Dauricine alleviates cognitive impairment in Alzheimer's disease mice induced by D-galactose and AlCl3 via the Ca2+/CaM pathway.

Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly. Dysregulation of intracellular Ca2+ homeostasis plays a critical role in the pathological development of AD. Dauricine (DAU) is a bisbenzylisoquinoline alkaloid isolated from Menispermum dauricum DC., which can prevent the influx of extracellular Ca2+ and inhibit the release of Ca2+ from the endoplasmic reticulum. DAU has a potential for anti-AD. However, it is unclear whether DAU can exert its anti-AD effect in vivo by regulating the Ca2+ related signaling pathways. Here, we investigated the effect and mechanism of DAU on D-galactose and AlCl3 combined-induced AD mice based on the Ca2+/CaM pathway. The results showed that DAU (1 mg/kg and 10 mg/kg for 30 days) treatment attenuated learning and memory deficits and improved the nesting ability of AD mice. The HE staining assay showed that DAU could inhibit the histopathological alterations and attenuate neuronal damage in the hippocampus and cortex of AD mice. Studies on the mechanism indicated that DAU decreased the phosphorylation of CaMKII and Tau and reduced the formation of NFTs in the hippocampus and cortex. DAU treatment also reduced the abnormally high expression of APP, BACE1, and Aß1-42, which inhibited the deposition of Aß plaques. Moreover, DAU could decrease Ca2+ levels and inhibit elevated CaM protein expression in the hippocampus and cortex of AD mice. The molecular docking results showed that DAU may have a high affinity with CaM or BACE1. DAU has a beneficial impact on pathological changes in AD mice induced by D-galactose and AlCl3 and may act by negative regulation of the Ca2+/CaM pathway and its downstream molecules such as CaMKII and BACE1.

Total alkaloids from the seed embryo of Nelumbo nucifera Gaertn. improve cognitive impairment in APP/PS1 mice and protect Aß-damaged PC12 cells.

The seed embryo of Nelumbo nucifera Gaertn. is a famous traditional Chinese medicine and food which is considered conducive to the prevention of Alzheimer's disease (AD). In this study, the effect and mechanism of TASENN (total alkaloids from the seed embryo of Nelumbo nucifera Gaertn.) on AD mice and amyloid-ß (Aß) injured PC12 cells were evaluated. HPLC-UV analysis showed that the extracted TASENN (purity = 95.6%) mainly contains Liensinine, Isoliensinine, and Neferine (purity was 23.01, 28.02, and 44.57%, respectively). In vivo, oral treatment with TASENN (50 mg/kg/day for 28 days) improved the learning and memory functions of APP/PS1 transgenic mice, ameliorated the histopathological changes of cortical and hippocampal neurons, and inhibited neuronal apoptosis. We found that TASENN reduced the phosphorylation of Tau and the formation of neurofibrillary tangles (NFTs) in APP/PS1 mouse brain. Moreover, TASENN down-regulated the expression of APP and BACE1, ameliorated Aß deposition, and inhibited microglial proliferation and aggregation. The elevated protein expression of CaM and p-CaMKII in APP/PS1 mouse brain was also reduced by TASENN. In vitro, TASENN inhibited the apoptosis of PC12 cells injured by Aß25-35 and increased the cell viability. Aß25-35-induced increase of cytosolic free Ca2+ level and high expression of CaM, p-CaMKII, and p-Tau were decreased by TASENN. Our findings indicate that TASENN has a potential therapeutic effect on AD mice and a protective effect on PC12 cells. The anti-AD activity of TASENN may be closely related to its negative regulation of the CaM pathway.

Newly excysted juveniles of Fasciola gigantica trigger the release of water buffalo neutrophil extracellular traps in vitro.

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes and are among the first line of immune system defense. PMNs can form neutrophil extracellular traps (NETs) in response to some pathogens. The release of NETs plays an important role in trapping and killing invading parasites. However, the effects of NETs on parasitic trematode infections remain unclear. In the present study, water buffalo NET formation, triggered by the newly excysted juveniles (NEJs) of Fasciola gigantica, was visualized by scanning electron microscopy. The major components of the structure of NETs were characterized by immunofluorescence. Viability of flukes incubated with water buffalo PMNs were examined under light microscopy. The results revealed that F. gigantic juveniles triggered PMN-mediated NETs. These NETs were confirmed to comprise the classic characteristics of NETs: DNA, histones, myeloperoxidase and neutrophil elastase. Although NETs were formed in response to viable larvae, the larvae were not killed in vitro. These results suggest that NET formation may serve as a mechanism to hamper the migration of large larvae to facilitate immune cells to kill them. This study demonstrates, for the first time, that parasitic trematode juveniles can trigger NET formation.

Protective effects of Liensinine, Isoliensinine, and Neferine on PC12 cells injured by amyloid-ß.

Excessive accumulation of amyloid-ß (Aß) is the leading cause of Alzheimer's disease (AD). Liensinine, Isoliensinine, and Neferine are main alkaloids in lotus seed embryos. In this paper, the protective effects of Liensinine, Isoliensinine, and Neferine on Aß25-35 -injured PC12 cells were studied. It was found that Liensinine, Isoliensinine, and Neferine could improve the viability and reduce the apoptosis of PC12 cell induced by Aß25-35 . These three alkaloids could also reduce the level of intracellular free Ca2+ and CaM expression in Aß25-35 -treated cells, thereby inhibiting the phosphorylation of CaMKII and tau. In addition, these three compounds can inhibit the production of ROS in PC12 cells injured by Aß25-35 . Our results suggest for the first time that Liensinine, Isoliensinine, and Neferine can inhibit hyperphosphorylation of tau protein by inhibiting the Ca2+ -CaM/CaMKII pathway, thereby reducing the apoptosis and death of PC12 cells damaged by Aß25-35 . PRACTICAL APPLICATIONS: This study highlighted the protective effects and mechanisms of three main active ingredients (Liensinine, Isoliensinine, and Neferine) in the lotus embryo on a typical cell model of Alzheimer's disease (AD). The results revealed that three alkaloids in this healthy food might exert therapeutic potential for AD.

Selenoprotein SELENOK Enhances the Migration and Phagocytosis of Microglial Cells by Increasing the Cytosolic Free Ca2+ Level Resulted from the Up-Regulation of IP3R.

Enhancing the migration and phagocytosis of microglial cells is of great significance for the reducing of the risk of the neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The effect of mouse selenoprotein K (mSELENOK) on the migration and phagocytosis of BV2 microglial cells and its mechanism were studied. The results showed that the over-expression of mSELENOK can increase the migratory and phagocytic abilities of the microglial cells, while the knockdown of mSELENOK can decrease the migratory and phagocytic abilities of the cells. The cytosolic free Ca2+ level and inositol trisphosphate receptor (IP3R) mRNA transcript and protein expression were also increased significantly as the consequence of the over-expression of mSELENOK in the microglial cells. On the contrary, the level of cytosolic free Ca2+ and the mRNA transcript and protein expression of IP3R in mSELENOK knockdown cells were decreased significantly. 2-aminoethoxydiphenyl borate (2-APB), an antagonist of IP3R, could prevent the increased migration, phagocytosis, and cytosolic free Ca2+ level of mSELENOK over-expressed microglial cells, and knockdown of IP3R3 could reduce the increased cytosolic Ca2+ level in mSELENOK over-expressed microglial cells. Further studies revealed that selenium supplement (Na2SeO3) can increase the expression of mSELENOK in microglial cells significantly. In summary, these data suggest that mSELENOK can increase cytosolic free Ca2+ level of microglial cells by up-regulating the expression of IP3R, thus enhancing the migration and phagocytosis of microglial cells. Our results indicated that mSELENOK is an important selenoprotein, which plays a role in trace element selenium's functions and can enhance the migration and phagocytosis of microglial cells.

Comparison of inhibitory potency of three different curcuminoid pigments on nitric oxide and tumor necrosis factor production of rat primary microglia induced by lipopolysaccharide.

Microglia are the resident innate immune cells in the central nervous system. Evidence supports that the unregulated activation of microglia results in the production of pro-inflammatory cytokines and chemokines that propagate neuronal injury and finally cause neurodegenerative diseases. Curcuminn (Cur), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are curcuminoid pigments extracted from turmeric (Curcuma longa L.). Cur has been reported to suppress the activation of microglia by reducing toxic factors production, but little is known about whether the two natural demethoxy derivatives of Cur, DMC and BDMC, have the similar effects as Cur. In the present study, we found that all of the three curcuminoid pigments significantly suppressed nitric oxide (NO) production by LPS-activated microglia and the relative potency was DMC>BDMC>Cur. This result was verified by RT-PCR analysis of iNOS mRNA. The NO-scavenging abilities of three curcuminoid pigments are very weak, which suggested that the indirect effect may not be critical in inhibiting NO production by LPS-activated microglia. Moreover, these three curcuminoid pigments attenuated the expression of mRNA and proteins of tumor necrosis factor-alpha (TNF-alpha) in a concentration-dependent manner and the relative potency was also DMC>BDMC>Cur. In conclusion, Cur, DMC and BDMC were found as potent microglia-activation inhibitors, and DMC exhibited the strongest inhibitory activity on NO and TNF-alpha production. These results provided an interesting clue for designing new compounds which could have better potential therapeutic implications for various neurodegenerative diseases.

RV09, a novel resveratrol analogue, inhibits NO and TNF-alpha production by LPS-activated microglia.

Excessive activation of microglial cells has been implicated in various neurodegenerative diseases. Resveratrol, a polyphenolic compound found in grapes and wine, has been reported to reduce the activation of microglia. In the present study, 5-[2-(4-bromothiophen-2-yl)vinyl]benzene-1,3-diol (RV09), a novel resveratrol analogue, was found to suppress NO production by LPS-activated N9 microglial cell line and/or cultured rat cortical microglia. RV09 appeared to have a slight NO-scavenging activity in sodium nitroprusside (SNP) solution. The inhibition of iNOS was also observed, suggesting the blockage of transcriptional levels. Moreover, RV09 attenuated the expression of mRNA and protein of tumor necrosis factor-alpha (TNF-alpha) in a concentration-dependent manner. Further studies revealed that RV09 blocked IkappaBalpha phosphorylation and degradation, as well as reactive oxygen species (ROS) production in N9 microglial cells. It was also found that RV09 is a effective scavenger for 2,2-diphenyl-1-picrylhydrazyl (DPPH) used as a general free radical model. In the summary, these data suggest that, by blocking IkappaBalpha phosphorylation and degradation, RV09 acts to suppress the LPS-induced NO and TNF-alpha production in microglia, and this effect was mediated, at least in part, by inhibiting the generation of ROS. Our results suggested that RV09 is a novel anti-inflammatory agent which can inhibit proinflammatory responses of microglia.

Effects of resveratrol and its derivatives on lipopolysaccharide-induced microglial activation and their structure-activity relationships.

The inhibitory effects of 21 resveratrol derivatives on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in microglia and their structure-activity relationships were studied. It was found, for the first time, that certain resveratrol derivatives that have 3,5-dimethoxyl groups in the A-ring, such as (E)-4-(3,5-dimethoxystyryl)phenol (pterostilbene, compound 2), or have substituted the B-ring of resveratrol with quinolyl, such as (E)-5-[2-(quinolin-4-yl)vinyl]benzene-1,3-diol (compound 18) and (E)-4-(3,5-dimethoxystyryl)quinoline (compound 19), strongly inhibited NO production. Compounds 2, 18, and 19 reduced LPS-induced protein and mRNA expression of inducible NO synthase (iNOS), but did not display direct NO-scavenging activity up to 30 microM in sodium nitroprusside (SNP) solution. Moreover, compounds 2, 18, and 19 could also significantly inhibit the production of TNF-alpha by LPS-activated microglia. Further studies revealed that compounds 2, 18, and 19 inhibited LPS-induced NO and TNF-alpha production in microglia by blocking IkappaBalpha phosphorylation and degradation. The potent inhibitory effects of compounds 2, 18, and 19 on microglial activation suggest their potential for treatment of neurodegenerative diseases accompanied by microglial activation.

Differential metastasis-associated gene analysis of prostate carcinoma cells derived from primary tumor and spontaneous lymphatic metastasis in nude mice with orthotopic implantation of PC-3M cells.

The purpose of these studies was to explore the genes associated with invasion and metastasis of human prostatic carcinoma line PC-3M in nude mice. After PC-3M cells were inoculated in orthotopic site (prostate) in male nude mice for two months, tumor cells were isolated from primary tumor and lymph node metastasis in the same mouse, respectively. Cell invasion and adhesion ability in vitro were first compared between two cell lines. Then human metastasis-related genes differentially expressed between them were analyzed by utilizing cDNA microarray technique. The in vitro cell invasion and adhesion potential of tumor cells from lymph node metastasis was significantly higher than those from primary tumor, Metastasis-related genes differentially expressed between those two cell lines were identified, all of them were up-regulated in the tumor cells from lymph node metastasis and could be categorized as: (1) genes encoding cellular matrix-degrading proteolytic enzyme including cathepsin and MMP; (2) genes encoding transcription factors; (3) genes related to heterotypic adhesion of tumor cells; (4) genes encoding cell surface receptors. Moreover, Four genes were chosen for semi-quantitative RT-PCR analysis, they showed a consistent expression pattern with that of cDNA microarray analysis. We concluded that the lymph node metastasis in nude mice given an injection of PC-3M cells in the prostate is a selective process favoring the survival and growth of a special subpopulation derived from primary tumor with specific genetic alterations, which may play a pivotal role in the metastasis of prostate cancer. Identification and further characterization of these genes may allow a better understanding of lymphatic metastasis in prostate carcinoma.

[Adjuvant effect of CpG DNA recombinant plasmid on antigen of Cysticercus cellulosae].

In order to prove the adjuvant effect of CpG DNA recombinant plasmid, the total antibodies and their IgG2a subtype induced by antigen of Cysticercus cellulosae, and content of IL-4 and IFN-gamma secreted from splenic cell of mouse immunized were measured. The recombinant plasmids showed an adjuvant effect, and CpG2 was the best adjuvant among the plasmids. It is proved that the CpG DNA possesses a synergistic effect with AI(OH)3 and 206 adjuvant, and is an effective Th1 type adjuvant in mice.

[Construction and sequencing of full-length cDNA of peste des petits ruminants virus].

To develop a reverse genetics system of Peste des petits ruminants virus(PPRV), five pairs of oligonucleotide primers were designed on the basis of the full-length genomic sequence of PPRV Nigeria 75/ 1 strain. Using RT-PCR technique, five over-lapping cDNA fragments, designated as JF1, JF2, JF3, JF4 and JF5, respectively, were amplified, followed by cloning into pcDNA3.1(+)vector. An AscI restriction enzyme site and a T7 promoter sequence were introduced immediately upstream of 5'-end, while a PacI restriction enzyme site was engineered downstream of 3'-end. Using pok12 as a plasmid vector, the full-length cDNA clone pok12-PPRV of Nigeria 75/1 was assembled by connecting the five cDNA fragments via the unique restriction endonuclease site of PPRV genome. The resultant nucleotide sequence of the PPRV Nigeria 75/1 strain in the study was compared with other members of genus morbillivirus, and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that PPRV Nigeria 75/ 1 was antigenically closely related to Rinderpest virus and Measles virus. Successful construction of full-length cDNA clone of PPRV Nigeria 75/1 strain lays the basis rescuing PPRV effectively and enables further research of PPRV at molecular level.

[Construction, expression and immunogenicity of eukaryotic vectors based on goat pox virus P32 gene].

The full-length P32 gene and the truncated P32 gene (MP-32) were amplified from the recombinant plasmid pMD-P32 by polymerase chain reaction (PCR) and cloned into pcDNA3. 1(+) and pcDNA3.1-CpG respectively. The recombinant plasmids (pcDNA3.1-P32, pcDNA3.1-CpG-P32 and pcDNA3. 1-CpG-MP32) were transfected into BHK-21 cells by using lipofectin. The expressed P32 protein was confirmed by indirect immunofluorescence assay (IFA). The BALB/c mice were immunized with these recombinant plasmids by intramuscular injection. The specific antibodies aginst CPV were detected by ELISA kit weekly. The murine splenic T lymphocyte subgroups CD4+ and CD8+ were detected by flow cytometry. Results showed that the P32 protein was expressed successfully in vitro. After 2 weeks post im munization, the specific IgG antibodies against CPV were detected in the vaccinated mice. The percentage of CD4+ /CD8+ T cells was significantly higher than that of the control. In conclusion, these constructed eukaryotic vectors could induce humoral and celluar immune responses in mice.