Mechanism of Sijunzi Decoction in treatment of Alzheimer's disease based on UPLC-Q-TOF-MS, network pharmacology, and experimental verification / 中国中药杂志

The study identified the blood-entering components of Sijunzi Decoction after gavage administration in rats by UPLC-Q-TOF-MS/MS, and investigated the mechanism of Sijunzi Decoction in treating Alzheimer's disease by virtue of network pharmacology, molecular docking, and experimental verification. The blood-entering components of Sijunzi Decoction were identified based on the mass spectra and data from literature and databases. The potential targets of the above-mentioned blood-entering components in the treatment of Alzheimer's disease were searched against PharmMapper, OMIM, DisGeNET, GeneCards, and TTD. Next, STRING was employed to establish a protein-protein interaction(PPI) network. DAVID was used to perform the Gene Ontology(GO) annotation and the Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment. Cytoscape 3.9.0 was used to carry out visual analysis. AutoDock Vina and PyMOL were used for molecular docking of the blood-entering components with the potential targets. Finally, the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway enriched by the KEGG analysis was selected for validation by animal experiments. The results showed that 17 blood-entering components were detected in the serum samples after administration. Among them, poricoic acid B, liquiritigenin, atractylenolide Ⅱ, atractylenolide Ⅲ, ginsenoside Rb_1, and glycyrrhizic acid were the key components of Sijunzi Decoction in treating Alzheimer's disease. HSP90AA1, PPARA, SRC, AR, and ESR1 were the main targets for Sijunzi Decoction to treat Alzheimer's disease. Molecular docking showed that the components bound well with the targets. Therefore, we hypothesized that the mechanism of Sijunzi Decoction in treating Alzheimer's disease may be associated with the PI3K/Akt, cancer treatment, and mitogen-activated protein kinase(MAPK) signaling pathways. The results of animal experiments showed that Sijunzi Decoction significantly attenuated the neuronal damage in the hippocampal dentate gyrus area, increased the neurons, and raised the ratios of p-Akt/Akt and p-PI3K/PI3K in the hippocampus of mice. In conclusion, Sijunzi Decoction may treat Alzheimer's disease by activating the PI3K/Akt signaling pathway. The findings of this study provide a reference for further studies about the mechanism of action and clinical application of Sijunzi Decoction.

Effect of Erjing Pills on alleviating neuroinflammation of AD rats based on TLR4/NF-κB/NLRP3 pathway and its mechanism / 中国中药杂志

This paper aimed to study the effect of Erjing Pills on the improvement of neuroinflammation of rats with Alzheimer's di-sease(AD) induced by the combination of D-galactose and Aβ_(25-35) and its mechanism. SD rats were randomly divided into a sham group, a model control group, a positive drug group(donepezil, 1 mg·kg~(-1)), an Erjing Pills high-dose group(9.0 g·kg~(-1)), and an Erjing Pills low-dose group(4.5 g·kg~(-1)), with 14 rats each group. To establish the rat model of AD, Erjing Pills were intragastrically administrated to rats for 5 weeks after 2 weeks of D-galactose injection. D-galactose was intraperitoneally injected into rats for 3 weeks, and then Aβ_(25-35) was injected into the bilateral hippocampus. The new object recognition test was used to evaluate the learning and memory ability of rats after 4 weeks of intragastric administration. Tissues were acquired 24 h after the last administration. The immunofluorescence method was used to detect the activation of microglia in the brain tissue of rats. The positive expressions of Aβ_(1-42) and phosphory protein Tau~(404)(p-Tau~(404)) in the CA1 area of the hippocampus were detected by immunohistochemistry. The levels of inflammatory factors interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in the brain tissue were determined by enzyme-linked immunosorbent assay(ELISA). Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptors 3(NLRP3) pathway-associated proteins in the brain tissue were determined by Western blot. The results showed that as compared with the sham group, the new object recognition index of rats in the model control group decreased significantly, the deposition of Aβ_(1-42) and p-Tau~(404) positive protein in the hippocampus increased significantly, and the levels of microglia activation increased significantly in the dentate gyrus. The levels of IL-1β, TNF-α, and IL-6 in the hippocampus of the model control group increased significantly, and the expression levels of TLR4, p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus increased significantly. Compared with the model control group, the Erjing Pill groups enhanced the new object recognition index of rats, decreased the deposition of Aβ_(1-42) and the expression of p-Tau~(404) positive protein in the hippocampus, inhibited the activation of microglia in the dentate gyrus, reduced the levels of inflammatory factors IL-1β, TNF-α, and IL-6 in the hippocampus, and down-regulated the expression levels of TLR4, p-NF-κB P65/NF-κB P65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus. In conclusion, Erjing Pills can improve the learning and memory ability of the rat model of AD presumably by improving the activation of microglia, reducing the expression levels of neuroinflammatory factors IL-1β, TNF-α, and IL-6, inhibiting the TLR4/NF-κB/NLRP3 neuroinflammation pathway, and decreasing hippocampal deposition of Aβ and expression of p-Tau, thereby restoring the hippocampal morphological structure.

Mechanism analysis of repeatedly steamed and sundried Rehmanniae Radix Praeparata in delaying brain aging in ovariectomized mice based on proteomics / 中国中药杂志

The present study explored the effect and mechanism of repeatedly steamed and sundried Rehmanniae Radix Praeparata(RRP) in delaying brain aging in ovariectomized mice. After ovariectomy, the mice were randomly divided into a model group, an estradiol valerate group(0.3 mg·kg~(-1)), and low-(1.0 g·kg~(-1)), medium-(2.0 g·kg~(-1)), and high-dose(4.0 g·kg~(-1)) RRP groups, and a sham operation group was also set up, with 15 mice in each group. One week after the operation, intragastric administration was carried out for 15 consecutive weeks. The step-down test and Morris water maze test were used to detect the behavioral changes of mice. HE staining and Nissl staining were used to observe the morphological changes of mouse brain tissues. Immunohistochemistry was used to detect the expression of Aβ and ER_β in mouse brain tissues. The serum estrogen levels and cholinesterase and cholinesterase transferase levels in brain tissues of mice were detected by assay kits. The extracted hippocampal protein was detected by the Nano-ESI-LC-MS system, identified by the Protein Discovery, and analyzed quantitatively and qualitatively by the SIEVE. The PANTHER Classification System was used for GO analysis and KEGG pathway enrichment analysis of the differential proteins. Compared with the sham operation group, the model group showed decreased learning and memory ability, shortened step-down latency(P<0.05), prolonged escape latency(P<0.05), reduced platform crossings and residence time in the target quadrant, scattered nerve cells in the hippocampus with enlarged intercellular space, increased expression of Aβ-positive cells(P<0.05), declining expression of ER_β-positive cells and estrogen level(P<0.05), and weakened cholinergic function(P<0.05). Compared with the model group, the RRP groups showed improved learning and memory ability, prolonged step-down latency(P<0.05), increased estrogen level(P<0.05), neatly arranged nerve cells in the hippocampus with complete morphology, declining Aβ-positive cells, and elevated expression of ER_β-positive cells. A total of 146 differential proteins were screened out by proteomics, and KEGG pathway enrichment yielded 75 signaling pathways. The number of proteins involved in the dopaminergic synapse signaling pathway was the largest, with 13 proteins involved. In summary, RRP can delay brain aging presumedly by increasing the level of estrogen, mediating the dopaminergic synapse signaling pathway, and improving cholinergic function.

Proteasome inhibitor bortezomib sensitizes Hep-2 human laryngeal squamous cell carcinoma cells to ionizing radiation in vitro and in vivo / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To determine if proteasome inhibitor bortezomib leads to enhanced radiation sensitivity of Hep-2 human laryngeal cancer cells and the relative mechanisms.</p><p><b>METHODS</b>Hep-2 cells with or without bortezomib were irradiated at 0, 2, 4, 6, 8, and 10 Gy. Growth and clonogenic survival data were obtained to assess effects of treatment on radiosensitization. In vitro results were tested in vivo using a Hep-2 xenograft model. Nuclear factor-kappaB (NF-kappaB) activation was determined by Trans AM NF-kappaB P65 kit. The distribution of cell cycle and apoptosis were evaluated by flow cytometry. Morphological evidence of apoptotic cells were observed with Hochest 33342.</p><p><b>RESULTS</b>It decreased cell growth and clonogenic survival. A 34% increase in radiosensitivity was observed for cells treated with bortezomib and radiation. Enhancement factor (EF) was 1.46 in Hep-2 xenografts receiving radiation and bortezomib. NF-kappaB activation was induced by radiation and inhibited by pretreatment with bortezomib, and was in a dose-dependent manner (r = 0.989, P < 0.05). Hep-2 cells treated with 100 nmol/L Bortezomib were arrested at G2-M phase (t = 22.31, P = 0.000) and resulted in all increased apoptosis with and without irradiation (P < 0.01). Morphological evidence of apoptotic cells could be distinguished under the fluorescence microscope after staining with Hochest 33342. Many nuclear fragments were observed in Hep-2 cells with bortezomib.</p><p><b>CONCLUSION</b>Bortezomib could enhanced the radiosensitivity of Hep-2 laryngeal cancer cells by regulation of the distribution of cell cycle.</p>

Expression of S100A6 in primary and metastatic human gastric cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>Some members of the S100 gene family have been suggested to be associated with cancer development and metastasis. Our previous cDNA micro-array studies have showed S100A6 expression is elevated in gastric cancer compared with that in paired normal mucosa. To validate our previous results and further investigate the possible role of S100A6 gene in gastric cancer, we carried out this detailed S100A6 expression analysis in more matched gastric cancer samples.</p><p><b>METHODS</b>S100A6 expression was detected in 20 paired fresh surgical samples of gastric tumor tissue and matched non-cancerous mucosa by QRT-PCR. A gastric cancer tissue microarray (TMA) containing 1020 duplicate matched normal mucosa, gastric cancer tissue and metastatic lymph node tissue cores from 208 gastric cancer patients was constructed. S100A6 expression was detected by immunohistochemistry and the correlation between S100A6 expression with clinicopathological factors and survival was analyzed.</p><p><b>RESULTS</b>As quantitated by QRT-PCR, S100A6 transcript level was elevated in 73.7% of the primary cancer lesions with an average 2.25-fold up-regulation than that in matched non-neoplastic mucosa. As displayed by immunohistochemistry, the positive rate of S100A6 in non-neoplastic mucosa, tumor lesions and metastatic lymph nodes was 34.3%, 84.1% and 90.9%, respectively. S100A6 expression level in cancer and metastatic lymph node was significantly higher than their matched non-neoplastic mucosa (P < 0.05). 65.5% of patients showed an increased S100A6 expression in cancer tissue compared with that in matched normal mucosa. S100A6 overexpression was associated with larger tumor size and deeper invasion (P = 0.022 and P = 0.009). No evidence was found for an association between S100A6 expression level and other variables, including tumor grade, nodal metastases, and TNM stage. There was no association between S100A6 expression level and survival. But compared with paired non-neoplastic mucosa, an increased S100A6 expression in tumor lesion predicated a decreasing suvival if compared with a decreased S100A6 expression, though the difference was statistically not significant.</p><p><b>CONCLUSION</b>Elevated expression of S100A6 gene may be an early event in the development and progression of gastric cancer. Further study of this gene may be helpful for understanding the nature of gastric carcinoma.</p>

Analysis of LRP16 gene promoter activity / 中国实验血液学杂志

The study was aimed to analyze the characteristics of LRP16 gene promoter and its activity in order to explore the possible regulation mechanism of LRP16 gene expression. A 2.6 kb genomic DNA sequence of LRP16 5'-end was obtained from NCBI by BLAST software. The 7 target sequences between 0.2 - 2.6 kb from a healthy blood donor DNA sample were amplified by PCR, then identified by DNA sequencing and semi-nest PCR. The verified sequences were analyzed on-line. The results showed that the 7 target sequences were about 400 bp different from each other. All 7 sequences were the same to these GenBank described. At last, all 7 promoter sequences were ligated with luciferase vector, and then the luciferase activity was analyzed in HeLa cells. A known gene promoter sequence can be freely obtained from NCBI database. It is concluded that LRP16 promoter is a standard type II promoter and its activity is strongest in the region from -200 to -600 bp.

Taxol-producing fungi: a new approach to industrial production of taxol / 生物工程学报

Produced by and purified from Taxus brevifolia, Taxol (paclitaxel) has become a widely used cancer drug in clinic. Due to the rapid growing market, current industrial production of taxol by semi-synthesis that consumes large amount of Taxus trees cannot meet the requirement of the market. The discovery of taxol-producing fungus Taxomyces andeanae, an endophyte of T. brevifolia, by Stierle et al (1993), paves a new way to the production of the drug, i.e. employing large-scale fungal fermentation to make Taxol at lower cost and yet higher yield. This review discusses the present problems in taxol production in pharmaceutical industry, the finding and research progress on taxol-producing fungi, and the potential application of fungal fermentation to manufacture this important drug.

Artificial transcription factors as tools for gene expression manipulation / 生物工程学报

In this new era of the genome, the complete sequences of various organisms (from the simplest to the most complex such as human) are now available, which provides new opportunities to study biology and to develop therapeutic strategies. But the paucity of research tools that manipulate specific genes in vivo represents a major limitation of functional genomic studies. In nature, the expression of genes is regulated at the transcriptional level primarily by proteins that bind to nucleic acids. Many of these proteins, which are termed transcription factors, are typically consist of two essential yet separable modules: DNA-binding domain (DBD) and effector domain (ED). Attempts to control the gene expression by artificial transcription factors are based on the application of this rule. Among the many naturally occurring DNA-binding domains, the Cys2-His2 zinc-finger domain has demonstrated the greatest potential for the design of novel sequence-specific DNA-binding proteins. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. Ideally, these artificial DNA binding proteins could be designed to specifically target and regulate one single gene within a genome as complex as that found in human. Such proteins would be powerful tools in basic and applied research.

RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells / 生物工程学报

SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.

Construction of a SV40 promoter specific artificial transcription factor / 生物工程学报

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.

Construction of eukaryotic expression vector using neomycin-resistance gene mutant as selectable marker / 生物工程学报

Neomycin-resistance gene is widely used as a selectable marker in eukaryotic expression vector. It codes neomycin phosphotransferase II (NPT II) which confers resistance to various aminoglycoside antibiotic such as G418 and kanamycine. In this work, by site-directed mutagenesis the neo gene mutant was obtained. The expression vector pmDNA using the neo gene mutant as selectable marker has been constructed. After inserting interest luciferase gene, the expression plasmid pmDNAluc + was stably transfected CHO-K1 cells. As a result, the expression positive ratio reaches to approximate 95% and the ratio of high expression colonies is apparently higher than the controls.