Rhizophagus Irregularis regulates flavonoids metabolism in paper mulberry roots under cadmium stress.

Broussonetia papyrifera is widely found in cadmium (Cd) contaminated areas, with an inherent enhanced flavonoids metabolism and inhibited lignin biosynthesis, colonized by lots of symbiotic fungi, such as arbuscular mycorrhizal fungi (AMF). However, the physiological and molecular mechanisms by which Rhizophagus irregularis, an AM fungus, regulates flavonoids and lignin in B. papyrifera under Cd stress remain unclear. Here, a pot experiment of B. papyrifera inoculated and non-inoculated with R. irregularis under Cd stress was carried out. We determined flavonoids and lignin concentrations in B. papyrifera roots by LC-MS and GC-MS, respectively, and measured the transcriptional levels of flavonoids- or lignin-related genes in B. papyrifera roots, aiming to ascertain the key components of flavonoids or lignin, and key genes regulated by R. irregularis in response to Cd stress. Without R. irregularis, the concentrations of eriodictyol, quercetin and myricetin were significantly increased under Cd stress. The concentrations of eriodictyol and genistein were significantly increased by R. irregularis, while the concentration of rutin was significantly decreased. Total lignin and lignin monomer had no alteration under Cd stress or with R. irregularis inoculation. As for flavonoids- or lignin-related genes, 26 genes were co-regulated by Cd stress and R. irregularis. Among these genes, BpC4H2, BpCHS8 and BpCHI5 were strongly positively associated with eriodictyol, indicating that these three genes participate in eriodictyol biosynthesis and were involved in R. irregularis assisting B. papyrifera to cope with Cd stress. This lays a foundation for further research revealing molecular mechanisms by which R. irregularis regulates flavonoids synthesis to enhance tolerance of B. papyrifera to Cd stress.

Immuno-genomic-radiomics to predict response of biliary tract cancer to camrelizumab plus GEMOX in a single-arm phase II trial.

Background & Aims: Immunotherapy is an option for the treatment of advanced biliary tract cancer (BTC), although it has a low response rate. In this post hoc analysis, we investigated the predictive value of an immuno-genomic-radiomics (IGR) analysis for patients with BTC treated with camrelizumab plus gemcitabine and oxaliplatin (GEMOX) therapy. Methods: Thirty-two patients with BTC treated with camrelizumab plus GEMOX were prospectively enrolled. The relationship between high-throughput computed tomography (CT) radiomics features with immuno-genomic expression was tested and scaled with a full correlation matrix analysis. Odds ratio (OR) of IGR expression for objective response to camrelizumab plus GEMOX was tested with logistic regression analysis. Association of IGR expression with progression-free survival (PFS) and overall survival (OS) was analysed with a Cox proportional hazard regression. Results: CT radiomics correlated with CD8+ T cells (r = -0.72-0.71, p = 0.004-0.047), tumour mutation burden (TMB) (r = 0.59, p = 0.039), and ARID1A mutation (r = -0.58-0.57, p = 0.020-0.034). There was no significant correlation between radiomics and programmed cell death protein ligand 1 expression (p >0.96). Among all IGR biomarkers, only four radiomics features were independent predictors of objective response (OR = 0.09-3.81; p = 0.011-0.044). Combining independent radiomics features into an objective response prediction model achieved an area under the curve of 0.869. In a Cox analysis, radiomics signature [hazard ratio (HR) = 6.90, p <0.001], ARID1A (HR = 3.31, p = 0.013), and blood TMB (HR = 1.13, p = 0.023) were independent predictors of PFS. Radiomics signature (HR = 6.58, p <0.001) and CD8+ T cells (HR = 0.22, p = 0.004) were independent predictors of OS. Prognostic models integrating these features achieved concordance indexes of 0.677 and 0.681 for PFS and OS, respectively. Conclusions: Radiomics could act as a non-invasive immuno-genomic surrogate of BTC, which could further aid in response prediction for patients with BTC treated with immunotherapy. However, multicenter and larger sample studies are required to validate these results. Impact and implications: Immunotherapy is an alternative for the treatment of advanced BTC, whereas tumour response is heterogeneous. In a post hoc analysis of the single-arm phase II clinical trial (NCT03486678), we found that CT radiomics features were associated with the tumour microenvironment and that IGR expression was a promising marker for tumour response and long-term survival. Clinical trial number: Post hoc analysis of NCT03486678.

Current Progress and Future Perspectives of Immune Checkpoint Inhibitors in Biliary Tract Cancer.

Biliary tract cancer (BTC) is an uncommon and aggressive neoplasm, with most patients presenting in an advanced stage. Systemic chemotherapy is the limited treatment available but is unsatisfactory, while targeted therapy is still awaiting validation from clinical trials. Given the potential effect of immune checkpoint inhibitors (ICIs) in the treatment of BTC, this review aims to summarize the evidence-based benefits and predictive biomarkers for using inhibitors of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) ligand, or programmed cell death protein-1 and its ligand (PD-1 and PD-L1) as monotherapy or combined with other anti-tumor therapies, while also pointing out certain pitfalls with the use of ICIs which need to be addressed.

The inhibition mechanism of the uptake of lamivudine via human organic anion transporter 1 by Stellera chamaejasme L. extracts.

Stellera chamaejasme L. is a traditional Chinese medicine with a long history to treat stubborn skin ulcer, and it also has antiviral and antitumor effects. Neochamaejasmine B (NCB), Neochamaejasmine A (NCA) and Chamaechromone (CMC) are the major components in dried roots of Stellera chamaejasme L.. Our studies suggested that NCB, NCA and CMC are inhibitors of Organic anion transporter 1 (OAT1). OAT1 is encoded by solute carrier family 22 member 6 gene (SLC22A6) in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney. Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments. The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L. and lamivudine via OAT1 both in vitro and in vivo. In vitro, the uptake studies in Madin-Darby canine kidney (MDCK) cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC. NCB was a noncompetitive and competitive inhibitor interaction with OAT1. IC50 values of NCB, NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46, 8.35 and 0.61 µmol·L-1, respectively. In vivo, the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve (AUC0-∞) and maximal plasma concentration (Cmax) of lamivudine after co-administration is increased 2.94-fold and 1.87-fold, respectively, compared to lamivudine administration alone. The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L. extracts via OAT1 in vivo are consistent with studies in vitro. The inhibition of OAT1-mediated uptake of lamivudine by NCB, NCA and CMC is the possible mechanism for Stellera chamaejasme L. extracts improving the oral bioavailability of lamivudine in rats.

[Effect of low-intensity pulsed ultrasound combined with triamcinolone acetonide on oral mucosal ulcer healing in syrian hamster].

PURPOSE: To evaluate the effect of low-intensity pulsed ultrasound（LIPUS） combined with triamcinolone acetonide on oral mucosal ulcer in syrian hamster in several ways, including healing time, contents of superoxide dismutase（SOD）and malondialdehyde (MDA). METHODS: Sixty syrian hamsters were randomly divided into 5 groups, including a baseline group (containing a normal baseline group and a model baseline group, n=6) and 4 experimental groups (LIPUS processing and drug use group, LIPUS group, drug group and a normal control group without any processing, n=12). Four experimental groups and model baseline group were given oxygen free radicals to model the oral mucosal ulcer. At 24 h after the last treatment, the healing time of ulcer, content of SOD and MDA were compared between each group. SPSS 20.0 software package was used for statistical analysis. RESULTS: Compared with LIPUS group,drug group and control group, the healing time of oral mucosal ulcer in LIPUS and drug combined group was shortened. At 24 h after the last treatment, the activity of SOD showed that the LIPUS and drug combined group[(2.32±0.30) U/mgprot] were significantly higher than the model baseline group[(1.48±0.29) U/mgprot], the LIPUS group[(1.83±0.15) U/mgprot], the drug group[(1.76±0.25) U/mgprot] and control group[(1.71±0.18) U/mgprot] (P<0.05). The results of MDA content showed that the LIPUS and drug combined group [(8.17±0.21) nmol/mgprot] were significantly lower than the model baseline group[(9.41±0.22) nmol/mgprot], the LIPUS group[(9.00±0.44) nmol/mgprot], the drug group [(9.04±0.43) nmol/mgprot] and control group[(9.03±0.46) nmol/mgprot] (P<0.05). After oral mucosal ulcer healing, the activity of SOD and MDA showed that the LIPUS and drug combined group, the LIPUS group, the drug group and control group were not significantly different from the normal baseline group (P>0.05). CONCLUSIONS: Low-intensity pulsed ultrasound combined with triamcinolone acetonide can effectively improve the activity of SOD and reduce the contents of MDA in ulcerated tissues, and therefore accelerate the process of ulcer healing．.

[Chemical constituents from roots of Caragana stenophylla and their anti-tumor activities].

Fourteen compounds were isolated from the 80% ethanol extract of Caragana stenophylla root, by using a combination of various chromatographic approaches, including silica gel sephadex LH-20 column chromatography, and preparative HPLC. On the basis of their physical and chemical properties and spectroscopic data, their structures were elucidated as 2-(4-hydroxy-3-methoxy lphenyl)-3-methoxyl benzofuran-6-ol (1), mucodianin C (2), isopterofuran (3), formononetin (4), afromosin (5), calycosin (6), acacetin (7), 3-O-methylkaempferol (8), liquiritigenin (9), isoliquiritigenin (10), variabilin (11), resveratrol (12), zhebeiresinol (13), and 2, 3-dicarboxy-6, 7-dihydroxy-1-(3', 4'-dihydroxy)-phenyl-1, 2-dihydronaphthalen (14). Compound 1 is a new benzofuran derivative, named as mucodianin S; compounds 2, 3, 11, 13, 14 were isolated from the genus Caragana for the first time, and compounds 4-10 were firstly isolated from Caragana stenophylla. MTT assay was used to determine their cytotoxicity of the isolated compounds against human tumor cell lines, and 2 showed cytotoxicity against human hepato cellular cancer (HepG2) and human cervical (HeLa) lines, with IC50 values of (16.18±0.95), (3.75±0.08) µmol•L ⁻¹, respectively.

Flavonoids from Caragana pruinosa roots.

A new pterocarpan derivative, pruinosanone D (1), a new isoflavonoid, pruinosanone E (2), and a new chalcone, pruinosanone F (3), were isolated from Caragana pruinosa roots, along with four known analogues (4-7), identified as 2,4-dihydroxy-3'-methoxy-4'-ethoxychalcone, 7,4-dihydroxyflavanone, butin and scutellaprostin C, respectively. Their structures were elucidated by detailed analyses of NMR, IR, and MS data. The ability of the isolated compounds to prevent nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophages was also studied. Compound 1 were among the most potent NO production inhibitor, with IC50 value of 0.62µM.

Pruinosanones A-C, anti-inflammatory isoflavone derivatives from Caragana pruinosa.

Pruinosanone A (1), a novel spirochromone, was isolated from the roots of Caragana pruinosa. Two biogenetically related isoflavone intermediates, pruinosanones B and C (2 and 3), were also isolated, together with five known analogs identified as 3-hydroxy-9-methoxypterocarpan (4), 7,2'-dihydroxy-4'-methoxyisoflavanol (5), retusin-8-methylether (6), 7,2'-dihydroxy-8,4'-dimethoxy isoflavone (7) and 7,3'-dihydroxy-8,4'-dimethoxy isoflavone (8). The structures of 1-3 were elucidated based on extensive spectroscopic methods. Notably, 1 is the first example of a spirochromone possessing an unprecedented pentacyclic skeleton containing a spiro[benzo[d][1,3]dioxole-2,3'-chroman]-4'-one motif, which was confirmed by X-ray diffraction analysis. A plausible biosynthetic pathway for 1 was also proposed. Compounds 1-8 were tested for their ability to inhibit nitric oxide (NO) production in LPS-induced RAW 264.7 macrophages, and compounds 1-3 were the most potent inhibitors of NO production, with IC50 values of 1.96, 1.93 and 1.58 µM, respectively. A structure-activity relationship analysis revealed that the fused 2-isopropenyl-2,3-dihydrofuran moiety plays a vital role in the potency of these compounds. Moreover, 1 was found to significantly inhibit inducible nitric oxide synthase (iNOS) protein expression, which accounts for the potent inhibition of NO production by this spirochromone.

(125)I-labeled anti-bFGF monoclonal antibody inhibits growth of hepatocellular carcinoma.

AIM: To investigate the inhibitory efficacy of (125)I-labeled anti-basic fibroblast growth factor (bFGF) monoclonal antibody (mAb) in hepatocellular carcinoma (HCC). METHODS: bFGF mAb was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with (125)I through the chloramine-T method, bFGF mAb was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of (125)I-bFGF mAb. The murine H22 HCC xenograft model was established and randomized to interventions with control (phosphate-buffered saline), (125)I-bFGF mAb, (125)I plus bFGF mAb, bFGF mAb, or (125)I. The ratios of tumor inhibition were then calculated. Expression of bFGF, fibroblast growth factor receptor (FGFR), platelet-derived growth factor, and vascular endothelial growth factor (VEGF) mRNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction. RESULTS: The purified bFGF mAb solution was 8.145 mg/mL with a titer of 1:2560000 and was stored at -20 °C. After coupling, (125)I-bFGF mAb was used at a 1: 1280000 dilution, stored at 4 °C, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the (125)I-bFGF mAb group than in the other groups (P < 0.05). Expression of bFGF and FGFR mRNA in the (125)I-bFGF mAb group was significantly decreased in comparison with other groups (P < 0.05). Groups under interventions revealed increased expression of VEGF mRNA (except for (125)I group) compared with the control group. CONCLUSION: (125)I-bFGF mAb inhibits growth of HCC xenografts. The coupling effect of (125)I-bFGF mAb is more effective than the concomitant use of (125)I and bFGF mAb.

Cyclosporin A affects the bioavailability of ginkgolic acids via inhibition of P-gp and BCRP.

Ginkgolic acids (GAs) in natural product Ginkgobiloba L. are the pharmacological active but also toxic components. Two compounds, GA (C15:1) and GA (C17:1) are the most abundant GAs. In this study, several in vitro and in vivo models were applied to investigate transport mechanism of GAs. A rapid and sensitive LC-MS/MS method for the simultaneous determination of GA (C15:1) and GA (C17:1) was applied to analyze the biological specimens. The Papp(AP→BL) values of GA (C15:1) and GA (C17:1) were 1.66-2.13×10(-)(6)cm/s and 1.34-1.85×10(-)(6)cm/s determined using MDCK and MDCK-MDR1 cell monolayers, respectively. The Papp(BL→AP) were remarkably greater in the MDCK-MDR1 cell line, which were 6.77-11.2×10(-)(6)cm/s for GA (C15:1) and 4.73-5.15×10(-)(6)cm/s for GA (C17:1). Similar results were obtained in LLC-PK1 and LLC-PK1-BCRP cell monolayers. The net efflux ratio of GA (C15:1) and GA (C17:1) in both cell models was greater than 2 and markedly reduced by the presence of Cyclosporin A (CsA) or GF120918, inhibitors of P-gp and BCRP, suggesting that GAs are P-gp and BCRP substrates. The results from a rat bioavailability study also showed that co-administrating CsA intravenously (20mg/kg) could significantly increase GA (C15:1) and GA (C17:1) AUC0-t by 1.46-fold and 1.53-fold and brain concentration levels of 1.43-fold and 1.51-fold, respectively, due to the inhibition of P-gp and BCRP efflux transporters by CsA.

Oleanene triterpenes from Sedum lineare Thunb.

One new olean-13(18)-ene-3,12,19-trione (1), and two known oleanene triterpenes δ-amyrone (2), and δ-amyrine acetate (3) were isolated from the petroleum ether fraction from an alcoholic extract of the whole plant of Sedum linear Thunb. The new compound was characterized by means of spectroscopic methods including 1D, 2D NMR and HR-ESI-MS, and was further confirmed by X-ray diffraction analysis. The known ones were established on the basis of comparing their NMR data with those of the corresponding compounds in the literature. In addition, the inhibitory effects of the compounds isolated on the TNF-α and NO production were examined in vitro.