Pharmaceuticals

11 pages, 1300 KB

Open AccessArticle

Protective Role of Alpha-Lipoic Acid Against Methotrexate-Induced Osteotoxicity: Mechanisms of Oxidative Stress Regulation and MAPK Pathway Inhibition

by Ahmet Can Haskan, Muhammed Said Altun, Osman Fatih Arpağ, Fariz Selimli, Soner Mete, Percin Pazarci and Halil Mahir Kaplan

Pharmaceuticals 2026, 19(5), 729; https://doi.org/10.3390/ph19050729 - 5 May 2026

Abstract

Background/Objectives: Osteotoxicity is a severe complication of Methotrexate (MTX) chemotherapy, characterized by oxidative stress and disrupted bone remodeling. The primary objective of this study was to investigate the cytoprotective mechanisms of the antioxidant Alpha-Lipoic Acid (ALA) against MTX-induced osteotoxicity, specifically focusing on [...] Read more.

Background/Objectives: Osteotoxicity is a severe complication of Methotrexate (MTX) chemotherapy, characterized by oxidative stress and disrupted bone remodeling. The primary objective of this study was to investigate the cytoprotective mechanisms of the antioxidant Alpha-Lipoic Acid (ALA) against MTX-induced osteotoxicity, specifically focusing on its modulation of oxidative stress, apoptosis, and Mitogen-Activated Protein Kinase (MAPK) signaling pathways. Methods: Murine osteocyte-like MLO-Y4 cells were cultured and exposed to a fixed dose of MTX (10⁻⁵ M), either alone or concurrently with ALA (50 μmol/L) for 48 h. Biochemical profiling was performed using specific enzyme-linked immunosorbent assays (ELISA) and colorimetric kits to evaluate pro- and anti-apoptotic proteins (Caspase-3, Bax, Bcl-2, Wee1, GRP78, GADD153, AIF), active MAPK components (p-JNK, p-ERK), and standard oxidative stress parameters (TAS, TOS, SOD, GPx). Results: MTX treatment induced significant cellular stress, evidenced by elevated Caspase-3, Bax, p-JNK, and p-ERK levels, alongside a critical reduction in Bcl-2 expression. MTX also markedly increased TOS while depleting TAS, SOD, and GPx levels. Conversely, co-treatment with ALA significantly mitigated these cytotoxic responses. ALA restored the Bax/Bcl-2 balance, effectively downregulated both p-JNK and p-ERK activation, and substantially reinforced the cellular antioxidant defense system by enhancing TAS, SOD, and GPx activities, although recovery to baseline control levels was partial. Conclusions: ALA exerts robust in vitro cytoprotective effects against MTX-induced osteotoxicity in MLO-Y4 cells by counteracting oxidative stress and inhibiting aberrant apoptotic and MAPK signaling. These findings establish a mechanistic baseline, underscoring the need for subsequent in vivo dose–response studies to validate ALA’s therapeutic potential in chemotherapy management. Full article

(This article belongs to the Section Pharmacology)

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26 pages, 4316 KB

Open AccessArticle

Protective Effects of Licorice (Glycyrrhiza uralensis) Against Vancomycin-Induced Nephrotoxicity In Vivo and In Vitro

by Jianping Zhang, Yan Zhou, Ruirui Cui, Lijun Wang, Sijia Wang, Wenhan Rao and Xinan Wu

Pharmaceuticals 2026, 19(5), 728; https://doi.org/10.3390/ph19050728 - 4 May 2026

Abstract

Background: Vancomycin (VAN)-induced nephrotoxicity limits its clinical application. Licorice (Glycyrrhiza uralensis Fisch.) and its bioactive constituents have been reported to protect against nephrotoxicity induced by various nephrotoxic agents. This study aimed to evaluate the protective effects of licorice against VAN-induced nephrotoxicity and [...] Read more.

Background: Vancomycin (VAN)-induced nephrotoxicity limits its clinical application. Licorice (Glycyrrhiza uralensis Fisch.) and its bioactive constituents have been reported to protect against nephrotoxicity induced by various nephrotoxic agents. This study aimed to evaluate the protective effects of licorice against VAN-induced nephrotoxicity and to explore the underlying mechanisms both in vivo and in vitro. Methods: Seven groups of male C57BL/6 mice received different treatments for 7 consecutive days. Blood, fecal and renal tissue samples were collected for the assessment of serum creatinine, renal histopathology, mitochondrial ultrastructure, oxidative stress markers, kidney injury molecule-1 (Kim-1), short-chain fatty acids (SCFAs), and uremic toxins. In human proximal tubular epithelial cells (HK-2 cells), the effects of licorice on cell viability, oxidative stress, inflammatory markers, and mitochondrial membrane potential (MMP) were further investigated. Results: Licorice significantly attenuated VAN-induced nephrotoxicity and restored glutathione peroxidase (GSH-Px) activity while reducing malondialdehyde (MDA) levels. In addition, licorice markedly ameliorated VAN-induced renal histopathological injury, as demonstrated by hematoxylin and eosin staining and transmission electron microscopy. Licorice also reversed VAN-induced intestinal microbiota dysbiosis and increased the relative abundance of SCFA-producing bacteria, including Bacteroides. Moreover, licorice treatment increased fecal SCFA contents and modulated multiple uremic toxins in both serum and renal tissue. Consistently, licorice protected HK-2 cells against VAN-induced cytotoxicity by regulating GSH, interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and MMP. Conclusions: These findings demonstrate that licorice exerts protective effects against VAN-induced nephrotoxicity in vivo and in vitro, suggesting the potential involvement of oxidative stress, mitochondrial structure and function, inflammation, intestinal microbiota-SCFAs and uremic toxins. Full article

(This article belongs to the Special Issue Advances in the Mechanism of Action and the Therapeutic Role of Phytopharmaceuticals)

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22 pages, 1109 KB

Open AccessReview

Phage Therapy in Combating Multidrug-Resistant Gram-Negative Pathogens: A Scoping Review

by Asif Sukri, Bruno Silvester Lopes and Alfizah Hanafiah

Pharmaceuticals 2026, 19(5), 727; https://doi.org/10.3390/ph19050727 - 3 May 2026

Abstract

Background: The emergence of multidrug-resistant (MDR) Gram-negative pathogens, namely Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Helicobacter pylori, necessitates urgent therapeutic alternatives. This scoping review aimed to summarize the current evidence on the efficacy of lytic bacteriophages against these critical [...] Read more.

Background: The emergence of multidrug-resistant (MDR) Gram-negative pathogens, namely Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Helicobacter pylori, necessitates urgent therapeutic alternatives. This scoping review aimed to summarize the current evidence on the efficacy of lytic bacteriophages against these critical MDR pathogens, and to identify existing research gaps and implementation challenges. Methods: The literature search was conducted by searching PubMed, Web of Science, and Scopus AI for studies published from 2015 to 2025. The inclusion criteria focused on experimental and human studies evaluating phage therapy against MDR, extensively drug-resistant (XDR), or pan-drug-resistant (PDR) strains in the four target species. A total of 172 articles were included. Results: A number of studies showed an increasing trend (2015–2025), focusing mainly on K. pneumoniae (n = 65), P. aeruginosa (n = 55), and A. baumannii (n = 48). No eligible studies for MDR H. pylori were found. All 172 studies confirmed lytic activity, with phage cocktails showing superior antibacterial activity than single phages in four studies. Phages also demonstrated antibiofilm activity (n = 44). Most animal studies reported successful bacterial reduction in animals treated with phages, and 87.5% of 23 human case studies reported patient improvement or infection clearance. However, heterogeneity in the types of animal models used and in dosage and administration routes in human studies was notable. Conclusions: Lytic bacteriophages exhibit strong potential as a new therapeutic option. Key challenges include the lack of data for MDR H. pylori, heterogeneity in animal models, and a paucity of large-scale human clinical trials. Future research must prioritize standardization, mechanistic studies, and conducting robust human trials to enable clinical translation and regulatory acceptance. Full article

(This article belongs to the Special Issue Antibiotic Resistance in Gram-Negative Bacteria: The Threat from the Pink Corner, 2nd Edition)

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28 pages, 10066 KB

Open AccessArticle

Pharmacological Mechanisms of Ursolic Acid Derivative Against Prostate Cancer via Regulating Cytoskeletal Homeostasis and Apoptotic Pathways

by Huiyue Shen, Zhaolan Ni, Haibo Guo, Xiaofeng Liu, Yaru Zhao, Xuan He, Yinghan Liu, Yan Zhao and Hongbo Teng

Pharmaceuticals 2026, 19(5), 726; https://doi.org/10.3390/ph19050726 - 2 May 2026

Abstract

Background: Ursolic acid (UA) is a natural pentacyclic triterpenoid with notable antitumor activity, yet its poor water solubility and insufficient targeting restrict clinical translation. Methods: Forty novel ursolic acid-phosphine derivatives bearing seven distinct lipophilic cationic moieties were synthesized via C28 modification [...] Read more.

Background: Ursolic acid (UA) is a natural pentacyclic triterpenoid with notable antitumor activity, yet its poor water solubility and insufficient targeting restrict clinical translation. Methods: Forty novel ursolic acid-phosphine derivatives bearing seven distinct lipophilic cationic moieties were synthesized via C28 modification and structurally characterized by ¹H NMR and ¹³C NMR. Their antitumor activities in PC3-M cells were evaluated via in vitro assays. Mechanistic investigations were performed using transcriptomic analysis and Western blot. Molecular docking was performed to predict the binding profile of Compound 25 with FGFR1. In vivo antitumor efficacy and biosafety were assessed in RM-1 xenograft models in C57BL/6 mice. Results: Compound 25 (bearing a tris(3,5-dimethylphenyl)phosphine group at the C28 position with an alkyl chain length of five methylene units) exhibited the most potent activity against PC3-M cells, dose-dependently inhibiting proliferation, migration, and invasion and inducing apoptosis. It triggered mitochondrial apoptosis via ROS accumulation and disrupted cytoskeletal homeostasis by suppressing the FGFR1/KRAS/RAC1/PIP4K2 axis. Molecular docking results suggested its strong binding affinity and specificity. In vivo studies confirmed its significant antitumor effect and favorable safety. Conclusions: These results highlight the potential of Compound 25 as a promising lead compound and provide valuable insights for further medicinal chemistry optimization and the development of novel anticancer drugs derived from ursolic acid. Full article

(This article belongs to the Special Issue Natural Products for the Treatment of Prostate Cancer)

29 pages, 1146 KB

Open AccessSystematic Review

Back to the Roots: Safety and Tolerability of Standardised Ashwagandha (Withania somnifera) Root Extract in Healthy Adults—A Systematic Review of Biomarkers and Adverse Events

by Olivia C. Coope, Mark E. T. Willems, Alex Levington, Mark J. Tallon, Blanca Roman-Viñas and Tilly J. Spurr

Pharmaceuticals 2026, 19(5), 725; https://doi.org/10.3390/ph19050725 - 2 May 2026

Abstract

Background: Standardised Ashwagandha root extract (SARE), characterised by its content of bioactive withanolides, is widely used for its antioxidant and adaptogenic properties; however, recent case reports have raised safety concerns, primarily involving non-standardised or multi-ingredient formulations. This systematic review evaluated the safety and [...] Read more.

Background: Standardised Ashwagandha root extract (SARE), characterised by its content of bioactive withanolides, is widely used for its antioxidant and adaptogenic properties; however, recent case reports have raised safety concerns, primarily involving non-standardised or multi-ingredient formulations. This systematic review evaluated the safety and tolerability of SARE in healthy adults, with a focus on clinical biomarkers and adverse event reporting. Methods: Randomised trials were identified through searches of PubMed, Web of Science and Google Scholar, published from 2010 to April 2026. Studies administering single-ingredient, standardised root-only extracts to generally healthy populations were included. Risk of bias was assessed using the Cochrane RoB 2 tool. Results: Twenty-three studies with a total of 2317 participants met the inclusion criteria, with doses ranging from 125 to 600 mg/day and intervention durations from a single dose to 180 days. Across studies, hepatic, renal, haematological, endocrine, and cardiovascular biomarkers remained within normal clinical ranges, with no clinically meaningful adverse alterations reported. Reductions in cortisol were consistently observed, while increases in testosterone remained within physiological ranges. No serious adverse events attributable to SARE were reported. Mild adverse events, including gastrointestinal discomfort, headache, and transient drowsiness, were infrequently reported and occurred in both intervention and comparator groups. Conclusions: SARE was well tolerated in healthy adults at the studied doses and durations. However, limited long-term data (>180 days) and heterogeneity in study design and reporting warrant further large-scale, standardised trials to confirm safety across extended use and diverse populations. The review is registered in the PROSPERO database with ID CRD420261337116. Full article

(This article belongs to the Section Medicinal Chemistry)

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20 pages, 831 KB

Open AccessArticle

A Three-Arm, Tiered Comparability Strategy Bridging Post-Approval Process Changes for an Omalizumab Biosimilar (CMAB007)

by Chenguang Wang, Chaoxin Zhou, Sheng Hou, Wenqiang Fan, Weizhu Qian, Yule Ren, Xiyuan Chen, Chenhong Pan, Qingcheng Guo, Huaizu Guo and Yajun Guo

Pharmaceuticals 2026, 19(5), 724; https://doi.org/10.3390/ph19050724 - 2 May 2026

Abstract

Background: Post-approval manufacturing changes for biologics require rigorous comparability assessments to ensure uninterrupted quality and clinical performance. CMAB007 (Aomaishu^®), a China-approved (2023) omalizumab biosimilar, underwent process enhancements—including media optimization and anion-exchange chromatography substitution—yielding a 5-fold increase in production without altering the [...] Read more.

Background: Post-approval manufacturing changes for biologics require rigorous comparability assessments to ensure uninterrupted quality and clinical performance. CMAB007 (Aomaishu^®), a China-approved (2023) omalizumab biosimilar, underwent process enhancements—including media optimization and anion-exchange chromatography substitution—yielding a 5-fold increase in production without altering the host cell line. Methods: A novel three-arm tiered strategy was adopted to compare post-change CMAB007, pre-change CMAB007, and reference (Xolair^®) products. Critical quality attributes (CQAs) were classified into tiers based on risk impact, with tier-specific acceptance criteria. Comprehensive analytics assessed structure, post-translational modifications, purity/impurities, activity, and Fc-mediated functions. Forced degradation (lyophilized/reconstituted states) and accelerated stability studies were evaluated. Based on the high degree of CMC similarity and to prevent “biological drift”, the pharmacokinetic (PK) and safety comparability of the post-change CMAB007 versus the reference product (Xolair^®) was confirmed in a randomized, double-blind, two-arm study in healthy males (N = 114; single 150 mg subcutaneous administration). The pre-change product was not included in this clinical PK study. Results: Post-change CMAB007 exhibited analytical similarity within tiered acceptance criteria for all CQAs. Stability studies confirmed enhanced robustness under stress conditions. PK equivalence was demonstrated for AUC_0–inf (GMR: 99.82%; 90% CI: 91.46~108.94%), AUC_0–t (99.54%; 91.40~108.41%), and C_max (101.88%; 95.21~109.01%). Immunogenicity (ADA incidence: 10.5% vs. 12.5%, p = 0.742) and safety profiles were comparable. Conclusions: This study pioneers a tiered three-arm comparability strategy for post-approval changes, integrating advanced analytics, risk-based quality assessment, and clinical validation. The approach mitigates “biological drift” risks, ensuring biosimilar quality, efficacy, and safety while enabling sustainable production scalability. Full article

(This article belongs to the Section Pharmacology)

31 pages, 6870 KB

Open AccessReview

Decoding the Role of MDSCs in Bone Metastasis: Multicellular Interactions and Clinical Implications

by Samaa Alotab, Mariam Zainab, Labibah Labib Khamies, Rasha Alissa and Khalid Said Mohammad

Pharmaceuticals 2026, 19(5), 723; https://doi.org/10.3390/ph19050723 - 2 May 2026

Abstract

Bone metastasis remains a major cause of morbidity in advanced cancer, driven not only by tumor–bone crosstalk but also by profound immune remodeling within the marrow. Myeloid-derived suppressor cells (MDSCs), including polymorphonuclear (PMN-MDSC) and monocytic (M-MDSC) subsets, are increasingly recognized as central effectors [...] Read more.

Bone metastasis remains a major cause of morbidity in advanced cancer, driven not only by tumor–bone crosstalk but also by profound immune remodeling within the marrow. Myeloid-derived suppressor cells (MDSCs), including polymorphonuclear (PMN-MDSC) and monocytic (M-MDSC) subsets, are increasingly recognized as central effectors of this process, integrating inflammatory signals with metabolic and stromal cues to enforce immune suppression and support skeletal colonization. In this review, we synthesize current evidence that bone metastases transform the bone marrow into an “MDSC amplifier,” where vascular and endosteal niches, CXCL12-rich stromal compartments, hypoxia, and adipocyte-derived lipids collectively promote MDSC recruitment, persistence, and functional maturation. We discuss the dominant suppressive programs deployed by MDSCs in bone (e.g., arginase-1 activity, reactive oxygen/nitrogen species, and checkpoint ligand expression), and how these mechanisms converge to impair cytotoxic T-cell and NK-cell responses while fostering regulatory T-cell dominance. Importantly, because the marrow is a hematopoietic organ, bone lesions can also generate systemic consequences through myeloid spillover, providing a mechanistic basis for reduced responsiveness to immune checkpoint blockade in bone-dominant disease. We then evaluate pharmacologic strategies to target MDSCs in the context of bone metastasis, including approaches that block trafficking (e.g., CCR2/CXCR2 axes), deplete or reprogram suppressive myeloid states (e.g., STAT3-directed strategies, differentiation therapy), and disrupt bone-resorptive feedback loops (e.g., receptor activator of NF-κB ligand (RANKL) inhibition and bisphosphonates), emphasizing rational combinations and sequencing to limit marrow toxicity. Finally, we highlight emerging single-cell and spatial profiling tools that can resolve bone-specific heterogeneity in MDSCs and guide biomarker-driven, mechanism-informed therapeutic development. Full article

(This article belongs to the Special Issue Tumor Immunopharmacology, 2nd Edition)

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21 pages, 2909 KB

Open AccessReview

Potential of Silver Nanoparticles in Imaging Diagnostics and Image-Guided Applications: A Narrative Review

by Vera Gledacheva and Stoyanka Nikolova

Pharmaceuticals 2026, 19(5), 722; https://doi.org/10.3390/ph19050722 - 1 May 2026

Abstract

Background/Objectives: Silver nanoparticles (AgNPs) are highly valuable nanomaterials due to their unique optical and physicochemical properties. AgNPs have a lot of promise as contrast-enhancing and diagnostic agents in image-guided treatment. With a focus on their incorporation into image-guided and theranostic approaches, this [...] Read more.

Background/Objectives: Silver nanoparticles (AgNPs) are highly valuable nanomaterials due to their unique optical and physicochemical properties. AgNPs have a lot of promise as contrast-enhancing and diagnostic agents in image-guided treatment. With a focus on their incorporation into image-guided and theranostic approaches, this narrative review attempts to assess the current function of AgNPs in imaging diagnostics. Methods: Using major scientific databases, such as PubMed, Web of Science, and Scopus, a narrative literature review has been conducted with an emphasis on recent preclinical and experimental research examining AgNP-based systems for diagnostic imaging applications. The design of the NPs, surface functionalization, imaging modality, and diagnostic performance of the evaluated studies were analyzed. Results: Due to their surface plasmon resonance and tunable physicochemical properties, AgNPs show great promise in a variety of imaging techniques, such as optical imaging, computed tomography (CT), and multimodal platforms, according to the reviewed literature. Functionalized AgNPs emerged as agents in image-guided therapy due to their improved target selectivity, enhanced imaging contrast, and signal amplification in tissues. Conclusions: AgNPs are appealing nanoscale platforms for image-guided methods and imaging diagnostics. Despite their encouraging preclinical results, some key issues, such as toxicity, biocompatibility, and clinical translation, remain critical. AgNP-based therapeutic and diagnostic systems will need to overcome these constraints in the future. Full article

(This article belongs to the Section Pharmacology)

18 pages, 337 KB

Open AccessReview

Is Short Therapy an Appropriate Regimen for Children and Young Adolescents with Drug-Susceptible Tuberculosis?

by Susanna Esposito, Valentina Fainardi, Beatrice Rita Campana, Gaia Giorgia Arnesano and Nicola Principi

Pharmaceuticals 2026, 19(5), 721; https://doi.org/10.3390/ph19050721 - 1 May 2026

Abstract

Background: Tuberculosis (TB) remains a major cause of morbidity and mortality among children worldwide, with approximately one million new pediatric cases annually. The conventional treatment for drug-susceptible TB has long relied on a 6-month multidrug regimen, which is highly effective but associated with [...] Read more.

Background: Tuberculosis (TB) remains a major cause of morbidity and mortality among children worldwide, with approximately one million new pediatric cases annually. The conventional treatment for drug-susceptible TB has long relied on a 6-month multidrug regimen, which is highly effective but associated with challenges in adherence, toxicity, and healthcare burden. Objectives: To evaluate whether short-course therapy is an appropriate regimen for children and young adolescents with drug-susceptible TB, with particular focus on its efficacy, safety, and applicability in different clinical contexts. Methods: A structured narrative review of the literature was conducted, including randomized controlled trials, observational studies, and international guidelines addressing treatment duration in children and young adolescents with drug-susceptible TB. Evidence was synthesized focusing on children and young adolescents <16 years with drug-susceptible TB treated with short-course regimens compared to standard therapy. Results: A shorter treatment regimen, particularly 4-month courses, has been investigated as an alternative to standard therapy in the pediatric population with drug-susceptible TB. Children often present with paucibacillary and non-severe forms of TB, providing a biological rationale for treatment shortening. Evidence from a randomized controlled trial has demonstrated that a 4-month regimen is non-inferior to the standard 6-month therapy in children and young adolescents with non-severe, drug-susceptible TB. These findings have informed recent international guideline updates, which now recommend short therapy in carefully selected patients. However, a short regimen is not appropriate for infants younger than 3 months, children with severe or complicated TB, extrapulmonary disease such as central nervous system involvement, or those with drug-resistant TB. The overall quality of evidence remains moderate, and long-term relapse data are still emerging. Conclusions: Short-course therapy represents a promising but selective strategy in pediatric drug-susceptible TB management. It offers potential advantages, including improved adherence, reduced drug toxicity, and lower healthcare costs. However, its safe implementation requires accurate patient selection, access to appropriate diagnostic tools, and structured follow-up. Careful application within clearly defined clinical criteria is essential to ensure optimal outcomes. Full article

(This article belongs to the Special Issue Advanced Nanosystems and Emerging Therapies: Innovations in Tuberculosis Treatment and Drug Resistance)

20 pages, 926 KB

Open AccessArticle

Methotrexate Exposure and Inflammatory–Metabolic Biomarker Networks in Hospitalized Patients with Psoriasis: A Network Analysis Approach

by Laura-Florina Nistor, Ruxandra-Cristina Marin, Laura Maria Endres, Gabriela S. Bungau, Ada Radu, Diana Alina Bei and Delia Mirela Tit

Pharmaceuticals 2026, 19(5), 720; https://doi.org/10.3390/ph19050720 - 1 May 2026

Abstract

Background: Psoriasis is a chronic immune-mediated inflammatory disorder strongly associated with cardiometabolic comorbidities. Although methotrexate (MTX) is widely used for moderate-to-severe disease, its influence on the relationships between inflammatory and metabolic biomarkers remains insufficiently characterized. Methods: This retrospective observational study included 132 hospitalized [...] Read more.

Background: Psoriasis is a chronic immune-mediated inflammatory disorder strongly associated with cardiometabolic comorbidities. Although methotrexate (MTX) is widely used for moderate-to-severe disease, its influence on the relationships between inflammatory and metabolic biomarkers remains insufficiently characterized. Methods: This retrospective observational study included 132 hospitalized adult patients with psoriasis, stratified into untreated (n = 101) and MTX-treated (n = 31) groups. Inflammatory markers, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), neutrophil-to-lymphocyte ratio (NLR), and systemic immune-inflammation index (SII), and metabolic indices, triglyceride–glucose index (TyG), metabolic score for insulin resistance (METS-IR), and atherogenic index of plasma (AIP), were analyzed. Group comparisons were performed using Mann–Whitney U and χ² tests. Spearman correlation matrices and regularized partial correlation networks (EBICglasso, γ = 0.5) were constructed separately for each group to explore inflammatory–metabolic connectivity. Results: MTX-treated patients exhibited lower NLR (p = 0.035) and fasting glucose levels (p = 0.004), while CRP, ESR, and composite metabolic indices did not differ significantly. In untreated patients, correlation analysis showed multiple significant cross-domain associations between inflammatory and metabolic markers. In contrast, fewer such associations reached statistical significance in the MTX-treated group. Network analysis indicated a less densely connected structure in the MTX group (9 vs. 12 non-zero edges); however, formal network comparison did not identify statistically significant differences between groups. Conclusions: Although fewer statistically significant cross-domain correlations were observed in MTX-treated patients, no statistically significant differences in network structure were detected between groups. These findings are exploratory and hypothesis-generating, not indicative of methotrexate-related modification of network structure, and are limited by the small size of the MTX-treated subgroup. Full article

(This article belongs to the Section Pharmacology)

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29 pages, 1997 KB

Open AccessArticle

Kun-Ling Wan Formula Ameliorates Postmenopausal Osteoporosis and Adipose Accumulation by Suppressing mTOR Signaling in Mesenchymal Stem Cells

by Xiaoqing Lu, Tingting Xie, He Lan, Yaqi Fan, Jie Yang, Qianzan Liao, Yuxin Jin, Yaoxuan Zhu, Jingxin Zhang, Dexin Li, Chunshui Pan, Quan Li, Kai Sun, Xinmei Huo, Ting Yuwen, Jing-Yan Han and Yin Li

Pharmaceuticals 2026, 19(5), 719; https://doi.org/10.3390/ph19050719 - 30 Apr 2026

Abstract

Background: Postmenopausal osteoporosis is a common metabolic bone disorder characterized by decreased bone mass and microstructural deterioration, often accompanied by increased bone marrow adiposity and systemic fat accumulation. Kun-Ling Wan Formula (KLW) is a compound Chinese medicine clinically used for gynecological disorders, [...] Read more.

Background: Postmenopausal osteoporosis is a common metabolic bone disorder characterized by decreased bone mass and microstructural deterioration, often accompanied by increased bone marrow adiposity and systemic fat accumulation. Kun-Ling Wan Formula (KLW) is a compound Chinese medicine clinically used for gynecological disorders, though its effects on postmenopausal osteoporosis and associated fat accumulation remain unclear. Distinct from previous herbal formulation studies that primarily focused on bone outcomes, our study uniquely integrates bone protection, marrow adiposity reduction, systemic metabolic improvement, and multi-omics mechanistic dissection in a high-fat diet-fed ovariectomized mouse model. Methods: KLW chemical composition was analyzed by UPLC-Q-TOF/MS. Ovariectomized (OVX) C57BL/6J mice fed high-fat or normal diet were treated with KLW at clinically equivalent or double doses, with estrogen and active compounds as controls. Bone microstructure was assessed by micro-CT, bone marrow fat by MRI-PDFF, and metabolism by OGTT, ITT, and metabolic cages. Network pharmacology, proteomics, molecular docking, and dynamics simulations identified core targets. C3H10T1/2 cells were used to assess osteogenic/adipogenic differentiation and mTOR pathway activation. Results: Twelve compounds were identified in KLW. In OVX mice, KLW significantly improved bone mineral density and trabecular microstructure, reduced adiposity and bone marrow fat, and enhanced glucose tolerance and insulin sensitivity. In vitro, KLW promoted osteogenesis and suppressed adipogenesis in C3H10T1/2 cells. Integrative analyses identified mTOR as a central target, with chrysophanol, pyrogallol, and apigenin showing high-affinity binding. KLW inhibited mTOR/S6K phosphorylation during differentiation, an effect reversible by leucine. Conclusions: KLW ameliorates osteoporosis and reduces fat accumulation in OVX mice by shifting mesenchymal stem cell differentiation toward osteogenesis via mTOR pathway modulation. Full article

(This article belongs to the Section Natural Products)

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26 pages, 4155 KB

Open AccessArticle

Revealing the Pharmacological Mechanism of Tibetan Medicine Wugeng San in Treating Rheumatoid Arthritis Through an Integrated Strategy of Chemical Composition Analysis, Network Pharmacology, Machine Learning, and In Vivo Experiments

by Zixian Chen, Yu Zhang, Shuangqi Chen, Chunxia Zhang, Rui Gu and Shaohui Wang

Pharmaceuticals 2026, 19(5), 718; https://doi.org/10.3390/ph19050718 - 30 Apr 2026

Abstract

Background: Wugeng San (WGS) is a traditional Tibetan medicinal preparation that has long been used to treat inflammatory and arthritic conditions. However, its contemporary pharmacological validation and the mechanisms underlying its action in rheumatoid arthritis (RA) have not been fully investigated. Objective: For [...] Read more.

Background: Wugeng San (WGS) is a traditional Tibetan medicinal preparation that has long been used to treat inflammatory and arthritic conditions. However, its contemporary pharmacological validation and the mechanisms underlying its action in rheumatoid arthritis (RA) have not been fully investigated. Objective: For the first time, this study aimed to systematically investigate the therapeutic effects of WGS on RA, identify its potential targets, and elucidate its action mechanisms. Methods: This study, as the first comprehensive investigation of WGS in RA, employed integrated multiple approaches including chemical component identification via UPLC-Q-TOF/MS, network pharmacology, bioinformatics, machine learning, and in vivo efficacy assessment and mechanism verification in a collagen-induced arthritis (CIA) rat model, a widely accepted experimental model that mimics the key pathological features of RA. Results: The results demonstrated that WGS reduced the severity of arthritis in a dose-dependent manner, as evidenced by decreased paw swelling, normalized body weight, and restored levels of pro- and anti-inflammatory cytokines. The high dose of WGS (252 mg/kg) showed an effect comparable to that of methotrexate (0.2 mg/kg). Histological analysis revealed that WGS reduced synovial hyperplasia, cartilage erosion and bone destruction, decreased osteoclast numbers, and promoted osteoblast activity. Eighty-four compounds were identified using UPLC-Q-TOF/MS. Network pharmacology and machine learning analyses indicated SYK as a key target enriched in the NF-κB signaling and osteoclast differentiation pathways. Experimental validation confirmed that WGS suppressed the phosphorylation of SYK and NF-κB pathway components (p65, IκBα, and IKKα/β), decreased MMP1/MMP3 levels, and modulated the Bax/Bcl-2 ratio to promote apoptosis. Conclusions: In conclusion, WGS exhibits strong anti-arthritic effects through “multi-component, multi-target, and multi-pathway” mechanisms, likely attributable to the inhibition of the SYK/NF-κB signaling axis, suppression of matrix degradation, and regulation of cellular apoptosis. This research offers a pharmacological basis for repurposing WGS as a promising natural candidate for RA therapy. Full article

(This article belongs to the Section Pharmacology)

30 pages, 4316 KB

Open AccessArticle

Coumarin– and Dipicolylamine–Terpenoid Hybrids as Selective Carbonic Anhydrases IX and XII Inhibitors: Mechanistic Insights and Selective Anti-Cancer Potential

by Venkatesan Saravanan, Andrea Angeli, Francesco Melfi, Nicola Amodio, Ilenia Valentino, Massimo Gentile, Ilaria D'Agostino, Kathiravan Muthukumaradoss, Gokhan Zengin, Davide Moi, Rahime Simsek, Claudiu T. Supuran and Simone Carradori

Pharmaceuticals 2026, 19(5), 717; https://doi.org/10.3390/ph19050717 - 30 Apr 2026

Abstract

Background: Carbonic Anhydrases (CAs) represent regulators of cell adaptation to hypoxia, pH regulation, and metabolic fitness. Among cancers, multiple myeloma (MM) is a plasma cell malignancy sustained by hypoxia-driven metabolic adaptation, extracellular acidification, and redox imbalance. Tight regulation of tumor extracellular pH, [...] Read more.

Background: Carbonic Anhydrases (CAs) represent regulators of cell adaptation to hypoxia, pH regulation, and metabolic fitness. Among cancers, multiple myeloma (MM) is a plasma cell malignancy sustained by hypoxia-driven metabolic adaptation, extracellular acidification, and redox imbalance. Tight regulation of tumor extracellular pH, mediated by Carbonic Anhydrases IX and XII, is crucial for myeloma survival, progression, and stemness, making these isoforms attractive therapeutic targets. Methods: We designed and synthesized a library of terpenoid-based hybrids by derivatizing chlorothymol and 4-isopropyl-3-methylphenol with either the natural coumarin umbelliferon or the 2,2′-dipicolylamine (DPA) scaffold. This chemical strategy aimed to selectively inhibit tumor-associated CAs IX/XII through coumarin- or DPA-mediated recognition, while terpenoid fragments were introduced to enhance lipophilicity, membrane permeability, and potential redox-modulating properties. The compounds were tested by a Stopped-Flow assay for CA inhibition, in cell-based assays for antiproliferative properties and by means of several antioxidant assays. Results: The most active compounds, connecting the coumarin core to a terpenoid tail, inhibited the targeted CAs in the nanomolar range, showing up higher selectivity over off-target isoforms (I and II). In studies performed on MM cell lines, selected derivatives reduced viability (IC₅₀ = 15.8–85.4 µM) and displayed favorable selectivity over normal cells. In silico investigations suggested that the compounds were able to interact selectively with the target enzymes. Conclusions: Collectively, these results support a dual-targeting strategy in which selective inhibition of tumor-associated CAs, combined with redox modulation, interferes with adaptive mechanisms of MM cells, providing a rational framework for the development of multifunctional agents against metabolically resilient hematological malignancies. Full article

(This article belongs to the Special Issue Enzyme Inhibitors: Potential Therapeutic Approaches, 2nd Edition)

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25 pages, 2846 KB

Open AccessArticle

Copper (Cu²⁺) Inhibits Voltage-Dependent Ionic Currents While Enhancing Neurotransmitter Release in Bovine Chromaffin Cells

by Víctor Varea-Tierno, Victoria Jiménez Carretero, Minerva Reyes Almodóvar, Javier Hernández Campano, María Arribas Tejedor, Ricardo de Pascual and Jesús M. Hernández-Guijo

Pharmaceuticals 2026, 19(5), 716; https://doi.org/10.3390/ph19050716 - 30 Apr 2026

Abstract

Background/Objectives: Copper (Cu²⁺) is an essential trace element that participates as a cofactor in key metabolic enzymes such as cytochrome c oxidase and superoxide dismutase. However, excessive copper exposure can be toxic and disturbances in copper homeostasis have been associated [...] Read more.

Background/Objectives: Copper (Cu²⁺) is an essential trace element that participates as a cofactor in key metabolic enzymes such as cytochrome c oxidase and superoxide dismutase. However, excessive copper exposure can be toxic and disturbances in copper homeostasis have been associated with neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. Despite growing evidence linking copper to neuronal dysfunction, the cellular mechanisms by which Cu²⁺ affects neuronal excitability and neurotransmission remain poorly understood. The aim of this study was to investigate the effects of acute Cu²⁺ exposure on ionic currents involved in cellular excitability and neurotransmitter release in bovine chromaffin cells. Methods: Primary cultures of bovine chromaffin cells were used as a neuroendocrine model to study cellular excitability. Voltage-dependent ionic currents were recorded using the whole-cell patch-clamp technique in voltage-clamp configuration. Catecholamine secretion was monitored by amperometry, and cytosolic Ca²⁺ dynamics were measured in fluo-4-loaded cells during depolarization induced by high K⁺ stimulation. Results: Acute Cu²⁺ exposure produced a concentration-dependent enhancement of depolarization-evoked catecholamine release. In parallel, Cu²⁺ inhibited voltage-dependent calcium (ICa), sodium (INa), potassium (IKv), and calcium/voltage-dependent potassium (IKCa-v) currents in a concentration-dependent and partially reversible manner. In addition, Cu²⁺ increased basal cytosolic Ca²⁺ levels while reducing the amplitude of depolarization-evoked Ca²⁺ transients. Conclusions: Acute Cu²⁺ exposure exerts a dual effect in bovine chromaffin cells, inhibiting the ionic currents that support cellular excitability while potentiating catecholamine secretion. This apparent paradox is consistent with a disruption of intracellular Ca²⁺ homeostasis, in which elevated basal cytosolic Ca²⁺ may facilitate exocytosis despite reduced depolarization-evoked Ca²⁺ entry. These findings provide new insight into the mechanisms by which copper may alter neuronal signaling and contribute to neurotoxicity. Full article

(This article belongs to the Section Medicinal Chemistry)

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27 pages, 4094 KB

Open AccessArticle

ComTarget: Small-Molecule Target Prediction with Combinatorial Modeling

by Yuzhu Li, Qingyi Shi, Xingjie Lu, Daiju Yang, Dilixiati Yeerken, Huizi Jin and Qingyan Sun

Pharmaceuticals 2026, 19(5), 715; https://doi.org/10.3390/ph19050715 - 30 Apr 2026

Abstract

Background: Identifying potential targets for bioactive compounds is crucial for elucidating the mechanisms of action and drug development. Methods: This study presents ComTarget, a computational tool that integrates 3D molecular shape similarity analysis (based on combined 3D descriptors, C3DD) with reverse [...] Read more.

Background: Identifying potential targets for bioactive compounds is crucial for elucidating the mechanisms of action and drug development. Methods: This study presents ComTarget, a computational tool that integrates 3D molecular shape similarity analysis (based on combined 3D descriptors, C3DD) with reverse docking to predict protein targets for small molecules. ComTarget screens against a library of 4429 unique protein targets derived from 26,272 PDB complexes. Results: Validation on benchmark datasets (DEKOIS 2.0 and DUDE-Z) demonstrated that the C3DD molecular similarity calculation method effectively enriches active ligands by capturing critical 3D shape information not evident from chemical topology alone. It outperformed conventional 2D fingerprint methods and offered a favorable balance between shape sensitivity and computational efficiency, serving as a rapid pre-screening filter within the integrated workflow. For FDA-approved drugs (e.g., Imatinib, Aspirin) and natural products (e.g., Berberine). ComTarget identified targets consistent with reported therapeutic targets or putative off-targets in the literature, while also revealing potential targets aligned with the compounds’ pharmacological mechanisms. Conclusions: As a local program, ComTarget offers flexibility in computational resources customization and is freely available for polypharmacology studies, drug repurposing, and adverse reaction prediction. Full article

(This article belongs to the Special Issue Computer-Aided Drug Design and Drug Discovery, 2nd Edition)

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16 pages, 1048 KB

Open AccessArticle

A Safe Natural Alternative to Phenylthiourea: Ethyl Acetate Extract of Alchemilla vulgaris for Zebrafish Embryo Depigmentation

by Muhammad Farooq Khan and Mohammad Ahmad Wadaan

Pharmaceuticals 2026, 19(5), 714; https://doi.org/10.3390/ph19050714 - 30 Apr 2026

Abstract

Background: Zebrafish (Danio rerio) embryos are transparent in early stages of embryonic development; however, pigment formation at later stages hinders internal organ visualization during imaging. Chemicals such as 1-phenyl-2-thiourea (PTU) and kojic acid, used to block pigmentation, pose significant toxicity [...] Read more.

Background: Zebrafish (Danio rerio) embryos are transparent in early stages of embryonic development; however, pigment formation at later stages hinders internal organ visualization during imaging. Chemicals such as 1-phenyl-2-thiourea (PTU) and kojic acid, used to block pigmentation, pose significant toxicity risks to human health. Therefore, effective and risk-free depigmentation agents are needed. This study investigates the efficacy of Alchemilla vulgaris (Lady’s mantle) as a safe, natural alternative for zebrafish embryo depigmentation. Methods: A. vulgaris was extracted using four solvents of varying polarities and evaluated for depigmentation efficacy and toxicity. Gas chromatography–mass spectrometry (GC–MS) was used to identify major constituents of the extract. Results: Ethyl acetate extract was more effective at removing pigments than all other extracts, and exhibited the lowest toxicity compared to PTU and kojic acid. Ethyl acetate extract of A. vulgaris remained effective even when administered 48 h post fertilization (post-pigmentation), making it suitable for long-term experiments requiring optical clarity. GC-MS revealed that this extract was rich in linoleic acid, various fatty acid esters, and phenolics, which likely contributed to its depigmentation activity. Conclusions: Based on these findings, we propose that ethyl acetate extract of A. vulgaris is a safer, natural alternative to PTU and kojic acid for depigmenting zebrafish embryos, particularly in long-term imaging experiments. The extract exhibits high efficacy at low concentrations, accompanied by a favorable toxicity profile, demonstrating potential as a depigmentation agent during early zebrafish development. However, further studies are needed to elucidate its mechanism of action. Full article

(This article belongs to the Special Issue Application of Zebrafish Model in Pharmacology and Toxicology)

21 pages, 4404 KB

Open AccessArticle

Evidence for Potentiation of M-Type Potassium Current by Flavonoid Corylin (3-(2,2-Dimethylchromen-6-yl)-7-hydroxychromen-4-one)

by Sheng-Nan Wu, Rasa Liutkevičienė and Sheng-Che Lin

Pharmaceuticals 2026, 19(5), 713; https://doi.org/10.3390/ph19050713 - 30 Apr 2026

Abstract

Background: Corylin (3-(2,2-dimethylchromen-6-yl)-7-hydroxychromen-4-one), a bioactive flavonoid, has been reported to exercise anti-inflammatory, antineoplastic, and antioxidant effects, and may also possess lifespan-extending properties. Objectives: Any modifications of transmembrane ionic currents produced by corylin remain largely unknown. Methods: The patch-clamp technique and docking prediction were [...] Read more.

Background: Corylin (3-(2,2-dimethylchromen-6-yl)-7-hydroxychromen-4-one), a bioactive flavonoid, has been reported to exercise anti-inflammatory, antineoplastic, and antioxidant effects, and may also possess lifespan-extending properties. Objectives: Any modifications of transmembrane ionic currents produced by corylin remain largely unknown. Methods: The patch-clamp technique and docking prediction were used in this study. Results: In pituitary GH₃ somatolactotrophs, corylin concentration-dependently increased the magnitude of the M-type K⁺ current (I_K(M)), with an EC₅₀ of 3.8 μM. Concurrently, the activation time constant of I_K(M) was shortened. The addition of linopirdine (10 μM), an I_K(M) inhibitor, suppressed the current amplitude. Corylin also induced a leftward shift in the steady-state activation curve and enhanced I_K(M) during pulse-train stimulation. Moreover, corylin increases the hysteretic strength of I_K(M) evoked by a long-lasting triangular ramp pulse; this effect was attenuated by linopirdine. The stimulatory effect of corylin on I_K(M) was not altered by carvedilol or iberiotoxin but was reduced by dapagliflozin. In contrast, depolarization-activated I_K(M) was not affected by 17β-estradiol alone. In cell-attached recordings, corylin increased M-type K⁺ (K_M)-channel activity with minimal change in single-channel amplitude, while prolonging the mean open time. This stimulatory effect was reversed by linopirdine or dapagliflozin. Additionally, corylin slightly inhibited the erg-mediated current. Docking analysis further suggested that corylin potentially interacts with residues in KCNQ2 or KCNH2 channels via hydrogen bonding and hydrophobic interactions. Conclusions: These findings suggest that corylin modulates ionic currents, primarily through K_M (KCNQ/K_V7) channels, which may underlie its in vivo actions and those of related flavonoids. These effects may contribute to the regulation of functional activities of neuronal, neuroendocrine, and endocrine cells. Full article

(This article belongs to the Topic Biological and Pharmacological Activity of Natural Products)

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18 pages, 416 KB

Open AccessArticle

Genetic Polymorphisms in SCN1A Gene (rs6432860) and Pharmacoresistance to Antiepileptic Drugs Among Jordanian Patients with Epilepsy

by Hanen Al-Sadir, Ayat Al-Farhood, Al-Motassem Yousef, Rami Abduljabbar, Shayma Abdullah, Ali Abuhaliema and Violet Kasabri

Pharmaceuticals 2026, 19(5), 712; https://doi.org/10.3390/ph19050712 - 30 Apr 2026

Abstract

Background: We investigated whether common variants in SCN1A are associated with antiepileptic drug (AED) non-response in Jordanian patients with epilepsy. Methods: We recruited 114 patients (105 successfully genotyped) and Sanger-sequenced five loci spanning rs6432860 and its flanking region of rs1531380, rs1531379, rs1531378, and [...] Read more.

Background: We investigated whether common variants in SCN1A are associated with antiepileptic drug (AED) non-response in Jordanian patients with epilepsy. Methods: We recruited 114 patients (105 successfully genotyped) and Sanger-sequenced five loci spanning rs6432860 and its flanking region of rs1531380, rs1531379, rs1531378, and rs10198801. Genotype–response associations were tested using contingency analyses and multivariable logistic regression adjusting for age at the time of the interview, number of AEDs, and carbamazepine use. Pre-specified secondary analyses included (i) stratification by AED class (voltage-gated sodium channel [VGSC]-acting vs. non-VGSC agents) and (ii) sensitivity analyses using alternative non-response thresholds (seizures > 0/year and ≥4/year). Linkage disequilibrium (LD) and exact Hardy–Weinberg equilibrium (HWE) tests were evaluated. Cohort minor allele frequencies (MAFs) were compared with global population estimates. Results: The four upstream previously cataloged intronic variant SNPs (rs1531380, rs1531379, rs1531378, and rs6432860) were in a complete pattern of LD association in this population (D′ = 1; r^{2 =} 1) whereas each upstream variant with rs10198801 showed D′ = 1 with inverse correlation (r ≈ −0.53). All loci conformed to the exact HWE tests. Upstream variants had novel associations with a non-response in unadjusted analyses and remained significant after adjustment (genotype aOR = 2.8; 95% CI = 1.1–7.2; p value = 0.03), alongside independent effects of carbamazepine use (aOR = 3.3; 95% CI = 1.3–8.0; p value = 0.009) and a number of AEDs (aOR = 0.17; 95% CI = 0.06–0.50; p value = 0.002). In AED-class stratification, upstream additional intronic variants had novel associations with a non-response among VGSC-treated patients (OR = 3.8; 95% CI = 1.1–13.6; p value = 0.03) whereas rs10198801 was associated among non-VGSC patients (OR = 7.9; 95% CI = 0.9–70; p value = 0.04). Findings were robust using a ≥4 seizures/year threshold (recessive model significant) but not using any seizures > 0/year. Cohort MAFs for upstream variants (~48.6%) exceeded European, African, and Asian estimates. Significance: SCN1A upstream intronic variation has a novel association with AED non-response in the Jordanian cohort, shows mechanism-aligned patterns by AED class, persists after covariate adjustment and under a clinically used seizure-frequency threshold, and warrants ancestry-informed replication and functional validation. Full article

(This article belongs to the Special Issue Pharmacogenomics and Ethnic Diversity: Optimizing Drug Response Across Populations)

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21 pages, 7314 KB

Open AccessArticle

Neuroprotective Effects of Rosa roxburghii Tratt Juice Concentrate Powder in Parkinson’s Disease Mice via the PI3K/AKT Signaling Pathway

by Tong Jin, Long Liu, Faguang Kuang, Mingjie Chen, Haonan Chen, Jiapan Deng, Yikai Yang, Baofei Sun and Heng Luo

Pharmaceuticals 2026, 19(5), 711; https://doi.org/10.3390/ph19050711 - 30 Apr 2026

Abstract

Background: The absence of disease-modifying treatments for Parkinson’s disease (PD)—a neurodegenerative condition with escalating global incidence—represents a critical unmet medical need. Traditionally utilized for both dietary consumption and medicinal preparations, the fruit derived from Rosa roxburghii Tratt demonstrates a remarkably rich profile [...] Read more.

Background: The absence of disease-modifying treatments for Parkinson’s disease (PD)—a neurodegenerative condition with escalating global incidence—represents a critical unmet medical need. Traditionally utilized for both dietary consumption and medicinal preparations, the fruit derived from Rosa roxburghii Tratt demonstrates a remarkably rich profile of biologically active compounds, with flavonoids, triterpenoids, and organic acids representing the predominant classes. Experimental evidence indicates that these compounds elicit robust antioxidative, anti-inflammatory, and neuroprotective effects, making them promising candidates for neurodegenerative disease modulation. This study aimed to systematically evaluate the neuroprotective effects of Rosa roxburghii Tratt juice concentrate powder (RRJCP) across the preventive, interventional, and therapeutic stages of PD and to elucidate its underlying molecular mechanisms. Methods: Rosa roxburghii Tratt juice was subjected to rotary evaporation concentration and vacuum freeze-drying to obtain the juice concentrate powder. C57BL/6 mice were randomly assigned to three main groups (prevention, intervention, and treatment), each containing subgroups including a normal control, an MPTP model group, low-, medium-, and high-dose RRCJP groups (50, 100, and 200 mg/kg), and a positive control Madopar group, totaling 18 subgroups. A chronic MPTP-induced PD mouse model was established. Motor function was assessed via the open field test, pole test, and wire hang test. Substantia nigra neuronal morphology was examined by hematoxylin and eosin staining. The area of tyrosine hydroxylase (TH)-positive regions was measured by immunohistochemistry. The levels of oxidative stress indicators in serum were measured using biochemical kits. Network pharmacology was employed to predict core targets, and the expression of PI3K/AKT pathway and apoptosis-related proteins was determined by Western blotting. Results: Compared with the MPTP model group, RRCJP (200 mg/kg) significantly increased the total distance traveled in the open field, shortened the pole climbing time, and improved the wire hang score. It attenuated the morphological disorganization and nuclear pyknosis of substantia nigra neurons, increased the TH-positive area and TH protein expression, reduced serum MDA content, and elevated the activities of SOD and GSH-Px. Network pharmacology analysis indicated that the PI3K/AKT signaling pathway was among the core targets. Western blotting results further showed that the juice concentrate powder upregulated the expression of p-PI3K, p-AKT, and Bcl-2, while downregulating Bax and Cleaved Caspase-3 levels, which was consistent with the network pharmacology prediction. Conclusions: RRCJP exerts neuroprotective effects across the preventive, interventional, and therapeutic stages in PD model mice, the mechanisms of which may be associated with activation of the PI3K/AKT signaling pathway, attenuation of oxidative stress, and inhibition of neuronal apoptosis. Full article

(This article belongs to the Section Natural Products)

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