RESUMO
The abnormal immune response mediated by malignant melanoma is related to PD1. Paeonol has pharmacological antitumor activity. Previous studies have indicated that paeonol induces tumor cell apoptosis, but its underlying mechanism in tumor immunity remains unknown. In this study, malignant melanoma was established in normal and thymectomized mice to determine the important role of the thymus in the antitumor effects of paeonol. Paeonol-treated thymocytes were cocultured with melanoma cell spheres to further evaluate the regulatory role of thymocytes in tumor immune dysfunction. Studies have shown that PD1 may be targeted by miR-139-5p. Our results revealed that tumor-induced thymic atrophy was significantly accompanied by high PD1 expression and low miR-139-5p expression. Interestingly, paeonol significantly reversed thymic atrophy and largely protected thymocytes against low PD1 expression and high miR-139-5p expression. Dual-luciferase assays indicated that miR-139-5p interacted with the 3' untranslated region (3'-UTR) of PD1. These results showed that paeonol alleviates PD1-mediated antitumor immunity by reducing miR-139-5p expression and demonstrated a novel mechanism for melanoma immunotherapy.
Assuntos
Melanoma , MicroRNAs , Animais , Camundongos , Regulação para Cima , MicroRNAs/genética , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Apoptose , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Melanoma Maligno CutâneoRESUMO
Inflammation is a biological process that exists in a large number of diseases. NF-κB has been proven to play a pivotal role in the development of inflammation. New drugs aimed at inhibiting the expression of NF-κB have gained attention from researchers. Sirt1 has an anti-inflammatory function, and the circRNA encoded by the Sirt1 gene may also play roles in the anti-inflammatory reaction of Sirt1. In the present study, LPS-treated RAW264.7 cells were used as an inflammatory cell model, and tanshinone IIA sodium sulfonate (TSS) was used as a therapeutic drug. We found that TSS downregulated LPS-induced TNF-α and IL-1ß expression nearly threefold. LPS reduced Circ-sirt1 mRNA expression by one-third, while TSS started this phenomenon. In addition, overexpression/knockdown of Circ-sirt1 neutralized the function of TSS by regulating the translocation of NF-κB. Thus, we proved that TSS has an anti-inflammatory function by upregulating circ-Sirt1 and subsequently inhibiting the translocation of NF-κB. An in vivo experiment was also performed to confirm the protective function of TSS on inflammation. These results indicated that TSS is a potential treatment for inflammation.
Assuntos
NF-kappa B/metabolismo , Fenantrenos/farmacologia , Salvia miltiorrhiza , Sirtuína 1/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos , Células RAW 264.7 , RNA Circular/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.
Assuntos
Antiparkinsonianos/farmacologia , Antiparkinsonianos/uso terapêutico , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson Secundária/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Extratos de Tecidos/química , alfa-Sinucleína/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , Hidroxidopaminas , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Células PC12 , Doença de Parkinson Secundária/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Estearatos/farmacologia , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , alfa-Sinucleína/efeitos dos fármacosRESUMO
Phagocytosis is a basic immune response to the pathogens invading. Immunosuppression may occur in diseases like sepsis and cancer, and cause a low phagocytic ability of phagocytes. High mobility group protein B1 (HMGB1) is a DNA chaperone which is closely related to the phagocytosis. Nonetheless, its influence on phagocytosis is still controversial. We found that paeonol could inhibit the translocation of HMGB1 from the nucleus to the cytoplasm, it may have an impact on phagocytosis. In the present study, we performed in vivo and in vitro experiments to investigate the influence of paeonol on phagocytosis. Zymosan was used to test the phagocytic function of macrophages. Our results showed that paeonol promotes the phagocytosis of macrophages through confining HMGB1 to the nucleus. Through interacting with P53, the nuclear HMGB1 keep it in the nucleus and decrease the negative influence of P53 on the phosphorylation of Focal Adhesion Kinase (FAK). The increasing of phosphorylated FAK promotes the formation of pseudopod and enhances the phagocytic ability of macrophages.
Assuntos
Acetofenonas/farmacologia , Núcleo Celular/efeitos dos fármacos , Proteína HMGB1/metabolismo , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Células RAW 264.7 , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
Phagocytosis is a basic immune response to the invasion of pathogens. High mobility group protein B1 (HMGB1) is a DNA chaperone that is associated with phagocytosis. However, its influence on phagocytosis is debated. In the present study, HMGB1-mutant, HMGB1-overexpressing and HMGB1-silenced RAW264.7 cells were constructed. In addition, HMGB1 conditional knockout mice were constructed to determine the influence of HMGB1 on phagocytosis. Lipopolysaccharide (LPS) was used to stimulate the translocation of HMGB1 from the nucleus to the cytoplasm. Zymosan particles were used to test the phagocytic function of the macrophages. Our results showed that the accumulation of HMGB1 in the nucleus enhances the phagocytic function of the macrophages. By interacting with P53, nuclear HMGB1 may remain in the nucleus and decrease the negative influence of P53 on the phosphorylation of focal adhesion kinase (FAK). The increase in phosphorylated FAK promotes the formation of pseudopods and enhances the phagocytic ability of macrophages.
Assuntos
Proteína HMGB1/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Transporte Proteico/fisiologia , Animais , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Fagocitose/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacosRESUMO
Inflammation is a common inducer of numerous severe diseases such as sepsis. The NF-κB signaling pathway plays a key role in the inflammatory process. Its activation promotes the release of pro-inflammatory mediators like inducible nitric oxide synthase and tumor necrosis factor alpha. Peroxisome proliferator-activated receptor gamma (PPAR-γ) inactivates nuclear factor kappa B (NF-κB) and subsequently attenuates inflammation. Rhein, an agent isolated from rhubarb, has been known to have anti-inflammatory effects. However, its influence on PPAR-γ remains largely unknown. In this study, an inflammation model was constructed by stimulating RAW264.7 cells with lipopolysaccharide. Rhein was used as a therapeutic agent, while rosiglitazone (PPAR-γ activator) and GW9662 (PPAR-γ inhibitor) were used as disrupters for in depth studies. The results demonstrated that rhein inhibits NF-κB activation and inflammatory factor release. However, GW9662 significantly reduced this effect, indicating that PPAR-γ is a critical mediator in the rhein-mediated anti-inflammatory process. Additionally, positive modulation of PPAR-γ expression and activity by rosiglitazone correspondingly influenced the effects of rhein on inflammatory factors and NF-κB expression. We also found that rhein could enhance PPAR-γ, NF-κB, and histone deacetylase 3 (HDAC3) binding. These results indicate that rhein exerts its anti-inflammation function by regulating the PPAR-γ-NF-κB-HDAC3 axis.
Assuntos
Antraquinonas/farmacologia , Anti-Inflamatórios/farmacologia , Lipopolissacarídeos/farmacologia , NF-kappa B/antagonistas & inibidores , PPAR gama/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antraquinonas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Células RAW 264.7RESUMO
Sepsis is a life-threatening disease caused by infection. Inflammation is a key pathogenic process in sepsis. Paeonol, an active ingredient in moutan cortex (a Chinese herb), has many pharmacological activities, such as anti-inflammatory and antitumour actions. Previous studies have indicated that paeonol inhibits the expression of HMGB1 and the transcriptional activity of NF-κB. However, its underlying mechanism is still unknown. In this study, microarray assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results confirmed that paeonol could significantly up-regulate the expression of miR-339-5p in RAW264.7 cells stimulated by LPS. Dual-luciferase assays indicated that miR-339-5p interacted with the 3' untranslated region (3'-UTR) of HMGB1. Western blot, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analyses indicated that miR-339-5p mimic and siHMGB1 both negatively regulated the expression and secretion of inflammatory cytokines (e.g., HMGB1, IL-1ß and TNF-α) in LPS-induced RAW264.7 cells. Studies have confirmed that IKK-ß is targeted by miR-339-5p, and we further found that paeonol could inhibit IKK-ß expression. Positive mutual feedback between HMGB1 and IKK-ß was observed when we silenced HMGB1 or IKK-ß. These results indicated that paeonol could attenuate the inflammation mediated by HMGB1 and IKK-ß by upregulating miR-339-5p expression. In addition, we constructed CLP model mice by cecal ligation and puncture. Paeonol was used to intervene to investigate its anti-inflammatory effect in vivo. The results showed that paeonol could improve the survival rate of sepsis mice and protect the kidney of sepsis mice.
Assuntos
Acetofenonas/farmacologia , Proteína HMGB1/genética , Inflamação/tratamento farmacológico , MicroRNAs/genética , Sepse/tratamento farmacológico , Acetofenonas/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Camundongos , NF-kappa B/genética , Paeonia/química , Células RAW 264.7 , Sepse/genética , Sepse/patologiaRESUMO
Shenfu injection (SFI), a Chinese herbal medicine with substances extracted from Ginseng Radix et Rhizoma Rubra and Aconiti Lateralis Radix Praeparata, is widely used as an anti-inflammatory reagent to treat endotoxin shock in China. However, the mechanism of SFI in endotoxin shock remains to be illuminated. High mobility group box 1 (HMGB1), a vital inflammatory factor in the late stage of endotoxin shock, may stimulate multiple signalling cascades, including κB (NF-κB), a nuclear transcription factor, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-1ß, among others in the overexpression of downstream proinflammatory cytokines. An investigation into the effects of SFI on the inhibition of the HMGB1-NF-κB pathway revealed the contribution of SFI to acute lung injury (ALI) in a rat model of endotoxin shock. To assess the anti-inflammatory activity of SFI, 5 ml/kg, 10 ml/kg, or 15 ml/kg of SFI was administered to different groups of rats following an injection of LPS, and the mean arterial pressure (MAP) at 5 h and the survival rate at 72 h were measured. 24 h after LPS injection, we observed pathological changes in the lung tissue and measured the mRNA expression, production, translocation, and secretion of HMGB1, as well as the expression of the NF-κB signal pathway-related proteins inhibitor of NF-κB (IκB)-α, P50, and P65. We also evaluated the regulation of SFI on the secretion of inflammatory factors including interleukin-1 beta (IL-1ß) and TNF-α. SFI effectively prevented the drop in MAP, relieved lung tissue damage, and increased the survival rate in the endotoxin shock model in dose-dependent manner. SFI inhibited the transcription, expression, translocation, and secretion of HMGB1, increased the expression of toll-like receptor (TLR4), increased the production of IκB-α, and decreased the levels of P65, P50, and TNF-α in the lung tissue of endotoxin shock rats in a dose-dependent manner. Furthermore, SFI decreased the secretion of proinflammatory cytokines TNF-α and IL-1ß. In summary, SFI improves the survival rate of endotoxin shock, perhaps through inhibiting the HMGB1-NF-κB pathway and thus preventing cytokine storm.
RESUMO
High-mobility group box 1 (HMGB1) is a highly conserved DNA-binding nuclear protein that facilitates gene transcription and the DNA repair response. However, HMGB1 may be released by necrotic cells as well as activated monocytes and macrophages following stimulation with lipopolysaccharide (LPS), interleukin-1ß (IL-1ß), or tumor necrosis factor-α (TNF-α). Extracellular HMGB1 plays a critical role in the pathogenesis of acute lung injury (ALI) through activating the nuclear transcription factor κB (NF-κB) P65 pathway, thus, it may be a promising therapeutic target in shock-induced ALI. Paeonol (Pae) is the main active component of Paeonia suffruticosa, which has been used to inhibit the inflammatory response in traditional Chinese medicine. We have proven that Pae inhibits the expression, relocation and secretion of HMGB1 in vitro. However, the role of Pae in the HMGB1-NF-κB pathway remains unknown. We herein investigated the role of Pae in LPS-induced ALI rats. In this study, LPS induced a marked decrease in the mean arterial pressure (MAP) and survival rate (only 25% after 72â¯h), and induced severe pathological changes in the lung tissue of rats, which was accompanied by elevated expression of HMGB1 and its downstream protein NF-κB P65. Treatment with Pae significantly improved the survival rate (>60%) and MAP, and attenuated the pathological damage to the lung tissue in ALI rats. Western blotting revealed that Pae also inhibited the total expression of HMGB1, NF-κB P65 and TNF-α in the lung tissue of ALI rats. Moreover, Pae increased the expression of HMGB1 in the nucleus, inhibited the production of HMGB1 in the cytoplasm, and decreased the expression of P65 both in the nucleus and cytoplasm of lung tissue cells in LPS-induced ALI rats. The results were in agreement with those observed in the in vitro experiment. These findings indicate that Pae may be a potential treatment for ALI through its repression of the HMGB1-NF-κB P65 signaling pathway.
Assuntos
Acetofenonas/uso terapêutico , Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Proteína HMGB1/metabolismo , Pulmão/patologia , Medicina Tradicional Chinesa , Lesão Pulmonar Aguda/imunologia , Animais , Reparo do DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Proteína HMGB1/genética , Humanos , Lipopolissacarídeos/imunologia , Pulmão/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Sepsis is a life-threatening disease. Inflammation is a major concomitant symptom of sepsis Chrysophanol, an anthraquinone derivative isolated from the rhizomes of rheumpalmatum, has been reported to have a protective effect against lipopolysaccharide(LPS)-induced inflammation. However, the underlying molecular mechanisms are not well understood. The aim of this study was to explore the effect and mechanism of chrysophanol on lipopolysaccharide (LPS)-induced anti-inflammatory effect of RAW264.7 cells and its involved potential mechanism. The mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and inducible nitric oxide synthase (iNOS), nuclear factor kappa B (NF-κB) and PPAR-γ were measured by qRT-PCR and western blotting, the production of TNF-α, IL-1ß was evaluated by ELISA. Then, the phosphorylation of NF-κB p65 was also detected by western blotting. And NF-κB p65 promoter activity was analyzed by the Dual-Luciferase reporter assay system as well. Meanwhile, PPAR-γ inhibitor GW9662 was performed to knockdown PPAR-γ expression in cells. Our data revealed that LPS induced the up-regulation of TNF-α, IL-1ß, iNOS and NF-κB p65, the down-regulation of PPAR-γ were substantially suppressed by chrysophanol in RAW264.7 cells. Furthermore, our data also figured out that these effects of chrysophanol were largely abrogated by PPAR-γ inhibitor GW9662. Taken together, our results indicated that LPS-induced inflammation was potently compromised by chrysophanol very likely through the PPAR-γ-dependent inactivation of NF-κB in RAW264.7 cells.