Dihydromyricetin attenuates Escherichia coli lipopolysaccharide-induced ileum injury in chickens by inhibiting NLRP3 inflammasome and TLR4/NF-κB signalling pathway.

Lipopolysaccharide (LPS) as a major component of Escherichia coli cell wall can cause inflammation and cell death. Dihydromyricetin (ampelopsin, DHM) is a natural flavonoid compound with anti-inflammatory, anti-oxidant and anti-bacterial effects. The preventive effects of DHM against ileum injury remain unclear. Here, we explored the protective role of DHM against LPS-induced ileum injury in chickens. In this study, DHM significantly attenuated LPS-induced alteration in diamine oxidase, malondialdehyde, reduced glutathione, glutathione peroxidase and superoxide dismutase levels in chicken plasma and ileum. Histology evaluation showed that the structure of blood vessels in ileum was seriously fragmented and presence of necrotic tissue in the lumen in the LPS group. Scanning electron microscopic observation revealed that the surface of the villi was rough and uneven, the structure was chaotic, and the normal finger shape was lost in the LPS group. In contrast, 0.05% and 0.1% DHM treatment partially alleviated the abnormal morphology. Additionally, DHM maintained the barrier function by restoring the protein expression of occludin, claudin-1 and zonula occludens protein-1. DHM inhibited apoptosis through the reduction of the expression of bax and caspase-3 and restored the expression of bcl-2. Importantly, DHM could reduce ileum NLR family pyrin domain-containing 3 (NLRP3), caspase-1, interleukin (IL)-1ß and IL-18 expression to protect tissues from pyroptosis and inhibited toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signalling pathway. In summary, DHM attenuated the ileum mucosal damage, oxidative stress and apoptosis, maintained barrier function, inhibited NLRP3 inflammasome and TLR4/NF-κB signalling pathway activation triggered by Escherichia coli LPS.

JNK Signaling Pathway Mediates Acetaminophen-Induced Hepatotoxicity Accompanied by Changes of Glutathione S-Transferase A1 Content and Expression.

Acetaminophen (APAP) is an analgesic-antipyretic drug and widely used in clinics. Its overdose may cause serious liver damage. Here, we examined the mechanistic role of c-Jun N-terminal kinase (JNK) signaling pathway in liver injury induced by different doses of APAP. Male mice were treated with APAP (150 and 175 mg·kg-1), and meanwhile JNK inhibitor SP600125 was used to interfere APAP-induced liver damage. The results showed that JNK signaling pathway was activated by APAP in a dose-dependent manner. C-Jun N-terminal kinase inhibitor decreased JNK and c-Jun activation significantly (P < 0.01) at 175 mg·kg-1 APAP dose, and phosphorylation levels of upstream proteins of JNK were also decreased markedly (P < 0.05). In addition, serum aminotransferases activities and hepatic oxidative stress increased in a dose-dependent manner with APAP treatment, but the levels of aminotransferases and oxidative stress decreased in mice treated with JNK inhibitor, which implied that JNK inhibition ameliorated APAP-induced liver damage. It was observed that apoptosis was increased in APAP-induced liver injury, and SP600125 can attenuate apoptosis through the inhibition of JNK phosphorylation. Meanwhile, glutathione S-transferases A1 (GSTA1) content in serum was enhanced, while GSTA1 content and expression in liver reduced significantly with administration of APAP (150 and 175 mg·kg-1). After inhibiting JNK, GSTA1 content in serum decreased significantly (P < 0.01); meanwhile, GSTA1 content and expression in liver enhanced. These findings suggested that JNK signaling pathway mediated APAP-induced hepatic injury, which was accompanied by varying GSTA1 content and expression in liver and serum.

Acetaminophen-induced hepatocyte injury: C2-ceramide and oltipraz intervention, hepatocyte nuclear factor 1 and glutathione S-transferase A1 changes.

Acetaminophen (APAP) is an antipyretic and analgesic, which is commonly associated with drug-induced hepatic injury. C2-ceramide plays a key role in mediating cell life activities, and oltipraz was extensively studied as a cancer chemopreventive agent. Glutathione S-transferase A1 (GSTA1) acts as a vital liver detoxification enzyme. Hepatocyte nuclear factor 1 (HNF-1) regulates various cellular signaling pathways. In this study, we investigated the effects of C2-ceramide and oltipraz on APAP-induced hepatocyte injury and the changes of HNF-1 and GSTA1. Results showed that C2-ceramide (6 µmol/L) exacerbated APAP-induced hepatocyte injury and caused a significant decrease (P < .01) in HNF-1 and GSTA1 expressions. Meanwhile, GSTA1 content in supernatant was significantly increased (P < .01). In contrast, oltipraz (8 µmol/L) reduced the injury and significantly elevated (P < .01) HNF-1 and GSTA1 expressions while GSTA1 content in supernatant was significantly decreased (P < .01). In conclusion, these findings revealed that C2-ceramide inhibited HNF-1 and GSTA1 expression and exacerbated hepatocyte injury, while oltipraz treatment results in the reduction of hepatocyte injury, and promoted HNF-1 and GSTA1 expression. Additionally, the changes in HNF-1 and GSTA1 were related to APAP-induced hepatocyte injury. These results were useful to investigate the mechanism of an antipyretic and analgesic drug combination.

Acetaminophen-induced reduction in glutathione-S-transferase A1 in hepatocytes: A role for hepatic nuclear factor 1α and its response element.

The role of hepatic nuclear factor 1α (HNF-1α) and its response element in the expression of glutathione S-transferase A1 (GSTA1) was investigated in hepatocytes cells injury induced by acetaminophen (APAP). Treatment of hepatocytes with C2-ceramide exacerbated cells injury with GSTA1 mRNA level reducing. Contrastingly, administration of oltipraz alleviated cells damage with GSTA1 mRNA level elevating relative to hepatotoxicity induced by APAP. Western blot analysis showed that C2-ceramide decreased the translocation of HNF-1α and expression of GSTA1 protein, while oltipraz increased nuclear HNF-1α level and transactivation of GSTA1. The role of HNF-1α on GSTA1 expression was confirmed by transfection experiment and dual-luciferase reporter assay system. In the cells transfected with pGSTA1-1298-LUC vector in which HNF-1 response element (HRE) was contained, the luciferase activity decreased with reduction of nuclear HNF-1α and increased with elevation of nuclear HNF-1α. However, the luciferase activity had no change with the variation of nuclear HNF-1α when the cells transfected with the plasmid of pGSTA1-ΔHNF1-LUC in which the HRE was mutated. In conclusion, HNF-1α could affect the transcription of GSTA1 and HNF-1 response element in the GSTA1 promoter region, which is functionally active for the GSTA1 transcription.

Combination of berberine and ciprofloxacin reduces multi-resistant Salmonella strain biofilm formation by depressing mRNA expressions of luxS, rpoE, and ompR.

Bacterial biofilms have been demonstrated to be closely related to clinical infections and contribute to drug resistance. Berberine, which is the main component of Coptis chinensis, has been reported to have efficient antibacterial activity. This study aimed to investigate the potential effect of a combination of berberine with ciprofloxacin (CIP) to inhibit Salmonella biofilm formation and its effect on expressions of related genes (rpoE, luxS, and ompR). The fractional inhibitory concentration (FIC) index of the combination of berberine with CIP is 0.75 showing a synergistic antibacterial effect. The biofilm's adhesion rate and growth curve showed that the multi-resistant Salmonella strain had the potential to form a biofilm relative to that of strain CVCC528, and the antibiofilm effects were in a dose-dependent manner. Biofilm microstructures were rarely observed at 1/2 × MIC/FIC concentrations (MIC, minimal inhibition concentration), and the combination had a stronger antibiofilm effect than each of the antimicrobial agents used alone at 1/4 × FIC concentration. LuxS, rpoE, and ompR mRNA expressions were significantly repressed (p ＜ 0.01) at 1/2 × MIC/FIC concentrations, and the berberine and CIP combination repressed mRNA expressions more strongly at the 1/4 × FIC concentration. The results indicate that the combination of berberine and CIP has a synergistic effect and is effective in inhibiting Salmonella biofilm formation via repression of luxS, rpoE, and ompR mRNA expressions.

Effects of C2-Ceramide and Oltipraz on Hepatocyte Nuclear Factor-1 and Glutathione S-Transferase A1 in Acetaminophen-Mediated Acute Mice Liver Injury.

In this study, acetaminophen (APAP)-induced acute liver injury mice model was used to investigate the effects of C2-ceramide and oltipraz on hepatocyte nuclear factor 1 (HNF-1) and glutathione S-transferase A1 (GSTA1). Notably, C2-ceramide caused alteration in mice serum transaminases and liver tissue indexes, and aggravated hepatic injury, while oltipraz alleviated hepatic injury. By screening, the optimal concentrations of C2-ceramide and oltipraz were confirmed to be 120 and 150 µmol/L, respectively. In histopathology, karyolysis and more necrotic cells and bleeding spots were appeared on administration of C2-ceramide, but only a small amount of inflammatory cells infiltration was seen after oltipraz treatment. In addition, RT-PCR and western blot results revealed that the mRNA and protein expression levels of HNF-1 and GSTA1 in liver were significantly decreased (p < 0.01) with the administration of 120 µmol/L C2-ceramide. Meanwhile, GSTA1 content in serum increased up to 1.27-fold. In contrast, 150 µmol/L oltipraz incorporation to APAP model mice resulted in obvious elevation (p < 0.01) in the mRNA and protein expression levels of HNF-1 and GSTA1 in liver, and serum GSTA1 content decreased up to 0.77-fold. In conclusion, C2-ceramide could down-regulate the expression of HNF-1 and GSTA1 which exacerbated hepatic injury, while oltipraz could up-regulate the expression of HNF-1 and GSTA1 which mitigated hepatic injury.

Evaluation of hepatoprotective activity of Syringa oblata leaves ethanol extract with the indicator of glutathione S-transferase A1

Abstract The leaves of Syringa oblata Lindl., Oleaceae, had been extensively used as a folk medicine to treat various infections, heal inflammations, icteric hepatitis and acute mastitis. The study was designed to evaluate the hepatoprotective activity of S. oblata leaves ethanol extract against CCl4-induced hepatotoxicity in primary hepatocytes and mice with the indicator of glutathione S-transferase alpha 1. The hepatoprotective effects of S. oblata leaves ethanol extract were evaluated on the basis of liver histopathology and biochemical parameters as well as hepatic oxidative stress markers. The results showed that CCl4 negatively modulated biochemical parameters and liver antioxidant activities. However, the use of S. oblata leaves ethanol extract restored altered-serum biochemical parameters and liver antioxidant activities in a dose-dependent manner. Importantly, the trends in S-transferase alpha 1 were similar to alanine aminotransferase and aspartate aminotransferase level, and S-transferase alpha 1 was suggested to be a marker for the evaluation of hepatoprotective activity of S. oblata leaves ethanol extract. Histopathological examination showed that CCl4 causes significant hepatic injury relative to control group. The above findings suggested that S. oblata leaves ethanol extract has hepatoprotective effects against CCl4-induced hepatic injury and S-transferase alpha 1 may be an indicator to evaluate the protective effects of S. oblata leaves ethanol extract.

Expression of glutathione S-transferase A1, a phase II drug-metabolizing enzyme in acute hepatic injury on mice.

In the present study, three models of acute liver injury in mice were induced via the administration of CCl4 (35 mg/kg, 24 h), acetyl-para-aminophenol (APAP; 200 mg/kg, 12 h) and ethanol (14 ml/kg, 8 h) to study the effect of glutathione S-transferase A1 (GSTA1) on acute liver injury. The serum levels of alanine transaminase, aspartate transaminase and liver homogenate indicators (superoxide dismutase, glutathione and glutathione peroxidase) were significantly lower in model groups compared with the control group (P<0.01), whereas the liver homogenate indicator malondialdehyde was significantly increased (P<0.01). The expression of GSTA1 in liver was significantly decreased in the model groups compared with the control group (P<0.01). GSTA1 protein content was 3.8, 1.3 and 2.6 times lower in the CCl4, APAP and ethanol model groups, respectively. Furthermore, GSTA1 mRNA expression levels decreased by 4.9, 2.1 and 3.7 times in the CCl4, APAP and ethanol model groups, respectively. Among the three models, the injury induced by CCl4 was the most marked, followed by ethanol and finally APAP. These results suggest that GSTA1 may be released by the liver and serve as an antioxidant in the prevention of liver damage.

Hepatoprotective effects of ethanol extracts from Folium Syringae against acetaminophen-induced hepatotoxicity in vitro and in vivo.

BACKGROUND: The leaves of Folium Syringae (FS) have been long used as a traditional Chinese folk medicine for their anti-inflammatory effect, utilized as an antibacterial and antiviral treatment. The purpose of this study was to investigate the potential hepatoprotective effects of FS on acetaminophen-induced hepatic injury in primary hepatocytes and mice. METHODS: Hepatocytes obtained by the inverse perfusion method were divided randomly into five groups. Prior to acetaminophen exposure, 3 different doses of FS ethanol extracts were given to hepatocytes and mice, respectively. Thereafter, transaminases, glutathione S-transferase A1 (GSTA1) and some hepatic indices were determined. RESULTS: FS ethanol extracts (200 µg/mL) pretreatment prevented all of the alterations, returning their levels to nearly those levels observed in the control group in vitro. Treatment with FS ethanol extracts (200 mg/kg) significantly reduced the toxicity induced by acetaminophen in vivo, which manifested as a decrease in transaminases, and the hepatoprotective effects of FS were similar to Silymarin (positive group). GSTA1 represented the same change trend as transaminases and hepatic indices, and at a dose of 100 µg/mL FS ethanol extracts in vitro and 100 mg/kg in vivo, GSTA1 content changed significantly (p < 0.01), but transaminases were insignificant (p > 0.05). CONCLUSION: The results of our investigation suggested that FS ethanol extracts possess significant protective effects against hepatotoxicity induced by acetaminophen both in vitro and in vivo. In addition, GSTA1 could be used as an indicator assessing the extents of hepatic injury, which is more sensitive than transaminases.

Glutathione S-transferase A1 (GSTA1) as a marker of acetaminophen-induced hepatocyte injury in vitro.

Acetaminophen (APAP) overdose causes serious hepatocyte injury, and new markers are needed to predict APAP-induced hepatic injury. Glutathione S-transferase A1 (GSTA1) plays a significant role in the metabolism of APAP. Primary mouse hepatocytes were isolated by a two-step perfusion in situ. An APAP-induced hepatocyte injury model was used to characterize GSTA1 in APAP treated cells and determine whether GSTA1 could be a prognostic marker in vitro. A significant increase (p < .05) in GSTA1 in cell culture supernatant was detected at 6 h after APAP treatment, while alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) showed marked differences (p < .05) at 8 h after APAP exposure, 2 h later than GSTA1. Furthermore, GSTA1 increased in a dose-dependent manner with APAP treatment. GSTA1 increased significantly (p < .05) at a concentration of 5.0 mmol/L APAP, while the marked changes in ALT, AST and other indexes were undetectable until the concentration of APAP reached 7.5 mmol/L. These results suggest that increased GSTA1 can be more sensitive than ALT and other indexes as a marker of APAP-induced hepatic injury, which provide novel diagnostic index for APAP-induced hepatic injury and supply valuable information to further understand the pathogenesis of liver damage.

Hepatoprotective effects of Solanum nigrum against ethanol-induced injury in primary hepatocytes and mice with analysis of glutathione S-transferase A1.

BACKGROUND: Solanum nigrum is a herbaceous perennial plant, which is widely used in traditional medicine systems for its antioxidant, antiulcerogenic, antitumorigenic, and anti-inflammatory characteristics. The purpose of this study was to investigate the protective effects of S. nigrum against alcoholic liver damage in primary hepatocytes and mice, using glutathione S-transferase alpha 1 (GSTA1) as an indicator. METHODS: Primary hepatocytes were obtained by the inverse perfusion method improved on Seglen two-step perfusion in situ. RESULTS: In the presence of S. nigrum aqueous extracts (100 µg/mL), no hepatocytic damage was observed in cells treated with ethanol, compared with the model group, and GSTA1 (p < 0.01) was more sensitive than alanine aminotransferase and aspartate aminotransferase (p < 0.05). Mice that received S. nigrum aqueous extracts (150 mg/kg) with ethanol showed marked attenuation of ethanol-induced hepatotoxicity, as evidenced by significant reductions of serum transaminases (p < 0.01), and variation of hepatic oxidative indices (p < 0.05) and GSTA1 (p < 0.05), compared with the model group and mice that received S. nigrum aqueous extracts (200 mg/kg). All the detection indexes were significantly different (p < 0.01) from those of the model group, and the protective effects were almost the same as that of the positive drug group. CONCLUSION: These results suggested that S. nigrum has hepatoprotective effects against ethanol-induced injury both in vitro and in vivo, and can protect the integrity of hepatocytes and thus reduce the release of liver GSTA1, which contributes to improved liver detoxification.