Multienzyme Active Nanozyme for Efficient Sepsis Therapy through Modulating Immune and Inflammation Inhibition.

Sepsis, a life-threatening condition caused by a dysregulated immune response to infection, leads to systemic inflammation, immune dysfunction, and multiorgan damage. Various oxidoreductases play a very important role in balancing oxidative stress and modulating the immune response, but they are stored inconveniently, environmentally unstable, and expensive. Herein, we develop multifunctional artificial enzymes, CeO2 and Au/CeO2 nanozymes, exhibiting five distinct enzyme-like activities, namely, superoxide dismutase, catalase, glutathione peroxidase, peroxidase, and oxidase. These artificial enzymes have been used for the biocatalytic treatment of sepsis via inhibiting inflammation and modulating immune responses. These nanozymes significantly reduce reactive oxygen species and proinflammatory cytokines, achieving multiorgan protection. Notably, CeO2 and Au/CeO2 nanozymes with enzyme-mimicking activities can be particularly effective in restoring immunosuppression and maintaining homeostasis. The redox nanozyme offers a promising dual-protective strategy against sepsis-induced inflammation and organ dysfunction, paving the way for biocatalytic-based immunotherapies for sepsis and related inflammatory diseases.

Exploring Potential Targets and Pathways of Toxicity Attenuation Through Serum Pharmacochemistry and Network Pharmacology in the Processing of Aconiti Lateralis Radix Praeparata.

Aconiti Lateralis Radix Praeparata (Fuzi, FZ) is a frequently utilized traditional Chinese medicine (TCM) in clinical settings. However, its toxic and side effects, particularly cardiac injury, are apparent, necessitating processing before use. To investigate the mechanism of toxicity induced by absorbed components and the mitigating effect of processed FZ, we established a comprehensive method combining serum pharmacochemistry and a network pharmacology approach. In total, 31 chemical components were identified in the plasma, with a general decrease in response intensity observed for these components in processed FZ. Subsequently, four components were selected for network pharmacology analysis. This analysis revealed 150 drug action targets and identified 1162 cardiac toxicity targets. Through intersection analysis, 41 key targets related to cardiac toxicity were identified, along with 9 significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The most critical targets identified were AKT1, MTOR, and PARP1. The key biological pathways implicated were adrenergic signaling in cardiomyocytes, proteoglycans in cancer, and the calcium signaling pathway. Significant differences were observed in histological staining and biochemical indicators in the cardiac tissue of rats treated with FZ, indicating that processing could indeed reduce its cardiotoxicity. Indeed, this article presents a valuable strategy for elucidating the toxification mechanism of toxic TCM.

Clinical and genetic analysis of essential hypertension with mitochondrial tRNAMet 4435A>G and YARS2 mutation. / 携带线粒体tRNAMet 4435A>Gå’Œæ ¸åŸºå› YARS2çªå˜çš„åŽŸå‘æ€§é«˜è¡€åŽ‹å®¶ç³»é—ä¼ å­¦åˆ†æž.

OBJECTIVES: To investigate the role of m.4435A>G and YARS2 c.572G>T (p.G191V) mutations in the development of essential hypertension. METHODS: A hypertensive patient with m.4435A>G and YARS2 p.G191V mutations was identified from previously collected mitochondrial genome and exon sequencing data. Clinical data were collected, and a molecular genetic study was conducted in the proband and his family members. Peripheral venous blood was collected, and immortalized lymphocyte lines constructed. The mitochondrial transfer RNA (tRNA), mitochondrial protein, adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) in the constructed lymphocyte cell lines were measured. RESULTS: Mitochondrial genome sequencing showed that all maternal members carried a highly conserved m.4435A>G mutation. The m.4435A>G mutation might affect the secondary structure and folding free energy of mitochondrial tRNA and change its stability, which may influence the anticodon ring structure. Compared with the control group, the cell lines carrying m.4435A>G and YARS2 p.G191V mutations had decreased mitochondrial tRNA homeostasis, mitochondrial protein expression, ATP production and MMP levels, as well as increased ROS levels (all P<0.05). CONCLUSIONS: The YARS2 p.G191V mutation aggravates the changes in mitochondrial translation and mitochondrial function caused by m.4435A>G through affecting the steady-state level of mitochondrial tRNA and further leads to cell dysfunction, indicating that YARS2 p.G191V and m.4435A>G mutations have a synergistic effect in this family and jointly participate in the occurrence and development of essential hypertension.

An exploration of the value of NLR, PLR, LMR, and WBC × CRP for the diagnosis and treatment of influenza B in adults.

The aim of the study was to study the diagnostic and therapeutic utility of NLR (neutrophil-to-lymphocyte ratio), LWR (lymphocyte-to-monocyte ratio), PLR (platelet-to-lymphocyte ratio), and WBC × CRP (WBC: white cell count, CRP: C-reactive protein) in patients with influenza B. This retrospective study included 122 adult patients with influenza B, 176 adult patients with bacterial infection, and 119 adult healthy physical examinees for routine blood examination and CRP testing, calculation of NLR, LMR, PLR, and WBC × CRP for relevant statistical analysis, monitoring of NLR, LMR, PLR and WBC × CRP in patients with influenza B during relevant treatment. All indicators, except for WBC and NLR, had no statistical differences between the influenza B group, the normal control group, and the influenza B group and bacterial infection group, respectively, and showed no statistical significance for the differences between the groups. The diagnostic effect of LMR and WBC × CRP was deemed good or excellent in patients with influenza B, healthy people, and patients with a bacterial infection. Conversely, NLR and PLR could only distinguish patients with influenza B from healthy people but remained unable to identify different pathogens. Moreover, many false negatives were noted for WBC and CRP during the diagnosis of influenza B. Also, NLR, LMR, PLR, and WBC × CRP exerted a good effect in evaluating curative effect and conditions for influenza B. LMR and WBC × CRP have a relatively high value in the early diagnosis of adults suffering from influenza B. Also, NLR and PLR excelled at differentiating adult patients with influenza B from healthy people. Therefore, NLR, PLR, LMR, and WBC × CRP can all be used for disease course monitoring and efficacy evaluation.

Research on improving the accuracy of laser-induced breakdown spectroscopy analysis by considering plasma attenuation rate characteristics.

BACKGROUND: Laser-induced breakdown spectroscopy (LIBS) is widely applied in various fields, but accuracy issues limit its further development. Signal uncertainty is the main reason that affects the accuracy of LIBS measurements, but the signal uncertainty caused by different plasmas exhibiting different radiation attenuation rates during the integration time is often neglected. There is a need for a method to correct LIBS signals by quantifying the radiation attenuation rate. RESULTS: In order to reduce the uncertainty due to different plasma attenuation rates, the attenuation rates of the energy level radiation emitted by plasma are described as attenuation coefficients, which are obtained by linearly fitting the logarithm of the time series of line intensities. The calibration curve was corrected by attenuation coefficients for 4 major elements in 7 standard samples. The results showed that the line intensities corrected by attenuation coefficients showed better linearity with elemental concentrations. SIGNIFICANCE: This study is important for improving the accuracy of LIBS measurements, and is also significant for modeling the plasma radiative attenuation of laser-induced plasma, and is expected to be applied to spectrometers that can obtain time series spectra of the same plasma to improve the accuracy of in-situ fast LIBS analysis.

A Muti-Substrate Flavonol O-glucosyltransferases from Safflower.

To explore the complete biosynthesis process of flavonoid glycosides in safflower, specifically the key glycosyltransferase that might be involved, as well as to develop an efficient biocatalyst to synthesize flavonoid glycosides, a glycosyltransferase CtUGT4, with flavonoid-O-glycosyltransferase activity, was identified in safflower. The fusion protein of CtUGT4 was heterologously expressed in Escherichia coli, and the target protein was purified. The recombinant protein can catalyze quercetin to form quercetin-7-O-glucoside, and kaempferol to form kaempferol-3-O in vitro, and a series of flavones, flavonols, dihydroflavones, chalcones, and chalcone glycosides were used as substrates to generate new products. CtUGT4 was expressed in the tobacco transient expression system, and the enzyme activity results showed that it could catalyze kaempferol to kaempferol-3-O-glucoside, and quercetin to quercetin-3-O-glucoside. After overexpressing CtUGT4 in safflower, the content of quercetin-3-O-rutinoside in the safflower florets increased significantly, and the content of quercetin-3-O-glucoside also tended to increase, which preliminarily confirmed the function of CtUGT4 flavonoid-O-glycosyltransferase. This work demonstrated the flavonoid-O-glycosyltransferase function of safflower CtUGT4 and showed differences in the affinity for different flavonoid substrates and the regioselectivity of catalytic sites in safflower, both in vivo and in vitro, providing clues for further research regarding the function of UGT genes, as well as new ideas for the cultivation engineering of the directional improvement of effective metabolites in safflower.

Emerging nanotechnology for Alzheimer's disease: From detection to treatment.

Alzheimer's disease (AD), one of the most common chronic neurodegenerative diseases, is characterized by memory impairment, synaptic dysfunction, and character mutations. The pathological features of AD are Aß accumulation, tau protein enrichment, oxidative stress, and immune inflammation. Since the pathogenesis of AD is complicated and ambiguous, it is still challenging to achieve early detection and timely treatment of AD. Due to the unique physical, electrical, magnetic, and optical properties of nanoparticles (NPs), nanotechnology has shown great potential for detecting and treating AD. This review provides an overview of the latest developments in AD detection via nanotechnology based on NPs with electrochemical sensing, optical sensing, and imaging techniques. Meanwhile, we highlight the important advances in nanotechnology-based AD treatment through targeting disease biomarkers, stem-cell therapy and immunotherapy. Furthermore, we summarize the current challenges and present a promising prospect for nanotechnology-based AD diagnosis and intervention.

Catalytic nanotechnology of X-ray photodynamics for cancer treatments.

Photodynamic therapy (PDT) has been applied in cancer treatment because of its high selectivity, low toxicity, and non-invasiveness. However, the limited penetration depth of the light still hampers from reaching deep-seated tumors. Considering the penetrating ability of high-energy radiotherapy, X-ray-induced photodynamic therapy (X-PDT) has evolved as an alternative to overcome tissue blocks. As the basic principle of X-PDT, X-rays stimulate the nanoparticles to emit scintillating or persistent luminescence and further activate the photosensitizers to generate reactive oxygen species (ROS), which would cause a series of molecular and cellular damages, immune response, and eventually break down the tumor tissue. In recent years, catalytic nanosystems with unique structures and functions have emerged that can enhance X-PDT therapeutic effects via an immune response. The anti-cancer effect of X-PDT is closely related to the following factors: energy conversion efficiency of the material, the radiation dose of X-rays, quantum yield of the material, tumor resistance, and biocompatibility. Based on the latest research in this field and the classical theories of nanoscience, this paper systematically elucidates the current development of the X-PDT and related immunotherapy, and highlights its broad prospects in medical applications, discussing the connection between fundamental science and clinical translation.

Both Two CtACO3 Transcripts Promoting the Accumulation of the Flavonoid Profiles in Overexpressed Transgenic Safflower.

The unique flavonoids, quinochalcones, such as hydroxysafflor yellow A (HSYA) and carthamin, in the floret of safflower showed an excellent pharmacological effect in treating cardiocerebral vascular disease, yet the regulating mechanisms governing the flavonoid biosynthesis are largely unknown. In this study, CtACO3, the key enzyme genes required for the ethylene signaling pathway, were found positively related to the flavonoid biosynthesis at different floret development periods in safflower and has two CtACO3 transcripts, CtACO3-1 and CtACO3-2, and the latter was a splice variant of CtACO3 that lacked 5' coding sequences. The functions and underlying probable mechanisms of the two transcripts have been explored. The quantitative PCR data showed that CtACO3-1 and CtACO3-2 were predominantly expressed in the floret and increased with floret development. Subcellular localization results indicated that CtACO3-1 was localized in the cytoplasm, whereas CtACO3-2 was localized in the cytoplasm and nucleus. Furthermore, the overexpression of CtACO3-1 or CtACO3-2 in transgenic safflower lines significantly increased the accumulation of quinochalcones and flavonols. The expression of the flavonoid pathway genes showed an upward trend, with CtCHS1, CtF3H1, CtFLS1, and CtDFR1 was considerably induced in the overexpression of CtACO3-1 or CtACO3-2 lines. An interesting phenomenon for CtACO3-2 protein suppressing the transcription of CtACO3-1 might be related to the nucleus location of CtACO3-2. Yeast two-hybrid (Y2H), glutathione S-transferase (GST) pull-down, and BiFC experiments revealed that CtACO3-2 interacted with CtCSN5a. In addition, the interactions between CtCSN5a and CtCOI1, CtCOI1 and CtJAZ1, CtJAZ1 and CtbHLH3 were observed by Y2H and GST pull-down methods, respectively. The above results suggested that the CtACO3-2 promoting flavonoid accumulation might be attributed to the transcriptional activation of flavonoid biosynthesis genes by CtbHLH3, whereas the CtbHLH3 might be regulated through CtCSN5-CtCOI1-CtJAZ1 signal molecules. Our study provided a novel insight of CtACO3 affected the flavonoid biosynthesis in safflower.

Atomically precise silver clusterzymes protect mice from radiation damages.

BACKGROUND: As we know, radiotherapy plays an irreplaceable role in the clinical management on solid tumors. However, due to the non-specific killing effects of ionizing radiation, normal tissues damages would be almost simultaneous inevitably. Therefore, ideal radioprotective agents with high efficiency and low toxicity are always desirable. In this work, atomically precise Ag14 clusterzymes were developed, and their applications in radioprotection were studied in vitro and in vivo for the first time. METHODS: The ultra-small glutathione supported Ag14 clusterzymes were synthesized by convenient sodium borohydride (NaBH4) reduction of thiolate-Ag (I) complexes and then they were purified by desalting columns. The enzyme-like activity and antioxidant capacity of Ag14 clusterzymes have been tested by various commercial kits, salicylic acid method and electron spin resonance (ESR). Next, they were incubated with L929 cells to evaluate whether they could increase cell viability after γ-ray irradiation. And then Ag14 clusterzymes were intravenously injected into C57 mice before 7 Gy whole-body γ-ray irradiation to evaluate the radioprotection effects in vivo. At last, the in vivo toxicities of Ag14 clusterzymes were evaluated through biodistribution test, hematological details, serum biochemical indexes and histological test in female Balb/c mice with intravenous injection of Ag14 clusterzymes. RESULTS: Our studies suggested atomically precise Ag14 clusterzymes were potential radioprotectants. Ag14 clusterzymes exhibited unique superoxide dismutase (SOD)-like activity, strong anti-oxidative abilities, especially on •OH scavenging. The Ag14 clusterzymes could effectively improve cell viability through eliminating ROS and prevent DNA damages in cells dealt with γ-ray irradiation. In vivo experiments showed that Ag14 clusterzymes could improve the irradiated mice survival rate by protecting hematological systems and repairing tissue oxidative stress damage generated by γ-ray irradiation. In addition, bio-distribution and toxicological experiments demonstrated that the ultrasmall Ag14 clusterzymes could be excreted quickly from the body by renal clearance and negligible toxicological responses were observed in mice up to 30 days. CONCLUSION: In summary, atomically precise, ultrasmall and water soluble Ag14 clusterzymes with SOD-like activity were successfully developed and proved to be effective both in vitro and in vivo for radioprotection. Furthermore, with atomically precise molecular structure, Ag14 clusterzymes, on aspect of the catalytic and optical properties, may be improved by structure optimization on atom-scale level for other applications in disease diagnosis and treatment.

Ligand-Modulated Catalytic Selectivity of Ag Clusterzyme for Relieving Multiorgan Injury via Inhabiting Acute Oxidative Stress.

The artificial enzymes at the atomic level have shown great potential in chemical biology and nanomedicine, and modulation of catalytic selectivity is also critical to the application of nanozymes. In this work, atomic precision Ag25 clusterzymes protected by single- and dual-ligand were developed. Further, the catalytic activity and selectivity of Ag25 clusterzymes were modulated by adjusting doping elements and ligand. The Ag24Pt1 shows more prominent antioxidant activity characteristics in the dual-ligand system, while the Ag24Cu1 possesses the superoxide dismutase-like (SOD-like) activity regardless of the single- or dual-ligand system, indicating modulated catalytic selectivity. In vitro experiments showed the Ag24Pt1-D can recover radiation induced DNA damages and eliminate the excessive reactive oxygen species (ROS) generated from radiation. Subsequent in vivo radiation protection experiments reveal that Ag24Cu1-S and Ag24Pt1-D can improve the survival rate of irradiated mice from 0 to 40% and 30%, respectively. The detailed biological experiments confirm that the Ag24Cu1-S and Ag24Pt1-D can recover the SOD and 3,4-methylenedioxyamphetamine (MDA) levels via suppressing the chronic inflammation reaction. Nearly 60% of Ag24Cu1-S and Ag24Pt1-D can be excreted after a 1 day injection, and no obvious toxicological reactions were observed 30 days after injection.

Diterpenoid glycosides from the flower of Trollius chinensis Bunge and their nitric oxide inhibitory activities.

Trolliusditerpenosides A-Q (1-17), seventeen new labdane-diterpenoid glycosides, were isolated from the dried flowers of Trollius chinensis Bunge, a plant that has been commonly used as both an anti-inflammatory folk medicine and a healthcare tea for its therapeutic and anti-viral and antibacterial properties. Their structures were corroborated via comprehensive spectroscopic analysis, ECD calculations, and single-crystal X-ray diffraction analysis. Furthermore, the inhibitory activities on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages of all compounds (1-17) were evaluated in vitro. Compounds 3, 6, 7, and 11 displayed significant inhibitory activities against NO production, with IC50 values ranging from 1.6 ± 0.1 to 14.4 ± 0.2 µM. In addition, compounds 3, 6, 7, and 11 all down-regulated the mRNA expression of iNOS, COX-2, and IL-1ß in RAW 264.7 cells mediated by LPS. These findings not only support the chemical context of genus Trollius but also the exploration of new chemical entities with pharmacological significance from this genus.

Autophagy is involved in the neuroprotective effect of nicotiflorin.

ETHNOPHARMACOLOGICAL RELEVANCE: Nicotiflorin is a flavonoid glycoside derived from the traditional Chinese medicine FlosCarthami, dried petals of Carthamus tinctorius L., and has been confirmed to be a promising novel drug candidate for ischemic stroke. Yet, the exact role of nicotiflorin in cerebral I/R injury is uncharacterized and the possible mechanisms have not been clearly expounded. AIM OF THE STUDY: The present study was designed to determine the effect of nicotiflorin on cerebral ischemia/reperfusion (I/R) injury and its relationship with autophagy. MATERIALS AND METHODS: Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation and reintroduction (OGD/R) in SH-SY5Y cells were established in in vivo and in vitro models, respectively. The severity of MCAO was assessed by brain infarct size, neurological scores and survival rate. The severity of OGD/R was evaluated by cell viability, lactate dehydrogenase (LDH) release and cell apoptosis. The level of autophagy was evaluated both in vivo and in vitro. Autophagosomes were observed using transmission electron microscopy and autophagic flux was measured using mRFP-GFP-tandem fluorescent LC3 adenovirus. Autophagy-related proteins (LC3-II/I, SQSTM1, beclin-1, Phospho-mTOR/mTOR) were measured by immunoblot. Autophagy-related mRNA levels (Becn1, Atg7) were detected by Real-Time PCR. Inhibition of autophagy was implemented by 3-Methyladenine (3-MA) or chloroquine in vitro. RESULTS: In vivo, nicotiflorin treatment alleviated brain damage and neurological deficit while it dramatically increased 72 h survival rate in rats. In vitro, nicotiflorin treatment also ameliorated the severity of OGD/R. Moreover, nicotiflorin treatment increased ischemic penumbra autophagy (autophagosomes, BECN1, LC3-II/I ratio, SQSTM1, Phospho-mTOR/mTOR, Atg7). In vitro, nicotiflorin likewise enhanced autophagy and promoted autophagy flux. Furthermore, the blockade of autophagy by 3-MA or chloroquine disabled the efficacic of nicotiflorin in preventing cell damage upon OGD/R insult. CONCLUSION: These findings suggest that autophagy plays a significant role in the protective effect of nicotiflorin against ischemic stroke.

Multiplexed CRISPR/Cas9-Mediated Knockout of Laccase Genes in Salvia miltiorrhiza Revealed Their Roles in Growth, Development, and Metabolism.

Laccases are multicopper-containing glycoproteins related to monolignol oxidation and polymerization. These properties indicate that laccases may be involved in the formation of important medicinal phenolic acid compounds in Salvia miltiorrhiza such as salvianolic acid B (SAB), which is used for cardiovascular disease treatment. To date, 29 laccases have been found in S. miltiorrhiza (SmLACs), and some of which (SmLAC7 and SmLAC20) have been reported to influence the synthesis of phenolic acids. Because of the functional redundancy of laccase genes, their roles in S. miltiorrhiza are poorly understood. In this study, the CRISPR/Cas9 system was used for targeting conserved domains to knockout multiple genes of laccase family in S. miltiorrhiza. The expressions of target laccase genes as well as the phenolic acid biosynthesis key genes decrease dramatically in editing lines. Additionally, the growth and development of hairy roots was significantly retarded in the gene-edited lines. The cross-sections examination of laccase mutant hairy roots showed that the root development was abnormal and the xylem cells in the edited lines became larger and looser than those in the wild type. Additionally, the accumulation of RA as well as SAB was decreased, and the lignin content was nearly undetectable. It suggested that SmLACs play key roles in development and lignin formation in the root of S. miltiorrhiza and they are necessary for phenolic acids biosynthesis.

Catalytic Nanozyme for Radiation Protection.

Radiotherapy has been widely used in clinical cancer treatment. However, the ionizing radiation required to kill the tumor will inevitably cause damage to the surrounding normal tissues. To minimize the radiation damage and side effects, small molecular radioprotective agents have been used as clinical adjuvants for radiation protection of healthy tissues. However, the shortcomings of small molecules such as short circulation time and rapid kidney clearance from the body greatly hinder their biomedical applications. In recent years, nanozymes have attracted much attention because of their potential to treat a variety of diseases. Nanozymes exhibit catalytic properties and antioxidant capabilities to provide a potential solution for the development of high-efficiency radioprotective agents in radiotherapy and nuclear radiation accidents. Therefore, in this review, we systematically summarize the catalytic nanozymes used for radiation protection of healthy tissues and discuss the challenges and future prospects of nanomaterials in the field of radiation protection.

Chemical constituents from the roots of Lindera aggregata and their biological activities.

Three new benzylisoquinoline alkaloids, (1'S)-12'-hydroxyl-linderegatine (1), (1S)-5'-O-p-hydroxybenzoyl norreticuline (2), (1R, 1'R)-11,11'-biscoclaurine (3), along with 18 known compounds were isolated from the roots of Lindera aggregata (Sims) Kosterm. Their structures were determined on the basis of extensive spectroscopic analysis (IR, UV, HR-ESI-MS, 1D and 2D NMR). The absolute configurations of three new compounds were determined by comparing their experimental and calculated ECD for the first time. Compounds (4) and (9) showed cytotoxic activities against human colon carcinoma cell line (HCT-116), with IC50 values of 51.4 and 27.1 µM, respectively. Furthermore, compounds (10) and (11) showed inhibitory activities on nitric oxide production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells, with IC50 values of 37.8 and 38.7 µM, respectively.

Integrating molecular characterization and metabolites profile revealed CtCHI1's significant role in Carthamus tinctorius L.

BACKGROUND: As a traditional Chinese herb, safflower (Carthamus tinctorius L.) is valued for its florets to prevent cardiovascular and cerebrovascular diseases. Basing on previous chemical analysis, the main active compounds are flavonoids in its florets. Although flavonoid biosynthetic pathway has been well-documented in many model species, unique biosynthetic pathway remains to be explored in safflower. Of note, as an important class of transitional enzymes, chalcone isomerase (CHI) has not been characterized in safflower. RESULTS: According to our previous research, CHIs were identified in a safflower transcriptome library built by our lab. To characterize CHI in safflower, a CHI gene named CtCHI1 was identified. A multiple sequences alignment and phylogenetic tree demonstrate that CtCHI1 shares 92% amino acid identity and close relationship with CHI to Saussurea medusa. Additionally, subcellular localization analysis indicated CtCHI1-GFP fusion protein was mainly in the cell nucleus. Further, we purified CtCHI1 protein from E. coli which can effectively catalyze isomerization of 2',4',4,6'-tetrahydroxychalcone into naringenin in vitro. Via genetic engineer technology, we successfully obtained transgenic tobacco and safflower lines. In transgenic tobacco, overexpression of CtCHI1 significantly inhibited main secondary metabolites accumulation, including quercetin (~ 79.63% for ovx-5 line) and anthocyanins (~ 64.55% for ovx-15 line). As shown in transgenic safflower, overexpression of CtCHI1 resulted in upstream genes CtPAL3 and CtC4H1 increasing dramatically (up to ~ 3.9fold) while Ct4CL3, CtF3H and CtDFR2 were inhibited. Also, comparing the whole metabolomics database by PCA and PLS-DA between transgenic and control group, 788 potential differential metabolites were marked and most of them displayed up-regulated trends. In parallel, some isolated secondary metabolites, such as hydroxysafflor yellow A (HSYA), rutin, kaempferol-3-O-ß-rutinoside and dihydrokaempferol, accumulated in transgenic safflower plants. CONCLUSIONS: In this study, we found that CtCHI1 is an active, functional, catalytic protein. Moreover, CtCHI1 can negatively and competitively regulate anthocyanins and quercetin pathway branches in tobacco. By contrast, CtCHI1 can positively regulate flavonol and chalcone metabolic flow in safflower. This research provides some clues to understand CHI's differential biochemical functional characterization involving in flavonoid pathway. More molecular mechanisms of CHI remain to be explored in the near future.

CtACO1 Overexpression Resulted in the Alteration of the Flavonoids Profile of Safflower.

BACKGROUND: Flavonoids with various structures play a vital role in plant acclimatization to varying environments as well as in plant growth, development, and reproduction. Exogenous applications of ethylene and 1-aminocyclopropane carboxylic acid (ACC), could affect the accumulation of flavonoids. Very few attempts have been made to investigate the effect of 1-aminocyclopropane carboxylic acid oxidase (ACO), a unique enzyme that catalyzes ACC to ethylene, on genes and metabolites in the flavonoid biosynthetic pathway. In this study, two ACOs in safflower (CtACOs) were cloned, and then transgenic safflower with overexpressed CtACO1 was generated through the Agrobacterium-mediated floral dipping method. RESULTS: CtACO1 and CtACO2 were both characterized by the 2-oxoglutarate binding domain RxS and the ferrous iron binding site HxDxnH as ACOs from other plants. However, the transcript levels of CtACO1 in flowers at stages I, II, III, and IV were all higher than those of CtACO2. At the cellular level, by using electroporation transformation, CtACO1 was found to be localized at the cytomembrane in onion epidermal cells. CtACO1 overexpression had varying effects on genes involved in the ethylene and flavonoid biosynthetic pathways. The metabolites analysis showed that CtACO1 overexpression lines had a higher accumulation of quercetin and its glycosylated derivatives (quercetin 3-ß-d-glucoside and rutin). In contrast, the accumulation of quinochalcones (hydroxysafflor yellow A and carthamin), kaempferol glycosylated derivatives (kaempferol-3-O-ß-rutinoside and kaempferol-3-O-ß-d-glucoside), apigenin, and luteolin in CtACO1 overexpression lines were decreased. CONCLUSION: This study confirmed the feasibility of applying the floral dipping method to safflower and showed a novel regulatory effect of CtACO1 in the flavonoid biosynthetic pathway. It provides hypothetical and practical groundwork for further research on regulating the overall metabolic flux of flavonoids in safflower, particularly hydroxysafflor yellow A and other quinochalcones, by using appropriate genetic engineering strategies.

Simultaneous determination of the novel anti-tumor candidate drug MDH-7 and 5-fluorouracil in rat plasma by LC-MS/MS: Application to pharmacokinetic interactions.

MDH-7 (2,3,9-tri-O-acetyl-5,6-dideoxy-1,10-di-[N4'-pentoxycarbonyl-5'-fluoro cytosine]-4-ulose 1,4: 7,10-difuranose-4,8-pyranose) is a novel anti-tumor drug candidate. To study the pharmacokinetic interaction between MDH-7 and 5-fluorouracil (5-FU), a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine the concentrations of MDH-7 and 5-fluorouracil (5-FU) in rat plasma. Plasma samples were prepared by simple liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters XBridge™ C18 column (5 µm, 2.1 mm × 150 mm) with the mobile phase of methanol and H2O (80:20, v/v). The ESI positive and negative ion switch was operated in the multiple reactions monitoring (MRM) mode. The calibration curves showed good linearity (r2 > 0.99) over the ranges of 50-8000 ng/mL for MDH-7 and 10-2000 ng/mL for 5-FU, respectively. The lower limit of quantitations (LLOQs) was 50 ng/mL (MDH-7) and 10 ng/mL (5-FU) with relative standard deviation (RSD) < 13.0%. The proposed method was successfully applied to simultaneous assessment of pharmacokinetic drug-drug interaction between MDH-7 and 5-FU in rats.

Celosins inhibit atherosclerosis in ApoE-/- mice and promote autophagy flow.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen celosiae is a traditional Chinese medicine for purging hepatic pathogenic fire and removing nebula to improve eyesight, treating hepatopyretic vertigo and hypertension. It possesses a serial of potential bioactivities such as hepatoprotection, anti-tumor and anti-inflammatory, anti-diabetes. The triterpenoid saponins celosins from it were proved to have hepatoprotection, lipid lowing and anti-inflammatory. However, the anti-atherosclerosis activities were not reported to date. AIM OF THE STUDY: This study was designed to examine the therapeutic effects of celosins (CES), the active constituents extracted from Semen celosiae. MATERIALS AND METHODS: Atherosclerosis model by feeding high fat diet for 12 weeks in ApoE-/- mice and foam cell model by ox-LDL-treated peritoneal macrophages were performed. The lipid plaque was measured by histopathological analysis. The LC3 dots in the aortic root lesion examined through tissue immunofluorescence. The peritoneal macrophage phagocytosis, formation of foam cells, genes associated protein expression and autophagy flux were measured on foam cell model by oxidized low-density lipoprotein (Ox-LDL) stimulating peritoneal macrophages. The mRNA expression of CD36, SR-A1, ABCA1 and ABCG1 were determined by Real-Time PCR method. The expressions of LC3 and beclin 1 were measured using Western blot. RESULTS: CES (10, 30, 90mg/kg; p.o.) administrated for 4 weeks significantly reduced the prevalence of the relative area of plaque in mouse aorta, and showed the therapeutic effect on atherosclerosis. In the tissue section of immunofluorescence for aortic root, compared with high fat diet model group, the number of autophagy bodies in CES group increased significantly, suggesting that inhibiting atherosclerosis effect of CES may be related to its promoting autophagy. In vitro, CES significantly reduced phagocytosis of macrophages on lipid and formation rate of foam cells. CES down-regulated the mRNA expression of CD36 and SR-A1 while up-regulated mRNA expression of ABCA1 and ABCG1. Further, CES increased the autophagy specific protein LC3 and beclin 1, and it also increased the level of autophagy in the cells, and promoted the process of autophagy. CONCLUSIONS: The therapeutic effect of CES on atherosclerosis may be related to the promotion of autophagy.