Highly pathogenic coronavirus N protein aggravates inflammation by MASP-2-mediated lectin complement pathway overactivation.

Excessive inflammatory responses contribute to the pathogenesis and lethality of highly pathogenic human coronaviruses, but the underlying mechanism remains unclear. In this study, the N proteins of highly pathogenic human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were found to bind MASP-2, a key serine protease in the lectin pathway of complement activation, resulting in excessive complement activation by potentiating MBL-dependent MASP-2 activation, and the deposition of MASP-2, C4b, activated C3 and C5b-9. Aggravated inflammatory lung injury was observed in mice infected with adenovirus expressing the N protein. Complement hyperactivation was also observed in SARS-CoV-2-infected patients. Either blocking the N protein:MASP-2 interaction, MASP-2 depletion or suppressing complement activation can significantly alleviate N protein-induced complement hyperactivation and lung injury in vitro and in vivo. Altogether, these data suggested that complement suppression may represent a novel therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses.

Role of AhR and Foxo1 in skin inflammation in burn animal model via MAPK signaling pathway.

Burn generally refers to thermal damage, including tissue damage caused by hydrothermal (water, soup, oil, etc.), flame, steam, high-temperature gas, hot metal liquid or solid (such as molten steel, ingot). However, little is known about the pathogenesis and inducement of skin inflammation in burned rats. Therefore, this study has carried out an in-depth analysis of the related causes of skin inflammation in burned rats. We analyzed the gene expression and the differentially expressed genes co-expression in burned rats. Subsequently, a set of functional dysfunction modules about inflammation of skin tissue in burned rats were obtained by comprehensive enrichment analysis. In addition, based on related network prediction analysis, we identified a number of regulatory factors, such as endogenous genes, nCRNAs, and transcription factors that have potential monitoring effects on skin inflammation in burned rats. Firstly, we obtained 2679 differentially expressed genes and 7 disease-related dysfunction modules in burned rats. Secondly, we identified a series of regulators related to skin inflammation in burned rats, including 117 ncRNAs (including miR-17-5p, miR-122-5p, and miR-140-5p), 31 transcription factors (including AhR, Foxo1 and Sp1) and 10 endogenous genes (including Il5, Atp5d, and Cox4i1). Core transcription factors AhR and Foxo1 may induce skin inflammation in burned rats through the cascade of MAPK signals. According to the results of this study, we can show a new method for biologists and pharmacists to reveal the inducement of skin inflammation in burned rats and provide a valuable reference for different treatment options.

Identification of Metabolites of the Cardioprotective Alkaloid Dehydrocorydaline in Rat Plasma and Bile by Liquid Chromatography Coupled with Triple Quadrupole Linear Ion Trap Mass Spectrometry.

Dehydrocorydaline (DHC), a quaternary alkaloid from Corydalis yanhusuo, has been demonstrated to be the active constituent in the treatment of coronary heart disease. In this study, a high-performance liquid chromatography-electrospray ionization-triple quadrupole linear ion trap mass spectrometry (HPLC-ESI-QTRAP MS) technique was used to identify DHC metabolites in plasma and bile after oral administration of DHC to rats. A total of 18 metabolites (M1 to M18) were identified and characterized by LC-MS/MS in the positive ion mode. These 18 metabolites were all present in rat bile, while only 9 were detected in plasma. O-demethylation, hydroxylation, di-hydroxylation, glucuronidation of O-demethyl DHC, sulfation of O-demethyl DHC and di-hydroxylation of dehydro-DHC were the major metabolic pathways of DHC. This is the first time that these metabolites of DHC have been identified in rat plasma and bile, which provides useful information for further analysis of the biotransformation of DHC and other quaternary protoberberine-type alkaloids.

The pharmacokinetic and pharmacodynamic properties of site-specific pegylated genetically modified recombinant human interleukin-11 in normal and thrombocytopenic monkeys.

In order to improve the pharmacokinetic and pharmacodynamic properties of recombinant human interleukin-11 mutein (mIL-11) and to reduce the frequency of administration, we examined the feasibility of chemical modification of mIL-11 by methoxy polyethylene glycol succinimidyl carbonate (mPEG-SC). PEG-mIL-11 was prepared by a pH controlled amine specific method. Bioactivity of the protein was determined in a IL-11-dependent in vitro bioassay, its pharmacodynamic and pharmacokinetic properties were investigated by using normal and thrombocytopenic monkey models. N-terminus sequencing and peptide mapping analysis revealed that Lys33 is the PEGylated position for PEG-mIL-11. Bioactivity of PEG-mIL-11 assessed by B9-11 cell proliferation assay was comparable to that of mIL-11. More than 79-fold increase in area-under-the curve (AUC) and 26-fold increase in maximum plasma concentration (Cmax) was observed in pharmacokinetic analysis. Single dose administration of the PEG-mIL-11 induced blood platelets number increase and the effect duration were comparable to that of 7 to 10 consecutive daily administration of mIL-11 to the normal and thrombocytopenic monkey models. PEG-mIL-11 is a promising therapeutic for thrombocytopenia.

LC-MS/MS determination and pharmacokinetic study of dehydrocorydaline in rat plasma after oral administration of dehydrocorydaline and Corydalis yanhusuo extract.

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC-MS/MS) was developed and validated for determination of dehydrocorydaline (DHC) in rat plasma using nitidine chloride as an internal standard. The analytes were solid-phase extracted and eluted on a C18 chromatography column using a mobile phase of acetonitrile and water (containing 0.8% formic acid and 10 mM ammonium acetate) (28:72, v/v). Detection was performed using positive ion electrospray ionization in multiple reaction monitoring modes. The assay was linear over the concentration range 0.625-250 ng/mL with a quantification limit of 0.625 ng/mL. The precision was <13.7%, the accuracy >93.1%, and extraction recovery ranged from 92.1% to 107%. The validated method was successfully applied to the pharmacokinetics and excretion study of DHC in rat plasma after oral administration of pure DHC and an effective fraction of Corydalis yanhusuo (EFY). The pharmacokinetic parameters showed that DHC from EFY was absorbed more rapidly and eliminated more slowly than pure DHC. The result suggests that the differences might be due to the presence of P-glycoprotein (P-gp) inhibitors and that other alkaloids co-existing in the EFY may compete with DHC for transportation by P-gp, metabolization by P450, and binding to plasma proteins.

LC-MS/MS method for determination of geldanamycin derivative GM-AMPL in rat plasma to support preclinical development.

A LC-MS/MS method for determining the concentration of the small molecule Hsp90 inhibitor, GM-AMPL, has been developed and validated in rat plasma to support preclinical development. 17-[2-(morpholine-4-yl)ethyl]amino-17-demethoxygeldanamycin (GM-AMPL) and the internal standard 17-allylamino-17-demethoxygeldanamycin (17-AAG) were sufficiently separated on a Venusil MP C18 column that was eluted with 80% methanol in water at 40°C. Quantification studies were performed with a multiple reaction monitoring (MRM) transition of m/z 657.3→614.3 and 584.3→541.3 for GM-AMPL and IS, respectively, in the negative ion mode. The present method exhibited good linearity (R>0.999) over the concentration range of 2-600ng/mL for GM-AMPL in rat plasma with a lower limit of quantification (LLOQ) of 2ng/mL. The intra-batch and inter-batch assay coefficients of variation (CV) were in range of 1.56-6.84% and 1.62-6.98%, respectively. The plasma samples were extracted with methanol to precipitate protein with extraction recovery in range of 84.09-95.25%. The matrix effect was determined as internal substance (IS) normalized matrix factor of 1.09, 1.18 and 1.05 for samples with three concentration levels of 4, 40 and 400ng/mL, respectively. This validated method was further applied to successfully determine the pharmacokinetic parameters and oral availability of GM-AMPL in Sprague-Dawley rats following intravenous injection and oral administration.

[Identification of foliumn pyrrosiae from different habitats and species by HPLC fingerprint].

OBJECTIVE: To establish HPLC fingerprint of Folium Pyrrosiae for identification. METHODS: The HPLC method was developed with Diamonsil C18 column (250 mm x 4.6 mm, 5 microm),and a mixture liquid of acetonitrile-0.8% acetic acid solution as mobile phase in a gradient elution. HPLC fingerprints of44 samples were analyzed by similarity, cluster and principal component analysis. RESULTS: The HPLC fingerprint common pattern of Pyrrosia petiolosa, Pyrrosia lingua and common pattern of Pyrrosia sheareri were set up separately. Samples from different species were classified based on the result of cluster and principal component analysis. Fingerprints of Pyrrosia sheareri and Pyrrosia lingua have high degree of similarity, but were different from Pyrrosia petiolosa, while Pyrrosia calvata and Pyrrosia assimlis were classified as adulterants with their dissimilar fingerprints. CONCLUSION: The method is stable and reliable with a good reproducibility and provides a reference standard for identifying Folium Pyrrosiae from different habitats and species.

[Determination of chlorogenic acid and mangiferin in Folium Pyrrosiae from different habitats and species by HPLC].

An HPLC method for simultaneous determination of chlorogenic acid and mangiferin in original medicinal materials and decoction pieces of Pyrrosiae Folium was developed. The assay was performed on a Diamonsil C18 (4.6 mm x 250 mm, 5 microm) column eluted with a mobile phase consisted of acetonitrile and 0.5% phosphoric acid solution in gradient elution at a flow rate of 1.0 mL x min(-1). The column temperature was set at 25 degrees C. The detection wavelength was 320 nm. The results showed that The linear ranges of chlorogenic acid and mangiferin were 5.2-130 mg x L(-1) (r = 0.9999) and 1.2-18 microg x mL(-1) (r = 0.9999), and the average recoveries (n=6) were 97.9% (RSD 1.9%) and 99.6% (RSD 2.9%), respectively. The method was simple, reproducible and valid. It can be used for quality evaluation and control of original medicinal materials and decoction pieces of Pyrrosiae Folium.

[Identification and analysis of absorbed components in rat plasma after oral administration of active fraction of Corydalis yanhusuo by LC-MS/MS].

To analyze and identify the constituents in rat plasma after oral administration of the active fraction of Corydalis yanhusuo, a LC-MS/MS method was established. The constituents absorbed into blood, their original crude drugs and their metabolites were identified either by comparing the retention time and mass spectrometry data with that of reference compounds or by mass spectrometry analysis and retrieving the reference literatures. Nine species are the original form in Corydalis yanhusuo, moreover, some metabolites in blood identified as glucuronide were found. The constituents absorbed into blood and the possible metabolites which demonstrate to originate from the active fraction of Corydalis yanhusuo are responsible for the observed efficacy. Its serum pharmacochemistry should be subjected to complete investigation so as to illuminate the pharmacology and active mechanism of the active fraction of Corydalis yanhusuo.