Effect of Wenyang Jiedu Huayu Granule on TLR4 mRNA, TNF-α and Hepatocyte Apoptosis in IETM Rats with Hepatic Failure / 中国实验方剂学杂志

Objective::To investigate the relationship between Toll-like receptor 4 (TLR4) mRNA and its downstream inflammatory factor-tumor necrosis factor-α (TNF-α) and hepatocyte apoptosis in mice with intestinal endotoxemia (IETM) of hepatic failure, and explore the regulatory mechanism of Wenyang Jiedu Huayu granule on endotoxin-induced hepatocyte apoptosis. Method::The 85 SPF male SD rats were randomly divided into normal group, model group, TLR4 monoclonal antibody group and Wenyang Jiedu Huayu granule group. D-galactosamine (D-Gal) intraperitoneal injection was performed to establish the IETM model of hepatic failure. The TLR4 monoclonal antibody group and the Wenyang Jiedu Huayu granule group were given Wenyang Jiedu Huayu granule solution by gavage 5 days before the modeling. The normal group and the model group were given isovolumetric distilled water. Each group was given by gavage until sacrifice. Rats in each group were randomly sacrificed at 24, 48, 72 h, respectively, and samples were collected. The levels of serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected at 24, 48, 72 h. The pathological changes of liver tissue were observed by hematoxylin and eosin(HE) staining. The expression of TLR4 in liver tissue was detected by real-time fluorescence quantitative PCR (Real-time PCR). The expression of TNF-α in liver tissue was detected by enzyme-linked immunosorbent assay (ELISA). The apoptotic rate of hepatocytes was detected by flow cytometry. Result::Compared with the normal group, ALT and AST were increased in model group, while the pathological injury degree of liver tissue was significantly increased. The expressions of TLR4 mRNA and TNF-α were increased (P<0.05, P<0.01), whereas the apoptosis rate of liver cells was significantly increased (P<0.05, P<0.01). Compared with the model group, ALT and AST were decreased in Wenyang Jiedu Huayu granule group (P<0.01), and the degree of pathological injury of liver tissue was significantly reduced (P<0.05). The expressions of TLR4 mRNA and TNF-α were significantly decreased (P<0.01), and the apoptosis rate of liver cells was also reduced (P<0.01). Conclusion::TLR4 mRNA and TNF-α are positively correlated with hepatocyte apoptosis in liver failure. Wenyang Jiedu Huayu granule can improve liver function, alleviate liver cell injury and reduce liver cell apoptosis in IETM mice with hepatic failure. The mechanism may be related to its ability to down-regulate the expression of liver TLR4 mRNA, inhibit the release of TNF-α, and reduce the rate of hepatocyte apoptosis.

Effect of Warming and Heat-clearing Method on Expressions of FoxP3 and ROR-γt in Colon Tissue of Rats with Intestinal Endotoxemia Caused by Acute-on-chronic Liver Failure / 中国实验方剂学杂志

Objective::To study the effect of warming and heat-clearing method (Wenyang Jiedu Huayu decoction) on the expressions of Forkhead box P3 (FoxP3), Retinoic acid-related orphan receptor gamma t (ROR-γt) in colon tissue of mice with acute-on-chronic liver failure (ACLF), in order to explore the possible regulatory mechanism on intestinal endotoxemia (IETM) in liver failure mice. Method::The 130 SD rats were randomly divided into normal group (10 rats) and model group (120 rats). The ACLF mice model was established through the subcutaneous injection with bovine serum albumin and the intraperitoneal injection with D-galactosamine(D-Gal) and lipopolysaccharide (LPS). The model mice were randomly divided into model group, heat-clearing group (Yinchenhao decoction, 6.68 g·kg-1), warming group (Yinchen Zhufu decoction, 7.09 g·kg-1) and warming and heat-clearing group (Wenyang Jiedu Huayu decoction, 19.53 g·kg-1). The normal group and the model group were given distilled water by gastric lavage, while the other groups were given equal volume of corresponding Chinese herbal medicines for a week. The value of each index at 1, 12 and 24 h was measured. The ratio of Treg/Th17 cell in peripheral blood were detected and calculated by flow cytometry. Real-time fluorescence quantitative PCR (Real-time PCR) was used to detect the expressions of FoxP3 and ROR-γt in colon tissues of mice at different time points. In situ hybridization and immunohistochemistry were used to observe the expressions of FoxP3 and ROR-γt genes and proteins. Result::Compared with normal group, the ratio of Treg/Th17 in the model group decreased significantly at each time point (P<0.01). Compared with the model group, the Treg/Th17 ratio increased only in the warming and heat-clearing method group (P<0.05). Compared with normal group, the expression of ROR-γt in the model group was significantly higher (P<0.01), and the expression of ROR-γt in the model group was higher than FoxP3.Compared with the model group, the expressions of FoxP3 and ROR-γt mRNA in the heat-clearing group and the warming group decreased at each time point (P<0.05), and the expressions of FoxP3 and ROR-γt in the warming and heat-clearing method group decreased significantly (P<0.01). The expressions of FoxP3 and ROR-γt mRNA in warming and heat-clearing group decreased compared with those in the warming group and heat-clearing group (P<0.05). Conclusion::The mechanism of the warming and heat-clearing method on IETM in liver failure may be related to the regulation of FoxP3 and ROR-γt expressions.

Linderae radix ethanol extract attenuates alcoholic liver injury via attenuating inflammation and regulating gut microbiota in rats

This study aimed to explore the influence of gut microbiota alterations induced by Linderae radix ethanol extract (LREE) on alcoholic liver disease (ALD) in rats and to study the anti-inflammatory effect of LREE on ALD through the lipopolysaccharide (LPS) toll-like receptor 4 (TLR4)-nuclear factor kappa B (NF-κB) pathway. ALD rat models were established by intragastric liquor [50% (v/v) ethanol] administration at 10 mL/kg body weight for 20 days. Rats were divided into six groups: normal group (no treatment), model group (ALD rats), Essentiale group (ALD rats fed with Essentiale, 137 mg/kg), and LREE high/moderate/low dose groups (ALD rats fed with 4, 2, or 1 g LREE/kg). NF-κB and LPS levels were evaluated. Liver pathological changes and intestinal ultrastructure were examined by hematoxylin and eosin staining and transmission electron microscopy. The gut microbiota composition was evaluated by 16S rDNA sequencing. Expression levels of TLR4 and CD68 in liver tissue, and occludin and claudin-1 in intestinal tissue were measured. LREE treatment significantly reduced NF-κB and LPS levels, improved liver pathological changes, and ameliorated intestinal ultrastructure injury. Meanwhile, LREE-fed groups showed a higher abundance of Firmicutes and a lower abundance of Bacteroidetes than the rats in the model group. Administration of LREE suppressed TLR4 overexpression and promoted the expression of occludin and claudin-1 in intestine tissue. Thus, LREE could partly ameliorate microflora dysbiosis, suppress the inflammatory response, and attenuate liver injury in ALD rats. The protective effect of LREE might be related to the LPS-TLR4-NF-κB pathway.

Theǎn soup of Miao nationality in Guizhou province improving intestinal barrier function in the acute liver failure rats / 重庆医学

Objective To investigate the protective effect of ǎn soup of Miao nationality on the intestinal barrier function in rats with acute liver failure ,in order to provide effective diet measures for hepatic failure patients .Methods A total of 50 male SD rats were randomly assigned to five groups :control group(group A) ,acute liver failure model group(group B) ,Bifidobacterium tri‐ple probiotics group(group C) ,high‐doseǎn soup group(group D) and low‐doseǎn soup group(E) ,10 cases in each group .The last four groups were subjected to the acute liver failure model by hypodermic injection with thioacetamide twice .In addition ,the last three groups were respectively intragastrically perfused with Bifidobacterium triple probiotics ,6 mL of ǎn soup and 1 .5 mL of ǎn soup before and during building the acute liver failure model .28 hours after the second injection ,femoral arterial blood to was drew to test serum endotoxin(ETX) ,diamine oxidase(DAO) ,D(‐)‐lactate(D‐lac) ,alanine aminotransferase (ALT) and aspartate amin‐otransferase(AST) .At the same time ,hepatic tissue and ileal tissue within 3 cm away from the ileocecal region were collected to do pathological examination .Results Pathological examination results showed that hepatic cord in hepar arranged mussily ,hepatic lob‐ules structure disordered ,hepatocyte focal necrosis or with large necrotic areas in which a large number of inflammatory cell infiltra‐tion in the acute liver failure model group .The pathology damage of liver in the other groups was almost in the same extent .The ile‐um mucosa in the group A was morphologically intact with clear structure of villi and lined up ,while that of group B was disorder with sparse villi ,epithelial cells in different degree of loss ,missing and necrosis ,lamina propria obviously hyperemia and there were large amount of inflammatory cellular infiltration .Intestinal mucosa injury in the other intervention groups was lighter than that in the group B .In particular ,levels of serum ETX ,D‐Lac ,DAO ,ALT and AST in the group B and other intervention groups were sig‐nificantly higher than that in the group A(P0 .05) .However ,there was no signif‐icant difference between group C and group D (P>0 .05) ,when obvious difference was observed between group C and group E(P<0 .05) .There was significant difference between group E and roup D (P<0 .05) .Conclusion Results demonstrated that ǎn soup protected intestinal barrier function of acute liver failure rats by reducing the production and release of serum endotoxin content in liver failure rats ,lowering intestinal endotoxemia (IETM ) ,which seems to prevent subsequent liver injury caused by IETM and have certain dietotherapy effect on liver failure .

Effect of Wen-Yang Jie-Du Hua-Yu Granules on TLR4 and NF-кB Expression of Rat Hepatic Failure Model with Intestinal Endotoxemia / 世界科学技术-中医药现代化

phological changes in rat liver tissues. TLR4 and NF-κB expression in the liver tissues were measured by im-munohistochemistry . The results showed that compared with the normal group , the serum levels of transaminase ( ALT , AST ) and endotoxin of the model group were higher ( P < 0 . 01 ); and the degree of liver pathology injury was significantly increased; the TLR4 and NF-кB expression were increased (P < 0.01). Compared with the model group, the serum levels of transaminase (ALT, AST) and endotoxin of the experimental group were lower (P < 0.01), the degree of liver pathology injury was significantly lighter; the TLR4 and NF-кB expression were significantly lower (P < 0.01). It was concluded that the WYJDHY granules has a good role in the prevention and treatment of liver injury of rat model of hepatic failure IETM through the downregulation of liver expression of TLR4 and NF-кB in rat liver tissues , reducing serum levels of endotoxin , which may be one of the mecha-nisms on hepatic failure treatment .

Anti-histamine treatment on intestinal endotoxemia and liver inflammation in experimental chronic hepatitis rats / 中华临床感染病杂志

Objective To evaluate the effect of anti-histamine treatment on intestinal endotoxemia and liver inflammation in experimental chronic hepatitis rats.Methods Thirty Wistar rats (15 males and 15 females) were randomly divided into control group (n =8),chronic hepatitis group (n =12) and hepatitis + anti-histamine group (n =10).Chronic hepatitis was induced by subcutaneous injection with 40％ of CCl4,and feeding with low protein,low choline,high cholesterol and high alcohol diet.Antihistamine treatment was given 1 week after the modeling by intragastric administration of ketotifen (1.25 mg/kg).All rats were sacrificed 4 weeks later.Plasma endotoxin,alanine aminotransferase (ALT),total bilirubin (TBil),tryptase,histamine,interferon-γ (IFNγ),iuterleukin (IL)-12,IL-10 and IL-4levels were detected,and the changes in liver histology,the morphology and ultrastructure of mast cells were observed.SPSS 13.0 software package was used for statistical analysis.ANOVA was used for the comparison of measurement data,and SNK method was used for pairwise comparison.Results Plasma endotoxin,ALT,TBil,tryptase,plasma and liver tissue histamine concentrations were (81 ± 19) pg/mL,(186 ± 140) U/L,(10.2±6.2) μmol/L,(0.75 ±0.21) mg/mL,(145 ±52) ng/mL,and (107 ±43) ng/100 mg in chronic hepatitis group,while the above parameters were significantly lower in anti-histamine group except TBil (P ＜ 0.05).Under light microscope,fatty degeneration and fibrosis were formed in liver of chronic hepatitis rats,the hepatic injury was attenuated in anti-histamine group.Toluidine blue stain showed that there was many degranulating and degranulated mast cells filled with purple granula around liver blood vessels and in fiber-interval in chronic hepatitis group,and there were few purple granula in anti-histamine group.The number of mast cells in anti-histamine group was (6.5 ± 1.5)/HP,which was significantly lower than chronic hepatitis group [(10.9 ± 1.6)/HP,P =0.000],but was still higher than that in the control group [(2.2 ± 0.9)/HP,P =0.000].Under electron microscope,the phenomenon of degranulation was severe in chronic hepatitis group and moderate in the anti-histamine group.Compared with the chronic hepatitis group,IL-4 and IL-10 in anti-histamine group were significantly decreased (P ＜0.05),IL-12 was increased (P ＜0.05),but the level of IFN-γ had no significant change (P ＞ 0.05).Conclusion Anti-histamine therapy can significantly improve liver inflammation and alleviate intestinal endotoxemia.