Carboxymethylated Abrus cantoniensis polysaccharide prevents CTX-induced immunosuppression and intestinal damage by regulating intestinal flora and butyric acid content.

As a Chinese folk health product, Abrus cantoniensis exhibits good immunomodulatory activity because of its polysaccharide components (ACP), and carboxymethylation of polysaccharides can often further improve the biological activity of polysaccharides. In this study, we explored the impact of prophylactic administration of carboxymethylated Abrus cantoniensis polysaccharide (CM-ACP) on immunosuppression and intestinal damage induced by cyclophosphamide (CTX) in mice. Our findings demonstrated that CM-ACP exhibited a more potent immunomodulatory activity compared to ACP. Additionally, CM-ACP effectively enhanced the abundance of short-chain fatty acid (SCFA)-producing bacteria in immunosuppressed mice and regulated the gene expression of STAT6 and STAT3 mediated pathway signals. In order to further explore the relationship among polysaccharides, intestinal immunity and intestinal flora, we performed a pseudo-sterile mouse validation experiment and fecal microbiota transplantation (FMT) experiment. The findings suggest that CM-FMT and butyrate attenuate CTX-induced immunosuppression and intestinal injury. CM-FMT and butyrate show superior immunomodulatory ability, and may effectively regulate intestinal cell metabolism and repair the damaged intestine by activating STAT6 and STAT3-mediated pathways. These findings offer new insights into the mechanisms by which CM-ACP functions as functional food or drug, facilitating immune response regulation and maintaining intestinal health.

Lyophilization enhances the stability of Panax notoginseng total saponins-loaded transfersomes without adverse effects on ex vivo/in vivo skin permeation.

Transfersomes (TFSs) have been extensively investigated to enhance transdermal drug delivery. As a colloidal dispersion system, TFSs are prone to problems such as particle aggregation and sedimentation, oxidation and decomposition of phospholipids. To enhance the stability of panax notoginseng saponins (PNS)-loaded transfersomes (PNS-TFSs) without adverse influences on their skin permeation, we prepared lyophilized PNS-loaded transfersomes (PNS-FD-TFSs), clarified their physicochemical characteristics and investigated their in vitro drug release, ex vivo skin permeation/deposition and in vivo pharmacokinetics. In this study, a simple, fast and controllable process was developed for preparing lyophilized PNS-TFSs. In the optimized PNS-FD-TFS formulation, sucrose and trehalose were added to the PNS-TFS dispersion with a mass ratio of trehalose, sucrose, and phospholipid of 3:2:1, and the mixture was frozen at -80 °C for 12 h followed by lyophilization at -45 °C and 5 Pa for 24 h. The optimized formulation of PNS-FD-TFSs was screened based on the appearance and reconstitution time of the lyophilized products, vesicle size, and PDI of the freshly reconstituted dispersions. It maintained stable physicochemical properties for at least 6 months at 4 °C. The vesicle size of PNS-FD-TFSs was below 100 nm and homogenous with a polydispersity index of 0.2 after reconstitution. The average encapsulation efficiencies of the five index saponins notoginsenoside R1 (NGR1), ginsenoside Rg1 (GRg1), ginsenoside Re (GRe), ginsenoside Rb1 (GRb1) and ginsenoside Rd (GRd) in PNS-FD-TFSs were 68.41 ± 5.77%, 68.95 ± 6.08%, 65.46 ± 10.95%, 91.50 ± 5.62% and 95.78 ± 1.70%, respectively. The reconstituted dispersions of PNS-FD-TFSs were similar to PNS-TFSs in in vitro release, ex vivo skin permeation, and deposition. The pharmacokinetic studies showed that, compared with the PNS liposomes (PNS-LPS), the PNS-FD-TFS-loaded drug could permeate through the skin and enter the blood rapidly. It can be concluded that the lyophilization process can effectively improve the stability of PNS-TFSs without compromising their transdermal absorption properties.

Magnolol alleviates pulmonary fibrosis inchronic obstructive pulmonary disease by targeting transient receptor potential vanilloid 4-ankyrin repeat domain.

Transient receptor potential vanilloid 4 (TRPV4) plays a role in regulating pulmonary fibrosis (PF). While several TRPV4 antagonists including magnolol (MAG), have been discovered, the mechanism of action is not fully understood. This study aimed to investigate the effect of MAG on alleviating fibrosis in chronic obstructive pulmonary disease (COPD) based on TRPV4, and to further analyze its mechanism of action on TRPV4. COPD was induced using cigarette smoke and LPS. The therapeutic effect of MAG on COPD-induced fibrosis was evaluated. TRPV4 was identified as the main target protein of MAG using target protein capture with MAG probe and drug affinity response target stability assay. The binding sites of MAG at TRPV4 were analyzed using molecular docking and small molecule interaction with TRPV4-ankyrin repeat domain (ARD). The effects of MAG on TRPV4 membrane distribution and channel activity were analyzed by co-immunoprecipitation, fluorescence co-localization, and living cell assay of calcium levels. By targeting TRPV4-ARD, MAG disrupted the binding between phosphatidylinositol 3 kinase γ and TRPV4, leading to hampered membrane distribution on fibroblasts. Additionally, MAG competitively impaired ATP binding to TRPV4-ARD, inhibiting TRPV4 channel opening activity. MAG effectively blocked the fibrotic process caused by mechanical or inflammatory signals, thus alleviating PF in COPD. Targeting TRPV4-ARD presents a novel treatment strategy for PF in COPD.

Psoralen prevents the inactivation of estradiol and treats osteoporosis via covalently targeting HSD17B2.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoralea corylifolia L. seeds (P. corylifolia), popularly known as Buguzhi in traditional Chinese medicine, are often used to treat osteoporosis in China. Psoralen (Pso) is the key anti-osteoporosis constituent in P. corylifolia, however, its targets and mechanism of action are still unclear. AIM OF THE STUDY: The purpose of this study was to explore the interaction between Pso and 17-ß hydroxysteroid dehydrogenase type 2 (HSD17B2), an estrogen synthesis-related protein that inhibits the inactivation of estradiol (E2) to treat osteoporosis. MATERIALS AND METHODS: Tissue distribution of Pso was analyzed by in-gel imaging after oral administration of an alkynyl-modified Pso probe (aPso) in mice. The target of Pso in the liver was identified and analyzed using chemical proteomics. Co-localization and cellular thermal shift assays (CETSA) were used to verify the key action targets. To detect the key pharmacophore of Pso, the interaction of Pso and its structural analogs with HSD17B2 was investigated by CETSA, HSD17B2 activity assay, and in-gel imaging determination. Target competitive test, virtual docking, mutated HSD17B2 activity, and CETSA assay were used to identify the binding site of Pso with HSD17B2. A mouse model of osteoporosis was established by ovariectomies, and the efficacy of Pso in vivo was confirmed by micro-CT, H&E staining, HSD17B2 activity, and bone-related biochemical assays. RESULTS: Pso regulated estrogen metabolism by targeting HSD17B2 in the liver, with the α, ß-unsaturated ester in Pso being the key pharmacophore. Pso significantly suppressed HSD17B2 activity by irreversibly binding to Lys236 of HSD17B2 and preventing NAD+ from entering the binding pocket. In vivo studies in ovariectomized mice revealed that Pso could inhibit HSD17B2 activity, prevent the inactivation of E2, increase levels of endogenous estrogen, improve bone metabolism-related indices, and play a role in anti-osteoporosis. CONCLUSIONS: Pso covalently binds to Lys236 of HSD17B2 in hepatocytes to prevent the inactivation of E2, thereby aiding in the treatment of osteoporosis.

Ligustilide attenuates airway remodeling in COPD mice by covalently binding to MH2 domain of Smad3 in pulmonary epithelium, disrupting the Smad3-SARA interaction.

Airway remodeling is one of the hallmarks of chronic obstructive pulmonary disease (COPD) and is closely related to the dysregulation of epithelial-mesenchymal transition (EMT). Smad3, an important transcriptional regulator responsible for transducing TGF-ß1 signals, is a promising target for EMT modulation. We found that ligustilide (Lig), a novel Smad3 covalent inhibitor, effectively inhibited airway remodeling in cigarette smoke (CS) combined with lipopolysaccharide (LPS)-induced COPD mice. Oral administration of an alkynyl-modified Lig probe was used to capture and trace target proteins in mouse lung tissue, revealing Smad3 in airway epithelium as a key target of Lig. Protein mass spectrometry and Smad3 mutation analysis via in-gel imaging indicated that the epoxidized metabolite of Lig covalently binds to the MH2 domain of Smad3 at Cys331/337. This irreversible bonding destroys the interaction of Smad3-SARA, prevents Smad3 phosphorylation activation, and subsequently suppresses the nuclear transfer of p-Smad3, the EMT process, and collagen deposition in TGF-ß1-stimulated BEAS-2B cells and COPD mice. These findings provide experimental support that Lig attenuates COPD by repressing airway remodeling which is attributed to its suppression on the activation of EMT process in the airway epithelium via targeting Smad3 and inhibiting the recruitment of the Smad3-SARA heterodimer in the TGF-ß1/Smad3 pathway.

Effects of the Probiotic, Lactobacillus delbrueckii subsp. bulgaricus, as a Substitute for Antibiotics on the Gastrointestinal Tract Microbiota and Metabolomics Profile of Female Growing-Finishing Pigs.

Lactobacillus delbrueckii subsp. bulgaricus (LDB) is an approved feed additive on the Chinese 'Approved Feed Additives' list. However, the possibility of LDB as an antibiotic replacement remains unclear. Particularly, the effect of LDB on microbiota and metabolites in the gastrointestinal tract (GIT) requires further explanation. This study aimed to identify the microbiota and metabolites present in fecal samples and investigate the relationship between the microbiota and metabolites to evaluate the potential of LDB as an antibiotic replacement in pig production. A total of 42 female growing-finishing pigs were randomly allocated into the antibiotic group (basal diet + 75 mg/kg aureomycin) and LDB (basal diet + 3.0 × 109 cfu/kg LDB) groups. Fecal samples were collected on days 0 and 30. Growth performance was recorded and assessed. 16S rRNA sequencing and liquid chromatography-mass spectrometry-based non-targeted metabolomics approaches were used to analyze the differences in microbiota and metabolites. Associations between the differences were calculated using Spearman correlations with the Benjamini−Hochberg adjustment. The LDB diet had no adverse effect on feed efficiency but slightly enhanced the average daily weight gain and average daily feed intake (p > 0.05). The diet supplemented with LDB increased Lactobacillus abundance and decreased that of Prevotellaceae_NK3B31_group spp. Dietary-supplemented LDB enhanced the concentrations of pyridoxine, tyramine, D-(+)-pyroglutamic acid, hypoxanthine, putrescine and 5-hydroxyindole-3-acetic acid and decreased the lithocholic acid concentration. The Lactobacillus networks (Lactobacillus, Peptococcus, Ruminococcaceae_UCG-004, Escherichia-Shigella, acetophenone, tyramine, putrescine, N-methylisopelletierine, N1-acetylspermine) and Prevotellaceae_NK3B31_group networks (Prevotellaceae_NK3B31_group, Treponema_2, monolaurin, penciclovir, N-(5-acetamidopentyl)acetamide, glycerol 3-phosphate) were the most important in the LDB effect on pig GIT health in our study. These findings indicate that LDB may regulate GIT function through the Lactobacillus and Prevotellaceae_NK3B31_group networks. However, our results were restrained to fecal samples of female growing-finishing pigs; gender, growth stages, breeds and other factors should be considered to comprehensively assess LDB as an antibiotic replacement in pig production.

Ursolic acid alleviates tetrandrine-induced hepatotoxicity by competitively binding to the substrate-binding site of glutathione S-transferases.

BACKGROUND: Tetrandrine (TET), a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra S. Moore, is the only approved medicine in China for silicosis. However, TET-induced hepatotoxicity has raised safety concerns. The underlying toxic targets and mechanism induced by TET remain unclear; there are no targeted detoxification strategies developed for TET-induced hepatotoxicity. Ursolic acid (UA), a pentacyclic triterpene with liver protective effects, may have detoxification effects on TET-induced hepatotoxicity. PURPOSE: This study aims to explore toxic targets and mechanism of TET and present UA as a potential targeted therapy for alleviating TET-induced hepatotoxicity. METHODS: A TET-induced liver-injury model was established to evaluate TET toxicity and the potential UA detoxification effect. Alkenyl-modified TET and UA probes were designed to identify potential liver targets. Pharmacological and molecular biology methods were used to explore the underlying toxicity/detoxification mechanism. RESULTS: TET induced liver injury by covalently binding to the substrate-binding pocket (H-site) of glutathione S-transferases (GSTs) and inhibiting GST activity. The covalent binding led to toxic metabolite accumulation and caused redox imbalance and liver injury. UA protected the liver from TET-induced damage by competitively binding to the GST H-site. CONCLUSION: The mechanism of TET-induced hepatotoxicity is related to irreversible binding with the GST H-site and GST-activity inhibition. UA, a natural antidote, competed with TET on H-site binding and reversed the redox imbalance. This study revealed the hepatotoxic mechanism of TET and provided a targeted detoxifying agent, UA, to alleviate hepatotoxicity caused by GST inhibition.

Psoralen induces hepatotoxicity by covalently binding to glutathione-S-transferases and the hepatic cytochrome P450.

BACKGROUND: Psoraleae Fructus has been widely used in China and its surroundings; however, Psoraleae Fructus and its compound preparation have been reported recently to cause liver injury in clinics. Thus, its safe use has attracted increasing attention. The possible mechanism is related to the metabolism of psoralen, but it still needs further clarification. PURPOSE: The present study was designed to evaluate the toxicity of psoralen and investigate the potentially related molecular mechanisms using chemical biology methods combined with animal experiments to provide evidence for the rational clinical use of psoralen. METHODS: An in vivo experiment was conducted with a time series of 20-80 mg/kg psoralen to verify its toxic performance. Target capture and click reactions were used to investigate direct targets of psoralen. Selectivity for different glutathione-S-transferase (GST) subtypes in the liver and inhibition of cytochrome P450 (CYP450) were also detected. RESULTS: Psoralen build-up in the liver is the primary cause of liver damage. Our study revealed the mechanism by which psoralen induces liver injury. Psoralen can bind directly to CYP2D6, CYP3A4, GST-α, and GST-µ and inhibit their activities, causing the depletion of glutathione (GSH) in vivo, which in turn induces hepatic damage. The special structure of α,ß-unsaturated lactones in psoralen facilitates its attachment to its target; therefore, complementing psoralen with GSH can efficiently protect the liver from damage. CONCLUSIONS: Psoralen causes a disorder in drug metabolism by inhibiting the activity of CYPs and GSTs, causing exhaustion of GSH, and subsequently leading to liver damage. The co-administration of GSH and psoralen is an effective way to avoid liver injury in clinical settings.

Exploration of the Mechanism of the Control of Coccidiosis in Chickens Based on Network Pharmacology and Molecular Docking With the Addition of Modified Gegen Qinlian Decoction.

Gegen Qinlian Decoction is a long-established Chinese herbal compound for the treatment of diarrhea and dysentery, while Magnolia officinalis has been demonstrated to have some anthelmintic activity. The preliminary screening of this study showed that the addition of Modified Gegen Qinlian Decoction has some effective on the prevention and treatment of coccidiosis in chickens. However, the mechanism of its treatment of chicken coccidiosis is not clear. The network pharmacology study was based on the screening of chemical components and related targets from TCMSP and PharmMapper server databases. Genes related to chicken coccidiosis were obtained from the SRA database, and those genes that intersected with the target genes of Modified Gegen Qinlian Decoction were screened. By exploring the target interactions through the String system and enrichment analysis by the Metascape system, the mechanism of action of Modified Gegen Qinlian Decoction in chicken coccidiosis was identified. Using real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the mRNA levels of the relevant factors in chicken coccidiosis, molecular docking was used to reveal the extent of binding of the key target genes predicted in the network pharmacology by the action of Modified Gegen Qinlian Decoction. Compound and target screening suggested that the 99 chemical targets of Modified Gegen Qinlian Decoction were involved in chicken coccidiosis, and the enrichment results of KEGG pathway suggested that Modified Gegen Qinlian Decoction was significantly associated with PI3K/AKT signaling pathway in chicken coccidiosis. The Hubba gene module in Cytoscape_v3.7.1 software was used to analyze the network topology to obtain the Hubba gene SRC, STAT3, and PPARG, etc. The molecular docking results showed that SRC, STAT3, and PPARG were key targets in the treatment of coccidiosis in chickens by Modified Gegen Qinlian Decoction, which was in agreement with the RT-qPCR results. Through network pharmacology, molecular docking and in vitro experiments, it was confirmed that Modified Gegen Qinlian Decoction fights against chicken coccidiosis through key targets such as SRC, STAT3, and PPARG.

Sinigrin Enhanced Antiasthmatic Effects of Beta Adrenergic Receptors Agonists by Regulating cAMP-Mediated Pathways.

Millions of patients suffer from asthma worldwide. However, the first-line drugs used to treat asthma, namely, the beta-adrenergic receptors agonists (ß-agonists), are not recommended for use as monotherapy because of their severe dose-related side effects. This limitation has prompted the search for new therapies, which can be used in conjunction with ß--agonists so that lower doses can be administered. Sinigrin is a major compound found in many antiasthmatic medicinal plants. In this study, we explored the antiasthmatic activity of sinigrin when used in combination with ß-agonists and its underlying mechanism. Sinigrin enhanced the asthma-relieving effects of isoproterenol and reduced the effective isoproterenol dose in an acute-asthma model in guinea pigs. Mechanistically, sinigrin enhanced the cAMP levels induced by ß-agonists by inhibiting PDE4. The resulting increase in cAMP levels stimulated the activity of the downstream effector protein kinase A, which would be expected to ultimately induce the relaxation of airway smooth muscle. In conclusion, sinigrin enhances the asthma-relieving effects of ß-agonists by regulating the cAMP signaling pathway and represents a potential add-on drug to ß-agonists for the treatment of asthma.

The Screening Research of NF-κB Inhibitors from Moutan Cortex Based on Bioactivity-Integrated UPLC-Q/TOF-MS.

Inflammation is a common and important pathological process, and nuclear factor-κB (NF-κB) is a key mediator of it. Moutan Cortex (MC), the dried root cortex of Paeonia suffruticosa Andr., is widely used as a remedy for the treatment of inflammatory diseases in Asian region. However, there are few studies on the systematic identification of NF-κB inhibitors of MC. In this study, the effect of inhibiting NF-κB activation of MC was assessed at the cellular level using a tumor necrosis factor-α (TNF-α) induced inflammatory model. Subsequently, ultra-performance liquid chromatography-quadrupole/time of flight-mass spectrometry (UPLC-Q/TOF-MS) combined with biological activity assay was established to screen and identify potential anti-inflammatory ingredients in MC. The results revealed that MC significantly inhibited the activation of NF-κB. Seven potential NF-κB inhibitors were screened from MC, including oxypaeoniflorin, paeoniflorin, galloylpaeoniflorin, benzoyloxypaeoniflorin, mudanpioside C, gallic acid, and paeonol. Among them, the NF-κB inhibitor activity of galloylpaeoniflorin, benzoyloxypaeoniflorin, and mudanpioside C is first reported here. In conclusion, the anti-inflammatory activity of MC was associated with the seven components mentioned above. And the bioactivity-integrated UPLC-Q/TOF which contains both chemical and bioactive details is suitable for screening active ingredients from natural medicines.

Searching for calcium antagonists for hypertension disease therapy from Moutan Cortex, using bioactivity integrated UHPLC-QTOF-MS.

INTRODUCTION: Calcium channel blockers (CCBs) are currently the most commonly used drugs for the treatment of hypertension. Moutan Cortex (MC), a traditional Chinese herb, has been found to have an anti-hypertensive effect. However, its potential mechanisms in the regulation of intracellular calcium concentration ([Ca2+ ]i ) remain poorly understood. OBJECTIVE: The main objective of this work was to identify the potential calcium antagonists from MC and study their molecular mechanisms. METHODS: Ultra-high performance liquid chromatography-quadrupole-time-of-fight-mass spectrometry (UHPLC-QTOF-MS) analysis combined with a dual-luciferase reporter assay was utilised to systematically screen the calcium antagonistic active ingredients in the methanol extract of MC. Additionally, the molecular mechanism of these compounds was further studied using live-cell imaging analysis with the calcium ion (Ca2+ ) probe dye fluo-4/AM to monitor changes in [Ca2+ ]i . RESULTS: Three monoterpenoids (paeoniflorin, benzoylpaeoniflorin and mudanpioside C), one phenolic acid (paeonol) and one gallotannin (1,2,3,4,6-O-pentagalloylglucose) were screened out as potential calcium antagonists in MC. Among them, the calcium antagonistic activity of benzoylpaeoniflorin, mudanpioside C and 1,2,3,4,6-O-pentagalloylglucose is first reported. Additionally, paeoniflorin, benzoylpaeoniflorin, mudanpioside C and paeonol can effectively block voltage-operated Ca2+ channels (VOCCs) to exert calcium antagonism, while 1,2,3,4,6-O-pentagalloylglucose plays a role in blocking inositol 1,4,5-trisphosphate receptors (IP3Rs). CONCLUSION: This work indicated that the anti-hypertensive efficacy of MC acted through multiple components selectively antagonising multiple cell signalling pathways to regulate [Ca2+ ]i . Furthermore, they could be considered as a reference standard for controlling the quality of Chinese medicinal materials.