Glycyrrhiza uralensis is a traditional herbal medicine with significant bioactivity. This study investigated the effect of G. uralensis crude water extract (GU-CWE) on nitric oxide synthase 2 (NOS2) expression during myogenesis. GU-CWE treatment increased myoblast differentiation by downregulating NOS2 and upregulating myogenic regulatory factors (MYOD, MYOG, and MYH). Notably, this effect was supported by an observed decrease in NOS2 expression in the gastrocnemius tissues of mice treated with GU-CWE. In addition, GU-CWE treatment and NOS2 knockdown were associated with reductions in reactive oxygen species levels. We further elucidate the role of the NOS2 gene in myoblast differentiation, demonstrating that its role was expression dependent, being beneficial at low expression but detrimental at high expression. High NOS2 gene expression induced oxidative stress, whereas its low expression impaired myotube formation. These findings highlight that the modulation of NOS2 expression by G. uralensis can potentially be use for managing muscle wasting disorders.

Excessive reactive oxygen species stress due to salinity poses a significant threat to the growth of Glycyrrhiza uralensis Fisch. To adapt to salt stress, G. uralensis engages in alternative splicing (AS) to generate a variety of proteins that help it withstand the effects of salt stress. While several studies have investigated the impact of alternative splicing on plants stress responses, the mechanisms by which AS interacts with transcriptional regulation to modulate the salt stress response in G. uralensis remain poorly understood. In this study, we utilized high-throughput RNA sequencing data to perform a comprehensive analysis of AS events at various time points in G. uralensis under salt stress, with exon skipping (SE) being the predominant AS type. KEGG enrichment analysis was performed on the different splicing genes (DSG), and pathways associated with AS were significantly enriched, including RNA transport, mRNA surveillance, and spliceosome. This indicated splicing regulation of genes, resulting in AS events under salt stress conditions. Moreover, plant response to salt stress pathways were also enriched, such as mitogen-activated protein kinase signaling pathway - plant, flavonoid biosynthesis, and oxidative phosphorylation. We focused on four differentially significant genes in the MAPK pathway by AS and qRT-PCR analysis. The alternative splicing type of MPK4 and SnRK2 was skipped exon (SE). ETR2 and RbohD were retained intron (RI) and alternative 5\'splice site (A5SS), respectively. The expression levels of isoform1 of these four genes displayed different but significant increases in different tissue sites and salt stress treatment times. These findings suggest that MPK4, SnRK2, ETR2, and RbohD in G. uralensis activate the expression of isoform1, leading to the production of more isoform1 protein and thereby enhancing resistance to salt stress. These findings suggest that salt-responsive AS directly and indirectly governs G. uralensis salt response. Further investigations into AS function and mechanism during abiotic stresses may offer novel references for bolstering plant stress tolerance.

This study aims to evaluate the in vivo function of Fusarium oxysporum in Glycyrrhiza uralensis by salt tolerance,indoleacetic acid(IAA) production capacity, phosphate-dissolving capacity, and iron carrier production capacity. The stable genetic transformation system of the F. oxysporum was established by Agrobacterium tumefaciens-mediated genetic transformation( ATMT)technology, and the stability and staining efficiency of transformants were detected by the cloning of the marker gene green fluorescent protein(GFP) and the efficiency of β-glucuronidase staining(GUS). Efficient and stable transformants were selected for restaining G. uralensis and evaluating its influence on the growth of the G. uralensis seedlings. The results show that F. oxysporum has good salt tolerance and could still grow on potato glucose agar(PDA) medium containing 7% sodium chloride, but the growth rate slows down with the increase in sodium chloride content in PDA medium. F. oxysporum has the function of producing indoleacetic acid, and the concentration of IAA in its fermentation broth is about 3. 32 mg · m L~(-1). In this study, the genetic transformation system of F. oxysporum is successfully constructed, and the ATMT system is efficient and stable. One transformant with both high staining efficiency and genetic stability is selected, and the restaining rate of the transformant in G. uralensis is 76. 92%, which could significantly improve the main root length of one-month-old G. uralensis seedlings and promote the growth and development of G. uralensis seedlings. The results of this study can lay the foundation for the development of biological bacterial fertilizer and the growth regulation of high-quality G. uralensis.

Zhigancao decoction is a traditional prescription for treating irregular pulse and palpitations in China. As the monarch drug of Zhigancao decoction, the bioactive molecules of licorice against heart diseases remain elusive. We established the HRESIMS-guided method leading to the isolation of three novel bicyclic peptides, glycnsisitins A-C (1-3), with distinctive C-C and C-O-C side-chain-to-side-chain linkages from the roots of Glycyrrhiza uralensis (Licorice). Glycnsisitin A demonstrated stronger cardioprotective activity than glycnsisitins B and C in an in vitro model of doxorubicin (DOX)-induced cardiomyocyte injury. Glycnsisitin A treatment not only reduced the mortality of heart failure (HF) mice in a dose-dependent manner but also significantly attenuated DOX-induced cardiac dysfunction and myocardial fibrosis. Gene set enrichment analysis (GSEA) of the differentially expressed genes indicated that the cardioprotective effect of glycnsisitin A was mainly attributed to its ability to maintain iron homeostasis in the myocardium. Mechanistically, glycnsisitin A interacted with transferrin and facilitated its binding to the transferrin receptor (TFRC), which caused increased uptake of iron in cardiomyocytes. These findings highlight the key role of bicyclic peptides as bioactive molecules of Zhigancao decoction for the treatment of HF, and glycnsisitin A constitutes a promising therapeutic agent for the treatment of HF.

A continuous chemical-free green approach was investigated for the comprehensive reutilization of all components in herbal extraction residues (HERs), taking Glycyrrhiza uralensis residue (GUR) as an example. The GUR structural changes induced by mechanical extrusion which improve the specific surface area and enzyme accessibility of GUR. With 3 % pretreated GUR loading of high-tolerance Penicillium oxalicum G2. The reducing sugar yield of 11.45 g/L was achieved, along with an 81.06 % in situ enzymatic hydrolysis. Finally, 8.23 g/L bioethanol (0.40 g/g total sugar) was produced from GUR hydrolysates after 24 h fermentation of Pichia stipitis G32. The amount of functional medicinal ingredients extracted from GUR after hydrolysis (39.63 mg/g) was 37.69 % greater than that of un-pretreated GUR. In total, 1.49 g flavonoids, 294.36 U cellulase, and 14.13 g ethanol could be produced from 100 g GUR using this process, illustrating that this green and efficient process has the potential for industrial production.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is a refractory respiratory disease mainly attributed to multiple pathological factors such as oxidative stress, infectious inflammation, and idiopathic fibrosis for decades. The medicinal plant Glycyrrhiza uralensis extract (ULE) was widely used to control respiratory diseases in China. However, the regulatory mechanism of scientific evidence to support the therapeutic benefits of ULE in the management of COPD is greatly limited.
OBJECTIVE: This study aims to discover the potential protection mechanism of ULE on COPD via a muti-targets strategy.
METHODS: The present study set out to determine the potential protective effects of ULE on COPD through a multi-target strategy. In vivo and in vitro models of COPD were established using cigarette smoke and lipopolysaccharide to assess the protective effects of ULE. It was evaluated by measuring inflammatory cytokines and assessing pulmonary pathological changes. HPLC was used to verify the active compounds of the potential compounds that were collected and screened using HERB, works of literature, and ADME tools. The mechanisms of ULE in the treatment of COPD were explored using transcriptomics, connectivity-map, and network pharmacology approaches. The relevant targets were further investigated using RT-PCR, western blot, and immunohistochemistry. The HCK inhibitor (iHCK-37) was used to evaluate the potential mechanism of ULE\'s active compounds in the prevention of COPD.
RESULTS: ULE effectively protected the lungs of COPD mice from oxidative stress, inflammation, and fibrosis damage. After screening and verification using ADME properties and HPLC, 4 active compounds were identified in ULE: liquiritin (LQ), licochalcone B (LCB), licochalcone A (LCA), and echinatin (ET). Network pharmacology integrated with transcriptomics analysis showed that ULE mitigated oxidative stress, inflammation, and fibrosis in COPD by suppressing HCK. The combination of LCB and LQ was optimized for anti-inflammation, antioxidation, and anti-fibrosis activities. The iHCK-37 further validated the preventive treatment of LCB and LQ on COPD by inhibiting HCK to exert antioxidant, anti-inflammatory, and anti-fibrotic effects. The combination of LCB and LQ, in a 1:1 ratio, exerted synergistic antioxidative, anti-inflammatory, and anti-fibrotic effects in the treatment of COPD by downregulating HCK.
CONCLUSIONS: The combination of LCB and LQ performed a significant anti-COPD effect via downregulating HCK.

Knee osteoarthritis (KOA), a major health and economic problem facing older adults worldwide, is a degenerative joint disease. Glycyrrhiza uralensis Fisch. (GC) plays an integral role in many classic Chinese medicine prescriptions for treating knee osteoarthritis. Still, the role of GC in treating KOA is unclear. To explore the pharmacological mechanism of GC against KOA, UPLC-Q-TOF/MS was conducted to detect the main compounds in GC. The therapeutic effect of GC on DMM-induced osteoarthritic mice was assessed by histomorphology, μCT, behavioural tests, and immunohistochemical staining. Network pharmacology and molecular docking were used to predict the potential targets of GC against KOA. The predicted results were verified by immunohistochemical staining Animal experiments showed that GC had a protective effect on DMM-induced KOA, mainly in the improvement of movement disorders, subchondral bone sclerosis and cartilage damage. A variety of flavonoids and triterpenoids were detected in GC via UPLC-Q-TOF/MS, such as Naringenin. Seven core targets (JUN, MAPK3, MAPK1, AKT1, TP53, RELA and STAT3) and three main pathways (IL-17, NF-κB and TNF signalling pathways) were discovered through network pharmacology analysis that closely related to inflammatory response. Interestingly, molecular docking results showed that the active ingredient Naringenin had a good binding effect on anti-inflammatory-related proteins. In the verification experiment, after the intervention of GC, the expression levels of pp65 and F4/80 inflammatory indicators in the knee joint of KOA model mice were significantly downregulated. GC could improve the inflammatory environment in DMM-induced osteoarthritic mice thus alleviating the physiological structure and dysfunction of the knee joint. GC might play an important role in the treatment of knee osteoarthritis.

Drought stress has become the primary severe threat to global agriculture production, including medicinal plants. Plant growth-promoting bacteria (PGPB) and environmentally friendly element silicon (Si) have emerged as effective methods in alleviating drought stress in various plants. Here, the effects of the plant endophytic G5 interaction with Si on regulating nitrogen absorption, assimilation, and metabolism pathways were investigated in the morphophysiological and gene attributes of Glycyrrhiza uralensis exposed to drought. Results showed that G5+Si application improved nitrogen absorption and assimilation by increasing the available nitrogen content in the soil, further improving the nitrogen utilization efficiency. Then, G5+Si triggered the accumulation of the major adjustment substances proline, γ-aminobutyric acid, putrescine, and chlorophyll, which played an important role in contributing to maintaining balance and energy supply in G. uralensis exposed to drought. These findings will provide new ideas for the combined application of PGPR and Si on both soil and plant systems in a drought habitat.

BACKGROUND: Glycyrrhiza inflata Bat. and Glycyrrhiza uralensis Fisch. are both original plants of \'Gan Cao\' in the Chinese Pharmacopoeia, and G. uralensis is currently the mainstream variety of licorice and has a long history of use in traditional Chinese medicine. Both of these species have shown some degree of tolerance to salinity, G. inflata exhibits higher salt tolerance than G. uralensis and can grow on saline meadow soils and crusty saline soils. However, the regulatory mechanism responsible for the differences in salt tolerance between different licorice species is unclear. Due to land area-related limitations, the excavation and cultivation of licorice varieties in saline-alkaline areas that both exhibit tolerance to salt and contain highly efficient active substances are needed. The systematic identification of the key genes and pathways associated with the differences in salt tolerance between these two licorice species will be beneficial for cultivating high-quality salt-tolerant licorice G. uralensis plant varieties and for the long-term development of the licorice industry. In this research, the differences in growth response indicators, ion accumulation, and transcription expression between the two licorice species were analyzed.
RESULTS: This research included a comprehensive comparison of growth response indicators, including biomass, malondialdehyde (MDA) levels, and total flavonoids content, between two distinct licorice species and an analysis of their ion content and transcriptome expression. In contrast to the result found for G. uralensis, the salt treatment of G. inflata ensured the stable accumulation of biomass and total flavonoids at 0.5 d, 15 d, and 30 d and the restriction of Na+ to the roots while allowing for more K+ and Ca2+ accumulation. Notably, despite the increase in the Na+ concentration in the roots, the MDA concentration remained low. Transcriptome analysis revealed that the regulatory effects of growth and ion transport on the two licorice species were strongly correlated with the following pathways and relevant DEGs: the TCA cycle, the pentose phosphate pathway, and the photosynthetic carbon fixation pathway involved in carbon metabolism; Casparian strip formation (lignin oxidation and translocation, suberin formation) in response to Na+; K+ and Ca2+ translocation, organic solute synthesis (arginine, polyamines, GABA) in response to osmotic stresses; and the biosynthesis of the nonenzymatic antioxidants carotenoids and flavonoids in response to antioxidant stress. Furthermore, the differential expression of the DEGs related to ABA signaling in hormone transduction and the regulation of transcription factors such as the HSF and GRAS families may be associated with the remarkable salt tolerance of G. inflata.
CONCLUSIONS: Compared with G. uralensis, G. inflata exhibits greater salt tolerance, which is primarily attributable to factors related to carbon metabolism, endodermal barrier formation and development, K+ and Ca2+ transport, biosynthesis of carotenoids and flavonoids, and regulation of signal transduction pathways and salt-responsive transcription factors. The formation of the Casparian strip, especially the transport and oxidation of lignin precursors, is likely the primary reason for the markedly higher amount of Na+ in the roots of G. inflata than in those of G. uralensis. The tendency of G. inflata to maintain low MDA levels in its roots under such conditions is closely related to the biosynthesis of flavonoids and carotenoids and the maintenance of the osmotic balance in roots by the absorption of more K+ and Ca2+ to meet growth needs. These findings may provide new insights for developing and cultivating G. uralensis plant species selected for cultivation in saline environments or soils managed through agronomic practices that involve the use of water with a high salt content.

BACKGROUND: Licorice is widely used clinically as one of the most famous traditional Chinese herbs. Its herb roasted with honey is called honey-processed licorice (HPL). Modern studies have shown that HPL has a stronger cardioprotective ability compared to raw licorice (RL), however the material basis and mechanism of action of the potential cardioprotection have not been fully elucidated.
OBJECTIVE: To screen and validate the material basis of cardioprotection exerted by HPL and to preliminarily predict the potential mechanism of action.
METHODS: UPLC-QTOF-MS/MS was used to analyze HPL samples with different processing levels, and differential compounds were screened out through principal component analysis. Network pharmacology and molecular docking were applied to explore the association between differential compounds and doxorubicin cardiomyopathy and their mechanisms of action were predicted. An in vitro model was established to verify the cardioprotective effects of differential compounds.
RESULTS: Six differential compounds were screened as key components of HPL for potential cardioprotection. Based on network pharmacology, 113 potential important targets for the treatment of Dox-induced cardiotoxicity were screened. KEGG enrichment analysis predicted that the PI3K-Akt pathway was closely related to the mechanism of action of active ingredients. Molecular docking results showed that the six differential compounds all had good binding activity with Nrf2 protein. In addition, in vitro experiments had shown that five of the active ingredients (liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, and licochalcone A) can significantly increase Dox-induced H9c2 cell viability, SOD activity, and mitochondrial membrane potential, significantly reduces MDA levels and inhibits ROS generation.
CONCLUSIONS: Liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin and licochalcone A are key components of HPL with potential cardioprotective capabilities. Five active ingredients can alleviate Dox-induced cardiotoxicity by inhibiting oxidative stress and mitochondrial damage.