Screening and characterization of xenobiotics in rat bio-samples after oral administration of Shen-Wu-Yi-Shen tablet using UPLC-Q-TOF-MS/MS combined with a targeted and non-targeted strategy.

Shen-Wu-Yi-Shen tablet (SWYST), a well-known traditional Chinese medicine prescription (TCMP), has been effectively used for treating chronic kidney disease (CKD) in clinically. However, an in-depth study of in vivo metabolism of SWYST is lacking. In this study, a targeted and non-targeted strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) was developed to screen and characterize SWYST-related xenobiotics in rats. Based on the in-house library, a chemical database of SWYST including 215 constituents was constructed through "find by formula" and further verified by characteristic fragmentations or the literatures. Then the constructed chemical database was applied for the targeted screening of prototypes. As for metabolites, the non-targeted screening was achieved combined the peak picking using the function "find by auto-MS/MS" and peak filtration of the prototypes and endogenous components, while the targeted screening was performed using Metabolite ID according to the possible metabolic reactions. Furthermore, the potential metabolites were preliminarily identified by comparison of the parent compounds or references to the literatures. As a result, 201 exogenous components (87 prototypes and 121 metabolites) were characterized in rats after administration of SWYST, including 55 (17 prototypes and 38 metabolites) in plasma, 151 (52 prototypes and 99 metabolites) in urine, and 121 (74 prototypes and 47 metabolites) in feces. Finally, their possible metabolic pathways were summarized, and the metabolic reactions mainly involved phase I reactions (hydroxylation, deoxygenation, hydrogenation, methylation, oxidation, hydrolysis and esterification) and phase II reactions (glucuronidation and sulfation). The findings of this research reveal the potential active ingredients of SWYST, providing an important material basis for the pharmacokinetics and pharmacodynamics of SWYST.

The application of proteomics and metabolomics to reveal the molecular mechanism of Nutmeg-5 in ameliorating cardiac fibrosis following myocardial infarction.

BACKGROUND: Nutmeg-5, an ancient and classic formula in traditional Mongolian medicine comprising five kinds of traditional Chinese medicine, is widely used in the treatment of myocardial infarction (MI, called heart "Heyi" disease in Mongolian medicine). Cardiac fibrosis plays a critical role in the development and progression of heart failure after MI. However, the material basis and pharmacological mechanisms of the effect of Nutmeg-5 on cardiac fibrosis after MI remain unclear. OBJECTIVE: The aim of this study was to first explore the potential material basis and molecular mechanism of action of Nutmeg-5 in improving cardiac fibrosis after MI via a multiomics approach. METHODS: The constituents in Nutmeg-5 were identified by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). High-performance liquid chromatography (HPLC) and gas chromatography (GC)-based fingerprints of Nutmeg-5 were analysed, and characteristic peaks were identified by comparison to standard samples. A rat MI model was created by permanent ligation of the left anterior descending artery. The protective effect of Nutmeg-5 on cardiac fibrosis after MI was evaluated by tissue histology and measurement of the serum biomarkers of myocardial injury. Cardiac fibrosis levels were evaluated by Sirius red staining. Differentially expressed proteins in the myocardium and metabolites in the serum were explored by proteomic and untargeted metabolome analyses, respectively. Pearson correlation analysis was performed to explore the association between serum metabolites and myocardial proteins. RESULTS: A total of 67 constituents were identified in Nutmeg-5 by UPLC-MS/MS. Sixteen components were identified in the fingerprint of Nutmeg-5 by comparison with a standard sample. Six lactones were isolated from Nutmeg-5 and quantified by HPLC and GC. MI was significantly alleviated in Nutmeg-5-treated rats compared to MI rats, as demonstrated by their decreased mortality, improved cardiac function, and attenuated cardiac fibrosis and myocardial injury. A total of 252 significant differential metabolites were identified in plasma between model and Nutmeg-5-treated rats by untargeted metabolome analysis. Among these, 36 critical metabolites were associated with Nutmeg-5 activity. Proteomic analysis identified 338 differentially expressed proteins in the rat myocardium between MI and Nutmeg-5-treated rats, including 204 upregulated and 134 downregulated proteins. Protein set enrichment analysis revealed that Nutmeg-5 treatment significantly inhibited the extracellular matrix (ECM)-receptor interaction pathway, which was activated in the myocardium of MI rats. A significant decrease in collagen and alpha smooth muscle actin expression levels was found in the myocardium of Nutmeg-5-treated rats compared to MI rats. These results illustrated that Nutmeg-5 had a significant protective effect on cardiac fibrosis after MI. A significant correlation was found between the ECM-receptor interaction pathway in the myocardium and critical metabolites in the serum. In addition, there were positive correlations between the levels of critical metabolites and the expression levels of transforming growth factor (TGF)-ß1 and Smad2 in the rat myocardium. CONCLUSIONS: Nutmeg-5 alleviated cardiac fibrosis after MI in rats by inhibiting the myocardial ECM-receptor interaction pathway and TGF-ß1/Smad2 signalling, which was achieved by regulating plasma metabolites.

Kellerin from Ferula sinkiangensis exerts neuroprotective effects after focal cerebral ischemia in rats by inhibiting microglia-mediated inflammatory responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferula sinkiangensis K. M. Shen is a traditional Chinese medicine that has a variety of pharmacological properties relevant to neurological disorders and inflammations. Kellerin, a novel compound extracted from Ferula sinkiangensis, exerts a strong anti-neuroinflammatory effect by inhibiting microglial activation. Microglial activation plays a vital role in ischemia-induced brain injury. However, the potential therapeutic effect of kellerin on focal cerebral ischemia is still unknown. AIM OF THE STUDY: To explore the effect of kellerin on cerebral ischemia and clarify its possible mechanisms, we applied the middle cerebral artery occlusion (MCAO) model and the LPS-activated microglia model in our study. MATERIALS AND METHODS: Neurological outcome was examined according to a 4-tiered grading system. Brain infarct size was measured using TTC staining. Brain edema was calculated using the wet weight minus dry weight method. Neuron damage and microglial activation were observed by immunofluorescence in MCAO model in rats. In in vitro studies, microglial activation was examined by flow cytometry and the viability of neuronal cells cultured in microglia-conditioned medium was measured using MTT assay. The levels of pro-inflammatory cytokines were measured by qRT-PCR and ELISA. The proteins involved in NF-κB signaling pathway were determined by western blot. Intracellular ROS was examined using DCFH-DA method and NADPH oxidase activity was measured using the NBT assay. RESULTS: We found that kellerin improved neurological outcome, reduced brain infarct size and decreased brain edema in MCAO model in rats. Under the pathologic conditions of focal cerebral ischemia, kellerin alleviated neuron damage and inhibited microglial activation. Moreover, in in vitro studies of LPS-stimulated BV2 cells kellerin protected neuronal cells from being damaged by inhibiting microglial activation. Kellerin also reduced the levels of pro-inflammatory cytokines, suppressed the NF-κB signaling pathway, and decreased ROS generation and NADPH oxidase activity. CONCLUSIONS: Our discoveries reveal that the neuroprotective effects of kellerin may largely depend on its inhibitory effect on microglial activation. This suggests that kellerin could serve as a novel anti-inflammatory agent which may have therapeutic effects in ischemic stroke.

Bioactive sesquiterpene coumarins from the resin of Ferula sinkiangensis targeted on over-activation of microglia.

Nine undescribed (1-4, 6-10) sesquiterpene coumarins, together with a new natural one (5) and ten known ones (11-20), were isolated from the low polarity fraction of the 95% ethanol extract of the resin of Ferula sinkiangensis. Their structures were elucidated based on the comprehensive analysis of HRESIMS, 1D and 2D NMR data. The absolute configurations were determined by comparison of experimental and calculated ECD spectra. All the identified SCs were evaluated for their anti-neuroinflammatory activities in LPS-induced BV-2 cells. Ferusingensine G (8) displayed a significant inhibitory effect on nitric oxide (NO) production with an IC50 value of 1.2 µM. The results suggested that natural SCs might be served as potential neuroinflammatory inhibitors.

Kellerin alleviates cognitive impairment in mice after ischemic stroke by multiple mechanisms.

Ischemic stroke is a global disease with high disability and mortality rates. Cognitive impairment is one of the major clinical features of ischemic stroke, and microglia-mediated inflammation has been shown to be an important contributor to the pathogenesis of ischemic stroke. Kellerin, extracted from Ferula sinkiangensis, was previously shown to inhibit microglial activation and exert a strong anti-neuroinflammatory effect. However, there is no report of the potential therapeutic effect of kellerin on ischemic stroke by targeting microglial cells. In this study, we wanted to examine the effects of kellerin on ischemic stroke in the bilateral common carotid artery occlusion (BCCAO) model and the lipopolysaccharide (LPS)-activated microglia model. We found that kellerin alleviated cognitive impairment, decreased neuronal loss, suppressed microglial activation, and transformed microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype in BCCAO mice. Moreover, in in vitro studies, we found that kellerin regulated microglial polarization and inhibited the NLRP3 and MAPK signaling pathways after LPS treatment. These findings provide a new understanding of the function of kellerin in ischemic stroke, and suggest that kellerin could be a potential therapeutic agent for the treatment of ischemic stroke.

[Correlation Among Soil Organic Carbon, Soil Inorganic Carbon and the Environmental Factors in a Typical Oasis in the Southern Edge of the Tarim Basin].

We analyzed the differentiation among the environmental factors and soil organic/inorganic carbon contents of irrigated desert soil, brown desert soil, saline soil and aeolian sandy soil by classical statistics methods, and studied the correlation between soil carbon contents and the environmental factor by redundancy analysis (RDA) in a typical oasis of Yutian in the southern edge of the Tarim Basin. The results showed that the average contents of soil organic carbon and soil inorganic carbon were 2.51 g · kg⁻¹ and 25.63 g · kg⁻¹ respectively. The soil organic carbon content of the irrigated desert soil was significantly higher than those of brown desert soil, saline soil and aeolian sandy soil, while the inorganic carbon content of aeolian sandy soil was significantly higher than those of other soil types. The soil moisture and nutrient content were the highest in the irrigated desert soil and the lowest in the aeolian sandy sail. All soil types had high degree of salinization except the irrigated desert soil. The RDA results showed that the impacts of environmental factors on soil carbon contents ranked in order of importance were total nitrogen > available phosphorus > soil moisture > ground water depth > available potassium > pH > total salt. The soil carbon contents correlated extremely significantly with total nitrogen, available phosphorus, soil moisture and ground water depth (P < 0.01), and it correlated significantly with available potassium and pH (P < 0.05). There was no significant correlation between soil carbon contents and other environmental factors (P > 0.05).