N-Heterocyclic Carbene Enabled Functionalization of Inert C(Sp3)-H Bonds via Hydrogen Atom Transfer (HAT) Processes.

Developing methods to directly transform C(sp3) -H bonds is crucial in synthetic chemistry due to their prevalence in various organic compounds. While conventional protocols have largely relied on transition metal catalysis, recent advancements in organocatalysis, particularly with radical NHC catalysis have sparked interest in the direct functionalization of "inert" C(sp3) -H bonds for cross C-C coupling with carbonyl moieties. This strategy involves selective cleavage of C(sp3) -H bonds to generate key carbon radicals, often achieved via hydrogen atom transfer (HAT) processes. By leveraging the bond dissociation energy (BDE) and polarity effects, HAT enables the rapid functionalization of diverse C(sp3)-H substrates, such as ethers, amines, and alkanes. This mini-review summarizes the progress in carbene organocatalytic functionalization of inert C(sp3)-H bonds enabled by HAT processes, categorizing them into two sections: 1) C-H functionalization involving acyl azolium intermediates; and 2) functionalization of C-H bonds via reductive Breslow intermediates.

Analysis of prognostic biomarker models of TXNIP/NLRP3/IL1B inflammasome pathway in patients with acute myeloid leukemia.

Background: Exploring potential biomarkers for predicting clinical outcomes and developing targeted therapies for acute myeloid leukemia (AML) is of utmost importance. This study aimed to investigate the expression pattern of the thioredoxin-interacting protein (TXNIP)/nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) pathway and its role in the prognosis of AML patients. Methods: In this study, we examined the prognostic value of TXNIP/NLRP3 pathway in AML patients using microarray data from Gene Expression Omnibus (GEO) and transcriptome data from the Cancer Genome Atlas (TCGA) to develop a prognostic model and validated the results by quantitative real-time PCR (qRT-PCR) in a validation cohort of 26 AML patients and 18 healthy individuals from Jinan University (JNU) database. Results: Analysis of the GSE13159 database revealed that TXNIP, interleukin 1 beta (IL1B) within the TXNIP/NLRP3 pathway were significantly upregulated and caspase1 (CASP1) was downregulated in AML patients (TXNIP, P = 0.031; IL1B, P = 0.042; CASP1, P = 0.038). Compared to high NLRP3 expression, AML patients with low NLRP3 expression had a longer overall survival (OS) in the GSE12417 dataset (P = 0.004). Moreover, both the training and validation results indicated that lower TXNIP, NLRP3, and IL1B expression were associated with favorable prognosis (GSE12417, P = 0.009; TCGA, P = 0.050; JNU, P = 0.026). According to the receiver operating characteristic curve analysis, this model demonstrated a sensitivity of 84% for predicting three-year survival. These data might provide novel predictors for AML outcome and direction for further investigation of the possibility of using TXNIP/NLRP3/IL1B genes in novel targeted therapies for AML.

Nontargeted metabolomics integrated with 1 H NMR and LC-Q-TOF-MS/MS methods to depict a more comprehensive metabolic profile in response to chrysosplenetin and artemisinin co-treatment against artemisinin-sensitive and -resistant Plasmodium berghei K173.

Our previous work revealed mutual and specific metabolites/pathways in artemisinin-sensitive and -resistant Plasmodium berghei K173-infected mice. In this study, we further investigated whether chrysosplenetin, a candidate chemical to prevent artemisinin resistance, can regulate these metabolites/pathways by integrating nontargeted metabolomics with 1 H NMR and LC-Q-TOF-MS/MS spectrum. The nuclear magnetic resonance method generated specifically altered metabolites in response to co-treatment with chrysosplenetin, including: the products of glycolysis such as glucose, pyruvate, lactate and alanine; taurine, closely associated with liver injury; arginine and proline as essential amino acids for parasites; TMAO, a biomarker for dysbacteriosis and renal function; and tyrosine, which is used to generate levodopa and dopamine and may improve the torpor state of mice. Importantly, we noticed that chrysosplenetin might depress the activated glycolysis induced by sensitive parasites, but oppositely promoted the inhibited glycolysis to generate more lactate, which suppresses the proliferation of resistant parasites. Moreover, chrysosplentin possibly disturbs the heme biosynthetic pathway in mitochondria. The MS method yielded changed coenzyme A, phosphatidylcholine and ceramides, closely related to mitochondria ß-oxidation, cell proliferation, differentiation and apoptosis. These two means shared no overlapped metabolites and formed a more broader metabolic map to study the potential mechanisms of chrysosplenetin as a promising artemisinin resistance inhibitor.

Antimalarial activity and sensitization of chrysosplenetin against artemisinin-resistant genotype Plasmodium berghei K173 potentially via dual-mechanism of maintaining host P-glycoprotein homeostasis mediated by NF-κB p52 or PXR/CAR signaling pathways and regulating heme/haemozoin metabolism.

This study investigated antimalarial efficacy and sensitization of chrysosplenetin against artemisinin-resistant Plasmodium berghei K173 and potential molecular mechanism. Our data indicated a risk of artemisinin resistance because a higher parasitaemia% and lower inhibition% under artemisinin treatment against resistant parasites than those in the sensitive groups were observed. Two non-antimalarial components, verapamil and chrysosplentin, being P-gp inhibitors, possessed a strong efficacy against resistant parasites but it was not the case for Bcrp inhibitor novobiocin. Artemisinin-chrysosplenetin combination improved artemisinin susceptibility of resistant P. berghei. Artemisinin activated intestinal P-gp and Abcb1/Abcg2 expressions and suppressed Bcrp whereas chrysosplenetin reversed them. Resistant parasite infection led to a decreased haemozoin in organs or an increased heme in peripheral bloods compared with the sensitives; however, that in Abcb1-deficient knockout (KO)-resistant mice reversely got increased or decreased versus wild type (WT)-resistant animals. Chrysosplenetin as well as rifampin (nuclear receptor agonist) increased the transcription levels of PXR/CAR while showed a versatile regulation on hepatic and enternal PXR/CAR in WT- or KO-sensitive or -resistant parasites. Oppositely, hepatic and enteric NF-κB p52 mRNA decreased conformably in WT but increased in KO-resistant mice. NF-κB pathway potentially involved in the mechanism of chrysosplenetin on inhibiting P-gp expressions while PXR/CAR play a more complicated role in this mechanism.

Secondary Metabolite Analysis of Phomopsis sp. LGT-5 Based on the Dual Guidance of Whole-Genome Sequencing and HPLC-Q-ToF-MS/MS.

Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities. The Phomopsis sp. LGT-5 was obtained through tissue block and repeatedly crossed methods from Tripterygium wilfordii Hook. F. The antibacterial experiments of LGT-5 showed that it has high inhibitory activity against Staphylococcus aureus and Pseudomonas aeruginosa, and moderate inhibitory activity against Candida albicans. To research the generation of the antibacterial phenomenon of LGT-5 and provide support for further research and application, the whole genome sequencing (WGS) of LGT-5 was obtained by single-molecule real-time DNA sequencing platform Pacific Biosciences (PacBio) sequencing and Illumina paired-end sequencing. The final assembled LGT-5 genome is 54.79 Mb with a contig N50 of 290.07 kb; in addition, its secondary metabolites were detected through HPLC-Q-ToF-MS/MS. By comparing its MS/MS data, the secondary metabolites were analyzed based on visual network maps obtained on the Global Natural Products Social Molecular Networking (GNPS). The analysis results showed that the secondary metabolites of LGT-5 were triterpenes and various cyclic dipeptides.

Higher TIGIT+CD226- γδ T cells in Patients with Acute Myeloid Leukemia.

The diverse structural and functional heterogeneity of γδ T cells is related to their distinct role in cancer immunity. The different phenotypes of γδ T cells in patients with acute myeloid leukemia (AML) is far from clear. In particular, the expression pattern of co-inhibitory and co-stimulatory receptors on γδ T cells remains unknown. In this study, we analyzed the distribution of γδ T cell subsets by expression of the immune checkpoint co-inhibitor TIGIT (T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain) and its competing co-stimulatory receptor CD226 in AML patients of different clinical statuses (including de novo AML, AML in non-remission (NR), and AML in complete remission (CR)). Our data demonstrated an imbalanced distribution of TIGIT and CD226 on γδ T cells with a decrease in CD226+ γδ T cells and an increase in TIGIT+ γδ T cells in de novo AML patients, while TIGIT-CD226+ γδ T cells were restored in AML patients who achieved CR after chemotherapy. Moreover, the patients who had higher TIGIT+CD226- γδ T cells showed lower overall survival rate for non-M3 AML, which may be considered a novel prognostic immune biomarker. In conclusion, our study reveals for the first time that imbalance in the TIGIT/CD226 axis might be related to different clinical outcomes for AML patients.Abbreviations: AML: acute myeloid leukemia; CR: complete remission; ICs: immune checkpoints; PD-1: programmed death-1; γδ T cells: gamma delta T cells; TCR: T cell receptor; MHC: major histocompatibility complex; TIGIT: T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain; NK: natural killer; PB: Peripheral blood; NR: non-remission; FAB: French-American-British; WHO: World Health Organization; HIs: healthy individuals; OS: overall survival.

Design, synthesis and biological evaluation of a series of novel pyrrolo[2,3-d]pyrimidin/pyrazolo[3,4-d]pyrimidin-4-amine derivatives as FGFRs-dominant multi-target receptor tyrosine kinase inhibitors for the treatment of gastric cancer.

Gastric cancer is the second most lethal cancer across the world. With the progress in therapeutic approaches, the 5-year survival rate of early gastric cancer can reach > 95%. However, the prognosis and survival time of advanced gastric cancer is still somber. Therefore, more effective targeted therapies for gastric cancer treatment are urgently needed. FGFR, VEGFR and other receptor tyrosine kinases have recently been suggested as potential targets for gastric cancer treatment. We herein report the discovery of pyrrolo[2,3-d]pyrimidin/pyrazolo[3,4-d]pyrimidin-4-amine derivatives as a new class of FGFRs-dominant multi-target receptor tyrosine kinase inhibitors. SAR assessment identified the most active compounds 8f and 8k, which showed excellent inhibitory activity against a variety of receptor tyrosine kinases. Moreover, 8f and 8k displayed excellent potency in the SNU-16 gastric cancer cell line. Furthermore, 8f and 8k could inhibit FGFR1 phosphorylation and downstream signaling pathways as well as induce cell apoptosis. In vivo, 8f and 8k suppress tumor growth in the SNU-16 xenograft model without inducing obvious toxicity. These findings raise the possibility that compounds 8f and 8k might serve as potential agents for the treatment of gastric cancer.

Classification of diterpenoid alkaloids from Aconitum kusnezoffii Reichb. by liquid chromatography-tandem mass spectrometry-based on molecular networking.

Trace amounts of components in traditional Chinese medicine are considered pharmacological active substances used for treating many serious diseases. However, purifying all the trace substances and making clear their structures are not easy. In this context, high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry based molecular networking was applied to investigate the chemical constituents of the roots of Aconitum kusnezoffii Reichb., which led to the identification of 33 nodes in different groups (N1-N33). Based on the excremental fragmentation pathway of known diterpenoid alkaloids (1-9) and comparisons of characteristic ions and characteristic loss of analogs in literature, the structures of unknown ions were deduced. This work lays a foundation for the evaluation of the clinical basis and mechanism of traditional Chinese medicine from the aspects of chemistry. In this paper, the method speculation of unknown natural products by means of molecular network method is expected to be applied in the discovery and change law of relevant active components in clinical pharmacology and the change of complex systems caused by trace active compounds.

Screening for proteins related to the biosynthesis of hispidin and its derivatives in Phellinus igniarius using iTRAQ proteomic analysis.

BACKGROUND: Hispidin (HIP) and its derivatives, a class of natural fungal metabolites, possess complex chemical structures with extensive pharmacological activities. Phellinus igniarius, the most common source of HIP, can be used as both medicine and food. However, the biosynthetic pathway of HIP in P. igniarius remains unclear and we have a limited understanding of the regulatory mechanisms related to HIP. In this work, we sought to illustrate a biosynthesis system for hispidin and its derivatives at the protein level. RESULTS: We found that tricetolatone (TL) is a key biosynthetic precursor in the biosynthetic pathway of hispidin and that its addition led to increased production of hispidin and various hispidin derivatives. Based on the changes in the concentrations of precursors and intermediates, key timepoints in the biosynthetic process were identified. We used isobaric tags for relative and absolute quantification (iTRAQ) to study dynamic changes of related proteins in vitro. The 270 differentially expressed proteins were determined by GO enrichment analysis to be primarily related to energy metabolism, oxidative phosphorylation, and environmental stress responses after TL supplementation. The differentially expressed proteins were related to ATP synthase, NAD binding protein, oxidoreductase, and other elements associated with electron transfer and dehydrogenation reactions during the biosynthesis of hispidin and its derivatives. Multiple reaction monitoring (MRM) technology was used to selectively verify the iTRAQ results, leading us to screen 11 proteins that were predicted to be related to the biosynthesis pathways. CONCLUTION: These findings help to clarify the molecular mechanism of biosynthesis of hispidin and its derivatives and may serve as a foundation for future strategies to identify new hispidin derivatives.

[A new 9,19-cycloartane glycoside from Asplenium ruprechtii].

Chemical constituents of water extracts of Asplenium ruprechtii were investigated. Five compounds were isolated by silica gel, Sephadex LH-20 gel column chromatographies and preparative HPLC, and their structures were identified by various spectral analyses as aspleniumside G(1), trans-p-coumaric acid(2), trans-p-coumaric acid 4-O-ß-D-glucoside(3), cis-p-coumaric acid 4-O-ß-D-glucoside(4), and(E)-ferulic acid-4-O-ß-D-glucoside(5). Among them, compound 1 is a new 9,19-cycloartane glycoside.

Structural determination and in vitro tumor cytotoxicity evaluation of five new cycloartane glycosides from Asplenium ruprechtii Sa. Kurata.

Five new cycloartane glycosides, named aspleniumside A - E, were discovered and characterized by re-investigated the remaining extracts of the whole plant of Asplenium ruprechtii Sa. Kurata, a famous folk medicine for treating thromboangitis obliterans in China, Japan, and Korea. Compounds 3-5 possessed the 9,19-seco-cycloartane-9,11-en triterpene aglycone with 3,7(or 23),24,25,30-highly oxidized methylene, methylene or quaternary carbons, that was found in this species for the first time. The stereo-chemistry of all new compounds were fully discussed by extensive analysis of the 1D and 2D NMR data, and comparisons with those data of known compounds. 24R configuration was determined here which indicated the different growing areas of the same species could influence the secondary metabolic behavior, leading to the differences in chemical composition. All glycoside groups were determined as ß-d-glucopyranosyl by 1H coupling constant of anomeric protons and co-TLC of the acid hydrolysate with d-glucose. All the cycloartane glycosides were evaluated against HL-60 and HepG2 cells for cytotoxicity, compounds 1-3, showed potential cytotoxicity with the IC50 in range of 18-60 µM, while the standard sorafenib showed IC50 value of 10.61 ± 0.43 and 13.43 ± 1.12 µM against HL-60 and HepG2, respectively. The results attained in this study indicated that cycloartane glycosides should be the cytotoxicity substance in A. ruprechtii Sa. Kurata, and had the potential to be developed as tumor cytotoxicity agent applied in clinic.

Structural Determination of a New Cycloartane Glycoside from Asplenium ruprechtii.

We characterized a new cycloartane glycoside, herein known as aspleniumside F (1), along with five known compounds as kaempferol-3-O-[(6-O-(E)-feruloyl)-ß-D-glucopyranosyl]-(1→2)-ß-D-galacopyranoside (2), quercetin-3-O-[(6-O-(E)-feruloyl)-ß-D-glucopyranosyl]-(1→2)-ß-D-glucopyranoside (3), kaempferol-3-O-[(6-O-(E)-caffeoyl)-ß-D-glucopyranosyl]-(1→2)-ß-D-glucopyranoside (4), kaempferol-3-O-[(6-O-(E)-caffeoyl)-ß-D-glucopyranosyl]-(1→2)-ß-D-glucopyranosyl-7-O-ß-D-glucopyranoside (5), and kaempferol-3-O-[(6-O-p-coumaroyl)-ß-D-glucopyranosyl]-(1→2)-ß-D-glucopyranosyl-7-O-ß-D-glucopyranoside (6), from Asplenium ruprechtii Sa. Kurata, a folk medicine widely used to treat Thromboangiitis obliterans in China, Japan, and Korea. Based on spectroscopic, mainly 1D-, 2D-NMR and (+)-HR-ESI-MS, analyses as well as through comparisons with previous reports, its chemical structure was determined as 3ß,24,30-tri-ß-D-glucopyranosyl-23,25-dihydroxycycloartane (= (23R,24R)-3ß,24-bis-(ß-D-glucopyranosyloxy)-23,25-dihydroxy-9ß-9,19-cyclolanostan-29-yl ß-D-glucopyranoside). According to the 1 H coupling constant of anomeric protons and co-TLC of the acid hydrolysate with D-glucose, all three glycoside groups in 1 were revealed as ß-D-glucopyranosyl. Furthermore, SOD-like antioxidant activity evaluation via IC50 of 12.43, 6.78, 9.12, 6.94 and 4.85 µM revealed that compounds 2-6 had bioactivity.

Disordered Metabolic Profiling in Plasma and Tissues of Mice Infected with Artemisinin-Sensitive and -Resistant Plasmodium berghei K173 Determined by 1H NMR Spectroscopy.

Artemisinin resistance has inevitably emerged in several malaria-endemic areas and led to an incremental clinical failure rate for artemisinin-based combination therapy (ACT), which is strongly recommended by the World Health Organization (WHO). Genetically resilient malaria parasites have evolved antimalarial drug-evasion mechanisms; meanwhile, the metabolic cross-talk between the malaria parasites and the host is of significance during the invasion. The intention of this work, therefore, is to propose a feasible method to discover the systematic metabolic phenotypes of mice invaded with artemisinin-sensitive or -resistant Plasmodium berghei K173 when compared with healthy mice. Biological samples, including plasma, liver, spleen, and kidney, of mice collected after euthanasia at day 7 were subjected to 1H nuclear magnetic resonance spectroscopy. Multivariable data analysis was utilized to estimate the metabolic characteristics of these samples from uninfected and infected mice. In contrast with healthy mice, both sensitive and resistant malaria-parasite-infected models displayed distinct metabolic profiles. Parasite invasion significantly changed the glycolysis, Kreb's cycle, and amino acid metabolism in plasma and tissues. Decreased N, N-dimethylglycine and glycine levels in plasma from the artemisinin-sensitive P. berghei-infected group and increased lactate, lipid, and aspartate in the artemisinin-resistant P. berghei-infected group were observed, respectively. In the liver, the artemisinin-sensitive group up-regulated the glutamate level and down-regulated glutamine. Artemisinin-resistant parasite exposure decreased ethanol and allantoin levels. The levels of myo-inositol and valine in the spleen were increased due to artemisinin-sensitive P. berghei infection, together with decreased trimethylamine N-oxide, phosphocholine, ß-glucose, and acetoacetic acid. In the artemisinin-resistant group, the spleen showed a remarkably increased phosphocholine content along with decreased dimethylglycine and arginine levels. In the kidney, artemisinin-sensitive P. berghei K173 caused increased lysine, glutamate, creatine, and 2-hydroxybutyrate as well as decreased ethanol. Artemisinin-resistant P. berghei led to low glycerophosphorylcholine and high acetate, betaine, and hypoxanthine. Mutual and specific altered metabolites and, accordingly, metabolic pathways induced by the infection of artemisinin-sensitive or -resistant P. berghei were therefore screened out. This should be considered a preliminary study to establish a direct relationship with the host metabolic background and artemisinin resistance.

Gene expression pattern of TCR repertoire and alteration expression of IL-17A gene of γδ T cells in patients with acute myocardial infarction.

BACKGROUND: γδ T cells are associated with the pathogenesis of coronary atherosclerotic heart disease, but the relationship between the development of acute myocardial infarction (AMI) and γδ T cells is not clear. So we attempt to investigate the expression pattern and clonality of T cell receptor (TCR) repertoire of γδ T cells in AMI patients, analyze the expression levels of regulatory genes Foxp3 and IL-17A, and characterize the correlation between γδ T cells and the pathogenesis of AMI. METHODS: 25 patients diagnosed with ST-segment-elevation AMI were enrolled and 14 healthy individuals were recruited as the controls. RT-PCR and GeneScan were used to analyze the complementarity-determining region 3 sizes of TCR γδ repertoire genes in sorted γδ T cells from peripheral blood mononuclear cells (PBMCs). RQ-PCR was used to detect the gene expression levels of Foxp3, IL-17A and TCR Vγ subfamilies in sorted γδ T cells. All the patients were followed up for recordings of clinical endpoints. RESULTS: The mRNA gene expression levels of TCR Vγ1, Vγ2, and Vγ3 subfamilies in AMI patients were significantly higher than those in healthy controls. The expression pattern was Vγ1 > Vγ2 > Vγ3 in AMI patients, while Vγ1 > Vγ3 > Vγ2 in healthy controls. The significantly restricted expression of TCR Vδ subfamilies were also found in AMI patients. The expression frequencies of TCR Vδ7 and TCR Vδ6 in AMI patients were significantly lower than those in healthy controls. The high clonal expansion frequencies of the TCR Vδ8, Vδ4 and Vδ3 were determined in AMI patients. High expression of Foxp3 gene was found in AMI PBMCs, while high expression of IL-17A was found in AMI γδ+ cells. CONCLUSIONS: Restrictive expression of TCR γδ repertoire and alteration expression of IL-17A gene are the important characteristics of γδ T cells in AMI patients, which might be related to the immune response and clinical outcome. γδ T cells might play a key role in the pathological progress of AMI and associated with the IL-17A mediated pathway.

Regulatory γδ T cells induced by G-CSF participate in acute graft-versus-host disease regulation in G-CSF-mobilized allogeneic peripheral blood stem cell transplantation.

BACKGROUND: The immunomodulatory effects of granulocyte colony-stimulating factor (G-CSF) on T cells result in a low incidence of acute graft-versus-host disease (aGVHD) in G-CSF-mobilized allogeneic peripheral blood stem cell transplantation (G-PBSCT). However, the exact mechanism remains unclear. Regulatory γδ T cells (γδTregs), characterized by the presence of TCRγδ and Foxp3, have aroused great concern in the maintenance of immune tolerance. We hypothesized that γδTregs might involve in the immunomodulatory effects of G-CSF mobilization. METHODS: The expression and immunomodulatory function of γδTreg subsets in peripheral blood of donors before and after G-CSF treatment in vivo and in vitro were evaluated by flow cytometry and CFSE assays. To investigate the effects of γδTregs on aGVHD, the association between γδTreg subsets in grafts and aGVHD in recipients was estimated. RESULTS: The proportions of Vδ1Tregs, CD27+Vδ1Tregs and CD25+Vδ1Tregs were significantly increased in peripheral blood after G-CSF treatment in vivo. γδTregs could be generated in vitro by stimulating with anti-TCRγδ in the presence of G-CSF. The immune phenotype, proliferation suppression function, and cytokine secretion of G-CSF-induced γδTregs were similar to that of transforming growth factor-ß (TGF-ß)-induced γδTregs. The clinical data demonstrated that the proportion of CD27+Vδ1Tregs in grafts was significantly lower in the patients who experienced aGVHD than in those who did not develop aGVHD (P = 0.028), and the proportions of other γδTreg subsets in grafts did not differ significantly between the two groups. The best cutoff value for CD27+Vδ1Treg proportion in grafts in prediction of aGVHD was 0.33%, with an area under the curve value of 0.725 (P = 0.043). Eight patients (26.7%) were classified as the low-CD27+Vδ1Treg group (< 0.33%), and 22 patients (73.3%) as the high-CD27+Vδ1Treg group (≥ 0.33%). The incidence of aGVHD was higher in the low-CD27+Vδ1Treg group than in the high-CD27+Vδ1Treg group (75.0% versus 22.7%, P = 0.028). CONCLUSIONS: G-CSF could induce the generation of γδTregs in vivo and in vitro, and γδTregs might participate in aGVHD regulation in G-PBSCT.

[Basic Research of the Adenovirus-mediated hCTR1 Transfection on the Treatment of Cisplatin Resistant Cervical Cancer].

OBJECTIVE: To induce cisplatin-resistant cervical squamous carcinoma cell line and investigate the drug resistant mechanisms and adenovirus trans-gene therapeutical treatment. METHODS: The cisplatin-resistant subline,designated C-33A/cis,was originated by growing parental C-33A cells with gradually increasing doses of cisplatin. The cytotoxicity of cisplatin was determined by CCK-8 assay,and the CTR1 expression was measured by Western blot. Subcutaneous xenograft cervical tumor model was established by cisplatin-resistant C-33A/cis cell line. Recombinant adenovirus ad-hCTR1 was transfected into tumor by intratumoral injection and combined with cisplatin chemotherapy. The changes in the volume of tumor were observed and the mice were executed at 10th day after the last injection,and the expression of CTR1 in tumor tissues was detected by immunohistochemistry. RESULTS: Cisplatin-resistant cervical carcinoma C-33A/cis cells were successfully induced by gradually increased concentration of cisplatin. The cytotoxic IC50 value of cisplatin on C-33A/cis had been upgraded from (1.86±0.08) to (8.11±0.21) µmol/L,while the CTR1 was found decreased by Western blot assay. Immunohistochemical analysis indicated that CTR1 expression was increased by intratumoral injection of adenovirus ad-hCTR1, and the tumor growth of C-33A/cis drug-resistant cervical carcinoma xenograft was inhibited by ad-hCTR1 transfection combined with cisplatin. CONCLUSION: The combination therapy of ad-hCTR1 transfection and cisplatin was effective to inhibit the growth of drug-resistant C-33A/cis tumor.

A New Anti-Inflammatory Alkaloid from Roots of Heracleum dissectum.

Using various chromatographic methods, a new piperidinone alkaloid, (3S)-3-{4-[(1E)-3-hydroxyprop-1-en-1-yl]-2-methoxyphenoxy}piperidin-2-one (1), together with 10 known compounds, bergapten (2), xanthotoxol (3), isopimpinellin (4), isobergapten (5), heratomol-6-O-ß-d-glucopyranoside (6), scopoletin (7), apterin (8), 3-methoxy-4-ß-d-glucopyranosyloxypropiophenone, (praeroside; 9), tachioside (10) and coniferin (11), were isolated from roots of Heracleum dissectum Ledeb. Their structures were elucidated on the basis of physicochemical properties and the detailed interpretation of various spectroscopic data. All the isolated compounds were screened for anti-inflammatory activity in vitro. As the results, compound 1 and 8 showed significantly inhibitory activity on nitric oxide production in RAW264.7 cells.

Chrysosplenetin inhibits artemisinin efflux in P-gp-over-expressing Caco-2 cells and reverses P-gp/MDR1 mRNA up-regulated expression induced by artemisinin in mouse small intestine.

CONTEXT: CYP3A4 and P-gp together form a highly efficient barrier for orally absorbed drugs and always share the same substrates. Our previous work revealed that chrysosplenetin (CHR) significantly augmented the rat plasma level and anti-malarial efficacy of artemisinin (ART), partially due to the uncompetitive inhibition effect of CHR on rat CYP3A. But the impact of CHR on P-gp is still unknown. OBJECTIVE: The present study investigates whether CHR interferes with P-gp-mediated efflux of ART and elucidates the underlying mechanism. MATERIALS AND METHODS: P-gp-over-expressing Caco-2 cells were treated with ART (10 µM) or ART-CHR (1:2, 10:20 µM) in ART bidirectional transport experiment. ART concentration was determined by UHPLC-MS/MS method. Healthy male ICR mice were randomly divided into nine groups (n = 6) including negative control (0.5% CMC-Na solution, 13 mL/kg), ART alone (40 mg/kg), verapamil (positive control, 40 mg/kg), ART-verapamil (1:1, 40:40 mg/kg), CHR alone (80 mg/kg), ART-CHR (1:0.1, 40:4 mg/kg), ART-CHR (1:1, 40:40 mg/kg), ART-CHR (1:2, 40:80 mg/kg) and ART-CHR (1:4, 40:160 mg/kg). The drugs were administrated intragastrically for seven consecutive days. MDR1 and P-gp expression levels in mice small intestine were examined by performing RT-PCR and western blot analysis. ABC coupling ATPase activity was also determined. RESULTS: After combined with CHR (1:2), Papp (AP-BL) and Papp (BL-AP) of ART changed to 4.29 × 10 - 8 (increased 1.79-fold) and 2.85 × 10 - 8 cm/s (decreased 1.24-fold) from 2.40 × 10 - 8 and 3.54 × 10 - 8 cm/s, respectively. Efflux ratio (PBA/PAB) declined 2.21-fold (p < 0.01) versus to ART alone. ART significantly up-regulated both MDR1 mRNA and P-gp levels compared with vehicle, while CHR in combination ratio of 0:1, 0.1:1, 1:1, 2:1 and 4:1 with ART, reversed them to normal levels as well as negative control (p < 0.05). The ATPase activities in ART-CHR 1:4 and CHR alone groups achieved a slight increase (p < 0.05) when compared with ART alone. DISCUSSION AND CONCLUSION: These results confirm that CHR inhibited P-gp activity and reverse the up-regulated P-gp and MDR1 levels induced by ART. It suggested that CHR potentially can be used as a P-gp reversal agent to obstruct ART multidrug resistance.

Characterization of γδ regulatory T cells from peripheral blood in patients with multiple myeloma.

γδ regulatory T cells are able to inhibit the activation and function of T cells involved in antigen-specific immune responses. This study aimed to investigate the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in patients diagnosed as multiple myeloma (MM). We measured the levels of γδ T cells, the distribution and clonally amplified TCR Vγ and VδT cells in peripheral blood of healthy donors, patients recently diagnosed with MM, and MM patients in remission cohorts. In addition, we evaluated the ability of γδ regulatory T cells to inhibit the proliferation of CD4+CD25- T cells and detected the expression of immunoregulatory-associated molecules. We found that the levels of γδ regulatory T cells from the peripheral blood in patients of MM were significantly higher than those in healthy donors. Comparison of γδT regulatory cells function in MM and healthy donors showed similarly inhibitory effects on the proliferation of T cells. Additionally, TLR8 expression level increased significantly in MM patients compared to healthy donors, while the expression levels of Foxp3, CD25, CTLA4, GITR, GATA3 and Tbet in MM patients and healthy donors showed no significant difference. Taken together, our study reveals the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in MM patients.

A self-contrast approach to evaluate the inhibitory effect of chrysosplenetin, in the absence and presence of artemisinin, on the in vivo P-glycoprotein-mediated digoxin transport activity.

In this study, we used a self-contrast method, which excluded the individual difference, to evaluate the inhibitory effect of chrysosplentin (CHR) in the presence or absence of artemisinin (ART) on the P-glycoprotein (P-gp) transport activity. A sensitive and rapid UHPLC-MS/MS method was applied for quantification of digoxin, a P-gp-specific substrate, in rat plasma. A pharmacokinetic study was carried out: first after an oral administration of digoxin at a dose of 0.09 mg/kg (first period), followed by a 20-day wash-out, then after another administration of digoxin (second period). During the second period, test compounds were orally given three times per day for seven consecutive days. Results showed that the t1/2 of digoxin in all the groups had no significant difference between the first and second periods. The AUC0-24 , Cmax , tmax , and Clz /F of the negative control and ART alone groups showed no difference. However, the AUC0-24 and Cmax in the CHR alone, CHR-ART (1:2) and verapamil (positive control) groups showed 2.34-, 3.04-, 1.79-, and 1.81-, 1.99-, 2.06-fold increases along with 3.50-, 3.84- and 4.76-fold decreases for CLz /F, respectively. The tmax in the CHR-ART (1:2) group increased 3.73-fold. In conclusion, our self-contrast study suggested that CHR, especially when combined with ART in a ratio of 1:2, inhibited P-gp activity while ART alone has no effect. Copyright © 2016 John Wiley & Sons, Ltd.