Effect of Xuanfuhua decoction on a mouse model of nonalcoholic steatohepatitis induced by high-fat, high-fructose, and high-cholesterol diet / 临床肝胆病杂志

Objective To investigate the intervention effect of Xuanfuhua decoction on mice with nonalcoholic steatohepatitis (NASH) induced by high-fat, high-fructose, and high-cholesterol diet. Methods A total of 32 male C57/BL6J mice were randomly divided into normal group, model group, Xuanfuhua decoction group, and obeticholic acid group, with 8 mice in each group. Since week 24 of modeling using high-fat, high-fructose, and high-cholesterol diet, each group was given the corresponding drug for intervention at a dose of 14.19 g/kg by gavage for the Xuanfuhua decoction group and 10 mg/kg by gavage for the obeticholic acid group and a volume of 20 mL/kg for gavage, once a day for 6 consecutive weeks. HE staining, oil red O staining, Sirius Red staining, and Masson staining were used to observe the pathological changes, lipid deposition, and collagen deposition of liver tissue; related kits were used to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and glucose, as well as the content of TG and hydroxyproline (Hyp) in liver tissue; quantitative real-time PCR was used to measure the expression of genes associated with lipid metabolism, inflammation, and fibrosis in liver tissue; immunohistochemical staining was used to observe the positive expression of F4/80 and α-SMA in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the normal group, the model group had significant increases in body weight, liver wet weight, and serum levels of AST, ALT, TC, TG, LDL-C and glucose (all P < 0.01). HE staining showed hepatocyte steatosis, a large number of fat vacuoles, hepatocyte ballooning degeneration, and inflammatory cell infiltration in liver tissue of the mice in the model group, and the model group had a significant increase in NAFLD activity score (NAS) compared with the normal group ( P < 0.01). Oil red O staining showed the deposition of a large number of red lipid droplets with different sizes in hepatocytes of the mice in the model group, and compared with the normal group, the model group had significant increases in the area percentage of oil red O staining and the content of TG in the liver ( P < 0.01). Sirius Red staining and Masson staining showed significant collagen fiber hyperplasia in the perisinusoidal area, the central vein, and the portal area in the model group, and the model group had a significant increase in the content of Hyp in liver tissue compared with the normal group ( P < 0.05). Compared with the model group, the Xuanfuhua decoction group had significant reductions in the serum levels of AST, ALT, TC, TG, LDL-C, and glucose (all P < 0.05), significant improvements in hepatic steatosis, inflammatory infiltration, lipid droplet deposition, and collagen fiber hyperplasia, and significant reductions in NAS score, area percentage of oil red O staining, and content of TG and Hyp in the liver (all P < 0.05). Compared with the normal group, the model group had significant increases in the mRNA expression levels of lipid metabolism-related genes (SREBP-1c, FASN, SCD-1, PPAR-γ, and CD36), inflammation-related genes (F4/80, TNF-α, CCL2, and CD11b), and the fibrosis-related gene α-SMA (all P < 0.05), and immunohistochemical staining showed significant increases in the positive expression of F4/80 and α-SMA ( P < 0.01). Compared with the model group, the Xuanfuhua decoction group had significant reductions in the mRNA expression levels of SREBP-1c, FASN, SCD-1, PPAR-γ, CD36, F4/80, TNF-α, CCL2, CD11b, and α-SMA in liver tissue (all P < 0.05), and immunohistochemical staining showed significant reductions in the positive expression of F4/80 and α-SMA ( P < 0.01). Conclusion Xuanfuhua decoction has a good intervention effect on mice with NASH induced by high fat, high fructose, and high-cholesterol diet and can significantly inhibit hepatic lipid deposition, inflammatory response, and liver fibrosis.

Expression and significance of response gene to complement 32 in liver regeneration after partial hepatectomy in mice / 临床肝胆病杂志

ObjectiveTo investigate the expression and role of response gene to complement 32 （RGC32） in liver regeneration after partial hepatectomy （PH）. MethodsA total of 42 male C57BL/6 mice， aged 10 weeks， were randomly divided into control group， postoperative day 1 group （1－d group）， postoperative day 2 group （2－d group）， postoperative day 4 group （4－d group）， postoperative day 6 group （6－d group）， postoperative day 8 group （8－d group）， and postoperative day 10 group （10－d group）， with 6 mice in each group. In the control group， the complete liver of the mice was resected for weighing and photography as the normal control group （sham group）； further， the left and middle lobes of the liver were resected for weighing and photography as the surgical control group （0－day group）； the sham group and the 0－day group shared the same group of mice. After successful modeling by PH， the mice were sacrificed on days 1， 2， 4， 6， 8， and 10 after surgery， and the liver was collected to measure the change in size. HE staining and oil red O staining were used to evaluate liver histomorphological changes； serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were measured to evaluate the changes in liver function； immunohistochemical staining was used to measure the expression of proliferating cell nuclear antigen （PCNA） and Ki67 and analyze the change in cell proliferation during liver regeneration； quantitatie real－time PCR and immunohistochemical staining were uused to measure the expression and subcellular distribution of RGC32 during liver regeneration； EdU cell proliferation assay was used to analyze the effect of RGC32 overexpression or knocknout on hepatocyte proliferation in L02 cells. For continuous data， comparison between multiple groups was made by analysis of variance， and further pairwise comparisons were conducted using the LSD－t test. The independent samples t－test was used for comparison of continuous data between two groups. A Pearson correlation analysis was performed. ResultsThe liver gradually enlarged after PH， and the liver/body weight ratio rose to the peak from days 0 to 6， with significant differences between different time points （all P<0.05）， while there was no significant change in liver size from days 6 to 10. The number of liver lipid droplets significantly increased after PH surgery and gradually decreased with liver regeneration， with a significant difference between the portal vein region and the central vein region （all P<0.05）. Compared with the sham group， the 1d group had significant increases in the serum levels of ALT and AST （all P<0.05）， which gradually returned to the levels of the sham group on day 6 and day 2 after surgery， respectively （P>0.05）. Immunohistochemical staining showed that there were rapid increases in the numbers of PCNA－ and Ki67－positive liver parenchymal cells after PH surgery， with the highest numbers of 86±5 and 89±5， respectively， on day 2， which then gradually decreased； however， there were gradual increases in the numbers of PCNA－ and Ki67－positive nonparenchymal cells， with the peak numbers of 34±5 and 25±3， respectively， on day 6， which then gradually decreased. The total expression of RGC32 increased to the highest level on day 2 after PH surgery and then gradually decreased， and the changing trend of RGC32 expression in cytoplasm was consistent with that of total RGC32 expression； however， the expression of RGC32 in nucleus decreased to the lowest level on day 2 after PH surgery and then increased gradually. The correlation analysis showed that the expression of RGC32 in nucleus was negatively correlated with the proliferation of liver parenchymal cells （R2=0.308 3， P=0.016 7）， and the expression of RGC32 in cytoplasm was positively correlated with the proliferation of liver parenchymal cells （R2=0.808 6， P<0.000 1）. Cell experiments showed that compared with the control group， the EdU－positive rate was reduced by 15.6% after RGC32 overexpression （P<0.01） and was increased by 19.2% after RGC32 knockdown （P<0.01）. ConclusionLiver parenchymal cells and nonparenchymal cells show asynchronous proliferation and participate in liver regeneration together. During liver regeneration after hepatectomy， there are differences in the expression of RGC32 between nucleus and cytoplasm， and RGC32 in nucleus may inhibit hepatocyte proliferation.

Counteractive effect of mouse dermal fibroblasts during their adipogenic differentiation against Staphylococcus aureus infection and its mechanisms / 中华皮肤科杂志

Objective:To investigate the counteractive effect of mouse dermal fibroblasts (MdFBs) during their adipogenic differentiation against Staphylococcus aureus infection, and to explore its mechanisms. Methods:MdFBs were obtained from newborn C57BL/6 mice, and their adipogenic differentiation was induced by culture in an adipogenic medium for 48 hours. Real-time fluorescence-based quantitative PCR (RT-PCR) was performed to determine the mRNA expression of cathelicidin antimicrobial peptide (CAMP) on days 0-6 during the adipogenic differentiation of MdFBs, and Western blot analysis to determine the protein expression of CAMP in the culture supernatant of MdFBs during their adipogenic differentiation. MdFBs were divided into 4 groups: co-stimulation group stimulated by S. aureus suspensions and cultured in an adipogenic medium, adipogenic control group cultured in an adipogenic medium, S. aureus-stimulation group stimulated by S. aureus suspensions and cultured in a common medium, and control group stimulated by phosphate-buffered saline and cultured in a common medium; Western blot analysis and RT-PCR were conducted to determine the protein and mRNA expression of CAMP. S. aureus (5 × 10 4 CFU/ml) was cultured with the culture supernatant of MdFBs after 5-day adipogenic differentiation (adipogenic group), and the growth activity was evaluated every 2 hours during 10 - 24 hours after the start of co-culture; S. aureus cultured with the culture supernatant of MdFBs in a common medium served as the normal control group, and that cultured with cell-free culture supernatant served as the negative control group. Differences between groups were assessed using unpaired t-test or analysis of variance. Results:Significant differences were observed in the relative mRNA expression of CAMP among different time points (days 0, 1, 2, 4, and 6) during the adipogenic differentiation of MdFBs (1.14 ± 0.74, 68.04 ± 12.72, 683.12 ± 38.06, 1 390.68 ± 226.21, 454.57 ± 204.12, F = 50.08, P < 0.001) ; the CAMP mRNA expression was significantly higher on days 1, 2, 4, and 6 than on day 0 ( t = 9.09, 31.03, 10.63, 3.85, respectively, all P < 0.05), and showed an initial rise and subsequent fall during days 0 - 6. The CAMP protein expression in the culture supernatant of MdFBs peaked on days 2-5 and subsequently decreased. Significant differences were observed in the mRNA and protein expression of CAMP among the control group, S. aureus-stimulation group, adipogenic control group and co-stimulation group (mRNA: 0.08 ± 0.02, 0.38 ± 0.10, 0.49 ± 0.11, 0.80 ± 0.03, respectively, F = 43.25, P < 0.05; protein: 0.433 ± 0.176, 0.574 ± 0.176, 1.007 ± 0.176, 1.217 ± 0.176, respectively, F = 46.79, P < 0.05), and the relative mRNA and protein expression of CAMP was significantly higher in the co-stimulation group than in the adipogenic control group, S. aureus-stimulation group and control group (all P < 0.05). At 10 hours during culture, the growth activity of S. aureus was significantly lower in the adipogenic group (0.053 ± 0.015) than in the normal control group and negative control group (0.109 ± 0.015, 0.106 ± 0.015, t = 11.30, 13.26, respectively, both P < 0.05) ; during 10 - 24 hours, the growth activity of S. aureus also showed a significant decrease in the adipogenic group compared with the normal control group and negative control group (all P < 0.05) . Conclusion:MdFBs secreted CAMP during the adipogenic differentiation, and could inhibit the proliferation of S. aureus.

Effect of Compound Lingdan Capsule on liver function and fibrosis degree in mice with liver fibrosis / 临床肝胆病杂志

Objective To investigate the effect of Compound Lingdan Capsule on serum biochemical parameters and liver fibrosis degree in a mouse model of liver fibrosis. Methods A total of 125 specific pathogen-free male C57BL/6 mice were randomly divided into normal control group with 5 mice, CCl 4 model group with 15 mice, low-, middle-, and high-dose CCl 4 groups (0.8, 1.6, and 3.2 mg·g -1 ·d -1 ) with 15 mice in each group, DDC model group with 15 mice, and low-, middle-, and high-dose DDC groups (0.8, 1.6, and 3.2 mg·g -1 ·d -1 ) with 15 mice in each group. After successful modeling, the mice in the administration groups were given Compound Lingdan Capsule suspension at the respective doses by gavage, and those in the normal control group and the model group were given an equal volume of normal saline by gavage, for 4 consecutive weeks. Blood samples were collected from the eyeballs, and serum was used to measure aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and bilirubin. Liver tissue samples were collected at the same site of the right lobe of the liver for pathological observation, Sirius Red staining, α-SMA antibody staining, and COL1A1 antibody staining. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Bonferroni method was used for further comparison between two groups. Results Compared with the model group, each dose group had significant reductions in the serum level of ALT and a significant increase in the serum level of albumin after the administration of Compound Lingdan Capsule (all P < 0.05), the levels of AST and bilirubin in the middle and high dose groups were lower (all P < 0.05), and the difference of each index in the high dose group was more significant than that in the low dose group (all P < 0.05). Each dose group had varying degrees of improvement in the pathological changes of the liver and a significant reduction in the number of Sirius Red staining-positive cells, as well as varying degrees of reduction in the protein expression of α-SMA and COL1A1. Conclusion Compound Lingdan Capsule can improve liver function and reduce liver fibrosis degree in mice with liver fibrosis.

Effect of metformin on liver fibrosis in a mouse model of Budd-Chiari syndrome / 临床肝胆病杂志

Objective To investigate the effect of metformin on liver fibrosis in a mouse model of Budd-Chiari syndrome and its mechanism. Methods A total of 30 male C57 mice were randomly divided into sham-operation group (SHAM group) with 6 mice, sham operation+ metformin group (SHAM+M group) with 5 mice, Budd-Chiari model group (BCS group) with 10 mice, and Budd-Chiari model+metformin group (BCS+M group) with 9 mice. The mice in the model group were treated with partial ligation of the inferior vena cava, those in the SHAM group were not treated with ligation, and those in the metformin group were given 0.1% metformin in drinking water besides modeling. The mice were sacrificed after 6 weeks. HE staining and picrosirius red staining were used to observe liver histopathology and collagen deposition; immunohistochemistry was used to measure the expressions of α-smooth muscle actin (α-SMA) and fibrinogen; quantitative real-time PCR was used to measure the mRNA expression of hypoxia-inducible factor 1α (HIF-1α) and type Ⅰ collagen (collagen 1), and Western blot was used to measure the relative protein expression levels of HIF-1α, vascular endothelial growth factor (VEGF), fibrinogen, α-SMA, and collagen 1. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Pathological staining showed that compared with the SHAM group, the BCS group had significant liver fibrosis, disordered arrangement of hepatocytes near the central vein, sinusoidal expansion with red blood cell deposition and a small amount of inflammatory cell infiltration, and collagen deposition. The BCS group had significant increases in the mRNA expression levels of HIF-1α and collagen 1 and the protein expression levels of α-SMA, collagen 1, HIF-1α, VEGF, and fibrinogen (all P < 0.05); compared with the BCS group, the BCS+M group had significant alleviation of liver fibrosis, red blood cell deposition, and collagen deposition and significant reductions in the mRNA expression levels of HIF-1α and collagen 1 and the protein expression levels of α-SMA, collagen 1, HIF-1α, VEGF, and fibrinogen (all P < 0.05). Conclusion Metformin can improve congestive liver fibrosis caused by Budd-Chiari syndrome, possibly by reducing microthrombus in hepatic sinusoid and inhibiting the HIF-1α/VEGF pathway.

Influence of mitochondria-targeted antioxidant SS-31 on acute liver injury in a mouse model of sepsis / 临床肝胆病杂志

Objective To investigate the effect of the mitochondria-targeted antioxidant SS-31 on acute liver injury in a mouse model of sepsis. Methods A total of 24 adult male C57BL/6J mice were randomly divided into control group, control+SS-31 group, lipopolysaccharide (LPS) group, and LPS+SS-31 group, with 6 mice in each group. The mice were given intraperitoneal injection of LPS (10 mg/kg) to establish a model of sepsis and acute liver injury, followed by intraperitoneal injection of SS-31 (10 mg/kg) for treatment, and the mice in the control group were given intraperitoneal injection of an equal volume of PBS solution, followed by intraperitoneal injection of an equal volume of normal saline. After 12 hours, ELISA was used to measure the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), reactive oxygen species (ROS), superoxide dismutase (SOD), tumor necrosis factor-α (TNFα), interleukin-1β(IL-1β), and interleukin-6 (IL-6), and HE staining was used to observe liver histopathological changes. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the LPS group, the LPS+SS-31 group had significant reductions in the serum levels of ALT (140.05±12.22 U/L vs 102.64±8.75 U/L, P < 0.05) and AST (80.22±4.71 U/L vs 69.26±5.37 U/L, P < 0.05) and the levels of ROS (1 030.21±115.87 pg/mL vs 847.84±63.65 pg/mL, P < 0.05), TNFα (767.18±60.60 ng/mL vs 698.89±16.99 ng/mL, P < 0.05), IL-1β (29.97±1.37 ng/mL vs 26.70±3.09 ng/mL, P < 0.05), and IL-6 (59.13±7.09 pg/mL vs 49.29±3.41 pg/mL, P < 0.05) in liver tissue. Compared with the control group based on HE staining, the LPS group showed destruction of hepatic lobular structure, inflammatory cell infiltration, ambiguous intercellular space, and hepatocyte swelling, while the LPS+SS-31 group showed alleviation of inflammatory cell infiltration and hepatocyte swelling. Conclusion The mitochondria-targeted antioxidant SS-31 can reduce the production of ROS, downregulate the highly expressed inflammatory factors in sepsis, and alleviate sepsis-related acute liver injury in mice.

Study of muscle fibers of the extensor digitorium longus and soleus muscles of C57BL/6 females exposed to glyphosate during pregnancy and lactation / Estudo das fibras musculares dos músculos extensor longo dos dedos e sóleo de fêmeas C57BL/6 expostas ao glifosato durante a prenhez e a lactação

ABSTRACT Objective To evaluate the morphology and morphometry of the muscles extensor digitorium longus and soleus of C57BL/6 females, who were exposed to glyphosate during pregnancy and lactation. Methods Twelve female mice from the C57BL/6 lineage were used. After detection of pregnancy, they were divided into a Control Group, which received only water, and a Glyphosate Group, which received water with 0.5% glyphosate during pregnancy and lactation. Both groups received ad libitum standard diet. After weaning, the females were euthanized and weighed; naso-anal length was measured, and fats were collected and weighed. The muscles extensor digitorium longus and soleus were collected, and their length and weight were measured. Then, the muscles were fixed in Methacarn to perform the histological study of muscle fibers. Results Glyphosate Group presented lower weight gain during pregnancy and also lower final body weight and naso-anal length; however, the other body parameters evaluated did not present a significant difference in relation to the Control Group. Significant differences were also not observed in the analysis of muscle fibers and connective tissue. Conclusion Exposure to 0.5% glyphosate during pregnancy and lactation resulted in lower weight gain during pregnancy, final weight, and naso-anal length. Despite not directly altering the morphology of muscle tissue, these results may indicate enough exposure to interfere with animal metabolism.

RESUMO Objetivo Avaliar a morfologia e a morfometria dos músculos extensor longo dos dedos e sóleo de fêmeas C57BL/6 expostas ao glifosato durante a prenhez e lactação. Métodos Foram utilizados 12 camundongos fêmeas da linhagem C57BL/6. Após detecção da prenhez, foram separadas em Grupo Controle, que recebeu somente água, e Grupo Glifosato, que recebeu água com 0,5% de glifosato durante a prenhez e lactação. Ambos os grupos receberam dieta padrão ad libitum. Após o desmame, as fêmeas foram eutanasiadas e pesadas; o comprimento nasoanal foi mensurado, e as gorduras foram coletadas e pesadas. Os músculos extensor longo dos dedos e sóleo foram coletados, e seu comprimento e peso foram mensurados. Em seguida, os músculos foram fixados em Methacarn para a realização do estudo histológico das fibras musculares. Resultados O Grupo Glifosato apresentou menor ganho de peso durante a prenhez e também menor peso corporal final e comprimento nasoanal, entretanto os demais parâmetros corporais avaliados não apresentaram diferença significativa em relação ao Grupo Controle. Na análise das fibras musculares e do tecido conjuntivo, também não foram observadas diferenças significativas. Conclusão A exposição a 0,5% de glifosato durante a prenhez e lactação resultou em menor ganho de peso na gestação, peso final e comprimento nasoanal, o que pode indicar que, apesar de não alterar a morfologia do tecido muscular diretamente, a exposição foi suficiente para interferir no metabolismo dos animais.

Effect of fecal microbiota transplantation on intestinal flora in mice with acute-on-chronic liver failure / 临床肝胆病杂志

ObjectiveTo investigate the protective effect of fecal microbiota transplantation (FMT) on mice with acute-on-chronic liver failure (ACLF) and its effect on intestinal flora. MethodsA total of 40 mice were randomly divided into control group (CON group), model group (MOD group), FMT group (feces of the mice in the CON group were used as fecal microbiota donor), and FMT model group (ANFMT group, with feces of the mice in the MOD group as fecal microbiota donor), with 10 mice in each group. All mice were observed in terms of body weight, death, liver histopathology, and changes in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and intestinal flora. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the SNK-q test was used for further comparison between two groups. ResultsCompared with the CON group, the MOD group had a significant reduction in body weight and significant increases in AST and ALT (all P＜0.05), as well as large patchy necrosis of hepatocytes, significant increases in Verrucomicrobia, Akkermansia, and Erysipelatoclostridium, and significant reductions in Dubosiella and Duncaniella (all P＜0.05). Compared with the CON group, the ANFMT group had a significant increase in AST (P＜0.05), hepatocyte swelling and mild ballooning degeneration, significant increases in Unclassified and Faecalibaculum, and significant reductions in Patescibacteria, Deferribacteres, Muribaculum, Candidatus_Saccharimonas, Rikenella, Odoribacter, Mucispirillum, and Lachnospiraceae_unclassified (all P＜0.05). Compared with the MOD group, the FMT group had significant reductions in AST and ALT (both P＜0.05), mild hepatocellular necrosis and marked ballooning degeneration, significant increases in Paramuribaculum and Bilophila, and significant reductions in Firmicutes, Rikenella, and Absiella (all P＜0.05). ConclusionIntestinal flora disturbance is observed in ACLF mice, and dysbacteriosis may lead to liver injury. FMT can alleviate liver inflammation in ACLF mice and thus exert a protective effect.

Effect of macrophages on the differentiation of mouse induced pluripotent stem cells into hepatic progenitor cells / 临床肝胆病杂志

ObjectiveTo investigate the effect of macrophages (MCs) on the differentiation of mouse induced pluripotent stem cells (iPSCs) into hepatic progenitor cells (HPCs). MethodsA total of 24 C57BL/6N mice were used to obtain MCs by peritoneal irrigation, and the supernatant was collected to obtain the conditioned medium of MCs (MC-CDM). Activin A, bone morphogenetic protein 4, and fibroblast growth factor were used to induce the differentiation of mouse iPSCs into HPCs. The differentiation of HPCs were randomly divided into control group (normal medium) and experimental group (MC group; use of MC-CDM medium on day 5 of induction). Morphology, immunofluorescence assay, and Western blot were used to compare the morphology of HPCs and the expression of related proteins between the control group and the MC group. The t-test was used for comparison of continuous data between two groups. ResultsHPCs derived from iPSCs were established in vitro, and HPCs had the potential to differentiate into hepatocytes. Immunofluorescence assay showed that compared with the D12 control group, the D12 MC group had a significant increase in the protein expression of the HPC-specific protein CK19 (0.901±0.072 vs 0.686±0.097, t=-3.093, P＜0.05). Western blot showed that compared with the D12 control group, the D12 MC group had a significant increase in the protein expression of the HPC-related protein CK19 (1.922±0.103 vs 1.448±0.012, t =-7.881, P ＜005), as well as a significant increase in the protein expression of the autophagy-related protein LC3 (1.392±0.042 vs 1.101±0048, t =-5.978, P＜005). ConclusionMCs can promote the differentiation of mouse iPSCs into HPCs, possibly by increasing the autophagy level of HPCs.

Therapeutic effect of Taohong Siwu decoction on a mouse model of carbon tetrachloride-induced liver fibrosis and its mechanism / 临床肝胆病杂志

Objective To investigate the therapeutic effect of Taohong Siwu decoction on a mouse model of carbon tetrachloride (CCl 4 )-induced liver fibrosis and its mechanism of action. Methods A total of 24 male C57BL/6 mice were randomly divided into normal group, model group, and Taohong Siwu decoction group, with 8 mice in each group. The mice in the model group and the Taohong Siwu decoction group were given intraperitoneal injection of 10% CCl 4 , and Taohong Siwu decoction was given by gavage since week 3 for 4 consecutive weeks. Liver function [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] was measured, and liver pathomorphology was observed. Real-time PCR was used to measure the mRNA expression of α-smooth muscle actin (α-SMA), hyaluronic acid synthase-2 (HAS-2), and collagen type Ⅰ(Col1), and Western blotting was used to measure the protein expression of α-SMA, Col1, and HAS-2. Primary hepatic stellate cells (HSCs) were isolated, and HAS-2 was silenced by siRNA to observe its influence on HSC activation. The t -test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the SNK or least significant difference t -test was used for further comparison between two groups. Results Compared with the normal group, the model group had significant increases in serum liver function parameters (ALT, AST) and the Taohong Siwu decoction group had significant reductions in the serum levels of ALT and AST (all P < 0.01). Pathological staining showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers, and the Taohong Siwu decoction group had loose fibrous septa and alleviated inflammatory cell infiltration. Compared with the model group, the Taohong Siwu decoction group had significant reductions in the mRNA and protein expression of α-SMA and Col1(all P < 0.001). Compared with the normal group, the model group had a significant increase in the mRNA expression level of HAS-2 in liver tissue ( t =6.14, P < 0.05), and compared with the model group, the Taohong Siwu decoction group had a significant reduction in the protein expression level of HAS-2 (0.29±0.10 vs 1.00±0.12, t =70.73, P < 0.001). After HAS-2 was silenced by siRNA, the Si HAS-2+transforming growth factor β (TGFβ) group (treated with TGFβ) had significant reductions in the mRNA expression levels of α-SMA and Col-Ⅰ compared with the NC+TGFβ group ( P < 0.01). Conclusion Taohong Siwu decoction exerts a marked therapeutic effect on CCl 4 -induced liver fibrosis in mice by inhibiting HAS-2.

A transcriptomic analysis of acute hepatotoxicity induced by aristolochic acid Ⅰ in mice / 临床肝胆病杂志

Objective To investigate the molecular mechanism of aristolochic acid Ⅰ (AAⅠ) inducing acute hepatotoxicity in mice. Methods A total of 15 male C57BL/6 mice were randomly divided into normal group with 6 mice and treatment group with 9 mice. The mice in the treatment group were given intraperitoneal injection of AAⅠ at a dose of 20 mg/kg for 5 consecutive days and were sacrificed to collect samples on day 6. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and HE staining was used to observe liver histological changes; three liver tissue samples were randomly selected from each group, and RNA was extracted for high-throughput transcriptome sequencing. Bioinformatics analysis and functional prediction were used to screen out differentially expressed genes, and quantitative real-time PCR (qRT-PCR) was used for validation. The t -test was used for comparison of continuous data between two groups. Results Compared with the normal group, the treatment group had significant increases in the activities of ALT and AST ( t =4.331 and 4.947, both P 2 and P < 0.05, among which there were 703 upregulated genes and 649 downregulated genes. The GO and KEGG enrichment analyses of these differentially expressed genes showed significant enrichment in GO terms (such as small molecular catabolism, immune response involving neutrophils, cytoplasmic vesicle lumen in secretory granules, cytoplasmic vesicle lumen, extracellular structural organization, and extracellular matrix) and KEGG pathways (such as chemical carcinogenesis, retinol metabolism, arachidonic acid metabolism, steroid hormone biosynthesis, transcriptional dysregulation in cancer, protein digestion and absorption, regulation of TRP channel by inflammatory mediators, drug metabolism, complement and coagulation cascade, glutathione metabolism, and the PPAR signaling pathway). A cluster analysis ( P < 0.05) showed that significantly downregulated genes included Srd5a1, Lipc, Aqp8, Hba-a1, Slco1a1, and Pklr, which were validated by qRT-PCR (all P < 0.05). Conclusion AA Ⅰ can lead to significant acute hepatotoxicity, which mainly involves the processes such as chemical carcinogenesis, retinol metabolism, arachidonic acid metabolism, steroid hormone biosynthesis, and transcriptional dysregulation in cancer.

A transcriptomic analysis of acute hepatotoxicity induced by aristolochic acid Ⅰ in mice / 临床肝胆病杂志

Objective To investigate the molecular mechanism of aristolochic acid Ⅰ (AAⅠ) inducing acute hepatotoxicity in mice. Methods A total of 15 male C57BL/6 mice were randomly divided into normal group with 6 mice and treatment group with 9 mice. The mice in the treatment group were given intraperitoneal injection of AAⅠ at a dose of 20 mg/kg for 5 consecutive days and were sacrificed to collect samples on day 6. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and HE staining was used to observe liver histological changes; three liver tissue samples were randomly selected from each group, and RNA was extracted for high-throughput transcriptome sequencing. Bioinformatics analysis and functional prediction were used to screen out differentially expressed genes, and quantitative real-time PCR (qRT-PCR) was used for validation. The t -test was used for comparison of continuous data between two groups. Results Compared with the normal group, the treatment group had significant increases in the activities of ALT and AST ( t =4.331 and 4.947, both P 2 and P < 0.05, among which there were 703 upregulated genes and 649 downregulated genes. The GO and KEGG enrichment analyses of these differentially expressed genes showed significant enrichment in GO terms (such as small molecular catabolism, immune response involving neutrophils, cytoplasmic vesicle lumen in secretory granules, cytoplasmic vesicle lumen, extracellular structural organization, and extracellular matrix) and KEGG pathways (such as chemical carcinogenesis, retinol metabolism, arachidonic acid metabolism, steroid hormone biosynthesis, transcriptional dysregulation in cancer, protein digestion and absorption, regulation of TRP channel by inflammatory mediators, drug metabolism, complement and coagulation cascade, glutathione metabolism, and the PPAR signaling pathway). A cluster analysis ( P < 0.05) showed that significantly downregulated genes included Srd5a1, Lipc, Aqp8, Hba-a1, Slco1a1, and Pklr, which were validated by qRT-PCR (all P < 0.05). Conclusion AA Ⅰ can lead to significant acute hepatotoxicity, which mainly involves the processes such as chemical carcinogenesis, retinol metabolism, arachidonic acid metabolism, steroid hormone biosynthesis, and transcriptional dysregulation in cancer.

Damage of blue-light exposure to retinal morphology and function in mice / 中华实验眼科杂志

Objective To study the damage effects of blue-light exposure on retinal morphology and function in mouse.Methods Twenty 8-week-old clean C57BL/6J mice were randomly divided into blue-light exposure group and normal control group by coin tossing method.The mice in the blue-light exposure group was exposed to 10 000 lx blue light for 5 days after dark adaptation for 24 hours,and the mice in the normal control group was kept under the normal light intensity for 5 days at 12-hour light/12-hour darkness cycles.The retinal thickness was detected by optical coherence tomography (OCT),and retinal function was evaluated by electroretinogram (ERG).The mice was sacrificed and the frozen section and flat mount of eyeball wall was created at 24 hours after irradiation.The expressions of rhodopsin (Rho),zonula occludens-1 (ZO-1) and β-catenin in the retinas were detected by immunofluorescent staining.The use and care of the experimental animals adhered to ARVO Statement by American Society of Visual and Ophthalmological Sciences (No.IACUC-1803029).Results The thickness of the retinal outer nuclear layer at 200,400,600,800 and 1 000 μm from the superior and inferior to optic nerves were thinned in the mice of the blue-light exposure group compared with those of the normal control group,showing significant differences between the two groups (all at P＜0.05).The b-wave amplitude of the scotopic and photopic ERG was (305.50±41.52) μV and (119.50±6.67)μV in the blue-light exposure group,respectively,which was significantly reduced in comparison with (415.50±28.77) μV and (139.75±8.26) μV of the normal control group (both at P＜ 0.05).Immunofluorescent staining showed that the retinal pigment epithelial (RPE) cells of the mice exhibited hexagonal shape with regular arrangement,retinal morphology was regular,and the expressions of Rho,ZO-1 and β-catenin proteins showed stronger fluorescence in the retinas of normal control group.However,structural disorder,diminishing fluorescence intensity of Rho,ZO-1 and β-catenin were found in the blue-light exposure group.The morphology of the retina was disordered while the cell structure was destroyed.Conclusions Blue-light irradiation results in morphological and functional damages of retina.

Damage of blue-light exposure to retinal morphology and function in mice / 中华实验眼科杂志

Objective@#To study the damage effects of blue-light exposure on retinal morphology and function in mouse.@*Methods@#Twenty 8-week-old clean C57BL/6J mice were randomly divided into blue-light exposure group and normal control group by coin tossing method.The mice in the blue-light exposure group was exposed to 10 000 lx blue light for 5 days after dark adaptation for 24 hours, and the mice in the normal control group was kept under the normal light intensity for 5 days at 12-hour light/12-hour darkness cycles.The retinal thickness was detected by optical coherence tomography (OCT), and retinal function was evaluated by electroretinogram (ERG). The mice was sacrificed and the frozen section and flat mount of eyeball wall was created at 24 hours after irradiation.The expressions of rhodopsin (Rho), zonula occludens-1 (ZO-1) and β-catenin in the retinas were detected by immunofluorescent staining.The use and care of the experimental animals adhered to ARVO Statement by American Society of Visual and Ophthalmological Sciences (No.IACUC-1803029).@*Results@#The thickness of the retinal outer nuclear layer at 200, 400, 600, 800 and 1 000 μm from the superior and inferior to optic nerves were thinned in the mice of the blue-light exposure group compared with those of the normal control group, showing significant differences between the two groups (all at P<0.05). The b-wave amplitude of the scotopic and photopic ERG was (305.50±41.52)μV and (119.50±6.67)μV in the blue-light exposure group, respectively, which was significantly reduced in comparison with (415.50±28.77)μV and (139.75±8.26)μV of the normal control group (both at P<0.05). Immunofluorescent staining showed that the retinal pigment epithelial (RPE) cells of the mice exhibited hexagonal shape with regular arrangement, retinal morphology was regular, and the expressions of Rho, ZO-1 and β-catenin proteins showed stronger fluorescence in the retinas of normal control group.However, structural disorder, diminishing fluorescence intensity of Rho, ZO-1 and β-catenin were found in the blue-light exposure group.The morphology of the retina was disordered while the cell structure was destroyed.@*Conclusions@#Blue-light irradiation results in morphological and functional damages of retina.

Effect of triptolide on fibrosis in a mouse model of cerulean-induced chronic pancreatitis / 临床肝胆病杂志

ObjectiveTo investigate the effect of triptolide on fibrosis in mice with cerulein-induced chronic pancreatitis (CP) based on the nuclear factor-kappa B (NF-κB)/interleukin-6 (IL-6) signaling pathway. MethodsA total of 15 male C57BL/6 mice were randomly divided into control group (treated with normal saline), cerulein group (model mice with cerulein-induced CP), and triptolide group (induced by cerulean and treated with triptolide), with 5 mice in each group. Samples were collected for detection after 6 weeks. The weight of the pancreas was measured; HE staining, picrosirius red staining, and Masson staining were used to observe pancreatic histomorphology and collagen deposition; ELISA was used to measure the expression of IL-6 in serum; immunohistochemistry was used to measure the expression of alpha-smooth muscle actin (α-SMA) and NF-κB/p65; Western blot was used to measure the expression of NF-κB/p65, IL-6, and α-SMA. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsThere were significant differences between the three groups in the weight of the pancreas, IL-6 concentration, pathological score, picrosirius red staining results, Masson staining results, and mean optical density of positive α-SMA signal and positive NF-κB/p65 signal(F=64.87, 15.85, 145.33, 141.80, 121.77, 250.22, and 69.22, all P＜0.001). Compared with the cerulein group, the triptolide group had a significant increase in the weight of the pancreas and significant reductions in the expression of IL-6, pancreatic fibrosis, collagen deposition, and the expression of α-SMA and NF-κB/p65 (all P＜0.05). Western blotting showed that there were significant differences in the expression of NF-κB/p65, IL-6, and α-SMA between the three groups (F=8.86, 6.74, and 16.23, all P＜0.05), and compared with the cerulein group, the triptolide group had significantly lower expression of NF-κB/p65, IL-6, and α-SMA in the pancreatic tissue (all P＜0.05). ConclusionTriptolide alleviates fibrosis in mice with cerulein-induced CP and inhibit the protein expression of NF-κB/p65 and IL-6.

Protective effect of pinocembrin in a mouse model of liver injury induced by acetaminophen / 临床肝胆病杂志

ObjectiveTo investigate the protective effect of pinocembrin (PIN) in a mouse model of liver injury induced by acetaminophen (APAP). MethodsA total of 50 healthy male C57BL/6J mice were randomly divided into blank group, PIN (50 mg/kg) group, APAP (300 mg/kg) model group, PIN (30 mg/kg)+APAP (300 mg/kg) experimental group, and PIN (50 mg/kg)+APAP (300 mg/kg) experimental group, with 10 mice in each group. The mice in the blank group and the model group were given an equal volume of normal saline by gavage, and those in the PIN group and the PIN+APAP groups were given PIN by gavage once a day, for 7 consecutive days. At 2 hours after the last administration, the mice in the model group and the PIN+APAP groups were given intraperitoneal injection of APAP 300 mg/kg once, and those in the blank group and the PIN group were given intraperitoneal injection of an equal volume of normal saline. Serum samples were collected to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST); liver tissue homogenate was prepared to measure the biochemical parameters of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH); HE staining was used to observe liver histopathology. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the blank group, the APAP (300 mg/kg) model group had significant increases in the activities of ALT and AST (P＜0.01), suggesting that a model was successfully established, while the PIN (30 mg/kg)+APAP (300 mg/kg) group and the PIN (50 mg/kg)+APAP (300 mg/kg) group had significant reductions in the levels of ALT and AST (P＜0.01). Compared with the blank group, the APAP (300 mg/kg) model group had a significant increase in the level of MDA and significant reductions in SOD activity and GSH level in the liver (P＜001); compared with the APAP (300 mg/kg) model group, the PIN (30 mg/kg)+APAP (300 mg/kg) group and the PIN (50 mg/kg)+APAP (300 mg/kg) group had a significant reduction in the level of MDA and significant increases in SOD activity and GSH level in the liver (P＜0.05). Histopathological observation showed that PIN significantly improved liver injury caused by APAP and helped to maintain normal liver histomorphology. ConclusionPIN exerts a marked protective effect on liver injury induced by APAP, possibly by inhibiting oxidative stress in the liver.

Effect of fenofibrate on the diversity of intestinal flora in a mouse model of nonalcoholic fatty liver disease / 临床肝胆病杂志

ObjectiveTo investigate the effect of fenofibrate on the diversity of intestinal flora in mice with nonalcoholic fatty liver disease (NAFLD). MethodsA total of 30 mice were randomly divided into normal group, high-fat group, and fenofibrate treatment group, with 10 mice in each group. The mice in the high-fat group and the fenofibrate treatment group were given high-fat diet intervention for 14 weeks, and those in the normal group were given normal diet for 14 weeks. After 10 weeks of dietary intervention, the mice in the fenofibrate treatment group were given fenofibrate by gavage and high-fat diet for another 4 weeks, and the change in body weight was monitored during the whole process. Fecal samples were collected after 14 weeks, and high-throughput sequencing 16S rRNA was used to investigate the diversity and difference of intestinal flora. Liver tissue samples were collected, and HE staining and oil red O staining were performed to observe the degree of steatosis. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsAfter 10 weeks of intervention, the fenofibrate treatment group had a significantly lower body weight than the high-fat group (P＜0.05). The results of HE staining and oil red O staining showed that the fenofibrate treatment group had a significantly lower degree of fat deposition than the high-fat group. There was a significant difference in intestinal flora between the fenofibrate treatment group and the high-fat group, while there was no significant difference in intestinal flora between the fenofibrate treatment group and the normal group. The fenofibrate treatment group had significant increases in the abundance of Bacteroidetes, Verrucomicrobia, Faecalibaculum, Muribaculaceae_norank, and Akkermansia and significant reductions in the abundance of Firmicutes, Actinobacteria, Clostridium, Turicibacter, and Bifidobacterium. ConclusionThe increases in the abundance of Bacteroidetes and Verrucomicrobia and the reductions in the abundance of Firmicutes and Actinobacteria suggest that fenofibrate may have positive significance in the treatment of NAFLD.

Preliminary Study on Relationship Between Terminal Ileal Histone Deacetylase 3 Expression and Obesity and Intestinal Flora / 胃肠病学

Background: Histone deacetylase 3 (HDAC3), which is expressed in liver, muscle and adipose tissue, is closely related to fat metabolism. Furthermore, HDAC3 expressed in intestinal epithelial cells is a critical factor that regulates host-commensal flora relationship and maintains intestinal homeostasis. Aims: To investigate the relationship between HDAC3 expression in terminal ileum and obesity and intestinal flora. Methods: Eight SPF C57BL/6 mice and eight germ-free C57BL/6 mice aged six weeks were randomly placed on a standard chow or a high-fat chow, respectively, for 5 weeks. Changes in body weight were recorded weekly, and the terminal ileum was obtained for detection of HDAC3 protein and mRNA expressions by immunohistochemistry and real-time PCR. Results: Increase of body weight of mice in SPF high-fat diet group was much more than that in SPF normal diet group (P0.05). No significant differences were found in HDAC3 protein and mRNA expressions between germ-free high-fat diet group and germ-free normal diet group (P>0.05). Conclusions: Expression of HDAC3 in terminal ileum might be regulated by intestinal flora and participates in the occurrence of obesity.

Differential expression and bioinformation analysis of retinal proteins in mice with experimental autoimmune uveitis / 中华实验眼科杂志

Objective@#To observe the expression of retinal proteins in experimental autoimmune uveitis (EAU) mice and to explore the possible molecular mechanism of autoimmune uveitis.@*Methods@#Twelve female C57BL/6J mice were randomly divided into model group and normal control group, 6 mice in each group.In the model group, the EAU model was established by subcutaneous injection of human interphotoreceptor retinoid-binding protein (IRBP) 651-670.The fundal change of EAV mice was assessed by direct ophthalmoscope, OCT and histopathological staining.At 18 days after immunization, the retinas of the two groups were taken for retinal protein extraction, protein restriction enzyme digestion, mass spectrometry detection, data analysis, and bioinformatics analysis.This study was approved by the experimental animal Ethics Committee of Tianjin Medical University Eye Hospital (TJYY2018070113). The feeding and use of experimental animals follow the ARVO statement.@*Results@#The EAU mouse model was successfully established.At 10 days after immunitation, the retina of EAV mouse was damaged.At 18 days after immunization, retinal edema and infiltration of inflammatory cells into vitreous were observed.Proteomic results showed that a total of 4 458 proteins were identified in this study, of which 522 were differentially-expressed proteins (fold change>1.5, P<0.05). Among these differentially-expressed proteins, 147 proteins including Stat1 and Stat3 were up-regulated and 375 proteins including Gucy2f and Rho were down-regulated.These differentially-expressed proteins were closely related to platelet activation, integrin signaling, T cell activation, the phototransduction cascade, Toll-like receptor and so on.@*Conclusions@#EAU is related to the abnormal expression of Stat1, Stat3 and other proteins, as well as the abnormal regulation of platelet activation and integrin signal pathway.

Expression of heterogeneous nuclear ribonucleo-protein A2B1 in mouse cerebellar development and human medulloblastoma / 中华病理学杂志

Objective@#To investigate the expression and potential role of heterogeneous nuclear ribonucleo-protein A2B1 (HNRNPA2B1) in mouse cerebellar development and the significance of HNRNPA2B1 in human medulloblastoma.@*Methods@#The data of HNRNPA2B1 RNA expression in mouse and human cerebella were obtained from databases. Western blot and immunohistochemical staining were performed to detect the protein level of HNRNPA2B1 in mouse cerebella at different ages. The expression level of HNRNPA2B1 in control human cerebellum and medulloblastoma was detected by immunohistochemical staining. m6A-IP-qPCR method was applied to confirm whether HNRNPA2B1 RNA in Daoy cells was modified with m6A.Western blot was used to detect the effect of MG132 treatment on the HNRNPA2B1 protein level in Daoy cells.@*Results@#The level of HNRNPA2B1 protein in postnatal mouse cerebella was higher than that in adult mouse cerebella, with weak HNRNPA2B1 staining in external granular cells while strong staining in mature Purkinje cells and molecular layer. Compared with control normal human cerebella, the RNA expression level of HNRNPA2B1 increased in medulloblastoma, while immunohistochemical staining showed that the mean intensity of HNRNPA2B1 decreased in medulloblastoma. HNRNPA2B1 RNA in medulloblastoma and Daoy cells was modified by m6A. The HNRNPA2B1 protein level in Daoy cells increased upon MG132 treatment.@*Conclusions@#HNRNPA2B1 is dynamically expressed during mouse cerebellar development. Compared with normal human cerebella, HNRNPA2B1 is significantly up-regulated at transcriptional level but obviously down-regulated at translational level in medulloblastoma. These results indicate that HNRNPA2B1 may be involved in cerebellar development process and medulloblastoma tumorigenesis. The m6A methylation in HNRNPA2B1 transcript and protein ubiquitin-proteasome pathway may account for the down-regulation of HNRNPA2B1 at protein level.