[Mouse immune responses elicited by eukaryotic expression plasmid and prokaryotically expressed protein of Brugia malayi myosin 29 epitope].

Objective: To determine the immune responses elicited in BALB/c mice by vaccination with eukaryotic expression plasmid pcDNA3.1(+)-BmM29 containing Brugia malayi myosin 29(BmM29) epitode and prokaryotically expressed recombinant BmM2 protein(rBmM29) respectively. Methods: rBmM29 protein was expressed in E. coli strain BL21, purified as recombinant protein vaccine, and administered via multiple subcutaneous injections. The purified recombinant plasmid pcDNA3.1(+)-BmM29 was used as the nucleic acid vaccine and injected into the tibialis anterior muscle. Sixty BALB/c mice were randomized to receive three immunizations(with intervals of 2 weeks) with PBS (100 µg, group A), pcDNA3.1(+)/CpG (100 µg/30 µg, group B), pcDNA3.1(+)-BmM29/CpG (100 µg/30 µg, group C), rBmM29/CpG(50 µg/30 µg, group D), or pcDNA3.1(+)-BmM29/rBmM29/CpG (two injections of pcDNA3.1(+)-BmM29/CpG 100 µg/30 µg followed by a rBmM29/CpG 50 µg/30 µg). Serum was prepared through ophthalmectomy at week 4, 6, and 8 after primary immunization, and the serum IgG titer was determined by ELISA. The mice were sacrificed at week 8, splenocyte suspension cultured for 48 h, and levels of INF-γ and IL-4 in the supernatant detected by ELISA. Results: ELISA results showed that the A490 values of serum IgG in groups A-E were 0.038 ± 0.050,0.053 ± 0.009,0.360 ± 0.035,0.456 ± 0.025,0.370 ± 0.025 at week 4,0.045 ± 0.003,0.045 ± 0.005,0.510 ± 0.018,0.548 ± 0.010,0.552 ± 0.018 at week 6, and 0.041 ± 0.004,0.044 ± 0.009,0.606 ± 0.047,0.674 ± 0.042,0.770 ± 0.041 at week 8, significantly higher in groups C, D and E than in groups A and B (P < 0.05) at all time points, and significantly higher in group E than in groups C and D(P < 0.05) at week 8. The IFN-γ levels in splenocyte culture supernatant at week 8 after primary immunization were (47.72 ± 8.94),(50.43 ± 2.81),(304.78 ± 8.42),(242.28 ± 5.99), and(426.52 ± 6.76) pg/ml in groups A-E, respectively, significantly higher in groups C-D than in groups A and B(P < 0.05), and in group E than in groups C and D(P < 0.05). The IL-4 levels in splenocyte culture supernatant were(60.00 ± 11.14),(57.71 ± 15.95),(93.17 ± 12.56),(96.67 ± 11.48), and (101.17 ± 5.81) pg/ml, significantly higher in groups C-D than in groups A and B(P < 0.05). Conclusion: Both the recombinant plasmid pcDNA3.19(+)-BmM29 and rBmM29 protein could elicit specific humoral and cellular immune responses in mice. Combined immunization with nucleic acid vaccine and protein vaccine is superior to each of the two alone.

Anticoagulation therapy is harmful to large-sized cerebellar infarction.

AIM: Anticoagulants are commonly used to treat ischemic stroke. Its impact on cerebellar infarction has not been fully understood. METHODS: In the clinical study, we reviewed a consecutive series of patients with large-sized cerebellar infarction (diameter > 3 cm, n = 30) treated with or without anticoagulation. In animal study, cerebellar infarction operation was performed in 12 Cynomolgus monkeys. Then the animals were administrated with low molecular weight heparin (LMWH) or vehicle for 14 days. RESULTS: Six patients died during the following treatment. All the subjects that died received anticoagulation therapy, while nobody in the survival group received such a therapy. Compared with sham-operated animals, all monkeys with cerebellar infarction have obvious neurological deficits. The number and size of the Purkinje cells in the cerebellar area were also reduced. Two animals in the LMWH group (33%) died, while all animals in the vehicle control group survived. Compared with the vehicle group, the neurological score in the LMWH group was significantly increased (P < 0.05). The water content in the cerebella was also significantly higher (P < 0.05). Edema, hemorrhage, and subarachnoid hemorrhage occurred in the cerebella as well as brainstem of all the LMWH treated animals. CONCLUSIONS: These results indicated the harmful effects of anticoagulation therapy on large-sized cerebellar infarction.

Total bakkenolides protects neurons against cerebral ischemic injury through inhibition of nuclear factor-κB activation.

Total bakkenolides is the major component of the rhizome of Petasites trichinous Franch.. In this study, we investigated its neuroprotective effects in a rat transient focal cerebral ischemia-reperfusion model, and in an in vitro cerebral ischemia model, oxygen-glucose deprivation of cultured nerve cells. Oral administration of total bakkenolides immediately after reperfusion at doses of 5, 10 and 20 mg/kg markedly reduced brain infarct volume and neurological deficits. Total bakkenolides significantly attenuated cell death and apoptosis in primarily cultured neurons subject to 1-h hypoxia followed by 24-h reoxygenation. Morphologic observations directly confirmed its protective effect on neurons. We also demonstrated that total bakkenolides could inhibit nuclear factor-κB (NF-κB) activation by blocking the classic activation pathway through suppression of phosphorylation of IκB-kinase complex, NF-κB/p65 and inhibitor protein IκB, inducing nuclear translocation of NF-κB/p65 and degradation of IκB. Further, total bakkenolides inhibited the activation of Akt and the extracellular signal-regulated kinase 1/2, two important upstream activators of NF-κB. In conclusion, our results provide a strong pharmacological basis for further understanding the potential therapeutic role of total bakkenolides in cerebral ischemic disease and shed new light on its neuroprotective mechanism.

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.

Interventional effect of Ginkgo biloba extract on the progression of gastric precancerous lesions in rats.

OBJECTIVE: To investigate the effect of Ginkgo biloba extract on gastric precancerous lesions in rats. METHODS: 80 4-week-old Wistar rats were randomly divided into four groups: a control group, a model group, a low and a high dose Ginkgo biloba extract intervention group; 20 in each group. Gastric precancerous lesions were induced by giving them 100 mg/L N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) solution to drink ad libitum for 20 weeks. In addition to the MNNG, the intervention groups were lavaged with Ginkgo biloba extract (0.5 mg/kg/d in the low dose group, 1.5 mg/kg/d in the high dose group) for 20 weeks. Starting from week 21 all the rats were fed with normal rat chow and tap water. At the end of week 30 the rats were killed. The histopathological changes of their gastric mucosa, ISA, NGI, the serum and gastric mucosal SOD/MDA and the expressions of oncogenes were studied. RESULTS: The incidence of mild to severe intestinal metaplasia and dysplasia were significantly lower in the intervention groups than those in the model group (P < 0.01). The ISA and NGI in the intervention groups were significantly lower than those in the model group (P < 0.01). In the intervention groups the activity of SOD was increased and the concentration of MDA was decreased (P < 0.01). Expressions of Bcl-2, c-myc and FasL decreased in the intervention groups, whereas the expression of Fas increased. When compared with the model group, the differences were statistically significant (P < 0.01, P < 0.05, respectively). CONCLUSION: Ginkgo biloba extract can increase anti-oxidative activity and inhibit the progression of gastric precancerous lesions via the regulation of cell proliferation and apoptosis.