Scutellarein alleviates the dysfunction of inner blood-retinal-barrier initiated by hyperglycemia-stimulated microglia cells.

AIM: To investigate the alleviation of scutellarein (SN) against inner blood-retinal-barrier (iBRB) dysfunction in microglia cells stimulated by hyperglycemia and to elucidate the engaged mechanism. METHODS: Microglia BV2 cells were stimulated by using 25 mmol/L D-glucose. The same concentration of mannitol (25 mmol/L) was applied as an isotonic contrast. Real-time PCR, Western-blot assay and immunofluorescence staining assay was performed. The dysfunction of iBRB in vitro was detected by using transendothelial electrical resistance (TEER) assay. Additionally, the leakage of fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa) was detected. RESULTS: SN abrogated microglia BV2 cells activation and reduced the phosphorylated activation of extracellular signal-regulated protein kinase (ERK)1/2. SN also decreased the transcriptional activation of nuclear factor κB (NFκB) and the elevated expression of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1ß in BV2 cells treated with D-glucose (25 mmol/L). SN attenuated iBRB dysfunction in human retinal endothelial cells (HRECs) or choroid-retinal endothelial RF/6A cells when those cells were treated with TNFα, IL-1ß or IL-6, or co-cultured with microglia cells stimulated by D-glucose. Moreover, SN restored the decreased protein expression of tight junctions (TJs) in TNFα-treated HRECs and RF/6A cells. CONCLUSION: SN not only alleviate iBRB dysfunction via directly inhibiting retinal endothelial injury caused by TNFα, IL-1ß or IL-6, but also reduce the release of TNFα, IL-1ß and IL-6 from microglia cells by abrogating hyperglycemia-mediated the activation of microglia cells.

An observation on the long-term efficacy of recombinant hepatitis B vaccines among the newborns / 预防医学

Objective evaluate the long-term efficacy of the recombinant hepatitis B vaccines (HBV) among the newbornswith vaccination at birth. Methods During 1996-1997, 135 newborns were selected from Deqing according to the inclusioncriterion. They were divided into 2 groups: a group of 35 newborns whose mother was HBsAg positive) and a group of 100newbornswhose mother was HBsAg negative. All 135 newborns routinely received 3 doses of yeast -derived hepatitis Bvaccines (i.e. the first dose at birth, the second dose at 1 month old, and the third dose at 6 months old) . Serologicalmarkers to HBV were repeatedly assessed at 3 follow-up stages (i.e. the first follow-up at 12 months, the second follow-upat 2010, the third follow-up at 2012) . Results Participants remained in the study at 3 follow-upstages were 123(91.11%), 95(70.37%) and 46(34.07%) respectively. Participants' serum HBsAg were negative at all 3 follow-upstages. Among participants whose mothers were HBsAg positive, 3 participants were found to be HBcAb positive in 2010,and no new HBcAb positive participants were found in 2012. The rates of HBsAb positive at 3 follow-up stages were 88.89%, 81.48%, and 80.00% respectively. The HBsAb geometric mean concentrations (GMCs) of participants at their 12 monthsold were significantly positively associated with those in 2010 and those in 2012(P<0.05) . Among participants whosemothers were HBsAg negative, no HBcAb positive participants were found. The rates of HBsAb positive at 3 follow-up stageswere 91.18%, 54.41%, and 52.78% respectively. No correlation was found among HBsAb GMCs of participants at 12 monthsold, in 2010 and in 2012. No correlation was found between boost vaccination and the rate of HBsAg positive, afteradjustment of the HBsAg status of their mothers. Conclusion The efficacy of the yeast-derived HBV could sustain for at least13-15 years, and the general population do not need booster immunization. After the 3-dose immunization, the HBsAblevels of the healthy mothers' 12-months-old children were related to those of their adolescence.

The Development of Diabetic Retinopathy in Goto-Kakizaki Rat and the Expression of Angiogenesis-Related Signals.

The Goto-Kakizaki (GK) rat is a genetic model of type 2 diabetes. Diabetic retinopathy (DR) is a common complication of diabetes. In this study, we observed the development of DR in GK rats and the expression of some angiogenesis-related signals. GK rats were housed for 5, 6 and 7 months. Results of retinal vessels stained by cluster of differentiation 31 (CD31) showed that the number of retinal vessels was increased in GK rats at both 6 and 7 months. Retinal histological observation also evidenced such increase. Retinal mRNA expression of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), VEGFB and its receptors (VEGFR1/2), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) A/B was increased in GK rats at both 6 and 7 months. Retinal mRNA expressions of matrix metalloproteinase (MMP) 2/9 and insulin-like growth factor 1 (IGF-1) were increased at 7 months. Retinal mRNA expression of pigment epithelium-derived factor (PEDF) was increased in GK rats at 6 months. Serum contents of VEGF, bFGF, PDGFA, MMP2/9, IGF-1, PEDF were increased in GK rats at both 6 and 7 months, while PDGFB was increased at 7 months. In summary, our results indicate that retinal angiogenesis occurred in GK rats at 6 and 7 months, and the expressions of some angiogenesis related factors were increased during the development of DR in GK rats.

An evaluation on immunization effect and the influencing factors after revaccination with 60 μg hepatitis B vaccine among non-response adults / 预防医学

Objective To explore vaccine immune effects of 60μg hepatitis B on adults who have no response to hepatitis B vaccination.Methods A Total of 689 healthy adults who were HBsAg and anti -HBs negative were selected,and they were received 3 doses of hepatitis B vaccine at dosage 10 μg.Those with anti-HBs<10 IU/L detected after full series vaccination were given 1 dose of hepatitis B vaccine at dosage 60 μg.One month later,anti-HBs detection was conducted for them.Results The non -responsiveness rate was 17.71% after primary hepatitis B vaccination.The multivariate logistic regression analysis showed that risk factors for primary non-responsiveness were the history of surgical operation, immunization schedule and "anti-HBc alone".The anti-HBs positive rate was 95.5 1% in 89 re-vaccinated people, and the geometric mean titers of anti -HBs(GMT)was 585.39 IU/L.Conclusion Re -immunization of hepatitis B vaccine at dosage 60 μg has good immunogenicity in people who have no response to primary vaccination,and weight control may improve the effect of hepatitis B re-immunization.

Chlorogenic acid prevents acetaminophen-induced liver injury: the involvement of CYP450 metabolic enzymes and some antioxidant signals.

Chlorogenic acid (CGA), a polyphenolic compound, is abundant in fruits, dietary vegetables, and some medicinal herbs. This study investigated the prevention of CGA against acetaminophen (AP)-induced hepatotoxicity and its engaged mechanisms. CGA reversed the decreased cell viability induced by AP in L-02 cells in vitro. In addition, CGA reduced the AP-induced increased serum levels of alanine/aspartate aminotransferase (ALT/AST) in vivo. The effect of CGA on cytochrome P450 (CYP) enzymatic (CYP2E1, CYP1A2, and CYP3A4) activities showed that CGA caused very little inhibition on CYP2E1 and CYP1A2 enzymatic activities, but not CYP3A4. The measurement of liver malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels showed that CGA prevented AP-induced liver oxidative stress injury. Further, CGA increased the AP-induced decreased mRNA expression of peroxiredoxin (Prx) 1, 2, 3, 5, 6, epoxide hydrolase (Ephx) 2, and polymerase (RNA) II (DNA directed) polypeptide K (Polr2k), and nuclear factor erythroid-2-related factor 2 (Nrf2). In summary, CGA ameliorates the AP-induced liver injury probably by slightly inhibiting CYP2E1 and CYP1A2 enzymatic properties. In addition, cellular important antioxidant signals such as Prx1, 2, 3, 5, 6, Ephx2, Polr2k, and Nrf2 also contributed to the protection of CGA against AP-induced oxidative stress injury.

The involvement of p62-Keap1-Nrf2 antioxidative signaling pathway and JNK in the protection of natural flavonoid quercetin against hepatotoxicity.

Quercetin, one of the most abundant dietary flavonoids, is reported to have protective function against various hepatotoxicant-induced hepatotoxicity. The present study aims to investigate the critical role of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidative signaling pathway in the protection of quercetin against hepatotoxicity. Quercetin prevented the cytotoxicity induced by a variety of hepatotoxicants including clivorine (Cliv), acetaminophen (APAP), ethanol, carbon tetrachloride (CCl4), and toosendanin (TSN) in human normal liver L-02 cells. Quercetin induced the nuclear translocation of Nrf2, along with the increased expression of the antioxidant responsive element (ARE)-dependent genes like catalytic or modify subunit of glutamate-cysteine ligase (GCLC/GCLM), and heme oxygenase-1 (HO-1). In addition, the HO-1 inhibitor zinc protoporphyrin (ZnPP) and the GCL inhibitor L-buthionine-(S,R)-sulfoximine (BSO) both reduced the hepatoprotection induced by quercetin. Quercetin had no effect on kelch-like ECH-associated protein-1(Keap1) expression, but molecular docking results indicated the potential interaction of quercetin with the Nrf2-binding site in Keap1 protein. Quercetin increased the expression of p62, and p62 siRNA decreased quercetin-induced hepatoprotection. Quercetin induced the activation of c-Jun N-terminal kinase (JNK) in hepatocytes. JNK inhibitor SP600125 and JNK siRNA both reduced quercetin-induced hepatoprotection. SP600125 and JNK siRNA decreased the increased p62 expression induced by quercetin. In addition, SP600125 also decreased the increased mRNA and protein expression of GCLC, GCLM, and HO-1 induced by quercetin. Taken together, our present study demonstrates that quercetin prevents hepatotoxicity by inducing p62 expression, inhibiting the binding of Keap1 to Nrf2, and thus leading to the increased expression of antioxidative genes dependent on Nrf2. Meanwhile, our study indicates that JNK plays some regulation in this process.

Protection of Flos Lonicerae against acetaminophen-induced liver injury and its mechanism.

This study aims to observe the protective action of Flos Lonicerae (FL) aqueous extract against acetaminophen (AP)-induced liver injury and its mechanism. Results show that FL decreases AP-increased serum alanine/aspartate transaminases (ALT/AST) activity, as well as total bilirubin (TB) amount, in mice. Histological evaluation of the liver further confirms the protection of FL against AP-induced hepatotoxicity. TdT-mediated biotin-dUTP nick-end labeling (TUNEL) assay shows that FL reduces AP-increased apoptotic cells. Furthermore, AP-decreased liver glutamate-cysteine ligase (GCL) enzymatic activity and glutathione (GSH) amount are both reversed by FL because of the increased expression of the catalytic subunit of GCL (GCLC) protein. The amount of chlorogenic acid (CGA), caffeic acid, and luteolin, the main active compounds in FL, is detected by high-performance liquid chromatography (HPLC). In addition, cell viability assay demonstrates that polyphenols in FL, such as CGA, caffeic acid, as well as isochlorogenic acids A, B, and C, can reverse AP-induced cytotoxicity. In conclusion, FL can prevent AP-induced liver injury by inhibiting apoptosis. The cellular antioxidant enzyme GCL is also involved in such protection. Polyphenols may be the main active hepato-protective ingredients in FL.

Ferulic acid prevents liver injury and increases the anti-tumor effect of diosbulbin B in vivo.

The present study is designed to investigate the protection by ferulic acid against the hepatotoxicity induced by diosbulbin B and its possible mechanism, and further observe whether ferulic acid augments diosbulbin B-induced anti-tumor activity. The results show that ferulic acid decreases diosbulbin B-increased serum alanine transaminase/aspartate transaminase (ALT/AST) levels. Ferulic acid also decreases lipid peroxide (LPO) levels which are elevated in diosbulbin B-treated mice. Histological evaluation of the liver demonstrates hydropic degeneration in diosbulbin B-treated mice, while ferulic acid reverses this injury. Moreover, the activities of copper- and zinc-containing superoxide dismutase (CuZn-SOD) and catalase (CAT) are decreased in the livers of diosbulbin B-treated mice, while ferulic acid reverses these decreases. Further results demonstrate that the mRNA expressions of CuZn-SOD and CAT in diosbulbin B-treated mouse liver are significantly decreased, while ferulic acid prevents this decrease. In addition, ferulic acid also augments diosbulbin B-induced tumor growth inhibition compared with diosbulbin B alone. Taken together, the present study shows that ferulic acid prevents diosbulbin B-induced liver injury via ameliorating diosbulbin B-induced liver oxidative stress injury and augments diosbulbin B-induced anti-tumor activity.

Ethanol extract of Dendrobium chrysotoxum Lindl ameliorates diabetic retinopathy and its mechanism.

Diabetic retinopathy (DR) is the most common and serious complication of diabetes mellitus (DM). The present study investigates the amelioration of ethanol extract of Dendrobium chrysotoxum Lindl (DC) on streptozotocin (STZ)-induced DR and its engaged mechanism. Retinal immunofluorescence staining with cluster of differentiation 31 (CD31) demonstrated that DC (30-300 mg/kg) decreased the increased retinal vessels in STZ-induced diabetic rats. Retinal histopathological observation also showed that retinal vessels were decreased in DC-treated diabetic rats. DC decreased the increased retinal mRNA expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) in diabetic rats, and DC also decreased the elevated serum VEGF level. Immunohistochemical staining further evidenced that DC decreased VEGF and VEGFR2 expression in retinas. Retinal mRNA expression of matrix metalloproteinase (MMP) 2/9 was decreased in DC (300 mg/kg)-treated diabetic rats. Serum levels of MMP 2/9, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A/B, insulin-like growth factor 1 (IGF-1), interleukin 1ß (IL-1ß), and IL-6 were all decreased in DC-treated diabetic rats. In addition, DC decreased the increased phosphorylation of p65 and the increased expression of intercellular adhesion molecule-1 (ICAM-1). In conclusion, DC can alleviate retinal angiogenesis during the process of DR via inhibiting the expression of VEGF/VEGFR2, and some other pro-angiogenic factors such as MMP 2/9, PDGF A/B, bFGF, IGF-1. In addition, DC can also ameliorate retinal inflammation via inhibiting NFκB signaling pathway.

The long-term efficacy of recombinant hepatitis B vaccines for children / 预防医学

Objective To observe long-term efficacy of recombinant hepatitis B vaccines for children received fundamental immunization or booster dose. Methods 493 school students from Deqing county with complete information of immunization history were investigated and their serological markers were detected. 430 students received booster dose vaccines at 3-11 years old and were defined as booster group while the remaining 63 students were defined as fundamental group. Results All vaccines the 493 students received for fundamental immunization were recombinant. Compared with fundamental group(57. 14%),booster group had significantly higher Anti-HBs positive rate of 91. 40%(P<0. 01). And the Anti-HBs positive rate had a high level of 94. 99% when 0-5 years after booster immunization while it declined to 64. 71% after 8-15 years and showed no significant difference compared with fundamental group. Conclusion Anti-HBs level and anti -HBs positive rate can significantly increase for short periods after booster immunization,and may decline considerably for long-term. Immunologic amnesia rate is low when 3 -11 years after fundamental immunization among infants.

Prevalence of occult hepatitis B virus infection and its phylogenetic features among mother-teenager pairs / 中华流行病学杂志

Objective Prevalence of occult hepatitis B virus (HBV) infection (OBI) was investigated in a paired mother-teenager population and HBV S gene variation including overt and occult HBV,was determined.Methods A follow-up study based on an initial survey of 135 mother-teenager pairs was carried out through collection of questionnaires and blood samples HBsAg were detected by ELISA method,viral load by PCR amplification and HBV S gene by phylogenetic analysis.Results 102 pairs of subjects were followed-up.Blood samples from 94 mothers and 101children were collected.OBI prevalence in mothers was 10.0％ (6/60),significantly higher than 2.0％(2/101) in teenagers.Medians of viral load were 399.9 IU/ml and 247.6 IU/ml in overt and occult HBV strains,but without significant difference.1 occult HBV strain belonged to genotype B with serotype adw while the other 7 were genotype C with serotype adr.15 of the overt HBV strains belonged to genotype B with serotype adw and the other 8 were genotype C with serotype adr.Proportions of genotype-C strains were significantly higher in occult HBV strains than in overt HBV strains.Conclusion OBI was seen in teenage-mother population.

A neuroprotective mechanism of YGY-E in cerebral ischemic injury in rats.

AIMS: To investigate the anticerebral ischemic properties of YGY-E (apigenin-7-O-ß-D-glucopyranosy l-4'-O-α-L-rhamnopy-ranosid, a flavonoid glycoside extracted from plant phoenix-tail fern), focusing on its effects on neuronal apoptosis. METHODS: In vitro YGY-E treatment was examined in primary cultured rat hippocampal neurons subjected to hypoxia-reoxygenation (H-R) injury. In addition, in vivo effects of YGY-E on neuronal apoptosis were measured by Hoechst staining and in situ DNA end labeling (TUNEL). Finally, B cell lymphoma/lewkmia-2 (Bcl-2) level in ischemic rat brain tissue was evaluated with immunohistochemistry and western blot analyses. RESULTS: In vitro YGY-E (50-100 µg/mL) treatment increased the survival rate compared to that of the vehicle-treated group (P < 0.05 and P < 0.01, respectively). In in vivo experiments, YGY-E (2.5-10 mg/kg) decreased the percentage of apoptotic neurons (P < 0.01), increased Bcl-2 (P < 0.01) in ischemic rat brain tissue. These effects were dose dependent. CONCLUSIONS: Our findings indicate that YGY-E's neuroprotective effects may be because of its inhibition of neuronal apoptosis by increasing Bcl-2 expression.

The difference of glutathione antioxidant system in newly weaned and young mice liver and its involvement in isoline-induced hepatotoxicity.

Cellular glutathione antioxidant system plays important roles in counteracting hepatotoxins-induced oxidative stress injury. The present study was designed to observe the differences of this system in newly weaned and young mice liver and its involvement in the susceptibility to isoline-induced liver injury. Our results showed that liver reduced glutathione (GSH) amounts were higher in newly weaned mice than young mice. Glutamate-cysteine ligase (GCL) activity was higher in newly weaned mice due to the higher expression of catalytic subunit of GCL (GCLC) protein and mRNA. However, the activities of glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) were higher in young mice liver, which might be due to the higher expression of GR, GPx-1, and GST-Pi proteins. Next, the results of AST analysis and histopathological evaluation showed that newly weaned mice demonstrated more severe liver injury induced by isoline. Furthermore, liver GSH amounts and the activities of GR, GPx, and GST were all lower in newly weaned mice than young mice after treated with isoline. Depletion of cellular GSH by D,L -buthionine-(S, R)-sulfoximine (BSO) aggravated isoline-induced cytotoxicity, while N-acetyl-l cysteine (NAC) ameliorated such cytotoxicity. Furthermore, the inhibitors of GR, GPx, and GST all aggravated isoline-induced cytotoxicity. In conclusion, our results demonstrated the differences of glutathione antioxidant system between newly weaned and young mice liver. Meanwhile, our results also revealed age-dependent liver injury induced by isoline for the first time, which might be due to the different responses of glutathione antioxidant system to isoline between newly weaned and young mice.

Pyrrolizidine alkaloid clivorine induces apoptosis in human normal liver L-02 cells and reduces the expression of p53 protein.

Clivorine, a pyrrolizidine alkaloid, is a potent toxic compound extracted from a Chinese medicinal plant Ligularia hodgsonii Hook. We have previously shown that clivorine inhibited human normal liver L-02 cells proliferation and induced p38 mitogen-activated protein kinase phosphorylation. The aim of this study is to further observe the mechanism of clivorine-inhibited L-02 cells growth. In this paper we show here that clivorine can induce apoptosis in L-02 cells, reduce the expression of p53 protein but has no effect on the expression of Bcl-2 protein, and clivorine also induces the cleavage of the poly(ADP-ribose) polymerase in L-02 cells. Our results suggest that the anti-proliferative function of clivorine in L-02 cells may be due to its inducing cell apoptosis, and clivorine-induced cell apoptosis is independent of p53 protein.