inhibition on the expression of serum exosomal miR-122 and acute liver injury. METHODS The acute alcoholic liver injury models were established by the intragastric administration of ethanol (5 g/kg) in ICR mice. Intervention group received 4-phenylbutyric acid (PBA, endoplasmic reticulum stress inhibitor; 75 mg/kg and 150 mg/kg, intraperitoneal) 12 and 24 hours before intragastric administration. Mice treated with saline were used as controls. RESULTS The ethanol treated mice exhibited significantly elevated hepatosomatic index (liver weight/body weight) and alanine aminotransferase (ALT), compared with those in the control group (P < 0.05). The ERS inhibitor 4-phenylbutyric acid protected against ethanol induced acute liver injury and hepatocyte necrosis, and PBA 150 mg/kg significantly attenuated ethanol induced hepatic ER stress-related proteins (GRP78, pIRE1α and pIF2α) (P < 0.05). Moreover, PBA 150 mg/kg markedly alleviated ethanol induced elevation of hepatic and serum exosomal miR-122 and pri-miR-122 (P < 0.05). CONCLUSIONS These findings suggest that ER stress inhibitor PBA attenuated ethanol induced acute liver injury and serum exosomal miR-122, and provides a potential therapy strategy for acute alcoholic liver injury.

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.

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, β-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/β-catenin pathway was activated in acetaldehyde-activated HSC-T6 cells and CD73 silencing or overexpression could regulate Wnt/β-catenin signaling pathway. Collectively, our study unveils the role of CD73 in HSCs activation, and Wnt/β-catenin signaling pathway might be involved in this progression.

Extracellular adenosine triphosphate (ATP) is a danger signal released by dying and damaged cells, and it functions as an immunostimulatory signal that promotes inflammation. The ectonucleotidases CD39/ectonucleoside triphosphate diphosphohydrolase-1 and CD73/ecto-5'-nucleotidase are cell-surface enzymes that breakdown extracellular ATP into adenosine. This drives a shift from an ATP-driven proinflammatory environment to an anti-inflammatory milieu induced by adenosine. The CD39-CD73-adenosine pathway changes dynamically with the pathophysiological context in which it is embedded. Accumulating evidence suggests that CD39 and CD73 play important roles in liver disease as critical components of the extracellular adenosinergic pathway. Recent studies have shown that the modification of the CD39-CD73-adenosine pathway alters the liver's response to injury. Moreover, adenosine exerts different effects on the pathophysiology of the liver through different receptors. In this review, we aim to describe the role of the CD39-CD73-adenosine pathway and adenosine receptors in liver disease, highlighting potential therapeutic targets in this pathway, which will facilitate the development of therapeutic strategies for the treatment of liver disease.

Activated hepatic stellate cells (HSCs) are the major cell type involved in the deposition of extracellular matrix (ECM) during the development of hepatic fibrosis. In this study, we revealed that left-right determination factor 2 (LEFTY2), one of the proteins belonging to the transforming growth factor-β (TGF-β) protein superfamily, was remarkedly decreased in human hepatic fibrosis tissues and in a carbon tetrachloride (CCl4)-induced liver fibrosis mouse model. In addition, TGF-β1 treatment markedly reduced the level of LEFTY2 in HSCs. Importantly, overexpression of LEFTY2 suppressed the activation and proliferation of HSCs. LEFTY2 inhibited the expression of TGF-β1-induced fibrosis-associated genes (α-SMA and COL1a1) in human (LX-2) and rat (HSC-T6) HSC cell lines in vitro. Mechanistically, we demonstrated, for the first time, the role of LEFTY2 in inhibiting TGF-β1/Smad3 signaling, suggesting that there is a mutual antagonism between LEFTY2 and TGF-β1/Smad3 signaling during liver fibrosis. Similarly, we observed that LEFTY2 has a negative effect on its downstream genes, including c-MYC, CDK4, and cyclin D1, in liver fibrosis. Collectively, our data strongly indicated that LEFTY2 plays an important role in controlling the proliferation and activation of HSCs in the progression of liver fibrosis and this could be a potential therapeutic target for its treatment.

Acute lung injury is an acute inflammatory disease with high morbidity rate and high mortality rate. However, there is still no effective clinical treatment to date. Our previous studies found that NLRC5 was significantly increased in acute liver injury model induced by LPS to reduce the secretion of IL-6 and TNF-α. Nevertheless, there is no report on the role of NLRC5 in regulating the development of acute lung injury. In this study we successfully established a model of acute lung injury induced by tracheal instillation of LPS in mice, and found NLRC5 expression was apparently elevated in mouse lung tissue and primary alveolar macrophages. NLRC5 overexpression negatively regulated secretion of inflammatory cytokines in murine alveolar macrophage cells through NF-κB and p38 MAPK pathway inhibition. There is a positively feedback between NLRC5 and NF-κB or p38 MAPK pathway. This study may provide some new ideas for clinical prevention of lung injury.

Macrophages play a crucial role in the progression of hepatic fibrosis (HF). In macrophages, epigenetic mechanisms are increasingly being recognized as crucial controllers of their phenotype. However, the functions of macrophage DNA methylation in experimental models of hepatic fibrosis have not been fully addressed. Here, we analyzed isolated hepatic macrophages DNA methylation from CCL4-induced (4 weeks) mice using reduced representation bisulfite sequencing (RRBS). We identified and validated the methylation status of 26 gene promoter regions associated with CpG islands. We further investigated the function of PSTPIP2 in HF by hepatic-adeno-associated virus (AAV9)-PSTPIP2 overexpression. The molecular mechanisms underlying PSTPIPS2-regulated HF were further explored in mice and RAW264.7 cell line. RRBS results show hypermethylation of PSTPIP2 (chr18: 77,843,840-77,843,968) in the 5'-UTR region. PSTPIP2 expression was significantly decreased in isolated hepatic macrophages from CCL4-induced mice. PSTPIP2 hypermethylation is mediated by the methyltransferases DNMT3a and DNMT3b in LPS-induced RAW264.7 cell line. Further investigation indicated that specific overexpression of PSTPIP2 in C57BL/6 mice reduced the inflammatory response and ameliorated liver fibrosis. These data indicated that hypermethylation of PSTPIP2 caused a mixed induction of hepatic classical macrophage (M1) and alternative macrophage (M2) biomarkers in CCL4-induced HF mice. Furthermore, overexpression of PSTPIP2 inhibited the expression of M1 markers by suppressing STAT1 activity, and enhanced the expression of M2 markers by promoting STAT6 activity. In contrast, knockdown of PSTPIP2 promoted M1 polarization and suppressed M2 polarization in vitro. Adding PSTPIP2 expression alleviates liver fibrosis and hepatic inflammation in mice by regulating macrophage polarization.

Acute kidney injury (AKI), characterized by aggressive inflammatory responses and destruction of renal resident cells, can cause abrupt kidney dysfunction. To date, effective therapy for AKI is lacking. In this study, we evaluated the renoprotective effect of wogonin, an herbal active compound, using a cisplatin-induced AKI mouse model. In vivo results show that wogonin substantially suppressed the increased levels of serum creatinine and blood urea nitrogen (BUN) almost to the normal level. Wogonin also attenuated tubular damage, shown by PAS staining, electron microscopy and molecular analysis of KIM-1. In addition, wogonin suppressed kidney inflammation as indicated by a >60% decrease in macrophage infiltration, a >50% reduction in inflammatory cytokine production and inhibited NF-κB activation in the injured kidney. Mechanistically, molecular docking results show that wogonin effectively inhibited RIPK1 by occupying the ATP-binding pocket of the enzyme, which is a key regulator of necroptosis. Moreover, inhibition of RIPK1, or RIPK3, reversed the protective effects of wogonin in cisplatin-treated HK2 cells, indicating wogonin works in a RIPK1/RIPK3-dependent manner. Surprisingly, wogonin enhanced the anti-proliferative effect of cisplatin on human hepatoma HepG2 cells. Thus, our findings suggest wogonin may be a renoprotective adjuvant for cisplatin-based anticancer therapy.

The goal of this study was to elucidate the functional role of Nox4 during acute kidney injury (AKI). NADPH oxidases are a major source of reactive oxygen species (ROS) in the kidney in normal and pathological conditions. Among NADPH oxidase isoforms, NADPH oxidase4 (Nox4) is highly expressed in the kidney and has an important role in kidney diseases, such as diabetic nephropathy and renal carcinoma. We previously found that Nox4 expression significantly increased in the toxic AKI model. However, its functional role and mechanism of action in AKI are still unknown. We scavenged ROS with apocynin in vitro and in vivo and found it attenuated cisplatin-triggered renal function decline. It also alleviated programmed cell death and renal inflammation, indicating a critical role for ROS in mediating AKI. Nox4 protein and mRNA levels were substantially upregulated by cisplatin in vivo and in vitro. Nox4 knockdown alleviated cisplatin-induced cell death and inflammatory response, while Nox4 overexpression aggravated them. Moreover, N-acetyl-L-cysteine (NAC)-mediated inhibition of ROS suppressed cell injury led by Nox4 overexpression, indicating Nox4-mediated ROS generation may be the key mediator in cisplatin-induced nephrotoxicity. Mechanistically, excessive expression of Nox4 induced programmed cell death, especially RIP-mediated necroptosis. Finally, we tested whether Nox4 is a potential therapeutic target using an AKI mouse model by injecting a lentivirus-packaged Nox4 shRNA plasmid through tail vein. Disruption of Nox4 led to renal function recovery, kidney damage relief and reduced inflammation. We conclude that Nox4 aggravates cisplatin-induced nephrotoxicity by promoting ROS-mediated programmed cell death and inflammation. Thus Nox4 may serve as a potential therapeutic target in the treatment of AKI.

CD39 is associated with diverse physiological and pathological processes, including cell proliferation and differentiation. Adenosine triphosphate (ATP) is hydrolysed to adenosine by different enzymes including ecto-nucleoside triphosphate diphosphohydrolase-1/ENTPD1 (CD39) and ecto-5'-nucleotidase (CD73), regulating many physiological and pathological processes in various diseases, but these changes and functions in alcoholic liver disease are generally unknown. In this study, an alcoholic liver disease model in vivo was induced by ethanol plus carbon tetrachloride(CCl4) administered to C57BL/6 mice, who were the intraperitoneally injected with the CD39 inhibitor sodium polyoxotungstate (POM1) or colchicine from the 5th week to the 8th week. Meanwhile, hepatic stellate cells were stimulated by acetaldehyde to replicate alcoholic liver fibrosis models in vitro. Exogenous ATP and POM1 were added in turn to the culture system. Pharmacological blockade of CD39 largely prevents liver damage and collagen deposition. We found that blockade or silencing of CD39 prevented acetaldehyde-induced proliferation of HSC-T6 cells and the expression of fibrogenic factors. Moreover, blockade or silencing of CD39 could block the activation of the adenosine A2A and adenosine A2B receptors and the TGF-β/Smad3 pathway, which are essential events in HSC activation. Thus, blockade of CD39 to inhibit the transduction of ATP to adenosine may prevent HSC activation, alleviating alcoholic hepatic fibrosis. The findings from this study suggest ATP-adenosine signalling is a novel therapeutic and preventive target for alcoholic liver disease.

Objective Challenges persist in the reconstruction of the ankle and the foot with exposed tendons, joints, and bones as a result of severe burns and trauma. In flap elevation involving the sensitive superficial nerve, the local nerve was always sacrificed to obtain an anesthetic donor site; however, such a procedure introduced the possibility of painful neuromas. In this study, we present a desired clinical application of a modified reversed superficial peroneal artery flap, in which the superficial peroneal nerve is preserved. Methods From 2008 to 2015, 12 patients with ankle or foot defects were treated with the modified reversed superficial peroneal artery flap. The defects of the patients were caused by hot liquid scald (one patient), electrical injury (five patients), and trauma (six patients). The flap was utilized for covering defects on the ankle (seven patients) and the foot (five patients). The size of the flaps ranged from 4.0 cm × 6.0 cm to 18.0 cm × 10.0 cm. The superficial peroneal artery was involved in the flap, whereas the superficial peroneal nerve was spared by dedicate dissection. The reverse-flow flap was nourished by the superficial peroneal artery through the terminal peroneal artery perforator. Results The obtained outcomes were satisfactory functionally and aesthetically. The flaps in 11 patients survived completely without complications, whereas partial necrosis occurred in a 78-year-old patient when the flap survived a week later during follow up. CT angiography revealed the stenosis of the popliteal artery. The wound healed after interventional treatment involving placing a stent and changing the dressings. Basic functions and configurations were salvaged in all cases. All patients were completely satisfied with the proposed flap and suffered no paresthesia in their lower leg. Conclusion Exhibiting beneficial characteristics such as reliable blood supply, favorable thickness, wide rotating arc, and retention of major vessels and the superficial peroneal nerve, the modified reversed superficial peroneal artery flap is useful in the reconstruction of ankle and foot defects that would not cause any hypoesthesia of the foot.

Long non-coding RNAs (lncRNAs) are increasingly recognized as major players in regulating various biological processes. LncRNA HOX transcript antisense RNA (Hotair) has been extensively studied in cancer. However, the role of Hotair in liver fibrosis remains unknown. Here we observed that Hotair expression was significantly increased in CCl4-induced mouse liver fibrosis models, human fibrotic livers and activated hepatic stellate cells (HSCs) by TGF-β1 stimulation. Enforced expression of Hotair in LX-2 cells promoted cell proliferation and activation while inhibition of its expression had an opposite effect. Furthermore, we found that Hotair may act as an endogenous 'sponge' of miR-148b, which regulates expression of the DNMT1/MEG3/p53 pathways in HSCs. Intriguingly, Hotair enhanced polycomb repressive complex 2 (PRC2) occupancy and histone H3K27me3 repressive marks, specifically at the MEG3 promoter region. Finally, we found that Hotair forms an RNA/DNA hybrid and recruits PRC2 to MEG3 promoter. These data suggest that Hotair inhibition may represent a promising therapeutic option for suppressing liver fibrosis.

Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately.

Background: Activation of macrophages and infiltration are key events in acute liver injury (ALI). Kv1.3 plays an important role in regulating immunologic functions of macrophages and is extensively recognized as a potential ion channel for immunological diseases. Objective: We hypothesized that blockage of Kv1.3 may influence ALI by inhibiting macrophages infiltration in damaged liver tissues. Methods: Margatoxin was administered into the peritoneal cavity of ALI mice. The impact of this treatment on ALI and macrophage migration in vivo and in vitro was determined using immunohistochemistry, transwell migration, and wound healing assays. Results: MgTX treatment alleviated ALI in mice, as evidenced by reduced macrophage infiltration in liver tissues and lower serum levels of liver ALT and AST. RNA-seq profiling analysis showed that the most obvious change by MgTX treatment was downregulation of δ-catenin, a protein known to be associated with macrophage migration. The effect of MgTX on macrophage migration and involvement of δ-catenin was confirmed by transwell and wound healing assays. Overexpression of δ-catenin in RAW264.7 cells promoted migration, an event that was suppressed upon silencing of δ-catenin. Mechanistically, the expression of RhoA was regulated by the overexpression or knockdown of δ-catenin. Conclusion: These findings suggest a role for blockage of Kv1.3 channel in macrophage migration and reveal a new target in the treatment of ALI.

Hepatic fibrosis is the most common pathological feature of most chronic liver diseases, and its continuous deterioration gradually develops into liver cirrhosis and eventually leads to liver cancer. At present, there are many kinds of drugs used to treat liver fibrosis. However, Western drugs tend to only target single genes/proteins and induce many adverse reactions. Most of the mechanisms and active ingredients of traditional Chinese medicine (TCM) are not clear, and there is a lack of unified diagnosis and treatment standards. Natural products, which are characterized by structural diversity, low toxicity, and origination from a wide range of sources, have unique advantages and great potential in anti-liver fibrosis. This article summarizes the work done over the previous decade, on the active ingredients in natural products that are reported to have anti-hepatic fibrosis effects. The effective anti-hepatic fibrosis ingredients identified can be generally divided into flavonoids, saponins, polysaccharides and alkaloids. Mechanisms of anti-liver fibrosis include inhibition of liver inflammation, anti-lipid peroxidation injury, inhibition of the activation and proliferation of hepatic stellate cells (HSCs), modulation of the synthesis and secretion of pro-fibrosis factors, and regulation of the synthesis and degradation of the extracellular matrix (ECM). This review provides suggestions for the development of anti-hepatic fibrosis drugs.

There are multiple causes of liver fibrosis, common ones include ethanol, toxins, and cholestasis. However, whether these different etiologies lead to the same pathological outcomes contain common genetic targets or signaling pathways, the current research has not attracted widespread attention. GSE40041 and GSE55747 were downloaded from the Gene Expression Omnibus (GEO) database. GSE40041 and GSE55747 represent the differential expression profiles in the liver of mice with bile duct ligation (BDL) and carbon tetrachloride (CCl4) induced liver fibrosis models, respectively. By using GEO2R, 701 differential expression genes (DEGs) in GSE40041 and 6540 DEGs in GSE55747 were identified. 260 co-DEGs were shared and extracted for gene ontology (GO) analysis. Through GO analysis, it was found that the regulation of cell migration in biological processes (BPs) was closely related to the pathogenesis of liver fibrosis, and the genes involved in this process include a key gene, chemokine (C-X-C motif) ligand 14 (CXCL14). Subsequently, further bioinformatic analysis showed that CXCL14 may be regulated by miR-122 to participate in the progression of liver fibrosis. Then real-time PCR and western blotting were performed to validate the expression of CXCL14 in liver tissue after liver fibrosis caused by different etiologies (ethanol, CCl4). The expression of CXCL4 in liver fibrosis induced by BDL was verified in another GEO dataset. Basically consistent with our bioinformatics results, our experimental results showed that the expression of CXCL14 was most significantly increased in alcoholic liver fibrosis model, followed by CCl4-induced liver fibrosis, which was also significantly increased in the BDL-induced model. Thus, CXCL14 can act as a common potential genetic target for different liver fibrosis diseases.

Hepatic stellate cells (HSCs) activation represents an essential event during alcoholic liver fibrosis (ALF). Previous studies have demonstrated that the rat HSCs could be significantly activated after exposure to 200 μmol/L acetaldehyde for 48 h, and the cAMP/PKA signaling pathways were also dramatically upregulated in activated HSCs isolated from alcoholic fibrotic rat liver. Exchange protein activated by cAMP (EPAC) is a family of guanine nucleotide exchange factors (GEFs) for the small Ras-like GTPases Rap, and is being considered as a vital mediator of cAMP signaling in parallel with the principal cAMP target protein kinase A (PKA). Our data showed that both cAMP/PKA and cAMP/EPAC signaling pathways were involved in acetaldehyde-induced HSCs. Acetaldehyde could reduce the expression of EPAC1 while enhancing the expression of EPAC2. The cAMP analog Me-cAMP, which stimulates the EPAC/Rap1 pathway, could significantly decrease the proliferation and collagen synthesis of acetaldehyde-induced HSCs. Furthermore, depletion of EPAC2, but not EPAC1, prevented the activation of HSC measured as the production of α-SMA and collagen type I and III, indicating that EPAC1 appears to have protective effects on acetaldehyde-induced HSCs. Curiously, activation of PKA or EPAC perhaps has opposite effects on the synthesis of collagen and α-SMA: EPAC activation by Me-cAMP increased the levels of GTP-bound (activated) Rap1 while PKA activation by Phe-cAMP had no significant effects on such binding. These results suggested that EPAC activation could inhibit the activation and proliferation of acetaldehyde-induced HSCs via Rap1.

Few studies have paid attention to the performances of non-invasive models in diagnosing stages of liver fibrosis and inflammation, which are critical for early and accurate assessment of prognostication and decisions on antiviral treatment in chronic hepatitis B infection patients with high hepatitis B virus DNA and normal or mildly elevated alanine transaminase levels (≤2 times upper limit of normal (ULN)). This study aimed to investigate the value of routine serum markers in evaluation of liver inflammation and fibrosis in these patients. A total of 370 consecutive chronic hepatitis B virus-infected patients who underwent liver biopsy were retrospectively analyzed. The Scheuer scoring system was adopted as the pathological standard for diagnosing liver inflammation and fibrosis. The receiver-operating characteristic curves (ROC) and the area under the ROC curves (AUROCs) were used to analyze the performances of the models, including aspartate transaminase to platelet ratio index (APRI), fibrosis index based on the 4 factors (FIB-4), red cell volume distribution width-to-platelet ratio (RPR), globulin-platelet model (GP), and gamma-glutamyl transpeptidase to platelet ratio index (GPR). To predict significant inflammation (G ≥2), the AUROC of APRI was higher than that of FIB-4 (0.705 vs 0.629, P = .001), RPR (0.705 vs 0.593, P

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-1β (IL-1β), 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.

The present study was undertaken to investigate the mechanism by which adenosine receptors (ARs)-mediated the cAMP/PKA/CREB signal pathway regulates the activation of acetaldehyde-induced hepatic stellate cells (HSCs). Primary HSCs were isolated from SD rats, cultured in vitro, and activated with different concentrations of acetaldehyde at different time points. Quantitative real-time PCR and Western blotting were used to quantify both protein and mRNA levels of the four AR (A1R, A2AR, A2BR, and A3R) in rat HSCs. Selective inhibitors of PDEs and the Gi/o protein pathway, general AR agonists, and AR subtype specific agents were used to study the AR signaling. The level of cAMP was measured by radio-immunoassay, and the expression of α-SMA, collagen type I and III, PKA and p-CREB were also detected by Western blotting. Acetaldehyde could significantly promote HSC proliferation, with a maximum stimulatory effect observed at 48 h after exposure to 200 μM acetaldehyde. All four AR subtypes could be present in rat HSCs, and the mRNA and protein expression levels for A2AR and A1R in much greater abundance than those for A2BR and A3R. The expression of A2AR and A1R was significantly increased in acetaldehyde-induced HSCs as compared with that of control group, whereas the expression of A2BR and A3R remained unaffected by the addition of acetaldehyde. Curiously, there is coupling of A2AR to the Gs-AC signaling, as well as coupling of A1R to the Gi/o-AC signaling pathway in acetaldehyde-induced HSCs. Both the A2AR and A1R antagonists could suppress the activation of HSC, although they have opposing effects on cAMP signal transduction. These results suggested that a combination of cAMP/PKA/CREB signals via A2AR and A1R likely mediate the activation of acetaldehyde-induced HSCs, and A1R coupled to the Gi/o-AC signaling pathway may be masked by the more predominant A2AR that coupled to the Gs-AC signaling pathway.

Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in alveolar macrophages (AMs), activates several intracellular signaling pathways, including mitogen-activated protein kinases (MAPK). In the present study, we investigated the MAPK pathway in AMs of chronic bronchitis (CB) rats. CB was induced by endotracheal instillation of LPS followed by Bacillus Calmette Guerin injection through the caudal vein 1 week later. Specific inhibitors were used and protein phosphorylations were detected by Western blot. We found that Genistein (PTK inhibitor) could inhibit protein kinase C (PKC), phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt or PKB) MAPK signaling pathway with different degrees, LY294002 (PI3K inhibitor) could not only inhibit phospho-PI3K/Akt expression, but also inhibit p38 and c-Jun NH2-terminal kinases (JNK) phosphorylation. Calphostin C (PKC inhibitor) could inhibit phospho-PKC expression and exerted significant effects on extracellular signal-regulated kinases (ERK) phosphorylation, however, it had no impact on p38 and JNK phosphorylation. These results demonstrated that the LPS mediated signaling pathway of MAPK in AMs of CB rats could be described as follows: PTK-PI3K-Akt-JNK/p38 or PTK-PI3K-PKC-ERK, and PI3K may have a negative regulation on the activation of downstream proteins.

Objective A systematic review and meta-analysis of randomized controlled trials (RCTs) were performed to understand the effectiveness of medication adherence (MA) interventions among Chinese patients with hypertension. Methods A literature search was conducted with three English databases (PubMed, Web of Science and Embase) and three Chinese databases (China National Knowledge Infrastructure, Wanfang and VIP Database for Chinese Technical Periodicals) for the period from 1970 to October 2017. Only both RCTs with a minimum of 10 participants in each intervention group and Chinese patients with hypertension as participants were included. A random-effects model was applied to calculate pooled effect sizes with 95% CI. Subgroup analysis was conducted to identify potential sources of heterogeneity from duration of intervention, type of intervener, methods of intervention and sites of intervention. Funnel plots and Egger's test were used to evaluate for publication bias. Key findings A total of 48 studies met criteria for the meta-analysis, including 14 568 participants, testing 57 independent comparisons. Overall, the effect size revealed that interventions significantly improved MA (pooled relative risk = 1.59, 95% CI: 1.43 to 1.78; pooled Cohen's d = 1.42, 95% CI: 0.976 to 1.876). Interventions were found to significantly reduce blood pressure (BP) (systolic BP: Cohen's d = −0.85, 95% CI: −1.11 to −0.60 and diastolic BP: Cohen's d = −0.73, 95% CI: −1.00 to −0.46). Longer duration of intervention gave better effectiveness. Physician as interventionist, regular follow-up visits and interventions conducted at a hospital were associated with better effectiveness. Conclusion Adherence interventions improve MA and reduce uncontrolled BP among Chinese patients with hypertension. In the future, investigators should adopt a skill set to address the problem of poor MA.

Liver disease is a primary cause of liver-related morbidity and mortality worldwide. Currently, histological examination is the gold standard for diagnosis and classification of liver disease; however, due to its several drawbacks, including the risk of complications and sampling variability, noninvasive diagnostic options are favorable. Exosomal miRNAs have recently been considered as an important source of medical biomarkers due to being widely distributed in body fluids. This review summarizes existing concepts related to the origin, mode of transportation and possible functions of exosomal miRNAs, and recent findings on the role of exosomal miRNAs in liver diseases and development of exosomal miRNA-based diagnostic biomarkers in the primary forms of liver diseases.

Long noncoding RNAs (lncRNAs) are being increasingly recognized as major players in governing fundamental biological processes through diverse mechanisms. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a lncRNA correlated with several human cancers. Recently, the methylation-dependent downregulation of MEG3 has been described in liver cancers. However, its biological functional role in liver fibrosis remains unknown. In our study, MEG3 levels were remarkably decreased in CCl4-induced mouse liver fibrosis models and human fibrotic livers as demonstrated by real-time quantitative PCR. Moreover, the expression of MEG3 was downregulated in human hepatic stellate cell lines LX-2 cells in response to transforming growth factor-β1 (TGF-β1) stimulation in dose and time-dependent manner. Enforced expression of MEG3 in LX-2 cells inhibited TGF-β1-induced cell proliferation, while promoting cell apoptosis. In addition, hypermethylation of MEG3 promoter was identified by methylation-specific PCR and MEG3 expression was robustly increased by the inhibition of methylation with either 5-aza-2-deoxycytidine (5-azadC), or siRNA to DNA methyltransferase 1 (DNMT1) in TGF-β1-induced LX-2 cells. More importantly, overexpression of MEG3 could activate p53 and mediate cytochrome c release, subsequently leading to caspase-3-dependent apoptosis in TGF-β1-treated LX-2 cells. These findings suggested that MEG3 may play an important role in stellate cell activation and liver fibrosis progression and act as a novel potential therapeutic target for liver fibrosis.

Systemic lupus erythematosus (SLE) is a systemic multi-organ autoimmune disease with different immunological characteristics and clinical manifestations characterized by an autoantibody response to nuclear and cytoplasmic antigens; the etiology of this disease remains largely unknown. Most recent genome-wide association studies demonstrate that genetics significantly predispose to SLE onset, but the incomplete disease concordance rates between monozygotic twins indicates a role for other complementary factors in SLE pathogenesis. Recently, much evidence strongly supports other molecular mechanisms involved in the regulation of gene expression ultimately causing autoimmune disease, and several studies, both in clinical settings and experimental models, have demonstrated that epigenetic modifications may hold the key to a better understanding of SLE initiation and development. DNA methylation changes the structure of chromatin, being typically able to modulate the fine interactions between promoter-transcription factors and encoding genes within the transcription machinery. Alteration in DNA methylation has been confirmed as a major epigenetic mechanism that may potentially cause a breakdown of immune tolerance and perpetuation of SLE. Based on recent findings, DNA methylation treatments already being used in oncology may soon prove beneficial to patients with SLE. We herein discuss what we currently know, and what we expect in the future.

Conversion of hepatic stellate cells (HSCs) into hepatic myofibroblasts is a necessary event during the development of liver fibrosis. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of profibrotic genes, is selectively induced in myofibroblasts from diseased livers. Treatment of HSC with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-azadC), prevented TGF-β1-induced proliferation and alpha-smooth muscle actin (α-SMA) and collagen expression. 5-AzadC also rescued TGF-β1-induced suppression of Smad7 expression which occurs during HSC activation. Similarly, silencing the expression of the DNMT1 gene ameliorated the suppression of Smad7 expression by TGF-β1. In addition, DNMT1 inhibition, by 5-azadC or DNMT1 silencing, prevented the phosphorylation of Smad2 and Smad3. These studies suggest that epigenetic repression of Smad7 promotes the phosphorylation of Smad2 and Smad3 that may be an important molecular mechanism for perpetuated HSC activation and liver fibrosis.

Systemic lupus erythematosus (SLE) is a chronic, multi-system autoimmune disease characterized by disorder of the generation of auto-antibodies to components of cell nucleus, and the exact etiology of this disease is unknown. MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs about 21 nucleotides in length that play a critical role in the regulation of host genome expression at the post-transcriptional level. Emerging evidence has shown that aberrant miRNAs influence a wide range of biological processes including immune cell lineage commitment, differentiation, maturation, immune homeostasis and normal function. Moreover, miRNAs can be detected in a variety of sources, such as tissues, serum, and other body fluids. The role of miRNAs is evident in various malignant and nonmalignant diseases including cancer, inflammatory, and autoimmune diseases, and there is accumulating evidence also for an important role of miRNAs in SLE. MiRNAs can be aberrantly expressed even in the different stages of SLE, allowing miRNAs to be important biomarkers, to monitor disease activity and effects of treatment, and to help understand the pathogenesis of the disease. Thus, miRNAs are emerging as potential targets for new therapeutic strategies of SLE. During the last several years, there has been rapidly accumulating evidence of an important role of miRNAs in SLE. This review focuses on the current understanding of miRNA biogenesis, the role of miRNAs in the regulation of SLE pathogenesis, with special emphasis on new advances and the association of miRNAs with DNA methylation. The field of miRNAs in mammalian gene regulation has tremendous potential research prospect. Identification of specific miRNA expression patterns in SLE and further a comprehensive understanding of the role of miRNAs in the disease pathogenesis offer promise of not only novel clinical diagnostic markers, but also new gene therapy strategies for patients with SLE.

Inflammatory cells, macrophages induced by lipopolysaccharide (LPS) stimulation, lead to the production of inflammatory cytokines, which are crucial to host defense. MicroRNAs are short noncoding RNAs that regulate key biological processes via suppression of gene expression at posttranscriptional levels. Recently, miR-146a has been shown to be involved in the regulation of immune and inflammatory responses. However, the role of miR-146a in LPS-induced RAW264.7 macrophage cells remains unclear. In this study, we found that the expression of miR-146a was upregulated in RAW264.7 macrophage cells in response to LPS stimulation in a dose- and time-dependent manner by one-step real-time quantitative PCR. In addition, miR-146a mimics decreased, while miR-146a inhibitor increased, the expression of inflammatory cytokine interleukin-6, but did not affect tumor necrosis factor-α expression in LPS-stimulated RAW264.7 macrophage cells. Bioinformatics analyses predict that Notch1 is a potential target of miR-146a. Moreover, miR-146a overexpression in LPS-treated RAW264.7 macrophage cells did significantly decrease Notch1 mRNA and protein levels. These results suggested that miR-146a may function as a novel feedback negative regulator to LPS-induced production of inflammatory cytokines, at least in part, via inhibiting the expression of Notch1.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by the rheumatoid factor and anti-citrullinated peptide antibody (ACPA) against common autoantigens that are widely expressed within and outside the joints. Many factors participate in the pathogenesis of RA, such as cytokine imbalance, Wnt pathway activation, matrix production, and osteoprotegerin on osteoclasts. Fibroblast-like synoviocytes (FLS) activation has an important role in RA pathogenesis. The methyl-CpG-binding protein (MeCP2) which promoted repressed chromatin structure was selectively detected in synovium of diseased articular in rats. Overexpression of this protein results in an up-regulation of global methylation levels and transcriptional suppression of specific genes. There were increased MeCP2 and decreased secreted frizzled-related protein 4 (SFRP4) in synovium as well as the FLS isolated from the synovium of RA rats. Knockdown of MeCP2 using siRNA technique enhanced SFRP4 expression in both mRNA and protein levels in FLS. These results indicated that epigenetic modification was involved in differential expression of SFRP4. To further explore the underlying molecular mechanisms, we hypothesized that the SFRP4 down-regulation in synovium was caused by DNA methylation. Treatment of FLS with DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-azadC) blocked the cell proliferation and increased the SFRP4 expression. Increased SFRP4 down-regulated the key gene β-catenin, the downstream effectors gene ccnd1 and fibronectin expression in canonical Wnt pathway at the same time. MeCP2 and DNA methylation may provide molecular mechanisms for canonical Wnt pathway activation in RA. Combination of 5-azadC and MeCP2 may be a promising treatment strategy for individuals with RA in which SFRP4 is inactivated.

DNA methylation refers to a heritable alteration in the pattern of gene expression that is regulated by a mechanism specifically not owing to changes in the primary nucleotide sequence. The transcriptional silencing caused by DNA methylation affects genes involved in the main cellular pathways: cell cycle control, Ras signaling, apoptosis, and detoxification. Recent studies have shown that methylation modifications orchestrate the activation of hepatic stellate cells (HSCs) characterized by excessive accumulation of extracellular matrices (ECMs). The activation of HSCs is mediated by multiple signal transduction pathways and is generally regarded as the major ECM producer responsible for liver fibrosis. In addition, aberrant methylation of specific gene involved in the activation of multiple signal transduction pathways in liver fibrosis. The aim of this review is to compile recent information on aberrant DNA methylation in hepatic fibrosis and to highlight key genes and molecular pathways in hepatic fibrosis formation.

The activation of hepatic stellate cells (HSCs) is an essential part in the development of alcoholic liver fibrosis (ALF). In this study, stimulated HSCs with 200μM acetaldehyde for 48h was used to imitate alcoholic liver fibrosis in vitro. The western blot and qRT-PCR results showed that P2X7R expression was significantly increased in the activation of HSCs after acetaldehyde treatment. Interestingly, activation of P2X7R by stimulating with P2X7R agonist BzATP significantly promoted acetaldehyde-induced CyclinD1 expression, cell proportion in S phase, inflammatory response, and the protein and mRNA levels of α-SMA, collagen I. In contrast, blockage of P2X7R by stimulating with the inhibitor A438079 or transfecting with specific siRNA dramatically suppressed acetaldehyde-induced HSCs activation. Furthermore, PKC activation treated with PMA could obviously up-regulate the expression of α-SMA and collagen I and the phosphorylation of GSK3β, while inhibition of PKC significantly reduced GSK3β activation. Moreover, GSK3β inhibition harvested a dramatic decrease of the mRNA and protein levels of α-SMA and collagen I by suppressing GSK3β phosphorylation. Taken together, these results suggested that purinergic P2X7R mediated acetaldehyde-induced activation of HSCs via PKC-dependent GSK3β pathway, which maybe a novel target for limiting HSCs activation.

Cisplatin is a classic chemotherapeutic agent widely used to treat different types of cancers including ovarian, head and neck, testicular and uterine cervical carcinomas. However, cisplatin induces acute kidney injury by directly triggering an excessive inflammatory response, oxidative stress, and programmed cell death of renal tubular epithelial cells, all of which lead to high mortality rates in patients. In this study, we examined the protective effect of protocatechuic aldehyde (PA) in vitro in cisplatin-treated tubular epithelial cells and in vivo in cisplatin nephropathy. PA is a monomer of Traditional Chinese Medicine isolated from the root of S. miltiorrhiza (Lamiaceae). Results show that PA prevented cisplatin-induced decline of renal function and histological damage, which was confirmed by attenuation of KIM1 in both mRNA and protein levels. Moreover, PA reduced renal inflammation by suppressing oxidative stress and programmed cell death in response to cisplatin, which was further evidenced by in vitro data. Of note, PA suppressed NAPDH oxidases, including Nox2 and Nox4, in a dosage-dependent manner. Moreover, silencing Nox4, but not Nox2, removed the inhibitory effect of PA on cisplatin-induced renal injury, indicating that Nox4 may play a pivotal role in mediating the protective effect of PA in cisplatin-induced acute kidney injury. Collectively, our data indicate that PA blocks cisplatin-induced acute kidney injury by suppressing Nox-mediated oxidative stress and renal inflammation without compromising anti-tumor activity of cisplatin. These findings suggest that PA and its derivatives may serve as potential protective agents for cancer patients receiving cisplatin treatment.

Background An increase in alcohol consumption and related harmful effects has been reported among the elderly population in Asia. Of note, it is important to monitor patterns of alcohol use, and to establish a valid and reliable evaluation system when screening for risky consumption in this age group. Objective The aim of the current study was to evaluate the possible alcoholic liver disease (ALD) risk factors of a local population in elderly Chinese adults. Methods A questionnaire was sent to 3393 Chinese adults over 40 years old in Hefei. Alcohol consumption was determined based on the AUDIT questionnaire. ALD was defined by ALD diagnostic standards. Adjusted odds ratios and 95% confidence intervals (95% CI) derived from multiple logistic regression models were used to assess the relationship between ALD and sociodemographic variables. Results Among 2545 total interviewees, 448 (17.6%) reported a history of alcohol consumption in the previous 12 months. Of these drinkers, 46.7% were male and 53.3% were female. The overall Cronbach's alpha coefficient for AUDIT was 0.648. The rate of ALD was 6.83%. Alcohol abuse was significantly associated with ALD. In the logistical model, alcohol abuse was independently associated with ALD (OR = 6.17, 95% CI: 3.69–15.24; p Conclusions Alcohol use, sex, age, and facial flushing were risk factors for ALD. These results provide important evidence for the prevention and therapy of ALD.

Renal fibrosis, a common feature and leading cause for End Stage Renal Disease, still lacks effective therapy. In the current study, we detected and compared the anti-fibrotic effects of wogonin and wogonoside, two major components of Scutellaria baicalensis Georgi, in TGF-β1-treated tubular epithelial cells of human and murine origins. Results consistently showed that compared with wogonoside, wogonin inhibits TGF-β1-induced upregulated mRNA and protein levels of collagen I and α-SMA with more efficiency, which was further confirmed by the immunofluorescence results that wogonin decreased the percentage of collagen I and α-SMA positive cells in TGF-β1-treated tubular epithelial cells. Mechanistically, wogonin mainly decreased Smad3 phosphorylation, but had marginal effect on non-canonical TGF-β signaling pathways, such as p38 and ERK MAP Kinase. Furthermore, in the cells deficient for TGF-β signaling or downstream Smad3, results demonstrated that even high concentration of wogonin failed to further decrease the level of collagen I and α-SMA, indicating the essential role of TGF-β/Smad3 signaling inhibition in the therapeutic action of wogonin in TGF-β1-stimulated tubular epithelial cells. Collectively, our results indicated that wogonin may be utilized as a potential anti-fibrotic Traditional Chinese Medicine monomer in the treatment of renal fibrosis.

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a negative regulator of this process. PTEN promoter hypermethylation is a major epigenetic silencing mechanism in tumors. The present study aimed to investigate whether PTEN promoter methylation was involved in HSC activation and liver fibrosis. Treatment of activated HSCs with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) decreased aberrant hypermethylation of the PTEN gene promoter and prevented the loss of PTEN expression that occurred during HSC activation. Silencing DNA methyltransferase 1 (DNMT1) gene also decreased the PTEN gene promoter methylation and upregulated the PTEN gene expression in activated HSC-T6 cells. In addition, knockdown of DNMT1 inhibited the activation of both ERK and AKT pathways in HSC-T6 cells. These results suggest that DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the activation of the PI3K/AKT and ERK pathways, resulting in HSC activation.

Alcoholic fatty liver (AFL) is a reversible condition, but it can potentiate the development of alcoholic hepatitis and even cirrhosis by increasing oxidant generation, which is one of the key pathogenic factors and could result in alcoholic liver disease (ALD). Total flavonoids from Litsea coreana (TFLC), an active component extracted from Litsea coreana leve, have been shown to have therapeutic effects on hyperlipidemia. The present study was to evaluate the protective effects of TFLC on alcoholic fatty liver (AFL) in rats, and investigate the potential mechanism. An AFL model in rats was established by intaking different doses of alcohol (concentration from 5% to 40%) over 12 weeks. Serum levels of TG, TC, LDL-C, HDL-C, TNF-α, insulin, and glucose were measured, histopathologic changes were determined, and expression of adipose differentiation-related protein (ADRP) in the liver were evaluated by Western blotting and immunohistochemistry, respectively. The results showed that treatment with TFLC resulted in decreased serum levels of TG, TC, LDL-C, TNF-α, glucose and insulin, as well as improved liver index. Morphological evaluation revealed rats in model group developed a severe steatosis, but the severities of liver steatosis were effectively ameliorated in TFLC (200 and 400 mg/kg) treated groups. Expression of hepatic ADRP were increased in model group, and suppressed in TFLC treated groups. These results suggest that TFLC had a protective effect on AFL rats; the mechanism may be involved in regulation serum lipid profiles via down-regulation of hepatic expression of ADRP in AFL rats.

Hepatic stellate cells (HSCs) activation is an essential event during liver fibrogenesis. A major pathway is the transition of HSCs into hepatic myofibroblasts. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. Overexpression of this protein results in an increase of global methylation levels. Treatment of HSCs with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) blocks the cell proliferation. 5-azadC also prevents loss of Ras GTPase activating-like protein 1 (RASAL1) expression that occurs during HSCs proliferation. To further explore the underlying molecular mechanisms, we hypothesized that this perpetuation of fibrogenesis was caused by DNA methylation. Results demonstrated that hypermethylation of RASAL1 is associated with the perpetuation of fibroblast activation and fibrogenesis in the liver. knockdown of MeCP2 using siRNA technique increased RASAL1 in both mRNA and protein level in myofibroblasts. These studies demonstrated that MeCP2 and DNA methylation may provide molecular mechanisms for perpetuated fibroblast activation and fibrogenesis in the liver.