Alkaloids from Fritillaria przewalskii Bulbs and Their Anti-Alzheimer's Disease Activities.

Three undescribed isosteroidal alkaloids, przewalskines A-C (1-3), as well as seven known alkaloids (4-10) were obtained from Fritillaria przewalskii bulbs. Their structures were deduced by extensive HRESIMS, 1D NMR, and 2D NMR analyses, and their bioactivities were evaluated involving the anti-inflammatory and inhibitory potencies on AChE, BChE, and Aß aggregation. Compound 4 revealed the potent effect on inhibiting Aß aggregation activity with IC50 value of 33.1 µM, AChE activity with IC50 value of 6.9 µM, and also showed NO release inhibitory acitivity with IC50 value of 32.6 µM. These findings contribute new multi-.target anti-AD agents and embody the chemical diversity of F. przewalskii.

Systematic Comparison of Structural Characterization of Polysaccharides from Ziziphus Jujuba cv. Muzao.

To investigate the structural information differences of Ziziphus Jujuba cv. Muzao polysaccharides, ten samples were successfully extracted from aqueous and alkaline solutions, prepared via DEAE-Sepharose Fast Flow through different eluents and Sephacryl S-300 columns, and systematically analyzed. Their characteristics were studied and then compared using chemical testing, high-performance gel permeation chromatography (HPGPC), gas chromatography (GC), methylation analysis, and NMR spectroscopy. The data achieved demonstrated that different jujube polysaccharide fractions possessed different structural characteristics, and most of them belonged to pectic polysaccharides. Overall, the structural information difference of jujube polysaccharides was preliminarily illuminated, which could not only promote the potential application of Z. Jujuba cv. Muzao polysaccharides but also provide an effective way to analyze the structures of polysaccharides from other genera jujube fruit.

MEF2C ameliorates learning, memory, and molecular pathological changes in Alzheimer's disease in vivo and in vitro

Myocyte enhancer factor 2C (MEF2C) is highly expressed in the nervous system, and regulates neuro-development, synaptic plasticity, and inflammation. However, its mechanism in Alzheimer's disease (AD) is underestimated. In this study, the role and mechanism of MEF2C were investigated in the brain tissue specimens from patients with AD, APPswe/PSEN1dE9 double transgenic (APP/PS1_DT) mice, and SH-SY5Y cells treated with ß-amyloid peptide (Aß). The results indicated that the expression of MEF2C is significantly reduced, and the expression of MEF2C/Aß in different parts of brain is negatively correlated in patients with AD. Knockdown of MEF2C promotes cell apoptosis and the level of ß-amyloid precursor protein cleaving enzyme 1 (BACE) but reduces BACE2 expression. In addition, knockdown of enhances the generation and aggregation of Aß in the cortex of APP/PS1_DT mice, reduces the expression of synaptic proteins, exacerbates the ability of learning and memory of APP/PS1_DT mice, damages the structure of mitochondria, increases the oxidative stress (OS) level, and inhibits the expression levels of members of the Nrf2-ARE signal pathway. In summary, inhibition of MEF2C exacerbates the toxic effect of Aß and , damages synaptic plasticity, reduces the ability of learning and memory of APP/PS1 mice, and increases the level of OS via the Nrf2-ARE signal pathway.

Synaptotagmin-11 inhibits spontaneous neurotransmission through vti1a.

Recent work has revealed that spontaneous release plays critical roles in the central nervous system, but how it is regulated remains elusive. Here, we report that synaptotagmin-11 (Syt11), a Ca2+ -independent Syt isoform associated with schizophrenia and Parkinson's disease, suppressed spontaneous release. Syt11-knockout hippocampal neurons showed an increased frequency of miniature excitatory post-synaptic currents while over-expression of Syt11 inversely decreased the frequency. Neither knockout nor over-expression of Syt11 affected the average amplitude, suggesting the pre-synaptic regulation of spontaneous neurotransmission by Syt11. Glutathione S-transferase pull-down, co-immunoprecipitation, and affinity-purification experiments demonstrated a direct interaction of Syt11 with vps10p-tail-interactor-1a (vti1a), a non-canonical SNARE protein that maintains spontaneous release. Importantly, knockdown of vti1a reversed the phenotype of Syt11 knockout, identifying vti1a as the main target of Syt11 inhibition. Domain analysis revealed that the C2A domain of Syt11 bound vti1a with high affinity. Consistently, expression of the C2A domain alone rescued the phenotype of elevated spontaneous release in Syt11-knockout neurons similar to the full-length protein. Altogether, our results suggest that Syt11 inhibits vti1a-containing vesicles during spontaneous release.

Identification of alkali-tolerant candidate genes using the NGS-assisted BSA strategy in rice.

Rice (Oryza sativa L.) is a saline-alkali-sensitive crop. Saline-alkali environments can seriously affect the growth, development, and yield of rice. The mechanisms of salt tolerance and alkali tolerance in rice are different; thus, it is very important to study and explore the alkali-tolerant gene loci to improve the saline-alkali tolerance of rice varieties. In this study, the japonica rice varieties Dongnong 425 (DN425) and Changbai 10 (CB10) and a hybridized recombinant inbred line (RIL) population were used as materials to be irrigated with Na2CO3 solution under field test conditions. A resistant pool (R-pool) and a sensitive pool (S-pool) were constructed by selecting the lines with extremely high and extremely low 1000-grain weight (TGW), respectively, from the RIL population under alkali treatment. Four candidate TGW regions on chromosomes (Chr.) 2 and 3 were associated using the bulked segregant analysis (BSA) strategy assisted by next-generation sequencing (NGS) technology (NGS-assisted BSA). Using the linkage analysis, QTL-qATGW2-2 in the candidate region was mapped within a range of 116 Kb between the SSR marker RM13592 and the Indel marker Indel3 of Chr. 2, which contained 18 predictive genes. The BSA sequencing results showed that Os02g39884 contained a nonsynonymous substitution mutation SNP (nsSNP), leading to the transformation of a residue from arginine (cGg) to glutamine (cAg); thus, Os02g39884 was inferred to be the candidate gene of qATGW2-2. The results of the qRT-PCR analysis also confirmed this. This paper provides important information for the rapid and accurate identification of the alkali-tolerant gene loci in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01228-x.

Stress-Sensitive Protein Rac1 and Its Involvement in Neurodevelopmental Disorders.

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a small GTPase that is well known for its sensitivity to the environmental stress of a cell or an organism. It senses the external signals which are transmitted from membrane-bound receptors and induces downstream signaling cascades to exert its physiological functions. Rac1 is an important regulator of a variety of cellular processes, such as cytoskeletal organization, generation of oxidative products, and gene expression. In particular, Rac1 has a significant influence on certain brain functions like neuronal migration, synaptic plasticity, and memory formation via regulation of actin dynamics in neurons. Abnormal Rac1 expression and activity have been observed in multiple neurological diseases. Here, we review recent findings to delineate the role of Rac1 signaling in neurodevelopmental disorders associated with abnormal spine morphology, synaptogenesis, and synaptic plasticity. Moreover, certain novel inhibitors of Rac1 and related pathways are discussed as potential avenues toward future treatment for these diseases.

Colorimetric determination of cysteine by a paper-based assay system using aspartic acid modified gold nanoparticles.

A method is described for cysteine (Cys) determination on paper-based analytical devices using aspartic acid modified gold nanoparticles (Asp-AuNPs). The Asp-AuNPs were characterized by their size, zeta potential, and UV-visible absorption spectrum. After the addition of Cys, it will interact with Asp-AuNPs selectively and leads to the aggregation of Asp-AuNPs. A color change from red to blue can be observed on the paper-based analytical devices. The results were recorded using a cell phone and subsequently analyzed using the Photoshop software. The ratiometric color intensity at red channel and blue channel (Red/Blue) increased linearly in the range 99.9-998.7 µM for Cys (R = 0.9984), and the limit of detection was 1.0 µM. The effects of assay conditions have been investigated and are discussed. The Cys concentration was determined as (0.27 ± 0.02 mM) in human plasma, and the recovery was from 99.2 to 101.1%. Graphical abstract Schematic representation of the paper-based assay system using aspartic acid modified gold nanoparticles (Asp-AuNPs). The ratiometric color intensity method was used for the cysteine (Cys) determination.

Retracted: microRNA-129-5p involved in the neuroprotective effect of dexmedetomidine on hypoxic-ischemic brain injury by targeting COL3A1 through the Wnt/ß-catenin signaling pathway in neonatal rats.

Our study aims to elucidate the mechanisms how microRNA-129-5p (miR-129-5p) involved in the neuroprotective effect of dexmedetomidine (DEX) on hypoxic-ischemic brain injury (HIBI) by targeting the type III procollagen gene (COL3A1) through the Wnt/ß-catenin signaling pathway in neonatal rats. A total of 120 rats were obtained, among which 15 rats were selected as sham group and rest rats as model, DEX, DEX + negative control (DEX + NC), DEX + miR-129-5p mimics, DEX + miR-129-5p inhibitors, DEX + XAV-939, and DEX + miR-129-5p inhibitors + XAV-939 groups. A dual-luciferase reporter assay was performed for the target relationship between miR-129-5p and COL3A1. Weight rate and water content of cerebral hemisphere were detected. Quantitative real-time polymerase chain reaction and Western blot analysis were conducted to detect miR-129-5p expression and expressions of COL3A1, E-cadherin, T-cell factor (TCF)- 4, and ß-catenin. The DEX, DEX + miR-129-5p mimics, DEX + XAV-939 groups had increased weight rate of the cerebral hemisphere, but decreased water content of left cerebral hemisphere, levels of COL3A1, ß-catenin, TCF-4, and E-cadherin in the hippocampus compared with the model and DEX + miR-129-5p inhibitors groups. COL3A1 was verified as the target gene of the miR-129-5p. Compared with the DEX + NC and DEX + miR-129-5p inhibitors + XAV-939 groups, the DEX + XAV-939 and DEX + miR-129-5p mimics groups had elevated weight rate of the cerebral hemisphere, but reduced water content of left cerebral hemisphere, levels of COL3A1, ß-catenin, TCF-4, and E-cadherin in the hippocampus. Our findings demonstrate that miR-129-5p improves the neuroprotective role of DEX in HIBI by targeting COL3A1 through the Wnt/ß-catenin signaling pathway in neonatal rats.

Upregulated circular RNA circ_0007534 indicates an unfavorable prognosis in pancreatic ductal adenocarcinoma and regulates cell proliferation, apoptosis, and invasion by sponging miR-625 and miR-892b.

Circular RNAs (circRNAs) have been regarded as critical regulators of human diseases and biological markers in some types of malignancies, including pancreatic ductal adenocarcinoma (PDAC). Recently, circ_0007534 has been identified as a novel cancer-related circRNA. Nevertheless, its clinical relevance, functional roles, and mechanism have not been studied in PDAC. In the current study, real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of circ_0007534 in 60-paired PDAC tissue samples and different cell lines. Loss-of-function and gain-of-function assays were performed to detect cell proliferation, apoptosis, and metastatic properties affected by circ_0007534. An animal study was also carried out. The luciferase reporter assay was performed to uncover the underlying mechanism of circ_0007534. As a result, circ_0007534 was overexpressed not only in PDAC tissues but also in a panel of PDAC cell lines, and this overexpression is closely associated with advanced tumor stage and positive lymph node invasion. In addition, circ_0007534 may be regarded as an independent prognostic factor for patients with PDAC. For the part of functional assays, circ_0007534 significantly increased cell proliferation, migratory, and invasive potential of PDAC cells. Circ_0007534 could inhibit cell apoptosis partly via a Bcl-2/caspase-3 pathway. The xenograft study further confirmed the cell growth promoting the role of circ_0007534. Mechanistically, miR-625 and miR-892b were sponged by circ_0007534. The oncogenic functions of circ_0007534 is partly dependent on its regulation of miR-625 and miR-892b. In conclusion, our study illuminates a novel circRNA that confers an oncogenic function in PDAC.

A successful bronchial thermoplasty procedure in a "very severe" asthma patient with rare complications: a case report.

Introduction: Bronchial thermoplasty (BT) is a unique bronchoscopic treatment for severe asthma that utilizes radiofrequency ablation to reduce smooth muscle in the bronchial walls. Current studies mainly focused on uncontrolled severe asthma with a forced expiratory volume in 1 second (FEV1) above 60% and associated complications, no human studies have performed on "very severe" asthma as well as its complications. Case study: We present a 60-year-old male with more than 15 years history of very severe asthma, who underwent BT. His FEV1 was only 20.4% predicted, which would have excluded him from all prior clinical trials of BT. The first BT procedure occurred without an issue. After the second BT procedure, he experienced severe dyspnea due to an infection with a non-flu respiratory virus. This illness was complicated by the formation of a pulmonary cyst. During recovering from the third procedure, he developed stomach stones. This is mainly related with taking large amounts of hawthorn previously, also cannot exclude the role of thermal energy injury on gastrointestinal nerve function. Results: Despite these unexpected complications, his quality of life greatly improved after BT, yet his lung function did not improve. Conclusion: This case is the first to describe BT procedures in patient with this level of lung function compromise, although accompanied with rare complications; our report indicates BT may be an opportunity and choice for the "very severe" asthma patients.

Monitoring and Control of Medical Air Disinfection Parameters of Nosocomial Infection System Based on Internet of Things.

To include the two key parameters of circulating air volume and instantaneous ultraviolet illuminance, as well as the minor influencing factors such as temperature, humidity, comings and goings of personnel and other parameters into the scope of conventional monitoring, and monitor and alarm each parameter, the key problem is to design the data transmission of sensors of existing products and debug the network management system, and solve every problem in the research process through the cycle of experiment-trial-experiment. The specific functions of a single instrument and system software can be achieved by solving the key links such as type selection of various sensors, circuit interface, guarantee measures for measurement accuracy of various parameters, research and development of networked air disinfection management system software, design of WiFi interface, cost control of single machine and system and so on. And ultraviolet luminance sensors can be used to monitor the ultraviolet intensity in the machine in real time, and monitor 7 parameters, including circulating air volume, ozone concentration, comings and goings of personnel, temperature, humidity and leaked ultraviolet intensity.

Quantitation of a Therapeutic Antibody in Serum Using Intact Sequential Affinity Capture, Trypsin Digestion, and LC-MS/MS.

Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents. The reagents were selective for the heavy and light chain of a monoclonal antibody, which when used consecutively, ensures that only the intact form of the antibody is represented by the surrogate peptide. The approach provided comparable results to a traditional sandwich IA assay indicating similar capture populations. The use of an initial ISAC assessment of affinity capture purification, should add a degree of confidence in the use of a single IA-LC-MS/MS quantitation assay.

Development of Anti-CD74 Antibody-Drug Conjugates to Target Glucocorticoids to Immune Cells.

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

Sr2+ has low efficiency in regulating spontaneous release at the Calyx of Held synapses.

It has been known that Ca2+ plays an essential role in mediating different modes of neurotransmitter release via different sensing mechanisms. Synaptotagmin 1, 2, and 9 were found to act as the Ca2+ sensors for synchronous release and synaptotagmin 7 and Doc-2 were proposed as the Ca2+ sensors for asynchronous release. Comparatively, the Ca2+ sensor for spontaneous release remains a mystery. At the Calyx of Held synapse, the Ca2+ sensor for spontaneous release was found not identical to the sensor for synchronous release, synaptotagmin 2. As Ca2+ sensors have different sensitivity to Sr2+ and Ca2+ and induce significantly different rate of vesicle release, Sr2+ is traditionally used as a tool to examine the intrinsic properties of different Ca2+ sensors. Here, we employed cell-attached patch recording and presynaptic/postsynaptic whole-cell recording at the Calyx of Held synapses of synaptotagmin 2 knock-out mice to assay the Sr2+ and Ca2+ influx into the nerve terminal at resting potential and observed the effects of Ca2+ and Sr2+ on spontaneous neurotransmitter release. We found that the dwell time of single voltage gated Ca2+ channel opening increased around threefold for Sr2+ than Ca2+ with the channel conductance unchanged; the divalent cation sensing machinery in regulating spontaneous release has much lower sensitivity to Sr2+ than Ca2+ . Thus, our study reveals some of the intrinsic properties of Ca2+ sensor(s) of spontaneous transmitter release and provided an insight into the underlying mechanisms.

Spatial Compartmentalization Specializes the Function of Aurora A and Aurora B.

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice.

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies.

[Lipid metabolomics in serum of hyperuricemic rats induced by fructose based on UPC²-Q/TOF-MS].

To explore the differences in lipid metabolites in serum of hyperuricemic rats induced by fructose and normal rats by using lipid metabolomics technology, and screen the potential biomarkers related to hyperuricemia. The metabolic fingerprint spectrum of the serum in hyperuricemic rats(model group) and normal rats(control group) was obtained and analyzed by using ultra performance convergence chromatography-tandem-Q-time of flight mass spectrometry(UPC ² -Q/TOF-MS) method and the differences of metabolic spectra between two groups were compared via the multivariate statistical methods to screen differential metabolites. The results indicated that there was significant difference in metabolic spectra between model group and control group, and 11 differential metabolites were screened. Then eight potential biomarkers such as arachidonic acid, palmitic acid, oleic acid and linoleic acid were tentatively identified by using the exact mass number and secondary mass spectrometry(MS/MS spectrum). Therefore, a new research method for lipid metabolomics in serum of hyperuricemic rats induced by fructose was established successfully based on UPC ² -Q/TOF-MS. What's more, it was speculated that the abnormal metabolism of fatty acid might be associated with the pathogenesis of hyperuricemia, which would provide scientific basis for early detection and prevention of hyperuricemia.

Purification and Characterisation of a Fibrinolytic Enzyme from Rhizopus microsporus var. tuberosus.

Extracellular fibrinolytic enzyme from Rhizopus microsporus var. tuberosus was purified and characterised. The microorganism was isolated in a distillery from daqu, a fermentative agent used in the production of Chinese liquor and vinegar at different temperatures. The fibrinolytic enzyme was partially purified by ammonium sulphate precipitation, dialysis, DEAE Sepharose® Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of the fibrinolytic enzyme was estimated to be 24.5 kDa by SDS-PAGE. The purified enzyme showed optimal activity at pH=7.0 and 37 °C by fibrin plate method. It showed stronger resistance to the inhibition by trypsin and was stable at 37 °C retaining 96.1% residual activity after 4 h of incubation. The fibrinolytic activity of the enzyme was enhanced by Na+, Ca2+, Mg2+ and Mn2+. Conversely, Zn2+ and Cu2+ partly inhibited enzymatic activity. Using fibrin plate method, we found that the enzyme not only degrades fibrin directly, but also activates plasminogen into plasmin to degrade fibrin. The results indicate that the pure enzyme has a potential in dissolving blood clot, and the possibility for application in the treatment of thrombosis.

Etanercept ameliorates inflammation and pain in a novel mono-arthritic multi-flare model of streptococcal cell wall induced arthritis.

BACKGROUND: The impact of anti-TNF, corticosteroid and analgesic therapy on inflammation and pain was evaluated in a novel mono-arthritic multi-flare rat Streptococcal Cell Wall (SCW) model using Etanercept, Dexamethasone and Buprenorphine. METHODS: Multiple flares of arthritis were induced with an intra-articular injection of SCW in the hind ankle on day 1, followed by intravenous challenges on days 21 and 42. Inflammation and pain were monitored in the hind paws. Cytokine profiling, cell phenotyping, bioluminescence imaging and histopathological evaluation were also performed. RESULTS: Local injection of SCW caused a rapid onset of inflammation and pain in the injected ankle which resolved within 4 days (Flare 1). Intravenous injection 20 days after sensitization resulted in an increase in ankle diameter and pain, which partially resolved in 8 days (Flare 2). The subsequent intra-venous injection in the same animals 14 days after resulted in a more chronic disease with inflammation and pain persisting over a period of 10 days (Flare 3). In Flare 2, therapeutic administration of Dexamethasone inhibited paw swelling (95%; P<0.001) and pain (55%; P<0.05). Therapeutic administration of Buprenorphine inhibited pain (80%; P<0.001) without affecting paw swelling (0%). Prophylactic administration of Etanercept in Flare 2 inhibited paw swelling (≥60%; P<0.001) and pain by ≥30%. Expression of IL-1ß, IL-6, MCP-1 and CINC was reduced by >50% (P<0.001). Treatment with Etanercept in Flare 3 inhibited paw swelling by 60% (P<0.001) and pain by 25%. Prior treatment with Etanercept in Flare 2 followed by re-administration in Flare 3 led to a complete loss in the efficacy of Etanercept. Systemic exposure of Etanercept corroborated with lack of efficacy. Dexamethasone inhibited inflammation and pain in both Flares 2 and 3 (P<0.001). CONCLUSIONS: We established a novel multi-flare SCW arthritis model enabling drug intervention in different stages of disease. We show for the first time the evaluation of inflammation and pain simultaneously in this model. Etanercept and Dexamethasone inhibited inflammation, pain and proinflammatory cytokines in this model. Taken together, this model facilitates the assessment of anti-rheumatic agents targeting inflammation and pain in the multiple flare paradigm and offers a powerful tool for drug discovery.

[Establishment of culture system of Silybum marianum hairy roots and determination of silybin].

This research uses six Agrobacterium rhizogenes R1601, R15384, R1000, A4, R1025 and R1 to infect silymarin explants to induce hairy roots and silibin. All of the six A. rhizogenes can induce Silybum marianum to generate hairy roots and the A. rhizogene A4 shows comparatively high infection on the plant. This research determines the condition to induce silymarin hairy roots by the factors of infection time, pre-culturing, co-culturing and pH value. The fact that MS liquid medium fits the proliferation of silymarin hairy roots is determined. Through PCR molecular identification, it can be seen that the DNA plasmids in the A. rhizogenes are successfully integrated into the genome of transformed roots. Using liquid chromatography, it is determined that the silibin content in silymarin hairy roots is 2.5 times that in the plant In this research, the silymarin hairy roots culturing system is established, which lays a foundation for the study of culturing silymarin hairy roots and producing silibin.