Effect of Astragaloside Ⅳ Combined with Notoginseng Total Saponins on Angiogenesis After Transplantation of BMSCs in Rats with Cerebral Ischemia / 中国实验方剂学杂志

Objective:To investigate the effect of astragaloside Ⅳ（AST Ⅳ）and Notoginseng total saponins （NTS） combined with bone marrow mesenchymal stem cell （BMSC） transplantation on neural repair and angiogenesis in rats with cerebral ischemia. Method:The rats were randomly divided into a sham operation group， a model group， low- and high-dose AST Ⅳ + NTS groups， a BMSC infusion group， and low- and high-dose BMSC infusion+AST Ⅳ （10 and 20 mg·kg^-1） + NTS group （25， 50 mg·kg^-1）. BMSCs were isolated and purified by whole bone marrow adherent culture. The positive expression of surface markers of BMSCs （CD29， CD90， CD34， and CD45） was detected by flow cytometry. The focal cerebral ischemia model was established by middle cerebral artery occlusion （MCAO）. The PKH26-labeled BMSCs were injected into the tail vein of rats in the BMSC infusion group， once a day. The rats in the combination groups received BMSC injection once a day and intragastric administration of drugs twice a day. Other groups were administered twice a day by gavage. The sham operation group and the model group received the same amount of normal saline. Symptoms and signs of neurological deficits were assessed by the Longa method and the cerebral infarction rate was determined by TTC staining. The survival and vascularization ［double positive expression of PKH26/vascular endothelial growth factor （VEGF）］ after transplantation of BMSCs were observed by the immunofluorescence method. The protein expression of Ang1 and TGF-<italic>β</italic>₁ was measured by Western blot. Result:BMSCs were properly isolated and cultured. The identification of surface markers CD29， CD90， CD34， and CD45 was consistent with the characteristics of BMSCs. The neurological deficit score and cerebral infarction rate of the model group were significantly increased （<italic>P</italic><0.01）. All drugs and cell transplantation could alleviate the above pathological changes in varying degrees. The strongest effect was observed in high-dose BMSC infusion+AST Ⅳ+NTS group （<italic>P</italic><0.01）， which was superior to those in the AST Ⅳ+NTS groups or the BMSC infusion group. BMSC injection helped cells survive in the ischemic brain tissues and promoted angiogenesis， and this effect could be enhanced by the combination with drugs. After cerebral ischemia， the expression of Ang1 and TGF-<italic>β</italic>₁ was increased， and the effect in the BMSC infusion+AST Ⅳ+NTS groups was the strongest （<italic>P</italic><0.01）. Conclusion:AST Ⅳ combined with NTS can promote the survival of transplanted BMSCs and facilitate angiogenesis after target repair of damaged blood vessels after cerebral ischemia. The mechanism may be related to the improvement of the local microenvironment in the brain after cerebral ischemia and the promotion of the survival and differentiation of transplanted stem cells.

Construction of NTCP-Huh7 cell lines and their susceptibility to HBV / 医学研究生学报

ObjectiveThe transient expression of the functional receptor sodium taurocholate cotransporting polypeptide (NTCP) leads to the inefficiency and instability of the hepatitis B virus (HBV)-associated hepatocellular carcinoma cell model. The aim of this study was to construct NTCP-Huh7 cell lines by transfecting GV358-NTCP into Huh7 cells and identify their susceptibility to HBV.MethodsThe recombinant plasmid GV358-NTCP was obtained by ligation of GV358 and NTCP, and then transfected into Huh7 cells for the construction of NTCP-Huh7 cells. The NTCP-Huh7 cells (NTCP-Huh7 group) and Huh7 cells (Huh7 group) were infected with HBV and co-incubated with HBV for 18 hours, and the co-incubation was continued after change of the culture medium. At 2, 4, 6, 8, 10, and 12 days of incubation, the supernatant and cells were collected for measurement of the contents of HBsAg, HBeAg, HBcAg and HBV DNA in the supernatant and HBV cccDNA in the cells as well as for determination of HBV susceptibility of the NTCP-Huh7 recombinant cells.ResultsWestern blot showed stably expressed NTCP proteins in the NTCP-Huh7 cells. At 8 days of incubation, the levels of HBsAg and HBeAg were significantly higher in the NTCP-Huh7 group (0.866±0.040 and 0.603±0.053) than in the Huh7 group (0.237±0.063 and 0.209±0.112) (P<0.05), reaching the peak at 8 days and also remarkably higher in the former than in the latter group at 4, 6 and 10 days (P < 0.05). So were the levels of HBV DNA and HBV cccDNA in the NTCP-Huh7 than in the Huh7 group at 4, 6, 8, 10 and 12 days (P < 0.05). Immunofluorescence assay revealed the core antigen of HBV in the NTCP-Huh7 but not in Huh7 cells.ConclusionNTCP-Huh7 cells obtained by transfection of the GV358-NTCP recombinant plasmid into Huh7 cells are susceptible to HBV infection.

Relationship Between Salivary Pepsin Concentration and Esophageal Mucosal Integrity in Patients With Gastroesophageal Reflux Disease

BACKGROUND/AIMS: Increased salivary pepsin could indicate an increase in gastro-esophageal reflux, however, previous studies failed to demonstrate a correlation between salivary pepsin concentrations and 24-hour esophageal acid exposure. This study aims to detect the salivary pepsin and to evaluate the relationship between salivary pepsin concentrations and intercellular spaces (IS) in different gastroesophageal reflux disease phenotypes in patients. METHODS: A total of 45 patients and 11 healthy volunteers were included in this study. All subjects underwent upper gastrointestinal endoscopy, 24-hour ambulatory multichannel impedance-pH (MII-pH) monitoring, and salivary sampling at 3-time points during the 24-hour MII-pH monitoring. IS were measured by transmission electron microscopy, and salivary pepsin concentrations were determined by enzyme-linked immunosorbent assay. RESULTS: The IS measurements were greater in the esophagitis (EE), non-erosive reflux disease (NERD), and hypersensitive esophagus (HO) groups than in the functional heartburn (FH) and healthy volunteer groups, and significant differences were indicated. Patients with NERD and HO had higher average pepsin concentrations compared with FH patients. A weak correlation was determined between IS and salivary pepsin among patients with NERD (r = 0.669, P = 0.035). CONCLUSIONS: We confirmed the presence of a higher level of salivary pepsin in patients with NERD than in patients with FH. Salivary pepsin concentrations correlated with severity of mucosal integrity impairment in the NERD group. We suggest that in patients with NERD, low levels of salivary pepsin can help identify patients with FH, in addition the higher the pepsin concentration, the more likely the severity of dilated IS.

Clinical efficacy of compound glycyrrhizin tablets in the treatment of children with nonalcoholic fatty liver disease / 中国当代儿科杂志

Department of Pediatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. zhuchuanlong@jsph.org.cn.

Identification of two novel mitochondrial DNA deletions induced by ionizing radiation / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>We identify ionizing radiation-induced mitochondrial DNA (mtDNA) deletions in human lymphocytes and their distribution in normal populations.</p><p><b>METHODS</b>Long-range polymerase chain reactions (PCR) using two pairs of primers specific for the human mitochondrial genome were used to analyze the lymphoblastoid cell line following exposure to 10 Gy (60)Co γ-rays. Limited-condition PCR, cloning and sequencing techniques were applied to verify the mtDNA deletions detected with long-range PCR. Human peripheral blood samples were irradiated with 0, 2 and 6 Gy (60)Co γ-rays, and real-time PCR analysis was performed to validate the mtDNA deletions. In order to know the distribution of mtDNA deletions in normal population, 222 healthy Chinese adults were also investigated.</p><p><b>RESULTS</b>Two mtDNA deletions, a 7455-bp deletion (nt475-nt7929 in heavy strand) and a 9225-bp deletion (nt7714 -nt369 in heavy strand), occurring between two 8-bp direct repeats, were identified in lymphoblastoid cells using long-range PCR, limited-condition PCR and sequencing. These results were also observed for (60)Co γ-rays irradiated human peripheral blood cells.</p><p><b>CONCLUSION</b>Two novel mtDNA deletions, a 7455-bp deletion and a 9225-bp deletion, were induced by ionizing radiation. The rate of the mtDNA deletions within a normal population was related to the donors' age, but was independent of gender.</p>

Determination and clinical significance of tissue inhibitors of metalloproteinase-1 and -2 in serum of children with nonalcoholic fatty liver disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To examine serum tissue inhibitors of metalloproteinases (TIMP) -1 and -2 levels in children with nonalcoholic fatty liver disease and to investigate possible roles of the two markers.</p><p><b>METHODS</b>One hundred and five obese children were classified into 4 groups: simple obesity (n=44), simple nonalcoholic fatty liver (SNAFL, n=25), and nonalcoholic steatohepatitis (NASH, n=36). Serum TIMP-1 and -2 levels were measured using ELISA. Serum ALT and gamma-GT levels were measured with totally automatic enzymatic method.</p><p><b>RESULTS</b>Serum levels of TIMP-1 and gamma-GT increased with the disease development from simple obesity to SNAFL and NASH (P<0.05). Both serum TIMP-1 and -2 levels were positively correlated with gamma-GT levels (r=0.534, P<0.01; r=0.351, P<0.05, respectively). Ninety-seven percent of children in the NASH group had serum TIMP-1 levels over 2 standard deviations of healthy controls (83.35 microg/ L) compared with 76% in the SNAFL group (P<0.05). There were no significant differences in the case proportion with TIMP-2 levels over 2 standard deviations of healthy controls between the NASH and the SNAFL groups.</p><p><b>CONCLUSIONS</b>Both TIMP-1 and -2 may reflect the state of liver fibrosis in children with nonalcoholic fatty liver disease, and serum TIMP-1 appears to be more reliable.</p>

Excessive copper induces hepatocyte apoptosis and affects Bax and Bcl-2 expression in rat liver / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To establish a hepatolenticular degeneration rat model with excessive copper, and investigate the effects of excessive copper deposits in the liver on hepatocyte apoptosis and Bax and Bcl-2 expression.</p><p><b>METHODS</b>Rat model of hepatolenticular degeneration was established by administering forages containing 1g/kg of copper sulfate and drinking water containing 0.185% copper sulfate. Copper level in the liver and serum and alanine aminotransferase (ALT) level in serum were measured using an atomic absorption spectrophotometer. The terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) method was used to detect hepatocyte apoptosis. Bax and Bcl-2 expression was observed by RT-PCR and imunohistochemistry staining. Quantification of positive cells was performed by image analyzer.</p><p><b>RESULTS</b>With more prolonged excessive copper ingestion, copper level in the liver and serum as well as ALT level in serum rose, and more apoptosis cells appeared in the liver. Bax and Bcl-2 expression increased significantly compared with controls fed a normal diet and progressively increased with more prolonged excessive copper ingestion. The progressively increased extent of Bcl-2 expression was lower than that of Bax expression, so the ratio of Bcl-2/Bax decreased with increasing excessive copper ingestion time.</p><p><b>CONCLUSIONS</b>Excessive copper deposits in the liver can induce hepatocyte apoptosis through an up-regulation of Bax expression.</p>

Curcumin attenuated the lipid peroxidation and apoptotic liver injury in copper-overloaded rats / 中华儿科杂志

<p><b>OBJECTIVE</b>Hepatolenticular degeneration (Wilson disease, WD) is an autosomal recessive disorder of copper metabolism. The clinical manifestations are dominated by the neuropsychiatric and hepatic symptoms due to copper deposition. Investigation of mechanism of copper injury should be helpful for elucidating the pathogenesis and treatment of WD. Curcumin, a plant-derived polyphenol, exhibits the properties of anti-oxidant, anti-inflammation and has no evident side effects, therefore, today curcumin is studied by more and more researchers in pharmacologic action and clinical application especially for its protective effect on liver diseases. The present study was designed to investigate the lipid peroxidation and apoptotic liver injury in copper-overloaded rats, and to explore the protective effects of curcumin.</p><p><b>METHODS</b>Wistar rats, male, were randomly divided by copper-overloaded groups and curcumin treatment groups and control group. Copper-overloaded rat model was established by feeding with forage containing 1 g/kg copper sulfate and water with 0.185% copper sulfate for 8 weeks or 12 weeks. In the treatment groups, curcumin was administered orally either 50 mg/kg or 200 mg/kg for 2 weeks and 4 weeks and 8 weeks and fed with copper sulfate at the same time until the 12th week. Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) in liver homogenates were measured to reflect the copper induced lipid peroxidation. The apoptosis of liver cell was detected by electron microscope (EM) and TUNEL assay. The expressions of TNF-alpha mRNA and IL-8 mRNA were observed by RT-PCR. Contents of TNF-alpha and IL-8 in liver homogenates were measured by ELISA.</p><p><b>RESULTS</b>The MDA concentrations were significantly increased and the GSH and SOD levels were decreased in the copper-overloaded rats. The apoptosis index displayed from (2.2 +/- 1.2)% in control rats to (16.7 +/- 2.5)% in the copper treated animals. Expression of TNF-alpha mRNA and IL-8 mRNA were enhanced in the copper-overloaded rats. Curcumin significantly attenuated the increase of MDA concentrations and recovered the GSH and SOD levels. The apoptosis index decreased to (10.4 +/- 1.2)% in the copper-overloaded rats with curcumin treatment. Curcumin down-regulated the expressions of TNF-alpha mRNA and IL-8 mRNA and content of TNF-alpha and IL-8. Histological changes induced by copper in liver, such as mitochondrial swelling and endoplasmic reticulum distention and increased lysosomal granules in the model rats, were also improved significantly by curcumin treatment as evidenced by EM examination.</p><p><b>CONCLUSION</b>Copper-overloading caused lipid peroxidatic injury and induced significant apoptosis in liver. TNF-alpha and IL-8 might be involved in liver injury in this model. Curcumin exhibited protective effects and possibly acted through its antioxidant and anti-apoptotic properties.</p>