Analyzing the molecular mechanisms of classic traditional Chinese medicine (TCM) formulas is essential for interpreting TCM principles through modern scientific frameworks, and serves as a cornerstone for establishing dominant disease categories of TCM. However, the lack of correlation with disease progression characteristics and clinical efficacy has become a scientific issue in this field that requires urgent resolution. To address this issue, this paper employs the classic formulas "Yuquan Pill" and "Xiaoke Prescription" for the treatment of "Xiaoke", a dominant disease in TCM, as a demonstration. Adhering to an "originating from clinic-returning to clinic" strategy, omics integrated with generative artificial intelligence is employed to identify active molecules and their biological associations in the formula, thereby uncovering integrated feature PANEL of disease progression and outcomes. This approach bridges the "clinical-basic" gap, enabling the characterization of emergent pharmacodynamic features that arise from metabolic network oscillations and chaotic dynamics. It reveals the synergistic principles of active molecules and their biological mechanisms in classic formulas. Returning to clinical practice, the focus is on validating the active molecules and discovering novel multi-component formulas with superior efficacy to that of classical formulas. This approach establishes a new paradigm for clearly elucidating and comprehensively clarifying the complex active molecules in classic TCM formulas.

Harpagoside (HPG) is an iridoid compound derived from Scrophularia ningpoensis Hemsl. This study aimed to investigate the therapeutic effect of HPG on metabolic dysfunction-associated steatohepatitis (MASH) and elucidate its underlying mechanisms. MASH was induced in mice using a methionine-choline-deficient diet (MCD). The efficacy of HPG in improving MASH was assessed through serum biochemical indexes, liver pathological changes, oxidative stress markers, and inflammatory responses (this experiment was approved by the Laboratory Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine, with approval number PZSHUTCM2305180005). To further explore the mechanism of action, Western blot (WB), quantitative real-time polymerase chain reaction (qPCR), and in vitro cell models were employed. The results demonstrated that HPG (30 and 60 mg·kg^-1) significantly mitigated liver damage (P < 0.05, P < 0.001), reduced hepatic lipid accumulation (P < 0.05, P < 0.001), lowered the elevated non-alcoholic fatty liver disease (NAFLD) activity score (NAS) (P < 0.05, P < 0.01, P < 0.001), alleviated hepatic oxidative stress injury, and suppressed hepatic inflammation in MCD-induced MASH mice (P < 0.05, P < 0.01, P < 0.001). WB and qPCR analyses revealed that HPG promoted the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) in the livers of MCD-fed mice and in AML-12 cells (P < 0.05). Additionally, it reduced the nuclear accumulation of nuclear factor kappa B (NF-κB) in the livers of MCD-fed mice and in THP-1 cells (P < 0.05). Collectively, these findings suggest that HPG improves MASH through alleviating liver oxidative and inflammatory injury by activating Nrf2 antioxidant signaling pathway and inhibiting NF-κB inflammatory signaling pathway.

Skin aging is a complex pathological process mediated by a combination of endogenous metabolic dysregulation and exogenous ultraviolet (UV) radiation, with oxidative stress due to excessive accumulation of reactive oxygen species (ROS) as the central driver. Rare ginsenoside Rh1 is a natural compound with antioxidant and anti-inflammatory properties, but its mechanism of action in UV-induced skin photoaging remains unclear. This study aims to investigate the protective effect of Rh1 against UV-induced skin aging and the underlying molecular mechanisms. All animal experiments were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (approval number: 20241230001). To evaluate the anti-photoaging effect of Rh1, we established photoaging models in BALB/c mice and human dermal fibroblasts (HDF). In vivo experiments showed that Rh1 (25 and 50 μmol·L^-1) significantly alleviated UV-induced skin thickening, erythema, and moisture loss, reduced the expression of pro-apoptotic protein Bax, and up-regulated anti-apoptotic protein Bcl-2. In vitro experiments indicated that Rh1 (12.5, 25, and 50 μmol·L^-1) reduced UVB (UV wavelength 280-320 nm)-induced ROS accumulation, oxidative damage, and the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells. Mechanistic studies demonstrated that Rh1 promotes nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation by activating sirtuin 3 (Sirt3), thereby upregulating downstream heme oxygenase-1 (HO-1) expression. Sirt3 siRNA experiments confirmed that gene silencing can blocks Rh1 from activating the Nrf2/HO-1 pathway, weakening its ability to inhibit ROS production and delay cellular aging. In summary, Rh1 alleviates UV-induced oxidative damage and skin aging through the Sirt3/Nrf2/HO-1 signaling axis, providing a new strategy for the development of anti-photoaging drugs targeting the antioxidant pathway.

α6/α3β2β3 nicotinic acetylcholine receptors (nAChRs) subtype is widely expressed in mesolimbic dopaminergic pathway and other regions, which plays a vital role in modulating the release of multiple neurotransmitters and drug reward mechanisms. It has become a potential therapeutic target for the treatment of various neuropsychiatric disorders and drug addiction. However, most ligands identified with high affinity and selectivity targeting α6/α3β2β3 subtype showed limited subtype discrimination against the closely related α3β2 subtype. Furthermore, the molecular mechanisms underlying ligands interact with α6/α3β2β3 nAChR remain absent. In this study, the related approaches of site-directed mutagenesis of receptor, in vitro transcription and electrophysiological assay were utilized to establish the drug screening models of wild-type α6/α3β2β3 nAChR and its α6/α3 subunit single-point mutants. The effects led by single amino acid substitution to the function of α6/α3β2β3 nAChR were also evaluated. The animal experimental protocol involved in this study has been approved by the Ethics Committee of Guangxi University (Approval No: GXU-2023-0249). As the results showed, the half maximal effective concentration (EC₅₀) for ACh activating α6/α3β2β3 nAChR was 18.5 μmol·L^-1. All 7 single-point mutations of α6/α3 subunit resulted in increase of the agonist sensitivity, while the EC₅₀ of ACh towards mutants α6/α3[V112E]β2β3、α6/α3[T147S]β2β3、α6/α3[E152K]β2β3 nAChR decreased to the greatest extent (EC₅₀s were 3.5, 4.4, and 2.0 μmol·L^-1, respectively, which were 0.1-0.2-fold relative to that of the wild-type receptor). These results established models for the research of structure-function relationship of α6/α3β2β3 nAChRs and molecular mechanism of ligand-receptor specific binding, as well as provided theoretical foundation for the design and optimization of selective drug leads targeting α6/α3β2β3 nAChRs.

To explore the protective effect of benzoylpaeoniflorin (BP), an active component of Paeonia lactiflora, on cerebral ischemic reperfusion injury (CIRI) in rats and its molecular mechanism, middle cerebral artery occlusion and reperfusion (MCAO/R) model was established, and oxygen-glucose deprivation/recovery (OGD/R) model was established by using rat adrenal pheochromocytoma nerve cells (PC12) to carry out experimental research. The results show that BP can significantly improve the neuronal apoptosis induced by CIRI, reduce the area of cerebral infarction, the neuronal damage in hippocampus, the loss of Nissl bodies and the mitochondrial damage. Western blot results showed that BP could significantly down-regulate the expression of pro-apoptosis proteins (Caspase3, Bax) and up-regulate the expression of anti-apoptosis protein (Bcl-2). qRT-PCR showed that BP down-regulated the gene expression levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and pro-apoptosis proteins (Caspase3, Bax), and up-regulated the expression level of anti-apoptosis protein (Bcl-2). Transcriptome analysis showed that the differential genes regulated by BP were mainly concentrated in TNF signaling pathway, rheumatoid arthritis, IL-17 signaling pathway and so on. To sum up, BP can reduce inflammatory reaction and oxidative stress, inhibit neuronal apoptosis, and alleviate the nerve injury of CIRI in rats, which provides experimental basis for the clinical application of BP in the treatment of ischemic stroke. All experimental procedures were approved by the Experimental Animal Ethics Committee of Zhejiang Chinese Medical University (approval number: 20241209-15).

This study aims to observe the effects of Guben Fangxiao Decoction (GBFXD) on inhibit ferroptosis of airway epithelial cells by regulating iron metabolism, thereby playing a role in the prevention and treatment of the asthma remission. The animal experiments have been approved by the Ethics Committee of the Animal Experiment Center of Nanjing University of Chinese Medicine (approval number: 202401A044). Female Balb/c mice were randomly divided into control group, model group, GBFXD low-, medium-, and high-dose groups [12, 24, 36 g·kg^-1 (crude drug)], and montelukast sodium group (2.6 mg·kg^-1). Except for the control group, the other groups of mice were used to establish a mouse model of the remission period of asthma with ovalbumin. Hematoxylin-eosin staining (HE) of the lung tissue of mice was observed and the inflammation score was conducted; the levels of interleukin 33 (IL-33), tumor necrosis factor-α (TNF-α), and high mobility histone B1 (HMGB1) in the lung tissue were detected by enzyme-linked immunosorbent assay (ELISA); the expression of solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and long-chain acyl-CoA synthase 4 (ACSL4) in the lung tissue was observed by immunohistochemistry; the content of lipid peroxides (LPO) and ferrous ions (Fe²⁺) in the lung tissue was detected. The protein expression levels of SLC7A11, GPX4, ACSL4, transferrin receptor 1 (TFR1), ferritin heavy chain 1 (FTH1), nuclear receptor co-activator 4 (NCOA4), and ferroportin 1 (FPN1) in 16HBE of each group were detected by Western blot; the expression of Fe²⁺ in 16HBE of each group was detected. The results of HE staining of lung tissue showed that GBFXD could reduce inflammatory infiltration in the periairway region of mice (P < 0.05). The results of ELISA showed that GBFXD down-regulated the levels of IL-33, TNF-α and HMGB1 in lung tissue of mice (P < 0.05). Immunohistochemical results showed that GBFXD could up-regulate the expression of SLC7A11 and GPX4 in the lung tissue of mice, and down-regulate the expression of ACSL4 (P < 0.05). Biochemical results showed that GBFXD treatment reduced LPO accumulation in lung tissue of mice and Fe²⁺ accumulation in lung tissue and 16HBE cells (P < 0.05). Western blot results showed that GBFXD up-regulated the expressions of SLC7A11, GPX4, FTH1, FPN1 in 16HBE cells, and down-regulated the expressions of ACSL4, TFR1 and NCOA4 in 16HBE cells (P < 0.05). These results suggest that GBFXD can inhibit ferroptosis of airway epithelial cells, reduce airway epithelial damage, alleviate airway inflammation, and prevent and treat the remission period of asthma. Its mechanism of inhibiting ferroptosis may be related to the regulation of iron metabolism-related protein expression and the reduction of Fe²⁺ accumulation.

Gastric cancer is a leading global malignancy with high incidence and mortality rates, highlighting the urgent need for novel therapeutic agents. As a natural product in the traditional Chinese medicine Arnebiae Radix, the anti-gastric cancer effects of β,β-dimethylacrylalkannin (β,β-DAL) have not been reported. This study aimed to evaluate the in vitro anti-gastric cancer effects of β,β-DAL and elucidate its underlying mechanisms. Through CCK-8 assay, colony formation experiment, and EdU staining, we demonstrated that β,β-DAL significantly suppressed the proliferation of human gastric cancer AGS and HGC-27 cells in a dose-dependent manner, with 48-hour IC₅₀ values of approximately 0.28 and 0.15 μmol·L^-1, respectively. RNA sequencing analysis indicated that the anti-gastric cancer effect of β,β-DAL is associated with the regulation of ferroptosis. Flow cytometry analysis revealed that β,β-DAL upregulated the ROS levels in AGS and HGC-27 cells. In addition, β,β-DAL increased the level of lipid peroxidation in AGS and HGC-27 cells, and the ferroptosis inhibitor ferrostatin-1 reduced their sensitivity to β,β-DAL. Western blot analysis showed that β,β-DAL downregulated the protein expression of stearoyl-CoA desaturase 1 (SCD1) and nuclear factor erythroid 2-related factor 2 (Nrf2), as well as inhibited the ratio of SCD1 to acyl-CoA synthetase long-chain family member 4 (ACSL4) in human gastric cancer AGS and HGC-27 cells. In summary, β,β-DAL may exert anti-gastric cancer effects by inducing ferroptosis in human gastric cancer cells through upregulating ROS levels, increasing intracellular lipid peroxidation, and inhibiting the antioxidant SCD1/Nrf2 pathway. This study provides a candidate drug for gastric cancer treatment and lays the groundwork for further research into the anti-gastric cancer effects of β,β-DAL.

The study aims to investigate the interventional effect of peroxydehydrotumulosic acid (PHA) in Poria cocos on allergic asthma mice, and to investigate its mechanism of action based on metabolomics. Male BALB/c mice were randomly divided into normal group (CON), model group (M), positive drug montelukastna group (Y, 1.3 mg·kg^-1), PHA low-dose group (PHA-L, 15 mg·kg^-1), and PHA high-dose group (PHA-H, 30 mg·kg^-1). The mouse model of allergic asthma was established by intraperitoneal injection of ovalbumin (OVA) and aluminium hydroxide adjuvant for sensitisation and OVA nebulisation for excitation. The corresponding drugs were given by gavage 30 min before modelling, and the drugs were administered for 7 consecutive days. 24 h after the last administration of the drugs, the mice were tested for cough and asthma indexes, lung function and lung coefficient, the mice were stained with hematoxylin-eosin (HE) to observe the pathological changes of the lung histology, and the mice were stained with Masson to observe the level of collagen fibre deposition, and the levels of inflammatory factors in the serum and alveolar lavage fluid of the mice were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry to determine the levels of inflammatory factors in the mice. Apoptotic cell levels in lung tissue and Th17 and Tregs cell levels in blood were detected by flow cytometry, and small molecule metabolites were analysed by liquid-liquid mass spectrometry (LC-MS) in serum samples of mice of different treatment groups. The metabolic pathway enrichment analysis was used to investigate the biometabolic pathways involved in the characterised metabolites. The results indicated a comparison with group M, PHA significantly improved lung function, reduced lung coefficient, attenuated lung histopathological damage and collagen fibre deposition, significantly down-regulated the levels of interleukin 4 (IL4), immunoglobulin E (IgE), interleukin 17A (IL17A) in blood and tumor necrosis factor-α (TNF-α) in alveolar lavage fluid, inhibited apoptosis and the generation of Th17 cells, and increased the level of Tregs cells in lung tissue. Metabolomics analysis of endogenous substances in the M and PHA groups separated significantly, and 63 differential metabolites were screened, mainly involving arachidonic acid metabolism, retinol metabolism, biosynthesis of phenylalanine, tryptophan, and tyrosine, glycerophospholipid metabolism, and tyrosine metabolism. PHA can improve the pathological state of mice with asthma by inhibiting the production of inflammatory factors IL4 and IL17A, hindering the recruitment of inflammatory cells, and regulating some differential metabolites and metabolic pathway disorders. The animal experiment was approved and supervised by the Animal Ethics Committee of Henan University of Traditional Chinese Medicine (ethics number: DWLL2018080003).

This study evaluated the effects of Shanyu Pills on the skeletal muscle mass, motor function, and metabolites of the gastrocnemius muscle in sarcopenia mice, aiming to explore the scientific essence of Shanyu Pills in treating sarcopenia. All animal experiments were approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (approval No.: FJTCM IACUC 2022123). Eight-month-old rapidly aging mice SAMP8 (senescence accelerated mouse prone/8, SAMP8) were used as the animal model for sarcopenia. The biological effects of Shanyu Pills in improving the skeletal muscle mass and motor function of mice with sarcopenia were evaluated by evaluating of the degree of senescence of mice, muscle volume of the hindlimbs, grip strength, hanging net duration time, rotarod performance time, free movement assessment, biochemical indicators of spleen deficiency, and histopathology of gastrocnemius muscle tissue. The metabolites in the gastrocnemius muscle of mice were analyzed using LC-MS/MS. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was used to screen out the differential metabolites among different comparison groups. The differential metabolites were analyzed for metabolic pathway enrichment using the MetaboAnalyst 6.0 database and the "Compound-Reaction-Enzyme-Gene" network of the differential metabolites was constructed using Cytoscape 3.10.0 software. The results showed that Shanyu Pills could significantly reduce the degree of senescence of sarcopenia mice; increase the muscle volume of the hindlimbs, grip strength, hanging net duration time, rotarod performance time, free movement assessment; improve the biochemical indicators of spleen deficiency; and alleviate the phenomenon of gastrocnemius muscle fiber atrophy. The non-targeted metabolomics analysis of the gastrocnemius muscle showed that Shanyu Pills mainly affected 14 differential metabolites such as arachidonic acid, leukotriene A4, carnosine, methylimidazoleacetic acid. The potential target metabolic pathways enriched were the arachidonic acid metabolic pathway. In summary, Shanyu Pills can effectively improve the skeletal muscle mass and motor function of sarcopenia mice, and its mechanism may be closely related to the regulation of arachidonic acid metabolism.

The chemical constituents of 50% acetone extract of the dried roots bark of Paeonia suffruticosa Andr. (Moutan Cortex) were isolated and purified using various column chromatographic methods, yielding 16 compounds. According to their physiochemical properties and spectral data, the structures of the isolated compounds were further identified as suffruticoside N (1), 1, 1′-[(1R)-2, 2′-dihydroxy-6, 6′-dimethoxy[1, 1′-biphenyl]-3, 3′-diyl]bis[ethanone] (2), 5-(1, 2-dihydroxy-1-methylethyl)-4-methylbicyclo[3.1.0]hexan-2-one (3), 4-(1, 2-dihydroxy-1-methylethyl)-1-cyclohexene-1-carboxylic acid (4), teutenone A (5), dihydroactinidiolide (6), cadina-4, 11-dien-14-oic acid (7), adenosine (8), uridine (9), methyl palmitate (10), 1-(3-hydroxy-4-methoxyphenyl)ethanone (11), 2, 4-dihydroxyacetophenone (12), methyl gallate (13), 2, 3-dihydroxybenzoic acid (14), benzoic acid (15), and pinoresinol (16). Compound 1 was a new biphenyl compound. Compounds 2 and 3 were two new natural products, and compounds 4 and 5 were firstly isolated from Moutan Cortex. Additionally, in preliminary vitro bioassays, all the isolated compounds were tested their inhibitory effects on acetylcholinesterase (AChE). As a result, compound 2 exhibited a moderate AChE with IC₅₀ value of 15.7 ± 2.5 μmol·L^-1.