Gustative Roussy Immune Score is a Predictor for Major Pathological Response in Rectal Cancer: A Result from the Preoperative Intraarterial Chemoembolization Combined with Radiotherapy (PCAR) Study.

Despite the emergence of various treatment strategies for rectal cancer based on neoadjuvant chemoradiotherapy, there is currently a lack of reliable biomarkers to determine which patients will respond well to neoadjuvant chemoradiotherapy. Through collecting hematological and biochemical parameters data of patients prior to receiving neoadjuvant chemoradiotherapy, we evaluated the predictive value of systemic inflammatory indices for pathological response and prognosis in rectal cancer patients. We found that baseline GRIm-Score was an independent predictor for MPR in rectal cancer patients. However, no association was observed between several commonly systemic inflammation indices and long-term outcome.

Thymosin ß4 reverses phenotypic polarization of glial cells and cognitive impairment via negative regulation of NF-κB signaling axis in APP/PS1 mice.

BACKGROUND: Thymosin ß4 (Tß4) is the most abundant member of the ß-thymosins and plays an important role in the control of actin polymerization in eukaryotic cells. While its effects in multiple organs and diseases are being widely investigated, the safety profile has been established in animals and humans, currently, little is known about its influence on Alzheimer's disease (AD) and the possible mechanisms. Thus, we aimed to evaluate the effects and mechanisms of Tß4 on glial polarization and cognitive performance in APP/PS1 transgenic mice. METHODS: Behavior tests were conducted to assess the learning and memory, anxiety and depression in APP/PS1 mice. Thioflavin S staining, Nissl staining, immunohistochemistry/immunofluorescence, ELISA, qRT-PCR, and immunoblotting were performed to explore Aß accumulation, phenotypic polarization of glial cells, neuronal loss and function, and TLR4/NF-κB axis in APP/PS1 mice. RESULTS: We demonstrated that Tß4 protein level elevated in all APP/PS1 mice. Over-expression of Tß4 alone alleviated AD-like phenotypes of APP/PS1 mice, showed less brain Aß accumulation and more Insulin-degrading enzyme (IDE), reversed phenotypic polarization of microglia and astrocyte to a healthy state, improved neuronal function and cognitive behavior performance, and accidentally displayed antidepressant-like effect. Besides, Tß4 could downregulate both TLR4/MyD88/NF-κB p65 and p52-dependent inflammatory pathways in the APP/PS1 mice. While combination drug of TLR4 antagonist TAK242 or NF-κB p65 inhibitor PDTC exerted no further effects. CONCLUSIONS: These results suggest that Tß4 may exert its function by regulating both classical and non-canonical NF-κB signaling and is restoring its function as a potential therapeutic target against AD.

PNU282987 alleviates Aß-induced anxiety and depressive-like behaviors through upregulation of α7nAChR by ERK-serotonin receptors pathway.

Patients with Alzheimer's disease often undergo anxiety and depression. Our previous studies have shown that α7nAChR protects against Aß-induced neurotoxicity via downregulation of p38 and JNK MAPKs, but the role of α7nAChR on Aß-induced anxiety and depressive-like behaviors and the effect of α7nAChR on the regulation of MAPKs pathways remain unknown. To examine the effects of α7nAChR and MAPKs pathways on Aß-induced anxiety and depression-like behaviors and to explore their relationships between them, elevated plus maze, open field and forced swim tests were performed. Protein levels of 5-HT1A receptor, 5-HT2C receptor, α7nAChR, t-ERK1/2 and p-ERK1/2 in the amygdala were analyzed by western blotting and immunostaining. Our study found out that Aß oligomers induced anxiety and depression-like behaviors in C56BL/6 mice with open field, elevated plus maze and forced swim tests. However, activation of α7nAChR or inhibition of ERK pathways showed significant antidepressant and anxiolytic-like effects on Aß-injected mice. Moreover, Aß significantly decreased the level of 5-HT1A receptor but increased the level of 5-HT2C receptor in the basolateral amygdala. Treatment with α7nAChR agonist PNU282987 or ERK inhibitor U0126 reversed Aß-induced 5-HT1A and 5-HT2C receptor changes. Moreover, activation of α7nAChR inhibited ERK pathway in the amygdala of Aß1-42-injected mice. Our study provides a new insight into the mechanism of α7nAChR in Aß-induced depression and anxiety-related symptoms through the regulation of ERK1/2 pathway and the potential association with serotonin receptors. Together, our data suggests that α7nAChR is protective against Aß-induced anxiety and depression-like behaviors in mice.

5-HT1AR alleviates Aß-induced cognitive decline and neuroinflammation through crosstalk with NF-κB pathway in mice.

The 5-hydroxytryptamine (5-HT) receptor is significant for the regulation of mood and memory. However, the role of 5-HT1AR in ß-Amyloid protein (Aß)-induced cognitive decline, neuroinflammation and the possible mechanism remains elusive. Thus, we aimed to evaluate the effects of 5-HT1AR on Aß-induced learning and memory decline and neuroinflammation in mice. Novel object recognition and Morris water maze tests were performed to observe learning and memory behavior in mice. Protein levels of Iba1, GFAP, MAP2, TNF-α, Tß4, C-fos, IKK-ß, IKB-α, NF-κBp65, phospho-NF-κBp65 in the hippocampus were examined by immunostaining or western blotting. Aß1-42-treatment inducing learning and memory decline was shown in novel object recognition and Morris water maze tests; neuroinflammation shown in immunostaining. Our study found out that 5-HT1AR inhibitor WAY100635 showed significant improvement in Aß-induced learning and memory decline. Moreover, WAY100635 decreases levels of Iba1, GFAP, and TNF-α in the hippocampus, which were related to neuroinflammation. While treatment with 5-HT1AR agonist 8-OH-DPAT or ERK inhibitor U0126 exerted no effects or even aggravated Aß-induced learning and memory decline. In addition, WAY100635 could downregulate phospho-NF-κB in the hippocampus of Aß1-42-injected mice. These results provide new insight into the mechanism, for 5-HT1AR in Aß-induced cognitive impairments through crosstalk with the NF-κB signaling pathway. Our data indicated that WAY100635 was involved in the protective effects against neuroinflammation and improvement of learning and memory in Alzheimer's disease.

Modulation of the MAPKs pathways affects Aß-induced cognitive deficits in Alzheimer's disease via activation of α7nAChR.

Cognitive impairment in Alzheimer's disease (AD) is characterized by being deficient at learning and memory. Aß1-42 oligomers have been shown to impair rodent cognitive function. We previously demonstrated that activation of α7nAChR, inhibition of p38 or JNK could alleviate Aß-induced memory deficits in Y maze test. In this study, we investigated whether the effects of α7nAChR and MAPKs on Y maze test is reproducible with a hippocampus-dependent spatial memory test such as Morris water maze. We also assessed the possible co-existence of hippocampus-independent recognition memory dysfunction using a novel object recognition test and an alternative and stress free hippocampus-dependent recognition memory test such as the novel place recognition. Besides, previous research from our lab has shown that MAPKs pathways regulate Aß internalization through mediating α7nAChR. In our study, whether MAPKs pathways exert their functions in cognition by modulating α7nAChR through regulating glutamate receptors and synaptic protein, remain little known. Our results showed that activation of α7nAChR restored spatial memory, novel place recognition memory, and short-term and long-term memory in novel object recognition. Inhibition of p38 restored spatial memory and short-term and long-term memory in novel object recognition. Inhibition of ERK restored short-term memory in novel object recognition and novel place recognition memory. Inhibition of JNK restored spatial memory, short-term memory in novel object recognition and novel place recognition memory. Beside this, the activation of α7nAChR, inhibition of p38 or JNK restored Aß-induced levels of NMDAR1, NMDAR2A, NMDAR2B, GluR1, GluR2 and PSD95 in Aß-injected mice without influencing synapsin 1. In addition, these treatments also recovered the expression of acetylcholinesterase (AChE). Finally, we found that the inhibition of p38 or JNK resulted in the upregulation of α7nAChR mRNA levels in the hippocampus. Our results indicated that inhibition of p38 or JNK MAPKs could alleviate Aß-induced spatial memory deficits through regulating activation of α7nAChR via recovering memory-related proteins. Moreover, p38, ERK and JNK MAPKs exert different functions in spatial and recognition memory.

Activation of α7 nicotinic acetylcholine receptor alleviates Aß1-42-induced neurotoxicity via downregulation of p38 and JNK MAPK signaling pathways.

Amyloid ß peptide 1-42 (Aß1-42) could induce cognitive deficits through oxidative stress, inflammation, and neuron death in Alzheimer's disease (AD). MAPK pathways have been thought to mediate Aß1-42-induced neuroinflammation responses, neuron death and cognitive decline in AD. The α7 nicotinic acetylcholine receptor (α7nAChR) exerts a neuroprotective effect. However, whether α7nAChR alleviates Aß1-42-induced neurotoxicity through MAPKs (p38, ERK, JNK) in vivo remains unclear. In our study, memory was assessed in C57BL/6 mice using a Y-maze test. Cell death was assessed by Nissl and Hoechst staining and Bax, Bcl-2, Caspase 3, and Cytochrome C levels using Western blotting. Oxidative stress was assayed by superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels. Inflammation was examined with GFAP and Iba1 using immunohistochemistry. The Aß degrading enzymes insulin degrading enzyme (IDE) and neprilysin (NEP) were tested using Western blotting. We found that activating α7nAChR or inhibiting p38 or JNK pathway alleviated Aß1-42-induced cognitive deficits and neuron loss and death by reducing oxidative stress. In addition, activating α7nAChR or inhibiting p38 or JNK pathway also reduced inflammation, which was observed as reduced GFAP and Iba1 levels with different effects on Aß degrading enzymes. Finally, we found that the activation of α7nAChR led to the downregulation of pp38 and pJNK levels. Conversely, the inhibition of p38 or JNK resulted in the upregulation of α7nAChR levels in the hippocampus and cortex. Our data indicate that the activation of α7nAChR alleviates Aß1-42-induced neurotoxicity, and this protective effect might act through the downregulation of p38 and JNK MAPKs.

The p38 mitogen activated protein kinase regulates ß-amyloid protein internalization through the α7 nicotinic acetylcholine receptor in mouse brain.

Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. Intracellular ß-amyloid protein (Aß) is an early event in AD. It induces the formation of amyloid plaques and neuron damage. The α7 nicotinic acetylcholine receptor (α7nAChR) has been suggested to play an important role in Aß caused cognition. It has high affinity with Aß and could mediate Aß internalization in vitro. However, whether in mouse brain the p38 MAPK signaling pathway is involved in the regulation of the α7nAChR mediated Aß internalization and their role in mitochondria remains little known. Therefore, in this study, we revealed that Aß is internalized by cholinergic and GABAergic neurons. The internalized Aß were found deposits in lysosomes/endosomes and mitochondria. Aß could form Aß-α7nAChR complex with α7nAChR, activates the p38 mitogen activated protein kinase (MAPK). And the increasing of α7nAChR could in return mediate Aß internalization in the cortex and hippocampus. In addition, by using the α7nAChR agonist PNU282987, the p38 phosphorylation level decreases, rescues the biochemical changes which are tightly associated with Aß-induced apoptosis, such as Bcl2/Bax level, cytochrome c (Cyt c) release. Collectively, the p38 MAPK signaling pathway could regulate the α7nAChR-mediated internalization of Aß. The activation of α7nAChR or the inhibition of p38 MAPK signaling pathway may be a beneficial therapy to AD.

Tongxinluo improves cognition by decreasing ß-amyloid in spontaneous hypertensive rats.

ß-Amyloid (Aß) accumulation in the brain is the major pathophysiology of Alzheimer disease (AD). Hypertension is a risk factor for AD by promoting Aß deposition. Traditional Chinese medicinal compound tongxinluo (TXL) can improve blood circulation and endothelium-dependent vasodilation. This study investigates the effects of TXL on cognition and Aß using spontaneously hypertensive rats (SHRs). TXL was intragastrically administered to SHRs at low-dose, mid-dose and high-dose for 15, 30 or 60days. Cognition was evaluated with a Morris Water Maze (MWM). Aß in the brain was detected by western blot, ELISA and Thioflavin-S staining. Western blot and RT-PCR were employed to exam the expression of receptor for advanced glycation end products (RAGE), low-density lipoprotein receptor-related protein-1 (LRP-1) and amyloid precursor protein (APP). After TXL treatment for 60days, compared with the vehicle, the number of crossed platform and the time spent in the target quadrant increased in parallel with the increasing length of treatment in MWM. Moreover, the Aß in the hippocampus significantly decreased compared to the vehicle group, both in western blot and ELISA. Additionally, TXL reduced RAGE expression in a dose- and time-depended manner, but LRP-1 expression had no difference between TXL groups and vehicle groups. Furthermore, the ß-secretase expression was significantly decreased compared to the vehicle group, but APP expression had no difference. In conclusion, TXL improved cognition and decreased Aß in SHRs in a dose- and time-dependent manner, the underlying mechanism may involved in inhibiting RAGE and ß-secretase expression.

The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated ß-amyloid protein internalization in mouse brain.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular ß-amyloid protein (Aß) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aß internalization may provide new insight for regulating Aß levels. In the present study, the regulation of Aß internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aß1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aß1-42-LRP1 complex was formed during Aß1-42 internalization, and the p38 MAPK signaling pathway was activated by Aß1-42 via LRP1. Aß1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aß1-42 internalization in mouse brain. The results of this investigation demonstrated that Aß1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aß1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aß1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo.

Puerarin attenuates learning and memory impairments and inhibits oxidative stress in STZ-induced SAD mice.

Puerarin (PUE), an isoflavone purified from the root of Pueraria lobata (Chinese herb), has been reported to attenuate learning and memory impairments in the transgenic mouse model of Alzheimer's disease (AD). In the present study, we tested PUE in a sporadic AD (SAD) mouse model which was induced by the intracerebroventricular injection of streptozotocin (STZ). The mice were administrated PUE (25, 50, or 100mg/kg/d) for 28 days. Learning and memory abilities were assessed by the Morris water maze test. After behavioral test, the biochemical parameters of oxidative stress (glutathione peroxidase (GSH-Px), superoxide dismutases (SOD), and malondialdehyde (MDA)) were measured in the cerebral cortex and hippocampus. The SAD mice exhibited significantly decreased learning and memory ability, while PUE attenuated these impairments. The activities of GSH-Px and SOD were decreased while MDA was increased in the SAD animals. After PUE treatment, the activities of GSH-Px and SOD were elevated, and the level of MDA was decreased. The middle dose PUE was more effective than others. These results indicate that PUE attenuates learning and memory impairments and inhibits oxidative stress in STZ-induced SAD mice. PUE may be a promising therapeutic agent for SAD.

Mitogen-activated protein kinase signaling pathways promote low-density lipoprotein receptor-related protein 1-mediated internalization of beta-amyloid protein in primary cortical neurons.

Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aß). Reuptake of extracellular Aß is believed to contribute significantly to the intraneuronal Aß pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aß1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aß internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aß1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aß internalization in neurons. We found that extracellular Aß1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in cortical neurons. Aß1-42 and LRP1 were also found co-localized in neurons during Aß1-42 internalization, and they could form Aß1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aß1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aß1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aß1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aß levels and served a potential therapeutic target for AD.

[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.

The effects of valsartan on cognitive deficits induced by aluminum trichloride and d-galactose in mice.

OBJECTIVES: Valsartan has been reported to reduce brain beta-amyloid protein levels and improve spatial learning in the Tg2576 transgenic mouse model of Alzheimer's disease (AD). However, the exact mechanism of neuroprotective effects of valsartan has not been properly studied especially in cholinergic function and oxidative damage, the essential factors that undergo impairment in AD. Therefore, the present study examined the effects of valsartan on memory impairment, cholinergic dysfunction, and oxidative stress in aluminum trichloride (AlCl3) and d-galactose (d-gal)-induced experimental sporadic dementia of Alzheimer's type. METHODS: Valsartan was administered intragastrically (i.g.) (20 mg/kg/day) for 60 days after mice were given AlCl3 (10 mg/kg/day) and d-gal (150 mg/kg/day) intraperitoneally (i.p.) once daily for 90 days. Then, memory function was evaluated by Morris water maze test. Acetylcholinesterase (AChE), superoxide dismutases (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) level in cortex and hippocampus were also assessed with biochemical technique. RESULTS: Chronic administration of valsartan not only improved learning and memory but also restored the elevation of AChE activity induced by AlCl3 and d-gal in cortex and hippocampus. In addition, valsartan significantly restored SOD and GSH-Px activities and reduced MDA level in cortex and hippocampus indicating attenuation of oxidative stress. DISCUSSION: Our results indicate that valsartan prevents AlCl3- and d-gal-induced cognitive decline partly to restore cholinergic function and attenuate oxidative damage. These findings further support the potential of valsartan to be used in AD treatment.

The effects of perindopril on cognitive impairment induced by d-galactose and aluminum trichloride via inhibition of acetylcholinesterase activity and oxidative stress.

Preclinical and clinical studies indicate involvement of renin angiotensin system (RAS) in memory functions. However, exact role of RAS in cognition is still ambiguous. The present study investigated the effects of perindopril on dementia of Alzheimer's type induced by d-galactose (d-gal) and aluminum trichloride (AlCl3). Perindopril, an angiotensin converting enzyme inhibitor, was administered intragastrically (0.5mg/kg/day) for 60days after mice were given d-gal (150mg/kg/day) and AlCl3 (10mg/kg/day) intraperitoneally for 90days. Then, memory function was evaluated by Morris water maze test. The biochemical studies were conducted in cerebral cortex and hippocampus of mouse brain after the behavioral studies. d-Gal and AlCl3 caused significant memory impairment along with significant elevation of acetylcholinesterase (AChE) activity in cerebral cortex and hippocampus. Further, a significant reduction of superoxide dismutases (SOD) and glutathione peroxidase (GSH-Px) activities, and elevation of malondialdehyde (MDA) level in cerebral cortex and hippocampus were observed. Perindopril not only improved cognitive impairment but also restored the elevation of AChE activity induced by d-gal and AlCl3. In addition, perindopril significantly increased SOD and GSH-Px activities, reduced MDA level in cerebral cortex and hippocampus. Taken together, the above findings indicate that perindopril improves learning and memorizing probably by restoring cholinergic function and attenuating oxidative damage.

The protective effects of tanshinone IIA on neurotoxicity induced by ß-amyloid protein through calpain and the p35/Cdk5 pathway in primary cortical neurons.

The characteristic pathological change of Alzheimer's disease (AD) include deposits of ß-amyloid protein (Aß) in brain, neurofibrillary tangles (NFTs), as well as a few neuronal loss. Evidence shows that Aß causes calcium influx and induces the cleavage of p35 into p25. Furthermore, the binding of p25 to cyclin-dependent kinase 5 (Cdk5) constitutively activates Cdk5. The p25/Cdk5 complex then hyperphosphorylates tau. Tanshinone IIA (tanIIA), a natural product extracted from Chinese herbal medicine Salvia miltiorrhiza BUNGE, has been reported to exert antioxidative activity. However, its neuroprotective activity remains unclear. The present study determined whether tanIIA protects neurons against Aß(25-35)-induced cytotoxicity and detected the association of this protective effect with calpain and the p35/Cdk5 pathway. The results showed that tanIIA protected neurons against the neurotoxicity of Aß(25-35), increased the viability of neurons, decreased expression of phosphorylated tau in neurons induced by Aß(25-35), improved the impairment of the cell ultrastructure (such as nuclear condensation and fragmentation, and neurofibril collapse). Further more, we found that tanIIA maintained the normal expression of p35 on peripheral membranes, and decreased p25 expression in the cytoplasm. TanIIA also inhibited the translocation of Cdk5 from the nucleus into the cytoplasm of primary neurons induced by Aß(25-35). These data suggested that tanIIA possessed neuroprotective action and the protection may involve in calpain and the p35/Cdk5 pathway.

[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.

[Podocyte injury and expression of nephrin and VEGF in rat nephrosis model induced by adriamycin].

OBJECTIVE: To investigate podocyte injury and the expression of nephrin and VEGF in rat nephrosis model induced by adriamycin. METHODS: The rat adriamycin induced nephrosis model was established, while the biochemical indicators in blood and urine were measured and the pathological changes of the renal tissue were evaluated by light microscope and electron microscope. The podocyte number was counted, and the expression levels of nephrin, VEGF were examined at different time by means of immunohistochemistry. RESULTS: After second injected with adriamycin,the model group nephrin presented a weak signal in the end of the first week (P < 0.05), and the expression of VEGF started to increase at the end of the eighth week (P < 0.05). The podocyte number decreased at the end of the eighth week (P < 0.05). The expression of nephrin and the number of podocyte were negatively correlated with the 24-hour urine protein, blood urea nitrogen and serum creatinine; while the expression of VEGF was positively correlated with the 24-hour urine protein, blood urea nitrogen and serum creatinine. CONCLUSION: The decrease of nephrin expression and the change of its distribution might be the significant factors resulting in considerable proteinuria. VEGF participated in the process of proteinuria and glomerular sclerosis in the development of rat adriamycin nephrosis.

[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.