RESUMO
In reconstructive and plastic surgery, random-pattern skin flaps (RPSF) are often used to correct defects. However, their clinical usefulness is limited due to their susceptibility to necrosis, especially on the distal side of the RPSF. This study validates the protective effect of celastrol (CEL) on flap viability and explores in terms of underlying mechanisms of action. The viability of different groups of RPSF was evaluated by survival zone analysis, laser doppler blood flow, and histological analysis. The effects of CEL on flap angiogenesis, apoptosis, oxidative stress, and autophagy were evaluated by Western blot, immunohistochemistry, and immunofluorescence assays. Finally, its mechanistic aspects were explored by autophagy inhibitor and Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) inhibitor. On the seventh day after surgery, the survival area size, blood supply, and microvessel count of RPSF were augmented following the administration of CEL. Additionally, CEL stimulated angiogenesis, suppressed apoptosis, and lowered oxidative stress levels immediately after elevated autophagy in ischemic regions; These effects can be reversed using the autophagy inhibitor chloroquine (CQ). Specifically, CQ has been observed to counteract the protective impact of CEL on the RPSF. Moreover, it has also been discovered that CEL triggers the AMPK-mTOR-TFEB axis activation in the area affected by ischemia. In CEL-treated skin flaps, AMPK inhibitors were demonstrated to suppress the AMPK-mTOR-TFEB axis and reduce autophagy levels. This investigation suggests that CEL benefits the survival of RPSF by augmenting angiogenesis and impeding oxidative stress and apoptosis. The results are credited to increased autophagy, made possible by the AMPK-mTOR-TFEB axis activation.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Triterpenos Pentacíclicos , Serina-Treonina Quinases TOR , Autofagia/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Triterpenos Pentacíclicos/farmacologia , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Masculino , Retalhos Cirúrgicos/irrigação sanguínea , Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Camundongos , Triterpenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacosRESUMO
OBJECTIVE: To elucidate the role of LRRK2 in intervertebral disc degeneration (IDD) as well as its mitophagy regulation mechanism. METHODS: The expression of LRRK2 in human degenerative nucleus pulposus tissues as well as in oxidative stress-induced rat nucleus pulposus cells (NPCs) was detected by western blot. LRRK2 was knocked down in NPCs by lentivirus (LV)-shLRRK2 transfection; apoptosis and mitophagy were assessed by western blot, TUNEL assay, immunofluorescence staining and mitophagy detection assay in LRRK2-deficient NPCs under oxidative stress. After knockdown of Parkin in NPCs with siRNA transfection, apoptosis and mitophagy were further assessed. In puncture-induced rat IDD model, X-ray, MRI, hematoxylin-eosin (HE) and Safranin O-Fast green (SO) staining were performed to evaluate the therapeutic effects of LV-shLRRK2 on IDD. RESULTS: We found that the expression of LRRK2 was increased in degenerative NPCs both in vivo and in vitro. LRRK2 deficiency significantly suppressed oxidative stress-induced mitochondria-dependent apoptosis in NPCs; meanwhile, mitophagy was promoted. However, these effects were abolished by the mitophagy inhibitor, suggesting the effect of LRRK2 on apoptosis in NPCs is mitophagy-dependent. Furthermore, Parkin knockdown study showed that LRRK2 deficiency activated mitophagy by recruiting Parkin. In vivo study demonstrated that LRRK2 inhibition ameliorated IDD in rats. CONCLUSIONS: The results revealed that LRRK2 is involved in the pathogenesis of IDD, while knockdown of LRRK2 inhibits oxidative stress-induced apoptosis through mitophagy. Thus, inhibition of LRRK2 may be a promising therapeutic strategy for IDD.
Assuntos
Apoptose/genética , Degeneração do Disco Intervertebral/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mitofagia/genética , Núcleo Pulposo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Idoso , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Degeneração do Disco Intervertebral/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Pessoa de Meia-Idade , Núcleo Pulposo/citologia , Estresse Oxidativo/genética , RatosRESUMO
Osteoarthritis (OA) is an age-related degenerative disease and currently cannot be cured. Transcription factor EB (TFEB) is one of the major transcriptional factors that regulates autophagy and lysosomal biogenesis. TFEB has been shown to be an effective therapeutic target for many diseases including OA. The current study explores the therapeutic effects of 20-Deoxyingenol (20-DOI) on OA as well as its working mechanism on TFEB regulation. The in vitro study showed that 20-DOI may suppress apoptosis and senescence induced by oxidative stress in chondrocytes; it may also promote the nuclear localization of TFEB in chondrocytes. Knock-down of TFEB compromised the effects of 20-DOI on apoptosis and senescence. The in vivo study demonstrated that 20-DOI may postpone the progression of OA in mouse destabilization of the medial meniscus (DMM) model; it may also suppress apoptosis and senescence and promote the nuclear localization of TFEB in chondrocytes in vivo. This work suggests that 20-Deoxyingenol may alleviate osteoarthritis by activating TFEB in chondrocytes, while 20-DOI may become a potential drug for OA therapy.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/agonistas , Condrócitos/efeitos dos fármacos , Diterpenos/farmacologia , Osteoartrite/tratamento farmacológico , Envelhecimento/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Diterpenos/uso terapêutico , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Inflammation and neuronal apoptosis contribute to the progression of secondary injury after spinal cord injury (SCI) and are targets for SCI therapy; autophagy is reported to suppress apoptosis in neuronal cells and M2 polarization may attenuate inflammatory response in microglia, while both are negatively regulated by mTORC1 signalling. We hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy. In this study, we evaluate a novel inhibitor of mTORC1 signalling, Astragaloside IV (AS-IV), in vitro and in vivo. Our results showed that AS-IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo. AS-IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo, rats were intraperitoneally injected with AS-IV (10 mg/kg/d) after SCI, behavioural and histological evaluations showed that AS-IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS-IV may become a novel therapeutic medicine for SCI.
Assuntos
Inflamação/prevenção & controle , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Microglia/citologia , Neurônios/citologia , Recuperação de Função Fisiológica , Saponinas/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Triterpenos/farmacologia , Animais , Apoptose , Autofagia , Polaridade Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Traumatismos da Medula Espinal/etiologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Diabetes mellitus may lead to intervertebral disc degeneration (IVDD). Matrix metalloproteinase-13 (MMP-13) is one of the major catabolic factors in extracellular matrix (ECM) metabolism of nucleus pulposus cells (NPCs) and contributes to diabetic IVDD. Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal protein family and is implicated in chronic inflammation. Here, we report that the expression of BRD4 and MMP-13 was elevated in diabetic nucleus pulposus tissues as well as in advanced glycation end products (AGEs)-treated NPCs; also, the regulatory effect of BRD4 on MMP-13 was studied. We found that MMP-13 was regulated by MAPK and NF-κB signaling as well as autophagy in AGEs-treated NPCs. Next, we explored the role of BRD4 in regulation of MAPK, NF-κB signaling, and autophagy. The results showed that BRD4 is the upstream regulator of all of these 3 factors, and inhibition of BRD4 may suppress MAPK and NF-κB signaling while activating autophagy in AGEs-treated NPCs. Finally, we demonstrated that BRD4 inhibition may suppress MMP-13 expression in diabetic NPCs in vitro as well as in vivo; meanwhile, it may preserve ECM in diabetic rats. Our study demonstrates that inhibition of BRD4 may suppress MAPK and NF-κB signaling and activate autophagy to suppress MMP-13 expression in diabetic IVDD, and diabetic IVDD may be compromised by BRD4 inhibitors.-Wang, J., Hu, J., Chen, X., Huang, C., Lin, J., Shao, Z., Gu, M., Wu, Y., Tian, N., Gao, W., Zhou, Y., Wang, X., Zhang, X. BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration.
Assuntos
Autofagia/fisiologia , Proteínas de Ciclo Celular/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Adulto , Animais , Diabetes Mellitus/metabolismo , Feminino , Humanos , Disco Intervertebral/metabolismo , Masculino , Pessoa de Meia-Idade , Núcleo Pulposo/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Benzo(
Assuntos
Benzo(a)pireno/toxicidade , Crataegus/química , Dano ao DNA/efeitos dos fármacos , Mutagênicos/toxicidade , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Expressão Gênica/efeitos dos fármacos , Histonas/genética , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais/isolamento & purificação , Substâncias Protetoras/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Baço/efeitos dos fármacos , Baço/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The pathophysiology of spinal cord injury (SCI) involves primary injury and secondary injury. For the irreversibility of primary injury, therapies of SCI mainly focus on secondary injury, whereas inflammation is considered to be a major target for secondary injury; however the regulation of inflammation in SCI is unclear and targeted therapies are still lacking. In this study, we found that the expression of BRD4 was correlated with pro-inflammatory cytokines after SCI in rats; in vitro study in microglia showed that BRD4 inhibition either by lentivirus or JQ1 may both suppress the MAPK and NF-κB signalling pathways, which are the two major signalling pathways involved in inflammatory response in microglia. BRD4 inhibition by JQ1 not only blocked microglial M1 polarization, but also repressed the level of pro-inflammatory cytokines in microglia in vitro and in vivo. Furthermore, BRD4 inhibition by JQ1 can improve functional recovery and structural disorder as well as reduce neuron loss in SCI rats. Overall, this study illustrates that microglial BRD4 level is increased after SCI and BRD4 inhibition is able to suppress M1 polarization and pro-inflammatory cytokine production in microglia which ultimately promotes functional recovery after SCI.
Assuntos
Azepinas/farmacologia , Inflamação/tratamento farmacológico , Proteínas Nucleares/genética , Traumatismos da Medula Espinal/tratamento farmacológico , Fatores de Transcrição/genética , Triazóis/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/fisiopatologia , MAP Quinase Quinase 1/genética , Microglia/efeitos dos fármacos , Microglia/patologia , NF-kappa B , Proteínas Nucleares/antagonistas & inibidores , Ratos , Recuperação de Função Fisiológica/genética , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/fisiopatologia , Fatores de Transcrição/antagonistas & inibidoresRESUMO
Random-pattern skin flaps are important method for skin reconstruction after defect; however, the distal end of flaps is not easily viable due to inadequate nutrient supply. Erastin is a well-established ferroptosis inducer, but our study found that low-dose of erastin (2⯵M) may reduce nutrient deficiency induced cell death in human umbilical vein endothelial cells (HUVECs). RNA-seq analysis suggested that its role was related to autophagy regulation. Follow-up studies have shown that the use of autophagy inhibitors or the knockdown of TFEB in HUVECs can both reduce the anti-apoptotic effect of erastin in HUVECs. Mechanism study demonstrated that erastin can suppress mTORC1 and promote TFEB activity in HUVECs, suggesting that the effect of erastin on the survival of HUVECs under nutrient deprivation conditions is regulated by mTORC1/TFEB. Subsequently, we evaluated the effect of erastin on the survival of random-pattern skin flaps in mice in vivo. On the postoperative day 7, we observed a significant increase in flap survival area, blood perfusion, and microvascular density after erastin treatment; also, erastin treatment showed enhanced autophagy within the ischemic region. In summary, our study demonstrates that low-dose of erastin may suppress cell death in endothelial cells under nutrient deficiency condition, and its effects may relate to the mTORC1-TFEB medicated autophagy regulation, erastin treatment may be a potential therapy for random-pattern skin flaps.
Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Células Endoteliais da Veia Umbilical Humana , Alvo Mecanístico do Complexo 1 de Rapamicina , Autofagia/efeitos dos fármacos , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Humanos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Retalhos Cirúrgicos , Masculino , Sobrevivência de Enxerto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologiaRESUMO
Low back pain, primarily caused by intervertebral disc degeneration (IVDD), lacks effective pharmacological treatments. Oxidative stress has been identified as a significant contributor to IVDD. This study aims to establish an in vitro model of IVDD induced by oxidative stress and identify potential therapeutic agents and their underlying mechanisms. By screening the natural product library, fisetin emerged as the most promising compound in suppressing cell death induced by oxidative stress in nucleus pulposus cells (NPCs). Furthermore, our investigation revealed that the cell death induced by oxidative stress was predominantly associated with ferroptosis, and fisetin demonstrated the ability to inhibit ferroptosis in NPCs. Mechanistic exploration suggested that the impact of fisetin on ferroptosis may be mediated through the Nrf2/HO-1 (Nuclear factor erythroid 2-related factor 2/heme oxygenase-1) axis. Notably, the in vivo study demonstrated that fisetin could alleviate IVDD in rats. These findings highlight fisetin as a potential therapeutic option for IVDD and implicate the involvement of the Nrf2/HO-1 pathway in its mechanism of action.
Assuntos
Ferroptose , Flavonóis , Degeneração do Disco Intervertebral , Animais , Ratos , Ferroptose/efeitos dos fármacos , Flavonóis/farmacologia , Flavonóis/uso terapêutico , Degeneração do Disco Intervertebral/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
Spinal cord injury (SCI) can lead to permanent paralysis and various motor, sensory and autonomic nervous system dysfunction. The complex pathophysiological processes limit the effectiveness of many clinical treatments. Mitochondria has been reported to play a key role in the pathogenesis of SCI; while mitophagy is a protective mechanism against mitochondrial dysfunction. However, there is recently little drugs that may targeted activate mitophagy to treat SCI. In this study, we evaluated the role of 20-Deoxyingenol (20-DOI) in SCI and explored its potential mechanisms. We used a SCI rat model and evaluated the functional outcomes after the injury. Western blotting and immunofluorescence techniques were used to analyze the levels of mitophagy, apoptosis, and TFEB-related signaling pathways. Our research results show that 20-DOI significantly improves the apoptosis of neural cells after TBHP stimulation and functional recovery after spinal cord injury. In addition, mitophagy, TFEB levels, and apoptosis are related to the mechanism of 20-DOI treatment for spinal cord injury. Specifically, our research results indicate that 20-DOI restored the autophagic flux after injury, thereby inducing mitophagy, eliminating the accumulation of Cyto C, and inhibiting apoptosis. Further mechanism research suggests that 20-DOI may regulate mitophagy by promoting TFEB nuclear translocation. These results indicate that 20-DOI can significantly promote recovery after spinal cord injury, which may be a promising treatment method for spinal cord injury.
RESUMO
BACKGROUND: Repairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis. METHODS: The relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF+/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague-Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair. RESULTS: The pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF+/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects. CONCLUSION: NGF+/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF+/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs.
Assuntos
Células-Tronco Mesenquimais , Fator de Crescimento Neural , Osteogênese , Ratos Sprague-Dawley , Alicerces Teciduais , Animais , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Ratos , Alicerces Teciduais/química , Regeneração Óssea , Aloenxertos , Masculino , Engenharia Tecidual/métodos , Piroptose , Sulfonamidas/farmacologia , Diferenciação Celular , Transplante de Células-Tronco Mesenquimais/métodos , Transplante Ósseo/métodosRESUMO
Intervertebral disc degeneration (IVDD) is a prevalent degenerative disease with significant adverse implications for patients' quality of life and socioeconomic status. Although the precise etiology of IVDD remains elusive, the senescence of nucleus pulposus cells is recognized as the primary pathogenic factor of IVDD; however, drugs that may targetedly inhibit senescence are still lacking. In the current study, we evaluated the small-molecule active drug 20-Deoxyingenol(20-DOI) for its effects on combating senescence and delaying the progression of IVDD. In vitro experiments revealed that the administration of 20-DOI displayed inhibitory effects on senescence and the senescence-related cGAS-STING pathway of nucleus pulposus cells. Additionally, it exhibited the ability to enhance lysosome activity and promote autophagy flux within nucleus pulposus cells. Subsequent investigations elucidated that the inhibitory impact of 20-DOI on nucleus pulposus cell senescence was mediated through the autophagy-lysosome pathway. This effect was diminished in the presence of transcription factor EB (TFEB) small hairpin RNA (shRNA), thereby confirming the regulatory role of 20-DOI on the autophagy-lysosome pathway and senescence through TFEB. In vivo experiments demonstrated that 20-DOI effectively impeded the progression ofIVDD in rats. These findings collectively illustrate that 20-DOI may facilitate the autophagy-lysosomal pathway by activating TFEB, thereby suppressing the senescence in nucleus pulposus cells, thus suggesting 20-DOI as a promising therapeutic approach for IVDD.
Assuntos
Degeneração do Disco Intervertebral , Núcleo Pulposo , Humanos , Ratos , Animais , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Núcleo Pulposo/metabolismo , Qualidade de Vida , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismoRESUMO
The repair of bone defects remains a huge clinical challenge. M2 macrophage-derived exosomes (M2-Exos) can act as immunomodulators to promote fracture healing; however, how to retain the sustained release of exosomes to the target area remains a challenge. Here, we report a composite hydrogel loaded with M2-Exos aiming to accelerate bone defect healing. It was verified that the F127/HA-NB hydrogel had a dense network structure, tissue adhesiveness, and dual sensitivity to temperature and light. F127/HA-NB loaded with M2-Exos (M2-Exos@F127/HA-NB) exhibited good biocompatibility and achieved sustained release of exosomes for up to two weeks. The study showed that both M0-Exos and M2-Exos@F127/HA-NB significantly promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells. The mechanism study implied that M2-Exos activates the Wnt/ß-catenin signaling pathway to promote osteogenic differentiation of BMSCs. Finally, we evaluated the osteogenetic effects of M2-Exos@F127/HA-NB in a rat cranial defect model, and the results showed that M2-Exos@F127/HA-NB had superior bone regeneration-promoting effects. This study provides a new strategy for cell-free treatment of bone defects.
RESUMO
Osteoarthritis (OA) is the most common degenerative joint disorder with no effective drugs. Puerarin is a dietary supplement that has wide-ranging pharmacological effects. This study aimed to investigate the effects of Puerarin on OA. The effects of Puerarin on apoptosis, extracellular matrix (ECM) metabolism, and inflammation-related factors were assessed; also, the nuclear factor-κB (NF-κB) signaling pathway and Nrf2/HO-1 (nuclear factor (erythroid-derived 2)-like 2/heme oxygenase-1) axis were evaluated to elucidate the working mechanism of Puerarin. Mice were fed with Puerarin to evaluate the therapeutic effect of Puerarin on Osteoarthritis in vivo. The results showed that Puerarin suppressed inflammatory mediators and apoptosis induced by IL-1ß treatment in chondrocytes, it may also suppress ECM degradation in IL-1ß treated chondrocytes. The mechanism study revealed that Nrf2/HO-1 pathway is involved in Puerarin induced inhibition of NF-κB signaling pathway. Finally, in vivo study demonstrated that Puerarin could postpone the progression of OA in mice and relieve the symptoms of pain. In conclusion, Puerarin may potentially alleviate OA progression, and the mechanism may relate to the Nrf2/HO-1 pathway regulation.
Assuntos
Matriz Extracelular , Inflamação/metabolismo , Isoflavonas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Osteoartrite/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Heme Oxigenase-1/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Dor/metabolismoRESUMO
Osteoarthritis (OA), manifested as degeneration and damage of the articular cartilage is a progressive disease of joints. Previous studies have shown that extracellular matrix degradation and inflammation have quite a significant performance in the occurrence and development of OA. In various maladies, an anti-inflammatory effect has been demonstrated for Xanthohumol (XN); while OA is an inflammation related disease. The current in vivo and in vitro study aimed to investigate the therapeutic effect of XN on OA as well as its working mechanism. The results showed that XN has the capability to hinder the expression of nitric oxide synthase (INOS), IL-1ß-promoted inducible nitric oxide (NO), necrosis factor-α of tumor (TNF-α), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) in vitro. In addition, XN has been found to down-regulate the expression of matrix metalloproteinase-13 and prothrombin stimulated by IL-1ß and up-regulates type II collagen and Aggrecan expression. At the same time, it was discovered that XN activates nuclear factor (Nrf2) in chondrocytes stimulated by IL-1ß and inhibits nuclear factor B (NF-кB) signal transduction. The DMM model manifests that XN has an inhibitory impact on the progression of osteoarthritis and thus may be a candidate drug to slow down and delay the development of OA.
Assuntos
Anti-Inflamatórios/farmacologia , Condrócitos/efeitos dos fármacos , Flavonoides/farmacologia , Mediadores da Inflamação/metabolismo , Articulações/efeitos dos fármacos , Osteoartrite/tratamento farmacológico , Propiofenonas/farmacologia , Animais , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Articulações/metabolismo , Articulações/patologia , Masculino , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Transdução de SinaisRESUMO
Osteoarthritis (OA), a degenerative disorder, is considered to be one of the most common forms of arthritis. Limonin (Lim) is extracted from lemons and other citrus fruits. Limonin has been reported to have anti-inflammatory effects, while inflammation is a major cause of OA; thus, we propose that limonin may have a therapeutic effect on OA. In this study, the therapeutic effect of limonin on OA was assessed in chondrocytes in vitro in IL-1ß induced OA and in the destabilization of the medial meniscus (DMM) mice in vivo. The Nrf2/HO-1/NF-κB signaling pathway was evaluated to illustrate the working mechanism of limonin on OA in chondrocytes. In this study, it was found that limonin can reduce the level of IL-1ß induced proinflammatory cytokines such as INOS, COX-2, PGE2, NO, TNF-α, and IL-6. Limonin can also diminish the biosynthesis of IL-1ß-stimulated chondrogenic catabolic enzymes such as MMP13 and ADAMTS5 in chondrocytes. The research on the mechanism study demonstrated that limonin exerts its protective effect on OA through the Nrf2/HO-1/NF-κB signaling pathway. Taken together, the present study shows that limonin may activate the Nrf2/HO-1/NF-κB pathway to alleviate OA, making it a candidate therapeutic agent for OA.
Assuntos
Artrite Experimental/tratamento farmacológico , Condrócitos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Interleucina-1beta/toxicidade , Limoninas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Osteoartrite/tratamento farmacológico , Animais , Artrite Experimental/etiologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Regulação da Expressão Gênica , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoartrite/etiologia , Osteoartrite/metabolismo , Osteoartrite/patologiaRESUMO
Damaged deoxyribonucleic acid (DNA) is a primary pathologic factor for osteoarthritis (OA); however, the mechanism by which DNA damage drives OA is unclear. Previous research demonstrated that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) participates in DNA damage response. As a result, the current study aimed at exploring the role STING, which is the major effector in the cGAS-STING signaling casacde, in OA progress in vitro, as well as in vivo. In this study, the expression of STING was evaluated in the human and mouse OA tissues, and in chondrocytes exposed to interleukin-1 beta (IL-1ß). The influences of STING on the metabolism of the extracellular matrix (ECM), apoptosis, and senescence, were assessed in STING overexpressing and knocking-down chondrocytes. Moreover, the NF-κB-signaling casacde and its role in the regulatory effects of STING on ECM metabolism, apoptosis, and senescence were explored. The STING knockdown lentivirus was intra-articularly injected to evaluate its therapeutic impact on OA in mice in vivo. The results showed that the expression of STING was remarkably elevated in the human and mouse OA tissues and in chondrocytes exposed to IL-1ß. Overexpression of STING promoted the expression of MMP13, as well as ADAMTS5, but suppressed the expression of Aggrecan, as well as Collagen II; it also enhanced apoptosis and senescence in chondrocytes exposed to and those untreated with IL-1ß. The mechanistic study showed that STING activated NF-κB signaling cascade, whereas the blockage of NF-κB signaling attenuated STING-induced apoptosis and senescence, and ameliorated STING-induced ECM metabolism imbalance. In in vivo study, it was demonstrated that STING knockdown alleviated destabilization of the medial meniscus-induced OA development in mice. In conclusion, STING promotes OA by activating the NF-κB signaling cascade, whereas suppression of STING may provide a novel approach for OA therapy.
Assuntos
Condrócitos , Proteínas de Membrana/fisiologia , Osteoartrite/metabolismo , Idoso , Animais , Apoptose , Células Cultivadas , Senescência Celular , Condrócitos/metabolismo , Condrócitos/patologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Cultura Primária de CélulasRESUMO
OBJECTIVE: To investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats. METHODS: Three lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type â expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining. RESULTS: After lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced ( P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups ( P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type â expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant ( P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased. CONCLUSION: Silencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Lentivirus , Ratos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento NeuralRESUMO
Thermal processing is a traditional method for processing hawthorn into food or medicine. In this study, the compositions of free and bound phenolic compounds in raw hawthorn were analyzed by ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry, and the effect of thermal processing on phenolics and antioxidant activity was determined. Among the phenolics identified in unheated hawthorn, 26 were soluble, while only 10 were insoluble-bound. Thermal processing caused a significant reduction in total soluble phenolics content, but an increase in total insoluble-bound phenolics (p < 0.05). Procyanidin B2 and epicatechin showed the largest decreases in content, and were not detected in well-cooked hawthorn. The antioxidant activity also clearly decreased, with the chlorogenic acid, procyanidin B2, hyperoside, and isoquercetin contents correlating significantly (p < 0.05) with antioxidant activity. In general, the effect of thermal processes on phenolics and antioxidant activity was dependent on the types of phenolics and processing conditions.
Assuntos
Antioxidantes/química , Crataegus/química , Manipulação de Alimentos/métodos , Fenóis/química , Extratos Vegetais/química , Antioxidantes/isolamento & purificação , Cromatografia Líquida , Temperatura Alta , Fenóis/isolamento & purificação , Extratos Vegetais/isolamento & purificaçãoRESUMO
Osteoarthritis is a chronic degenerative disease characterized by cartilage destruction. It is the fourth most disabling disease worldwide and is currently incurable. Inflammation and extracellular matrix (ECM) degradation are considered to be substantial reasons for accelerating the progression of OA. ß-Hydroxyisoamylshikonin (ß-HIVS) is a natural naphthoquinone compound with anti-inflammatory and antioxidant activity. However, the effect of ß-HIVS on OA is still unclear. In this study, we found that ß-HIVS can down-regulate the expression of NO, PEG2, IL-6, TNF-α, COX-2, and iNOS, suggesting its anti-inflammatory effects in chondrocytes; we also found that ß-HIVS may down-regulate the expression of ADAMTS5 and MMP13 and up-regulate the expression of aggrecan and collagen II to inhibit the degradation of ECM. Mechanistically, ß-HIVS inhibited the NFκB pathway by activating the Nrf2/HO-1 axis, thereby exerting its anti-inflammatory and inhibitory effects on ECM degradation. In vivo experiments also proved the therapeutic effects of ß-HIVS on OA in mice, and Nrf2 is the target of ß-HIVS. These findings indicate that ß-HIVS may become a new drug for the treatment of OA.