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B lymphocytes are central drivers of pathogenic immune responses in autoimmune diseases and represent a promising target for therapeutic intervention. Sinomenine (SIN), a natural alkaloid derived from Sinomenium acutum, exhibits broad immunomodulatory properties and has long been clinically used for treating autoimmune diseases, however, its direct effects on B cells and underlying mechanisms are largely unexplored. To investigate the effects of SIN on the proliferation and hyperactivation of B lymphocytes both in vitro and in vivo, and elucidate its therapeutic mechanisms in autoimmune pathologies. Firstly, SIN was examined for its ability against lipopolysaccharides (LPS) or CpG oligodeoxynucleotide (CpG) induced B cell proliferation and hyperactivity. Then, the effects of SIN regulating B cell subsets and antibody response were studied in pristane-induced lupus and keyhole limpet hemocyanin (KLH)-immunized murine models. Furthermore, transcriptomics, small molecule-protein interaction assay (SPIA) and a series of validation experiments (including Western blotting, immunofluorescence, cellular thermal shift assay (CETSA), surface plasmon resonance (SPR), and flow cytometry) were performed to identify the key mechanisms involved. For the first time, our research revealed the significant inhibitory efficacy of SIN on B cell proliferation induced by LPS or CpG. Moreover, SIN significantly reduced CD69 expression, and attenuated IL-6 and IL-10 hypersecretion. Notably, SIN exerted a better inhibitory effect against CpG-mediated activation compared to LPS-induced responses. In the early immune response of pristane-induced lupus model, both SIN and prednisone significantly decreased the spleen coefficient and the proportion of CD19+CD69+ B cell subsets. In the KLH immunization model, SIN alleviated splenomegaly, lowered serum levels of IgG and IgM, and reduced the cell counts of B220+ cells and proportion of plasma cells (B220lowCD19-CD138+). Integration of transcriptomic analyses and in vitro verification revealed SIN's regulatory effects on cell cycle, the PI3K-AKT signaling pathway, along with its marked suppression of NF-κB protein activation following both LPS and CpG stimulation. In response to CpG stimulation, SIN also significantly decreased the protein levels of p-STAT3 and STAT3. Further research by SPIA identified the downregulation of interferon regulatory factor 5 (IRF5) by SIN. This effect was then corroborated by Western blot, immunofluorescence, molecular docking, CETSA, and SPR analyses, confirming SIN as a novel modulator of IRF5. Moreover, pharmacological inhibition of IRF5 by IRF5-IN-1 markedly significantly attenuated the proliferation and hyperactivation of B cells following LPS or CpG challenge, implying the critical role of IRF5 in B cells. Collectively, our findings suggest that SIN restrains the proliferation and hyperactivation of B lymphocytes via inhibiting IRF5 and modulating the PI3K-Akt, STAT3, and NFκB signaling cascades. These results uncover a previously unrecognized mechanism of B cell regulation and support a potential therapeutic approach for autoimmune diseases involving pathogenic B cells by SIN.
Atractylodes Macrocephala Rhizome (AMR), the dried rhizome of Atractylodes macrocephala Koidz (family Asteraceae), is a commonly used Chinese herbal medicine for treating ulcerative colitis (UC) and has extensive applications in both Traditional Chinese Medicine (TCM) and modern medicine. The therapeutic effects of its distinctive processed variety, rice-washed water processed Atractylodis Macrocephalae Rhizoma (Migan Shui Piao Baizhu, R-AMR), have yet to be reported, and its potential mechanisms of action require further exploration. This study aims to compare the therapeutic effects of R-AMR and raw AMR on UC mice and further explore their mechanisms of action, thereby providing insights into the processing mechanism of AMR using rice-washed water. Firstly, a DSS-induced mouse UC model was established in vivo. The efficacy of treatment with rice-washed water processed AMR in improving UC was evaluated by DAI scores, histopathological changes, serum inflammatory cytokines, tight junction proteins (ZO-1, Claudin-1), and mucin (MUC2) levels. Then, bioinformatics, machine learning, and molecular docking were integrated to identify core disease targets and explore the mechanism of R-AMR in treating UC. Nextly, an LPS-induced UC cell model was established using NCM460 cells, and LCN2 siRNA-transfected cell experiments were conducted. The biological effects and mechanisms of action of key targets were validated in vitro through immunofluorescence, WB, and RT-qPCR techniques. Finally, we validated the roles of the above-identified pathways in the treatment of UC with AMR through in vivo experiments. In in vivo efficacy experiments, both R-AMR and raw AMR effectively reduced DAI scores and histopathological scores in UC mice. They downregulated proinflammatory factors and upregulated anti-inflammatory factors while increasing tight junction protein expression (ZO-1, Claudin-1) to restore damaged intestinal epithelial mucosal barriers. R-AMR demonstrated superior therapeutic effects compared to raw AMR. Bioinformatics and machine learning results indicate that Lipocalin 2 (LCN2) and Tissue inhibitor of metalloproteinase 1 (TIMP1) is a key target in UC, with the NOD-like receptor signaling pathway being one of its representative pathways. Molecular docking results reveal that Atractylenolide I (Atr I) has a strong affinity for LCN2 and is one of the active components enhancing the efficacy of R-AMR. In vitro experiments demonstrate that Atr I restores tight junctions between LPS-induced NCM460 cells. Targeted inhibition of LCN2 reduces NLRP3 inflammasome, Caspase 1 activation, and GSDMD-N expression, decreases proinflammatory factor release (IL-1β, IL-18, TNF-α, IL-6), and alleviates UC inflammatory responses. In vivo experiments verified that the therapeutic effect of AMR on UC is associated with the LCN2/NLRP3/ Caspase 1/GSDMD pyroptosis pathway. Both raw AMR and R-AMR exhibit protective effects against UC, with R-AMR displaying superior therapeutic outcomes. Its bioactive constituent Atr I attenuates pyroptosis through targeted inhibition of LCN2, leading to preservation of the intestinal epithelial mucosal barrier and subsequent amelioration of UC symptoms.
Achyranthis bidentatae radix (ABR, Niu-Xi in Chinese) is a well-documented medicinal herb with a long history of use in Traditional Chinese Medicine (TCM). Traditional application records suggest that ABR may be widely used for treating conditions such as amenorrhea, dysmenorrhea, waist and knee pain, muscle and bone weakness, stranguria, edema, headache, dizziness, toothache, oral aphthae, hematemesis, and epistaxis. This paper provides a comprehensive review of ABR, encompassing its botany, processing methods, ethnopharmacology, phytochemistry, pharmacology, quality control, toxicity, and pharmacokinetics. It aims to offer an overview of current research to facilitate further exploration and utilization of ABR. This study used "Achyranthis bidentatae radix", "Achyranthes bidentata Bl." and "NiuXi" as the keywords to gather relevant information on ABR from different databases, including PubMed, CNKI, Web of Science, and Google Scholar. Literature on non-medicinal parts, including stems, leaves, fruits, and seeds, was excluded. To date, 204 compounds have been isolated and identified from ABR, including triterpenoid saponins, steroids, polysaccharides, alkaloids, flavonoids, polypeptides, volatile oils, and other chemical components. Triterpenoid saponins, steroids and polysaccharides exert the main pharmacological effects, including anti-osteoporosis, anti-inflammatory, immunomodulatory and anti-tumor activities. ABR also exhibits other pharmacological activities, including nephroprotective, antioxidant, neuroprotective, anti-diabetic, hepatoprotective, antiviral, and cardioprotective effects. Toxicological studies confirm ABR's general safety at conventionally recommended dosages, while pharmacokinetic investigations have clarified the in vivo behaviors of β-ecdysterone and triterpenoid saponins from ABR. In addition, this paper addresses the shortcomings of current research on ABR and proposes potential future research directions. ABR exhibits a diverse chemical composition and a wide range of pharmacological activities. However, studies on the single active compounds of ABR remain insufficient. Research on their in vivo mechanisms of action is limited and lacks clinical trial validation, while research on the mechanisms of multi-component synergistic effects remains incomplete. Future studies should therefore focus on the identification of bioactive constituents, along with systematic pharmacological and clinical evaluation. Quality control research on ABR and its processed products must be strengthened to ensure their safe and effective application. Furthermore, in-depth investigations into the toxicology and pharmacokinetics of ABR will provide a scientific basis for its clinical application.
Coptis teeta, Andrographis paniculata, and Nyctanthes arbor-tristis are widely used in herbal remedies for the treatment of various diseases in Southeast Asia. In Arunachal Pradesh, India, these plants are combined by traditional healers to treat malaria. The study aimed to evaluate the antimalarial potential of the traditionally used polyherbal combination through in vitro screening against chloroquine-sensitive (Pf3D7) and chloroquine-resistant (PfRKL9) strains of Plasmodium falciparum, and in vivo antimalarial activity against the P. berghei ANKA strain. The study also aimed to study the molecular interactions of putatively detected phytocompounds against P. falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) using molecular docking and dynamics simulations. Taxonomic identification was carried out at the Botanical Survey of India (BSI), Shillong. The cold maceration technique was used to prepare different combinations in different ratios for in-vitro antimalarial screening. Using two concentrations, a preliminary in vitro antimalarial screening was conducted to identify the most potent polyherbal combination. Following this, the most potent combination was subjected to in vitro antimalarial studies, cytotoxicity studies, a blood hemolysis assay, and in vivo studies in P. berghei-infected mice. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was performed on the individual extracts to detect putative phytocompounds. In silico studies were done to study the molecular interactions of the putatively identified phytocompounds with PfDHFR-TS. A combination of powdered rhizomes of C. teeta, leaves of N. arbor-tristis, and aerial parts (leaves and young shoots) of A. paniculata in the ratio 5:2:3 was identified as the most potent polyherbal combination against P. falciparum. The cytotoxicity and blood hemolysis assays confirmed that the combination has negligible toxicity and has no hemolytic effect. Acute toxicity studies showed that the LD50 of the polyherbal combination was greater than 2000 mg/kg body weight. The in-vivo antimalarial study revealed that the polyherbal combination exhibits greater parasite-suppressive activity than the individual extracts but could not outperform the standard drugs. Molecular docking and dynamics simulations revealed that the LC-HRMS-detected compounds (specifically, PubChem CIDs 23786445, 23874507, 38360299, 332427, and 38361380) exhibited stable interactions with PfDHFR-TS and may be one of the contributing factors to the observed antimalarial activity. This study reveals that the traditional practice of using polyherbal combinations rather than individual plants yields greater antimalarial activity. The findings of the study encourage the development of an antimalarial polyherbal formulation after performing pharmacokinetic profiling of these specific computational candidates and extended toxicity studies of the combined polyherbal extract.
As a traditional Chinese medicine formula effectively used for the clinical treatment of Knee Osteoarthritis (KOA), the mechanism of action of Jiawei Wutou Decoction (JWD) remains unclear. This study aims to preliminarily explore the potential mechanism of JWD in regulating mitochondrial oxidative stress for the treatment of KOA through the integration of UHPLC-MS/MS, network pharmacology, molecular docking technology and experimental validation. UHPLC-MS/MS was used to identify the main components of the water extract of JWD. HPLC was employed to determine the contents of Salidroside, Paeoniflorin, Calycosin- 7-O-β-D-glucopyranoside, Benzoylmesaconitine, Benzoylhypaconitine, Icariin and Glycyrrhizic Acid, which are the signature components of JWD. A DMM-induced KOA rat model was established. The pharmacodynamics, morphology and histopathological analysis were applied to preliminary determination of the preventive and therapeutic effects of JWD on KOA. A network of key compounds, core targets and key signaling pathways of JWD was constructed by combining network pharmacology and the binding activity between key components and core targets was verified by molecular docking techniques. Western Blot (WB), immunohistochemistry (IHC), CCK-8 assay and immunofluorescence techniques were employed to validate the therapeutic mechanism of JWD on KOA in both in vitro and in vivo experiments. A total of 55 compounds were identified in JWD by UHPLC-MS/MS. Animal experiments showed JWD had a protective effect on DMM-induced KOA rats model, mainly in the amelioration of sclerosis, cartilage damage, increasing the pain threshold, and inhibition of oxidative stress. The potential targets of 55 compounds from JWD were predicted using online compound target databases. Protein-Protein Interaction (PPI) network analysis revealed that key targets such as AKT1, IL6, TNF, TP53, BCL2 and STAT3 are potential therapeutic targets of JWD for KOA treatment via regulating mitochondrial oxidative stress. Functional enrichment analysis indicated that apoptosis, cellular senescence, and the JAK/STAT signaling pathway were significantly enriched. Molecular docking results demonstrated that 18-a glycyrrhetinic acid, icariin, benzoylmesaconine, glabrol, rhodiosin, and benzoylpaeoniflorin in JWD exhibited high binding activity to JAK2 and STAT3. Therefore, the JAK2/STAT3 signaling pathway was identified as the potential target for the experimental validation study. JWD could upregulate the expression of Collagen II, Aggrecan and Bcl-2, downregulate the expression of Bax, Caspase-3, IL-6 and JAK2/STAT3 signaling related proteins in KOA rats. Besides, JWD improved mitochondrial morphology, reduced ROS generation and decreased MMP levels, which was attributed to the activation of JAK/STAT signaling molecules in TBHP-induced chondrocytes. This study demonstrates that JWD exhibits potential therapeutic effects on KOA, which may be associated with the suppression of mitochondrial oxidative stress via the JAK/STAT signaling pathway.
Paederia foetida L. is traditionally used in Northeast India and Southeast Asia to treat inflammatory disorders. This study validates its traditional use and bridges ethno-medicinal knowledge with modern pharmacological evidence by providing mechanistic support for the anti-inflammatory and antioxidant activities of P. foetida and its bioactive constituent keracyanin. This study aimed to evaluate the anti-inflammatory, antioxidant, and protective effects of P. foetida extract and its bioactive phytoconstituent, keracyanin, through integrated in silico, in vitro, and in vivo approaches, and to elucidate the underlying molecular mechanisms with particular emphasis on the MAPK and NF-κB signaling pathways. Thirty-six polyphenolic compounds previously identified from the methanolic extract of P. foetida (PFME) by HR-LC-MS were retrieved from PubChem and evaluated as phytoligands. Molecular docking and 100 ns molecular dynamics simulations were performed against key inflammatory targets, including iNOS, MAPK, and TNF-α. The pharmacokinetic, toxicity, and biological activity profiles of the identified compounds were predicted using ADMET and PASS analyses. Based on the docking and simulation results, keracyanin was selected for further investigation. The in vitro anti-inflammatory and antioxidant activities of PFME and keracyanin were assessed using lipoxygenase (LOX), cyclooxygenase-2 (COX-2), and nitric oxide (NO) inhibition assays, with quercetin and celecoxib as reference compounds. In vivo anti-inflammatory activity was evaluated in a carrageenan-induced paw edema model in Wistar albino rats orally administered PFME (250 and 500 mg/kg) or keracyanin (25 and 50 mg/kg). Paw edema and percentage inhibition were recorded at different time points, and histopathological changes were examined using hematoxylin and eosin staining. Levels of inflammatory mediators, including CRP, NF-κB, iNOS, TNF-α, IL-1β, IL-6, p38-MAPK, MPO, COX-2, NO, and PGE2, the anti-inflammatory cytokine IL-10, oxidative stress markers (MDA and NO), and antioxidant parameters (TAOC, CAT, SOD, GPx, and GSH), were quantified using ELISA and biochemical assay kits. The expression of TNF-α, p38-MAPK, and NF-κB signaling proteins was further evaluated by Western blot analysis. Statistical significance was determined using one-way ANOVA followed by Tukey's post hoc test (p < 0.05). Molecular docking and molecular dynamics simulations demonstrated that keracyanin exhibited strong and stable interactions with key inflammatory targets, including iNOS, MAPK, and TNF-α, showing binding affinities superior to those of the standard inhibitors. PASS prediction suggested a high probability of anti-inflammatory activity, while ADMET analysis indicated favorable pharmacokinetic properties, good drug-likeness, and low toxicity. In vitro studies showed that keracyanin significantly suppressed LOX and COX-2 activities as well as NO production, with efficacy comparable to PFME and greater than that of the reference compounds quercetin and celecoxib. In vivo, PFME (250 and 500 mg/kg) and keracyanin (25 and 50 mg/kg) significantly attenuated carrageenan-induced paw edema in a dose-dependent manner, achieving 53.5-64.2% and 54.5-65.8% inhibition, respectively, at 5 h (p < 0.05), compared with 69.5% inhibition by diclofenac. Histopathological examination confirmed marked protection against carrageenan-induced tissue injury, evidenced by reduced inflammatory cell infiltration, diminished epithelial hyperplasia, minimal edema, and preservation of normal tissue architecture. Furthermore, PFME and keracyanin significantly decreased the levels of inflammatory mediators, while enhancing IL-10 production and restoring antioxidant defenses. These effects were accompanied by reduced lipid peroxidation and significant downregulation of the MAPK and NF-κB signaling pathways, as confirmed by ELISA and Western blot analyses. The integrated in silico, in vitro, and in vivo findings demonstrate that PFME and its bioactive constituent keracyanin exert potent anti-inflammatory and antioxidant effects through modulation of MAPK and NF-κB signaling pathways. These results provide mechanistic support for the traditional use of P. foetida in the management of inflammatory disorders and identify keracyanin as a key contributor to its therapeutic activity.
Jinhua Qinggan Granules (JHQG) is a traditional Chinese medicine formulation widely used for the treatment of viral respiratory infections. Traditional herbal formulations contain multiple phytochemical constituents that may act individually or synergistically to produce therapeutic effects. Although its clinical applications have been reported, the bioactive constituents underlying its antiviral activity and their mechanisms of action remain to be clarified. This study aimed to identify representative bioactive constituents of JHQG with antiviral activity against Zika virus (ZIKV) and to investigate their potential mechanisms of action. A panel of representative phytochemical constituents reported in JHQG was screened for antiviral activity against ZIKV in vitro. Antiviral efficacy and cytotoxicity were evaluated by determining EC50 and CC50 values. Viral replication was assessed using plaque assay, RT-qPCR, immunofluorescence, and Western blot analysis. Time-of-addition experiments were performed to explore stage-specific effects. Transcriptome analysis and quantitative PCR were conducted to examine host inflammatory gene expression. Intracellular reactive oxygen species (ROS) levels and NF-κB activation were evaluated in stimulated cells. In vivo antiviral efficacy was assessed in a ZIKV infection model using A129 mice. Molecular docking and molecular dynamics simulations were performed to explore potential interactions with the viral NS3 protease. Among the screened compounds, chlorogenic acid (CGA) exhibited notable antiviral activity and was selected for further investigation. CGA treatment was associated with reduced ZIKV replication, decreased viral protein expression, and lower production of infectious viral particles under the tested conditions. Time-of-addition experiments indicated that CGA exerted inhibitory effects during early stages and throughout the infection period. ZIKV infection induced the expression of inflammatory genes including CXCL8, CXCL10, and TNFAIP3, whereas treatment with JHQG or CGA was associated with reduced expression of these genes. CGA treatment also corresponded with decreased intracellular ROS levels and attenuation of NF-κB activation. In A129 mice, CGA administration was associated with improved survival, reduced viral RNA levels in brain and liver tissues, and partial preservation of tissue morphology. Computational analyses suggested potential interactions between CGA and the NS3 protease. These findings identify CGA as a representative bioactive constituent selected from the screened phytochemical components of JHQG that may contribute to its antiviral activity. The antiviral effects observed in this study, together with the modulation of host inflammatory responses, provide experimental evidence supporting further investigation of CGA as a candidate compound for the development of therapeutic strategies against ZIKV infection.
Hirudo (HO) is the medicinal name for the dried whole body of leeches, including species such as Whitmania pigra Whitman, Hirudo nipponica Whitman, and Whitmania acranulata Whitman, all of which belong to the family Hirudidae. It is characterized by a salty, bitter taste and a neutral nature with mild toxicity. HO is traditionally used to activate blood circulation, regulate menstruation, and eliminate blood stasis, making it effective in treating conditions like blood stagnation with menstrual irregularities, abdominal masses, stroke sequelae, and traumatic injuries. This review aims to provide a comprehensive overview of HO from the perspectives of its zoological characteristics, traditional applications, processing methods, chemical composition, pharmacological activities, quality control, and toxicity. The goal is to offer a reference for the rational use of HO and to lay the foundation for related clinical research. This review searched the literature on HO in databases including ResearchGate, Web of Science, Baidu Scholar, Google Scholar, CNKI, and other databases using the keywords HO, zoology, compounds, pharmacology, and quality control. More than 140 chemical constituents have been identified in HO., including 65 types of peptides (1-65), 8 types of pteridines (66-73), 34 types of lipids (74-107), 17 types of amino acids (108-124), and 18 other chemical compounds (125-142). Among these, peptides are the main active ingredients of HO, exhibiting pharmacological effects such as anticoagulation, antithrombosis, anti-atherosclerosis, anti-tumor, anti-inflammatory, and anti-fibrotic activities. Notably, HO has shown significant efficacy in anticoagulation and antithrombosis. It can treat cardiovascular diseases through mechanisms such as inhibiting blood coagulation, preventing platelet aggregation, and promoting fibrinolysis. While HO demonstrates remarkable pharmacological activities, its distinct fishy odor may affect patient compliance, requiring further processing to eliminate this unpleasant scent. This review also systematically outlines the quality control standards, toxicity, and clinical applications of HO. This review systematically summarizes the zoological characteristics, traditional uses, processing methods, chemical composition, pharmacological activity, quality control, and toxicity of HO. Current research indicates that peptides are the primary bioactive components responsible for HO's anticoagulant, antithrombotic, anti-inflammatory, and anti-fibrotic activities, particularly in the treatment of cardiovascular diseases. This review also emphasizes the importance of establishing a robust quality control system and further elucidating the pharmacological mechanisms of HO to support its safe and rational clinical application.
Liparis nervosa (Thunb.) Lindl. (L. nervosa), a species belonging to the genus Liparis within the Orchidaceae family, is known as "Jian-Xueqing" or "Jian-Xueqin" in traditional Chinese medicine (TCM). This plant has a long history of medicinal use in TCM, valued for its diverse therapeutic properties for treating hemoptysis, hematemesis, cough due to lung heat, rheumatic arthralgia, infantile convulsions, and abscesses. Externally, it is applied for traumatic bleeding, boils, contusions, dermatitis, and snakebites. However, no systematic review of L. nervosa has been reported so far. This paper aims to provide a comprehensive review of the research progress on L. nervosa regarding its botany, traditional uses, phytochemistry, pharmacological activities, toxicology, and clinical applications. By bridging traditional knowledge with modern scientific evidence, this review seeks to offer new perspectives for future research of L. nervosa. This paper uses "Liparis nervosa", "Jian-Xueqing" and "Jian-Xueqin" as the keywords to conduct all the relevant information from PubMed, Web of Science, SciFinder, CNKI, China Master's Doctoral Thesis, Wanfang, Google Scholar, and ancient books. We retrieved all literature (in Chinese and English) from the establishment date of the database up to February 2026, including articles, reviews, master's thesis, doctoral thesis, and technical reports, while excluding literature unrelated to L. nervosa. In total, 150 compounds have been isolated and identified from L. nervosa, including 36 benzoic acid derivatives, 27 alkaloids and their derivatives, 24 phenanthrenes, 21 phenylpropanoids, 7 flavonoids, 7 steroids and triterpenoids, 11 fatty acid derivatives, and 17 other compounds so far. Pharmacological studies have demonstrated that extracts and compounds derived from L. nervosa exhibit a broad range of bioactivities, such as hemostatic, anti-inflammatory, antinociceptive, antitumor, antioxidant, antibacterial, hypolipidemic, and immunosuppressive effects. In safety evaluations, no adverse effects or toxic activities were observed in acute toxicity studies. This review summarizes the recent advances in the phytochemistry, pharmacology, and clinical potentials of L. nervosa, and systematically categorizes its structure-activity-mechanism relationship. As a valuable traditional herb, L. nervosa contains diverse chemical constituents and exhibits various bioactivities. Several of its traditional uses have been supported by modern pharmacological research. However, most studies have been conducted only at the cellular and animal model levels, lacking more robust evidence from clinical studies. Furthermore, systematic safety data on L. nervosa are severely lacking. Further research is needed to elucidate the pharmacological mechanisms and ensure safe clinical applications. Moreover, as a valuable and endangered species, future investigations should focus on the cultivation techniques, product development, and sustainable utilization.
Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by hepatic steatosis accompanied by persistent inflammation and early fibrotic remodeling. In traditional Chinese medicine, Huanglian Wendan Decoction (HLWDD) is prescribed for phlegm-heat and damp-heat syndromes affecting the gallbladder and stomach and is traditionally used to clear heat, dry dampness, and resolve phlegm. It is commonly applied in the treatment of phlegm-heat-related metabolic disorders, including fatty liver disease. However, the therapeutic effects of HLWDD and the contributions of its key constituents to MASH remain to be further elucidated. This study aimed to evaluate the anti-inflammatory and lipid-regulatory effects of HLWDD and its key components in MASH and to explore the underlying molecular mechanisms. Male C57BL/6 J mice were given a methionine-choline-deficient (MCD) diet and received HLWDD in either low or high doses through oral gavage, with fenofibrate serving as a positive control. Body weight, liver index, serum levels of alanine aminotransferase and aspartate aminotransferase, serum lipid profiles, and hepatic triglyceride and total cholesterol contents were among the evaluated parameters. H&E, Oil Red O, and Masson's trichrome staining were used to evaluate histopathological changes. Hepatic macrophage infiltration was examined by immunofluorescence, inflammatory cytokines were measured by ELISA, and key signaling and lipid metabolism-related proteins were analyzed by western blotting. UPLC‒MS/MS was used to characterize the chemical profile of the HLWDD granules and identify their major constituents. Network pharmacology analysis integrating multiple databases, together with GO and KEGG enrichment analyses, was performed to predict potential targets and pathways. Molecular docking and molecular dynamics simulations were further used to investigate compound‒target interactions. Cell viability in vitro was measured with CCK-8 assays, protein levels were confirmed through western blotting, and intracellular lipid buildup was assessed using Oil Red O staining. UPLC‒MS/MS analysis revealed that berberine (BBR), an isoquinoline alkaloid, is a major bioactive component of HLWDD. Network pharmacology analysis suggested that HLWDD and BBR may exert anti-MASH effects by modulating multiple targets and pathways, including IL-6, PPARα, and the NF-κB/HDAC1/SREBP-1c axis. These predictions were supported by in vivo experiments, which confirmed the protective effects of HLWDD against MASH. Both in vivo and in vitro studies further revealed that BBR markedly ameliorated MASH-related phenotypes by suppressing the NF-κB/HDAC1/SREBP-1c axis. Additionally, simulations of molecular docking and dynamics revealed stable interactions between BBR and important proteins within this axis. Microscale thermophoresis (MST) assays further demonstrated direct binding of BBR to HDAC1. Collectively, these findings suggest that HLWDD and its key active constituent BBR alleviate MASH, at least in part, by inhibiting the NF-κB/HDAC1/SREBP-1c axis, which is closely associated with inflammatory responses and dysregulated lipogenesis. HLWDD markedly ameliorated the MASH phenotype by attenuating hepatic inflammation and lipogenesis, with the NF-κB/HDAC1/SREBP-1c axis emerging as a key mechanism linking inflammatory signaling to aberrant lipid synthesis. BBR, identified by UPLC‒MS/MS as a major active constituent of HLWDD, largely recapitulated these effects and directly bound to HDAC1, supporting its important contribution to the protective effects of HLWDD against MASH.
Cardiovascular diseases (CVDs) are the leading cause of hospitalization and death worldwide. The use of complementary therapies, particularly medicinal plants, has become increasingly prevalent as a therapeutic option. Petiveria alliacea L. (Phytolaccaceae) is a prominent species widely distributed across Brazil. Regarding its ethnopharmacological application, studies involving folk healers have documented the use of this species to treat various disorders of the cardiovascular system. To investigate the cardioprotective effects of an ethanol-soluble fraction obtained from the aerial parts of P. alliacea in hypertensive rats subjected to isoprenaline-induced cardiac stress. Aerial parts of P. alliacea were collected, and an aqueous extract was prepared via infusion. This extract was subsequently treated with ethanol to yield the ethanol-soluble fraction of P. alliacea (ESPA). Wistar-Kyoto and spontaneously hypertensive rats (SHR) were assigned to the following experimental groups: naïve, negative control (vehicle), metoprolol (10 mg/kg), and ESPA (30, 100, and 300 mg/kg). All hypertensive groups received isoprenaline (4.5 mg/kg, s.c.) once daily for four consecutive days. After a 28-day treatment period, renal function, electrocardiographic parameters, blood pressure, and mesenteric vascular bed reactivity were assessed. Biochemical markers, histopathology, and cardiac morphometry were also evaluated. Additionally, intracellular cyclic guanosine monophosphate (cGMP) levels were measured in SHR aortic rings. On the first day, treatment with ESPA (30 mg/kg) induced a significant natriuretic response, increasing urinary Na+ excretion by ∼ 18% compared to the negative control group; after 28 days, animals treated with 100 and 300 mg/kg of ESPA showed a reduction in systolic blood pressure by ∼ 21%, diastolic blood pressure by ∼ 31%, and lipid peroxidation by ∼ 93%, while nitrite levels rose by ∼ 49% compared to negative control animals. Furthermore, treatment with doses of 100 and 300 mg/kg attenuated plasma angiotensin-converting enzyme activity by approximately 38% and aldosterone levels by ∼ 26%, restoring them to values comparable to the naïve group. Additionally, ESPA (100 and 300 mg/kg) prevented increases in relative heart weight, left ventricular wall thickness, and interventricular septum thickness, specifically reducing left ventricular wall thickness by ∼ 67% compared to the negative control rats. Finally, incubation with ESPA (0.1 and 1 mg/mL) increased intracellular cGMP levels by approximately 42% and 102%, respectively, in SHR rat aortic rings, evidencing an activation of the nitric oxide (NO)/cGMP pathway. Long-term treatment (28 days) with ESPA exerts significant cardioprotective effects in isoprenaline-induced hypertensive rats. This effect appears to be dependent on the activation of the NO/cGMP pathway, coupled with the inhibition of angiotensin-converting enzyme.
Buyiniuxi pill (BYNX) is a classical traditional Chinese medicine (TCM) formula first recorded in Taiping Shenghui Fang (AD 992) and has been traditionally prescribed for hair loss and premature hair greying. Its constituent herbs remain relevant in contemporary ethnopharmacological practice for hair regeneration. This study aimed to evaluate the hair regrowth-promoting effects of BYNX in a dihydrotestosterone (DHT)-induced murine model of androgenetic alopecia (AGA) and to explore its potential molecular mechanisms. The chemical profile of the BYNX aqueous extract was characterized using UHPLC-Orbitrap-MS. Network pharmacology was used to explore potential targets and pathways associated with AGA. In vivo efficacy was assessed in a DHT-induced murine model following oral administration of BYNX at low or high doses. Hair regrowth was evaluated by hair length and hair coverage percentage, while histopathology, immunohistochemistry, ELISA, and RT-qPCR were performed to investigate underlying mechanisms. UHPLC-Orbitrap-MS analysis revealed a chemically complex profile of BYNX, and representative constituents were selected for subsequent network pharmacology analysis. Predicted targets were mainly enriched in inflammation-related biological processes and the PI3K-AKT signaling pathway. In vivo, BYNX significantly alleviated DHT-induced hair growth inhibition, as evidenced by increased hair length, improved hair coverage, and restoration of hair follicle morphology. These changes were accompanied by reduced IL-6 and IL-4 expression, enhanced activation of the AKT/BCL-2 signaling pathway, and increased Cyclin D1 and β-catenin expression in dorsal skin tissue. BYNX significantly promotes hair regrowth in a DHT-induced murine model of AGA. The hair-regenerative effects of BYNX were associated with modulation of inflammatory responses and activation of AKT/BCL-2-related survival signaling, suggesting a multi-target regulatory pattern consistent with its traditional ethnopharmacological use.
Ophiopogon japonicus (Thunb.) Ker Gawl. is a traditional Chinese medicinal herb commonly used to "nourish yin and moisten the lung", and has long been applied for the prevention and treatment of lung-related disorders in classical medical texts such as the Treatise on Typhoid Fever and Item Differentiation of Warm Febrile Diseases. Pulmonary fibrosis (PF) is a chronic progressive fibrotic lung disease with limited therapeutic options. Although Ophiopogon japonicus has shown anti-inflammatory and anti-fibrotic potential in respiratory diseases, its pharmacological mechanisms against PF remain unclear. This study aimed to evaluate the protective effects of Ophiopogon japonicus aqueous decoction against PF and to investigate whether its anti-fibrotic effects are associated with the modulation of hypoxia-inducible factor 1 subunit α (HIF1A)/tubulin beta 3 class III (TUBB3) axis-associated M2 macrophage polarization and arginine metabolic alterations. Potential bioactive constituents of Ophiopogon japonicus aqueous decoction were first screened using UHPLC-Q Exactive Orbitrap-HRMS combined with Lipinski's rule of five. A bleomycin (BLM)-induced mouse model of PF was then established, and Ophiopogon japonicus aqueous decoction was administered as an intervention. Body weight, pulmonary function testing (PFT), micro-CT, histopathology, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) were used to evaluate its pharmacodynamic effects. Network pharmacology, Gene Expression Omnibus (GEO) mining, weighted gene co-expression network analysis (WGCNA), machine learning (ML), transcriptomics, metabolomics, and single-cell transcriptomics combined with virtual knockout analysis were further integrated to identify candidate targets, related pathways, and metabolic alterations. Finally, flow cytometry, western blotting, immunofluorescence (IF) co-staining, AlphaFold3, molecular docking, and molecular dynamics simulation were performed to further assess phenotypic changes, predicted molecular associations, and candidate active constituents. Ophiopogon japonicus aqueous decoction significantly alleviated BLM-induced PF, and 16 potential bioactive constituents were identified. In vivo experiments showed that Ophiopogon japonicus aqueous decoction attenuated body weight loss, pulmonary dysfunction, micro-CT abnormalities, histopathological injury, collagen deposition, inflammatory responses, and fibrosis scores in PF mice. Bioinformatic analysis identified HIF1A as a candidate hub target and TUBB3 as a key feature gene. Further multi-omics analysis showed that the HIF1A/TUBB3 axis-associated module was closely associated with M2 macrophage polarization and arginine metabolic dysregulation; combined with virtual knockout analysis, these results further suggested that HIF1A may be located upstream of TUBB3 at the predicted regulatory-network level. Experimental analyses showed that Ophiopogon japonicus aqueous decoction intervention was associated with decreased HIF-1α and TUBB3 protein expression, reduced M2 macrophage polarization, and partial correction of arginine metabolic dysregulation. Structural simulation suggested that Ophiopogonin D could stably bind to HIF-1α and TUBB3. This study demonstrated that Ophiopogon japonicus aqueous decoction alleviates BLM-induced PF. Integrated multi-omics analysis and experimental evidence suggested that its anti-fibrotic effects may be associated with modulation of HIF1A/TUBB3 axis-associated M2 macrophage polarization and arginine metabolic dysregulation. Ophiopogonin D may be one of the candidate active constituents associated with these protective effects.
Solanum cernuum Vell. (Solanaceae), popularly known in Brazil as "panacéia", is widely employed in folk medicine to treat renal and urinary tract disorders. However, despite its ethnopharmacological relevance, the scientific basis for its anti-urolithiatic activity has yet to be established. The present study aims to validate the ethnopharmacological use of S. cernuum by investigating its anti-urolithiatic activity associated with its potential to modulate the inflammatory response. Additionally, a chemical characterization of the infusion was performed. The aqueous leaf infusion was prepared according to traditional use. Anti-urolithiatic activity was assessed in an in vitro human urine model to evaluate calcium oxalate (CaOx) crystal dissolution. Anti-inflammatory potential was investigated via an ex vivo human blood assay stimulated with LPS and calcium ionophore, followed by the assessment of PGE2 and LTB4 mediators. The chemical profiling was performed via high-resolution UHPLC-ESI-HRMS in positive ionization mode using a Data-Independent Acquisition (DIA) strategy. The infusion significantly reduced CaOx crystal counts in a dose-dependent manner, reaching an 86.6% reduction at the highest concentration tested (p ≤ 0.05), with efficacy comparable to positive controls. Furthermore, it inhibited the production of PGE2 (74.8%) and LTB4 (39.8%), suggesting a dual inhibition of the COX and LOX pathways, showing a more pronounced effect on the COX pathway. Chemical profiling via high-resolution UHPLC-ESI-HRMS led to the annotation of 18 metabolites (level 2, MSI), primarily O-glycosylated flavonoids and other bioactive classes. These findings provide scientific support for the traditional use of S. cernuum in renal disorders. The dual modulation of inflammatory mediators, combined with high crystal dissolution capacity, indicates that the infusion holds promising potential as a complementary resource for the management of urolithiasis and constitutes a valuable source of bioactive phytochemicals.
Cleome droserifolia (CD) has been employed traditionally by the Egyptians inhabiting desert for hyperglycemia treatment. We aimed to evaluate the capability of CD to lessen the detrimental consequences of cisplatin (CP) on testis. CD phytoconstituents were studied by LC-ESI-MS/MS. The in vivo study involved four groups of forty male rats. Group I had a normal saline intraperitoneal (IP) injection daily; groups II, III, and IV had a single IP injection of 7 mg/kg of CP. Groups III and IV were orally administered 100 and 200 mg/kg of CD, respectively. They were treated daily for 10 days, and a single dose of IP injection of CP was taken on the third day. The LC-ESI-MS/MS exposed presence of 33 different compounds in CD. Also, CD at 200 mg/kg considerably reestablished the testicular weight loss and serum testosterone in CP-treated rats. Treatment with CD improved the antioxidant capabilities via downregulating malondialdehyde and increasing catalase and glutathione peroxidase activity as well as nuclear factor erythroid 2-related factor 2 immunostaining. It exerted a significant anti-inflammatory action via its decreasing effect on nuclear factor kappa beta immunostaining, and testicular level of tumor necrosis factor-α. Moreover, it exhibited an anti-apoptotic effect by downregulation of p53 and caspase-3, besides enhancing the B-cell lymphoma 2 immunostaining. In addition, molecular docking studies revealed that the major phytochemicals in CD had favorable binding affinities to the key proteins of apoptosis. CD demonstrated promising antioxidant, anti-inflammatory, and anti-apoptotic properties.
Xinxue Granules (XXGR) is a traditional Chinese medicine formula derived from the classical prescription Zixue Powder, one of the three treasured formulas in traditional Chinese medicine. Based on the principle of heat-clearing and detoxification, it has long been used to treat respiratory diseases associated with heat-toxin accumulation. Owing to its multi-component nature, XXGR has been reported to modulate inflammatory responses and alleviate pulmonary injury. However, its protective effects and underlying mechanisms in pulmonary fibrosis-like injury remain unclear. To investigate the protective effects and mechanisms of XXGR in pulmonary fibrosis. A bleomycin-induced pulmonary fibrosis-like injury mice model was established after random grouping. The therapeutic effects were assessed by histopathological and biochemical analyses. Network pharmacology, metabolomics, and transcriptomics were integrated to identify potential pathways. Additional in vitro experiments using TGF-β1-induced MLE-12 cells, including EPAC inhibition with ESI-09, were performed to further explore the underlying mechanism. A bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model was established to evaluate the antifibrotic effects of XXGR. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to characterize the chemical constituents and serum-absorbed components of XXGR. Network pharmacology, transcriptomics, and non-targeted metabolomics were integrated to identify potential targets and pathways. In vivo experiments, including histopathology, biochemical assays, quantitative real-time PCR (qRT-PCR), and Western blotting (WB), were performed to validate key molecular mechanisms. In vitro experiments using TGF-β1-stimulated MLE-12 cells with EPAC inhibition (ESI-09) were performed to explore mechanisms. Molecular docking was conducted to evaluate compound-target interactions. All botanical names were verified according to the World Flora Online. UPLC-Q-TOF/MS identified 20 blood-absorbed components of XXGR, and network pharmacology revealed 222 potential targets, highlighting the cyclic adenosine monophosphate (cAMP) signaling pathway. XXGR reduced lung injury, inflammatory cytokine levels, fibroblast activation, and extracellular matrix deposition in BLM-induced mice. Mechanistically, XXGR increased EPAC expression and CREB phosphorylation while decreasing GLI3 expression in vivo and in vitro, consistent with activation of cAMP signaling. EPAC inhibition with ESI-09 partially reversed the effects of XXGR on fibrosis- and EMT-related markers. XXGR attenuated bleomycin-induced pulmonary fibrosis-like injury by suppressing inflammation and fibroblast activation. Its protective effects may be partially mediated via the cAMP/EPAC/CREB signaling pathway. These findings suggest that XXGR represents a promising multi-target therapeutic candidate for pulmonary fibrosis.
Parkinson's disease is a common neurodegenerative disorder with a complex pathogenesis, which limits the application of single-target inhibitors. At present, the main Western therapeutic drugs for Parkinson's disease include dopamine precursors, dopamine receptor agonists, monoamine oxidase inhibitors (MAO-B), catechol-O-methyltransferase (COMT) inhibitors and anticholinergic drugs. These treatments have limited efficacy and obvious toxic and side effects. In recent years, with the deepening of research on Parkinson's disease, traditional Chinese medicine (TCM) and its active components have played an increasingly important role in clinical treatment. A large number of studies have shown that TCM and its active components exert anti-Parkinsonian effects by inhibiting ferroptosis, regulating autophagy and apoptosis, exerting anti-inflammatory and antioxidant effects, and enhancing neuronal protection. To show that traditional medicine is a promising strategy for the treatment of Parkinson's disease, and to lay a foundation for exploring the specific mechanisms of traditional Chinese medicine in the prevention and treatment of this disease. This study conducted a bibliometric analysis of Chinese and foreign databases to screen the core traditional Chinese medicines currently used clinically for Parkinson's disease, and further analyzed the specific mechanisms of action of the selected core herbs in the treatment of Parkinson's disease. Bibliometric analysis identified core TCM herbs clinically used for Parkinson's disease. The specific mechanisms of these core herbs against Parkinson's disease were clarified at the molecular level, involving inhibition of ferroptosis, regulation of autophagy and apoptosis, anti-inflammatory and antioxidant effects, and enhancement of neuroprotection. This study provides data support for the subsequent research and clinical application of traditional Chinese medicine in Parkinson's disease, and deepens the molecular understanding of the efficacy of traditional Chinese medicine, verifying that traditional medicine is a promising therapeutic strategy for Parkinson's disease.
Heart failure (HF) is a complex clinical syndrome characterized by significant cardiac electrical conduction disturbances. Qishen paste (QSP), a modern herbal formulation consisting of the dried root of Astragalus membranaceus (Fisch.) Bge. (Fabaceae), the dried root and rhizome of Salvia miltiorrhiza Bunge (Lamiaceae), the processed lateral root of Aconitum carmichaelii Debeaux (Ranunculaceae), the dried root of Scrophularia ningpoensis Hemsl. (Scrophulariaceae), the dried flower bud of Lonicera japonica Thunb. (Caprifoliaceae), and the dried root and rhizome of Glycyrrhiza uralensis Fisch. ex DC. (Fabaceae), has shown therapeutic efficacy in HF, but its mechanisms in regulating immune homeostasis and electrical conduction remain unclear. This study aims to evaluate the therapeutic effect of QSP on HF from the perspective of immune homeostasis and electrical conduction. HF was induced in mice by left anterior descending coronary artery (LAD) ligation. After treatment with QSP, cardiac structure and function were assessed in each group. Electrical conduction was monitored by electrocardiography, a multi-channel electrical mapping system, and microelectrode arrays. Network pharmacology and RNA-seq were used to predict potential mechanisms. Flow cytometry, Western blot, quantitative real-time PCR, and immunofluorescence were employed to detect the proportion of cardiac resident macrophages (RCMs) and the expression of connexin43 (Cx43) in HF mice. QSP improved cardiac function, inflammation and fibrosis, and electrophysiological abnormalities in HF mice. In macrophage-cardiomyocyte (CM) co-cultures, it improved repolarization parameters, with Cx43 as a key target. In vivo experiments confirmed that QSP upregulated Cx43 expression in RCMs. QSP ameliorates HF by enhancing cardiac conduction through modulating Cx43 in RCMs, highlighting the therapeutic promise of targeting RCM-CM electrical coupling. However, future validation using multiple batches of QSP is required to confirm the reproducibility of these results.
The nine steaming and nine sun-drying method, a classic representative of traditional Chinese medicine (TCM) processing, is primarily used for processing precious tonic herbs. Its core value lies in its multi-faceted regulation of herbal properties, thereby providing a material basis for TCM syndrome differentiation and treatment. This review aims to systematically summarize the historical evolution and technical characteristics of this processing method, integrate representative herbs under a unified analytical framework, and interpret this traditional processing method from the perspectives of coordinated chemical transformation, bioactivity remodeling, and modernization potential. Available literature related to the nine steaming and nine sun-drying process was collected by searching PubMed, ScienceDirect, Web of Science, Scopus, and CNKI. Through repeated steaming and sun-drying, this process markedly alters the chemical composition of selected medicinal materials, such as Heshouwu, Huangjing, rhubarb, and Rehmanniae Radix. These transformations, mediated by mechanisms including pyrolysis and hydrolysis, oxidation-reduction, and Maillard reactions, may optimize the component profile and contribute to enhanced pharmacological activities (e.g., immunomodulation, neuroprotection, and hematinic effects), while attenuating toxicity-related or irritant effects in specific herbs, such as hepatotoxicity-related constituents in Heshouwu and gastrointestinal irritation in Huangjing or rhubarb. The nine steaming and nine sun-drying process achieves the optimization of herbal properties and potentiation of therapeutic effects, but challenges such as low production efficiency and difficulties in quality control persist in its modern application. In-depth research is expected to promote its inheritance and innovation.