Protective Effects of Red Ginseng Against Tacrine-Induced Hepatotoxicity: An Integrated Approach with Network Pharmacology and Experimental Validation.

Introduction: Tacrine, an FDA-approved acetylcholinesterase inhibitor, has shown efficacy in treating Alzheimer's disease, but its clinical use is limited by hepatotoxicity. This study investigates the protective effects of red ginseng against tacrine-induced hepatotoxicity, focusing on oxidative stress. Methods: A network depicting the interaction between compounds and targets was constructed for RG. Effect of RG was determined by MTT and FACS analysis with cells stained by rhodamine 123. Proteins were extracted and subjected to immunoblotting for apoptosis-related proteins. Results: The outcomes of the network analysis revealed a significant association, with 20 out of 82 identified primary RG targets aligning with those involved in oxidative liver damage including notable interactions within the AMPK pathway. in vitro experiments showed that RG, particularly at 1000µg/mL, mitigated tacrine-induced apoptosis and mitochondrial damage, while activating the LKB1-mediated AMPK pathway and Hippo-Yap signaling. In mice, RG also protected the liver injury induced by tacrine, as similar protective effects to silymarin, a well-known drug for liver toxicity protection. Discussion: Our study reveals the potential of RG in mitigating tacrine-induced hepatotoxicity, suggesting the administration of natural products like RG to reduce toxicity in Alzheimer's disease treatment.

Antioxidant Efficacy of Hwangryunhaedok-tang through Nrf2 and AMPK Signaling Pathway against Neurological Disorders In Vivo and In Vitro.

Alzheimer's disease (AD) is a representative cause of dementia and is caused by neuronal loss, leading to the accumulation of aberrant neuritic plaques and the formation of neurofibrillary tangles. Oxidative stress is involved in the impaired clearance of amyloid beta (Aß), and Aß-induced oxidative stress causes AD by inducing the formation of neurofibrillary tangles. Hwangryunhaedok-tang (HHT, Kracie K-09®), a traditional herbal medicine prescription, has shown therapeutic effects on various diseases. However, the studies of HHT as a potential treatment for AD are insufficient. Therefore, our study identified the neurological effects and mechanisms of HHT and its key bioactive compounds against Alzheimer's disease in vivo and in vitro. In a 5xFAD mouse model, our study confirmed that HHT attenuated cognitive impairments in the Morris water maze (MWM) test and passive avoidance (PA) test. In addition, the prevention of neuron impairment, reduction in the protein levels of Aß, and inhibition of cell apoptosis were confirmed with brain tissue staining. In HT-22 cells, HHT attenuates tBHP-induced cytotoxicity, ROS generation, and mitochondrial dysfunction. It was verified that HHT exerts a neuroprotective effect by activating signaling pathways interacting with Nrf2, such as MAPK/ERK, PI3K/Akt, and LKB1/AMPK. Among the components, baicalein, a bioavailable compound of HHT, exhibited neuroprotective properties and activated the Akt, AMPK, and Nrf2/HO-1 pathways. Our findings indicate a mechanism for HHT and its major bioavailable compounds to treat and prevent AD and suggest its potential.

Forsythiaside A Activates AMP-Activated Protein Kinase and Regulates Oxidative Stress via Nrf2 Signaling.

Forsythiaside A (FA) is an active constituent isolated from Forsythia suspensa, a beneficial herb used in traditional medicine known for its antioxidant and anti-inflammatory properties. Although various studies have suggested that FA has the protective effects, its impacts on arachidonic acid (AA) plus iron in vitro models and carbon tetrachloride (CCl4)-induced mouse liver damage in vivo have not been explored. In this study, HepG2 cells were subjected to AA + iron treatment to induce apoptosis and mitochondrial impairment and determine the molecular mechanisms. FA exhibited protective effects by inhibiting cell damage and reactive oxygen species (ROS) production induced by AA + iron, as assessed via immunoblot and flow cytometry analyses. Further molecular investigations revealed that FA resulted in the activation of extracellular-signal-related protein kinase (ERK), which subsequently triggered the activation of AMP-activated protein kinase (AMPK), a critical regulator of cellular oxidative stress. Additionally, FA modulated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which is a significant antioxidant transcription factor regulated by the AMPK pathway. For in vivo studies, mice were orally administered FA and then subjected to induction of CCl4-based hepatotoxicity. The protective effect of FA was confirmed via blood biochemistry and immunohistochemical analyses. In conclusion, our findings demonstrated the protective effects of FA against oxidative stress both in vitro and in vivo, thus indicating that FA is a potential candidate for liver protection. Our study sheds light on the mechanistic pathways involved in the antioxidant effects of FA, highlighting the hepatoprotective potential of naturally occurring compounds in traditional herbs, such as FA.

Unraveling the Antioxidant Capacity of Spatholobi caulis in Nonalcoholic Fatty Liver Disease: A Multiscale Network Approach Integrated with Experimental Validation.

Nonalcoholic fatty liver disease (NAFLD) is a global health problem that is closely associated with obesity and metabolic syndrome. Spatholobi caulis (SC) is a herbal medicine with potential hepatoprotective effects; however, its active compounds and underlying mechanisms have not been fully explored. In this study, we combined a multiscale network-level approach with experimental validation to investigate SC's antioxidant properties and their impact on NAFLD. Data collection and network construction were performed, and active compounds and key mechanisms were identified through multi-scale network analysis. Validation was conducted using in vitro steatotic hepatocyte models and in vivo high-fat diet-induced NAFLD models. Our findings revealed that SC treatment improved NAFLD by modulating multiple proteins and signaling pathways, including AMPK signaling pathways. Subsequent experiments showed that SC treatment reduced lipid accumulation and oxidative stress. We also validated SC's effects on AMPK and its crosstalk pathways, emphasizing their role in hepatoprotection. We predicted procyanidin B2 to be an active compound of SC and validated it using a lipogenesis in vitro model. Histological and biochemical analyses confirmed that SC ameliorated liver steatosis and inflammation in mice. This study presents SC's potential use in NAFLD treatment and introduces a novel approach for identifying and validating active compounds in herbal medicine.

Integrative approach to uncover antioxidant properties of Bupleuri Radix and its active compounds: Multiscale interactome-level analysis with experimental validation.

Acute and chronic liver disease are global problems with high morbidity and mortality. Bupleuri Radix (BR) is an herbal medicine that has been prescribed empirically in traditional Asian medicine to modulate liver metabolism. However, its active compounds and therapeutic mechanisms remain unclear. Here, we integrated a network-based approach and experimental validation to elucidate BR's therapeutic potential in treating oxidative liver injury. Our approach incorporated data collection and network construction utilizing bioinformatics tools, and identified active compounds and key mechanisms based on the multiscale interactome. The proposed mechanisms were validated using an in vitro oxidative stress model and an in vivo carbon tetrachloride-induced model. We found that BR ameliorated the oxidative hepatic damage by acting on multiple proteins (STAT3, TNF, and BCL2) and signaling pathways (AMPK and Hippo signaling pathways). Subsequent in vitro experiments confirmed that BR significantly inhibited oxidative stress and mitochondrial damage. We further validated the effect of BR on the AMPK and Hippo-YAP pathways; a key mechanism for the antioxidant properties of BR. We prioritized the active compounds in BR based on a multiscale interactome-based approach, and further experiments revealed that saikosaponin A was a key active compound involved in hepatocyte protection (EC50 = 50 µM), similar to the result using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide. Histochemistry and blood biochemistry established that BR significantly inhibited carbon tetrachloride-induced oxidative tissue damage in mice. Thus, BR can be used to develop novel therapeutics for oxidative liver injury. Moreover, we suggest a novel strategy to prioritize and validate the active compounds and key mechanisms of herbal medicine based on the multiscale interactome.

A Machine Learning Approach for Recommending Herbal Formulae with Enhanced Interpretability and Applicability.

Herbal formulae (HFs) are representative interventions in Korean medicine (KM) for the prevention and treatment of various diseases. Here, we proposed a machine learning-based approach for HF recommendation with enhanced interpretability and applicability. A dataset consisting of clinical symptoms, Sasang constitution (SC) types, and prescribed HFs was derived from a multicenter study. Case studies published over 10 years were collected and curated by experts. Various classifiers, oversampling methods, and data imputation techniques were comprehensively considered. The local interpretable model-agnostic explanation (LIME) technique was applied to identify the clinical symptoms that led to the recommendation of specific HFs. We found that the cascaded deep forest (CDF) model with data imputation and oversampling yielded the best performance on the training set and holdout test set. Our model also achieved top-1 and top-3 accuracies of 0.35 and 0.89, respectively, on case study datasets in which clinical symptoms were only partially recorded. We performed an expert evaluation on the reliability of interpretation results using case studies and achieved a score close to normal. Taken together, our model will contribute to the modernization of KM and the identification of an HF selection process through the development of a practically useful HF recommendation model.

Hair Growth Stimulation Effect of Centipeda minima Extract: Identification of Active Compounds and Anagen-Activating Signaling Pathways.

Centipeda minima (L.) A. Braun & Asch is a well-studied plant in Chinese medicine that is used for the treatment of several diseases. A recent study has revealed the effects of extract of Cetipeda minima (CMX) standardized by brevilin A in inducing hair growth. However, the mechanism of action of CMX in human hair follicle dermal papilla cells (HFDPCs) has not yet been identified. We aimed to investigate the molecular basis underlying the effect of CMX on hair growth in HFDPCs. CMX induced the proliferation of HFDPCs, and the transcript-level expression of Wnt family member 5a (Wnt5a), frizzled receptor (FZDR), and vascular endothelial growth factor (VEGF) was upregulated. These results correlated with an increase in the expression of growth-related factors, such as VEGF and IGF-1. Immunoblotting and immunocytochemistry further revealed that the phosphorylation of ERK and JNK was enhanced by CMX in HFDPCs, and ß-catenin accumulated significantly in a dose-dependent manner. Therefore, CMX substantially induced the expression of Wnt signaling-related proteins, such as GSK phosphorylation and ß-catenin. This study supports the hypothesis that CMX promotes hair growth and secretion of growth factors via the Wnt/ß-catenin, ERK, and JNK signaling pathways. In addition, computational predictions of drug-likeness, together with ADME property predictions, revealed the satisfactory bioavailability score of CMX compounds, exhibiting high gastrointestinal absorption. We suggest that CMX could be used as a promising treatment for hair regeneration and minimization of hair loss.

Investigating the Biomarkers of the Sasang Constitution via Network Pharmacology Approach.

Sasang constitutional (SC) medicine classifies people into Soeum (SE), Soyang (SY), Taeeum (TE), and Taeyang (TY) types based on psychological and physical traits. However, biomarkers of these types are still unclear. We aimed to identify biomarkers among the SC types using network pharmacology methods. Target genes associated with the SC types were identified by grouping herb targets that preserve and strengthen the requisite energy (Bomyeongjiju). The herb targets were obtained by constructing an herb-compound-target network. We identified 371, 185, 146, and 89 target genes and their unique biological processes related to SE, SY, TE, and TY types, respectively. While the targets of SE and SY types were the most similar among the target pairs of the SC types, those of TY type overlapped with only a few other SC-type targets. Moreover, SE, SY, TE, and TY were related to "diseases of the digestive system," "diseases of the nervous system," "endocrine, nutritional, and metabolic diseases," and "congenital malformations, deformations, and chromosomal abnormalities," respectively. We successfully identified the target genes, biological processes, and diseases related to each SC type. We also demonstrated that a drug-centric approach using network pharmacology analysis provides a deeper understanding of the concept of Sasang constitutional medicine at a phenotypic level.

Effects of estrogen inhibition formula herbal mixture for danazol-induced precocious puberty in female rats: an experimental study with network pharmacology.

BACKGROUND: This study aimed at determining the effect of the herbal mixture estrogen inhibition formula (EIF) and its possible mechanisms by precocious puberty animal models and network pharmacology-based analysis. METHODS: Precocious puberty animal models were established by a single injection of 300 µg danazol, then female rats were administered EIF, vaginal openings were monitored, uterus and pituitary indices were determined. The levels of ALP, E2, LH, and FSH were measured using ELISA kits. Real-time PCR was performed to evaluate the mRNA expression of GnRH, UNC5C, and netrin-1 in hypothalamic tissues. We applied network pharmacological analysis to predict potential targets and pathways of EIF. RESULTS: EIF delayed danazol-induced early vaginal opening. In the onset model, EIF reduced the increased levels of serum ALP, E2, LH, and FSH; as well as mRNA expressions of GnRH, Netrin-1, and UNC5C. Moreover, long-term administration of EIF not only diminished all impaired factors but also had no effect on the normal development of the animals. The gene set enrichment analysis showed that the targets of EIF are mainly associated with the GnRH signaling and ovarian steroidogenesis pathways. CONCLUSION: EIF could be used in preclinical research for the treatment of precocious puberty by the inhibition of HPGA pre-maturation.

Investigating the Systems-Level Effect of Pueraria lobata for Menopause-Related Metabolic Diseases Using an Ovariectomized Rat Model and Network Pharmacological Analysis.

This study was conducted to evaluate the biological activities of Pueraria lobata (PL) on menopause-related metabolic diseases and to explore the underlying mechanism of PL by network pharmacological analyses. We used ovariectomized (OVX) rats as a postmenopausal model and administered PL at different doses (50, 100, and 200 mg/kg). In OVX rats, decreased uterine weights and PPAR-γ (peroxisome proliferator-activated receptor-gamma) mRNA expression in the thigh muscle were significantly recovered after PL administration. PL also significantly alleviated OVX-induced increases in total cholesterol, triglyceride, alanine aminotransferase (ALT/GPT), and aspartate aminotransferase (AST/GOT) levels. To identify the systems-level mechanism of PL, we performed network pharmacological analyses by predicting the targets of the potential bioactive compounds and their associated pathways. We identified 61 targets from four potential active compounds of PL: formononetin, beta-sitosterol, 3'-methoxydaidzein, and daidzein-4,7-diglucoside. Pathway enrichment analysis revealed that among female sex hormone-related pathways, the estrogen signaling pathways, progesterone-mediated oocyte maturation, oxytocin signaling pathways, and prolactin signaling pathways were associated with multiple targets of PL. In conclusion, we found that PL improved various indicators associated with lipid metabolism in the postmenopausal animal model, and we also identified that its therapeutic effects are exerted via multiple female sex hormone-related pathways.

The Methodological Trends of Traditional Herbal Medicine Employing Network Pharmacology.

Natural products, including traditional herbal medicine (THM), are known to exert their therapeutic effects by acting on multiple targets, so researchers have employed network pharmacology methods to decipher the potential mechanisms of THM. To conduct THM-network pharmacology (THM-NP) studies, researchers have employed different tools and databases for constructing and analyzing herb-compound-target networks. In this study, we attempted to capture the methodological trends in THM-NP research. We identified the tools and databases employed to conduct THM-NP studies and visualized their combinatorial patterns. We also constructed co-author and affiliation networks to further understand how the methodologies are employed among researchers. The results showed that the number of THM-NP studies and employed databases/tools have been dramatically increased in the last decade, and there are characteristic patterns in combining methods of each analysis step in THM-NP studies. Overall, the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was the most frequently employed network pharmacology database in THM-NP studies. Among the processes involved in THM-NP research, the methodology for constructing a compound-target network has shown the greatest change over time. In summary, our analysis describes comprehensive methodological trends and current ideas in research design for network pharmacology researchers.

Uwhangchungsimwon, a traditional herbal medicine, protects brain against oxidative injury via modulation of hypothalamus-pituitary-adrenal (HPA) response in a chronic restraint mice model.

ETHNOPHARMACOLOGICAL RELEVANCE: Uwhangchungsimwon (UCW) is a representative traditional herbal medicine for central nervous system disorders in East Asia countries over thousand years. To evaluate the pharmacological effects of UCW against oxidative brain injury in a chronic restraint stress mice model. METHODS AND MATERIALS: C57BL/6 male mice underwent daily oral administration of distilled water, UCW or ascorbic acid 1h before induction of restraint stress (5h of immobilization daily for 14 days). Nitric oxide (NO), total reactive oxygen species (ROS) levels, malondialdehyde, protein carbonyl contents, and activities of antioxidant enzymes, and concentrations of corticosterone, adrenaline, noradrenaline, and dopamine, were measured in brain tissues or sera. RESULTS: Restraint stress notably increased NO and ROS levels, malondialdehyde and protein carbonyl contents in brain tissues, but decreased activities of catalase, glutathione reductase and glutathione peroxidase. These alterations were significantly ameliorated by UCW. UCW significantly attenuated the elevated serum concentrations of corticosterone, adrenaline and noradrenaline. UCW also significantly normalized the gene expressions in brain tissues altered by restraint stress; up-regulation of phenylethanolamine N-methyltransferase (PNMT) and N-methyl-d-aspartate type 1 receptor (NMDAR1), and down-regulation of gamma-Aminobutyric acid type A receptor (GABAAR), glutamate decarboxylase 1 (GAD 67), and glutamate decarboxylase 2 (GAD 65), respectively. Moreover, UCW considerably restored neurogenesis in the hippocampal regions which was disturbed by chronic restraint stress. CONCLUSIONS: These results evidenced that UCW has pharmacological properties for brain protection and neurogenesis in status of stress-associated oxidative damage, and the underlying mechanisms involve the regulation of HPA axis in stress responses.