[Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes].

The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley (SD) rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached confluence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate (70.2 ± 4.0)% being much higher than that of the 20 min group (P < 0.05). Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell proliferation was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 (P1) were higher than those of corresponding control groups, and the GFAP positive rate of 30 min + two-time-digestion group in P1 reached (98.1 ± 1.7)%, which was equivalent to that of the 20 min + control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentioned results, we conclude that 30 min + two-time of trypsin digestion effectively improves the purity of astrocytes and shortens the time of primary culture and purification, suggesting that it is a rapid and effective method to obtain astrocytes with high purity in vitro.

[Synergistic action of 4-hydroxynonenal and tumor necrosis factor involving the NF-kB/IkBa signaling pathway in alcohol-induced liver injury].

OBJECTIVE: To investigate the effects and mechanism of intracellular 4-hydroxynonenal (4-HNE) accumulation on tumor necrosis factor (TNF)-induced hepatotoxicity in alcoholic liver disease (ALD). METHODS: An ALD model was established in male C57BL/6 mice (6-8 weeks old) by feeding an ethanol-containing diet for 5 weeks; mice given regular (non-ethanol) diet served as controls. ALD-related changes in 4-HNE and TNF levels were detected by western blotting. The underlying mechanisms of this molecular effect were examined by pre-treating HepG2 cells with 4-HNE followed by exposure to various concentrations of TNF. Effects on cell death were evaluated by MTT assay. Effects on TNF-mediated upstream factors' expression were detected by ELISA, western blotting, and real-time PCR. Effects on the TNF-induced inhibitor of NF-kB (IkBa) activity (phosphorylation status) and its formation of adducts were detected by western blotting and immunoprecipitation. RESULTS: ALD mice showed increased hepatic 4-HNE and TNF levels, and the increases were associated with extent of liver injury. Cell culture studies revealed that 4-HNE, at non-toxic concentrations, sensitized hepatocytes to TNF killing, which was associated with suppressed NF-kB trans activity. Furthermore, 4-HNE prevented phosphorylation of IkBa without affecting upstream IkB kinase activity. The ALD-enhanced 4-HNE content was found to associated with increased formation of 4-HNE-IkBa adduction for both the 4-HNE - treated hepatocytes in culture and in the livers of ALD mice. CONCLUSION: Alcohol-induced increase in 4-HNE accumulation represents a potent and clinically relevant mechanism of sensitizing hepatocytes to TNF-induced toxicity. These data support the notion that decreasing or eliminating accumulated intracellular 4-HNE can serve as a potential therapeutic option for ALD.

IGF-1R/ß-catenin signaling axis is involved in type 2 diabetic osteoporosis.

Insulin-like growth factor-1 receptor (IGF-1R) is involved in both glucose and bone metabolism. IGF-1R signaling regulates the canonical Wnt/ß-catenin signaling pathway. In this study, we investigated whether the IGF-1R/ ß-catenin signaling axis plays a role in the pathogenesis of diabetic osteoporosis (DOP). Serum from patients with or without DOP was collected to measure the IGF-1R level using enzyme-linked immunosorbent assay (ELISA). Rats were given streptozotocin following a four-week high-fat diet induction (DOP group), or received vehicle after the same period of a normal diet (control group). Dual energy X-ray absorption, a biomechanics test, and hematoxylin-eosin (HE) staining were performed to evaluate bone mass, bone strength, and histomorphology, respectively, in vertebrae. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to measure the total and phosphorylation levels of IGF-1R, glycogen synthase kinase-3ß (GSK-3ß), and ß-catenin. The serum IGF-1R level was much higher in patients with DOP than in controls. DOP rats exhibited strikingly reduced bone mass and attenuated compression strength of the vertebrae compared with the control group. HE staining showed that the histomorphology of DOP vertebrae was seriously impaired, which manifested as decreased and thinned trabeculae and increased lipid droplets within trabeculae. PCR analysis demonstrated that IGF-1R mRNA expression was significantly up-regulated, and western blotting detection showed that phosphorylation levels of IGF-1R, GSK-3ß, and ß-catenin were enhanced in DOP rat vertebrae. Our results suggest that the IGF-1R/ß-catenin signaling axis plays a role in the pathogenesis of DOP. This may contribute to development of the underlying therapeutic target for DOP.

[Vasoactive intestinal peptide attenuates apoptosis and iNOS protein expression induced by cerebral ischemia in the rat].

OBJECTIVE: To explore the neuroprotective action of vasoactive intestinal peptide (VIP) on ischemia and reperfusion in the rat. METHODS: VIP was given via intracerebroventriclar injection after a 2 hour transient middle cerebral artery occlusion using filament model. The infarct volume was investigated with TTC stain. Apoptosis in the ischemic boundary zone were evaluated with TUNEL stain. Western blotting were used to analyze the iNOS protein expression as well. RESULTS: After VIP injection, the relative infarct volume of rats was significantly reduced by approximately 28% campared to that of the control groups at 1 day (P < 0.05). The number of TUNEL positive cells was significantly decreased in the ischemic boundary zone, and then the expression of iNOS was remarkablely decreased as well (P < 0.05). CONCLUSION: VIP has a neuroprotective effect on cerebral ischemia and reperfusion. The mechanism seems to involve decreasing the apoptosis and down-regulating the iNOS expression.

The binding of BmK abT, a unique neurotoxin, to mammal brain and insect Na(+) channels using biosensor.

The binding properties of BmK abT (a novel neurotoxic polypeptide abT from Chinese scorpion Buthus martensi Karsch), a unique neurotoxin from Chinese scorpion, on mammal brain and insect sodium channels were investigated using the BIAcore assay. Results showed that BmK abT could bind to rat brain synaptosomes with an association rate constant of about 2.49 x 10(6) M(-1) s(-1) and a dissociation rate constant of about 1.57 x 10(-4) s(-1), and to Heliothis nerve cord synaptosomes with an association rate constant of about 1.21 x 10(7) M(-1) s(-1) and a dissociation rate constant of about 0.99 x 10(-3) s(-1). The binding of BmK abT to rat brain synaptosomes could be partially inhibited by increasing the membrane potential, but not by BmK AS (a novel active polypeptide AS from B. martensi Karsch), BmK IT2 (a depressant insect-selective toxin IT2 from B. martensi Karsch), and BmK I (an alpha-like anti-mammal toxin I from B. martensi Karsch). Binding was not modulated by veratridine. In addition, the binding of BmK abT to Heliothis nerve cord synaptosomes was significantly enhanced by increasing the membrane potential and veratridine concentration and was inhibited by BmK IT2, but not by BmK AS or BmK I. The results suggest that BmK abT binds to a distinct receptor site on mammal brain Na(+) channels and associates with a related site for depressant insect-selective toxins on insect sodium channels.

[Chronic multiple stress enhances learning and memory capability in rats].

The present study aimed at investigating the effects of chronic multiple stress on learning and memory functions of rats. Adult male Wistar rats were randomly divided into stressed and control groups. Rats in the stressed group were irregularly and alternately exposed to the situation of vertical revolution, sleep deprivation, noise stimulation, and night illumination 6 h per day for 6 weeks to prepare a chronic multiple stressed model. Learning and memory performance of rats was measured by using Morris water maze first and Y-maze afterwards. Neurons in the dentate gyrus(DG), CA3 and CA1 regions of the hippocampus were stained by using Cresyl violet method and counted. The results showed that: (1) After chronic multiple stress, compared with the control rats, the escape latency to the hidden platform in Morris water maze was significantly shortened in stressed rats. In stressed and control groups, the escape latency periods were (15.89+/-9.15) s and (27.30+/-12.51) s, respectively, indicating that spatial memory of the stressed rats was stronger than that of the control ones. In brightness-darkness discrimination learning in the Y- maze, the correct trials and correct percentage of entering safe arm was remarkably increased in the stressed rats, the correct rates of stressed and control groups were (79.01+/-1.23)% and (66.12+/-1.61)%, respectively, indicating that brightness-darkness discrimination learning ability of the stressed rats was better than that of the control ones. (2) After chronic multiple stress, nerve cell density in DG, CA1 and CA3 of the hippocampus in stressed rats was higher than that of the control group, the cell densities in DG, CA1 and CA3 of the stressed and the control group were (223.78+/-26.52), (112.07+/-14.23) and (105.55+/-18.12) as well as (199.13+/-15.36), (92.89+/-13.69), and (89.02+/-15.77) respectively. These results suggest that the chronic multiple stress may enhance the capability of spatial memory and brightness-darkness discrimination learning of rats. Possible reasons for the chronic multiple stress-induced learning and memory enhancement of rats were also discussed.

[Immunization with recombinant HBcAg and ß-amyloid peptide fusion protein promotes clearance of intrahippocampally injected ß-amyloid peptide in rats].

OBJECTIVE: To study the effects of immunization with the fusion protein CAC (a product of prokaryotic expression of recombinant HBcAg and ß-amyloid peptide fusion gene) against the toxicity induced by intrahippocampal injection of aggregated ß-amyloid peptide (Aß) in rats. METHODS: SD rats were immunized intraperitoneally with the fusion protein CAC, and the titer of anti-Aß antibody was evaluated by ELISA. When the titers of the anti-Aß antibody reached 1:3 000, aggregated Aß was injected into the CA1 region of the rat hippocampus. Two weeks after Aß injection, the rats underwent morris water maze test before sacrificed to prepare the brain slices with Congo red and haematoxylin staining. RESULTS: The titer of anti-Aß antibody reached 1:3 000 after 5 immunizations with the fusion protein. After Aß injection, the saline-immunized rats showed a reduced cognitive behavior in the Morris water maze test compared to the CAC-immunized rats. In the saline-immunized rats, the neurons around the site of Aß injection exhibited obvious cell damages with Aß deposits and glial infiltration, whereas in CAC-immunized rats, Aß deposits were significantly reduced or even absent. CONCLUSION: Immunization with the fusion protein CAC can inhibit the toxicity induced by intrahippocampal aggregated Aß injection.

[Prokaryotic expression and immunogenicity analysis of the chimeric HBcAg containing APP beta cleavage site peptide and Aß(1-15);].

AIM: To construct the recombinant prokaryotic expression plasmid pET/c-ABCSP-Aß(15-c);, and evaluate the immunogenicity of the fusion protein expressed in E.coli. METHODS: The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The APP beta cleavage site peptide(ABCSP) and Aß(1-15); gene(ABCSP-Aß(15);) was amplified by PCR and inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-17-ABCSP-Aß(15); were connected with HBc88-144, yielding the recombinant gene c-ABCSP-Aß(15-c);. c-ABCSP-Aß(15-c); gene was subcloned into pET-28a(+).The fusion protein expressed in transformed E.coli BL21 was induced with IPTG and analyzed by SDS-PAGE. The virus-like particles (VLP) formed by fusion protein was observed with Transmission Electron Microscope (TEM). 4 Kunming (KM) mice received intraperitoneal injection (i.p) of fusion protein VLP. The antibody was detected by indirect ELISA. RESULTS: The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, fusion protein was expressed and mainly existed in the sediment of the bacterial lysate. The expression level was 40% of all the proteins in the sediment. The fusion protein could form VLP. After 5 times of immunization, the titer of anti-ABCSP and anti-Aßantibody in sera of KM mice reached up to 1:5 000 and 1:10 000 respectively, while the anti-HBc antibody was undetectable. CONCLUSION: Recombinant c-ABCSP-Aß(15-c); gene can be expressed in E.coli. The expressed protein could form VLP and has a strong immunogenicity. This study lays the foundation for the study of AD genetic engineering vaccine.

[Effect of [Gly14]-Humanin on Abeta(25-35)-induced PC12 cell apoptosis].

AIM: To investigate the effect of [Gly14]-Humanin overexpression on Abeta(25-35);-induced PC12 cell apoptosis. METHODS: Recombinant plasmid pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells by liposome method. The subclone cell lines were obtained by persistent G418 selection. The HNG gene expression of PC12 cells was detected by immunocytochemistry. After being treated with 25 micromol/L Abeta(25-35); for 24 h, cell viability was determined by MTT assay, and apoptosis rate was detected by using flow cytometric analysis. Hochest33258 staining was used to observe the morphological changes of cellular nuclei. RESULTS: PC12 cell lines stably expressing HNG gene was successfully selected. After being treated with 25 micromol/L Abeta(25-35); for 24 h, cell viability of PC12 cells overexpression HNG was significant elevated compared with empty plasmid transfected cells (P<0.05), and the apoptosis rate was lower significantly (P<0.05). By Hoechst 33258 staining, the nuclei of empty plasmid transfected PC12 cells exhibited highly condensed and fragmented nuclei morphology which was the typical characteristics of apoptosis, and the nuclei of PC12 cells overexpression HNG were round and homogeneously stained. CONCLUSION: Overexpression of HNG prevented the cell apoptosis induced by Abeta(25-35); in PC12 cells.

Protective effects of [Gly14]-Humanin on beta-amyloid-induced PC12 cell death by preventing mitochondrial dysfunction.

Mitochondrial dysfunction is a hallmark of beta-amyloid (Abeta)-induced neuronal toxicity in Alzheimer's disease (AD), and is considered as an early event in AD pathology. Humanin (HN) and its derivative, [Gly14]-Humanin (HNG), are known for their ability to suppress neuronal death induced by AD-related insults in vitro and in vivo. In the present study, we investigated the neuroprotective effects of HNG on Abeta(25-35)-induced toxicity and its potential mechanisms in PC12 cells. Exposure of PC12 cells to 25 microM Abeta(25-35) caused significant viability loss and cell apoptosis. In addition, decreased mitochondrial membrane potential and increased cytochrome c releases from mitochondria were also observed after Abeta(25-35) exposure. All these effects induced by Abeta(25-35) were markedly reversed by HNG. Pretreatment with 100 nM HNG 6h prior to Abeta(25-35) exposure significantly elevated cell viability, reduced Abeta(25-35)-induced cell apoptosis, stabilized mitochondrial membrane potential, and blocked cytochrome c release from mitochondria. Furthermore, HNG also ameliorated the Abeta(25-35)-induced Bcl-2/Bax ratio reduction and decreased caspase-3 activity in PC12 cells. These results demonstrate that HNG could attenuate Abeta(25-35)-induced PC12 cell injury and apoptosis by preventing mitochondrial dysfunction. Furthermore, these data suggest that mitochondria are involved in the protective effect of HNG against Abeta(25-35).

Ototoxicity on cochlear nucleus neurons following systemic application of gentamicin.

CONCLUSION: The gentamicin-induced pathological alteration in the cochlear nucleus is not exclusively a secondary consequence of the damage in the cochlea. Instead, the toxic effect of gentamicin on the cochlear nucleus may occur simultaneously or even earlier than that on the cochlea. OBJECTIVES: To investigate the pathological alteration of cochlear nucleus neurons in guinea pigs following systemic application of gentamicin. MATERIALS AND METHODS: Guinea pigs were injected with gentamicin for 1 day, 3 days, 1 week, 2 weeks, and 3 weeks, respectively. In gentamicin-treated animals, the hearing function was evaluated by measuring the auditory brainstem response (ABR). The number and cross-sectional area of substance P-positive neurons in the cochlear nucleus were also measured. RESULTS: The threshold of ABR and the number of substance P-positive neurons in the cochlear nucleus were significantly increased after 1 week and 3 days of injection of gentamicin, respectively. The cross-sectional area of substance P-positive neurons in the cochlear nucleus was significantly reduced after 1-day injection of gentamicin.

Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro.

Malignant gliomas are the main brain tumors notoriously resistant to currently available therapies, since they fail to undergo apoptosis upon anticancer treatment. Recent progress on enhanced studies of ion channels involved in glioma cells shed new light on the investigation of glioma cell growth and proliferation. Here we report BmK scorpion venom, a rich resource of various ion channels blockers/modulators, induces cell death of cultured malignant glioma U251-MG cells in vitro specifically at a dose of 10 mg/ml while shows no effect on human hepatocellular carcinoma cells and Chinese hamster ovary cells. The glioma cell death was then determined as apoptosis using 4,6-diamidino-2-phenylindole staining and fluorescence-activated cell sorting analysis. After incubation with BmK venom for 32 and 40 h, 36.20% and 63.08% of U251-MG cells showed apoptosis. Furthermore, BmK venom could significantly inhibit the tumor growth in vitro, which was assessed using U251-MG tumor xenografts on severe combined immunodeficiency mice. The tumor volume of the BmK venom treated mice is nearly 1/8 of that of control after 21 days, and the tumor weight is less than half of that of control. That BmK venom induces apoptosis and inhibits growth of glioma may result from the inhibition and/or modulation of various ion channels in glioma cells.

Martentoxin, a novel K+-channel-blocking peptide: purification, cDNA and genomic cloning, and electrophysiological and pharmacological characterization.

Martentoxin, a novel K+-channel-specific peptide has been purified and characterized from the venom of the East-Asian scorpion (Buthus martensi Karsch). The whole cDNA precursor sequence suggested that martentoxin was composed of 37 residues with a unique sequence compared with other scorpion neurotoxins. The genomic DNA of martentoxin showed an additional intron situated unexpectedly in the 5' UTR region, besides one located close to the C-terminal of the signal peptide. The patch-clamp recording found that martentoxin at the applied dose of 100 nm could strongly block large-conductance Ca2+-activated K+ (BKCa) currents in adrenal medulla chromaffin cells, and BKCa currents blocked by martentoxin could be fully recovered within 30 seconds after washing, which is at least 10 times faster than recovery after charybdotoxin. Meanwhile, a biosensor binding assay showed a fast association rate and a slow dissociation rate of martentoxin binding on rat brain synaptosomes. The binding of martentoxin on rat brain synaptosomes could be inhibited regularly by charybdotoxin, and gradually by toosendanin in a concentration-dependent manner, but not by either apamin or P03 from Buthus martensi. The results thus indicate that martentoxin is a new member in the family of K+-channel-blocking ligands.