Intraperitoneal Injection Of D-galactosamine Research Articles

Phase Preparation of Xiao-chai-hu Decoction and its Pharmacodynamics of Acute Liver Injury.

Self-emulsifying nano-phase of traditional Chinese medicine are a research hotspot. Xiao-Chai-Hu decoction is a commonly used compound decoction in clinical practice, which is of great research significance. The aim of this study was to isolate and characterize the self-emulsifying nano-phase and other phases of Xiao-Chai-Hu decoction, and to study the effects of each phase on acute liver injury. The liquid medicine was prepared employing centrifugation followed by dialysis. Single- factor investigation methodology was utilized to optimize the preparation parameters for both phases. Characterization of the formulated phase involved analyses such as surface morphology assessment, measurement of nanoparticle size and Zeta potential using an analyzer, observation of the Tyndall effect, conducting diffusion and dilution tests, examination under a microscope, and structural visualization via transmission electron microscopy (TEM). Furthermore, an acute liver injury model was established in rats through intraperitoneal injection of D-Galactosamine (D-Gal- N). To assess hepatic function and oxidative stress status, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content in liver tissue were quantified. The liver coefficients for each group were calculated as an additional parameter. For histopathological evaluation, liver tissue sections from the experimental group were stained with Hematoxylin and Eosin (H&E) and examined microscopically under light conditions. These revisions aim to enhance clarity, correct minor grammatical errors (such as capitalization of "HE" to "H&E"), and ensure a smoother flow of information without altering the scientific content of your original text. Successful establishment and separation of four distinct phases were achieved, including the self-emulsifying nano-phase, precipitation phase, suspension phase, and true solution phase. The self-emulsifying nano-phase was characterized as spherical particles with an average diameter of approximately 100 nm. Pharmacodynamic assessments revealed that both Xiao-Chai-Hu decoction and its self-emulsifying nano-phase significantly reduced liver coefficients and alanine aminotransferase (ALT) levels compared to controls (P<0.05). However, no statistically significant differences were observed in regards to aspartate aminotransferase (AST) concentrations, malondialdehyde (MDA) content, or superoxide dismutase (SOD) activity between the treatment groups and control (P>0.05). These findings indicate that both Xiao-Chai-Hu decoction and its self-emulsifying nano-formulation ameliorated D-GalN-induced acute liver injury, albeit without statistically distinguishable efficacy between them (P>0.05). The presence of a self-emulsifying nano-phase within Xiao-Chai-Hu decoction is confirmed, and this nano-phase emerges as a therapeutically efficacious component in mitigating acute liver injury.

Hyperactivation of succinate dehydrogenase promotes pyroptosis of macrophage via ROS-induced GSDMD oligomerization in acute liver failure

Acute liver failure (ALF) is a life-threatening disease with high mortality. Given excessive inflammation is one of the major pathogenesis of ALF, candidates targeting inflammation could be beneficial in the condition. Now the effect of hyperactivated succinate dehydrogenase (SDH) on promoting inflammation in lipopolysaccharide (LPS)-treated macrophages has been studied. However, its role and mechanism in ALF is not well understood. Here intraperitoneal injection of D-galactosamine and LPS was conducted in male C57BL/6 J mice to induce the ALF model. Dimethyl malonate (DMM), which inhibited SDH activity, was injected intraperitoneally 30 min before ALF induction. Macrophage pyroptosis was induced by LPS plus adenosine triphosphate (ATP). Pyroptosis-related molecules and proteins including GSDMD oligomer were examined by ELISA and western blot techniques, respectively. ROS production was assessed by fluorescence staining. The study demonstrated SDH activity was increased in liver macrophages from ALF mice. Importantly, DMM administration inhibited ROS, IL-1β, and pyroptosis-associated proteins levels (NLRP3, cleaved caspase-1, GSDMD-N, and GSDMD oligomers) both in the ALF model and in macrophages stimulated with LPS plus ATP. In vitro, ROS promoted pyroptosis by facilitating GSDMD oligomerization. Additionally, when ROS levels were increased through the addition of H2O2 to the DMM group, the levels of GSDMD oligomers were reverted. In conclusion, SDH hyperactivation promotes macrophage pyroptosis by ROS-mediated GSDMD oligomerization, suggesting that targeting this pathway holds promise as a strategy for treating ALF and other inflammatory diseases.

Upregulated microRNA-450b-5p represses the development of acute liver failure via modulation of liver function, inflammatory response, and hepatocyte apoptosis.

It has been evidenced that microRNAs (miRs) exert crucial effects on acute liver failure (ALF), while the detailed function of miR-450b-5p in ALF progression remained obscure. The purpose of this research was to unravel the regulatory mechanism of miR-450b-5p in ALF via modulating Mouse Double Minute 2 protein (MDM2). ALF was induced in mice by intraperitoneal injection of d-galactosamine ( d-GalN) and lipopolysaccharide (LPS). Adenoviruses containing overexpressed miR-450b-5p, MDM2 shRNA, and overexpressed MDM2 sequences were utilized to manipulate miR-450b-5p and MDM2 expression in the liver before the mice were treated with d-GalN/LPS-induced ALF. Subsequently, miR-450b-5p and MDM2 expression levels in liver tissues of ALF mice were examined. Serum biochemical parameters of liver function were tested, serum inflammatory factors were assessed, and the histopathological changes and hepatocyte apoptosis in liver tissues were observed. The relation between miR-450b-5p and MDM2 was verified. In ALF mice, miR-450b-5p was low-expressed while MDM2 was high-expressed. The upregulation of miR-450b-5p or downregulation of MDM2 could alleviate liver function, mitigate the serum inflammatory response and pathological changes in liver tissues, as well as inhibit the apoptosis of hepatocytes. MiR-450b-5p targeted MDM2. MDM2 overexpression reversed the repressive effects of elevated miR-450b-5p on ALF. The upregulated miR-450b-5p blocks the progression of ALF via targeting MDM2. This study contributes to affording novel therapeutic targets for ALF treatment.

Changes in Mitochondria-Related Gene Expression upon Acupuncture at LR3 in the D-Galactosamine-Induced Liver Damage Rat Model.

Hepatic diseases, such as hepatonecrosis, hepatitis, and hepatocirrhosis, are associated with mitochondrial dysfunction and increased reactive oxygen species generation and inflammation, ultimately leading to liver failure. In this study, we examined if acupuncture at LR3 can affect mitochondria-related gene expression in a liver damage model of experimentally induced acute liver failure (ALF). ALF was induced by the intraperitoneal injection of D-galactosamine (D-GalN) in experimental rats, who then received either sham (ALF), manual acupuncture (MA), electroacupuncture (EA), or silymarin (PC, positive control) treatment. Liver tissues were extracted from experimental and untreated control rats for histopathological analysis and expression profiling of genes involved in mitochondrial function. Of the 168 mitochondria-related genes profiled, two genes belonging to the solute-carrier transporter family (Slc25a15 and Slc25a25) and Ndufb7 were upregulated. Gamma-glutamylcysteine synthetase was more downregulated in MA than ALF. Furthermore, MA reversed D-GalN-induced inflammatory cell infiltration, destruction of hepatic cell plates, and increase in the levels of the proinflammatory cytokine TNF-α. MA at LR3 can reduce the risk of D-GalN-induced ALF by inducing the expression of metabolic and inflammation-related genes and regulating proinflammatory factor production in hepatic mitochondria.

The Protective Effects of a Modified Xiaohua Funing Decoction against Acute Liver Failure in Mice Induced by D-Gal and LPS.

Objective The aim of this study was to evaluate the effects of a modified Xiaohua Funing decoction (Xfd) on acute liver failure (ALF) and determine whether the protective mechanisms are related to alterations in the gut microbiota. Methods An animal model of ALF was induced by intraperitoneal injection of D-galactosamine (D-Gal, 0.5 g/kg) and lipopolysaccharide (LPS, 100 μg/kg). Male BALB/c mice were randomly divided into the following 4 groups: the control group (saline, Con), model group (D-Gal/LPS, Mod), silymarin pretreatment group (200 mg/kg, Sil), and modified Xfd pretreatment group (650 mg/kg, Xfd). The Sil and Xfd groups received the respective intervention orally for 14 days and 2 h before D-Gal/LPS treatment. The liver injury markers included alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and liver histology. 16S rRNA gene sequencing was performed to assess the effects on the caecum content. Results D-Gal/LPS treatment caused severe ALF, illustrating that the ALF model was successfully established. The administration of Sil and Xfd greatly reduced the serum ALT and AST levels and improved the pathological signs of liver injury. However, no significant difference was found between the two groups. In contrast to the Mod group, the Sil and Xfd groups showed a shift toward the Con group in terms of the gut microbiota structure. The abundances of Firmicutes and Bacteroidetes and the Bacteroidetes/Firmicutes ratio in the Mod group significantly differed from those in the Con group. The Sil and Xfd groups showed restoration of the disordered microbiota. Significantly increased relative abundances of Lachnospiraceae_NK4A136_group and Candidatus_Saccharimonas and a markedly decreased Muribaculaceae abundance were found in the Sil and Xfd mice compared with those in the Mod mice (P < 0.01, P < 0.05). Interestingly, a negative correlation was observed between the abundances of the gut microbiota constituents, specifically Clostridia_UCG-014, and ALT and AST levels. Conclusion In summary, our results indicate that Xfd may protect the liver and modify the gut microbiota in ALF mice.

Effect of Comparison between Qingchangligan Granules and Qingchangligan Decoction on D-GalN/LPS-Induced Liver Injury in Mice

<sec><title>Ethnopharmacological relevance</title> The Qingchangligan formula has two dosage forms, granules and decoction. Both of them have the same prescription; however, whether their protective effects are different remain elusive. </sec> <sec><title>Aim of the study</title> To compare and evaluate the protective effect of the granules and decoction of Qingchangligan formula on acute liver failure (ALF) mice induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). </sec> <sec><title>Materials and Methods</title> ALF was induced by intraperitoneal injection of D-GalN (700 mg/kg) plus LPS (10 μg/kg). The granules and decoction of Qingchangligan formula were administered to mice in the same dose and at the same time point, respectively, with three doses of 50 mg/kg (on day 1, day 2, and day 3) prior to D-GalN/LPS injection by intragastric administration. The mice in different groups were sacrificed at 6 h after D-GalN/LPS injection, and liver samples and blood were collected for analysis. </sec><sec><title>Results</title> The different processed methods for the Qingchangligan resulted in a little component difference between the granules and decoction: the content of some ingredients, including Catalpol, Caffeic acid, Naringin, Emodin and Chrysophanol in granules, are higher than in decoction. Both of the two Qingchangligan formulas administration ameliorated liver injury, as evidenced by the decreased the lethality in ALF mice, the reduced transaminase levels and well-preserved liver architecture. However, the granules seemed to be more effective than the decoction in the ALF model. Mice pretreatment with the Qingchangligan formula reduced the expression of inflammatory cytokines and promoted autophagy in ALF model. Moreover, the effect on inflammation and autophagy of granules is more obvious than the decoction. </sec><sec><title>Conclusions</title> Our findings demonstrated that the granules and decoction of Qingchangligan formula both exert a protective effect against the pathophysiology of ALF, especially the granules is more efficacious, which provide a rationale for using of the different formulations of Qingchangligan formula. </sec>

Probiotics enhance the efficacy of fecal microbiota transplantation in severe acute liver injury

Objective: To observe the changes of gut flora in mice, and explore the outcome of fecal microbiota transplantation combined with probiotics in the intervention of severe acute liver injury. Methods: Forty male BALB/c mice were selected and randomly divided into blank control group (10 mice), model group (10 mice), ordinary fecal microbiota transplantation group (10 mice), and fecal microbiota + probiotics transplantation group (10 mice). An intraperitoneal injection of d-galactosamine (D-GalN 3.0g/kg) was given to every group except the blank control group to induce severe acute liver injury model. Simultaneously, ordinary fecal microbiota transplantation group and fecal microbiota + probiotics transplantation group and modeling group were given enema solutions (once a day). After 48 hours, fetched serum was taken to detect alanine transaminase, aspartate transaminase and total bilirubin, and liver tissue was taken for pathological detection. The colonic content was used to extract DNA for 16S V3-V4 high-throughput sequencing. The results of sequencing were analyzed by using bioinformatics analysis; including OTU cluster analysis, α diversity analysis, β diversity analysis, and linear discriminant analysis effect size (Lefse) to find the bacteria with different colonic content characteristics in different groups of mice. Differences in clinical biochemical indicators between groups were compared using t-test, and the differences between 16S V3-V4 region sequencing results were compared using Wilcoxon test. Results: Model group mice serum biochemical parameters were higher than the other three groups, and the difference was statistically significant (P < 0.05). HE staining of liver sections showed severe inflammatory changes under the microscope in the model group. Ordinary fecal microbiota transplantation group and fecal microbiota + probiotic microbiota transplantation group had low levels of inflammation than the model group. The analysis results of 16S rRNA high-throughput sequencing showed that there was no statistically significant difference in Shannon's index between the blank control and the other three groups. Observed Species difference was statistically significant, and the gut flora composition varied greatly. Species number in the mice gut flora was increased with fecal microbiota transplantation. The results of β - diversity analysis showed that the difference between the blank control group and the other three groups was greater than that between the disease groups. The difference in the structure of the gut flora of the diseased mice in the fecal microbiota + probiotic transplantation group was mostly butyrate-producing bacteria. Conclusion: Fecal microbiota + probiotics enhance the therapeutic effect of fecal microbiota transplantation, improve liver inflammation, and increase the number of butyrate-producing bacteria in the gut.

Protective Activity of &amp;lt;i&amp;gt;Markhamia tomentosa&amp;lt;/i&amp;gt; (Benth.) K. Schum. (Bignoniaceae) Methanol Leaves Extract against D-Galactosamine/Lipopolysaccharide-Induced Acute Liver Injury in Mice

Markhamia tomentosa (Benth.) K. Schum. (Mt) is a Cameroonian medicinal plant, traditionally used to treat painful and inflammatory illness. This study aimed to examine the effects of methanol leaves extract (MLE) of Mt in D-galactosamine (D-GaIN)/lipopolysaccharide (LPS)-induced liver injury. The MLE (100 and 200 mg/kg), Ascorbic acid (10 mg/kg) and distilled water were administered 12 h and 1 h before intraperitoneal injection of D-GaIN (10 mg/mouse)/LPS (0.1 μg/g). Animals were sacrificed 6 h after D-GalN/LPS challenge. Liver injury was assessed biochemically by determination of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (ALP), superoxide dismutase (SOD) and catalase activities. Malondialdehyde (MDA), reduced glutathione (GSH), nitrites, total protein and bilirubin levels were explored. Histopathological examination of liver tissue was also performed. Liver enzymes (ALAT, ASAT, ALP) activity, nitrites, MDA and bilirubin levels were increased, while protein level, SOD and catalase activities were significantly reduced by D-GalN/LPS administration. MLE (100 or 200 mg/kg) protected mice against D-GalN/LPS-induced death. In addition, the plant extract significantly reduced ALAT and ALP activity, exhibiting 23.00% and 62.20% protection, respectively. SOD activity and total protein were significantly (p D-GaIN/LPS-induced liver injury. The obtained results showed that MLE of Mt may possess hepatoprotective effects. Protection afforded by MLE against D-GalN/LPS-induced fulminant liver injury may result from reduction of oxidative stress.

Bifidobacterium adolescentis CGMCC 15058 alleviates liver injury, enhances the intestinal barrier and modifies the gut microbiota in d-galactosamine-treated rats

Acute liver failure is a drastic, unpredictable clinical syndrome with high mortality. Various preventive and adjuvant therapies based on modulating the gut flora have been proposed for hepatic injury. We aimed to explore the preventive and therapeutic effects of Bifidobacterium adolescentis CGMCC15058 on rat liver failure, as well as the potential microecological and immunological mechanisms of those effects. B. adolescentis CGMCC15058 (3 × 109CFU), isolated from healthy human stool, was gavaged to Sprague-Dawley rats for 14days. Acute liver injury was induced on the 15th day by intraperitoneal injection of D-galactosamine. After 24h, liver and terminal ileum histology, liver function, plasma cytokines, bacterial translocation and gut microbiota composition were assessed. We found that pretreatment with B. adolescentis significantly relieved elevated serum levels of alanine aminotransferase (ALT), total bile acid and lipopolysaccharide-binding protein and enhanced the expression of mucin 4 and the tight junction protein zonula occludens-1. B. adolescentis exhibited anti-inflammatory properties as indicated by decreased levels of mTOR and the inflammatory cytokines TNF-α and IL-6, as well as elevated levels of the anti-inflammatory cytokine interleukins-10 in the liver. Similar anti-inflammatory signs were also found in plasma. B. adolescentis significantly altered the microbial community, depleting the common pathogenic taxon Proteus and markedly enriching the taxa Coriobacteriaceae, Bacteroidales and Allobaculum, which are involved in regulating the metabolism of lipids and aromatic amino acids. Our findings not only suggest B. adolescentis acts as a prospective probiotic against liver failure but also provide new insights into the prevention and treatment of liver disease.

Thymosin α1 treatment reduces hepatic inflammation and inhibits hepatocyte apoptosis in rats with acute liver failure.

The present study aimed to evaluate whether thymosin α1 (Tα1) increases survival rates through the improvement of immunofunction and inhibition of hepatocyte apoptosis in rats with acute liver failure (ALF). A total of 25 rats were randomly divided into the control group (CG), the model group (MG) and the treatment group (TG). The CG received an intraperitoneal injection of saline (2 ml). The ALF rat model was established by the intraperitoneal injection of D-galactosamine (700 mg/kg) and lipopolysaccharide (10 µg/kg). The TG received an intraperitoneal injection of Tα1 (0.03 mg/kg) 1 h prior to and 30 min after modeling. The survival rates of the rats were recorded. An additional 63 rats were randomly divided into a CG (n=3), MG (n=30) and TG (n=30). Three rats were sacrificed at 3, 6, 9 and 12 h after establishment of the rat model to detect plasma alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), tumor necrosis factor (TNF)-α and interleukin-10 (IL-10). Liver samples were stained with hematoxylin and eosin and TUNEL, and reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to detect B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in liver tissue. The results indicated that the survival rate of the TG was significantly higher compared with that of the MG at 24 h (P<0.05). Plasma ALT, AST and TBIL in the MG and TG increased over time (3–12 h), with ALT, AST and TBIL observed to be significantly lower in the TG compared with the MG at each time-point (P<0.05). Hepatocellular necrosis, hemorrhage and inflammatory cell infiltration of ALF were aggravated over time (3–12 h) in the MG and TG. Notably, in the Tα1-treated rats, the hepatocytes appeared healthier with fewer apoptotic cells compared with those from the MG at the same time-points. Hepatocyte apoptotic index increased in the TG and MG, but was significantly lower in the TG compared with the MG at each time-point (P<0.05) in TUNEL assays. Plasma TNF-α and IL-10 in the MG and TG increased over time (3–12 h), with TNF-α observed to be significantly lower in the TG compared with the MG at each time-point (P<0.05), however, IL-10 was observed to be significantly higher in the TG compared with the MG at each time-point (P<0.05). Bax mRNA expression was significantly lower in the TG compared with the MG at each time-point (P<0.05), whereas Bcl-2 was significantly higher (P<0.05). In conclusion, Tα1 improved survival rates in an ALF rat model by downregulating TNF-α and upregulating IL-10, leading to the attenuation of hepatic inflammation and hepatocyte apoptosis.

Induction of renal artery hyperresponsiveness by alpha1-adrenoceptor in hepatorenal syndrome.

ObjectiveTo investigate the potential role of alpha1-adrenoceptor (α1-AR) in the pathogenesis of hepatorenal syndrome.MethodsHepatorenal syndrome was induced in male rats by intraperitoneal injection of D-galactosamine and orally treatment with α1-AR antagonist tamsulosin. Hyperresponsiveness of the renal artery contraction was evaluated by the laser-Doppler flowmetry and multimyograph system, while renal blood flow (cortical and medullary perfusion) was simultaneously measured. Renal artery ring segment tone was recorded with the myograph system, and concentration-response curves were obtained by cumulative administration of agonists.ResultsThis model developed acute renal and liver failure without renal damage in pathology, accompanied by significant hyperresponsiveness of renal artery contraction. After hepatorenal syndrome, plasma concentrations of tumor necrosis factor-α increased by two-fold, and α1-AR was significantly activated in the renal artery. Concentration-dependent vasoconstriction induced by noradrenaline was significantly decreased in the renal arteries of hepatorenal syndrome rat because of gradually decreased renal blood flow. Administration of tamsulosin prevented renal failure when given before the onset of liver injury, but it had no effect on liver injury by itself.Conclusionα1-AR expression is positively associated with renal vasoconstriction induced by renal artery hyperresponsiveness in HRS. Therefore, α1-AR may be a potential target in the treatment of HRS.

Sirtuin-activating compounds (STACs) alleviate D-galactosamine/lipopolysaccharide-induced hepatotoxicity in rats: involvement of sirtuin 1 and heme oxygenase 1.

Sirtuin activating compounds (STACs) attenuate various type of liver insults through mechanisms which are not fully understood. In the present study, we investigated the ameliorative potential of quercetin (natural polyphenol) and SRT1720 (synthetic SIRT1 activator) against D-galactosamine/lipopolysaccharide-induced hepatotoxicity (an experimental model of acute liver failure). Moreover, we compared and contrasted the roles of stress responsive enzymes, sirtuin 1 (SIRT1) and heme oxygenase 1 (HO-1) in hepatoprotection/ hepatotoxicity. Liver injury was induced in male Wistar rats by intraperitoneal injection of D-galactosamine (400 mg/kg) and lipopolysaccharide (10 microg/kg). Some animals were pretreated with quercetin (50 mg/kg i.p.) or SRT1720 (5 mg/kg i.p.). Twenty-four hours later, the effects of these treatments were evaluated by biochemical studies and Western blot. D-GalN/LPS treatment upregulated HO-1 expression, downregulated SIRT1 expression, decreased AST: ALT ratio and markedly increased bilirubin, catalase and conjugated diene levels. Pretreatment of D-GalN/LPS rats with either quercetin or SRT1720 returned SIRT1 expression, HO-1 expression and all the aforementioned markers towards normal. Collectively, these findings suggest that elevated HO-1 and low SIRT1 expressions are involved in the pathogenesis of D-GalN/LPS-induced hepatotoxicity. Drugs that downregulate HO-1 and/or upregulate SIRT1 seem to have antihepatotoxic effects and need further exploration.

Microflora changes in upper digestive tract and bile in acute liver failure mice

Objective: To study the changes of upper digestive tract and bile flora associated with acute liver failure in mice. Methods: Mice were randomly divided into 2 groups: control group and acute liver failure group (group M). Acute liver failure in group M was induced by intraperitoneal injection of D-galactosamine (D-GaIN). The information of relative abundance and diversity were observed by high-throughput sequencing of V3 + V4 region in 16 S rDNA from bacteria of oral cavity, stomach, duodenal and bile.Bacterial translocation and changes associated with acute liver injury were identified by LEfSe (LDA effect size) analysis. Results: At the phylum level, compared with control group, the relative abundance of TM7 in oral cavity and stomach increased in group M. At the family level, compared with control group, group M resulted in a significant decrease in Pasteurellaceae in the stomach (P<0.05) and a increase in Lactobacillaceae in the bile (P<0.05). Conclusion: The diversity of bacteria and relative abundance of each bacterium in upper digestive tract and the bile are significantly changed during the process of acute liver injure in mice.

Genistein attenuates D-GalN induced liver fibrosis/chronic liver damage in rats by blocking the TGF-β/Smad signaling pathways

Background and aimGenistein is a major isoflavonoid abundantly found in soy. Earlier genistein has been reported to possess protective effect against a multitude of disorders including cancer. Previously we demonstrated the protective effects of Genistein in d-Galactosamine (D-GalN) induced fulminant hepatic failure (FHF) in rats. In present study, we evaluated the hepatoprotective activity of Genistein in rat model of chronic liver damage and liver fibrosis. MethodsLiver fibrosis was induced by intraperitoneal injection of D-GalN (250 mg/kg BW) twice a week for 12 weeks. Genistein (5 mg/kg BW) was given via intra-gastric route as co-treatment daily for 12 weeks. ResultsGenistein co-treatment significantly attenuated D-GalN-induced chronic liver damage and liver fibrosis as evident from a significant amelioration in functional impairment, including inhibition of the activation of Hepatic stellate cells (HSC), decreased expression in alpha smooth muscle actin (α-SMA) and accumulation of collagen matrix, and an elevation in serum alanine transaminase (ALT) and aspartate transaminase (AST) level. In addition Genistein co-treatment was associated with elevated expression of hepatic Smad7, which ultimately blunts the expression of TGF-β and the activation of TGF-β/Smad signaling. Furthermore Genistein significantly prevented the histopathological changes induced by D-GalN. ConclusionOur results suggest that Genistein could be a novel therapeutic/nutraceutical agent in treating chronic liver damage and liver fibrosis. In addition our study also suggests a possible mechanism of action in which Smad7-induced inhibition of TGF-β/Smad2/3 can be a central mechanism by which Genistein protects liver from chronic injury.

Role of neutrophils in treatment of rats with D-galactosamine-induced acute liver failure with bone marrow mesenchymal stem cells

Objective: To investigate the therapeutic effect of bone marrow mesenchymal stromal cell (BMSC) transplantation on D-galactosamine-induced acute liver failure in Sprague-Dawley (SD) rats, as well as the mechanism of neutrophils in this process. Methods: A total of 39 male SD rats were divided into control group (8 rats, intraperitoneal injection of isotonic saline), model group (10 rats, intraperitoneal injection of D-galactosamine), solvent group (9 rats, tail vein injection of isotonic saline at 2 hours after intraperitoneal injection of D-galactosamine), and treatment group (12 rats, tail vein injection of MSCs at 2 hours after intraperitoneal injection of D-galactosamine). The rats were sacrificed at 24 hours after the model of D-galactosamine-induced acute liver failure was established, and the blood and liver tissue were harvested. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and total bilirubin (TBil) were measured, and blood analysis was performed to measure the number and percentage of neutrophils in peripheral blood. Immunofluorescence assay was used to measure the expression of the neutrophil marker Ly6g in the liver, the myeloperoxidase (MPO) kit was used to measure the activity of MPO in liver, and RT-PCR was used to measure the mRNA expression of inflammatory cytokines and chemokines in the liver, i.e., tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interferon-γ(IFN-γ), interleukin-10 (IL-10), CXC chemokine ligand 1 (CXCL1), and CXC chemokine ligand 2 (CXCL2). Another 64 male SD rats were randomly divided into groups, and the survival rates of rats in each group were observed for 7 days. The independent samples t-test was used for comparison between any two groups (Levene homogeneity test of variance, and the corrected t-test was used for a P value of < 0.05), and the log-rank test was used for comparison of survival rates between any two groups. Results: At 24 hours after acute liver failure was induced by D-galactosamine in the SD rats, there were significant increases in the liver function parameters (ALT: 2884.1±541.0 U/L vs 45.4±11.0 U/L,P< 0.001; AST: 3634.9±755.9 U/L vs 143.9±23.7 U/L,P< 0.001; TBil: 44.4±8.4μmmol/L vs 0.9±0.2μmmol/L,P< 0.001) and the number and percentage of peripheral blood neutrophils [number: (4.7±1.1)×109 vs (1.4±0.4)×109,P< 0.001; percentage: 44.9%±8.0% vs 18.3%±4.4%,P< 0.001]. A large number of neutrophils aggregated in the liver tissue, and there were significant increases in the MPO activity (4.72±1.09 U/g vs 1.13±0.24 U/g,P< 0.001), inflammatory cytokines, and chemokines. Compared with the model group, the treatment group showed significant improvements in liver function (ALT: 1 823.9±389.2 U/L vs 2 884.1±541.0 U/L,P< 0.001; AST: 2173.0±567.3 U/L vs 3634.9±755.9 U/L,P< 0.001; TBil: 30.9±6.5μmmol/L vs 44.4±8.4μmmol/L,P< 0.001) and survival rate (50% vs 12.5%,P= 0.023). Meanwhile, the treatment group also showed significant reductions in the number and percentage of peripheral blood neutrophils [number: (3.5±1.0)×109 vs (4.7±1.1)×109,P= 0.012; percentage: 35.9%±8.9% vs 44.9%±8.0%,P= 0.021], number of neutrophils in the liver, and MPO activity (3.52±1.03 U/g vs 4.72±1.09 U/g,P= 0.040), as well as significantly inhibited expression of inflammatory cytokines and chemokines (TNF-α: 2.458±0.762 vs 3.778±1.046, P = 0.005; IL-1β: 2.498±0.547 vs 4.065 ± 0.953,P= 0.002; IFN-γ: 3.977±1.039 vs 5.418±1.255, P = 0.025; IL-10: 6.056±1.542 vs 3.368±0.952,P= 0.001; CXCL1: 7.988±1.911 vs 10.366±1.239,P= 0.010; CXCL2: 3.441±1.005 vs 4.847±1.113,P= 0.019). Conclusion: BMSC transplantation has a therapeutic effect on D-galactosamine-induced acute liver failure in rats, and this process is accompanied by reduced aggregation and activity of neutrophils in peripheral blood and liver. Inflammatory cytokines and chemokines may be involved in the mechanism of regulation of these two aspects.

Role of autophagy in acute liver failure induced by D-galactosamine/lipopolysaccharide in mice

Objective: To investigate the expression and role of autophagy in the progression of acute liver failure (ALF) using the mouse model of ALF induced by D-galactosamine/LPS (D-GalN/LPS). Methods: The C57BL/6 mice were used, and intraperitoneal injection of D-galactosamine (D-GalN) and lipopolysaccharide (LPS) was performed to establish the mouse model of ALF. The mice were divided into control group and 2-, 4-, and 6-hour D-GalN/LPS-induced ALF model groups. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess liver function, and the pathological changes in liver tissue were observed to evaluate the status of liver injury. Quantitative real-time PCR was used to measure the expression of autophagy-related genes, Western blot was used to measure the expression of autophagy-related proteins in liver tissue, and a fluorescence microscope was used to observe the expression of autophagosome in the progression of liver failure. A one-way ANOVA was used for comparison of means of multiple samples between any two groups (LSD-t test for data with homogeneity of variance and Games-Howell method for data with heterogeneity of variance).P< 0.05 was considered statistically significant. Results: The ALF model groups showed gradual liver impairment over the time of D-GalN/LPS stimulation. There were significant increases in ALT and AST after 4 hours; the pathological injury of liver tissue gradually aggravated over the time of D-GalN/LPS stimulation and fulfilled the criteria for ALF at 6 hours. The mRNA and protein expression of autophagy-related genes (ATG-7, ATG-5, Beclin-1, Lamp-1, and LC3a) increased in the early and medium stages of ALF (2 and 4 hours) and decreased after ALF progressed to liver failure (6 hours). As was observed via the fluorescence microscope, the 4-hour D-GalN/LPS-induced ALF model group showed the highest expression of autophagosome. Conclusion: The expression of autophagy gradually increases in the early and medium stages of ALF and decreases when ALF progresses to liver failure. Therefore, autophagy plays an important role in the pathogenesis of ALF.

Effects of Salmonella infection on hepatic damage following acute liver injury in rats

Acute liver injury is a common clinical disorder associated with intestinal barrier injury and disturbance of intestinal microbiota. Probiotic supplementation has been reported to reduce liver injury; however, it is unclear whether enteropathogen infection exacerbates liver injury. The purpose of this study was to address this unanswered question using a rat model. Oral supplementation with Salmonella enterica serovar enteritidis (S. enteritidis) was given to rats for 7 days. Different degrees of acute liver injury were then induced by intraperitoneal injection of D-galactosamine. The presence and extent of liver injury was assayed by measuring the concentrations of serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin. Histology was used to observe liver tissue damage. Additionally, we measured the changes in plasma endotoxin, serum cytokines and bacterial translocation to clarify the mechanisms underlying intestinal microbiota associated liver injury. The levels of liver damage and endotoxin were significantly increased in the Salmonella infected rats with severe liver injury compared with the no infection rats with severe liver injury (P<0.01); The peyer's patch CD3+ T cell counts were increased significantly when the Salmonella infection with severe injury group was compared with the normal group (P<0.05). S. enteritidis pretreatment enhanced intestinal barrier impairment and bacterial translocation. Oral S. enteritidis administration exacerbates acute liver injury, especially when injury was severe. Major factors of the exacerbation include inflammatory and oxidative stress injuries induced by the translocated bacteria and associated endotoxins, as well as over-activation of the immune system in the intestine and liver.

Lipoxin A4 exerts protective effects against experimental acute liver failure by inhibiting the NF-κB pathway.

Although rare, acute liver failure (ALF) is associated with high levels of mortality, warranting the development of novel therapies. Nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) play roles in ALF. Lipoxin A4 (LXA4) has been shown to alleviate inflammation in non-hepatic tissues. In the present study, we explored whether LXA4 exerted hepatoprotective effects in a rat model of ALF. A rat model of ALF was generated by intraperitoneal injections of D-galactosamine (300 mg/kg) and lipopolysaccharide (50 µg/kg). Animals were randomly assigned to: control group (no ALF); model group (ALF); and the groups treated with a low dose (0.5 µg/kg), medium dose (1 µg/kg), and high dose (2 µg/kg) of LXA4 (all with ALF); and pyrrolidine dithiocarbamate (PDTC)-treated group (ALF and 100 mg/kg PDTC, an inhibitor of NF-κB). Liver histology was measured using H&E staining, serum levels by ELISA, and liver mRNA expression was measured by RT-PCR for the detection of the pro‑inflammatory cytokines TNF-α and IL-6. Liver cell apoptosis (as measured using the TUNEL method and examining caspase-3 activity), and Kupffer cell NF-κB activity [using an electrophoretic mobility shift assay (EMSA)] were examined. Serum levels of transaminases, TNF-α and interleukin-6 (IL-6) were substantially higher in the model group compared to controls. In the model group, significant increases in TNF-α and IL-6 mRNA expression, TUNEL‑positive cells, and caspase-3 activity in the liver tissue were noted. LXA4 improved liver pathology and significantly decreased the indicators of inflammatory response and apoptosis in a dose-dependent manner. High-dose LXA4 provided better protection than PDTC. LXA4 administration significantly decreased NF-κB expression in hepatocytes and Kupffer cells. These results indicated that LXA4 inhibited NF-κB activation, reduced the secretion of pro-inflammatory cytokines, and inhibited apoptosis of liver cells, thereby exerting protective effects against ALF.

Association between protective effect of Liuwei Wuling tablets against acute liver injury and its inhibitory effect on cytoplasmic translocation of high-mobility group box-1 in hepatocytes in mice

To investigate the effect of Liuwei Wuling tablets on the cytoplasmic translocation and release of high-mobility group box-1 (HMGB1) in hepatocytes in mice with acute live injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). A Balb/c mouse model of acute liver injury was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (5 ug/kg). A total of 24 healthy mice were randomly and equally divided into acute liver injury control group and Liuwei Wuling tablet treatment group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in both groups at each time point within one week. Liver tissues were collected at 36 hours to perform pathological examination. The serum levels of HMGB1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), complement 3a (C3a), and complement 5a (C5a) were measured. Immunohistochemistry was used to determine the expression and cytoplasmic translocation of HMGB1 in hepatocytes. At 6, 12, and 24 hours, the Liuwei Wuling tablet treatment group had significantly lower serum levels of ALT than the control group (225.33±181.64 U/L vs 471.17±174.72 U/L, t = 3.38, P < 0.01; 1509.53±182.51 U/L vs 7149.52±734.25 U/L, t = 25.82, P < 0.01; 162.89±86.51 U/L vs 1318.16±557.71 U/L, t = 7.09, P < 0.01), as well as significantly lower serum levels of AST than the control group (179.22±94.57 U/L vs 561.91±209.6 U/L, t = 5.76, P < 0.01; 590.92±190.92 U/L vs 2266.48±705.64 U/L, t = 7.94, P < 0.01; 231.24±87.7 U/L vs 444.32±117.01 U/L, t = 5.05, P < 0.01). The treatment group had significantly lower levels of HMGB1 than the control group at 6 and 12 hours (54.21±11.89 ng/ml vs 72.07±13.65 ng/ml, t = 3.41, P < 0.01; 49.23±5.97 ng/ml vs 68.71±13.07 ng/ml, t = 4.70, P < 0.01). The treatment group had significantly lower levels of TNF-α, IL-1β, and IL-6 than the control group at 12 hours (163.62±9.12 pg/ml vs 237.09±51.47 pg/ml, t = 4.86, P < 0.01; 15.66±13.13 pg/ml vs 37.43±18.68 pg/ml, t = 3.30, P < 0.01; 7.10±3.06 pg/ml vs 21.42±8.23 pg/ml, t = 5.65, P < 0.01). The treatment group had significantly lower levels of C3a and C5a than the control group at 12 hours (2.52±1.27 pg/ml vs 9.83±2.96 ng/ml, t = 7.86, P < 0.01; 2.16±1.03 ng/ml vs 7.23±1.55 ng/ml, t = 9.67, P < 0.01). Compared with the control group, the treatment group had significantly reduced liver inflammation and necrosis, and a significantly lower cytoplasmic translocation rate of HMGB1 in hepatocytes (38.76%±7.37% vs 8.15%±2.11%, P < 0.01). Liuwei Wuling tablets can reduce the cytoplasmic translocation of HMGB1 in hepatocytes and relieve liver inflammation in mice with acute liver injury.

Aqueous extract from Aconitum carmichaelii Debeaux reduces liver injury in rats via regulation of HMGB1/TLR4/NF-ΚB/caspase-3 and PCNA signaling pathways

Ethnopharmacological relevanceAconitum carmichaelii Debeaux is a well-known Chinese herb that has been used to treat liver diseases for many years in China. We investigated the effects of aqueous extract from Aconitum carmichaelii Debeaux (AEACD) on acute liver failure and identified the possible mechanisms of these effects. Material and methodsSpecific pathogen-free (SPF) male Wistar rats were used to establish acute liver failure model by intraperitoneal injection of D-galactosamine (D-GalN) and treated with Stronger Neo-Minophagen C (SNMC) and AEACD by gavage. Then, the serum biochemical parameters, the pathological scores in the liver tissue, the mRNA expressions of toll- like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), high mobility group box 1 (HMGB1) and caspase-3, the proliferating cell nuclear antigen (PCNA) positive rates were analyzed. ResultsThe liver function was improved, the pathological scores were decreased, the expressions the TLR4, NF-κB, HMGB1, and caspase-3 were inhibited, and the PCNA positive rates were increased by both SNMC and AEACD, but AEACD induced greater effects. ConclusionsAEACD protected liver function by inhibiting inflammatory reaction, apoptosis and promoting liver tissue regeneration in the acute liver failure rats induced by D-galactosamine.