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Acetaminophen (APAP)-induced liver injury is a common hepatic disease resulting from drug abuse. Few targeted treatments are available clinically nowadays. The flower bud of Rosa rugosa has a wide range of biological activities. However, it is unclear whether it alleviates liver injury caused by APAP. Here, we prepared an ethanol extract of Rosa rugosa (ERS) and analyzed its chemical profile. Furthermore, we revealed that ERS significantly ameliorated APAP-induced apoptosis and ferroptosis in AML-12 hepatocytes and dampened APAP-mediated cytotoxicity. In AML-12 cells, ERS elevated Sirt1 expression, boosted the LKB1/AMPK/Nrf2 axis, and thereby crippled APAP-induced intracellular oxidative stress. Both EX527 and NAM, which are chemically unrelated inhibitors of Sirt1, blocked ERS-induced activation of LKB1/AMPK/Nrf2 signaling. The protection of ERS against APAP-triggered toxicity in AML-12 cells was subsequently abolished. As expression of LKB1 was knocked down, ERS still upregulated Sirt1 but failed to activate AMPK/Nrf2 cascade or suppress cytotoxicity provoked by APAP. Results of in vivo experiments showed that ERS attenuated APAP-caused hepatocyte apoptosis and ferroptosis and improved liver injury and inflammation. Consistently, ERS boosted Sirt1 expression, increased phosphorylations of LKB1 and AMPK, and promoted Nrf2 nuclear translocation in the livers of APAP-intoxicated mice. Hepatic transcriptions of HO-1 and GCLC, which are downstream antioxidant genes of Nrf2, were also significantly increased in response to ERS. Our results collectively indicated that ERS effectively attenuates APAP-induced liver injury by activating LKB1/AMPK/Nrf2 cascade. Upregulated expression of Sirt1 plays a crucial role in ERS-mediated activation of LKB1.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Leucemia Mieloide Aguda , Rosa , Animales , Ratones , Acetaminofén/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Rosa/metabolismo , Transducción de Señal , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Sirtuina 1/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hígado , Hepatocitos , Estrés Oxidativo , Leucemia Mieloide Aguda/metabolismoRESUMEN
Previous studies have demonstrated that the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib shows efficacy against multiple cancers, including hepatocellular carcinoma. However, whether celecoxib is effective in alleviating steatosis during hepatocarcinogenesis is unknown. In a rapid hepatocellular carcinoma (HCC) mouse model established via hydrodynamic transfection of activated forms of AKT and c-Met proto-oncogenes, we investigated the antisteatotic and anticarcinogenic efficacy of celecoxib in vivo. Multiple HCC cell lines were employed for in vitro evaluation. Additionally, immunoblotting, immunohistochemistry, hematoxylin and eosin staining and Oil Red O staining were applied for mechanistic investigation. The results revealed that if celecoxib was administered in the early stage of AKT/c-Met-induced HCC, it resulted in disease stabilization. Moreover, celecoxib could alleviate lipid accumulation in the HCC mice and in an oleic acid-induced in vitro hepatic steatosis model. Further evidence at the molecular level indicated that celecoxib down-regulated the expression of phospho-ERK (Thr202/Tyr204) and proliferating cell nuclear antigen (PCNA) in the HCC mice. In addition, celecoxib efficiently repressed the phosphor-Akt (Thr308)/fatty acid synthase (FASN) axis both in vivo and in vitro. Altogether, this study suggests that celecoxib exerts its antilipogenic efficacy by targeting a COX-2/AKT/FASN cascade, which contributes to its ability to delay hepatocarcinogenesis.
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Carcinoma Hepatocelular/prevención & control , Celecoxib/farmacología , Ciclooxigenasa 2/química , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Apoptosis , Carcinogénesis , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proliferación Celular , Inhibidores de la Ciclooxigenasa 2/farmacología , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado Graso/prevención & control , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Ratones , Ácido Oléico/toxicidad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
An electrochemiluminescence (ECL) sensing platform for ultrasensitive and highly selective detection of kanamycin (KANA) was developed based on the prepared Ru(bpy)32+-functionalized MOF (Ru@MOF) composites by hydrothermal synthesis and Ag+-dependent DNAzyme. In this sensor, the stem-loop DNA (HP) with the ferrocene (Fc) was used as substrate chain to quench the ECL emission generated by the Ru@MOF. Using the specific recognition effect between KANA and the KANA aptamer (Apt) and the DNAzyme dependence on Ag+, the KANA aptamer as the pendant strand of the DNAzyme was assembled on Ru@MOF/GCE with the aptamer. When both Ag+ and KANA were present simultaneously, KANA specifically was binded to KANA aptamer as a pendant chain. Subsequently, Ag+-dependent DNAzyme walker continuously cleaved the HP chain and released the modified end of Fc to restore the ECL signal of Ru@MOF composites, thus achieving selective and ultrasensitive detection of KANA. The constructed KANA biosensor exhibits a wide detection range (30 pM to 300 µM) accompanied by a low detection limit (13.7 pM). The KANA in seawater and milk samples are determined to evalute the practical application results of the sensor. This ECL detection strategy could be used for detecting other similar analytes and has broad potential application in biological analysis.
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Técnicas Biosensibles , ADN Catalítico , Kanamicina/análisis , Límite de Detección , Técnicas Biosensibles/métodos , ADN , Mediciones Luminiscentes , Oligonucleótidos , Técnicas Electroquímicas/métodosRESUMEN
A novel electrochemical gas sensor for sensitive detection of H2S at room temperature is constructed based on the Fe@Pt/C composite material. The core-shell structured Fe@Pt catalyst was synthesized by a two-step reduction method and physically dispersed in Vulcan XC-72 carbon powders. The core-shell structure increases the effective catalytic surface area of Pt while significantly reducing the usage of the noble metal Pt, leading to improved catalytic performance and decreased production costs. Additionally, the mature screen-printing process is used to coat the catalyst film. A waterproof and breathable PTFE film was used as the substrate and the parameters in the screen printing process were also optimized to achieve the best gas sensing performance of the electrode film. Through the detection of hydrogen sulfide (H2S) with different concentrations, it is found that the sensor strictly shows linear correlation in the range of 1-20 ppm, R2 = 0.99974. Notably, the sensor exhibits high sensitivity (658.45 nA ppm-1) and a low detection limit of 0.33 ppm. Moreover, the consistency and stability of the sensor are satisfactory. The constructed gas sensor is expected to be well applied to industrial H2S detection.
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ETHNOPHARMACOLOGICAL RELEVANCE: When left untreated, liver fibrosis (LF) causes various chronic liver diseases. Earthworms (Pheretima aspergillum) are widely used in traditional medicine because of their capacity to relieve hepatic diseases. AIM OF THE STUDY: This study aimed to explore the anti-LF effects of water extract of earthworms (WEE) and the underlying molecular mechanisms. MATERIALS AND METHODS: A CCl4-induced mouse model of LF was used to study the impact of WEE on LF in vivo. The anti-LF activity of WEE in mice was compared with that of silybin, which can be clinically applied in LF intervention and was used as a positive control. Activation of LX-2 hepatic stellate cells (HSCs) and apoptosis and ferroptosis of AML-12 hepatocytes induced by TGFß1 were used as in vitro models. RESULTS: WEE drastically improved LF in mice. WEE reduced markers of activated HSCs in mice and inhibited TGFß1-induced activation of LX-2 HSCs in vitro. Additionally, WEE suppressed CCl4-induced apoptosis and ferroptosis in mouse hepatocytes. Mechanistically, WEE induced Nrf2 to enter the nuclei of the mouse liver cells, and the hepatic levels of Nrf2-downstream antioxidative factors increased. LKB1/AMPK/GSK3ß is an upstream regulatory cascade of Nrf2. In the LF mouse model, WEE increased hepatic phosphorylated LKB1, AMPK, and GSK3ß levels. Similar results were obtained for the LX-2 cells. In AML-12 hepatocytes and LX-2 HSCs, WEE elevated intracellular Nrf2 levels, promoted its nuclear translocation, and inhibited TGFß1-induced ROS accumulation. Knocking down LKB1 abolished the impact of WEE on the AMPK/GSK3ß/Nrf2 cascade and eliminated its protective effects against TGFß1. CONCLUSIONS: Our findings reveal that WEE improves mouse LF triggered by CCl4 and supports its application as a promising hepatoprotective agent against LF. The potentiation of the hepatic antioxidative AMPK/GSK3ß/Nrf2 cascade by activating LKB1 and the subsequent suppression of HSC activation and hepatocyte apoptosis and ferroptosis are implicated in WEE-mediated alleviation of LF.
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Leucemia Mieloide Aguda , Oligoquetos , Animales , Ratones , Factor 2 Relacionado con NF-E2 , Proteínas Quinasas Activadas por AMP , Glucógeno Sintasa Quinasa 3 beta , Hígado , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Hepatocitos , Fibrosis , Células Estrelladas Hepáticas , Modelos Animales de Enfermedad , Antioxidantes/efectos adversos , Leucemia Mieloide Aguda/patologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Ischemia-reperfusion (IR) injury can result in acute renal failure. Oxidative stress is a major factor in IR-induced cell death in the kidneys. According to traditional Chinese medicine, earthworms (Pheretima aspergillum) can be used to treat various kidney diseases. AIM OF THE STUDY: The present study was designed to understand the protective effects of the water extract of earthworms (WEE) against oxidative stress on the kidneys and the crucial molecular events associated with its nephroprotective activity. MATERIALS AND METHODS: Cytotoxicity caused by H2O2 in HEK293, HK2, and primary mouse renal tubular epithelial cells (TECs) was used to investigate the effect of WEE on oxidative stress-induced renal injury in vitro. IR-induced kidney injury was established using rats as an in vivo model. The WEE-mediated protection of the kidneys against oxidative stress was compared with that of glutathione, a common antioxidant used as a positive control. RESULTS: In HEK293 cells, HK2 cells, and primary mouse TECs, WEE relieved H2O2-induced mitochondrial damage, apoptosis, and ferroptosis. In kidney cells, WEE increased the expression of Sirt1, boosted LKB1 and AMPK phosphorylation, and upregulated nuclear Nrf2. Suppression of Sirt1 and LKB1 knock down abrogated WEE-induced protection against H2O2. WEE ameliorated IR-induced kidney injury and intrarenal inflammation in rats. In rat kidneys, WEE mitigated mitochondrial damage and suppressed IR-induced apoptosis and ferroptosis. Mechanistically, WEE increased Sirt1 expression, enhanced the phosphorylation of LKB1 and AMPK, and increased intranuclear Nrf2 levels in IR kidneys. IR treatment resulted in considerable increase in renal MDA levels and a prominent decrease in antioxidative enzyme activity. These lesions were significantly alleviated by WEE. CONCLUSIONS: WEE mitigated H2O2-induced cytotoxicity in kidney cells in vitro and improved IR-induced kidney damage in rats. Mechanistically, WEE potentiated the Sirt1/Nrf2 axis and relieved mitochondrial damage in the kidney cells. These events inhibited the apoptosis and ferroptosis induced by oxidative stress. Our findings support the potential application of WEE for the clinical treatment of kidney diseases caused by intrarenal oxidative stress.
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Aim: Lysosomal pH changes are associated with drug resistance, cell growth and invasion of tumors, but effective and specific real-time monitoring of lysosomal pH compounds for cancer therapy is lacking. Materials & methods: Here, based on the covalent linkage of the anticancer drug palbociclib and fluorescent dye fluorescein isothiocyanate (FITC), we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Results & discussion: Pal-FITC fluoresces is 20-fold stronger than that of FITC and shows a linear response in the pH range of 4.0-8.2 (R2 = 0.9901). Pal-FITC blocks cells in G1 phase via Cyclin D-CDK4/6-Rb. Conclusion: Our study provides new strategies for tumor-targeted imaging and personalized therapy.
Based on the covalent linkage of the anticancer drug and the fluorescent dye, we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Pal-FITC responded linearly in the pH range of 4.08.2. In addition, Pal-FITC was able to effectively treat lung cancer without toxic side effects on normal cells. It has a significant cell cycle blocking phenomenon and blocks G1 phase cells via Cyclin D-CDK4/6-Rb. Our study provides a new strategy for tumor-targeted imaging and personalized therapy.
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Antineoplásicos , Lisosomas , Piperazinas , Piridinas , Humanos , Piridinas/química , Piridinas/farmacología , Lisosomas/metabolismo , Piperazinas/química , Piperazinas/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Colorantes Fluorescentes/síntesis química , Fluoresceína-5-Isotiocianato/química , Proliferación Celular/efectos de los fármacos , Concentración de Iones de Hidrógeno , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Estructura MolecularRESUMEN
OBJECTIVE: To explore the effect of bear bile powder (BBP) on acute lung injury (ALI) and the underlying mechanism. METHODS: The chemical constituents of BBP were analyzed by ultra-high-pressure liquid chromatography-mass spectrometry (UPLC-MS). After 7 days of adaptive feeding, 50 mice were randomly divided into 5 groups by a random number table (n=10): normal control (NC), lipopolysaccharide (LPS), dexamethasone (Dex), low-, and high-dose BBP groups. The dosing cycle was 9 days. On the 12th and 14th days, 20 µL of Staphylococcus aureus solution (bacterial concentration of 1 × 10-7 CFU/mL) was given by nasal drip after 1 h of intragastric administration, and the mice in the NC group was given the same dose of phosphated buffered saline (PBS) solution. On the 16th day, after 1 h intragastric administration, 100 µL of LPS solution (1 mg/mL) was given by tracheal intubation, and the same dose of PBS solution was given to the NC group. Lung tissue was obtained to measure the myeloperoxidase (MPO) activity, the lung wet/dry weight ratio and expressions of CD14 and other related proteins. The lower lobe of the right lung was obtained for pathological examination. The concentrations of inflammatory cytokines including interleukin (IL)-6, tumour necrosis factor α (TNF-α ) and IL-1ß in the bronchoalveolar lavage fluid (BALF) were detected by enzyme linked immunosorbent assay, and the number of neutrophils was counted. The colonic contents of the mice were analyzed by 16 sRNA technique and the contents of short-chain fatty acids (SCFAs) were measured by gas chromatograph-mass spectrometer (GC-MS). RESULTS: UPLC-MS revealed that the chemical components of BBP samples were mainly tauroursodeoxycholic acid and taurochenodeoxycholic acid sodium salt. BBP reduced the activity of MPO, concentrations of inflammatory cytokines, and inhibited the expression of CD14 protein, thus suppressing the activation of NF-κB pathway (P<0.05). The lung histopathological results indicated that BBP significantly reduced the degree of neutrophil infiltration, cell shedding, necrosis, and alveolar cavity depression. Moreover, BBP effectively regulated the composition of the intestinal microflora and increased the production of SCFAs, which contributed to its treatment effect (P<0.05). CONCLUSIONS: BBP alleviates lung injury in ALI mouse through inhibiting activation of NF-κB pathway and decreasing expression of CD14 protein. BBP may promote recovery of ALI by improving the structure of intestinal flora and enhancing metabolic function of intestinal flora.
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Activation of hepatic stellate cells (HSCs) constitutes a crucial etiological factor leading to liver fibrosis. Theaflavine (TF) is a characteristic bioactive compound in fermented tea. Here, we found that TF attenuated the activation of LX-2 HSCs induced by transforming growth factor-ß1 (TGF-ß1). TF potentiated nuclear factor erythroid 2-related Factor 2 (Nrf2) signaling. Knockdown of Nrf2 abrogated TF-mediated resistance to TGF-ß1. Liver kinase B1 (LKB1), AMP-activated kinase (AMPK), and glycogen synthase kinase-3ß (GSK3ß) are upstream regulators of Nrf2. TF modulated the LKB1/AMPK/GSK3ß axis. Inhibition of AMPK or knockdown of LKB1 crippled TF-mediated potentiation of Nrf2. Protein kinase A (PKA) catalyzes LKB1 phosphorylation. In LX-2 cells, TF increased the LKB1/PKA interaction without affecting their contents. Inhibition of PKA abolished TF-mediated potentiation of LKB1/Nrf2 and abrogated the inhibitory effects of TF on their activation. TF also enhanced direct binding between purified catalytic subunit α of PKA (PKA-Cα) and LKB1 proteins in vitro. Molecular docking indicated that TF showed binding activity with both LKB1 and PKA-Cα proteins. In mouse primary HSCs, TF elevated LKB1/PKA-Cα binding, boosted LKB1 phosphorylation, potentiated Nrf2 and suppressed their spontaneous activation. PKA inhibition or LKB1 knockdown eliminated TF-mediated induction of Nrf2 and suppression of HSC activation. Furthermore, TF considerably alleviated CCl4-induced mouse liver fibrosis. In mouse livers, TF increased the LKB1/PKA-Cα interaction, upregulated LKB1 phosphorylation and modulated its downstream AMPK/GSK3ß/Nrf2 cascade. Our findings collectively indicated that TF suppresses HSC activation. Mechanistically, TF elevated the LKB1/PKA interaction in HSCs, which increased LKB1 phosphorylation and subsequently modulated the downstream AMPK/GSK3ß/Nrf2 axis.
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Proteínas Quinasas Activadas por AMP , Proteínas Quinasas Dependientes de AMP Cíclico , Ratones , Animales , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Células Estrelladas Hepáticas/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Simulación del Acoplamiento Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Cirrosis Hepática/metabolismoRESUMEN
Ergothioneine (EGT) is a bioactive compound derived from certain edible mushrooms. The activation of hepatic stellate cells (HSCs) is critically involved in the etiology of liver fibrosis (LF). Here, we report that in LX-2 HSCs, EGT upregulates the expression of Hint1 and Smad7 and suppresses their activation provoked by TGFß1. The EGT-triggered inhibition of HSC activation is abolished by knocking down the expression of Hint1. Overexpression of Hint1 increases Smad7 and represses TGFß1-provoked activation of LX-2 HSCs. In silico predictions unveiled that in the promoter region of the human Hint1 gene, there are two conserved cis-acting elements that have the potential to interact with the transcription factor Foxa3 termed hFBS1 and hFBS2, respectively. The knockdown of Foxa3 obviously declined Hint1 expression at both mRNA and protein levels. Transfection of Foxa3 or EGT treatment increased the activity of the luciferase reporter driven by the Hint1 promoter in an hFBS2-dependent manner. The knockdown of Foxa3 eliminated EGT-mediated upregulation of Hint1 promoter activity. Additionally, EGT triggered the nuclear translocation of Foxa3 without obviously affecting its expression level. Molecular docking analysis showed that EGT has the potential to directly interact with the Foxa3 protein. Moreover, Foxa3 played a critical role in EGT-mediated hepatoprotection. EGT modulated the Foxa3/Hint1/Smad7 signaling in mouse primary HSCs and inhibited their activation. The gavage of EGT considerably relieved CCl4-induced LF in mice. Our data provide new insights into the anti-LF activity of EGT. Mechanistically, EGT triggers the nuclear translocation of Foxa3 in HSCs, which promotes Hint1 transcription and subsequently elevates Smad7.
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Ergotioneína , Ratones , Humanos , Animales , Ergotioneína/farmacología , Células Estrelladas Hepáticas/metabolismo , Simulación del Acoplamiento Molecular , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/genética , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Factor Nuclear 3-gamma del Hepatocito/genética , Factor Nuclear 3-gamma del Hepatocito/metabolismoRESUMEN
OBJECTIVE: To study the molecular mechanism of cyclooxygenase-2 (COX-2), one of effective ingredient of brucine, in inducing non-small cell lung cancer cell apoptosis. METHOD: COX-2 promoter, transcription factor deletion mutants and COX-2 mRNA 3'-UTR-containing report plasmids were transfected with Renillia to non-small cell lung cancer A549 cell, in order to detect the activity of report gene luciferase and minimum cis-acting element of COX-2 promoter inhibited by brucine. The influence of brucine on IkappaB phosphorylation and the nuclear translocation of p65 were detected by immunoblotting assay. RESULT: Brucine significantly suppressed LPS-induced COX-2 promoter activation, but revealed minor impact on COX-2 mRNA stability. NF-kappaB in the vicinity of COX-2 promoter-262 was an important cis-acting element of brucine for inhibiting the activity of COX-2 promoter. Brucine was found to inhibit the phosphorylation of IkappaBalpha as well as the nuclear translocation of p65. CONCLUSION: Brucine can improve A549 cells apoptosis by inhibiting the activity of NF-kappaB and the subsequent COX-2 gene expression.
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Ciclooxigenasa 2/genética , FN-kappa B/metabolismo , Estricnina/análogos & derivados , Transporte Biológico/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Humanos , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Estabilidad del ARN/efectos de los fármacos , Estricnina/farmacologíaRESUMEN
Liver fibrosis is a pathological process as a result of intrahepatic deposition of excessive ECM. EMT of hepatocytes and activation of HSCs both play important roles in the etiology of liver fibrosis. Here, we found that limonin repressed TGF-ß-induced EMT in AML-12 hepatocytes and activation of LX-2 HSCs. Limonin suppressed TGF-ß-provoked Smad2/3 C-terminal phosphorylation and subsequent nuclear translocation. However, limonin exerted few effects on Smad2/3 phosphorylation atlinker region. Mechanistically, limonin increased Smad7 in both AML-12 and LX-2 cells. Knockdown of Smad7 abrogated inhibitory effects of limonin on TGF-ß-induced changes in both two cells. Further studies revealed that limonin upregulated Smad7 and declined C-terminal phosphorylation and nuclear translocation of Smad2/3 to alleviate mouse CCl4-induced liver fibrosis. Our findings indicated that limonin inhibits TGF-ß-induced EMT of hepatocytes and activation of HSCs in vitro and CCl4-induced liver fibrosis in mice. Upregulated Smad7 which suppresses Smad2/3-dependent gene transcription is implicated in the hepatoprotective activity of limonin.
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Leucemia Mieloide Aguda , Limoninas , Animales , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/metabolismo , Leucemia Mieloide Aguda/patología , Limoninas/farmacología , Hígado/metabolismo , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Ratones , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Piperine (PIP) is an alkaloid derived from peppercorns. Herein, we assessed its effects on hepatocyte EMT and HSC activation in vitro and CCl4-elicited liver fibrosis in mice. Further experiments were performed to unveil the molecular mechanisms underlying the hepatoprotective activity of PIP. We found that PIP inhibited TGF-ß1-provoked AML-12 hepatocyte EMT and LX-2 HSC activation. Mechanistically, in AML-12 and LX-2 cells, PIP evoked Nrf2 nuclear translocation and increased transcriptions of Nrf2-responsive antioxidative genes. These events decreased TGF-ß1-induced production of ROS. Moreover, PIP increased the expression of Smad7, suppressed phosphorylation and nuclear translocation of Smad2/3, and decreased the transcriptions of Smad2/3-downstream genes. Knockdown of Nrf2 abrogated the protective activity of PIP against TGF-ß1. Modulatory effects of PIP on the TGF-ß1/Smad cascade were also crippled, which suggested that activation of Nrf2 played critical roles in the regulatory effects of PIP on TGF-ß1/Smad signaling. Experiments in vivo unveiled that PIP ameliorated mouse liver fibrosis provoked by CCl4. PIP modulated the intrahepatic contents of the markers of EMT and HSC activation. In mouse livers, PIP activated Nrf2 signaling and reduced Smad2/3-dependent gene transcriptions. Our findings collectively suggested PIP as a new chemical entity with the capacity of alleviating liver fibrosis. The activation of the Nrf2 cascade and subsequent suppression of the TGF-ß1/Smad axis are implicated in the hepatoprotective activity of PIP.
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Alcaloides/farmacología , Benzodioxoles/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Cirrosis Hepática/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Piperidinas/farmacología , Alcamidas Poliinsaturadas/farmacología , Animales , Tetracloruro de Carbono/efectos adversos , Línea Celular , Hepatocitos/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Liver fibrosis constitutes a pathologic condition resulting in a series of advanced liver diseases. Oleanane-type saponins are distinctive active constituents in the medicinal plant Panax japonicus C. A. Mey (P. japonicus). Herein, we assessed protective effects of a characterized saponin extract of rhizomes of P. japonicus (SEPJ) on hepatocyte EMT and HSC activation in vitro and liver fibrosis in mice. We also investigated molecular mechanisms underlying the hepatoprotective activity of SEPJ. METHODS: EMT of AML-12 hepatocytes was evaluated by observing morphology of cells and quantifying EMT marker proteins. Activation of LX-2 HSCs was assessed via scratch assay, transwell assay, and EdU-incorporation assay, and by quantifying activation marker proteins. Liver fibrosis in mice was evaluated by HE, SR, and Masson staining, and by measuring related serum indicators. Immunoblotting and RT-PCR were performed to study mechanisms underlying the action of SEPJ. RESULTS: SEPJ inhibited TGF-ß-induced EMT in AML-12 hepatocytes and activation of LX-2 HSCs. SEPJ elevated Akt phosphorylation at Ser473 and GSK3ß phosphorylation at Ser9 in these cells, giving rise to a descent of the catalytic activity of GSK3ß. These events increased levels of both total and nuclear Nrf2 protein and upregulated expressions of Nrf2-responsive antioxidative genes. In addition, enhanced phosphorylation of Akt and GSK3ß acted upstream of SEPJ-mediated activation of Nrf2. Knockdown of Nrf2 or inhibition of Akt diminished the protective activity of SEPJ against TGF-ß in both AML-12 and LX-2 cells. Our further in vivo experiments revealed that SEPJ imposed a considerable alleviation on CCl4-provoked mouse liver fibrosis. Moreover, hepatic Akt/GSK3ß/Nrf2 cascade were potentiated by SEPJ. Taken together, our results unveiled that SEPJ exerted protective effects against fibrogenic cytokine TGF-ß in vitro and ameliorated liver fibrosis in mice. Mechanistically, SEPJ regulated the Akt/GSK3ß/Nrf2 signaling which subsequently enhanced intracellular antioxidative capacity. CONCLUSIONS: SEPJ inhibits hepatocyte EMT and HSC activation in vitro and alleviates liver fibrosis in mice. Modulation of the Akt/GSK3ß/Nrf2 cascade attributes to its hepatoprotective effects. Our findings support a possible application of SEPJ in the control of liver fibrosis.
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Panax , Saponinas , Animales , Glucógeno Sintasa Quinasa 3 beta , Células Estrelladas Hepáticas/patología , Hepatocitos , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Ratones , Factor 2 Relacionado con NF-E2 , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas c-akt , Saponinas/farmacologíaRESUMEN
OBJECTIVE: To study the acute toxicity of the crude and processed products of Entada phaseoloides and their effects on gastrointestinal movement in mice. METHODS: Using the method of intragastric administration, to observe the acute toxicity of the crude and processed products of Entada phaseoloides in mice and determine their LD50. With the methods of charcoal propulsion of small intestine and methyl orange colorimetry of gastric emptying, to study the impact of the crude and processed products of Entada on gastrointestinal movement in mice. RESULTS: The oral LD50 of crude Entada phaseoloides, No. 1 and No. 2 processed products of Entada phaseoloides in mice were 27.17, 35.13, 42.18 g/kg body weight. Crude and processed products of Entada phaseoloides can significantly promote the enteric propulsion of normal mice, and can significantly counteract the depressing status induced by atropine, but have no influence on the overactive status induced by neostigmine. The high, middle and low-dose of groups showed significant inhibition of the gastric emptying in normal mice. CONCLUSION: Processed Entada phaseoloides showed effects on the enteric propulsion of normal and depressing mice, can restrain the gastric emptying under normal mice, but its safety is better than crude Entada phaseoloides.
Asunto(s)
Antiinflamatorios/farmacología , Medicamentos Herbarios Chinos/aislamiento & purificación , Medicamentos Herbarios Chinos/farmacología , Fabaceae/química , Enfermedades Gastrointestinales/patología , Motilidad Gastrointestinal/efectos de los fármacos , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/toxicidad , Atropina/administración & dosificación , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/toxicidad , Fabaceae/toxicidad , Femenino , Vaciamiento Gástrico/efectos de los fármacos , Enfermedades Gastrointestinales/inducido químicamente , Dosificación Letal Mediana , Masculino , Ratones , Ratones Endogámicos , Neostigmina/administración & dosificación , Distribución Aleatoria , Semillas/química , Tecnología Farmacéutica/métodosRESUMEN
We investigated the liver protective activity of dandelion polyphenols (DP) against acetaminophen (APAP; Paracetamol)-induced hepatotoxicity. Mice were acclimated for 1 week and randomly divided into the following groups (n = 9 per group): Control, APAP, APAP + DP (100 mg·kg-1), APAP + DP (200 mg·kg-1), and APAP + DP (400 mg·kg-1) groups. Mice were pretreated with DP (100, 200, and 400 mg·kg-1) by oral gavage for 7 d before being treated with 350 mg·kg-1 APAP for 24 h to induced hepatotoxicity. Severe liver injury was observed, and hepatotoxicity was analyzed after 24 h by evaluation of biochemical markers, protein expressions levels, and liver histopathology. Pretreatment with DP was able to restore serum liver characteristics (aspartate transaminase, AST; alanine aminotransferase, ALT; alkaline phosphatase, AKP), improve redox imbalance (superoxide dismutase, SOD; glutathione, GSH; malondialdehyde, MDA), and decrease inflammatory factors (tumor necrosis factor-α, TNF-α; interleukin-1ß, IL-1ß). Pretreatment with DP also significantly inhibited the expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, DP pretreatment could inhibit the apoptosis of liver cells caused by APAP through up-regulation of Bcl-2 and down-regulation of Bax and caspase-9 protein. DP also down-regulated p-JNK protein expression levels to inhibit APAP-induced mitochondrial oxidative stress and up-regulated the expression of Nrf-2 and its target gene HO-1. The histopathological staining demonstrated that DP pretreatment could inhibit APAP-induced hepatocyte infiltration, congestion, and necrosis. Our results demonstrate that DP pretreatment could protect against APAP-induced hepatic injury by activating the Nrf-2/HO-1 pathway and inhibition of the intrinsic apoptosis pathway.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Extractos Vegetales/farmacología , Polifenoles/farmacología , Taraxacum/química , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/sangre , Modelos Animales de Enfermedad , Hemo-Oxigenasa 1/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: In traditional use, Panax medicinal plants (ginseng, red ginseng, notoginseng, Panax japonicus, and Panacis majoris rhizoma) have different bioactivities from each other, even under different dosages, but their chemical compositions are very similar; so the question is, what is the primary effective substance induced the different efficacy, and how to identify them from a group of chemical constituents? AIM OF THE STUDY: The goal of this research was to provide a strategy to determine the effective substance in Panax genus medicinal plants responsible for the anticoagulant response. MATERIALS AND METHODS: This research used ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS) to analyze the saponin chemical compositions of different concentration ethanol extraction of notoginseng and the ginseng genus medical plant 75% ethanol extraction (Panax ginseng, red ginseng, Panax japonicus, Panacis majoris rhizome), and used four coagulation factors (PT, TT, APTT, Fib) to evaluate the anticoagulant activity of the extracts. Grey correlation analysis was applied to establish the spectral effect relationship and give the anticoagulant potency of different saponins. Network pharmacology and molecular docking were adopted to clarify and verify the possible mechanisms of anticoagulant action. RESULTS: The results showed that the blood physiological regulation activities of Panax medicinal plants were different according to the solvent concentration, processing, species and dosage. Overall, the most suitable solvent for extraction of SQ was 75% ethanol; At low dosage (10-100â¯mg/mL), the anticoagulant effect of Panax medical plants was: ZJSâ¯>â¯ZZSâ¯>â¯SQâ¯>â¯RSâ¯>â¯HS, and at high doses (100-1000â¯mg/mL) was: SQâ¯>â¯ZJSâ¯>â¯ZZSâ¯>â¯RSâ¯>â¯HS. GRA and molecular docking results showed the contribution of some components (NG-R2, NG-Fc/G-Ra1/G-Ra2, G-Rc, G-Rk3, and G-Rh4) to the whole anticoagulant activity of the drug were increased, while the effect of CS-IVa was just decreased with the increase of dosage; the anticoagulant effect of G-Rg3 (the main anticoagulant component) is mainly related to the targets F2, AR, RHO, ACR, MB, GZMB, B2M, CA2, CAT, and PAPOLA. CONCLUSION: This study determined the effective substance of anti-coagulation of ginseng genus herbal medicines and the regulation of different anticoagulant effects of TCM by changing various influencing conditions, including processing method, extraction method, and dose. It also provided an effective strategy for effective substances identification of multicomponent, multifunction, and multipurpose herbal medicine.
Asunto(s)
Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Simulación del Acoplamiento Molecular , Panax , Extractos Vegetales/farmacología , Plantas Medicinales , Saponinas/farmacología , Espectrometría de Masa por Ionización de Electrospray , Anticoagulantes/aislamiento & purificación , Pruebas de Coagulación Sanguínea , Humanos , Panax/química , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales/química , Saponinas/aislamiento & purificación , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Acetaminophen (APAP) overdose is a major cause of drug-induced liver injury worldwide. Our current study was performed to assess the potential protective effects of γ-oryzanol (ORY) on APAP-induced liver injury in mice and explore the underlying molecular mechanisms. We unveiled that ORY alleviated the APAP-induced death of HL-7702 hepatocytes in vitro and liver injury in mice. Moreover, ORY promoted the nuclear translocation of Nrf2, increased the expressions of Nrf2-downstream antioxidative enzymes, including HO-1, NQO1, GCLC, and GCLM, and thereby restrained APAP-induced oxidative stress in hepatocytes. Moreover, ORY modulated the AMPK/GSK3ß axis that acts upstream of Nrf2 in hepatocytes. Compound C, an inhibitor of AMPK, prevented the ORY-mediated activation of Nrf2 and protection against APAP toxicity in HL-7702 hepatocytes. Additionally, in the liver of mice receiving APAP, ORY suppressed the nuclear translocation of the NF-κB p65 subunit, downregulated the expressions of iNOS and COX-2, and reduced the levels of pro-inflammatory factors including TNF-α, IL-1ß, IL-6, and NO. Taken together, our findings revealed that ORY is capable of ameliorating APAP-induced liver injury. The modulation of AMPK/GSK3ß/Nrf2 and NF-κB signaling pathways is implicated in the hepatoprotective activity of ORY.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Acetaminofén/efectos adversos , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Fenilpropionatos/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Animales , Antioxidantes/farmacología , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Glutamato-Cisteína Ligasa/metabolismo , Hemo-Oxigenasa 1/metabolismo , Hepatocitos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Hígado/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Oxazinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Dauricine (Dau) is a natural alkaloid exhibiting anti-proliferative activity against several different types of malignant cell. However, effects of Dau on hepatocellular carcinoma (HCC) cells and the underlying molecular mechanisms have remained to be fully elucidated. In this study, we found that Dau elevated the sensitivities of HCC cells to chemotherapeutic reagents, including cisplatin, sorafenib, and isoliensinine. Moreover, Dau promoted apoptosis of HCC cells triggered by these chemotherapeutic reagents. Consistently, in a xenograft mouse model, Dau sensitized HCC cells to sorafenib. In HCC cells, Dau dose-dependently inhibited glucose glycolysis and increased oxidative phosphorylation. Mechanistically, Dau downregulated the expression of hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2). HK2 and PKM2 can be directly targeted by miR-199a. Dau dose-dependently increased miR-199a expression in HCC cells. Transfection of anti-miR-199a abrogated Dau-mediated suppression of HK2 and PKM2. Dau-induced metabolic shift was thereby severely crippled by anti-miR-199a. In addition, the incremental activity of Dau on sorafenib sensitivity of HCC cells was diminished in response to the transfection of anti-miR-199a. Taken together, our findings provided novel insights into the impact of Dau on HCC cells and supported considering Dau as an adjuvant reagent in the clinical treatment of HCC.
Asunto(s)
Antineoplásicos/uso terapéutico , Bencilisoquinolinas/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Proteínas Portadoras/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Glucólisis/efectos de los fármacos , Cinesinas/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Tetrahidroisoquinolinas/farmacología , Hormonas Tiroideas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Aerobiosis , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Fosforilación Oxidativa , Proteínas de Unión a Hormona TiroideRESUMEN
In this study, chemical properties of polysaccharides from rhizomes of Panax japonicus C. A. Mey (PSPJ) were investigated and the antitumor immunostimulatory activity of PSPJ was assessed in mice bearing H22 hepatoma cells. Chemical properties of PSPJ were determined by GC, FT-IR, 1H NMR and 13C NMR analysis. Furthermore, we showed that PSPJ repressed H22 tumor growth in vivo with undetectable toxic effects on tumor-bearing mice. PSPJ upregulated host thymus/spleen indexes and ConA/LPS-induced splenocyte proliferation. Cytotoxic activities of natural killer and CD8+ T cells against H22 hepatoma cells were also elevated. Tumor transplantation led to substantial apoptosis of CD4+ T cells and dysregulation of the cytokine profile secreted by CD4+ T cells. These abnormalities were alleviated by PSPJ in a dose-dependent manner. In tumor-associated macrophages (TAMs), PSPJ reduced the production of immunosuppressive factors such as TGF-ß, IL-10 and PEG2. In addition, M2-like polarization of TAMs was also considerably declined in response to PSPJ. Our findings clearly demonstrated the antitumor immunostimulatory activity of PSPJ and supported considering PSPJ as an adjuvant reagent in clinical treatment of malignant diseases.