A LC-MS/MS procedure for the analysis of 19 perfluoroalkyl substances in food fulfilling recent EU regulations requests.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are synthetic, stable, fluorinated molecules widely used in consumer products. They are non-biodegradable, persistent and bio-accumulating. In 2020 European Food Safety Authority (EFSA) lowered the Tolerable Weekly Intake (TWI) for the four PFASs (PFOA, PFOS, PFNA, PFHxS) and in 2022, the EU issued a Recommendation asking to monitor twenty-one PFASs in food. Since 1st January 2023 limits in food were set. Here we report a sensitive, straightforward and robust procedure enabling the determination of 19 PFAS in food matrices using a liquid chromatography mass spectrometer (LC-MS/MS). Few are the published methods applicable to the different food matrices and covering the molecules listed in Recommendation 2022/1431, fulfilling the LOQs requested. The method was satisfactory validated (UNI CEI EN ISO/IEC 17025:2018 and Regulation (EU) 2022/1428) and used to investigate hen egg samples, highlighting home-produced eggs as the more critical egg farming process for PFAS accumulation.

Comparison of Direct and Extraction Immunoassay Methods With Liquid Chromatography-Tandem Mass Spectrometry Measurement of Urinary Free Cortisol for the Diagnosis of Cushing's Syndrome.

Background: Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing's syndrome (CS). We compared UFC determination by both direct and extraction immunoassays using Abbott Architect, Siemens Atellica Solution, and Beckman DxI800 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we evaluated the value of 24-hr UFC measured by six methods for diagnosing CS. Methods: Residual 24-hr urine samples of 94 CS and 246 non-CS patients were collected. A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by direct assays (D) using Abbott, Siemens, and Beckman platforms and by extraction assays (E) using Siemens and Beckman platforms. Method was compared using Passing-Bablok regression and Bland-Altman plot analyses. Cut-off values for the six assays and corresponding sensitivities and specificities were calculated by ROC analysis. Results: Abbott-D, Beckman-E, Siemens-E, and Siemens-D showed strong correlations with LC-MS/MS (Spearman coefficient r=0.965, 0.922, 0.922, and 0.897, respectively), while Beckman-D showed weaker correlation (r=0.755). All immunoassays showed proportionally positive bias. The areas under the curve were 0.975 for Abbott-D, 0.972 for LC-MS/MS, 0.966 for Siemens-E, 0.948 for Siemens-D, 0.955 for Beckman-E, and 0.877 for Beckman-D. The cut-off values varied significantly (154.8-1,321.5 nmol/24 hrs). Assay sensitivity and specificity ranged from 76.1% to 93.2% and from 93.0% to 97.1%, respectively. Conclusions: Commercially available immunoassays for measuring UFC show different levels of analytical consistency compared to LC-MS/MS. Abbott-D, Siemens-E, and Beckman-E have high diagnostic accuracy for CS.

Jianghu decoction and its active component polydatin inhibit inflammation and fibrotic lesions in the lungs of ILD mice via the AMPK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Recently, interstitial lung disease (ILD) morbidity and mortality have been increasing with insidious epidemiological characteristics. Jianghu decoction (JH) is an effective Chinese medicine for ILD. AIM OF THE STUDY: We aimed to reveal the material basis and mechanism of action of JH in the treatment of ILD. MATERIALS AND METHODS: In this study, an ILD mouse model was constructed with bleomycin. HE staining, transcriptome analysis, parallel reaction monitoring-mass spectrometry (PRM-MS), UPLC‒MS, and western blotting assays were conducted. RESULTS: HE staining results showed that JH effectively reduced inflammation and fibrosis foci in the lungs of the ILD model. Furthermore, transcriptome analysis revealed that JH regulates a set of biological signaling pathways related to immune inflammation and fibrosis. PRM-MS combined with western blotting was applied to detect inflammation and fibrosis involving proteins in lung tissue. JH effectively reversed the aberrant expression of HMGB1, RAGE, SEPTIN4, ACTA2, and ITGAV proteins in the model group. AMPK was identified as the core upstream regulatory protein for JH-mediated ILD regulation. In addition, UHPLC‒MS technology was applied to determine the active ingredients of JH. A total of 80 components were identified from JH, and polydatin (PD) was identified as the active ingredient that effectively alleviated lung fibrosis and inflammatory injury in ILD mice. To illustrate the molecular regulatory network of JH and PD in alleviating lung fibrosis and inflammatory injury, we also examined inflammation and fibrosis-related molecules downstream of the AMPK pathway with RT‒qPCR and western blotting. CONCLUSIONS: The results showed that both JH and its active component PD exert synergistic inhibition on pulmonary fibrosis and inflammation. Specifically, the AMPK/PGC1α/PPARγ signaling pathway was activated, and the AMPK/HMGB1/RAGE signaling pathway was inhibited in ILD lungs responding to JH or PD administration.

Diosmin: A Daboia russelii venom PLA2s inhibitor- purified, and characterized from Oxalis corniculata L medicinal plant.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxalis corniculata L is a medicinal plant that belongs to the Oxalidaceae family. It is a little, slow-growing plant with a frail appearance typically found in mild temperate and tropical areas like Pakistan and India. This plant also includes many other bioactive substances, including alkaloids, flavonoids, terpenoids, cardiac glycosides, saponins, phlobatannins, and steroids. AIM OF THE STUDY: To investigate the anti-inflammatory effects of Compound diosmin, which is derived from Oxalis corniculata L, on VRV-PL-5 and VRV-PL-8a isolated from Vipera russelli. MATERIALS AND METHODS: Extraction, purification, and characterization of bioactive by TLC, HPTLC, FT-IR analysis, UV-Vis spectrophotometer, LC-MS/MS Analysis, NMR, XRD Analysis, In vitro evaluation, Circular dichroism spectroscopy, in vivo, and in silico studies. RESULTS: In this study, the extract of Oxalis corniculata was evaluated for its in vitro and in vivo anti-inflammatory effect against PLA2. The methanolic extract decreased hemolytic activity by about 60% at 1:75 w/w and neutralized the hemolytic activity completely at 1:100 w/w concentration. Diosmin inhibited VRV-PL-5 and VRV-PL-8a in a dose-dependent manner, with the extent of inhibition being about 56% for VRV-PL-5120 µM and VRV-PL-8a by 62% at the same concentration with IC50 concentrations of 87.08 µM for VRV-PL-5 and 82.08 µM for VRV-PL-8a, while at 75 µM. Diosmin inhibited the hemolytic activity of VRV-PL-5 by about 85%, and at the same concentration, VRV-PL-8a inhibited by about 75%. UV-CD spectra at the IC50 concentration of diosmin disrupted the secondary structure of VRV-PL-5 &VRV-PL-8a. In vivo, studies showed decreased myotoxicity and cardiotoxicity of the VRV-PL-5 &VRV-PL-8a, which was seen in the decrease in cytoplasmic markers LDH and CPK levels in the serum when incubated with diosmin. Furthermore, Histopathological studies of Muscles and lungs revealed that diosmin considerably protects against cellular abnormality caused by VRV-PL-5 & VRV-PL-8a. Molecular docking, MM/GBSA, and molecular dynamics simulation studies show that the diosmin is a potent inhibitor for VRV-PL-5 and VRV-PL-8a. CONCLUSION: This study shows that diosmin is a potentially effective VRV-PL-5 and VRV-PL-8a.

Network pharmacology analysis of the active ingredients of Corydalis hendersonii Hemsl. and their effects on eliminating neuroinflammation and improving motor functions in MPTP-intoxicated mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis hendersonii Hemsl. (CH), is a traditional Tibetan medicine used in highland areas for the treatment of alpine polycythemia, ulcers and various inflammatory diseases. Its antioxidant and anti-inflammatory effects have been demonstrated in experimental mice. Loss of dopaminergic neurons due to oxidative damage is thought to be an important factor in the development of PD, the potential antioxidant, anti-inflammatory effects of CH could potentially be used for PD treatment. AIM OF THE STUDY: To identify potential targets of CH using network pharmacology and to investigate the neuroprotective effects in cultured cell models and in MPTP-intoxicated mice. MATERIALS AND METHODS: The main chemical components of CH were analyzed by UPLC-MS/MS and their potential targets of action or signaling pathways were analyzed using network pharmacology. MPP + or LPS was added to SH-SY5Y or BV2 cells, respectively, to establish cellular models. MPTP was administered to C57BL/6J mice to induce inflammation and dopaminergic neuron loss as well as dyskinesia, followed by behavioral analysis to determine the role of CH in eliminating inflammation, avoiding neuron loss, and improving dyskinesia. RESULTS: CH contains 241 alkaloids, 213 flavonoids, 177 terpenoids and 114 phenolic compounds. The targets crossover between CH and PD yielded 210 potential therapeutic targets, especially growth factors and inflammatory pathway-related genes, such as BDNF, NF-κB, as potential key targets. In cultured cells, CHE eliminated MPP + -induced impairment of cell viability as well as LPS-induced inflammation, respectively. In mice, CHE ameliorated MPTP-induced dyskinesia and rescued the loss of dopaminergic neurons in the substantia nigra and striatum. Mechanistically, CHE effectively maintained the activity of the BDNF-TrkB/Akt signaling pathway, accordingly, inhibited inflammatory signaling pathways such as HIF-1α/PKM2 and Notch/NF-kB. CONCLUSIONS: CH performed well in eliminating inflammation and improving locomotor deficits in mice, and its potent active ingredients are worthy of subsequent research and development.

Comprehensive untargeted lipidomic analysis of sea buckthorn using UHPLC-HR-AM/MS/MS combined with principal component analysis.

Sea buckthorn is an important ecological and economic plant which has multiple bioactivities. The fruits and seeds of sea buckthorn are rich in oil. However, there are few studies on the differences of lipid profiles of sea buckthorn varieties. Herein, the lipidomic fingerprints of sea buckthorn was established. First, a mixture solvent of methanol and chloroform (2:1, v/v) was selected to extract the lipid of the flesh and seed of sea buckthorn. Then, global lipidomic analysis of different varieties of sea buckthorn was conducted. A total of 16 lipid classes and 112 lipid molecular species were determined. Several molecular species, such as PE (phosphatidylethanolamine) 18:1/18:3, PE18:0/18:1, PE18:0/18:2, etc. were selected as the potential biomarkers to classify the samples. Our study provides a scientific basis for quality control of sea buckthorn and promotes the development of sea buckthorn oil.

Development of a two-step pretreatment and UPLC-MS/MS-based method for simultaneous determination of acrylamide, 5-hydroxymethylfurfural, advanced glycation end products and heterocyclic amines in thermally processed foods.

A two-step pretreatment and UPLC-MS/MS-based method was established to extract and determine 17 thermal processing hazards (TPHs) simultaneously. The first step was to extract acrylamide (AA), 5-hydroxymethylfurfural (HMF) and free heterocyclic amines (HAs). The bound HAs and advanced glycation end products (AGEs) were released by acid hydrolysis in the second step. A fairly good separation was achieved within 7 min. Almost all TPHs showed high correlation coefficients (R2 >0.999) in their respective linear ranges. The accuracy ranged from 98.13 to 100.96%. LODs and LOQs were in the range of 0.01-0.89 µg/L and 0.02-2.96 µg/L, respectively. The method was successfully applied to four representative food products, including high-starch, high-protein, high-fat and high-sugar foods, showing acceptable recoveries, intra-day and inter-day precisions. Moreover, PCA was performed to visualize the association between TPHs and food matrices. The developed method provided technical support for the formation and control researches of TPHs.

Characterization and determination of casein glycomacropeptide in dairy products by UHPLC-MS/MS based on its characteristic peptide.

The ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method was built to quantify the casein glycomacropeptide (CGMP) in bovine dairy products accurately based on targeted proteomics. Qualitative analysis of theoretical peptides was carried out using high-resolution mass spectrometry (HRMS) and protein software. Isotope-labeled characteristic peptides were acquired via the labeled amino acid condensation method to correct the matrix effects. Peptide MAIPPK was the representative characteristic peptide for distinguishing the CGMP from κ-casein through trypsin digestion. After optimizing the pre-treatment conditions, the final 8% oxidant concentration was selected and the 10% formic acid concentration with 2.5 h oxidation time. Moreover, the results of methodological verification showed that the recovery rate was 103.7%, meanwhile the precision of inter-day and intra-day was less than 5%. In conclusion, the research demonstrated the characteristic peptide MAIPPK could quantitatively applied to detect CGMP in dairy products.

Integrated ESI-MS/MS and APCI-MS/MS based metabolomics reveal the effects of canning and storage on peach fruits.

The characterization of peach metabolites and carotenoids during canning and storage remains unclear. The present study identified 658 metabolites and 40 carotenoids in peach fruits throughout the canning and storage using ESI-MS/MS and APCI-MS/MS based metabolome approach. A total of 282 differentially accumulated metabolites were found, mainly including 78 phenolic acids, 74 lipids, 61 flavonoids. Five esterified carotenoids (rubixanthin palmitate, ß-cryptoxanthin oleate, ß-cryptoxanthin laurate, ß-cryptoxanthin palmitate, and ß-cryptoxanthin myristate) were the main peach carotenoids, with a proportion of approximately 90%, while free carotenoids accounted for 4.22-5.95% during the entire processing period. Moreover, the total carotenoid loss rates for canning and storage were 56.67% and 46.55%, respectively. Compared to the loss of free carotenoids, esterified carotenoids were more stable during storage, while canning led to a greater loss of esterified carotenoids. The results provided new insights into the maintenance of health-related phytochemicals from canning processes.

Label-Free quantitation of milk oligosaccharides from different mammal species and heat treatment influence.

Milk oligosaccharides (MOs) exhibit significant variations in concentrations and patterns among different species. However, there is limited knowledge about milk oligosaccharides in domestic animals and the impact of heat treatment on them. Here, we developed an LC-ESI-MS/MS method to analyze 11 milk oligosaccharides in 7 distinct species simultaneously. The results showed that human milk presented a completely different composition pattern of milk oligosaccharides from animals. In detail, animal milk predominantly contained sialylated oligosaccharides, and human milk had high levels of fucosylated neutral oligosaccharides. Notably, sheep milk exhibited similarities to human milk in terms of oligosaccharides composition. Then, the milk samples from dairy cows were treated with two common industrial heat treatments. We found that 65 °C treatment had no significant effect on the concentration of milk oligosaccharides, whereas 135 °C heating was associated with their decline, suggesting that high temperatures should be avoided in the processing of oligosaccharides supplemented/enriched products.

Untargeted metabolomics analysis of non-volatile metabolites and dynamic changes of antioxidant capacity in Douchi with edible mushroom by-products.

This study investigated the non-volatile metabolites and antioxidant activity of Douchi, an edible mushroom by-product. A total of 695 non-volatile metabolites were detected using UPLC-MS/MS-based metabolomics analysis, and the greatest impact on metabolite composition was observed during Koji-making and the first 5 days of post-fermentation. Throughout the fermentation process, 366 differential metabolites were identified, with flavonoids being the most prominent followed by amino acids and their derivatives, which were found to be important for the quality of edible mushroom by-product Douchi (EMD). The antioxidant capacity of EMD significantly increased with the longer fermentation time, which might be associated with the conversion of isoflavone glycosides to aglycones, amino acids and their derivatives, free fatty acids, group A saponins, and phenolic acids. These findings suggested that different fermentation phases of EMD significantly affected the non-volatile metabolite profile and antioxidant capacity.

Synephrine, as a scavenger and promoter, cooperates with hesperidin to reduce acrolein levels.

Acrolein (ACR) is a harmful and active aldehyde produced in processed food that endangers foods safety. We undertook this work to explore the ACR-trapping ability of hesperidin (HES) and synephrine (SYN) from the diet. After comparing their ACR-trapping abilities, the reaction pathways of HES and SNY were analyzed using LC-MS/MS, and two adducts (HES-ACR-1 and SNY-2ACR) were synthesized, and their structures were identified by NMR. Then, we not only evaluated the synergistic trapping effects of HES and SNY on ACR in the model through the Chou-Talalay method but verified it in the processing of roasted duck wings and cookies. Furthermore, based on the quantitative analysis of the ACR-adducts of HES and SNY, we demonstrated that SYN, as a promoter, could greatly improve the ACR-capturing ability of HES by forming more adducts (3-fold). Our findings could serve as a guide for using SNY and HES as new scavengers in food processing.

Changes in flavor and biological activities of Lentinula edodes hydrolysates after Maillard reaction.

This study aimed to elucidate how the Maillard reaction (MR) affects the flavor and bioactivities of Lentinula edodes hydrolysates (LEHs). Changes in flavor were investigated using non-targeted metabolomics techniques (GC-MS and LC-MS/MS) and sensory evaluation. Simultaneously, UV absorption, fluorescence, and FT-IR spectra were used to characterize the process of MR. We also evaluated the effects of MR on the antioxidant activity, hypoglycemic activity and antimicrobial activity of LEHs in vitro. The results revealed that MR produced many volatile aldehydes and ketones and decreased the content of most amino acids, sugars and flavonoids in the LEHs while increasing the content of l-theanine and succinic acid. MRPs had a strong caramel and like-meat flavor and an obvious improvement in umami, taste continuity, and total acceptability. Furthermore, MR improved the antioxidant and antimicrobial properties of LEHs. This research establishes a theoretical foundation for MR in the deep processing of edible mushrooms.

Comprehensive analysis of oxylipins using reverse phase liquid chromatography and data dependent acquisition workflow on LTQ-Orbitrap® Velos Pro.

Oxylipins - involved in inflammatory processes - are reported in several diseases, in biological, pharmacological, and physiological fields. To face the structural complexity of oxylipins, the study of isomers and isobars species relied on Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM) in tandem mass spectrometry such as triple quadrupole, quadrupole-Time of Flight (TOF). Unfortunately, false positive signals in cellular matrix could occur using MRM or SRM mode since the MS/MS spectrum of each molecule is not acquired with the previous mode to help molecule confirmation. Using the versatile ability of LTQ-Orbitrap® Velos Pro mass spectrometer, we developed a novel method based on data dependent acquisition (DDA) workflow for oxylipins analysis. To reach sufficient data points per peak and a better sensitivity to quantify oxylipins traces, an optimization of the acquisition frequency was carried out both on linear trap and Orbitrap analyzers. A segmentation of the chromatographic profile and an optimization of the collision energies by HCD (higher energy collision dissociation) for each eicosanoid increased the acquisition frequency significantly and the detection threshold: around 2 pg for some prostanoids and 0.02-2 pg for some leukotrienes and oxidized species. We validated our method in terms of specificity (RSD <10%), sensitivity, accuracy and precision. The intra and inter-day accuracy were between 86.56% and 114.93%. Besides, a relative standard deviation less than 15% as intra- and inter-day precision were obtained for almost all molecules. A linear range between 2.5 and 12,500 pg was reached. DDA approach on LTQ-Orbitrap® constitutes an alternative to MRM mode on triple quadrupole for eicosanoids quantification in complex matrices. Finally, this method helped us to compare for the first time the amount of prostanoids released by J774 and THP-1 macrophages under lipopolysaccharide (LPS) stimulation.

An eco-friendly sample preparation procedure based on air-assisted liquid-liquid microextraction for the rapid determination of phthalate metabolites in urine samples by liquid chromatography-tandem mass spectrometry.

Urinary phthalate metabolite (mPAEs) analysis is a reliable tool for assessing human exposure to phthalates. With growing interest in urinary biomonitoring of these metabolites, there is a need for fast and sensitive analytical methods. Therefore, a simple, rapid procedure for simultaneous determination of fifteen phthalate metabolites in human urine samples by liquid chromatography-tandem mass spectrometry was developed. The novelty of the present procedure is based on the use of diethyl carbonate as a green biobased extraction solvent and air-assisted liquid-liquid microextraction (AALLME) as a sample preparation step. A Plackett-Burman design was used for screening the factors that influence the AALLME extraction efficiency of mPAEs. The effective factors were then optimized by response surface methodology using a central composite rotatable design. Under the optimized conditions, good linearity can be achieved in a concentration range of 1.0-20.0 ng mL-1 with correlation coefficients higher than 0.99. The repeatability and reproducibility precision were in the range of 2-12% and 1-10% respectively. Recoveries ranging from 90% to 110%. This, and the low limits of detection, ranging from 0.01 to 0.05 ng mL-1, make the proposed procedure sensitive and suitable for human biomonitoring of phthalate exposures. For proof-of-principle, the new method was used to measure the urinary concentrations of mPAEs in 20 urine samples from Brazilian women. The high frequency of detections and in part high concentrations of mPAEs indicate to widespread exposure to several phthalates among Brazilian women.

High throughput LC-MS/MS method for steroid hormone analysis in rat liver and plasma - unraveling methodological challenges.

Comprehensive reference data for steroid hormones are lacking in rat models, particularly for early developmental stages and unconventional matrices as the liver. Therefore, we developed and validated an enzymatic, solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify a panel of 23 steroid hormones in liver and plasma from adult and neonatal rats. Our approach tackles methodological challenges, focusing on undesired byproducts associated with specific enzymatic treatment, and enables a thorough assessment of potential interferences in complex matrices by utilizing unstripped plasma and liver. We propose an optimized enzymatic hydrolysis protocol using a recombinant ß-glucuronidase/sulfatase mix (BGS mix) to efficiently deconjugate steroid phase II conjugates. The streamlined sample preparation and high-throughput solid phase extraction in a 96-well plate significantly accelerate sample processing for complex matrices and alarge number of samples. We were able to achieve the necessary sensitivity for accurately measuring the target analytes, particularly estrogens, in small sample sizes of 5-20 mg of liver tissue and 100 µL of plasma. Through the analysis of liver and plasma samples from adult and neonatal rats, including both sexes, our study showed a novel set of steroid hormone reference intervals. This study provides a reliable diagnostic tool for the quantification of steroids in rat models and gives insight in liver and plasma-related steroid hormone dynamics at early developmental stages. In addition, the method covers several pathway intermediates and extend the list of steroid hormones to be investigated.

Qualitative and quantitative determination of phenols and their metabolites in urine by in-syringe solid-phase extraction and LC-MS/MS analysis for evaluation of virgin olive oil metabolism.

To know the bioavailability of virgin olive oil (VOO) phenols and its impact on health, it is necessary to determine the levels of phenols excreted in urine. We present here a novel strategy for in-syringe solid-phase extraction and analysis of the extract by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using ammonium fluoride as ionization agent to enhance sensitivity. This approach allows avoiding additional steps such as solvent evaporation or analytes derivatization. The method can be used with a previous acid hydrolysis for quantitative determination of tyrosol and hydroxytyrosol to estimate metabolized phenols. We tested this application by analysis of a cohort of volunteers (n = 20) after a standardized intake of VOO. Additionally, the method can be used as such for metabolite profiling of phenolic derivatives in urine using LC-MS/MS in high-resolution data-independent acquisition (DIA). Information about the phenolic profile of the consumed VOO and the human metabolism is thus obtained. The proposed approach represents a simple and versatile tool for qualitative and quantitative characterization of VOO phenolic metabolism.

Network pharmacology and experimental validation on yangjing zhongyu decoction against diminished ovarian reserve.

ETHNOPHARMACOLOGICAL RELEVANCE: Diminished ovarian reserve (DOR) was considered a refractory reproductive endocrine condition that negatively affected female reproductivity. Yangjing Zhongyu Decoction (YJZYD) had effects on treating infertility. However, there were few studies on the mechanisms of YJZYD preserving ovarian reserve. AIM OF THE STUDY: To explore the possible mechanisms of YJZYD against DOR by UPLC-ESI-MS/MS, network pharmacology, and experimental validation. METHODS: The chemicals of YJZYD were measured by UPLC-ESI-MS/MS. The correlating targets of YJZYD and DOR were identified by the ETCM database, GeneCards database, and PubMed database. The common targets were employed with the DAVID database and visualized with the PPI network. GO and KEGG enrichment analyses were carried out to explore biological progression and pathways. In vivo experiments, energy production was assessed by ATP, and apoptosis rate was analyzed by TUNEL. The serum FSH, AMH, and E2 levels were evaluated by ELISA. Western blotting and immunohistochemistry were used to measure the expression of SIRT1, PGC1α, NRF1, COX IV, FSHR, CYP19A1, PI3K, p-Akt, Akt, Bcl-2, and Bax. RESULTS: 132 components in YJZYD were identified by UPLC-ESI-MS/MS. 149 overlapped targets were extracted from YJZYD and DOR, and the top 20 common targets included AKT1 and CYP19A1. ATP binding was involved in GO analysis. In the KEGG enrichment analysis, the metabolic pathway was the top, and the PI3K-Akt signaling pathway was included. In vivo experiments, YJZYD improved ovarian index and histomorphology. After YJZYD treatment, serum FSH, E2, and AMH were well-modulated, and the content of ATP was up-regulated. Besides, the expression of Bax was suppressed in ovarian tissue, while the expressions of SIRT1, PGC1α, NRF1, COX IV, FSHR, CYP19A1, PI3K, Bcl-2, and p-Akt/Akt were enhanced. CONCLUSION: YJZYD could attenuate reproductive endocrine disturbance and ovarian lesions in vivo by mediating steroidogenesis, energy metabolism, and cell apoptosis. This study uncovered the mechanisms of YJZYD against DOR, providing a theoretical basis for further study.

Chinese herbal medicine alleviates autophagy and apoptosis in ovarian granulosa cells induced by testosterone through PI3K/AKT1/FOXO1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Polycystic ovary syndrome (PCOS) is a common gynecological endocrine and metabolic disorder. Chinese herbal medicine has some advantages in the treatment of PCOS with its unique theoretical system and rich clinical practice experiences. AIM OF THE STUDY: The present study was to investigate the potential mechanisms of Bu-Shen-Jian-Pi Formula (BSJPF) on the treatment of PCOS. MATERIAL AND METHODS: The combination of ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS) rapid analysis, network pharmacology, molecular docking analysis and bio-experiments were firstly conducted to identify the main effective components of BSJPF, and to predict the potential mechanisms. The ovarian granulosa cell line (KGN) was treated with testosterone to construct the PCOS model in vitro, and the cells were further treated with the lyophilized powder of BSJPF. The levels of proliferation, autophagy and apoptosis were detected to explore the mechanisms of BSJPF on treating PCOS. RESULTS: Firstly, thirty-six active compounds were identified in BSJPF and thirty-one potential targets on PCOS were found. Then, PI3K and PDK1 were verified to have good binding activity with the active compounds through molecular docking analysis. In bio-experiments, BSJPF significantly alleviated the arrested proliferation of KGN cells in G0/G1 phase and reduced the active levels of autophagy and apoptosis of KGN cells induced by testosterone. Additionally, the inhibition of autophagy diminished apoptosis, while the repression apoptosis enhanced autophagy. Finally, BSJPF significantly decreased the FOXO1 expression levels induced by testosterone, especially for nuclear FOXO1, and significantly activated the PI3K/AKT pathway. CONCLUSIONS: BSJPF significantly alleviated the activated autophagy and apoptosis in KGN induced by testosterone through PI3K/AKT1/FOXO1pathway, which is an effective treatment for PCOS.

Shenhuangdan decoction alleviates sepsis-induced lung injury through inhibition of GSDMD-mediated pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Sepsis-induced lung injury is closely associated with it remarkable morbidity and mortality. Shenhuangdan (SHD) decoction, a traditional Chinese medicine prescription, has been clinically proven to be an effective treatment for sepsis. However, the mechanism of SHD decoction in treating sepsis remains unclear. AIM OF THE STUDY: This study aimed to evaluate the therapeutic effect of SHD decoction on sepsis-induced lung injury and its underlying mechanism. MATERIALS AND METHODS: In this study, we established a mouse model of sepsis by cecum ligation and puncture (CLP) surgery. Firstly, seven-day survival analysis and histological staining of lung tissue were used to evaluate the therapeutic effect of SHD decoction on lung injury in septic mice. Multifactor microarray was used to detect cytokine expression changes in serum and bronchoalveolar lavage fluid (BALF). Subsequently, the main components in medicated serum of SHD decoction were inspected by Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The material basis of SHD decoction and potential interaction mechanisms were analysed by systemic pharmacology. To confirm the reliability of network pharmacology for predicting outcomes, we used molecular docking techniques to identify interactions between the core components and targets of SHD. Finally, TUNEL staining, immunofluorescence and western blotting were used to explore the mechanism of SHD decoction through the inhibition of GSDMD-mediated pyroptosis in septic mice. RESULTS: SHD was found to be effective in reducing the mortality and alleviating lung pathological damage in septic mice. Multifactor microarray results showed that SHD can reduce the expression of inflammation factors (IL-18, IL-1ß, IL-5, IL-6 and TNF-α) in serum and BALF of septic mice. There were 22 major blood-entry components detected by UPLC-MS/MS. Then, combined with the network pharmacological analysis, it is evident that the main components of SHD for sepsis are Renshen-ginsenoside Rh2, Danshen-tanshinone IIA and Dahuang-rhein. The main targets were IL-1ß and caspase-1, which were related to GSDMD-mediated pyroptosis signalling pathway. Molecular docking exhibited that Renshen-ginsenoside Rh2, Danshen-tanshinone IIA and Dahuang-rhein can closely bind to GSDMD, IL-1ß and caspase-1. In addition, TUNEL staining and immunohistochemistry demonstrated that SHD alleviated the expression of GSDMD protein. The western blotting showed that SHD significantly inhibited the protein expression levels of NLRP3, GSDMD, GSDMD-N, cleved caspase-1, caspase-1 and ASC in lung tissue. CONCLUSIONS: Our study revealed that SHD improves CLP-induced lung injury by blocking the GSDMD-mediated pyroptosis signalling pathway in septic mice. This study provides evidence to support that SHD had a potential therapeutic effect on sepsis.