RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is widely used in the treatment of ulcerative colitis (UC) and has good antioxidant and anti-inflammatory effects, but its specific active ingredients and mechanisms of action are still unknown. THE PURPOSE OF THE STUDY: To elucidate the specific molecular mechanisms of licorice in the treatment of UC and to experimentally verify its activity. METHODS: Through network pharmacology, the active ingredients of licorice and the molecular targets of UC were identified. A traditional Chinese medicine (TCM)-components-target-disease network diagram was established, and the binding energies of the active ingredient and targets of licorice were verified by molecular docking. A BALB/c mice model of UC was established by treatment with 3% dextran sulfate sodium (DSS). The effect of licorice on colon tissue injury was histologically assessed. The expression of IL-6 and IL-17 in colon tissue was detected by immunohistochemistry (IHC). Transmission electron microscopy (TEM) was used to observe morphological changes in mitochondria in the colon. Caco2 cells were treated with lipopolysaccharide (LPS) for 24 h to establish the cell inflammatory damage model, and cells were exposed to different concentrations of drug-containing serum of Licorice (DCSL) for 24 h. In cells treated with the drug, the contents of oxidation markers were measured and ELISA was used to determine the levels of inflammatory factors in the cells. TEM was used to observe morphological changes in mitochondria. ZO-1 and occludin were detected by Western blotting. DCSL effects on autophagy were evaluated by treating cells with DCSL and autophagy inhibitor for 24 h after LPS injection. Small interfering ribonucleic acid (si-RNA) was used to silence Nrf2 gene expression in Caco2 cells to observe the effects of DCSL on autophagy through the Nrf2/PINK1 pathway. Nrf2, PINK1, HO-1, Parkin, P62, and LC3 were detected by Western blotting. RESULTS: Ninety-one active ingredients and 339 action targets and 792 UC disease targets were identified, 99 of which were overlapping targets. Molecular docking was used to analyze the binding energies of liquiritin, liquiritigenin, glycyrrhizic acid, and glycyrrhetinic acid to the targets, with glycyrrhetinic acid having the strongest binding energy. In the UC mouse model, licorice improved colon histopathological changes, reduced levels of IL-6 and IL-17 and repaired mitochondrial damage. In the LPS-induced inflammation model of Caco2 cells, DCSL decreased MDA, IL-1ß, Il-6, and TNF-α levels and increased those of Superoxide Dismutase (SOD), glutathione peroxidase (GSH-PX), and IL-10, and improved the morphological changes of mitochondria. Increased expression of Nrf2, PINK1, Parkin, HO-1, ZO-1, occludin, P62, and LC3 promoted autophagy and reduced inflammation levels. CONCLUSION: Licorice improves UC, which may be related to the activation of the Nrf2/PINK1 signaling pathway that regulates autophagy.
Assuntos
Colite Ulcerativa , Colite , Ácido Glicirretínico , Glycyrrhiza , Humanos , Animais , Camundongos , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Interleucina-17/metabolismo , Colo , Farmacologia em Rede , Células CACO-2 , Lipopolissacarídeos/toxicidade , Fator 2 Relacionado a NF-E2/metabolismo , Interleucina-6/metabolismo , Simulação de Acoplamento Molecular , Ocludina/metabolismo , Inflamação/patologia , Ácido Glicirretínico/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Quinases/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Colite/tratamento farmacológicoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is widely used in traditional Chinese Medicine (TCM) for compound compatibility, which could reduce toxicity and increase efficacy of certain herbal medicine, and its active components prominently effects of inhibit of inflammation and regulate of immunity. AIM OF THE STUDY: The study probed into the mechanism of the anti-inflammatory and immunomodulatory effects of licorice based on the domination of the T helper type 17/regulatory T cells (Th17/Treg) differentiation balance and the composition and structure of the intestinal flora through the nuclear factor kappa B (NF-κB) signaling pathway. MATERIALS AND METHODS: BALB/c mice were inoculated with dextran sulfate sodium (DSS) to establish animal models of ulcerative colitis (UC). For the pharmacodynamic study, UC mice were observed for the anti-inflammatory effect of licorice water extraction (LWE) in vivo, including clinical observation and measurement of colon length. Hematoxylin-eosin (HE) staining was used to evaluate pathological conditions. Immunohistochemistry (IHC) and transmission electron microscopy (TEM) were performed to observe the intestinal barrier of the colons. Inflammatory cytokine levels were measured using with enzyme-linked immunosorbent assay (ELISA) kits. The proportions of T helper (Th) cells in the colons was assessed using flow cytometry. Gut microbiota diversity was detected using 16S ribosomal (r)DNA sequencing. In addition, Western blot (WB) assays were used to verify ROR-γt, Foxp3, TLR4, MyD88 and NF-κB expression according to a standard protocol. RESULTS: LWE exerted a pharmacological anti-inflammatory effect by attenuating inflammation in the colonic tissues through affecting the protein expression of TLR4/MyD88/NF-κB, and increasing the expression of tight junction (TJ) protein in the colons, improving the integrity of the intestinal mucosal barrier in vivo. Moreover, LWE reversed the imbalance in Th17/Treg cells differentiation and influenced the protein expression of ROR-γt and Foxp3 in UC mouse colons. In particular, LWE significantly affected the diversity of the gut microbiota in UC mice, ameliorated the composition of dominant species, and significantly increased the type and quantity of probiotics. CONCLUSION: Licorice tends to reduce inflammation and enhance the protective action of the intestinal mucosal barrier via the TLR4/MyD88/NF-κB signal transduction pathway and alter the imbalance of Th-cell differentiation. Notably, licorice may affect the diversity of intestinal microbiota and the content of beneficial bacteria in the colon, which is a potential mechanism for understanding anti-inflammatory and immunomodulatory effects in UC mice in vivo.
Assuntos
Colite Ulcerativa , Colite , Microbioma Gastrointestinal , Glycyrrhiza uralensis , Animais , Anti-Inflamatórios , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismoRESUMO
Proline (Pro) metabolism is intimately associated with stress adaptation. The catabolism of Pro includes two dehydrogenation reactions catalyzed by proline dehydrogenase (ProDH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDh). P5CDh is a mitochondrial matrix NAD+-dependent dehydrogenase that is critical in preventing P5C-Pro intensive cycling and avoiding ROS production from electron run-off. Little is known about the roles of P5CDh in invertebrates, however. We cloned the P5CDh sequence in the Pacific white shrimp, Litopenaeus vannamei, and found that LvP5CDh is expressed predominantly in pleopod, hepatopancreas and gill. Subcellular localization analysis revealed that LvP5CDh protein was mainly found in the cytoplasm. In addition, overexpressing LvP5CDh in cells reduced ROS formation and inhibited apoptosis induced by LC50 Cd2+. Shrimp were exposed to various stress factors including infection with Vibrio alginolyticus, (½ LC50 and LC50) Cd2+, acid (pH 5.6) and alkali stress (pH 9.3). Both biotic and abiotic stress resulted in increased LvP5CDh expression and Pro accumulation; V. alginolyticus infection, pH 9.3 and LC50 Cd2+ stress apparently stimulated the Glu pathway of Pro synthesis, while pH 5.6 and ½ LC50 Cd2+ stress promoted the Orn pathway of Pro synthesis. Silencing of Lvp53 in shrimp attenuated LvP5CDh expression during Cd2+ stress, but had no effect on LvP5CDh mRNA levels if no Cd2+ stress was imposed. Our study contributes to the functional characterization of LvP5CDh in biotic and abiotic stress and reveals it to protect against ROS generation, damage to the cell, including the mitochondria, and apoptosis. Thus, LvP5CDh plays a critical role in immune defense and antioxidant responses.