The Antioxidant Salidroside Ameliorates the Quality of Postovulatory Aged Oocyte and Embryo Development in Mice.

Postovulatory aging is known to impair the oocyte quality and embryo development due to oxidative stress in many different animal models, which reduces the success rate or pregnancy rate in human assisted reproductive technology (ART) and livestock timed artificial insemination (TAI), respectively. Salidroside (SAL), a phenylpropanoid glycoside, has been shown to exert antioxidant and antitumor effects. This study aimed to investigate whether SAL supplementation could delay the postovulatory oocyte aging process by alleviating oxidative stress. Here, we show that SAL supplementation decreases the malformation rate and recovers mitochondrial dysfunction including mitochondrial distribution, mitochondrial membrane potential (ΔΨ) and ATP content in aged oocytes. In addition, SAL treatment alleviates postovulatory aging-caused oxidative stress such as higher reactive oxygen species (ROS) level, lower glutathione (GSH) content and a reduced expression of antioxidant-related genes. Moreover, the cytoplasmic calcium ([Ca2+]c) and mitochondrial calcium ([Ca2+]mt) of SAL-treated oocytes return to normal levels. Notably, SAL suppresses the aging-induced DNA damage, early apoptosis and improves spindle assembly in aged oocytes, ultimately elevating the embryo developmental rates and embryo quality. Finally, the RNA-seq and confirmatory experience showed that SAL promotes protective autophagy in aged oocytes by activating the MAPK pathway. Taken together, our research suggests that supplementing SAL is an effective and feasible method for preventing postovulatory aging and preserving the oocyte quality, which potentially contributes to improving the successful rate of ART or TAI.

Cable bacteria regulate sedimentary phosphorus release in freshwater sediments.

Previous studies have demonstrated that e-SOx can regulate the sedimentary release of phosphorus (P) in brackish and marine sediments. When e-SOx is active, an iron (Fe) and manganese (Mn) oxide rich layer is formed near the sediment surface, which prevents P release. When e-SOx becomes inactive, the metal oxide layer is reduced via sulfide-mediated dissolution, and P is subsequently released to the water column. Cable bacteria have been shown to also occur in freshwater sediments. In these sediments, sulfide production is limited, and the metal oxide layer would thus dissolve less efficiently, leaving the P trapped at the sediment surface. This lack of an efficient dissolution mechanism implies that e-SOx could play an important role in the regulation of P availability in eutrophied freshwater streams. To test this hypothesis, we incubated sediments from a eutrophic freshwater river to investigate the impact of cable bacteria on sedimentary cycling of Fe, Mn and P. High-resolution depth profiling of pH, O2 and ΣH2S complemented with FISH analysis and high-throughput gene sequencing showed that the development of e-SOx activity was closely linked to the enrichment of cable bacteria in incubated sediments. Cable bacteria activity caused a strong acidification in the suboxic zone, leading to the dissolution of Fe and Mn minerals and consequently a strong release of dissolved Fe2+ and Mn2+ to the porewater. Oxidation of these mobilized ions at the sediment surface led to the formation of a metal oxide layer that trapped dissolved P, as shown by the enrichment of P-bearing metal oxides in the top layer of the sediment and low phosphate in the pore and overlying water. After e-SOx activity declined, the metal oxide layer did not dissolve and P remained trapped at the surface. Overall, our results suggested cable bacteria can play an important role to counteract eutrophication in freshwater systems.

Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice.

Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1high CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.

A Luminol-Based Self-Illuminating Nanocage as a Reactive Oxygen Species Amplifier to Enhance Deep Tumor Penetration and Synergistic Therapy.

The dense extracellular matrix (ECM) in tumor tissues resists drug diffusion into tumors and leads to a poor prognosis. To address this problem, glucose oxidase (GOx)-modified ferritin loaded with luminol-curcumin was fabricated. Once delivered to the tumor, this luminol-based self-illuminating nanocage could actively convert glucose to reactive oxygen species (ROS) to achieve starvation therapy. Then, excessive ROS were transmitted to luminol, thereby emitting 425 nm blue-violet light. Momentarily, light was further absorbed by curcumin and ROS production was amplified. Abundant ROS helps break down the ECM network to penetrate deep into tumors. In addition, ROS produced after cell internalization can induce apoptosis of tumor cells by decreasing the mitochondrial membrane potential and can promote ferroptosis by consuming reduced glutathione. Effective penetration and multiple pathways inducing tumor cell death contributed to the efficient antitumor effect (tumor inhibition rate of GOx-modified ferritin loaded with luminol-curcumin: 71.73%). This study developed a glucose-driven self-illuminating nanocage for active tumor penetration via ROS-mediated destruction of the ECM and provided the synergetic mechanism of apoptosis and ferroptosis.

Activation of FGFR2 Signaling Suppresses BRCA1 and Drives Triple-Negative Mammary Tumorigenesis That is Sensitive to Immunotherapy.

Fibroblast growth factor receptor 2 (FGFR2) is a membrane-spanning tyrosine kinase that mediates FGF signaling. Various FGFR2 alterations are detected in breast cancer, yet it remains unclear if activation of FGFR2 signaling initiates tumor formation. In an attempt to answer this question, a mouse model berrying an activation mutation of FGFR2 (FGFR2-S252W) in the mammary gland is generated. It is found that FGF/FGFR2 signaling drives the development of triple-negative breast cancer accompanied by epithelial-mesenchymal transition that is regulated by FGFR2-STAT3 signaling. It is demonstrated that FGFR2 suppresses BRCA1 via the ERK-YY1 axis and promotes tumor progression. BRCA1 knockout in the mammary gland of the FGFR2-S252W mice significantly accelerated tumorigenesis. It is also shown that FGFR2 positively regulates PD-L1 and that a combination of FGFR2 inhibition and immune checkpoint blockade kills cancer cells. These data suggest that the mouse models mimic human breast cancers and can be used to identify actionable therapeutic targets.

Synergistic effect of tumor chemo-immunotherapy induced by leukocyte-hitchhiking thermal-sensitive micelles.

Some specific chemotherapeutic drugs are able to enhance tumor immunogenicity and facilitate antitumor immunity by inducing immunogenic cell death (ICD). However, tumor immunosuppression induced by the adenosine pathway hampers this effect. In this study, E-selectin-modified thermal-sensitive micelles are designed to co-deliver a chemotherapeutic drug (doxorubicin, DOX) and an A2A adenosine receptor antagonist (SCH 58261), which simultaneously exhibit chemo-immunotherapeutic effects when applied with microwave irradiation. After intravenous injection, the fabricated micelles effectively adhere to the surface of leukocytes in peripheral blood mediated by E-selectin, and thereby hitchhiking with leukocytes to achieve a higher accumulation at the tumor site. Further, local microwave irradiation is applied to induce hyperthermia and accelerates the release rate of drugs from micelles. Rapidly released DOX induces tumor ICD and elicits tumor-specific immunity, while SCH 58261 alleviates immunosuppression caused by the adenosine pathway, further enhancing DOX-induced antitumor immunity. In conclusion, this study presents a strategy to increase the tumor accumulation of drugs by hitchhiking with leukocytes, and the synergistic strategy of chemo-immunotherapy not only effectively arrested primary tumor growth, but also exhibited superior effects in terms of antimetastasis, antirecurrence and antirechallenge.

Molecular landscape and subtype-specific therapeutic response of nasopharyngeal carcinoma revealed by integrative pharmacogenomics.

Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer type with high morbidity in Southeast Asia, however the pathogenic mechanism of this disease is poorly understood. Using integrative pharmacogenomics, we find that NPC subtypes maintain distinct molecular features, drug responsiveness, and graded radiation sensitivity. The epithelial carcinoma (EC) subtype is characterized by activations of microtubule polymerization and defective mitotic spindle checkpoint related genes, whereas sarcomatoid carcinoma (SC) and mixed sarcomatoid-epithelial carcinoma (MSEC) subtypes exhibit enriched epithelial-mesenchymal transition (EMT) and invasion promoting genes, which are well correlated with their morphological features. Furthermore, patient-derived organoid (PDO)-based drug test identifies potential subtype-specific treatment regimens, in that SC and MSEC subtypes are sensitive to microtubule inhibitors, whereas EC subtype is more responsive to EGFR inhibitors, which is synergistically enhanced by combining with radiotherapy. Through combinational chemoradiotherapy (CRT) screening, effective CRT regimens are also suggested for patients showing less sensitivity to radiation. Altogether, our study provides an example of applying integrative pharmacogenomics to establish a personalized precision oncology for NPC subtype-guided therapies.

Fucoidan-based micelles as P-selectin targeted carriers for synergistic treatment of acute kidney injury.

Acute kidney injury (AKI) is a life-threatening disease without effective treatment. The utilization of curcumin (Cur) for the treatment of AKI is still facing challenges due to its poor water-solubility and low bioavailability. Herein, kidney-targeted octenyl succinic anhydride-grafted fucoidan loaded with Cur (OSA-Fucoidan/Cur) was fabricated for synergistic treatment of AKI. It was found that OSA-Fucoidan/Cur micelles had a sustained drug release behavior and excellent physicochemical stability. Cellular uptake studies demonstrated that the specific binding between fucoidan and P-selectin overexpressed on H2O2-stimulated HUVECs contributed to the higher internalization of OSA-Fucoidan/Cur micelles by the cells. In addition, OSA-Fucoidan micelles exhibited an ideal kidney-targeted characteristic in lipopolysaccharide (LPS)-induced AKI mice. In vivo studies showed that the combination of Cur and OSA-Fucoidan endowed the OSA-Fucoidan/Cur micelles with synergistically anti-inflammatory and antioxidant abilities, thereby largely enhancing the therapeutic efficacy of AKI. Therefore, OSA-Fucoidan/Cur micelles may represent a potential kidney-targeted nanomedicine for effective treatment of AKI.

Does Acupuncture Benefit Delayed-Onset Muscle Soreness After Strenuous Exercise? A Systematic Review and Meta-Analysis.

Purpose: This systematic review and meta-analysis was designed to evaluate the effects of acupuncture intervention on alleviating delayed onset of muscle soreness (DOMS) after intense exercise. Method: Randomized controlled trials (RCTs) were searched from online databases including Medline (PubMed), Cochrane Library, Web of Science, Embase, PsycINFO, China Knowledge Resource Integrated Database (CNKI), and Wanfang (Chinese) up to April 2019. Data points were extracted from the eligible RCTs at the time points of 24, 48, and 72 h post strenuous exercise-induced DOMS. The outcomes of muscle soreness rating (MSR), creatine kinase (CK), and maximal isometric force (MIF) were pooled into the meta-analysis to assess the acupuncture intervention on DOMS. Results: Six eligible RCTs were included in the meta-analysis, and the results showed that acupuncture intervention significantly decreased MSR [standardized mean difference (SMD) -0.49, 95%CI -0.73 to -0.24, P < 0.001, I 2 = 34%] and the serum level of CK (SMD -0.91, 95%CI -1.27 to -0.56, P < 0.001, I 2 = 30%), accompanied with the improvement of the muscle strength (MIF) (SMD 0.54, 95%CI 0.16 to 0.93, P = 0.006, I 2 = 51%) after intense exercise. At the same time, the findings also revealed that acupuncture intervention had a long-lasting effect and tended to accumulate the effect size and that it had the most efficacy on alleviating DOMS at the time point of 72 h post exercise. Conclusion: The current evidence indicates that acupuncture intervention after intense exercise could be effective for alleviating DOMS and improving muscle recovery. The long-lasting effect of acupuncture intervention on DOMS started from 24 h and would reach a peak on the time point of 72 h post exercise.

NIR-Triggered Sequentially Responsive Nanocarriers Amplified Cascade Synergistic Effect of Chemo-Photodynamic Therapy with Inspired Antitumor Immunity.

A desirable cancer therapeutic strategy is supposed to have effective ability to not only exert maximum anticancer ability but also inspire antitumor immunity for preventing tumor relapse and metastasis. During this research, multifunctional upconversion nanoparticles (UCNPs) coated by ROS-responsive micelles are prepared for tumor targeting and near-infrared (NIR)-triggered photodynamic therapy (PDT)-combined synergistic effect of chemotherapy. Moreover, both PDT and chemotherapy agents could activate antitumor immunity via inducing immunogenic cell death with CD8+ and CD4+ T cells infiltrating in tumors. Through the experiments, intravenous administration of multifunctional nanocarriers with noninvasive NIR irradiation destroys the orthotopic tumors and efficiently suppresses lung metastasis in a metastatic triple-negative breast cancer model by cascade-amplifying chemo-PDT and systemic antitumor immunity. In conclusion, this study provides prospective chemo-PDT with inspired antitumor immunity for metastatic cancer treatment.

pH and Thermal Dual-Sensitive Nanoparticle-Mediated Synergistic Antitumor Effect of Immunotherapy and Microwave Thermotherapy.

Cationic anticancer peptides, which can induce tumor cell immunogenic death and further promote systemic tumor-specific immune responses, have offered a promising solution to relieve the tumor immunosuppressive microenvironment. However, peptide drugs are easily degraded and lack of targeting ability when administered systemically, leading to limitations in their applications. Herein, we report a pH and thermal dual-sensitive bovine lactoferricin-loaded (one of the most widely studied cationic anticancer peptides) nanoparticles, which simultaneously exhibited antitumor and immune cell activated effects when applied with microwave thermotherapy, an auxiliary method of immunotherapy. The bovine lactoferricin could be delivered to the tumor site by nanoparticles, be immediately released from nanoparticles in the acidic environment of lysosomes and the thermal condition caused by microwave radiation, and ultimately induce tumor apoptosis with the release of damage-associated molecular patterns (DAMPs). It is worth noting that the strategy of bovine lactoferricin-loaded nanoparticles intravenous injection combined with local microwave thermotherapy not only showed excellent efficacy in relieving tumor growth but also resulted in strong antitumor immunities, which was due to the released bovine lactoferricin under stimulating conditions, and the pool of tumor-associated antigens generated by tumor destruction. In conclusion, this work presents a strategy for tumor treatment based on dual-sensitive bovine lactoferricin-loaded nanoparticles combined with microwave thermotherapy, which may provide a solution for cationic anticancer peptides delivery and improving antitumor immune responses.

Sialic Acid-Functionalized PEG-PLGA Microspheres Loading Mitochondrial-Targeting-Modified Curcumin for Acute Lung Injury Therapy.

Acute lung injury (ALI) is a serious illness without resultful therapeutic methods commonly. Recent studies indicate the importance of oxidative stress in the occurrence and development of ALI, and mitochondria targeted antioxidant has become a difficult and hot topic in the research of ALI. Therefore, a sialic acid (SA)-modified lung-targeted microsphere (MS) for ALI therapy are developed, with triphenylphosphonium cation (TPP)-modified curcumin (Cur-TPP) loaded, which could specifically target the mitochondria, increasing the effect of antioxidant. The results manifest that with the increase of microsphere, lung distribution of microsphere is also increased in murine mice, and after SA modification, the microsphere exhibits the ideal lung-targeted characteristic in ALI model mice, due to SA efficiently targeting to E-selectin expressed on inflammatory tissues. Further investigations indicate that SA/Cur-TPP/MS has better antioxidative capacity, decreases intracellular ROS generation, and increases mitochondrial membrane potential, contributing to a lower apoptosis rate in human umbilical vein endothelial cells (HUVECs) compared to H2O2 group. In vivo efficacy of SA/Cur-TPP/MS demonstrates that the inflammation has been alleviated markedly and the oxidative stress is ameliorated efficiently. Significant histological improvements by SA/Cur-TPP/MS are further proved via HE stains. In conclusion, SA/Cur-TPP/MS might act as a promising drug formulation for ALI therapy.

The Zn2Cys6-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in Aspergillus fumigatus.

Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.

Mild microwave activated, chemo-thermal combinational tumor therapy based on a targeted, thermal-sensitive and magnetic micelle.

The development of combinational anti-tumor therapy is of great value. Here, the thermal-sensitive and hepatic tumor cell targeting peptide-A54 modified polymer, A54-poly(ethylene glycol)-g-poly(acrylamide-co-acrylonitrile) (A54-PEG-g-p(AAm-co-AN)) can self-assemble into an 80 nm-sized micelle, which shows a thermal-sensitive behavior with an upper critical solution temperature (UCST) of 43 °C. This self-assembled and targeted A54-PEG-g-p(AAm-co-AN) micelle can co-encapsulate anti-tumor drug doxorubicin (DOX) and magnetic nanoparticles (MNPs) taking advantage of the hydrophobic core of the core-shell micellar structure, when the temperature is lower than 43 °C. A much higher accumulation of the MNPs@A54-PEG-g-p(AAm-co-AN) to the tumor navigated by the A54 targeting peptide is achieved. Due to the thermal-agent effect of the accumulated MNPs in tumor, the mild microwave (8 W) applied afterwards specifically elevates the local tumor temperature by 13 °C, compared to 6 °C without MNPs accumulation in 30 min. The greater temperature rise resulted from the thermal-agent effect of MNPs doesn't only activate the drug release inside tumor cells, but also achieve an augmented hyperthermia. A mild microwave activated, chemo-thermal combinational tumor therapy is thus developed.

Crystal Structure of Feline Infectious Peritonitis Virus Main Protease in Complex with Synergetic Dual Inhibitors.

UNLABELLED: Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (M(pro)s) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV M(pro) in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases. IMPORTANCE: Coronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (M(pro)) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. M(pro), conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV M(pro). We also solved the structure of FIPV M(pro) complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV M(pro) through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible peptidomimetic inhibitor.

Attitudes toward the American nutrition guidelines for the critically ill patients of Chinese intensive care physicians.

Nutrition therapy is essential for the management of critically ill patients. Some guidelines have been published to standardize and optimize the nutrition therapy. However, there are still many controversies in nutrition practice and there is a gap between guidelines and clinical nutrition therapy for patients in intensive care units (ICUs). This study aimed to assess attitudes and beliefs toward nutrition therapy of Chinese intensive care physicians by using the American guidelines as a surrogate. A questionnaire was sent to 45 adult ICUs in China, in which surveyed physicians were asked to rate their attitudes toward the American guidelines. A total of 162 physicians from 45 ICUs returned the questionnaires. Physicians were categorized into groups according to their professional seniority, hospital levels and whether they were members of Chinese Society for Parenteral and Enteral Nutrition (CSPEN). Overall, 94% of the respondents thought that nutrition therapy for critically ill patients was very important, and 80% mentioned that they used the American guidelines. There was diversity of opinion on the recommendations pertaining to nutrition assessment, supplemental parenteral nutrition and cutoff values for gastric residual volume, negative or neutral attitudes about these recommendations were 43%, 59% and 41%, respectively. Members of CSPEN were more likely to select a greater strength of recommendation than non-members. In conclusion, the overall attitudes of Chinese intensive care physicians toward the American guidelines were positive. Nevertheless, given the great guideline-practice gap, nutrition-focused education is warranted for many intensive care physicians in China.

tRNA synthetase counteracts c-Myc to develop functional vasculature.

Recent studies suggested an essential role for seryl-tRNA synthetase (SerRS) in vascular development. This role is specific to SerRS among all tRNA synthetases and is independent of its well-known aminoacylation function in protein synthesis. A unique nucleus-directing domain, added at the invertebrate-to-vertebrate transition, confers this novel non-translational activity of SerRS. Previous studies showed that SerRS, in some unknown way, controls VEGFA expression to prevent vascular over-expansion. Using in vitro, cell and animal experiments, we show here that SerRS intervenes by antagonizing c-Myc, the major transcription factor promoting VEGFA expression, through a tandem mechanism. First, by direct head-to-head competition, nuclear-localized SerRS blocks c-Myc from binding to the VEGFA promoter. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation. Thus, vertebrate SerRS and c-Myc is a pair of 'Yin-Yang' transcriptional regulator for proper development of a functional vasculature. Our results also discover an anti-angiogenic activity for SIRT2.DOI: http://dx.doi.org/10.7554/eLife.02349.001.

Quantitative evaluation of the in vitro effect and interactions of active fractions in Yaotongning-based formulae on prostaglandin E2 production.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) formula of Yaotongning Capsules (YTNC) is a common remedy to treat rheumatism (RA) in China and possesses diverse biological activities including anti-inflammation. However the effects of component material medicines (CMMs) in YTNC and different combinations of the CMMs on the efficacy of YTNC and the interactions of these CMMs have been being unclear due to ten CMMs and too many compounds involved in YTNC. Moreover, many TCM formulae are available for treating RA according to TCM theory. It is unknown if the YTNC prescription is better than other TCM formulae for treating RA or better efficacy could be obtained when some CMMs in YTNC are replaced by other herbs. Quantitatively investigate the in vitro effect of active fractions from the CMMs of YTNC and other eight herbs commonly used in the TCM formulae for RA treatment on anti-inflammatory activity of different combinations of the active fractions, the interactions of the active fractions to evaluate the reasonability, advantage (or disadvantage) of the YTNC prescription and to see if the prescription could be improved from the point of anti-inflammation. MATERIALS AND METHODS: Twenty-six active fractions, which were categorized as alkaloids, flavonoids, saponins, volatile oils/aqueous extracts and polysaccharides were prepared to design TCM samples by combining some of the active fractions, based on the YTNC formulating principle, combination chemistry concept and the importance of the active fractions in YTNC. The anti-inflammatory activities of the samples were evaluated by their half-maximal inhibitory concentration (IC50) values that inhibiting the production of prostaglandin E2 (PGE2) in ANA-1 murine macrophages (ANA-1 cells). The cells plated in 96-well plates were classified into blank group and test sample group. Each group was stimulated with lipopolysaccharides (LPS, 1 mg/mL) for 2h. ANA-1 cells were pretreated with different concentrations of test samples prior to the addition of arachidonic acid (10 µmol/L). The supernatants were collected and measured using PGE2 ELISA Kit, and the cytotoxicity was assayed by cell counting Kit-8 (CCK8)-based test. The interactions of the active fractions in YTNC were evaluated by comparing the experimental IC50 values of the samples derived from YTNC to their corresponding additive IC50 values. The effect of each active fraction on cellular anti-inflammation-PGE2 secretion inhibition activity, and the reasonability, advantages (or disadvantages) of YTNC were evaluated based on the comparison of IC50 values of the samples. RESULTS: The disassembled formulae consisted of some active fractions of YTNC and the whole prescription of YTNC consisted of the all active fractions from YTNC all demonstrate cellular anti-inflammatory activity, and there were no significant differences between these formulae. The vehicle of YTNC Chinese rice wine exhibits the ability to enhance the cellular anti-inflammation of YTNC. Synergistic effect exerts in the combination of alkaloids, flavonoids and saponins of YTNC, antagonistic or additive effects occur in the other combinations of active fractions from YTNC. The anti-inflammatory activities of some TCM samples which include some active fractions from the eight selected herbs are significantly higher than the samples derived from YTNC. The flavonoids of Carthamus tinctorius, the volatile oils of Cinnamomum cassia and Angelica pubescens perform better in cellular anti-inflammation than the flavonoids and volatile oils in YTNC. CONCLUSIONS: The prescription of YTNC is reasonable in the view of anti-inflammation. The saponins and polysaccharides from the CMMs of YTNC have better anti-inflammatory activities than the saponins and polysaccharides from the other eight herbs. Reducing the varieties of YTNC CMMs and replacing the flavonoids and volatile oils of YTNC with the flavonoids of Carthamus tinctorius and the volatile oils of Cinnamomum cassia (or Angelica pubescens) would improve the safety and anti-inflammatory activity of YTNC. Synthetically evaluating various pharmacological activities of TCM formulae designed in the present work may lead to develop more effective and safer TCM using YTNC as prototypes.

Quantitative transcript profiling reveals down-regulation of A sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants.

To investigate the dynamic fluctuation of terpenoid relevant transcriptomics in transgenic ARTEMISIA ANNUA plants that express the genomic integrated antisense squalene synthase gene ( ASSS), we have quantified the transcript levels of the sterol anabolic SS gene as well as artemisinin biogenetic amorphadiene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 reductase (CPR) genes by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). The SS mRNA level in transgenic plants sharply droped to 7.4 % - 55.3 % (i. e., 44.7 - 92.6 % reduction as the wild-type control), strongly implying that the expression of endogenous SS gene is significantly suppressed by the exogenous ASSS gene. In a synchronous fashion, ADS, CYP71AV1 and CPR mRNA levels elevated with the decline of SS mRNA level in transgenic plants, and the maximal ADS, CYP71AV1 and CPR mRNA levels in transgenic plants were 3.0-, 4.4- and 2.5-fold, respectively, higher than those in the control. Without a lethal effect but with a distinguishable impact on the organogenesis and morphology of transgenic plants, the down-regulation of SS gene has also led to the coordinated overexpression of ADS, CYP71AV1 and CPR genes together with the overproduction of artemisinin although no fully perfect correlation among the available experimental data has been shown.