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.

Performance and mechanism of a novel S-scheme heterojunction sonocatalyst CuS/BaWO4 for degradation of bisphenol A by ultrasonic activation.

A novel CuS/BaWO4 heterojunction catalyst was prepared and characterized. Taking bisphenol A as the target pollutant for catalytic degradation, the sonocatalytic activity of CuS/BaWO4 composite was evaluated, and the combination with persulfate improved the sonocatalytic degradation of bisphenol A. The results showed that CuS/BaWO4 composite had good sonocatalytic degradation activity for bisphenol A, and the degradation rate was 70.99% ± 1.46%. After combined with persulfate, the degradation rate was further increased to 95.34% ± 0.10%, and the reaction time was relatively shortened. The results of the trapping experiment and calculated energy band positions showed that the formation of S-scheme heterojunction and the formation of hydroxyl radicals and holes were the key to the catalytic degradation of bisphenol A by CuS/BaWO4 composite. In this study, a new CuS/BaWO4 heterojunction sonocatalyst was synthesized. The catalyst can efficiently remove bisphenol A from the water environment and can be used as a potential solution for endocrine disruptor pollution in the water environment.

Prominent Effects of Berbamine Hydrochloride on Alzheimer's Disease Model Mice.

Very few anti-Alzheimer's disease (AD) drugs are clinically available at present due to the complex mechanism of Alzheimer's disease. For the purpose of discovering potential anti-AD drugs in bisbenzylisoquinoline alkaloids, the anti-AD function and the mechanism of the function of berbamine hydrochloride (BBMH) were studied. Three kinds of AD model mice, double transgenic APP/PS1 AD mice, Gal-Alu AD mice induced by the intraperitoneal injection of d-galactose combined with the intragastric administration of aluminum trichloride, and Alu AD-like mice induced by stereotactic brain injection of aluminum trichloride, were administered with BBMH for 40 days at a dosage of 280 mg/kg/d. The effects of BBMH on the learning and memory behavior of the AD mice were studied through the Morris water maze experiment, and the influences of BBMH on the pathological features of AD, including the deposition of Aß, the lesions of pyramidal cells (neurons), and the formation of neurofibrillary tangles, were studied by the immunohistochemical staining, hematoxylin-eosin staining, and silver staining of the brain tissues of the mice. The water maze experiment showed that BBMH could significantly improve the learning and memory abilities of three kinds of treated mice. Immunohistochemical staining showed that BBMH could significantly reduce the deposition of Aß in the brain tissues of treated mice. Hematoxylin-eosin staining showed that BBMH could significantly alleviate the lesions of pyramidal cells in the hippocampal tissue of the mice. Silver staining showed that BBMH could significantly reduce the formation of neurofibrillary tangles in the hippocampal tissue of the mice. These results indicated that BBMH has significant anti-AD effects and the potential as an anti-AD drug. Western blot analysis of the brain tissue of the mice showed that the expression level of calpain, a Ca2+-dependent proteolytic enzyme, was significantly inhibited and the expression level of SelK, a selenoprotein mainly expressed in immune cells, was significantly increased. It is speculated that the anti-AD effect of BBMH is related to the improvement of the phagocytosis of microglial cells in brain tissues and macrophages migrated into the brain as well as the regulation of calcium homeostasis and calcium-dependent proteases in the brain tissues of the mice.

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.

A novel flavonoid with antioxidant activity from Patrinia villosa (Thunb.) Juss.

Patrinia villosa (Thunb.) Juss (P. villosa), a perennial herb, is widely used as a medicinal plant in Chinese folk. This study aims to isolate and identify the chemical constituents from P. villosa and evaluate their antioxidant activity. Normal silica column chromatography, ODS silica column chromatography, Sephadex LH-20 column chromatography and semi-preparative HPLC methods were used to obtain a new compound named 3-n-pentadecyl-4'-methoxyluteolin (1) and two known compounds including luteolin-7-O-ß-D-glucuronide methyl ester (2) and apigenin-7-O-ß-D-glucuronide methyl ester (3). The antioxidant activity of these compounds was determined by DPPH and ABTS methods and the IC50 values were calculated. The IC50 values of ABTS scavenging activity of 1, 2 and 3 were 12.99 ± 0.09 µM, 7.13 ± 0.07 µM and 5.15 ± 0.08 µM, respectively, and the IC50 values of DPPH scavenging activity of 1, 2 and 3 were 51.86 ± 0.41 µM, 23.95 ± 0.71 µM and 25.06 ± 0.65 µM, respectively. All the compounds exhibited good antioxidant activities in vitro.

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.

[Prokaryotic expression of Drosophila selenoprotein D-SelK and preparation of polyclonal antibody to D-SelK].

AIM: To clone and encode Drosophila selenoprotein D-SelK structure gene, express it in E.coli efficiently, and after purification, to generate its antibody in rabbits. METHODS: D-SelK gene segment amplified from pGM-T-D-SelK plasmid by PCR was inserted into pGEX-6p-1 to construct recombinant plasmid pGEX-6p-1-D-SelK. The recombinant plasmid was transfected into E.coli BL21(DE3) to express the recombinant protein D-SelK in E.coli under IPTG induction. The protein was purified by denaturation and electrophoresis, and then identified by SDS-PAGE and Western blotting. Polyclonal antibody to D-SelK was obtained by immunizing rabbits with the protein. Quality and quantity of the antibody was examined. RESULTS: D-SelK gene segment was successfully inserted into pGEX-6p-1 and the positive clones of the recombinant plasmid was identified by PCR screening and restriction endonuclease analysis. The target protein was effectively expressed in E.coli by the IPTG induction. Through immunizing rabbits with the purified target protein, we obtained the specific antibodies to D-SelK, the titer of which was more than 1:51 200. The polyclonal antibody had a good specificity to D-SelK. CONCLUSION: D-SelK recombinant protein and rabbit anti-D-SelK polyclonal antibody with high specificity were obtained, which provides good tools for further research on the functional characterization of D-SelK.

[Comparison of ICP-MS and two other analytical methods for determination of selenium content in Cordyceps militaris].

Samples were digested by microwave digestion. The selenium content in selenium enriched Cordyceps militaris was determined by ICP-MS method, HPLC/fluorometric method, and 3,3-diaminobenzidine method separately. And the detection conditions, the lowest detection limit and the relative standard deviation (RSD) of the three determination methods were compared. The detection conditions of the three methods for the detection of selenium content in selenium enriched Cordyceps militaris were established. It was showed that the lowest detection limit of ICP-MS method, HPLC/fluorometric method, and 3,3-diaminobenzidine method was 0.260 7, 0.182 1 and 10.485 9 microg x L(-1) respectively, and this means that the lowest detection limit of ICP-MS method was the lowest and that of 3,3-diaminobenzidine method was the highest. For the same sample the relative standard deviation (RSD) of ICP-MS method was the lowest and the RSD of 3,3-diaminobenzidine method was the highest. It was recommended that selenium content is determined by ICP-MS and HPLC/fluorometric method when the selenium in the sample is very low and by 3,3-diaminobenzidine method when the content is rather high.

G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein.

G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is the first selenoprotein identified in the Golgi apparatus.