RESUMEN

INTRODUCTION: Fu-Fang-Jin-Qian-Cao is a Chinese herbal preparation used to treat urinary calculi. Fu-Fang-Jin-Qian-Cao can protect renal tubular epithelial cells from calcium oxalateinduced renal injury by inhibiting ROS-mediated autopathy. The mechanism still needs further exploration. Metabonomics is a new subject; the combination of metabolomics and network pharmacology can find pathways for drugs to act on targets more efficiently. METHODS: Comprehensive metabolomics and network pharmacology to study the mechanism of Fu-Fang-Jin-Qian-Cao inhibiting autophagy in calcium oxalate-induced renal injury. Based on UHPLC-Q-TOF-MS, combined with biochemical analysis, a mice model of Calcium oxalateinduced renal injury was established to study the therapeutic effect of Fu-Fang-Jin-Qian-Cao. Based on the network pharmacology, the target signaling pathway and the protective effect of Fu- Fang-Jin-Qian-Cao on Calcium oxalate-induced renal injury by inhibiting autophagy were explored. Autophagy-related proteins LC3-II, BECN1, ATG5, and ATG7 were studied by immunohistochemistry. RESULTS: Combining network pharmacology and metabolomics, 50 differential metabolites and 2482 targets related to these metabolites were found. Subsequently, the targets enriched in PI3KAkt, MAPK and Ras signaling pathways. LC3-II, BECN1, ATG5 and ATG7 were up-regulated in Calcium oxalate-induced renal injury. All of them could be reversed after the Fu-Fang-Jin-Qian- Cao treatment. CONCLUSIONS: Fu-Fang-Jin-Qian-Cao can reverse ROS-induced activation of the MAPK signaling pathway and inhibition of the PI3K-Akt signaling pathway, thereby reducing autophagy damage of renal tubular epithelial cells in Calcium oxalate-induced renal injury.

Asunto(s)

Oxalato de Calcio , Medicamentos Herbarios Chinos , Ratones , Animales , Oxalato de Calcio/metabolismo , Oxalato de Calcio/farmacología , Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Farmacología en Red , Fosfatidilinositol 3-Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Riñón/metabolismo , Autofagia , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/metabolismo

RESUMEN

SARS-CoV-2 has developed a variety of approaches to counteract host innate antiviral immunity to facilitate its infection, replication and pathogenesis, but the molecular mechanisms that it employs are still not been fully understood. Here, we found that SARS-CoV-2 NSP8 inhibited the production of type I and III interferons (IFNs) by acting on RIG-I/MDA5 and the signaling molecules TRIF and STING. Overexpression of NSP8 downregulated the expression of type I and III IFNs stimulated by poly (I:C) transfection and infection with SeV and SARS-CoV-2. In addition, NSP8 impaired IFN expression triggered by overexpression of the signaling molecules RIG-I, MDA5, and MAVS, instead of TBK1 and IRF3-5D, an active form of IRF3. From a mechanistic view, NSP8 interacts with RIG-I and MDA5, and thereby prevents the assembly of the RIG-I/MDA5-MAVS signalosome, resulting in the impaired phosphorylation and nuclear translocation of IRF3. NSP8 also suppressed the TRIF- and STING- induced IFN expression by directly interacting with them. Moreover, ectopic expression of NSP8 promoted virus replications. Taken together, SARS-CoV-2 NSP8 suppresses type I and III IFN responses by disturbing the RIG-I/MDA5-MAVS complex formation and targeting TRIF and STING signaling transduction. These results provide new insights into the pathogenesis of COVID-19.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , Proteínas Adaptadoras del Transporte Vesicular/genética , Helicasa Inducida por Interferón IFIH1/genética , Interferones , SARS-CoV-2/metabolismo , Transducción de Señal

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a poor inducer of innate antiviral immunity, and the underlying mechanism still needs further investigation. Here, we reported that SARS-CoV-2 NSP7 inhibited the production of type I and III interferons (IFNs) by targeting the RIG-I/MDA5, Toll-like receptor (TLR3)-TRIF, and cGAS-STING signaling pathways. SARS-CoV-2 NSP7 suppressed the expression of IFNs and IFN-stimulated genes induced by poly (I:C) transfection and infection with Sendai virus or SARS-CoV-2 virus-like particles. NSP7 impaired type I and III IFN production activated by components of the cytosolic dsRNA-sensing pathway, including RIG-I, MDA5, and MAVS, but not TBK1, IKKε, and IRF3-5D, an active form of IRF3. In addition, NSP7 also suppressed TRIF- and STING-induced IFN responses. Mechanistically, NSP7 associated with RIG-I and MDA5 prevented the formation of the RIG-I/MDA5-MAVS signalosome and interacted with TRIF and STING to inhibit TRIF-TBK1 and STING-TBK1 complex formation, thus reducing the subsequent IRF3 phosphorylation and nuclear translocation that are essential for IFN induction. In addition, ectopic expression of NSP7 impeded innate immune activation and facilitated virus replication. Taken together, SARS-CoV-2 NSP7 dampens type I and III IFN responses via disruption of the signal transduction of the RIG-I/MDA5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways, thus providing novel insights into the interactions between SARS-CoV-2 and innate antiviral immunity.

Asunto(s)

COVID-19 , Interferón Tipo I , Humanos , SARS-CoV-2/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Transducción de Señal , Interferones , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , Antivirales , Proteínas Adaptadoras del Transporte Vesicular/genética

RESUMEN

A characteristic feature of COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the dysregulated immune response with impaired type I and III interferon (IFN) expression and an overwhelming inflammatory cytokine storm. RIG-I-like receptors (RLRs) and cGAS-STING signaling pathways are responsible for sensing viral infection and inducing IFN production to combat invading viruses. Multiple proteins of SARS-CoV-2 have been reported to modulate the RLR signaling pathways to achieve immune evasion. Although SARS-CoV-2 infection also activates the cGAS-STING signaling by stimulating micronuclei formation during the process of syncytia, whether SARS-CoV-2 modulates the cGAS-STING pathway requires further investigation. Here, we screened 29 SARS-CoV-2-encoded viral proteins to explore the viral proteins that affect the cGAS-STING signaling pathway and found that SARS-CoV-2 open reading frame 10 (ORF10) targets STING to antagonize IFN activation. Overexpression of ORF10 inhibits cGAS-STING-induced interferon regulatory factor 3 phosphorylation, translocation, and subsequent IFN induction. Mechanistically, ORF10 interacts with STING, attenuates the STING-TBK1 association, and impairs STING oligomerization and aggregation and STING-mediated autophagy; ORF10 also prevents the endoplasmic reticulum (ER)-to-Golgi trafficking of STING by anchoring STING in the ER. Taken together, these findings suggest that SARS-CoV-2 ORF10 impairs the cGAS-STING signaling by blocking the translocation of STING and the interaction between STING and TBK1 to antagonize innate antiviral immunity.

Asunto(s)

COVID-19 , Interferón Tipo I , Autofagia , Humanos , Inmunidad Innata , Interferón Tipo I/genética , Interferones , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Sistemas de Lectura Abierta , Proteínas Serina-Treonina Quinasas/genética , SARS-CoV-2 , Proteínas Virales/metabolismo

RESUMEN

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I-MAVS complex to attenuate the RIG-I-mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.

Asunto(s)

Proteasas 3C de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/genética , Proteína 58 DEAD Box/genética , ADN Helicasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas de Unión al ARN/genética , Receptores Inmunológicos/genética , SARS-CoV-2/genética , Gránulos de Estrés/genética , Animales , Chlorocebus aethiops , Proteasas 3C de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteína 58 DEAD Box/inmunología , ADN Helicasas/inmunología , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Evasión Inmune , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Poli I-C/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , Unión Proteica , ARN Helicasas/inmunología , Proteínas con Motivos de Reconocimiento de ARN/inmunología , ARN Bicatenario/genética , ARN Bicatenario/inmunología , Proteínas de Unión al ARN/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Virus Sendai/genética , Virus Sendai/inmunología , Transducción de Señal , Gránulos de Estrés/efectos de los fármacos , Gránulos de Estrés/inmunología , Gránulos de Estrés/virología , Células Vero , Vesiculovirus/genética , Vesiculovirus/inmunología

RESUMEN

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.

Asunto(s)

Proteína 58 DEAD Box/inmunología , Evasión Inmune/genética , Interferones/inmunología , Nucleotidiltransferasas/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2/inmunología , Receptor Toll-Like 3/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Animales , Chlorocebus aethiops , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteína 58 DEAD Box/genética , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/inmunología , Interferones/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Nucleotidiltransferasas/genética , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Plásmidos/química , Plásmidos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Receptores Inmunológicos/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 3/genética , Transfección , Células Vero , Replicación Viral/inmunología

RESUMEN

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has quickly spread worldwide and has affected more than 10 million individuals. A typical feature of COVID-19 is the suppression of type I and III interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism by which SARS-CoV-2 evades antiviral immunity remains elusive. Here, we reported that the SARS-CoV-2 membrane (M) protein inhibits the production of type I and III IFNs induced by the cytosolic dsRNA-sensing pathway mediated by RIG-I/MDA-5-MAVS signaling. In addition, the SARS-CoV-2 M protein suppresses type I and III IFN induction stimulated by SeV infection or poly (I:C) transfection. Mechanistically, the SARS-CoV-2 M protein interacts with RIG-I, MAVS, and TBK1, thus preventing the formation of the multiprotein complex containing RIG-I, MAVS, TRAF3, and TBK1 and subsequently impeding the phosphorylation, nuclear translocation, and activation of IRF3. Consequently, ectopic expression of the SARS-CoV-2 M protein facilitates the replication of vesicular stomatitis virus. Taken together, these results indicate that the SARS-CoV-2 M protein antagonizes type I and III IFN production by targeting RIG-I/MDA-5 signaling, which subsequently attenuates antiviral immunity and enhances viral replication. This study provides insight into the interpretation of SARS-CoV-2-induced antiviral immune suppression and illuminates the pathogenic mechanism of COVID-19.

Asunto(s)

COVID-19/metabolismo , Proteína 58 DEAD Box/metabolismo , Interferón Tipo I/biosíntesis , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferones/biosíntesis , SARS-CoV-2/metabolismo , Transducción de Señal , Proteínas de la Matriz Viral/metabolismo , Animales , COVID-19/genética , Chlorocebus aethiops , Proteína 58 DEAD Box/genética , Células HEK293 , Células HeLa , Humanos , Interferón Tipo I/genética , Helicasa Inducida por Interferón IFIH1/genética , Interferones/genética , Receptores Inmunológicos , SARS-CoV-2/genética , Células Vero , Proteínas de la Matriz Viral/genética , Interferón lambda

RESUMEN

Objective@#The purpose of our study was to investigate the predictive abilities of clinical and computed tomography (CT)features for outcome prediction in patients with coronavirus disease (COVID-19). @*Materials and Methods@#The clinical and CT data of 238 patients with laboratory-confirmed COVID-19 in our two hospitalswere retrospectively analyzed. One hundred sixty-six patients (103 males; age 43.8 ± 12.3 years) were allocated in thetraining cohort and 72 patients (38 males; age 45.1 ± 15.8 years) from another independent hospital were assigned in thevalidation cohort. The primary composite endpoint was admission to an intensive care unit, use of mechanical ventilation, ordeath. Univariate and multivariate Cox proportional hazard analyses were performed to identify independent predictors. Anomogram was constructed based on the combination of clinical and CT features, and its prognostic performance wasexternally tested in the validation group. The predictive value of the combined model was compared with models built on theclinical and radiological attributes alone. @*Results@#Overall, 35 infected patients (21.1%) in the training cohort and 10 patients (13.9%) in the validation cohortexperienced adverse outcomes. Underlying comorbidity (hazard ratio [HR], 3.35; 95% confidence interval [CI], 1.67–6.71;p < 0.001), lymphocyte count (HR, 0.12; 95% CI, 0.04–0.38; p < 0.001) and crazy-paving sign (HR, 2.15; 95% CI, 1.03–4.48;p = 0.042) were the independent factors. The nomogram displayed a concordance index (C-index) of 0.82 (95% CI, 0.76–0.88),and its prognostic value was confirmed in the validation cohort with a C-index of 0.89 (95% CI, 0.82–0.96). The combinedmodel provided the best performance over the clinical or radiological model (p < 0.050). @*Conclusion@#Underlying comorbidity, lymphocyte count and crazy-paving sign were independent predictors of adverseoutcomes. The prognostic nomogram based on the combination of clinical and CT features could be a useful tool for predictingadverse outcomes of patients with COVID-19.

RESUMEN

Pattern recognition receptors (PRRs) are sensors of exogenous and endogenous "danger" signals from pathogen-associated molecular patterns (PAMPs), and damage associated molecular patterns (DAMPs), while autophagy can respond to these signals to control homeostasis. Almost all PRRs can induce autophagy directly or indirectly. Toll-like receptors (TLRs), Nod-like receptors (NLRs), retinoic acid-inducible gene-I-like receptors (RLRs), and cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway can induce autophagy directly through Beclin-1 or LC3-dependent pathway, while the interactions with the receptor for advanced glycation end products (RAGE)/high mobility group box 1 (HMGB1), CD91/Calreticulin, and TLRs/HSPs are achieved by protein, Ca2+, and mitochondrial homeostasis. Autophagy presents antigens to PRRs and helps to clean the pathogens. In addition, the induced autophagy can form a negative feedback regulation of PRRs-mediated inflammation in cell/disease-specific manner to maintain homeostasis and prevent excessive inflammation. Understanding the interaction between PRRs and autophagy in a specific disease will promote drug development for immunotherapy. Here, we focus on the interactions between PRRs and autophagy and how they affect the inflammatory response.

Asunto(s)

Autofagia , Inflamación , Receptores de Reconocimiento de Patrones , Autofagia/inmunología , Humanos , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal

RESUMEN

The CO I gene sequences of Qianghuoyu, Pachytriton labiatus and Gehyra mutilata were achieved by PCR amplification and bi-directional sequencing. Furthermore, a pair of specific primers SJYW1 and SJYW2 in the non-conservative district were designed through sequence alignment. The PCR reaction condition was established by changing the annealing temperature and cycle numbers. The results showed that 350 bp DNA fragment was amplified from Qianghuoyu in PCR with annealed temperature at 54 °C and the cycle number was 25 cycles, whereas not any DNA fragment was amplified from P. labiatus and G. mutilata under the same reaction condition. This method is well-performed in the identification of Qianghuoyu for its excellent specificity and repeatability.

Asunto(s)

Animales , Contaminación de Medicamentos , Medicina Tradicional Tibetana , Reacción en Cadena de la Polimerasa , Métodos

RESUMEN

Acute kidney injury is a serious global health problem and determinant of morbidity and mortality. Recent advancements in the field of stem cell research raise hopes for stem cell-based regenerative approaches to treat acute kidney diseases. In this review, the authors summarized the latest research advances of the adult resident renal progenitor cells (ARPCs) on kidney repair, the role of ARPCs on tubular regeneration after acute kidney injury, the current understanding of the mechanisms related to ARPC activation and modulation, as well as the challenges that remain to be faced.

Asunto(s)

Lesión Renal Aguda/fisiopatología , Túbulos Renales/fisiopatología , Regeneración/fisiología , Células Madre/fisiología , Antígenos CD/metabolismo , Medicamentos Herbarios Chinos/farmacología , Humanos , Riñón/fisiopatología , Receptores CXCR/metabolismo , Daño por Reperfusión/fisiopatología

RESUMEN

Acute kidney injury is a serious global health problem and determinant of morbidity and mortality. Recent advancements in the field of stem cell research raise hopes for stem cell-based regenerative approaches to treat acute kidney diseases. In this review, the authors summarized the latest research advances of the adult resident renal progenitor cells (ARPCs) on kidney repair, the role of ARPCs on tubular regeneration after acute kidney injury, the current understanding of the mechanisms related to ARPC activation and modulation, as well as the challenges that remain to be faced.

Asunto(s)

Humanos , Lesión Renal Aguda , Antígenos CD , Metabolismo , Medicamentos Herbarios Chinos , Farmacología , Riñón , Túbulos Renales , Receptores CXCR , Metabolismo , Regeneración , Fisiología , Daño por Reperfusión , Células Madre , Fisiología

RESUMEN

Development of resistance to TNF-related apoptosis-inducing ligand (TRAIL) in tumor cells is one of the important problems in cancer treatment. Despite the previous report demonstrating that oligomycin suppressed TNF-induced apoptosis, in our screening of small molecules enhancing cancer cell death to TRAIL, oligomycin A (OMA) was found to enhance TRAIL-induced apoptosis in HeLa cells. CCAAT/enhancer-binding protein homologous protein (CHOP) was found to directly bind to death receptor 5 (DR5) promoter through endoplasmic reticulum stress (ER-stress) signaling and sensitize the cells to TRAIL. Among ER-stress associated proteins, OMA triggered the inositol-requiring enzyme 1 (IRE1) signaling pathway, leading to X-binding protein 1 (XBP1) splicing, CHOP expression and DR5 upregulation. In contrast, small-interfering RNA (siRNA) of CHOP reduced the number of apoptotic cells in response to the co-treatment of TRAIL and OMA. Collectively, our data suggest that OMA enhances apoptotic death of cervical cancer cells to TRAIL through upregulation of CHOP-mediated DR5 expression following ER-stress.

Asunto(s)

Apoptosis/efectos de los fármacos , Oligomicinas/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factor de Transcripción CHOP/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Estrés del Retículo Endoplásmico , Endorribonucleasas/metabolismo , Células HeLa/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Empalme de Proteína , ARN Interferente Pequeño/genética , Factores de Transcripción del Factor Regulador X , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Factor de Transcripción CHOP/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteína 1 de Unión a la X-Box

RESUMEN

OBJECTIVE: To explore the effects of erythropoietin (EPO) on the differentiation and secretion of cultured bone marrow-derived mesenchymal stem cells (BM-MSC) in the microenvironment of acute kidney injury (AKI). METHODS: C57BL/6 murine BM-MSC (mBM-MSC) were successfully isolated by the methods of Percoll density gradient centrifugation and adherence cultivation. The AKI murine model was induced by ischemia/reperfusion (I/R). The homogenate supernatants were prepared for normal and I/R murine kidney. P3-mBM-MSC were treated differently: Group A: low glucose DMEM medium with 10% fetal bovine serum, Group B: normal murine kidney homogenate supernatant intervention, Group C: I/R kidney homogenate supernatant intervention, Group D: I/R kidney homogenate supernatant plus different concentrations of EPO (1, 5, 10, 50 U/ml). Each group was incubated for 1, 3, 5 and 7 days. Inverted microscope was used to observe the morphological changes of these cells and their ultrastructural changes were observed by transmission electron microscope. Cytokeratin-18 was detected by flow cytometry. The levels of bone morphogenetic protein-7 (BMP-7), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) were detected by ELISA in culture medium. RESULTS: The cells yielded a high expression of CD29 and CD44 and a low expression of CD34 and CD45. Compared with Groups A and B, the cells of Group C presented oval and short fusiform shapes. After the intervention of EPO, Group D showed a cobble appearance. More organelles were also found. A trace expression of CK18 was found in Groups A and B. A positive expression of CK18 was significantly higher in Groups C and D than Groups A and B (P < 0.01). The expression of EPO 50 U/ml at Day 5 and 7 was higher than Group C of the same time (5 d: 35.22 ± 4.04 vs 8.72 ± 0.38, 7 d: 42.00 ± 5.39 vs 13.20 ± 1.14, both P < 0.01). The results of ELISA showed that the levels of BMP-7, HGF and VEGF in Group C decreased significantly (P < 0.01 or P < 0.05). CONCLUSION: The intervention of EPO may boost the differentiation of mBM-MSC but reverse its low secretion.

Asunto(s)

Lesión Renal Aguda/metabolismo , Eritropoyetina/farmacología , Células Madre Mesenquimatosas/citología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Células Cultivadas , Citometría de Flujo , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL

RESUMEN

Ginsenoside Rh2 is one of the most active components of red ginseng, controlling cancer and other metabolic diseases including osteoclast differentiation. However, the molecular mechanism underlying the inhibition of osteoclast differentiation by ginsenoside Rh2 remains poorly understood. In the present study, it was found that ginsenoside Rh2 suppressed osteoclast differentiation from bone marrow macrophages (BMMs) treated with receptor activator of nuclear factor κB ligand (RANKL) without any cytotoxicity. Ginsenoside Rh2 significantly reduced RANKL-induced expression of transcription factors, c-Fos and nuclear factor of activated T-cells (NFATc1), as well as osteoclast markers, TRAP and OSCAR. In defining the signaling pathways, ginsenoside Rh2 was shown to moderately inhibit NF-κB activation and ERK phosphorylation in response to RANKL stimulation in BMM cells without any effect on p38 and c-Jun N-terminal kinase (JNK). Finally, ginsenoside Rh2 blocked osteoporosis in vivo as confirmed by restored bone mineral density (BMD) and other markers associated osteoclast differentiation. Hence, it is suggested that ginsenoside Rh2 could suppress RANKL-induced osteoclast differentiation in vitro and in vivo through the regulation of c-Fos and NFATc1 expressions, not excluding the involvement of NF-κB and ERK. Ginsenoside Rh2 is also suggested to be developed as a therapeutic drug for prevention and treatment of osteoporosis.

Asunto(s)

Ginsenósidos/farmacología , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Secuencia de Bases , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Resorción Ósea/prevención & control , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cartilla de ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Genes fos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Factores de Transcripción NFATC/genética , Osteoclastos/citología , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Osteoporosis/prevención & control , Fosforilación , Ligando RANK/farmacología

RESUMEN

We exploited the biological activity of an antibiotic agent asperlin isolated from Aspergillus nidulans against human cervical carcinoma cells. We found that asperlin dramatically increased reactive oxygen species (ROS) generation accompanied by a significant reduction in cell proliferation. Cleavage of caspase-3 and PARP and reduction of Bcl-2 could also be detected after asperlin treatment to the cells. An anti-oxidant N-acetyl-L-cysteine (NAC), however, blocked all the apoptotic effects of asperlin. The involvement of oxidative stress in asperlin induced apoptosis could be supported by the findings that ROS- and DNA damage-associated G2/M phase arrest and ATM phosphorylation were increased by asperlin. In addition, expression and phosphorylation of cell cycle proteins as well as G2/M phase arrest in response to asperlin were significantly blocked by NAC or an ATM inhibitor KU-55933 pretreatment. Collectively, our study proved for the first time that asperlin could be developed as a potential anti-cancer therapeutics through ROS generation in HeLa cells.

Asunto(s)

Antibióticos Antineoplásicos/farmacología , Carcinoma/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Compuestos Epoxi/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Pironas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Antibióticos Antineoplásicos/aislamiento & purificación , Apoptosis , Aspergillus nidulans/química , Proteínas de la Ataxia Telangiectasia Mutada , Carcinoma/enzimología , Proteínas de Ciclo Celular/antagonistas & inhibidores , División Celular/efectos de los fármacos , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN/antagonistas & inhibidores , Compuestos Epoxi/aislamiento & purificación , Femenino , Fase G2/efectos de los fármacos , Células HeLa , Humanos , Morfolinas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pironas/aislamiento & purificación , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Neoplasias del Cuello Uterino/enzimología

RESUMEN

Adiponectin (ADN), an insulin-sensitizing adipokine, stimulates glucose uptake, inhibits gluconeogenesis, and plays an important role in improving insulin sensitivity. Since blood levels of ADN are low in type 2 diabetes mellitus (DM), this study was designed to investigate the therapeutic effectiveness of increasing the ADN level through injection of plasmid DNA encoding ADN in type 2 DM. A non-obese type 2 DM mouse model was established via combined administration of streptozotocin with nicotinamide and exhibited significantly higher plasma glucose concentration and insulin resistance compared with normal controls according to oral glucose tolerance and insulin challenge tests. Plasmid DNA encoding mouse ADN from differentiated NIH3T3 adipocytes was constructed in pVAX1 (pVAX/ADN). Transfection of pVAX/ADN into various cell lines including HeLa, HT22, HEK293, HepG2, and SK-Hep1 cells, increased ADN mRNA expression levels in a dose-dependent manner. The administration of pVAX/ADN into non-obese type 2 DM mice via tail vein significantly increased the blood level of ADN and decreased the plasma glucose concentration. Moreover, the parameters related to insulin resistance (HOMA-IR) and insulin sensitivity (QUICKI) were significantly improved. These results suggest that ADN gene therapy could be a clinically effective tool for the treatment of type 2 DM.

Asunto(s)

Adiponectina/administración & dosificación , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 2/terapia , Terapia Genética/métodos , Adiponectina/sangre , Adiponectina/genética , Animales , Glucemia/metabolismo , Línea Celular , Línea Celular Tumoral , ADN/administración & dosificación , Diabetes Mellitus Tipo 2/genética , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Niacinamida , Plásmidos , ARN Mensajero/metabolismo , Estreptozocina

RESUMEN

To study the chemical constituents of the Entada phaseoloides (L.) Merr., seeds of Entada phaseoloides were extracted with 70% ethanol at room temperature. Isolation and purification were performed by silica gel, reversed-phase silica gel column chromatography and semi-preparative HPLC. Structures of the pure compounds were established on the basis of spectral analysis. Four sulfur-containing amide compounds were isolated from the n-BuOH-soluble fraction and identified as entadamide A-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranoside (1), entadamide A (2), entadamide A-beta-D-glucopyranoside (3) and clinacoside C (4). Compound 1 is a new compound. Compound 4 is isolated from the genus Entada for the first time.

Asunto(s)

Acrilamidas , Química , Fabaceae , Química , Estructura Molecular , Plantas Medicinales , Química , Semillas , Química , Tioglucósidos , Química

RESUMEN

To study the chemical constituents from the root of Berchemia lineata (L.) DC., nine compounds were isolated from the EtOAc extract by using silica gel, RP-C18 silica gel column chromatography and preparative HPLC. Based on the spectroscopic analysis, their structures were identified as 5-hydroxy-7-(2'-hydroxypropyl)-2-methyl-chromone (1), (-)-(1'R, 2'S)-erythro-5-hydroxy-7-(1', 2'-dihydroxypropyl)-2-methyl-chromone (2), naringenin (3), eriodictyol (4), (+)-aromadendrin (5), (+)-taxifolin (6), (+)-catechin (7), (+)-epigallocatechin (8) and quercetin (9). Among them, compound 2 is a new chromone derivative. Compound 1 is a known chromone derivative and isolated from this genus for the first time. Compounds 3-9 are known flavonoids and isolated from this plant for the first time.

Asunto(s)

Catequina , Química , Cromonas , Química , Flavanonas , Química , Flavonoides , Química , Estructura Molecular , Raíces de Plantas , Química , Plantas Medicinales , Química , Quercetina , Química , Rhamnaceae , Química

RESUMEN

This study was designed to examine the anti-hypertensive effect of the combination therapy of captopril with losartan by oral administration using both independent and cross-over experimental protocols. In independent experimental protocols, four different groups of spontaneous hypertensive rats (SHR) were treated for 1 or 2 weeks: control, captopril (20 mg/kg/day), losartan (20 mg/kg/day), and combination captopril (10 mg/kg/day) with losartan (10 mg/kg/day). In cross-over protocols, each SHR received all four treatments for 1 or 3 days with an interval of several days between each injection for washing-out and return to high blood pressure (BP) levels. BP and heart rate (HR) were measured in conscious telemetered SHR. According to the results from the independent protocol, regardless of a 1- or 2-week administration period, combination therapy with low doses of captopril and losartan had a greater anti-hypertensive effect than individual high-dose monotherapy. Similarly, results from the cross-over protocol showed that regardless of 1-day or 3-day administration, the decrease in BP in the 11(th) and 12(th) hour after administration was greatest with the combination of low-dose captopril and losartan. Therefore, combination therapy with low doses of captopril with losartan lowered BP to a greater extent than a high dose of either individual monotherapy.

Asunto(s)

Antihipertensivos/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Captopril/administración & dosificación , Losartán/administración & dosificación , Administración Oral , Animales , Estudios Cruzados , Sinergismo Farmacológico , Quimioterapia Combinada , Masculino , Distribución Aleatoria , Ratas , Ratas Endogámicas SHR , Factores de Tiempo