RESUMO
Altered intestinal microbial composition promotes intestinal barrier dysfunction and triggers the initiation and recurrence of inflammatory bowel disease (IBD). Current treatments for IBD are focused on control of inflammation rather than on maintaining intestinal epithelial barrier function. Here, we show that the internalization of Gram-negative bacterial outer membrane vesicles (OMVs) in human intestinal epithelial cells promotes recruitment of caspase-5 and PIKfyve to early endosomal membranes via sorting nexin 10 (SNX10), resulting in LPS release from OMVs into the cytosol. Caspase-5 activated by cytosolic LPS leads to Lyn phosphorylation, which in turn promotes nuclear translocalization of Snail/Slug, downregulation of E-cadherin expression, and intestinal barrier dysfunction. SNX10 deletion or treatment with DC-SX029, a novel SNX10 inhibitor, rescues OMV-induced intestinal barrier dysfunction and ameliorates colitis in mice by blocking cytosolic LPS release, caspase-5 activation, and downstream signaling. Our results show that targeting SNX10 may be a new therapeutic approach for restoring intestinal epithelial barrier function and promising strategy for IBD treatment.
Assuntos
Membrana Externa Bacteriana/química , Caspases/metabolismo , Colite/patologia , Lipopolissacarídeos/metabolismo , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Animais , Células CACO-2 , Colite/induzido quimicamente , Colite/genética , Citosol/metabolismo , Modelos Animais de Doenças , Endossomos/metabolismo , Endossomos/transplante , Feminino , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismoRESUMO
The damage of integrated epithelial epithelium is a key pathogenic factor and closely associated with the recurrence of ulcerative colitis (UC). Here, we reported that vanillic acid (VA) exerted potent therapeutic effects on DSS-induced colitis by restoring intestinal epithelium homeostasis via the inhibition of ferroptosis. By the CETSA assay and DARTS assay, we identified carbonic anhydrase IX (CAIX, CA9) as the direct target of VA. The binding of VA to CA9 causes insulin-induced gene-2 (INSIG2) to interact with stromal interaction molecule 1 (STIM1), rather than SREBP cleavage-activating protein (SCAP), leading to the translocation of SCAP-SREBP1 from the endoplasmic reticulum (ER) to the Golgi apparatus for cleavage into mature SREBP1. The activation of SREBP1 induced by VA then significantly facilitated the transcription of stearoyl-CoA desaturase 1 (SCD1) to exert an inhibitory effect on ferroptosis. By inhibiting the excessive death of intestinal epithelial cells caused by ferroptosis, VA effectively preserved the integrity of intestinal barrier and prevented the progression of unresolved inflammation. In conclusion, our study demonstrated that VA could alleviate colitis by restoring intestinal epithelium homeostasis through CA9/STIM1-mediated inhibition of ferroptosis, providing a promising therapeutic candidate for UC.
Assuntos
Colite , Ferroptose , Humanos , Animais , Camundongos , Ácido Vanílico , Molécula 1 de Interação Estromal , Colite/induzido quimicamente , Colite/tratamento farmacológico , Homeostase , Mucosa Intestinal , Sulfato de Dextrana , Camundongos Endogâmicos C57BL , Anidrase Carbônica IX , Antígenos de Neoplasias , Proteínas de NeoplasiasRESUMO
Rhododendron principis leaves have been used as "Dama", a Traditional Tibetan Medicine for treating inflammatory diseases. R. principis crude polysaccharides with anticomplementary activity demonstrated promising anti-inflammatory effects on acute lung injury induced by lipopolysaccharide. R. principis crude polysaccharides significantly decreased the levels of TNF-α and interleukin-6 in both serum and blood and bronchoalveolar lavage fluid in lipopolysaccharide-induced acute lung injury mice by intragastric administration (100 mg/kg). A heteropolysaccharide, ZNDHP, was obtained from R. principis crude polysaccharides with successive anticomplementary activity-guided separation. ZNDHP was characterized as a branched neutral polysaccharide with a backbone composed of â 2)-ß-Glcp-(1â, â 2,6)-α-Glcp-(1â, â 6,3)-ß-Galp-(1â, â 2,6)-α-Galp-(1â, â 6,2)-ß-Glcp-(1â, â 4)-α-Glcp-(1â, â 5)-ß-Araf-(1â, â 3,5)-α-Araf-(1â, and â 4,6)-ß-Manp-(1â, and the backbone structure was further confirmed by partial acid hydrolysis. In addition to anticomplementary and antioxidant activities, ZNDHP exhibited potent anti-inflammatory activity by significantly inhibiting the secretion of nitric oxide, TNF-α, interleukin-6, and interleukin-1ß of lipopolysaccharide-treated RAW 264.7 cells. However, all of these activities decreased greatly after partially hydrolyzing, indicating the importance of the multibranched structure for its bioactivity. Therefore, ZNDHP might be an important component of R. principis for treating inflammation.
Assuntos
Rhododendron , Camundongos , Animais , Lipopolissacarídeos , Fator de Necrose Tumoral alfa , Interleucina-6 , Polissacarídeos/farmacologia , Polissacarídeos/química , Anti-Inflamatórios/farmacologiaRESUMO
RATIONALE: SNX10 (sorting nexin 10) has been reported to play a critical role in regulating macrophage function and lipid metabolism. OBJECTIVE: To investigate the precise role of SNX10 in atherosclerotic diseases and the underlying mechanisms. METHODS AND RESULTS: SNX10 expression was compared between human healthy vessels and carotid atherosclerotic plaques. Myeloid cell-specific SNX10 knockdown mice were crossed onto the APOE-/- (apolipoprotein E) background and atherogenesis (high-cholesterol diet-induced) was monitored for 16 weeks. We found that SNX10 expression was increased in atherosclerotic lesions of aortic specimens from humans and APOE-/- mice. Myeloid cell-specific SNX10 deficiency (Δ knockout [KO]) attenuated atherosclerosis progression in APOE-/- mice. The population of anti-inflammatory monocytes/macrophages was increased in the peripheral blood and atherosclerotic lesions of ΔKO mice. In vitro experiments showed that SNX10 deficiency-inhibited foam cell formation through interrupting the internalization of CD36, which requires the interaction of SNX10 and Lyn-AKT (protein kinase B). The reduced Lyn-AKT activation by SNX10 deficiency promoted the nuclear translocation of TFEB (transcription factor EB), thereby enhanced lysosomal biogenesis and LAL (lysosomal acid lipase) activity, resulting in an increase of free fatty acids to fuel mitochondrial fatty acid oxidation. This further promoted the reprogramming of macrophages and shifted toward the anti-inflammatory phenotype. CONCLUSIONS: Our data demonstrate for the first time that SNX10 plays a crucial role in diet-induced atherogenesis via the previously unknown link between the Lyn-Akt-TFEB signaling pathway and macrophage reprogramming, suggest that SNX10 may be a potentially promising therapeutic target for atherosclerosis treatment.
Assuntos
Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Reprogramação Celular/fisiologia , Macrófagos/fisiologia , Nexinas de Classificação/fisiologia , Animais , Apolipoproteínas E/genética , Aterosclerose/sangue , Aterosclerose/patologia , Antígenos CD36/metabolismo , Núcleo Celular/metabolismo , Progressão da Doença , Ácidos Graxos não Esterificados/metabolismo , Células Espumosas/citologia , Humanos , Lisossomos/fisiologia , Macrófagos/citologia , Camundongos , Mitocôndrias/metabolismo , Monócitos/citologia , Oxirredução , Proteínas Proto-Oncogênicas c-akt/metabolismo , Nexinas de Classificação/deficiência , Nexinas de Classificação/genética , Esterol Esterase/metabolismoRESUMO
The pathological features of inflammatory bowel disease necessitate therapeutic strategies aimed at restoring intestinal mucosal barrier function in addition to controlling inflammation. Paeoniflorin, a bioactive herbal constituent isolated from the root of Paeonia albiflora Pall, has been reported to protect against acute colitis in mice. However, the direct molecular target of paeoniflorin in preventing colitis remains elusive. Here, we evaluated the therapeutical effects of Paeoniflorin using IL-10-/- chronic colitis model, and explored the precise mechanism of action involved. Our results demonstrated that intragastric administration of Paeoniflorin significantly ameliorated inflammatory response and restored the aberrant intestinal proliferation and differentiation in IL-10-/-colitis mice. By utilizing a chemical biology approach, we identified C1qa, a crucial component of C1q, is the direct target of Paeoniflorin. Binding of Paeoniflorin to C1qa prevented the cleavage of C1q on macrophages, resulting in the aggregation of surface membrane-anchored C1q and the diminished C1q secretion. The excessive surface membrane-anchored C1q significantly enhanced the phagocytic capability of macrophages and promoted the elimination of infiltrated bacteria and inflammatory cells in mouse colon. The reduced C1q secretion conferred by Paeoniflorin dampened Wnt/ß-catenin signaling activation, thereby rectifying the aberrant proliferation and differentiation of intestinal stem cells (ISCs). In summary, our study demonstrates that Paeoniflorin can orchestrate mucosal healing and intestinal inflammation elimination through C1q-bridged macrophage-ISCs crosstalk, highlighting a novel strategy to treat chronic colitis by restoring mucosal homeostasis via targeting C1q.
Assuntos
Colite , Interleucina-10 , Animais , Proliferação de Células , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Complemento C1q/metabolismo , Complemento C1q/uso terapêutico , Sulfato de Dextrana , Modelos Animais de Doenças , Glucosídeos , Inflamação/metabolismo , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monoterpenos , Células-Tronco/metabolismoRESUMO
Sorting nexin 10 (SNX10) has been reported as a critical regulator in macrophage function, and germline SNX10 knockout effectively alleviated mouse colitis. Here, we investigated the precise role of SNX10 in inflammatory responses in macrophages in mouse colitis, and explored the druggability of SNX10 as a therapeutic target for inflammatory bowel disease (IBD). Our results revealed that myeloid-specific SNX10 deletion alleviated inflammation and pathological damage induced by dextran sulfate sodium (DSS). In vitro experiments showed that SNX10 deletion contributed to inflammation elimination by inhibiting PIKfyve-mediated TANK-binding kinase 1 (TBK1) /c-Rel signaling activation. Further study provided rational mechanism that SNX10 was required for the recruitment of PIKfyve to the TRIF-positive endosomes, through which PIKfyve activated TBK1/c-Rel for LPS-induced inflammation response. Based on the structure of SNX10, we discovered a new small-molecule inhibitor DC-SX029, which targeted SNX10 to block the SNX10-PIKfyve interaction, thereby decreased the TBK1/c-Rel signaling activation. Additionally, therapeutic efficiency of DC-SX029 was evaluated in both DSS-induced and IL10-deficient mouse colitis models. Our data demonstrate a new mechanism by which SNX10-PIKfyve interaction regulates LPS-induced inflammation response in macrophages via the TBK1/c-Rel signaling pathway. In vivo and in vitro pharmacological studies of SNX10 protein-protein interaction (PPI) inhibitor DC-SX029 demonstrate the feasibility of targeting SNX10 in IBD treatment.
Assuntos
Anti-Inflamatórios/uso terapêutico , Colite/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Transdução de Sinais/efeitos dos fármacos , Nexinas de Classificação/efeitos dos fármacos , Animais , Colite/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nexinas de Classificação/metabolismoRESUMO
BACKGROUND & AIMS: Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. However, the cellular defense mechanisms underlying ALD are not well understood. Recent studies highlighted the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Sorting nexin (SNX)-10 has a regulatory function in endolysosomal trafficking and stabilisation. Here, we investigated the roles of SNX10 in CMA activation and in the pathogenesis of alcohol-induced liver injury and steatosis. METHODS: Snx10 knockout (Snx10 KO) mice and their wild-type (WT) littermates fed either the Lieber-DeCarli liquid alcohol diet or a control liquid diet, and primary cultured WT and Snx10 KO hepatocytes stimulated with ethanol, were used as in vivo and in vitro ALD models, respectively. Activation of CMA, liver injury parameters, inflammatory cytokines, oxidative stress and lipid metabolism were measured. RESULTS: Compared with WT littermates, Snx10 KO mice exhibited a significant amelioration in ethanol-induced liver injury and hepatic steatosis. Both in vivo and in vitro studies showed that SNX10 deficiency upregulated lysosome-associated membrane protein type 2A (LAMP-2A) expression and CMA activation, which could be reversed by SNX10 overexpression in vitro. LAMP-2A interference confirmed that the upregulation of Nrf2 and AMPK signalling pathways induced by SNX10 deficiency relied on CMA activation. Pull-down assays revealed an interaction between SNX10 and cathepsin A (CTSA), a key enzyme involved in LAMP-2A degradation. Deficiency in SNX10 inhibited CTSA maturation and increased the stability of LAMP-2A, resulting in an increase in CMA activity. CONCLUSIONS: SNX10 controls CMA activity by mediating CTSA maturation, and, thus, has an essential role in alcohol-induced liver injury and steatosis. Our results provide evidence for SNX10 as a potential promising therapeutic target for preventing or ameliorating liver injury in ALD. LAY SUMMARY: Alcoholic liver disease is a major cause of morbidity and mortality worldwide. Recent studies highlight the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Our study reveals that deficiency of sorting nexin (SNX) 10 increases the stability of LAMP-2A by inhibiting cathepsin A maturation, resulting in the increase of CMA activity and, thus, alleviates alcohol-induced liver injury and steatosis.
Assuntos
Fígado Gorduroso/genética , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hepatopatias Alcoólicas/complicações , Estresse Oxidativo , RNA/genética , Nexinas de Classificação/genética , Animais , Autofagia , Western Blotting , Modelos Animais de Doenças , Etanol , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Hepatócitos/patologia , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Nexinas de Classificação/biossínteseRESUMO
BACKGROUND: Metastasis driven by epithelial-mesenchymal transition (EMT) remains a significant contributor to the poor prognosis of colorectal cancer (CRC), and requires more effective interventions. GPR81 signaling has been linked to tumor metastasis, while lacks an efficient specific inhibitor. PURPOSE: Our study aimed to investigate the effect and mechanism of Gentisic acid on colorectal cancer (CRC) metastasis. STUDY DESIGN: A lung metastasis mouse model induced by tail vein injection and a subcutaneous graft tumor model were used. Gentisic acid (GA) was administered by an intraperitoneal injection. HCT116 was treated with lactate to establish an in vitro model. METHODS: MC38 cells with mCherry fluorescent protein were injected into tail vein to investigate lung metastasis ability in vivo. GA was administered by intraperitoneal injection for 3 weeks. The therapeutic effect was evaluated by survival rates, histochemical analysis, RT-qPCR and live imaging. The mechanism was explored using small interfering RNA (siRNA), Western blotting, RT-qPCR and immunofluorescence. RESULTS: GA had a therapeutic effect on CRC metastasis and improved survival rates and pathological changes in dose-dependent manner. GA emerged as an GPR81 inhibitor, effectively suppressed EMT and mTOR signaling in CRC induced by lactate both in vivo and in vitro. Mechanistically, GA halted lactate-induce degradation of DEPDC5 through impeding the activation of Chaperone-mediated autophagy (CMA). CONCLUSION: CMA-mediated DEPDC5 degradation is crucial for lactate/GPR81-induced CRC metastasis, and GA may be a promising candidate for metastasis by inhibiting GPR81 signaling.
Assuntos
Neoplasias Colorretais , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Receptores Acoplados a Proteínas G , Animais , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Humanos , Camundongos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/tratamento farmacológico , Células HCT116 , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Masculino , Serina-Treonina Quinases TOR/metabolismoRESUMO
Three analogues of aristololactam â (AL â ), AL Aâ ¡, AL Fâ and AL Bâ ¡, had been isolated from Houttuynia cordata, a commonly used medicinal and edible plant with heat-removing and toxin-removing functions. Considering the significant nephrotoxicity of AL â , this study evaluated the toxicity of these three aristololactams (ALs) on human proximal tubular epithelial cells (HK-2) by MTT assay, ROS assay, ELISA tests and cytologic morphology observation. Furthermore, the distribution of the three ALs in H. cordata were investigated by UPLC-MSn recognition and quantitation in SIM mode, so as to estimate primarily the safety of the plant. The results showed that all the three ALs in H. cordata had comparative cytotoxicity as AL I with the IC50 values from 3.88 µM to 20.63 µM, caused high levels of cellular levels of reactive oxygen species (ROS) in HK-2 cells, exhibited the potential to cause renal fibrosis by remarkably increasing the levels of transforming growth factor-ß (TGF-ß1) and fibronectin (FN), and induced fibrous changes in morphology of HK-2 cells. The contents of the three ALs varied significantly in 30 batches of H. cordata from different regions and parts. Overall, the aerial part contained much more ALs (3.20 - 108.19 µg/g) than the underground part (0.95 - 11.66 µg/g), and flowers had the highest contents. Besides, no ALs were detected in the water extract of any part of H. cordata. This work revealed that the aristololactams in H. cordata had similar in vitro nephrotoxicity as AL â and were mainly distributed in the aerial part of the plant.
Assuntos
Houttuynia , Humanos , Espécies Reativas de Oxigênio , Cromatografia Líquida , Espectrometria de Massas em Tandem , Extratos Vegetais/farmacologiaRESUMO
Intestinal stem cell (ISC) is a promising therapeutic target for inflammatory bowel disease. Cholesterol availability is critical for ISC stemness. Low plasma cholesterol is a typical feature of Crohn's disease (CD); however, its impact on mucosal healing remains unclear. Here, we identified an essential role of sorting nexin 10 (SNX10) in maintaining the stemness of ISCs. SNX10 expression in intestinal tissues positively correlates with the severity of human CD and mouse colitis. Conditional SNX10 knockout in intestinal epithelial cells or ISCs promotes intestinal mucosal repair by maintaining the ISC population associated with increased intracellular cholesterol synthesis. Disassociation of ERLIN2 with SCAP by SNX10 deletion enhances the activation of SREBP2, resulting in increased cholesterol biosynthesis. DC-SX029, a small-molecule inhibitor of SNX10, was used to verify the druggable potential of SNX10 for the treatment of patients with CD. Our study provides a strategy for mucosal healing through SREBP2-mediated stemness restoration of ISCs.
Assuntos
Doenças Inflamatórias Intestinais , Nexinas de Classificação , Animais , Humanos , Camundongos , Mucosa Intestinal , Intestinos , Nexinas de Classificação/genética , Células-TroncoRESUMO
OBJECTIVE: To investigate the therapeutic effect of Yixin Ningshen Tablet (YXNS) on comorbidity of myocardial infarction (MI) and depression in rats and explore the underlying mechanism. METHODS: The Sprague-Dawley rats were randomly divided into 5 groups with 7 rats in each group according to their weights, including control, model, fluoxetine (FLXT, 10 mg/kg), low-dose YXNS (LYXNS, 100 mg/kg), and high-dose YXNS (HYXNS, 300 mg/kg) groups. All rats were pretreated with corresponding drugs for 12 weeks. The rat model of MI and depression was constructed by ligation of left anterior descending coronary artery and chronic mild stress stimulation. The echocardiography, sucrose preference test, open field test, and forced swim test were performed. Myocardial infarction (MI) area and myocardial apoptosis was also detected. Serum levels of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT), adrenocorticotrophic hormone (ACTH), corticosterone (CORT), and norepinephrine (NE) were determined by enzyme linked immunosorbent assay. The proteins of adenosine 5'-monophosphate -activated protein kinase (AMPK), p-AMPK, peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), and nuclear respiratory factor 1 (NRF1) in heart were detected by Western blot analysis. The expression levels of TNF-α, IL-6, indoleamine 2,3-dioxygenase (IDO1), kynurenine 3-monooxygenase (KMO), and kynureninase (KYNU) in hippocampus were detected by real-time quantitative polymerase chain reaction. RESULTS: Compared with the model group, the cardiac function of rats treated with YXNS improved significantly (P<0.01). Meanwhile, YXNS effectively reduced MI size and cardiomyocytes apoptosis of rats (P<0.01 or P<0.05), promoted AMPK phosphorylation, and increased PGC-1α protein expression (P<0.01 or P<0.05). HYXNS significantly increased locomotor activity of rats, decreased the levels of TNF-α, IL-6 and IL-1ß, and increased the serum levels of 5-HT, NE, ACTH, and CORT (all P<0.05). Moreover, HYXNS decreased the mRNA expressions of IDO1, KMO and KYNU (P<0.05). CONCLUSIONS: YXNS can relieve MI by enhancing myocardial energy metabolism. Meanwhile, YXNS can alleviate depression by resisting inflammation and increasing availability of monoamine neurotransmitters. It may be used as a potential drug to treat comorbidity of MI and depression.
Assuntos
Infarto do Miocárdio , Fator de Necrose Tumoral alfa , Proteínas Quinases Ativadas por AMP/metabolismo , Hormônio Adrenocorticotrópico , Animais , Comorbidade , Depressão/complicações , Depressão/tratamento farmacológico , Metabolismo Energético , Interleucina-6/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/patologia , Neurotransmissores , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Comprimidos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Our previous study found that deletion of Sorting nexin 10 (SNX10) can protect against colonic inflammation and pathological damage induced by dextran sulfate sodium (DSS). This inspired us that modulation of SNX10 expression in colonic epithelial cells might represent a promising therapeutic strategy for inflammatory bowel disease (IBD). METHODS: Effective delivery of siRNA/shRNA to silence genes is a highly sought-after means in the treatment of multiple diseases. Here, we encapsulated SNX10-shRNA plasmids (SRP) with polylactide-polyglycolide (PLGA) to make oral nanoparticles (NPs), and then applied them to acute and chronic IBD mice model, respectively. The characteristics of the nanoparticles were assayed and the effects of SRP-NPs on mouse IBD were evaluated. RESULTS: High-efficiency SNX10-shRNA plasmids were successfully constructed and coated with PLGA to obtain nanoparticles, with a particle size of 275.2 ± 11.4mm, uniform PDI distribution, entrapment efficiency of 87.6 ± 2.5%, and drug loading of 13.11 ± 1.38%, displayed dominant efficiency of SNX10 RNA interference in the colon. In both acute and chronic IBD models, SRP-NPs could effectively reduce the loss of mice body weight, relieve the intestinal mucosal damage and inflammatory infiltration, inhibit the expression of inflammatory cytokines IL-1ß, IL-23, TNF-α, and down-regulate the expression of toll-like receptors (TLRs) 2 and 4. CONCLUSION: Oral nanoparticles of SNX10-shRNA plasmid displayed dominant efficiency of SNX10 RNA interference in the colon and ameliorate mouse colitis via TLR signaling pathway. SNX10 is a new target for IBD treatment and nanoparticles of SNX10-shRNA plasmid might be a promising treatment option for IBD.
Assuntos
Colite/terapia , Portadores de Fármacos/química , Terapia Genética/métodos , Nanopartículas/química , Plasmídeos/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Administração Oral , Animais , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Mucosa Intestinal/metabolismo , Camundongos , Interferência de RNA , RNA Interferente Pequeno/química , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Inflammatory bowel disease (IBD) is an autoimmune disease. The pathogenesis of IBD is complicated and intestinal mucosal barrier damage is considered as the trigger factor for the initiation and recurrence of IBD. Total Glucosides of Paeony (TGP) has shown good inhibitory effects on immune-inflammation in clinic studies. However, its effect and mechanism on IBD are largely unknown. PURPOSE: The purpose of this study is to evaluate the effect and mechanism of TGP on IBD. STUDY DESIGN: DSS-induced colitis mouse model was used. TGP was given by gavage. Caco-2 cells were stimulated by outer membrane vesicles (OMV) to establish an in vitro model. METHODS: C57BL/6 mice were divided into normal control group, model group, mesalazine group, paeoniflorin (PA) group, high-dose group of TGP, and low-dose group of TGP. The model was induced with 2.5% DSS for 7 days, and TGP was intragastrically administered for 10 days. The therapeutic effect of TGP was evaluated by symptoms, histochemical analysis, RT-qPCR and ELISA. The mechanism was explored by intestinal permeability, Western blot and immunofluorescence in vivo and in vitro. RESULTS: Our results showed that TGP could significantly improve the symptoms and pathological changes, with reduced levels of TNF-α, IL-17A, IL-23 and IFN-γ in the colon tissues and serum under a dose-dependent manner. TGP also reduced the intestinal permeability and restored the protein expression of tight junction and adherens junction proteins of intestinal epithelial cells in vivo and in vitro. Furthermore, TGP could inhibit the expression of p-Lyn and Snail and prevent Snail nuclear localization, thereby maintaining tight and adherens junctions. CONCLUSION: TGP effectively improves the symptoms of DSS-induced colitis in mice, protects the intestinal epithelial barrier by inhibiting the Lyn/Snail signaling pathway, and maybe a promise therapeutic agent for IBD treatment.
Assuntos
Colite/tratamento farmacológico , Glucosídeos/farmacologia , Paeonia/química , Quinases da Família src/metabolismo , Animais , Células CACO-2 , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Glucosídeos/química , Humanos , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Monoterpenos/farmacologia , Permeabilidade , Fatores de Transcrição da Família Snail/metabolismo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Most cardiovascular diseases ultimately result in heart failure, an intractable problem in modern medicine. Yangxinshi tablet (YXS) is a Chinese medicine formula that is used clinically to treat coronary heart disease. However, the active compounds, potential targets, and pharmacological and molecular mechanism of its anti-heart failure activity remain unclear. Therefore, further investigation is required. AIM OF STUDY: Active ingredients and potential targets of YXS for treating heart failure have been reported previously. However, the molecular functions or biological processes of YXS in energy metabolism have not been discovered. To date, no experimental study to validate the potential anti-heart failure mechanism of YXS. The aim of this study was to study the therapeutic effect of YXS on rats with chronic ischemic heart failure by evaluating rat cardiac function and exercise tolerance, and to explore its potential mechanism by network pharmacology, western blotting, quantitative RT-PCR and histological analysis. MATERIALS AND METHODS: In this investigation, chronic ischemic heart failure rats were randomly assigned to five groups: control group (sham operation), model group (0.5% CMC-Na), trimetazidine group (positive control) and two YXS groups (low- and high-dose groups). Experimental rats were treated by gavage with 10â¯mg/kg/d (clinical equivalent dose) trimetazidine (TMZ), 500â¯mg/kg/d (clinical equivalent dose) YXS and 1000â¯mg/kg/d YXS, respectively, for 5 weeks. The cardiac functions of rats were detected by High-Resolution In Vivo Imaging System. We elucidated novel understanding of the active compounds of YXS in rat plasma and predicted the energy metabolism related targets and processes for heart failure. Then, we validated experimentally the targets and mechanism of YXS on these pathological processes in vivo. RESULTS: It was found that YXS was able to effectively improve cardiac LVIDs, LVEDV, LVESV and EF, decrease myocardial oxygen consumption and reduce myocardial infarct size in rats with chronic ischemic heart failure was similar to that of TMZ. We identified 63 major candidate targets for YXS that are closely to heart failure progression. Enrichment analysis revealed key targets for YXS associated to oxygen delivery, glucose utilization, and mitochondrial biogenesis. Meanwhile, we validated that YXS could promote the expression of downstream HIF-1α, PGC1α and GLUT4 by increasing phosphorylation of PI3K, Akt, mTOR, rpS6 and AMPK. The results show that YXS could activate related PI3K/Akt/mTOR/rpS6/HIF-1α and AMPK/PGC1α/GLUT4 signaling pathways in chronic ischemic heart failure rats. Further experiments demonstrated that YXS increased mitochondrial biogenesis in chronic ischemic heart failure rats and improved exercise tolerance CONCLUSION: YXS treated chronic ischemic heart failure through activating its targets which play pivotal roles in oxygen delivery, glucose utilization and mitochondrial biogenesis to improve energy metabolism through a multi-component, multi-level, multi-target, multi-pathway and multi-mechanism approaches.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Metabolismo Energético/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Fitoterapia , Ratos , Ratos Sprague-DawleyRESUMO
Sepsis is caused by a dysregulated host inflammatory response to serious infections resulting in life-threatening organ dysfunction. The high morbidity and mortality make sepsis still a major clinical problem. Here, we investigated the roles of Brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) in the pathogenesis process of sepsis and the underlying mechanisms. We found myeloid cell-specific BIG1 knockout (BIG1 cKO) significantly reduced the mortality and organ damage in LPS-induced and CLP-induced polymicrobial sepsis mouse model. The serum concentration and mRNA expression of pro-inflammatory cytokines including TNF-α, IL-6, IL-1ß, and IL-12 were obviously decreased in BIG1 cKO mice. In bone marrow-derived macrophages or THP-1 cells, BIG1 deficiency caused an inhibited ARF3 activation, which reduced PI(4,5)P2 synthesis and the recruitment of TIRAP to the plasma membrane through inhibiting the activation of PIP5K induced by LPS, and eventually resulted in the inhibitory activity of TLR4-MyD88 signaling pathway. These results reveal a crucial new role of BIG1 in regulating macrophage inflammation responses, and provide evidence for BIG1 as a potential promising therapeutic target in sepsis.
Assuntos
Citocinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Fatores de Ribosilação do ADP/farmacologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Lipopolissacarídeos/metabolismo , Transporte Proteico , Sepse/tratamento farmacológico , Sepse/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The non-receptor tyrosine kinase SRC is a key mediator of cellular protumorigenic signals. SRC is aberrantly over-expressed and activated in more than 80% of colorectal cancer (CRC) patients, therefore regulation of its stability and activity is essential. Here, we report a significant down regulation of SNX10 (sorting nexin 10) in human CRC tissues, which is closely related to tumor differentiation, TNM stage, lymph node metastasis and survival period. SNX10 deficiency in normal and neoplastic colorectal epithelial cells promotes initiation and progression of CRC in mice. SNX10 controls SRC levels by mediating autophagosome-lysosome fusion and SRC recruitment for autophagic degradation. These mechanisms ensure proper controlling of the activities of SRC-STAT3 and SRC-CTNNB1 signaling pathways by up-regulating SNX10 expression under stress conditions. These findings suggest that SNX10 acts as a tumor suppressor in CRC and it could be a potential therapeutic target for future development.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG12: autophagy related 12; CQ: chloroquine; CRC: colorectal cancer; CTNNB1: catenin beta 1; EBSS: Earle's balanced salt solution; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; mRNA: messenger RNA; PX: phox homology; RT-qPCR: real time quantitative polymerase chain reaction; siRNA: small interfering RNA; SNX10: sorting nexin 10; SQSTM1: sequestosome 1; SRC: SRC proto-oncogene, non-receptor tyrosine kinase; STAT3: signal transducer and activator of transcription 3; WT: wild type.
Assuntos
Autofagia/fisiologia , Neoplasias Colorretais/metabolismo , Nexinas de Classificação/metabolismo , Animais , Autofagia/genética , Movimento Celular/fisiologia , Neoplasias Colorretais/patologia , Regulação para Baixo , Humanos , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proto-Oncogene Mas , Nexinas de Classificação/deficiência , Nexinas de Classificação/genética , Regulação para CimaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Wang-bi tablet (WB) consists of 17 traditional Chinese medicines and has been used for treating rheumatoid arthritis (RA) in China for many years, however, its pharmacologic mechanism is not clear. AIM OF STUDY: The aim of this study was to investigate the therapeutic effect of WB on collagen-induced mouse arthritis and explored the underlying mechanism. MATERIALS AND METHODS: DBA/1 mice were used to establish a type II collagen-induced arthritis (CIA) model. From the day of arthritis onset, mice were treated daily by gavage with either total glucosides of paeony (TGP, 0.37â¯â¯g/kg/d) or WB at a lower (1.11â¯â¯g/kg/d, WBL) or higher dose of (3.33â¯â¯g/kg/d, WBH) for 8 weeks. The severity of arthritis, levels of cytokines and the activation of signaling pathways were determined. RESULTS: Our results revealed that WB treatment effectively alleviated inflammatory symptoms and prevented bone erosions and joint destructions. It obviously decreased the serum concentration of pro-inflammatory cytokines TNF-α, IL-6 and IL-17α, while increased the concentration of anti-inflammatory cytokine IL-10. Interestingly, the proportion of splenic Treg cells were increased significantly. In vitro experiments showed that WB inhibited the differentiation of osteoclasts. Consistently, the mRNA levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK), and the activation of NF-κB and JAK-STAT3 signaling pathways in the paws of CIA mice were inhibited by WB treatment. On the other hand, up-regulation of osteogenic genes Runx2, Osterix mRNA, and activation of Wnt/ß-catenin signaling pathway along with a decreased receptor activator of nuclear factor κB ligand (RANKL) expression were found in WB treated mice. CONCLUSION: Our results suggest that the therapeutic effect of Wang-bi tablet could be attributed to its inhibitory activity on NF-κB and STAT3 signaling pathway-mediated osteoclast differentiation, and its enhancement on Wnt/ß-catenin signaling pathway-mediated osteoblast functions.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Citocinas/imunologia , Medicamentos de Ervas Chinesas/farmacologia , Articulações/efeitos dos fármacos , Articulações/patologia , Masculino , Medicina Tradicional Chinesa , Camundongos Endogâmicos DBA , NF-kappa B/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Ligante RANK/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Chaperone-mediated autophagy (CMA) characterized by the selective degradation of target proteins has been linked with tumorigenesis in recent years. Here, we explored the function of sorting nexin 10 (SNX10), a protein involved in maintaining endosome/lysosome homeostasis, in mediating CMA activity and its impact on the progression of mouse inflammation-driven colorectal cancer. Our results revealed that SNX10 deficiency increased the activation of CMA by preventing the degradation of lysosomal LAMP-2A. In SNX10 KO cells, we disclosed that p21Cip1/WAF1, a master effector in various tumor suppressor pathways, is a substrate of CMA, and decrease of p21Cip1/WAF1 caused by SNX10-mediated CMA activation contributes to HCT116â¯cell proliferation and survival. Moreover, we found that SNX10 KO promoted tumorigenesis in the mouse colorectum which could be restored by SNX10 over-expression. Furthermore, SNX10 was remarkably down-regulated in human CRC tissues which showed the increased activity of CMA and decreased expression of p21Cip1/WAF1. These findings suggest that SNX10 acts as a tumor suppressor in the mouse colorectum and drives inflammation-associated colorectal cancer by a chaperone-mediated autophagy mechanism.
Assuntos
Autofagia , Carcinogênese/metabolismo , Neoplasias Colorretais/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Nexinas de Classificação/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Carcinogênese/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Progressão da Doença , Células HCT116 , Humanos , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Interferência de RNA , Nexinas de Classificação/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Rheumatoid arthritis (RA) severely affects the quality life of patients due to its high association with disability. Traditional Chinese medicines have been reported to exert notable therapeutic effects on RA. The Chinese medicinal prescription WangBi Tablet (WB) has been successfully used to clinically treat RA for many years; however, its pharmacological mechanism of action is largely unclear. In the present study, adjuvantinduced arthritis (AIA) rats were used to evaluate the antiinflammatory effects of WB and western blotting was used to explore the molecular mechanisms. The experimental results demonstrated that WB treatment significantly reduced arthritis score and hindpaw volume. Furthermore, synovial hyperplasia, inflammatory cell infiltration and joint destruction were ameliorated by WB. The expression levels of the proinflammatory cytokines interleukin (IL)1ß, tumor necrosis factorα and IL6, were reduced in the joints of WBtreated rats. Western blotting revealed that WB could also inhibit excessive activation of nuclear factor (NF)κB and Janus kinase (JAK)signal transducer and activator of transcription 3 (STAT3) signaling pathways. These results indicated that the therapeutic effects of WB on AIA may be accomplished through inhibition of the NFκB and JAKSTAT3 signaling pathways. These findings provide experimental evidence to support WB as a therapeutic agent for the treatment of patients with RA.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Medicamentos de Ervas Chinesas/uso terapêutico , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Animais , Artrite Experimental/patologia , Citocinas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Mediadores da Inflamação/metabolismo , Articulações/efeitos dos fármacos , Articulações/patologia , Lipopolissacarídeos , Masculino , Camundongos , Substâncias Protetoras/farmacologia , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Jiang-Xian HuGan (JXHG) formulated by five natural products including Freshwater clam (Corbicula fluminea), Curcuma longa L., Ligustrum lucidum, Eclipta prostrata (L.) L. and Paeonia lactiflora Pall., has exhibited a great hepatoprotective effect. AIM OF THIS STUDY: We investigated the effect of JXHG on concanavalin A (ConA)-induced acute live injury in mice, and to elucidate its underlying molecular mechanisms. MATERIALS AND METHODS: Jiangkanling Capsule (900â¯mg/kg), low-dose JXHG (LJXHG, 700â¯mg/kg), high-dose JXHG (HJXHG, 1400â¯mg/kg) were administered to mice by oral gavage daily for 20 days prior to a single intravenous injection of ConA (20â¯mg/kg). Liver injury was evaluated by measuring the serum levels of enzymes and cytokines as well as liver histological analysis. We also measured the hepatic expression of cytokines at mRNA levels and the proteins related to NF-κB and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathways. RESULT: Our results showed that JXHG pretreatment significantly alleviated ConA-induced live injury as evidenced by decreased serum levels of glutamic-pyruvic transaminase (ALT) and glutamic oxalacetic transaminase (AST), and reduced hepatocyte apoptosis and mortality. Furthermore, JXHG was able to significantly reduce the serum levels of proinflammatory cytokines, down-regulate the mRNA expression of interleukin-6 (IL-6) and interferon-γ (IFN-γ), and up-regulate IL-10 as well as superoxide-dimutase-1 (SOD1), glutathione reductase (GSR) and Glutathione peroxidase 2 (GPX2) mRNA in the liver tissues after Con A injection. In addition, JXHG pretreatment dramatically suppressed the phosphorylation of NF-κB p65 (p65), increased Nrf2 expression, and decreased the expression ratio of cleaved caspase-3/caspase-3 in liver tissues. CONCLUSION: These results suggest that JXHG protects against ConA-induced acute live injury through inhibiting NF-κB mediated inflammatory pathway and promoting Nrf2 mediated anti-oxidative stress signaling pathway.