[Role of mitochondrial autophagy and the curative effect of rehmannia glutinosa leaves total glycoside capsules on nucleos(t)ide drug-induced renal injury].

Objective: To study the curative effect of rehmannia glutinosa leaves total glycoside capsules and the role of mitochondrial autophagy on nucleos(t)ide drug-induced renal injury. Methods: Adefovir dipivoxil (ADV) was used to construct a hepatitis B virus (HBV) transgenic mouse model for renal injury. Renal function was measured in each group at one and two weeks of modeling. Mitochondrial autophagy indicators were measured at two weeks of modeling in renal tissue. Transmission electron microscopy was used to detect mitochondrial autophagy phenomena in renal tissue. The model was established for two weeks. Mouse with renal injury were treated with rehmannia glutinosa leaves total glycoside capsules or isotonic saline for eight weeks by intragastric administration. Renal function was measured. Renal tissue morphology was observed. Mitochondrial autophagy indicators were detected in renal tissue. The protective effect of different concentrations of verbascoside (the main active ingredient of rehmannia glutinosa capsule) was observed on HK-2 cell damage induced by ADV. HK-2 cells were divided into control, ADV, and ADV plus verbascoside groups. The effects of verbascoside at different times and concentrations were observed on the HK-2 mitochondrial autophagy indicators. Fifty patients with chronic hepatitis B were collected who presented with renal injury after treatment with nucleos(t)ide analogs. The random number method was used to divide 29 cases into a control group that received conventional treatment. The treatment group of 21 cases was treated with rehmannia glutinosa leaves total glycoside capsules on the basis of the control group. Serum creatinine (Scr) and urinary protein were detected at eight weeks.The χ(2) test or t-test was used for statistical analysis. Results: Compared with the control group, two weeks of modeling in the ADV group induced renal function injury in HBV mice. The expression of autophagy indicators was higher in the renal tissue of the ADV group than that of the control group. Transmission electron microscopy had revealed mitochondrial autophagy in the renal tissue of the ADV group. Compared with the control group, the renal function of HBV mice treated with rehmannia glutinosa leaves total glycoside capsules improved for two months, and the expressions of autophagy indicators were down-regulated.Verbascoside promoted proliferation in ADV-damaged HK-2 cells, and the expression of autophagy indicators was down-regulated compared with the ADV alone group. In 50 patients with renal function injury, the urinary protein improvement was significantly superior in the treatment group than that in the control group, with eighteen and three cases being effective and ineffective in the treatment group and 12 and 17 cases being effective and ineffective in the control group, with a statistically significant difference (χ(2) = 9.975 0, P = 0.001 6). Serum creatinine was decreased in the treatment group compared with the control group, with 11 and 10 cases being effective and ineffective in the treatment group and 12 and 17 cases being effective and ineffective in the control group, with no statistically significant difference (χ(2) = 0.593 5, P = 0.441 1). Conclusion: Rehmannia glutinosa leaves total glycoside capsule can improve the nucleos(t)ide drug-induced renal function injury in chronic hepatitis B, possibly playing a role via inhibiting PINK1/Parkin-mediated mitochondrial autophagy.

[Interventional effect and mechanism of fermentation liquid of Dendrobium officinale leaves on alcoholic hepatitis mice].

Objective: To explore the intervention effect and mechanism of Dendrobium officinale leaf fermentation liquid on alcoholic hepatitis (AH) mice. Methods: Seventy inbred C57BL/6J male mice aged 6-8 weeks were selected and randomly divided into normal group (NG), model group (MG), liquid feed control group (CG), silybum group (SI), low-dose group (DL), medium-dose group (DM), and high-dose group (DH) of Dendrobium officinale fermentation liquid, with 10 mice in each group. NG group was given common feed, CG group was given control feed (LB alcoholic liquid control feed), SI group was given LB alcoholic liquid feed and silybum by gavage, DL, DM and DH groups were given LB alcoholic liquid feed and 25%, 50% and 100% concentration of Dendrobium officinale leaf fermentation liquid by gavage. An AH model was established by feeding LB alcoholic liquid feed for 8 weeks.At week 8, alanine Transaminase (ALT), triglyceride (TG), transferrin (TRF), interleukin (IL)-6, IL-10, and IL-1ß, tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ) were detected in eye blood of mice. Liver tissues were stained with HE, Oil Red O, Prussian blue and immunofluorescence ROS. The contents of glutathione(GSH) and malondialdehyde (MDA) in liver tissue homogenate were detected. To analyze the intervention effect and mechanism of Dendrobium officinale leaf fermentation solution on AH mice, the mRNA and protein relative expression levels of adenylate activated protein kinase (AMPK), AMPKß1, phosphorylated AMPKß1 (p-AMPKß1), tumor suppressor gene p53 (p53), solsolic vector family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GXP4) were detected by polymerase chain reaction (PCR) and Western blot. Results: Compared with MG group, the serum ALT and TG levels in the DL, DM, and DH groups were all reduced [ALT: (45.94±19.85), (45.73±22.62), and (41.68±7.13) vs (75.51±17.76) U/L, respectively; TG: (0.90±0.23), (0.69±0.22) and (0.41±0.20) vs (1.28±0.19) mmol/L, respectively, all P<0.05]; IL-6, IL-1ß, TNF-α, IFN-γ were decreased (all P<0.05). The serum TRF and IL-10 levels in the DM and DH groups were increased (all P<0.05). Compared with MG group, the liver tissue MDA of mice in DL, DM and DH groups was decreased [(0.41±0.05), (0.40±0.03), and (0.43±0.14) vs (0.64±0.06)µmol/g, respectively], GSH was increased (all P<0.05). Compared with MG, mRNA expression levels of AMPK (1.36±0.11, 1.61±0.17, 1.68±0.11 vs 0.80±0.12, respectively), SLC7A11 (0.91±0.12, 0.97±0.12, 0.99±0.13 vs 0.60±0.14, respectively) and GPX4 (0.51±0.11, 0.63±0.17, 0.83±0.15 vs 0.42±0.14, respectively) in the liver tissue of DL, DM and DH groups were all increased (all P<0.05). Compared with MG group, DL, DM and DH groups showed the relative expression levels of AMPKß1, p-AMPKß1, SLC7A11 and GPX4 were increased in the liver tissue of mice, while the relative expression levels of p53 protein were decreased (all P<0.05). Compared with MG group, DL, DM and DH groups reduced the degree of hepatic steatosis and inflammation in the lobules, while the iron and ROS staining in the liver tissue became lighter. Conclusion: Dendrobium officinale leaf fermentation liquid can alleviate the severity of AH in mice, and its mechanism may be related to the up-regulation of AMPK to inhibiting the p53/SLC7A11/GPX4 mediated Ferroptosis pathway.

[Exploring effects of natural hyperoxic environment on liver lipid metabolism based on the bile acid-farnesoid X receptor pathway in sub-healthy rats].

Objective: To investigate the effect of natural hyperoxic environment on liver lipid metabolism and liver function based on the bile acid-farnesoid X receptor pathway in sub-healthy rats. Methods: Forty adult male Wistar rats were randomly divided into control group (n = 10) and sub-healthy model group (n = 30). The control group was fed a normal diet, and the model group was fed a high-fat-sugar diet with limited daily activities for 5 weeks. The sub-healthy model was successfully established and the feeding conditions were restored. The hyperoxic intervention group (healthy group) were placed in a natural hyperoxic environment for 7 days. The rats feeding status in the spontaneous recovery group were unchanged. The appearance and exhaustive swimming time were compared before and after in healthy rats. Peripheral blood was collected for biochemical measurement. The fluorescence intensity of FXR and peroxisome proliferator activated receptor α (PPAR α) in liver tissue was detected by fluorescence double staining. Real-time fluorescent semi-quantitative PCR and Western blot were used to detect the RNA and protein expression condition of bile acid-FXR signaling pathway related indicators (FXR, PPARα, and SREBP-1c) in liver tissues. Results: Compared with the control group, the model group had gained body weight, and the vitality was decreased, while triglycerides [TG, (1.18 ± 0.20) mmol/L vs. (0.65 ± 0.12) mmol/L] and total cholesterol [TC, (1.23 ± 0.29) mmol/L vs. (1.00 ± 0.25) mmol/L] level was increased, (P < 0.05), which suggests the presence of hepatic steatosis. TG and TC level in the healthy group and spontaneous recovery group were lower than the model group, and the differences between the healthy group and the model group were statistically significant (P < 0.05). Compared with the model group, the expression of FXR and PPARα in the liver of the healthy and the spontaneous recovery group was enhanced, while the expression of the sterol regulatory element binding protein 1c (SREBP-1c) was decreased. FXR and PPARα mRNA levels in the healthy group and the model group were (9.27 ± 0.26 vs. 6.77 ± 0.20), and (9.71 ± 0.21 vs. 7.09 ± 0.24), P < 0.01, respectively. Compared with the model group, spontaneous recovery group mRNA levels were 7.99 ± 0.30 and 8.44 ± 0.28, P < 0.05, respectively. FXR and SREBP-1c protein levels between the healthy group and the model group were (1.30 ± 0.19 vs.0.43 ± 0.28), and (1.56 ± 0.22 vs. 2.43 ± 0.19), P < 0.01, respectively. Compared with the model group, the FXR and SREBP-1c protein levels of the spontaneous recovery group were 0.81 ± 0.33 vs. 2.10 ± 0.38, P < 0.05, respectively. In addition, natural hyperoxic environment had enhanced liver lipid metabolism and improved lipid disorders. Conclusion: The natural hyperoxic environment have the ability to regulate liver lipid metabolism and can improve mild hyperlipidemia to a certain extent.

LncRNA DANCR promotes ATG7 expression to accelerate hepatocellular carcinoma cell proliferation and autophagy by sponging miR-222-3p.

OBJECTIVE: LncRNA differentiation antagonizing non-protein coding RNA (DANCR) is an oncogene in various malignant cancers, including hepatocellular carcinoma (HCC). Autophagy is an intracellular self-digestion mechanism that accelerates the progression of HCC via promoting cell survival. However, the role of lncRNA DANCR in HCC, and the mechanism of lncRNA DANCR in the regulation of autophagy in HCC remains unknown. Therefore, the aims of this study are the investigation of the role of lncRNA DANCR in HCC, and the exploration of the molecular mechanism of lncRNA DANCR in regulating autophagy of HCC cells. PATIENTS AND METHODS: In this study, the expression of lncRNA DANCR, miR-222-3p, and autophagy-related gene 7 (ATG7) was detected by qRT-PCR. The cell proliferation and colony formation were measured by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. And the autophagic flux was evaluated by mRFP-GFP-LC3B reporter. The autophagy related proteins were analyzed by Western blotting. Besides, the relationship between lncRNA DANCR and miR-222-3p, as well as between miR-222-3p and ATG7, was determined by Dual-Luciferase reporter system. RESULTS: We found high expression of lncRNA DANCR and ATG7, and low expression of miR-222-3p in HCC tissues and cell lines. And lncRNA DANCR positively correlated with poor survival of HCC patients. Moreover, the knockdown of lncRNA DANCR inhibited cell proliferation and autophagy of HCC cells. And we predicted and proved that lncRNA DANCR induced cell proliferation, colony formation and autophagy by increasing ATG7 and suppressing miR-222-3p. CONCLUSIONS: Our study demonstrates the promoting role of lncRNA DANCR in HCC, and indicates the regulatory effects of lncRNA DANCR on regulating autophagy of HCC.

[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, ß 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 ß - 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.

[Interventional effect of bicyclol on isoniazid-induced liver injury in rats and the expression of glucose-regulated protein 78, and growth arrest and DNA-damage-inducible gene 153].

Objective: To investigate the interventional effect of bicyclol on isoniazid-induced liver injury in rats and the expression of endoplasmic reticulum stress (ERS) protein, glucose regulatory protein 78 (GRP78), and growth arrest and DNA-damage-inducible gene 153(CHOP). Methods: Eighty Wistar rats were randomly divided into control group (8 rats) and model group (72 rats). After 10 days of intragastric administration of isoniazid, the model group rats were randomly divided into treatment group (A), natural recovery group (B), etiological persistence group (C) and etiological persistence plus treatment group (D). Sixteen rats from each group were sacrificed after 1 and 2 weeks of intervention with different methods. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected. Liver pathological morphology was observed. Apoptotic cells were detected by TUNEL assay. ERS protein expression was detected by Western blot. A t-test or randomized block analysis of variance, K-S test and Levene's test were used to analyze the normality and homogeneity of variance. Kruskal-Wallis rank sum test was used for data that did not suit the conditions of t-test and variance analysis. Results: ALT and AST were elevated in the model group, and liver pathological examination showed liver tissue damage. Apoptotic index was higher than control group (7.13% ± 1.55% vs. 0.75% ± 0.71%, Z = -3.411, P < 0.01), and the expression value of ERS protein in model group was significantly higher than control group (GRP78: 1.16 ± 0.30 vs. 0.23 ± 0.05, t = -6.008, P < 0.01; CHOP: 0.98±0.23 vs. 0.20 ± 0.10, t = -6.378, P < 0.01). Serum enzymes, apoptotic index and ERS protein expressions of rats were decreased after treatment with bicyclol, and the pathological damage was eased. Rats in natural recovery group recovered less than the treatment group. Conclusion: Isoniazid-induced liver injury is associated to ERS-related excessive apoptosis and the therapeutic effect of bicyclol on drug-induced liver injury may minimize ERS-induced apoptosis.

Cellular mechanism of Tß4 intervention in liver fibrosis by regulating NF-κB signaling pathway.

OBJECTIVE: To investigate the inhibitory effect of thymosin-ß4 (Tß4) on the activation of the human hepatic stellate cell line (HSC-LX2) induced by interleukin (IL)-1ß. MATERIALS AND METHODS: There were 5 groups in this study, i.e., blank control group, negative control group (SI-NC, empty plasmid), model group (20 ng/ml of IL-1ß), siRNA-Tß4 knockdown group (IL-1ß and si-Tß4) and Tß4 treatment group (IL-1ß and 1000 ng/ml of Tß4). Cell proliferation rate was measured using the Cell Counting Kit-8 (CCK-8) method. The cell cycle change and percentage of apoptotic cells were determined by Propidium Iodide (PI) DNA staining and Annexin V-fluorescein isothiocyanate (FITC) double staining. Cellular nucleic acid levels of p-IKB and nuclear factor-kappa B (NF-κB)/p65 proteins were measured by fluorescent quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Double immunofluorescence staining and Western blot were used to detect nuclear translocation of NF-κB and p65 and levels of cytoplasmic p-IKB protein and nuclear p65 protein. RESULTS: Due to the G0/G1 phase arrest, the number of cells in the Tß4 treatment group increased, compared with the model group and the siRNA-Tß4 knockdown group (p<0.01). In the same between-group comparison, apoptotic rate in the Tß4 treatment group increased significantly (p<0.05). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were markedly higher in the model group and the siRNA-Tß4 knockdown group than in the blank control group (p<0.01). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were remarkably lower in the Tß4 treatment group than in the siRNA-Tß4 knockdown group (p<0.01). The expression levels of NF-κB/p65 and NF-κB/p50 were significantly lower in the Tß4 treatment group. The expression levels of cytoplasmic p-IKB and nuclear NF-κB/p65 were lower in the Tß4 treatment group than in the model group (p<0.01). CONCLUSIONS: Tß4 significantly inhibited IL-1ß-induced HSC-LX2 cell proliferation. The mechanism may involve decreased activation of the NF-κB pathway, decreased expression of p-IKB and nuclear translocation of p65. Therefore, Tß4 had the effect of reversing liver fibrosis.

[Experimental study of silybin-phospholipid complex intervention on amiodarone-induced fatty liver in mice].

Objective: To probe into the mechanism and interventional effects of silybin-phospholipid complex on amiodarone-induced steatosis in mice. Methods: Eight-week-old male C57BL/6 mice were divided into three groups (5 mice in each group): a control group (WT) with normal diet, a model group with amiodarone 150mg/kg/d by oral gavage (AM), and an intervention group on amiodarone 150mg/kg/d combined with silybin-phospholipid complex(AM+SILIPHOS. All mice were fed their assigned diet for one week. Then, one week later, serum alanine aminotransferase, aspartate aminotransferase, triglyceride, total cholesterol and high-density lipoprotein were detected of each group. A liver pathological change was observed by oil red O and H&E staining. Ultrastructural pathological changes of hepatocytes were observed to evaluate the intervention effect by transmission electron microscopy. RT-q PCR was used to detect the expression of peroxisome proliferator-activated receptor alpha and its regulated lipid metabolism genes CPTI, CPTII, Acot1, Acot2, ACOX, Cyp4a10 and Cyp4a14 in liver tissues. Intra-group comparison was done by paired t-test. One-way ANOVA was used for comparison between groups and semi-quantitative data were tested using Mann-Whitney U test. Results: Oil Red O and H&E staining results of liver tissue in the intervention group showed that intrahepatic steatosis was significantly reduced when compared to model group. Transmission electron microscopy showed that the model group had pyknotic nuclei, mitochondrial swelling, structural damage, and lysosomal degradation whereas the intervention group had hepatic nucleus without pyknosis, reduced mitochondrial swelling and slight structural damage than that of model group. RT-q PCR results showed that the expression of peroxisome proliferator-activated receptor alpha, CPTI, CPTII, Acot1, Acot2, ACOX, Cyp4a10 and Cyp4a14 were increased in the model group but the expression of CPTI, Cyp4a14, Acot1 and peroxisome proliferator-activated receptor alpha were decreased in the intervention group (P < 0.05). Conclusion: Silybin-phospholipid complex can alleviate amiodarone-induced steatosis, and its mechanism may play a role in protecting mitochondrial function and regulating fatty acid metabolism. Thus, silybin-phospholipid complex has potential intervention effect on amiodarone-induced fatty liver.

[Effects of tenofovir and telbivudine on HBV RNA in pregnant women with different genotypes of HBeAg-positive hepatitis B in Guizhou Province].

Objective: To investigate whether HBV genotype influences HBV DNA and RNA responses to tenofovir(TDF) and telbivudine(LDT) in pregnant women with HBeAg-positive in Guizhou. Methods: This was a retrospective analysis of 75 pregnant women hepatitis B with HBsAg and HBeAg double-positive(19-38 years old, median age 26 years old), who were enrolled in the Department of Infectious Diseases and Obstetrics Clinic of the Affiliated Hospital of Guizhou Medical University from May 2016 to July 2017.Blood samples were collected at 12-24, 28-32 and 36-40 weeks of pregnancy for analyses of genotype, including hepatitis B surface antigen(HBsAg), hepatitis B e antigen (HBeAg), HBV DNA, HBV RNA and liver function, alanine transaminase(ALT), aspartate transaminase(AST), total bilirubin(TBiL), total bile acids(TBA), cholinesterase(CHE), alkaline phosphatase (ALP). Continuous variable was adopted by means of mean±standard deviation, and categorical variables were used for statistical analysis. Results: The HBV genotype was B in 64.0%(48/75)and C in 36.0%(27/75). The TDF and LDT groups showed no differences in demographic and clinical characteristics, including liver function tests, HBsAg, HBeAg, log(10)HBV DNA and log(10)HBV RNA.TDF groups, pre-treatment: HBV DNA (4.8±2.0), HBV RNA (6.4±1.1); at 4 weeks of treatment: HBV DNA (4.0±0.8), HBV RNA (6.0±0.9); at the end of treatment: HBV DNA (3.1±0.7), HBV RNA (5.5±0.8). LDT groups, pre-treatment HBV DNA (5.1±2.0), HBV RNA(6.5±0.9); at 4 weeks of treatment: HBV DNA (4.4±1.2), HBV RNA(6.5±0.8); at the end of treatment: HBV DNA(3.5±1.2), HBV RNA (6.1±0.7). Compared with pre-treatment (12-24 weeks), the TDF and LDT group showed significant reductions in log(10)(HBV DNA) and log(10)(HBV RNA) at 36-40 weeks ( P<0.05). Under the influence of excluding other variables, the genotype had a certain influence on the HBV RNA load.That was, HBV RNA in patients with the C genome decreased by 0.54 units(log(10)) at the end of the treatment compared to patients with the B genome, and the P value was less than 0.05. Conclusion: B and C genotypes are predominant in pregnant women with hepatitis B in Guizhou Province. B-type viruses are more easily controlled when different genotypes are treated with nucleotide analogues.

[Effects of blueberry on hepatic fibrosis and expression of nuclear transcription factor-кB in rats].

Objective: To observe the effects of blueberry and nuclear expression of transcription factor-кb (NF-кb) p65 in an experimental rat model of liver fibrosis. Methods: Forty-five Sprague-Dawley rats were randomly divided into isotonic saline control group (A); model group (B); blueberry juice prevention group (C, 15 g/kg); dan-shao-hua-xian capsule prevention group (D, 1 g/kg); and blueberry juice + dan-shao-hua-xian capsule prevention group (E). Rat liver fibrosis model was established by covalent compound carbon tetrachloride (CCl(4)). Each prevention group was given the corresponding dose of blueberry juice or (and) dan-shao-hua-xian capsule, and the rats were sacrificed after 8 weeks. The degree of liver fibrosis was evaluated by hematoxylin and eosin stain. A liver tissue of NF-κBp65 was detected by immunohistochemical method. The NF-κBp65 protein expression of liver tissue and transforming growth factor (TGF) ß1 was detected by Western blot. Data of multiple groups were compared by one-way analysis of variance, and rank sum test. Results: Immunohistochemistry detected that TGFß1 protein was mainly expressed in mesenchymal origin of hepatic stellate cells. The expression level of group A (3.75 ± 1.67) was low, while those of group B (9.00 ± 2.07), C (7.33 ± 1.00), D (6.00 ± 1.51), and E (3.5 ± 1.41) were high. However, the expression level of TGF-ß1 protein in hepatic tissues of group B was significantly higher than that of group C, D and E [group E: 3.5 ± 1.41, F = 18.350, P < 0.05]. In addition, group D was higher than group E (P < 0.05). The expression of NF- kappa Bp65 protein was very complex, and the expression patterns in different groups were different (Statistical calculation of experimental data were based on expression of liver cells). Compared with group B (4.37 ± 2.13), the relative expression levels of NF-κBp65 protein in-group A (0.46 ± 0.25), group C (2.76 ± 1.01), group D (2.13 ± 1.51), group E (1.88 ± 0.99) were significantly decreased (F = 27.490, P < 0.05), and the expression trend was consistent with TGFß1 protein. Western blot detected NF-κBp65 protein in liver tissues of rats. Compared with group A, levels in groups B, C, D and E were significantly increased (F = 96.983, P < 0.05), and groups C, D and E were significantly lower. The E group was significantly lower than the C group (F = 96.983, P < 0.05), and the degree of hepatic fibrosis was lower in each prevention group than in the B group (T = 24.1, P < 0.05). Conclusion: Blueberries have preventive effect on CCl4-induced hepatic fibrosis in rats, and its preventive mechanism may inhibit the expression and activation of NF-κBp65 in hepatocytes, thereby reducing TGFß1- mediated production or activation.

Effect of vitrification solutions on survival rate of cryopreserved Epinephelus moara embryos.

Embryo cryopreservation is important for long-term preservation of germplasm and assisted reproduction. However, it is still very difficult to obtain viable embryos from cryopreserved fish embryos. In this study, embryos of Epinephelus moara were used to investigate the effects of various cryopreservation methods. Embryos in stages 10 pairs somite (10S), 18 pairs somite (18S), 22 pairs somite (22S), tail-bud (TB), embryo twitching (ET) and pre-hatch (PH) were treated with five-step equilibrium penetration in 40% PMG3T vitrification solution, which contained 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Trehalose. We found that 18S, 22S, TB and ET stage embryos had higher survival rates and were more tolerant to the vitrification solution. Five-step equilibrium treatments on the embryos at the tail-bud stage were performed using two vitrification solutions: 40% PMG3T and 40% PMG3S, which consisted of 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Sucrose. The embryonic survival rate under PMG3S treatment (63.36%) was significantly higher than PMG3T treatment (43.93%) (P < 0.05). PMG3S and PMG3T with concentrations of 35%, 40% and 45% were tested on tail-bud stage embryos. Higher concentration of the vitrification solution led to significantly lower embryonic survival rate (P < 0.05). The survival rate was 36.79-72.05% in PMG3S, and 37.11-55.18% in PMG3T, and there were non-significant differences in embryonic development and malformation rates among the groups treated with different concentrations. The embryonic normal development rates in PMG3S and PMG3T were 21.27% and 11.04%, and the malformation rates were 36.13% and 31.04%, respectively. The optimum treatment condition was 40 min using 40% PMG3S on embryos at the tail-bud stage. Both PMG3S and PMG3T were used for cryopreserving embryos at 16 pairs somite, tail-bud and ET stage in liquid nitrogen, where we obtained 190 surviving embryos, and 44 fishes underwent normal development and hatched. The survival rate of cryopreserved embryos was 5.15%, the normal development rate was 1.31%, and the malformation rate was 3.66%. We found that PMG3S and PMG3T were effective for cryopreservation of Epinephelus moara embryos. The results provide a foundation for further explorations of fish embryo cryopreservation techniques.

[Clinical study on blocking mother-to-child transmission of hepatitis B virus with high viral load and HBeAg positivity during pregnancy in Guizhou province].

Objective: To observe the efficacy and safety related measures by blocking mother-to-child transmission of hepatitis B virus with high viral load and HBeAg positivity during pregnancy in Guizhou province. Methods: Outpatient and inpatient cases of the Department of Infectious Diseases and Obstetrics of Guizhou Medical University Affiliated Hospitals from May 2016 to July 2017 were retrospectively divided into intervention group, non-intervention group and non- hepatitis B pregnant women group; with 75 cases in each group. HBsAg and HBeAg were positive in the intervention group. Pregnant women with HBV DNA ≥10(6) IU/ml were treated with anti-HBV therapy for 24 to 28 weeks of gestation until delivery. According to oral drugs, they were divided into tenofovir (TDF) group or telbivudine (LDT) group, non-intervention group (HBsAg and HBeAg positive), HBV DNA positive pregnant women, pregnant women with no anti-HBV drugs, non-hepatitis B pregnant women (normal pregnant women without HBV infection). Infants and young children born to the three groups of women were immunized with the national viral hepatitis B action plan. The gestational weeks and Apgar scores at birth, delivery mode, feeding mode, sex and 7-months-old age were observed and counted. Serum hepatitis B markers (HBVM) and HBV DNA were quantitatively detected. HBVM was detected by time-resolved fluorescence immunoassay (TRFIA), and HBV DNA was detected by real-time PCR (FQ-PCR). The changes of liver parameters, HBsAg, HBeAg, HBV DNA, adverse drug reactions and treatment response of pregnant intervention group before medication (12-24 weeks of gestation), 4 weeks of medication (28-32 weeks of gestation), 36-40 weeks of gestation (36-40 weeks of gestation) were statistically calculated. A t-test was used to compare the data between the measurements. Data measurements within the groups were analyzed using rank -sum test. Results: In the intervention group, therapeutic medications showed no differences in demographic and clinical characteristics between TDF group and LDT group, including liver parameters, HBsAg, HBeAg and log10HBV DNA level. Compared with pre-treatment (TDF group: 4.84 ± 2.01; LDT group: 5.08 ± 1.99), TDF and LDT were significantly lower at the end of pregnancy (TDF group: 3.06 ± 0.66; LDT group: 3.51 ± 1.20). P < 0.05); and the treatment response rate was 100%. There were no serious adverse events in the intervention group. Infants and young children (7-months-old) in the intervention group had negative HBsAg, HBeAg and HBV DNA. The mother-to-child transmission rate of HBV was zero, with blocking rate of 100%. In addition, both infants and young children had different degrees of hepatitis B protective antibodies (anti-HBs, M: 144.33), and their antibody titers were higher than that of non-intervention group (anti-HBs, M: 65.91) and non-hepatitis B pregnant women (anti-HBs, M: 58.43). The difference was statistically significant (P < 0.05), and there was no significant correlation between the use of antiviral and the way of delivery and feeding. Outcomes of mother-to-child transmission of HBV infection in infants and young children (7-months-old) delivered by three groups of pregnant women in the non-intervention groups had 20.0% (15/75)/ 17.3% (13/75) HBsAg/HBeAg positivity rate, and 17.3% (13/75) HBV DNA positivity rate. Overall, mother-to-child transmission rate of HBV infection was 20% (15/75). Furthermore, the relationship between mother's HBV DNA load and infant HBV infection in the non-intervention group showed mother's HBV DNA ≥10(6) IU/ml. Conclusion: In the non-intervention group, mother-to-child transmission of HBV occurred, and infected mothers HBV DNA was ≥106 IU/ml before delivery. This suggests that HBeAg positive and high load HBV DNA replication were independent risk factors for mother-to-child transmission of hepatitis B. Therefore, prenatal drug intervention and postpartum standard immune blockade are necessary for high-risk pregnant women with hepatitis B to achieve zero mother-to-child transmission of hepatitis B in real- clinical practice.

[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.

[Components of human placental extract and its protective effect to rat from D-GalN induced acute liver injury].

Objective: To evaluate the effect of human placental extracts (HPE) on the protection from acute liver injury (ALI) induced by D-GalN and analyze the components of HPE. Methods: (1)Fourty male mice were randomly divided into five groups (Blank, Model, MgIG, HPE and HPE+ MgIG) for the ALI model and treatments.The serum alanine aminotransferase (ALT) and aspartate transaminase (AST) were determined by biochemical assays.Nitric monoxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) in serum and hepatic tissue were detected by assay kits.The extent of liver damage was evaluated by histological examination.(2)Relative molecular mass of HPE was determined by SDS-PAGE.(3) Component identification was performed by using LCMS-TOF.(4)Selected functional molecules in HPE were detected by protein array. Results: (1) A lower level of NO and MDA and a higher SOD and T-AOC were observed in rats treated with HPE compared to the non-treated rats in an acute liver failure disease model.(2) The size of HPE was about 1 200-4 600 by electrophores.(3) 7 peaks of HPE were identified, including uracil, hypoxanthine, tyrosine, phenylalanine, tryptophan, xanthine and thymine.(4) Comparable high concentrations of TGF-ß, IGF-1, IL-9, IL-29 and TNF-α of HPE were revealed by protein array. Conclusions: (1) HPE protects rat from liver damage induced by D-GalN. (2) HPE contains Uracil, hypoxanthine, xanthine, thymine, and functional proteins as TGF-ß, IGF-1, IL-9, IL-29 and TNF-α.

[Changes in the composition of intestinal microbiota in mice with acute liver failure induced by D-galactosamine].

Objective: To investigate the changes in the composition of intestinal microbiota in mice with acute liver failure and identify characteristic bacteria, and to provide a basis for the diagnosis and treatment of acute liver failure with intestinal microbiota disorders. Methods: A total of 30 specific pathogen-free male BALB/c mice were used in this study, including 25 mice in the model group and 5 mice in the control group. An acute liver failure model was induced by D-galactosamine. Microbial DNA was extracted from intestinal contents in different segments of the lower digestive tract (ileum and colon) and feces and then were amplified using PCR. The regions of 16S V3-V4 were subjected to high-throughput sequencing, followed by bioinformatics analyses, including OTU hierarchical clustering, species annotation, alpha-diversity analysis, and LEfSe (LDA Effect Size) analysis. Comparison of continuous data was made using t-test, while comparison of categorical data was made using χ2 test. Results: A total of 10 mice survived in the two groups, with 80% mortality rate in the model group. The alpha-diversity analysis revealed increased bacterial diversity and abundance in the ileum, increased bacterial diversity and reduced bacterial abundance in the colon, and reduced bacterial diversity and insignificantly changed bacterial abundance in feces in the model group compared with the control group. Based on the optimized classification level, significantly reduced abundance of Clostridiaceae (44.95% ± 19.28% vs 7.51% ± 16.57%, P = 0.011) in the ileum, whereas significantly increased abundance of Rikenellaceae (1.08% ± 1.01% vs 4.18% ± 2.39%, P = 0.028), S24-7 (4.75% ± 4.87% vs 22.77% ± 13.05%, P = 0.020), and F16 (0.24% ± 0.28% vs 2.18% ± 1.61%, P = 0.029) in the colon were found in model group compared with the control group. The LEfSe analysis demonstrated that there were significant differences in Staphylococcaceae and S24-7 between the two groups, and S24-7 could be defined as the characteristic bacteria. Conclusion: Intestinal microbiota disorders, especially the excessive growth of microbes in the ileum, are observed in mice with acute liver failure. Moreover, acute liver failure may be closely associated with the excessive growth of S24-7.

[Utilization of dental services for children: a review of the influencing factors and the possible improvements].

It has been reported that children's oral health conditions are correlated with their attendance to dental health services. Evaluating the influencing factors of utilization of dental services for children may give ways to improve the services per se, and furtherly the children's oral health. The present review retrieved and summarized domestic and foreign studies on the utilization of oral health services for children based on the Andersen behavior model. It was concluded that the utilization of dental services for children was affected by demographic characteristics, social structure, health belief, family factors, community factors and perceived/evaluated needs. To improve the utilization of dental services for children, effort should be made by means of changing caregivers' health belief, developing oral health insurance system, setting up regular oral health resources and increasing the financial support for oral health services by government.

Combining ultrasound and lactobacilli treatment for high-fat-diet-induced obesity in mice.

Chronic systemic lipopolysaccharide-induced inflammation can cause obesity. In animal experiments, lactobacilli have been shown to inhibit obesity by modifying the gut microbiota, controlling inflammation and influencing the associated gene expression. A previous study found that high-fat-diet-induced (HFD) obesity was suppressed by lactobacilli ingestion in rats via the inhibition of parasympathetic nerve activity. This study explored the combined use of lactobacilli ingestion and ultrasound (US) to control body weight and body fat deposition in HFD mice over an 8-week experimental period. Male C57BL/6J mice received an HFD during treatment and were randomly divided into four groups: (i) control group (H), (ii) lactobacilli alone (HB), (iii) US alone (HU) and (iv) lactobacilli combined with US (HUB). The US was targeted at the inguinal portion of the epididymal fat pad on the right side. At the 8th week, body weight had decreased significantly in the HUB group (15.56 ± 1.18%, mean ± SD) group compared with the HU (26.63 ± 0.96%) and H (32.62 ± 5.03%) groups (p < 0.05). High-resolution microcomputed tomography (micro-CT) scans revealed that the reduction in total body fat volume was significantly greater in the HUB group (69%) than in the other two experimental groups (HB, 52%; HU, 37%; p < 0.05). The reductions in the thickness of the subcutaneous epididymal fat pads were significantly greater in the HUB group (final thickness: 340 ± 7 µm) than in the H (final thickness: 1150 ± 21 µm), HB (final thickness: 1060 ± 18 µm) and HU (final thickness: 370 ± 5 µm) groups (all p < 0.05). Combination therapy with lactobacilli and US appears to enhance the reduction in body weight, total and local body fat deposition, adipocyte size and plasma lipid levels over an 8-week period over that achieved with lactobacilli or US alone in HFD mice. These results indicate that US treatment alone can reduce hyperlipidemia in HFD mice.

[Effect of cannabinoid receptor-2 agonist AM1241 on platelet-derived growth factor expression in the liver tissue of mice with hepatic fibrosis].

Objective: To investigate the effect of cannabinoid receptor-2 (CB2) agonist AM1241 on the mRNA and protein expression of platelet-derived growth factor (PDGF) and collagen-III (Col-III) in the liver tissue of mice with experimental liver fibrosis induced by carbon tetrachloride (CCl(4)). Methods: Totally 38 8-week-old male C57BL/6J mice were randomly divided into control group, model group, 3 mg/kg CB2 receptor agonist (AM1241) group, and 9 mg/kg AM1241 group. All mice, except for the control group, were treated with 30% CCl(4) (three times a week, 5 ml/kg body weight, 16 weeks) to establish a liver fibrosis model. Meanwhile, 3 and 9 mg/kg AM1421 was intraperitoneally injected for daily intervention, respectively. The dosage was adjusted according to actual body weight. The same solvent was given in the control group. The serum level of aspartate aminotransferase (AST) was measured by serum enzyme digestion. The liver inflammation and fibrosis were observed by HE staining of tissue slices. The mRNA and protein expression of PDGF and Col-III in hepatic tissue was determined by real-time PCR and immunohistochemistry. Results: Compared with the control group, the mice in model group showed severe liver fibrosis, significantly elevated serum AST level (742 ± 300.8 U/L vs 118.1 ± 31.1 U/L, P < 0.05), and significantly increased mRNA and protein expression of PDGF and Col-III in liver tissue (P < 0.05). Compared with the model group, the mice in 3 mg/kg AM1241 group and 9 mg/kg AM1241 group had less severe liver fibrosis, and significantly reduced serum AST levels (116.6 ± 13.68 U/L vs 742 ± 300.8 U/L, P < 0.05; 113.8 ± 16.01 U/L vs 742 ± 300.8 U/L, P < 0.05) and mRNA and protein expression of PDGF and Col-III in liver tissue (P < 0.05). Conclusion: CB2 receptor agonist AM1241 can inhibit the mRNA and protein expression of PDGF in the liver tissue of mice with hepatic fibrosis, and reduce extracellular matrix synthesis.