Ligustilide covalently binds to Cys703 in the pre-S1 helix of TRPA1, blocking the opening of channel and relieving pain in rats with acute soft tissue injury.

ETHNOPHARMACOLOGICAL RELEVANCE: The natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive. AIM OF THE STUDY: The objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel. METHODS: The therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca2+ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches. RESULTS: The administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca2+ influx through the channel opening and ultimately relieving pain. CONCLUSIONS: Lig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.

Catalpolaglycone disrupts mitochondrial thermogenesis by specifically binding to a conserved lysine residue of UCP2 on the proton leak tunnel.

BACKGROUND: Catalpol (CAT), a naturally occurring iridoid glycoside sourced from the root of Rehmannia glutinosa, affects mitochondrial metabolic functions. However, the mechanism of action of CAT against pyrexia and its plausible targets remain to be fully elucidated. PURPOSE: This study aimed to identify the specific targets of CAT for blocking mitochondrial thermogenesis and to unveil the unique biological mechanism of action of the orthogonal binding mode between the hemiacetal group and lysine residue on the target protein in vivo. METHODS: Lipopolysaccharide (LPS)/ carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced fever models were established to evaluate the potential antipyretic effects of CAT. An alkenyl-modified CAT probe was designed to identify and capture potential targets. Binding capacity was tested using in-gel imaging and a cellular thermal shift assay. The underlying antipyretic mechanisms were explored using biochemical and molecular biological methods. Catalpolaglycone (CA) was coupled with protein profile identification and molecular docking analysis to evaluate and identify its binding mode to UCP2. RESULTS: After deglycation of CAT in vivo, the hemiacetal group in CA covalently binds to Lys239 of UCP2 in the mitochondria of the liver via an ɛ-amine nucleophilic addition. This irreversible binding affects proton leakage and improves mitochondrial membrane potential and ADP/ATP transformation efficiency, leading to an antipyretic effect. CONCLUSION: Our findings highlight the potential role of CA in modulating UCP2 activity or function within the mitochondria and open new avenues for investigating the therapeutic effects of CA on mitochondrial homeostasis.

Astragaloside IV protects against lung injury and pulmonary fibrosis in COPD by targeting GTP-GDP domain of RAS and downregulating the RAS/RAF/FoxO signaling pathway.

BACKGROUND: Pulmonary fibrosis is a chronic progressive interstitial lung disease characterized by the replacement of lung parenchyma with fibrous scar tissue, usually as the final stage of lung injury like COPD. Astragaloside IV (AST), a bioactive compound found in the Astragalus membranaceus (Fisch.) used in traditional Chinese medicine, has been shown to improve pulmonary function and exhibit anti-pulmonary fibrosis effects. However, the exact molecular mechanisms through which it combats pulmonary fibrosis, especially in COPD, remain unclear. PURPOSE: This study aimed to identify the potential therapeutic target and molecular mechanisms for AST in improving lung injury especially treating COPD type pulmonary fibrosis both in vivo and in vitro. METHODS: Multi lung injury models were established in mice using lipopolysaccharide (LPS), cigarette smoke (CS), or LPS plus CS to simulate the processes of pulmonary fibrosis in COPD. The effect of AST on lung function protection was evaluated, and proteomic and metabolomic analysis were applied to identify the signaling pathway affected by AST and to find potential targets of AST. The interaction between AST and wild-type and mutant RAS proteins was studied. The RAS/RAF/FoxO signaling pathway was stimulated in BEAS-2B cells and in mice lung tissues by LPS plus CS to investigate the anti-pulmonary fibrosis mechanism of AST analyzed by western blotting. The regulatory effects of AST on the RAS/RAF/FoxO pathway dependent on RAS were further confirmed using RAS siRNA. RESULTS: RAS was predicted and identified as the target protein of AST in anti-pulmonary fibrosis in COPD and improving lung function. The administration of AST was observed to impede the conversion of fibroblasts into myofibroblasts, reduce the manifestation of inflammatory factors and extracellular matrix, and hinder the activation of epithelial mesenchymal transition (EMT). Furthermore, AST significantly suppressed the RAS/RAF/FoxO signaling pathway in both in vitro and in vivo settings. CONCLUSION: AST exhibited lung function protection and anti-pulmonary fibrosis effect by inhibiting the GTP-GDP domain of RAS, which downregulated the RAS/RAF/FoxO signaling pathway. This study revealed AST as a natural candidate molecule for the protection of pulmonary fibrosis in COPD.

Ginsenoside CK targeting KEAP1-DGR/Kelch domain disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage.

BACKGROUND: Panax ginseng and Panax notoginseng as traditional Chinese medicines, are widely used in the treatment of qi deficiency, viral or bacterial infection, inflammation and cancer. Ginsenoside CK, an active metabolite of protopanoxadiol among the ginseng saponins, has been shown in previous studies to improve the organism's oxidative balance by regulating the KEAP1-NRF2/ARE pathway, thus slowing the progression of diseases. However, the specific targets and mechanisms of CK in improving oxidative stress remain unclear. PURPOSE: The aim of this study was to determine the potential therapeutic targets and molecular mechanisms of CK in improving oxidative stress injury both in vitro and in vivo. METHODS: LPS was used to induce oxidative damage in RAW 264.7 cells to evaluate the regulatory effects of CK on the KEAP1-NRF2/ARE pathway. Drug affinity responsive target stability technology (DARTS) combined with proteomics was employed to identify CK's potential target proteins. CK functional probe were designed to analyze the target protein using click chemistry. Furthermore, small molecule and protein interaction technologies were used to verify the mechanism, and computer dynamic simulation technology was used to analyze the interaction sites between CK and the target protein. The pharmacological effects and mechanism of CK in improving oxidative damage were verified in vivo by LPS-induced acute injury in mice and physical mechanical injury in rat soft tissues. RESULTS: KEAP1 was identified as the target protein that CK regulates to improve oxidative damage through the KEAP1-NRF2/ARE pathway. CK competitively binds to the DGR/Kelch domain of KEAP1, disrupting the binding between DLG peptide in NRF2 and KEAP1, thereby inhibiting the occurrence of oxidative damage induced by LPS or physical mechanical stress. CONCLUSIONS: CK functions as a natural KEAP1-NRF2 inhibitor, disrupting the binding between KEAP1 and NRF2-DLG motifs by targeting the DGR/Kelch domain of KEAP1, activating the antioxidant transcriptional program of NRF2, and reducing oxidative stress damage.

Fuziline Ameliorates Glucose and Lipid Metabolism by Activating Beta Adrenergic Receptors to Stimulate Thermogenesis.

Radix aconiti carmichaeli is a widely used traditional Chinese medicine that has been found to be effective in treating cardiovascular diseases and metabolic disorders. Patients with these diseases often experience a heat generation disorder, which is characterized by chilliness and can worsen the progression of the disease. This study established an in vitro screening model combining the examination of cellular mitochondrial membrane potential and mitochondrial temperature to screen drugs with thermogenic activity. After differentiation and determination of the content of characteristic metabolites of the drug-containing serum blood components, it was found that Fuziline (FZL) is the key thermogenic property in Radix aconiti carmichaeli, responsible for its thermogenic effects with a high relative importance of 33%. Experiments were conducted to evaluate the thermogenic activity of Radix aconiti carmichaeli and FZL in vivo by assessing temperature changes in various organs, including the rectum, liver, and brown adipose tissue. Moreover, the effects of intracellular ß3-adrenergic receptor (ß3-AR) agonistic effects were evaluated using transient ß3-AR transfection and dual-luciferase assay systems. The molecular mechanism by which FZL promotes thermogenesis and improves mitochondrial function was investigated by verifying the ß-adrenergic receptors (ß-AR) downstream signaling pathway. The results suggest that FZL activates ß-AR nonselectively, which in turn activates the downstream cAMP-PKA signaling pathway and leads to an increase in liver glycogenolysis and triglyceride hydrolysis, accompanied by enhancing mitochondrial energy metabolism. Consequently, the liver and brown adipose tissue receive energy to generate heat. In summary, these findings provide insight into the therapeutic application of Radix aconiti carmichaeli for metabolic disorders associated with heat generation disorders.

Astragaloside IV targets PRDX6, inhibits the activation of RAC subunit in NADPH oxidase 2 for oxidative damage.

BACKGROUND: Radix Astragali Mongolici, as a traditional Chinese medicine, is widely used in the treatment of qi deficiency, viral or bacterial infection, inflammation and cancer. Astragaloside IV (AST), a key active compound in Radix Astragali Mongolici, has been shown to reduce disease progression by inhibiting oxidative stress and inflammation. However, the specific target and mechanism of action of AST in improving oxidative stress are still unclear. PURPOSE: This study aims to explore the target and mechanism of AST to improve oxidative stress, and to explain the biological process of oxidative stress. METHODS: AST functional probes were designed to capture target proteins and combined with protein spectrum to analyze target proteins. Small molecule and protein interaction technologies were used to verify the mode of action, while computer dynamics simulation technology was used to analyze the site of interaction with the target protein. The pharmacological activity of AST in improving oxidative stress was evaluated in a mouse model of acute lung injury induced by LPS. Additionally, pharmacological and serial molecular biological approaches were used to explore the underlying mechanism of action. RESULTS: AST inhibits PLA2 activity in PRDX6 by targeting the PLA2 catalytic triad pocket. This binding alters the conformation and structural stability of PRDX6 and interferes with the interaction between PRDX6 and RAC, hindering the activation of the RAC-GDI heterodimer. Inactivation of RAC prevents NOX2 maturation, attenuates superoxide anion production, and improves oxidative stress damage. CONCLUSION: The findings of this research indicate that AST impedes PLA2 activity by acting on the catalytic triad of PRDX6. This, in turn, disrupts the interaction between PRDX6 and RAC, thereby hindering the maturation of NOX2 and diminishing the oxidative stress damage.

Dietary naringin supplementation on hepatic yolk precursors formation and antioxidant capacity of Three-Yellow breeder hens during the late laying period.

In this study, the effects of naringin on hepatic yolk precursors formation and antioxidant capacity of Three-Yellow breeder hens during late laying period were evaluated. A total of 480 (54-wk-old) Three-Yellow breeder hens were randomly assigned to 4 groups (6 replicates of 20 hens): nonsupplemented control diet (C), and control diet supplemented with 0.1%, 0.2%, and 0.4% of naringin (N1, N2, and N3), respectively. Results showed that dietary supplemented with 0.1%, 0.2%, and 0.4% of naringin for 8 wk promoted the cell proliferation and attenuated the excessive fat accumulation in the liver. Compared with C group, increased concentrations of triglyceride (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C), and very low-density lipoprotein (VLDL), and decreased contents of low-density lipoprotein cholesterol (LDL-C) were detected in liver, serum and ovarian tissues (P < 0.05). After 8 wk of feeding with naringin (0.1%, 0.2%, and 0.4%), serum estrogen (E2) level, expression levels of proteins and genes of estrogen receptors (ERs) increased significantly (P < 0.05). Meanwhile, naringin treatment regulated expression of genes related to yolk precursors formation (P < 0.05). Furthermore, dietary naringin addition increased the antioxidants, decreased the oxidation products, and up-regulated transcription levels of antioxidant genes in liver tissues (P < 0.05). These results indicated that dietary supplemented with naringin could improve hepatic yolk precursors formation and hepatic antioxidant capacity of Three-Yellow breeder hens during the late laying period. Doses of 0.2% and 0.4% are more effective than dose of 0.1%.

Dietary naringin supplementation on laying performance and antioxidant capacity of Three-Yellow breeder hens during the late laying period.

In this study, the effects of 3 graded dietary levels (0.1%, 0.2%, and 0.4%) of naringin were studied in Three-Yellow breeder hens during the late laying period (55-62 wk). A total of 480 Three-Yellow breeder hens (54-wk-old) were randomly divided into 4 groups (6 replicates of 20 hens): basal diet group (C), and basal diets supplemented with 0.1%, 0.2%, and 0.4% of naringin (N1, N2, and N3), respectively. Results showed that dietary supplementation with 0.1%, 0.2%, and 0.4% of naringin for 8 wk increased the laying rate and egg mass, enhanced egg yolk color, and decreased the feed egg ratio (P < 0.05). Meanwhile, compared with hens in C group, there were more preovulatory follicles and higher ovarian index as well as an enhanced ovarian somatic cell proliferation in hens of N2 and N3 groups (P < 0.05). With 0.2% and 0.4% naringin, glutathione concentration, the activity of catalase and total superoxide dismutase, and the total antioxidant capacity of ovarian tissues and serum increased (P < 0.05), while the contents of malondialdehyde and hydrogen peroxide decreased (P < 0.05). Moreover, compared to C group, the transcription levels of antioxidant genes in ovarian tissues increased in hens from N2 and N3 groups (P < 0.05). In conclusion, supplementation with 0.2% and 0.4% naringin both could improve the laying rate, ovarian and serum antioxidant capacity of Three-Yellow breeder hens during the late laying period.

Ursolic acid inhibits the cholesterol biosynthesis and alleviates high fat diet-induced hypercholesterolemia via irreversible inhibition of HMGCS1 in vivo.

BACKGROUND: In hypercholesteremia, the concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are enhanced in serum, which is strongly associated with an increased risk of developing atherosclerosis. Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid, was found to alleviate hypercholesterolemia and hypercholesterolemia-induced cardiovascular disease. However, the specific targets and molecular mechanisms related to the effects of UA in reducing cholesterol have not been elucidated. PURPOSE: In this study, we aimed to illustrate the target of UA in the treatment of hypercholesterolemia and to reveal its underlying molecular mechanism. METHODS: Nontargeted metabolomics was conducted to analyze the metabolites and related pathways that UA affected in vivo. The main lipid metabolism targets of UA were analyzed by target fishing and fluorescence colocalization in mouse liver. Molecular docking, in-gel fluorescence scan and thermal shift were assessed to further investigate the binding site of the UA metabolite with HMGCS1. C57BL/6 mice were fed a high-fat diet (HFD) for 12 weeks to induce hypercholesteremia. Liver tissues were used to verify the cholesterol-lowering molecular mechanism of UA by targeted metabolomics, serum was used to detect biochemical indices, and the entire aorta was used to analyze the formation of atherosclerotic lesions. RESULTS: Our results showed that hydroxy­3-methylglutaryl coenzyme A synthetase 1 (HMGCS1) was the primary lipid metabolism target protein of UA. The UA metabolite epoxy-modified UA irreversibly bonds with the thiol of Cys-129 in HMGCS1, which inhibits the catalytic activity of HMGCS1 and reduces the generation of precursors in cholesterol biosynthesis in vivo. The contents of TC and LDL-C in serum and the formation of the atherosclerotic area in the entire aorta were markedly reduced with UA treatment in Diet-induced hypercholesteremia mice. CONCLUSION: UA inhibits the catalytic activity of HMGCS1, reduces the generation of downstream metabolites in the process of cholesterol biosynthesis and alleviates Diet-induced hypercholesteremia via irreversible binding with HMGCS1 in vivo. It is the first time to clarify the irreversible inhibition mechanism of UA against HMGCS1. This paper provides an increased understanding of UA, particularly regarding the molecular mechanism of the cholesterol-lowering effect, and demonstrates the potential of UA as a novel therapeutic for the treatment of hypercholesteremia.

Characterization of deglycosylated metabolites of platycosides reveals their biotransformation after oral administration.

Platycodon grandiflorus is a well-known edible and medicinal plant that has been developed for dietary supplements or functional foods to relieve pulmonary disorders. Platycosides are the main active constituents of P. grandiflorus with multiple pharmacological activities. However, their metabolic fates after dietary consumption are still unclear. Herein, 25 deglycosylated metabolites of platycosides were identified, most of which were identified in vivo for the first time. Notably, 3-O-ß-d-glucopyranosyl platycosides could be absorbed into the bloodstream, and their structures were unambiguously characterized with the aid of chemically prepared standards, including two new compounds (M3 and M11). These findings reveal that both intestinal bacterial metabolism and hydrolysis of ester linkage at C-28 by carboxylesterases in liver are the possible in vivo deglycosylation metabolism pathway of platycosides. This study greatly facilitated our understanding of the fate of the platycosides after dietary consumption of P. grandiflorus products.

Targeting thalamic circuits rescues motor and mood deficits in PD mice.

Although bradykinesia, tremor and rigidity are the hallmark motor defects in patients with Parkinson's disease (PD), patients also experience motor learning impairments and non-motor symptoms such as depression1. The neural circuit basis for these different symptoms of PD are not well understood. Although current treatments are effective for locomotion deficits in PD2,3, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking4-6. Here we found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN) and nucleus accumbens (NAc). Whereas PF→CPu and PF→STN circuits are critical for locomotion and motor learning, respectively, inhibition of the PF→NAc circuit induced a depression-like state. Whereas chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation (LTP) at PF→STN synapses restored motor learning behaviour in an acute mouse model of PD. Furthermore, activation of NAc-projecting PF neurons rescued depression-like phenotypes. Further, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

Differences in egg yolk precursor formation of Guangxi Ma chickens with dissimilar laying rate at the same or various ages.

Egg yolk formation and deposition are quite vital for oocyte maturation and egg production in hens. There are great differences in egg production and number of preovulatory follicles among individuals in Guangxi Ma chickens at the same or various ages, however, the causes of this phenomenon remain unclear. This study aimed to elucidate the differences in yolk precursors formation among Guangxi Ma hens at the same or various ages with dissimilar egg laying rate via comparing the synthesis and transportation of yolk precursors, serum estradiol (E2) levels, as well as the antioxidant capacity of the liver and serum from hens of 32-week-old (32W), 50-week-old with higher laying rate (50WH), 50-week-old with lower laying rate (50WL), 72-week-old with higher laying rate (72WH), and 72-week-old with lower laying rate (72WL). The results showed that the contents of triglyceride (TG), total cholesterol (T-CHO), high density lipoprotein cholesterol (HDL-C) and very low density lipoprotein cholesterol (VLDL-C) in the liver, serum and ovarian stroma, serum E2 levels, expression levels of proteins and genes related to yolk precursors formation, and the antioxidant capacity of liver and serum decreased significantly during aging process. TG, HDL-C and VLDL-C contents in serum and ovarian stroma, as well as HDL-C and VLDL-C levels in liver tissues of hens in 50WL and 72WL were significantly lower compared to hens in 50WH and 72WH, respectively. Meanwhile, expression levels of estrogen receptor ɑ and transcription levels of genes related to lipid transportation in liver tissues, and the antioxidant capacity of livers were remarkably lower in hens in 50WL and 72WL than those in 50WH and 72WH, respectively. However, no significant difference was detected in liver TG levels, serum E2 levels, genes related to fatty acids synthesis and serum antioxidant capacity between hens in 50WL and 50WH. In conclusion, the age-related decline of egg production in Guangxi Ma chicken maybe related to the age-related decreases in the synthesis and transportation of liver yolk precursor as well as the antioxidant capacity of liver and hens. And the non-age related decline of egg production may mainly attribute to the decreases in yolk precursors transportation ability and hepatic antioxidant capacity.

Multi-omics analysis reveals the mechanisms of action and therapeutic regimens of traditional Chinese medicine, Bufei Jianpi granules: Implication for COPD drug discovery.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is a serious public health challenge in the world. According to the treatment instructions by Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2020, bronchiectasis combine with inhaled corticosteroids and long-acting anti-muscarinic agents were recommended as the main prescription. However, this symptomatic treatment still has ineluctable limits because it ignored the most pathogenesis mechanism of COPD. As an alternative traditional Chinese medicine (TCM) for COPD, Bufei Jianpi granules (BJG) can reduce the frequency and duration of acute exacerbation in COPD patients and improve their quality of life. The evidence demonstrated BJG acts as therapeutics that retarding the airway remodeling process, eliminating phlegm, thrombolysis and improving mitochondrial function. However, the detailed molecular mechanism is still urgently revealed. PURPUSE: In this study, we aim to find out the active pharmacodynamic ingredients and reveal the treatment mechanism of active pharmacodynamic ingredients. METHODS: Based on the pharmacodynamic evaluation and chemomic profiling of BJG in COPD rats, an integrated multi-omics analysis was performed, including molecular networking, metabonomics, proteomics and bioinformatics. Moreover, focus on the active compounds, we verified the molecular core mechanism by molecular biology methods. RESULTS: Pachymic acid, shionone, peiminine and astragaloside A was verified as therapeutic agents for improving the condition of COPD by acting on the EGFR, ERK1, PAI-1 and p53 target, respectively. CONCLUSION: In this study, our findings indicated that some compounds in BJG alleviates the pathological process of COPD, which is related to regulating lung function, mucus production, pulmonary embolism and energy metabolism and this will be a benefit complementary to GOLD guidelines.

Computational study on novel natural inhibitors targeting c-MET.

ABSTRACT: This study was designed to select ideal lead compounds and preclinical drug candidates http://dict.youdao.com/w/eng/preclinical_drug_candidate/javascript:void (0); with inhibitory effect on c-MET from the drug library (ZINC database).A battery of computer-aided virtual techniques was used to identify possible inhibitors of c-MET. A total of 17,931 ligands were screened from the ZINC15 database. LibDock is applied for structure-based screening followed by absorption, distribution, metabolic, and excretion, and toxicity prediction. Molecular docking was conducted to confirm the binding affinity mechanism between the ligand and c-MET. Molecular dynamics simulations were used to assess the stability of ligand-c-MET complexes.Two new natural compounds ZINC000005879645 and ZINC000002528509 were found to bind to c-MET in the ZINC database, showing higher binding affinity. In addition, they were predicted to have lower rodent carcinogenicity, Ames mutagenicity, developmental toxicity potential, and high tolerance to cytochrome P4502D6. Molecular dynamics simulation shows that ZINC000005879645 and ZINC000002528509 have more favorable potential energies with c-MET, which could exist stably in the natural environment.This study suggests that ZINC000005879645 and ZINC000002528509 are ideal latent inhibitors of c-MET targeting. As drug candidates, these 2 compounds have low cytotoxicity and hepatotoxicity as well as important implications for the design and improvement of c-MET target drugs.

The rich pharmacological activities of Magnolia officinalis and secondary effects based on significant intestinal contributions.

ETHNOPHARMACOLOGICAL RELEVANCE: Magnolia officinalis Cortex (M. officinalis) is a traditional herbal drug widely used in Asian countries. Depending on its multiple biological activities, M. officinalis is used to regulate gastrointestinal (GI) motility, relieve cough and asthma, prevent cardiovascular and cerebrovascular diseases, and treat depression and anxiety. AIM OF THE REVIEW: We aimed to review the abundant form of pharmacodynamics activity and potential mechanisms of action of M. officinalis and the characteristics of the internal processes of the main components. The potential mechanisms of local and distance actions of M. officinalis based on GI tract was provided, and it was used to reveal the interconnections between traditional use, phytochemistry, and pharmacology. MATERIALS AND METHODS: Published literatures about M. officinalis and its main components were collected from several scientific databases, including PubMed, Elsevier, ScienceDirect, Google Scholar and Web of Science etc. RESULTS: M. officinalis was shown multiple effects including effects on digestive system, respiratory system, central system, which is consistent with traditional applications, as well as some other activities such as cardiovascular system, anticancer, anti-inflammatory and antioxidant effects and so on. The mechanisms of these activities are abundant. Its chief ingredients such as magnolol and honokiol can be metabolized into active metabolites in vivo, which can increase water solubility and bioavailability and exert pharmacological activity in the whole body. In the GI tract, M. officinalis and its main ingredient can regulate GI hormones and substance metabolism, protect the intestinal barrier and affect the gut microbiota (GM). These actions are effective to improve local discomfort and some distal symptoms such as depression, asthma, or metabolic disorders. CONCLUSIONS: Although M. officinalis has rich pharmacological effects, the GI tract makes great contributions to it. The GI tract is not only an important place for absorption and metabolism but also a key site to help M. officinalis exert local and distal efficacy. Pharmacodynamical studies on the efficacies of distal tissues based on the contributions of the GI tract hold great potential for understanding the benefits of M. officinalis and providing new ideas for the treatment of important diseases.

Integrated molecular network and HPLC-UV-FLD analysis to explore antioxidant ingredients in camellia nitidissima Chi.

At present, screening of active ingredients from natural products for pharmacological and clinical research is mostly time-consuming and costly. In this study, a molecular network (MN) guided high performance liquid chromatography-ultraviolet-fluorescence detector (HPLC-UV-FLD) method was carried out to profile the global antioxidant activity compounds, including the trace amount ingredients in Camellia nitidissima Chi (CNC). Firstly, HPLC-UV-FLD postcolumn derivatization system was utilized to screen the antioxidants. Then the MN of CNC was established via mass spectrometry (MS) data for getting the connection between ingredient structures. As a result, HPLC-UV-FLD indicated three antioxidant ingredients: gallic acid (126.3 mg/g), catechin (564.8 mg/g), and salicylic acid (24.3 mg/g). Combined with the MN, the actives' precise location and connection relationship were clarified based on the structural similarities. A new antioxidant ingredient, okicamelliaside, was suggested and evaluated at free radical scavenging and enzymatic protection. The novel method of activity and structural correlation analysis based on MN could provide a useful guide for screening trace active ingredients in natural products. PRACTICAL APPLICATION: Three main ingredients were screened out from Camellia nitidissima Chi by HPLC-UV-FLD postcolumn derivatization system. Integrated molecular network and HPLC-UV-FLD analysis, a new type of antioxidant okicamelliaside was selected. The novel method of activity and structural correlation analysis based on molecular network could provide a useful guide for screening trace active ingredients in natural products.

Integrated artificial neural network analysis and functional cell based affinity mass spectrometry for screening a bifunctional activator of Ca2+ and ß2AR in aconite.

Arrhythmia, a common heart disease, is an abnormal frequency or rhythm of heartbeat caused by the origin or conduction obstacle of the heart. Aconite (Fuzi) has been regarded as an effective cardiotonic agent in traditional Chinese medicine (TCM), but it sometimes induces cardiac toxicity, such as arrhythmia, in the clinic. It is still a challenge to identify the active ingredients related to the therapeutic effect or toxicity in the application of aconite. To clarify which ingredient or derivative plays a key role of reducing toxicity and improving efficiency in the use of aconite, a serum pharmacology approach integrated with metabolomics and artificial neural network (ANN) analysis was used to in vivo screen Ca2+ and ß2AR regulators from the extracts of Heishunpian (HSP), which a processed lateral root of aconite. In addition, ß2AR transfected CHO and myocardium H9C2 functional cells-based affinity mass spectrometry (AMS) screening tests were carried out for evaluating the active ingredients in vitro. The results demonstrated that the monoester diterpenoid alkaloids (MDAs) represented by fuziline were the key bifunctional activators that could activate Ca2+ and ß2AR simultaneously. The effective compatibility of the calcium antagonists could regulate the heart rhythm to alleviate arrhythmia while maintain their cardiotonic effect, which was induced by aconite. In this study, ANN analysis based on serum pharmacology combined with AMS screen, which utilized with functional cells presented a powerful analytical strategy to the discovery and evaluation of active ingredients from a complex system.

GluA1 in Central Amygdala Promotes Opioid Use and Reverses Inhibitory Effect of Pain.

Increasing evidence suggests that long-term opioids and pain induce similar adaptive changes in the brain's reward circuits, however, how pain alters the addictive properties of opioids remains poorly understood. In this study using a rat model of morphine self-administration (MSA), we found that short-term pain, induced by an intraplantar injection of complete Freund's adjuvant (CFA), acutely decreased voluntary morphine intake, but not food intake, only at a morphine dose that did not affect pain itself. Pre-treatment with indomethacin, a non-opioid inhibitor of pain, before the pain induction blocked the decrease in morphine intake. In rats with steady MSA, the protein level of GluA1 subunits of glutamate AMPA receptors (AMPARs) was significantly increased, but that of GluA2 was decreased, resulting in an increased GluA1/GluA2 ratio in central nucleus of the amygdala (CeA). In contrast, pain decreased the GluA1/GluA2 ratio in the CeA of rats with MSA. Microinjection of NASPM, a selective inhibitor of homomeric GluA1-AMPARs, into CeA inhibited morphine intake. Furthermore, viral overexpression of GluA1 protein in CeA maintained morphine intake at a higher level than controls and reversed the pain-induced reduction in morphine intake. These findings suggest that CeA GluA1 promotes opioid use and its upregulation is sufficient to increase opioid consumption, which counteracts the acute inhibitory effect of pain on opioid intake. These results demonstrate that the CeA GluA1 is a shared target of opioid and pain in regulation of opioid use, which may aid in future development of therapeutic applications in opioid abuse.

A natural AKT inhibitor swertiamarin targets AKT-PH domain, inhibits downstream signaling, and alleviates inflammation.

Swertiamarin (SW), a representative component in Flos Lonicerae Japonicae, has been reported to exert significant activity in preventing infections. In this research, we aim to clarify the details of SW and its target to explore SW's underlying anti-inflammatory mechanisms. An azide labeled SW probe was synthesized for protein target fishing, and the results demonstrated that AKT could be captured specifically. Immunofluorescence colocalization with AKT was implemented by a click reaction of the SW probe and alkynyl CY5. The result showed that AKT was one of the targets of SW. Then, a competitive combination experiment using a set of AKT inhibitors and a membrane translocation experiment confirmed that SW might target the pleckstrin homology (PH) domain of AKT. This specific binding directly deactivated the phosphorylation of AKT on both Ser473 and Thr308, which induced the dephosphorylation of IKK and NF-κB. Finally, proinflammatory cytokines (TNF-α, IL-6, and IL-8) were suppressed both in cells and in acute lung injury animal model by targeting AKT-PH domain. This study demonstrated that SW functions as a natural AKT inhibitor and presents significant anti-inflammatory activity by directly regulating the AKT-PH domain and inhibiting downstream inflammatory molecules.

Micro-PET Imaging Demonstrates 3-O-ß-D-Glucopyranosyl Platycodigenin as an Effective Metabolite Affects Permeability of Cell Membrane and Improves Dosimetry of [18F]-Phillygenin in Lung Tissue.

Platycodon grandiflorum, as a traditional medicinal plant, is commonly used in the treatment of pulmonary disease. Platycodon saponins are proposed as active ingredients. However, the role of secondary saponin metabolites (SSM) in the traditional use of Platycodon has not yet been fully clarified. In this study, [18F]-phillygenin ([18F]-PH) probe was synthesized and thereby used as a tracer for micro-positron emission tomography scanning to explore the effects of platycodon saponins. The membrane permeability with different SSM was evaluated in vitro based on the dye-carrying capacity of fluorescein isothiocyanate. The results showed that total platycodon saponins improved the dosimetry of [18F]-PH in the lung tissue, and an SSM named 3-O-ß-D-glucopyranosyl platycodigenin (GPD682) appreciably changed the distribution of drugs both in vitro and in vivo. We propose that GPD682 could be utilized as an important ingredient to help drug delivery to the lung tissue and improve the treatment of respiratory disease.