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Type I collagen (Col I) and hyaluronic acid (HA), derived from the extracellular matrix (ECM), have found widespread application in cartilage tissue engineering. Nevertheless, the potential of cell-free collagen-based scaffolds to induce in situ hyaline cartilage regeneration and the related mechanisms remain undisclosed. Here, we chose Col I and HA to construct Col I hydrogel and Col I-HA composite hydrogel with similar mechanical properties, denoted as Col and ColHA, respectively. Their potential to induce cartilage regeneration was investigated. The results revealed that collagen-based hydrogels could regenerate hyaline cartilage without any additional cells or growth factors. Notably, ColHA hydrogel stood out in this regard. It elicited a moderate activation, recruitment, and reprogramming of macrophages, thus efficiently mitigating local inflammation. Additionally, ColHA hydrogel enhanced stem cell recruitment, facilitated their chondrogenic differentiation, and inhibited chondrocyte fibrosis, hypertrophy, and catabolism, thereby preserving cartilage homeostasis. This study augments our comprehension of cartilage tissue induction theory by enriching immune-related mechanisms, offering innovative prospects for the design of cartilage defect repair scaffolds. STATEMENT OF SIGNIFICANCE: The limited self-regeneration ability and post-injury inflammation pose significant challenges to articular cartilage repair. Type I collagen (Col I) and hyaluronic acid (HA) are extensively used in cartilage tissue engineering. However, their specific roles in cartilage regeneration remain poorly understood. This study aimed to elucidate the functions of Col I and Col I-HA composite hydrogels (ColHA) in orchestrating inflammatory responses and promoting cartilage regeneration. ColHA effectively activated and recruited macrophages, reprogramming them from an M1 to an M2 phenotype, thus alleviating local inflammation. Additionally, ColHA facilitated stem cell homing, induced chondrogenesis, and concurrently inhibited fibrosis, hypertrophy, and catabolism, collectively contributing to the maintenance of cartilage homeostasis. These findings underscore the clinical potential of ColHA for repairing cartilage defects.
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Injectable, self-crosslinking collagen-based hydrogels are beneficial for chondrocytes to secrete matrix, positioning them as promising candidates for cartilage tissue engineering. However, previous studies lacked insight into the ability of cell-free collagen-based hydrogels to regenerate hyaline cartilage defect. Therefore, this study aimed to evaluate the potential of collagen-based hydrogels (Col and ColHA) to induce chondrogenic differentiation of stem cells and in situ hyaline cartilage regeneration. Both Col and ColHA hydrogels self-crosslinked in situ and exhibited similar physical properties. In vitro experiments showed they supported the survival, adhesion, spreading, and proliferation of bone marrow stem cells (BMSCs). Moreover, both hydrogels induced ectopic differentiation of BMSCs into chondrocytes when implanted subcutaneously into the back of nude mice. ColHA hydrogel notably enhanced type II collagen secretion. The results of repairing cartilage defects in situ revealed both hydrogels facilitated hyaline cartilage regeneration and maintained cartilage phenotype without exogenous BMSCs. Hydrogels encapsulating BMSCs expedited cartilage repair, and ColHA/BMSC constructs showed better mechanical properties, suggesting their potential for cartilage repair applications. This study implies that collagen-based hydrogels are good candidates for hyaline cartilage regeneration.
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Diferenciação Celular , Condrogênese , Colágeno , Cartilagem Hialina , Hidrogéis , Regeneração , Hidrogéis/química , Hidrogéis/farmacologia , Condrogênese/efeitos dos fármacos , Animais , Regeneração/efeitos dos fármacos , Camundongos , Colágeno/química , Colágeno/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Camundongos Nus , Engenharia Tecidual/métodos , Proliferação de Células/efeitos dos fármacosRESUMO
OBJECTIVE: Hepatic insulin resistance, which leads to increased hepatic gluconeogenesis, is a major contributor to fasting hyperglycemia in type 2 diabetes mellitus (T2DM). However, the mechanism of impaired insulin-dependent suppression of hepatic gluconeogenesis remains elusive. Delta/Notch-like epidermal growth factor (EGF)-related receptor (DNER), ï¬rstly described as a neuron-specific Notch ligand, has been recently identiï¬ed as a susceptibility gene for T2DM through genome-wide association studies. We herein investigated whether DNER regulates hepatic gluconeogenesis and whether this is mediated by enhanced insulin signaling. METHODS: The association between DNER, tribbles homolog 3 (TRB3) and Akt signaling was evaluated in C57BL/6J, ob/ob and db/db mice by western blot analysis. DNER loss-of-function and gain-of-function in hepatic gluconeogenesis were analyzed by western blot analysis, quantitative real-time PCR, glucose uptake and output assay in AML-12 cells and partially validated in primary mouse hepatocytes. Hepatic DNER knockdown mice were generated by tail vein injection of adenovirus to confirm the effects of DNER in vivo. The interaction between DNER and TRB3 was investigated by rescue experiments, cycloheximide chase analysis, co-immunoprecipitation and immunofluorescence. The potential insulin-stimulated phosphorylation sites of DNER were determined by co-immunoprecipitation, LC-MS/MS analysis and site-specific mutagenesis. RESULTS: Here we show that DNER enhanced hepatic insulin signaling in gluconeogenesis by inhibiting TRB3, an endogenous Akt inhibitor, through the ubiquitin-proteasome degradation pathway. In AML-12 hepatocytes, insulin-stimulated activation of Akt and suppression of gluconeogenesis are attenuated by DNER knockdown, but potentiated by DNER over-expression. In C57BL/6J mice, hepatic DNER knockdown is accompanied by impaired glucose and pyruvate tolerance. Furthermore, the in vitro effects of DNER knockdown or over-expression on both Akt activity and hepatic gluconeogenesis can be rescued by TRB3 knockdown or over-expression, respectively. In response to insulin stimulation, DNER interacted directly with insulin receptor and was phosphorylated at Tyr677. This site-specific phosphorylation is essential for DNER to upregulate Akt activity and then downregulate G6Pase and PEPCK expression, by interacting with TRB3 directly and inducing TRB3 proteasome-dependent degradation. CONCLUSIONS: Taken together, the crosstalk between insulin-Akt and DNER-TRB3 pathways represents a previously unrecognized mechanism by which insulin regulates hepatic gluconeogenesis.
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Proteínas de Ciclo Celular , Gluconeogênese , Insulina , Fígado , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Diabetes Mellitus Tipo 2/metabolismo , Hepatócitos/metabolismo , Insulina/metabolismo , Resistência à Insulina , Fígado/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Background: Dopamine receptors have been reported to be involved in pain, while the exact effects and mechanism in bone cancer pain have not been fully explored. Methods: Bone cancer pain model was created by implanting walker 256 mammary gland carcinoma into right tibia bone cavity. Primary cultured spinal neurons were used for in vitro evaluation. FLIPR, western-blot, immunofluorescence, and Co-IP were used to detect cell signaling pathway. Results: Our results indicated that spinal dopamine D1 receptor (D1DR) and spinal dopamine D2 receptor (D2DR) could form heteromers in TCI rats, and antagonizing spinal D1DR and D2DR reduced heteromers formation and alleviated TCI-induced bone cancer pain. Further results indicated that D1DR or D2DR antagonist induced antinociception in TCI rats could be reversed by D1DR, D2DR, and D1/D2DR heteromer agonists. And Gq, IP3, and PLC inhibitors also attenuated TCI-induced bone cancer pain. In vitro results indicated that D1DR or D2DR antagonist decreased the Ca2+ oscillations upregulated by D1DR, D2DR, and D1/D2DR heteromer agonists in activated primary cultured spinal neurons. Moreover, inhibition of D1/D2DR heteromers induced antinociception in TCI rats was partially mediated by the CaMKII and MAPKs pathway. In addition, a natural compound levo-Corydalmine (l-CDL), could inhibit D1/D2DR heteromers and attenuate bone cancer pain. Results: Inhibition of spinal D1/D2DR heteromers via l-CDL decreases excitability in spinal neurons, which might present new therapeutic strategy for bone cancer pain.
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Collagen and hyaluronic acid are commonly applied in cartilage tissue engineering, yet there has been limited investigation into their inflammatory response, a crucial factor in articular cartilage repair. This study aimed to evaluate the impact of components and physical properties of hydrogels on inflammatory response and cartilage repair. Three kinds of hydrogels with comparable storage moduli at low frequencies were designed and fabricated, namely, methacrylic anhydride-modified hyaluronic acid hydrogel (HAMA), methacrylic anhydride-modified type I collagen hydrogel (CMA) and unmodified type I collagen hydrogel (Col). HAMA hydrogel was unfavorable for adhesion and spreading of BMSCs. Furthermore, HAMA hydrogel stimulated rapid migration and pro-inflammatory M1 polarization of macrophages, leading to persistent and intense inflammation, which was unfavorable for cartilage repair. CMA and Col hydrogels possessed the same component and facilitated the adhesion, spreading and proliferation of BMSCs. Compared with CMA hydrogel, Col hydrogel induced rapid migration and moderate M1 polarization of macrophages at the early stage of injury, which was mainly influenced by its fast dissolution rate, small pore size fiber network structure and rapid stress relaxation. In addition, the phenotype of macrophages timely transformed into anti-inflammatory M2 due to the properties of the collagen component, which shortened the duration of inflammation and enhanced cartilage repair. The results indicated that moderate macrophage activation adjusted by hydrogel components and physical properties was critical in modulating inflammation and cartilage regeneration.
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Cartilagem Articular , Hidrogéis , Humanos , Hidrogéis/farmacologia , Hidrogéis/química , Condrócitos , Ácido Hialurônico/farmacologia , Ácido Hialurônico/química , Colágeno Tipo I , Colágeno/química , Inflamação/tratamento farmacológico , AnidridosRESUMO
Articular cartilage is refractory to self-healing due to the absence of vascular, nervous, and lymphatic systems, and its repair remains a clinical challenge. Tissue regeneration through in situ recruitment of stem cells via cell-free scaffolds is a promising alternative strategy. Herein, a kind of functional injectable hydrogel system (Col-Apt@KGN MPs), which is a collagen-based and microsphere-embedded cell-free scaffold, was designed to achieve spatiotemporal regulation of endogenous mesenchymal stem cells (MSCs) recruitment and their chondrogenic differentiation by respective release of aptamer 19S (Apt19S) and kartogenin (KGN). In vitro results confirmed that the Col-Apt@KGN MPs hydrogel had sequential release characteristics. Apt19S was rapidly released from the hydrogel within 6 days, while KGN was slowly released for 33 days via the degradation of poly(lactic-co-glycolic acid) (PLGA) microspheres. When cultured with MSCs, the Col-Apt@KGN MPs hydrogel supported the adhesion, proliferation, and chondrogenic differentiation of MSCs. In vivo results indicated that the Col-Apt@KGN MPs hydrogel effectively promoted the recruitment of endogenous MSCs in a rabbit full-thickness cartilage defect model; furthermore, the Col-Apt@KGN MPs hydrogel enhanced the secretion of cartilage specific extracellular matrix and achieved the reconstruction of subchondral bone. This study demonstrates that the Col-Apt@KGN MPs hydrogel possesses great potential in recruitment of endogenous stem cells and cartilage tissue regeneration.
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Doenças das Cartilagens , Cartilagem Articular , Células-Tronco Mesenquimais , Animais , Coelhos , Hidrogéis/metabolismo , Diferenciação CelularRESUMO
The suitable seeding cells and scaffolds are very important for tissue engineering to create functional cartilage. Although the physicochemical properties of scaffold and co-culture system of mesenchymal stem cells (MSCs) and chondrocytes could affect functional properties of engineered cartilage tissues respectively, the combined effects of them on chondrogenesis is currently unknown. Herein, methacrylated collagen (CMA30 and CMA80) hydrogels with different degradation rate and stiffness were prepared. The MSCs and chondrocytes were co-cultured or monocultured in collagen, CMA30 and CMA80 hydrogels in vitro or in vivo. The results demonstrated that cell spreading and proliferation was regulated by degradation rate and stiffness of hydrogels. Compared to single MSCs culture, co-culture cells in all collagen-based hydrogels significantly improved chondrogenesis. CMA30 hydrogel with moderate degradation rate and low storage modulus was the most effective for co-culture system to promote chondrogenesis compared to Col and CMA80 hydrogel in vitro culture, while there was no obvious difference between CMA30 and CMA80 hydrogel in vivo. Furthermore, the intercellular substance exchange was very important for co-culture system to maintain the positive effect on chondrogenesis. Overall, the current study highlights the synergistic effects of the physicochemical properties of collagen-based hydrogel and co-culture system on cartilage formation. STATEMENT OF SIGNIFICANCE: Scaffolds and cells play a key role in cartilage tissue engineering. The combined effects of physicochemical properties of collagen hydrogels and co-culture system (MSCs and chondrocytes) on chondrogenesis is unknown. In contrast to the studies that investigated the effect of single factor (scaffolds or cells) on cartilage formation, this manuscript explored the synergistic regulation of both scaffold properties and biological factors on chondrogenesis, and provided a promising strategy for cartilage tissue engineering.
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Condrogênese , Hidrogéis , Hidrogéis/química , Técnicas de Cocultura , Condrócitos , Colágeno/farmacologia , Colágeno/metabolismo , Engenharia Tecidual/métodos , Diferenciação Celular , Alicerces Teciduais , Células CultivadasRESUMO
Collagen is a promising material for tissue engineering, but the poor mechanical properties of collagen hydrogels, which tend to cause contraction under the action of cellular activity, make its application challengeable. In this study, the amino group of type I collagen (Col I) was modified with methacrylic anhydride (MA) and the photo-crosslinkable methacrylate anhydride modified type I collagen (CM) with three different degrees of substitution (DS) was prepared. The physical properties of CM and Col I hydrogels were tested, including micromorphology, mechanical properties and degradation properties. The results showed that the storage modulus and degradation rate of hydrogels could be adjusted by changing the DS of CM. In vitro, chondrocytes were seeded into these four groups of hydrogels and subjected to fluorescein diacetate/propidium iodide (FDA/PI) staining, cell counting kit-8 (CCK-8) test, histological staining and cartilage-related gene expression analysis. In vivo, these hydrogels encapsulating chondrocytes were implanted subcutaneously into nude mice, then histological staining and sulfated glycosaminoglycan (sGAG)/DNA assays were performed. The results demonstrated that contraction of hydrogels affected behaviors of chondrocytes, and CM hydrogels with suitable DS could resist contraction of hydrogels and promote the secretion of cartilage-specific matrix in vitro and in vivo.
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BACKGROUND: Galli gigeriae endothelium corneum (GGEC) has been effectively used for centuries for the treatment of functional dyspepsia (FD) in clinical practice in Asian countries. However, its potential mechanism and chemical composition remains undertermined. METHODS: In this study, the chemical profile of GGEC ethyl acetate extract (EAE) was evaluated by HPLC-Q-TOF-MS/MS. The effects of EAE on intestinal barrier function and inflammation were investigated in IEC-6 cells and RAW264.7 cells. RESULTS: The results showed that 33 compounds were tentatively identified, including 12 soy isoflavones, 7 bile acids for the first time in EAE. EAE significantly reinforced intestinal barrier function via increasing the tight junction protein levels of ZO-1 and Occludin, reducing the mRNA expression levels of interleukin (IL)-1ß and IL-6 in tumor necrosis factor alpha (TNF-α)-challenged IEC-6 cells. The scratch wound assay showed that EAE accelerated wound healing of IEC-6 cells. EAE evidently reduced the level of NO in a dose-dependent manner with an IC50 value of 18.12 µg/mL, and the mRNA expression of TNF-α, IL-1ß, IL-6, iNOS and COX-2 in LPS-treated RAW264.7 cells. CONCLUSION: This study revealed the intestinal barrier protective effects and chemical profile of GGEC, and the results indicated that GGEC strengthened the intestinal barrier by up-regulating protein expression of tight junctions and limiting inflammatory responses.
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Dopamine D1 receptor (D1DR) and D2 receptor (D2DR) are closely associated with pain modulation, but their exact effects on neuropathic pain and the underlying mechanisms remain to be identified. Our research revealed that intrathecal administration of D1DR and D2DR antagonists inhibited D1-D2DR complex formation and ameliorated mechanical and thermal hypersensitivity in chronic constriction injury (CCI) rats. The D1-D2DR complex was formed in the rat spinal cord, and the antinociceptive effects of D1DR and D2DR antagonists could be reversed by D1DR, D2DR, and D1-D2DR agonists. Gαq, PLC, and IP3 inhibitors also alleviated CCI-induced neuropathic pain. D1DR, D2DR, and D1-D2DR complex agonists all increased the intracellular calcium concentration in primary cultured spinal neurons, and this increase could be reversed by D1DR, D2DR antagonists and Gαq, IP3, PLC inhibitors. D1DR and D2DR antagonists significantly reduced the expression of p-PKC γ, p-CaMKII, p-CREB, and p-MAPKs. Levo-corydalmine (l-CDL), a monomeric compound in Corydalis yanhusuo W.T. Wang, was found to obviously suppress the formation of the spinal D1-D2DR complex to alleviate neuropathic pain in CCI rats and to decrease the intracellular calcium concentration in spinal neurons. l-CDL-induced inhibition of p-PKC γ, p-MAPKs, p-CREB, and p-CaMKII was also reversed by D1DR, D2DR, and D1-D2DR complex agonists. In conclusion, these results indicate that D1DR and D2DR form a complex and in turn couple with the Gαq protein to increase neuronal excitability via PKC γ, CaMKII, MAPK, and CREB signaling in the spinal cords of CCI rats; thus, they may serve as potential drug targets for neuropathic pain therapy.
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Neuralgia/etiologia , Neuralgia/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Células Receptoras Sensoriais/metabolismo , Traumatismos da Medula Espinal/etiologia , Traumatismos da Medula Espinal/metabolismo , Animais , Comportamento Animal , Biomarcadores , Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Suscetibilidade a Doenças , Antagonistas de Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2/farmacologia , Masculino , Complexos Multiproteicos/metabolismo , Neuralgia/diagnóstico , Medição da Dor , Fosforilação , Ligação Proteica , Ratos , Receptores de Dopamina D1/antagonistas & inibidores , Transdução de Sinais , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/diagnósticoRESUMO
INTRODUCTION: Spinal N-methyl-D-aspartate receptor (NMDAR) is vital in chronic pain, while NMDAR antagonists have severe side effects. NMDAR has been reported to be controlled by G protein coupled receptors (GPCRs), which might present new therapeutic targets to attenuate chronic pain. Dopamine receptors which belong to GPCRs have been reported could modulate the NMDA-mediated currents, while their exact effects on NMDAR in chronic bone cancer pain have not been elucidated. OBJECTIVES: This study was aim to explore the effects and mechanisms of dopamine D1 receptor (D1DR) and D2 receptor (D2DR) on NMDAR in chronic bone cancer pain. METHODS: A model for bone cancer pain was established using intra-tibia bone cavity tumor cell implantation (TCI) of Walker 256 in rats. The nociception was assessed by Von Frey assay. A range of techniques including the fluorescent imaging plate reader, western blotting, and immunofluorescence were used to detect cell signaling pathways. Primary cultures of spinal neurons were used for in vitro evaluation. RESULTS: Both D1DR and D2DR antagonists decreased NMDA-induced upregulation of Ca2+ oscillations in primary culture spinal neurons. Additionally, D1DR/D2DR antagonists inhibited spinal Calcitonin Gene-Related Peptide (CGRP) and c-Fos expression and alleviated bone cancer pain induced by TCI which could both be reversed by NMDA. And D1DR/D2DR antagonists decreased p-NR1, p-NR2B, and Gαq protein, p-Src expression. Both Gαq protein and Src inhibitors attenuated TCI-induced bone cancer pain, which also be reversed by NMDA. The Gαq protein inhibitor decreased p-Src expression. In addition, D1DR/D2DR antagonists, Src, and Gαq inhibitors inhibited spinal mitogen-activated protein kinase (MAPK) expression in TCI rats, which could be reversed by NMDA. CONCLUSIONS: Spinal D1DR/D2DR inhibition eliminated NMDAR-mediated spinal neuron activation through Src kinase in a Gαq-protein-dependent manner to attenuate TCI-induced bone cancer pain, which might present a new therapeutic strategy for bone cancer pain.
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Managing patients with refractory inflammatory bowel diseases (IBD) is a common clinical challenge. Galli Gigeriae Endothelium Corneum (GGEC), a chicken by-product, has been used for centuries in Asian countries as a functional food and supplement for the treatment of gastrointestinal disorders. In this study, a novel peptide (LNLYP, LP-5) with gastrointestinal stability that can enhance the intestinal barrier function that was first identified in GGEC. Our work demonstrated that aryl hydrocarbon receptor (AhR) activation by LP-5 could inhibit the Src kinase to increase tight junction protein levels and down-regulate the expression of inflammatory cytokines to protect the intestinal barrier and finally alleviate dextran sulfate sodium (DSS)-induced colitis. This study revealed that LP-5 had the potential to develop into a therapeutic agent for the treatment of colitis and provided new high-valued utilization of GGEC.
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BACKGROUND AND OBJECTIVES: Gap junctions play a pivotal role in contributing to the formation of astroglial networks and in chronic pain. However, the mechanisms underlying the dysfunction of astroglial gap junctions in chronic pain have not been fully elucidated. METHODS: Chronic constriction injury (CCI) of the sciatic nerve was used to establish rat neuropathic pain model. C6 cells were used to perform experiments in vitro. Von Frey hairs and Hargreave's method were used to determine the withdrawal threshold of rats. Protein expression was detected by immunofluorescence and western blotting. RESULTS: Astragaloside IV (AST IV) significantly attenuated neuropathic pain and suppressed the excitation of spinal astrocytes in rats with CCI. The antinociceptive effect of AST IV was reversed by the gap junction decoupler carbenoxolone (CBX). AST IV inhibited the high expression of phosphorylated connexin 43 (p-Cx43) and p-c-Jun N-terminal kinase (p-JNK) in spinal cord of rats with CCI. JNK inhibitor alleviated neuropathic pain, which was reversed by CBX. JNK inhibitor decreased the high expression of p-Cx43 in both rats with CCI and tumor necrosis factor-alpha (TNF-α)-treated C6 cells. Additionally, the analgesic effect of AST IV was reversed by the adenosine triphosphate-sensitive potassium (KATP) channel blocker, glibenclamide (Glib). Glib abolished the inhibitory effects of AST IV on p-JNK and p-Cx43 both in vivo and in vitro. KATP channel opener (KCO) mimicked the inhibitory effects of AST IV on p-JNK and p-Cx43 in TNF-α-treated C6 cells. CONCLUSION: Our results indicate that the sciatic nerve CCI induces the dysfunction of gap junctions in the spinal cord by activating KATP/JNK signaling to contribute to neuropathic pain. AST IV attenuates neuropathic pain via regulating the KATP-JNK gap junction axis.
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Neuralgia , Trifosfato de Adenosina , Animais , Junções Comunicantes , Hiperalgesia , Fatores Imunológicos , Neuralgia/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Saponinas , Transdução de Sinais , Medula Espinal , TriterpenosRESUMO
BACKGROUND: Anti-nerve growth factor (NGF) monoclonal antibodies (anti-NGF mAbs) have been reported to significantly attenuate pain, but the mechanism involved has not been fully elucidated, and the serious adverse events associated with mAbs seriously limit their clinical use. This study further investigated the mechanism by which peripheral NGF is involved in neuropathic pain and found safe, natural compounds that target NGF to attenuate neuropathic pain. METHODS: Nociception was assessed by the Von Frey hair and Hargreaves' methods. Western-blotting, qPCR and immunofluorescence were used to detect the cell signaling pathway. RAW264.7 macrophages and RSC96 Schwann cells were cultured for in vitro evaluation. RESULTS: Intraplantar administration of anti-NGF mAbs suppressed the expression of phosphorylated transforming growth factor-ß-activated kinase 1 (TAK1) in the dorsal root ganglion (DRG) and sciatic nerve. Intraplantar administration of a TAK1 inhibitor attenuated CCI-induced neuropathic pain and suppressed the expression of phosphorylated mitogen-activated protein kinases (MAPKs) in the DRG and sciatic nerve. Perisciatic nerve administration of levo-corydalmine (l-CDL) on the operated side obviously attenuated CCI-induced neuropathic pain and suppressed the expression of mNGF and proNGF. In addition, l-CDL-induced antinociception was reversed by intraplantar administration of NGF. Further results indicated that l-CDL-induced suppression of phosphorylated TAK1, MAPKs, and p65 and expression of the proinflammatory cytokines TNF-α and IL-1ß in the DRG and sciatic nerve were all abolished by NGF. In addition, in vitro experiments indicated that l-CDL suppressed the secretion of NGF and proNGF in RAW264.7 macrophages and RSC96 Schwann cells, which was abolished by AP-1 and CREB agonists, respectively. CONCLUSIONS: This study showed NGF inhibition suppressed TAK1 in the periphery to attenuate CCI-induced neuropathic pain through inhibition of downstream MAPK and p65 signaling. The natural compound l-CDL inhibited NGF secretion by macrophages and Schwann cells and downstream TAK1-MAPK/NF-κB signaling in the periphery to attenuate CCI-induced neuropathic pain. Video abstract Proposed mechanisms underlying the effect of l-CDL in periphery of CCI rats. In CCI rats, macropahages and Schwann cells could secret NGF to act on the receptors in the periphery to activate TAK1-MAPK/NF-κB axis and promote the release of proinflammatory cytokines, including TNF-α and IL-1ß to promote neuropathic pain. l-CDL decreased the secretion of NGF through inhibiting AP-1 and CREB respectively in RAW264.7 and RSC96 Schwann cells to attenuate CCI-induced neuropathic pain by inhibiting the TAK1-p38 MAPK/NF-κB signaling pathway.
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Anticorpos Monoclonais , MAP Quinase Quinase Quinases , Fator de Crescimento Neural , Neuralgia/tratamento farmacológico , Extratos Vegetais , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Corydalis/química , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/imunologia , Fator de Crescimento Neural/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
BACKGROUND AND OBJECTIVES: Neuropathic pain is partially refractory to currently available treatments. Although some studies have reported that apoptosis signal-regulating kinase 1 (ASK1) may inhibit chronic pain, the mechanisms underlying this process have not been fully elucidated. METHODS: Chronic constriction injury (CCI) of the rat sciatic nerve was used to establish a neuropathic pain model. Nociception was assessed using von Frey hair and Hargreaves' methods. Western blot and immunofluorescence were used to detect the cell signaling pathway. BV2 cell line was cultured for in vitro evaluation. RESULTS: Our results indicated that spinal ASK1 was co-expressed with the microglia marker ionized calcium binding adaptor 1. Additionally, intrathecal administration of ASK1 inhibitor suppressed the activation of spinal microglia and attenuated CCI-induced neuropathic pain. The ASK1 inhibitor also decreased the levels of phosphorylated ASK1 (p-ASK1), p65, p38 mitogen-activated protein kinase (MAPK) and tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) messenger RNA in lipopolysaccharide-stimulated BV2 microglia cells. Intragastric administration of levo-corydalmine (l-CDL) significantly attenuated CCI-induced neuropathic pain and inhibited the expression of p-ASK1 in the spinal cord. l-CDL conspicuously suppressed the activation of spinal microglia in vitro and in vivo. Translocation of nuclearfactor-kappa B (NF-κB) and upregulation of p-p65, TNF-α, IL-1ß were inhibited by l-CDL. Further, the analgesic effects of l-CDL were associated with reduced levels of phosphorylated protein kinase C (PKC γ), c-JunNH2-terminal kinase, and extracellular signal-regulated kinase. CONCLUSIONS: This study showed that the expression of ASK1 in spinal microglia and ASK1 inhibitor suppressed microglia activation via suppression of p38 MAPK/NF-κB, which ultimately attenuated CCI-induced neuropathic pain. l-CDL also inhibited the ASK1-P38 MAPK/NF-κB axis to attenuate CCI-induced neuropathic pain.
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Berberina/análogos & derivados , MAP Quinase Quinase Quinase 5/metabolismo , Microglia/metabolismo , NF-kappa B/metabolismo , Neuralgia/metabolismo , Medula Espinal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Berberina/farmacologia , Interleucina-1beta/metabolismo , Masculino , Neuralgia/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Nervo Isquiático , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Stephania species is one of the alkaloid-rich genus of the family Menispermaceae. Most plants of the genus Stephania possess medicinal value, whose main components are alkaloids. However, the non-medical species are often mistakenly used as herbs because of the difficulty in identification of the species. A systematic method which involved the combination of DNA barcoding, HPLC-QTOF-MS/MS and UHPLC was established for differentiation, chemical profiles and quality evaluation of medicinal Stephania species. Firstly, twenty batches of Stephania species samples were classified into five Stephania species by DNA barcoding. Secondly, 114 alkaloids including 22 tetrahydroprotoberberines, 13 protoberberines, 27 aporphines, 13 benzylisoquinolines, 12 hasubanans, 3 morphines and 24 other alkaloids were clearly or tentatively identified. Thirdly, thirteen representative components were simultaneously detected by UHPLC-DAD to characterize the differences of chemical compositions among five Stephania species. In conclusion, this method was comprehensive and effective for identification, chemical profiles and quality evaluation of medicinal Stephania species. It will provide a basis for holistic quality evaluation of medicinal Stephania species.
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Cromatografia Líquida/métodos , Código de Barras de DNA Taxonômico , DNA de Plantas/genética , Stephania/química , Espectrometria de Massas em Tandem/métodos , Alcaloides/química , Alcaloides/metabolismo , DNA Intergênico , Variação Genética , Humanos , Raízes de Plantas , Caules de Planta , Especificidade da EspécieRESUMO
OBJECTIVES: To characterize methyltransferases involved in the biosynthesis of benzylisoquinoline alkaloids in Stephania intermedia. RESULTS: Three N-methyltransferases, SiCNMT1, SiCNMT2, SiCNMT3, and O-methyltransferase SiSOMT were identified in Stephania intermedia. Then, four methyltransferase genes were cloned into the pGEX-6P-1 vector. The recombinant vectors were transformed into Escherichia coli BL21(DE3) for expression and were functionally tested. SiCNMT1, SiCNMT2, and SiCNMT3 could methylate (R)-coclaurine to produce (R)-N-methylcoclaurine. SiCNMT2 further methylated the product of (R)-N-methylcoclaurine to produce (R)-magnocurarine. Similarly, (R)-norcoclaurine was continuously catalyzed to yield (R)-N-methylnorcoclaurine and (R)-N, N-dimethylnorcoclaurine by SiCNMT2. Furthermore, SiSOMT was shown to catalyze the conversion of (S)-scoulerine to (S)-tetrahydropalmatine. CONCLUSIONS: The key methyltransferases, which were in the last step biosynthesis of (R)-magnocurarine, (R)-N, N-dimethylnorcoclaurine and (S)-tetrahydropalmatine were revealed and their activities were verified in vitro. Four novel methyltransferases will be promising candidates for methylation of benzylisoquinoline alkaloids.
Assuntos
Benzilisoquinolinas/química , Metiltransferases/química , Proteínas de Plantas/química , Stephania/enzimologia , Benzilisoquinolinas/metabolismo , Clonagem Molecular , Metiltransferases/biossíntese , Metiltransferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Stephania/genéticaRESUMO
Melatonin (Nacetyl5methoxytryptamine; MT) has been shown to have a protective effect against sepsisinduced renal injury, however, the mechanisms underlying the function of MT remain to be elucidated. Therefore, in the present study, the potential mechanisms underlying the preventive role of MT in sepsisinduced renal injury were investigated. Hematoxylin and eosin staining was used to detect the effect of MT on the reduction of renal tissue damage, and immunohistochemistry (IHC), ELISA and western blot analysis were performed to determine the influence of MT on the protein expression of PTENinduced putative kinase 1 (PINK1), nucleotideoligomerization binding domain and leucinerich repeat pyrin domaincontaining 3 (NLRP3), apoptosisassociated specklike protein containing a Cterminal caspase recruitment domain (ASC1), interleukin (IL)18, IL1ß, IL6 and cleaved caspase1. Finally, a TUNEL assay was used to compare the rate of apoptosis of renal tissues among the sham, cecal ligation and puncture (CLP), and CLP + MT groups. The extent of tissue damage in the CLP group was the highest and the extent of tissue damage in the sham group was the lowest. The IHC and western blot analysis showed that the sham group had the highest protein level of PINK1, whereas the CLP group had the lowest protein level of PINK1. By contrast, the sham group had the lowest protein level of NLRP, whereas the CLP group had the highest level of NLRP3. Furthermore, CLP treatment enhanced the protein expression of ASC1 and cleaved caspase1, whereas the administration of MT reduced the protein expression of ASC1 and cleaved caspase1 to a certain degree. Finally, the apoptotic rate was found to be the highest in the CLP group and the lowest in the sham group. Taken together, in evaluating the therapeutic effect of MT on sepsisinduced renal injury, the results of the present study showed that MT alleviated sepsisinduced renal injury by regulating the expression of PINK1, Parkin1, NLRP3, ASC and cleaved caspase1 in rats.
Assuntos
Nefropatias/etiologia , Nefropatias/metabolismo , Melatonina/farmacologia , Substâncias Protetoras/farmacologia , Proteínas Quinases/metabolismo , Sepse/complicações , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Biópsia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Nefropatias/tratamento farmacológico , Testes de Função Renal , RatosRESUMO
Synovitis is an aseptic inflammation that leads to joint effusion, pain and swelling. As one of the main drivers of pathogenesis in osteoarthritis (OA), the presence of synovitis contributes to pain, incidence and progression of OA. In our previous study, DC32 [(9α,12α-dihydroartemisinyl) bis(2'-chlorocinnmate)], a dihydroartemisinin derivative, was found to have an antirheumatic ability via immunosuppression, but the effect of DC32 on synovitis has not been fully illuminated. In this study, we chose to evaluate the effect and mechanism of DC32 on attenuating synovial inflammation. Fibroblast-like synoviocytes (FLSs) of papain-induced OA rats were isolated and cultured. And DC32 significantly inhibited the invasion and migration of cultured OA-FLSs, as well as the transcription of IL-6, IL-1ß, CXCL12 and CX3CL1 in cultured OA-FLSs measured by qPCR. DC32 remarkably inhibited the activation of ERK and NF-κB pathway, increased the expression of Nrf2 and HO-1 in cultured OA-FLSs detected by western blot. DC32 inhibited the degradation and phosphorylation of IκBα which further prevented the phosphorylation of NF-κB p65 and the effect of DC32 could be relieved by siRNA for Nrf2. In papain-induced OA mice, DC32 significantly alleviated papain-induced mechanical allodynia, knee joint swelling and infiltration of inflammatory cell in synovium. DC32 upregulated the mRNA expression of Type II collagen and aggrecan, and downregulated the mRNA expression of MMP2, MMP3, MMP13 and ADAMTS-5 in the knee joints of papain-induced OA mice measured by qPCR. The level of TNF-α in the serum and secretion of TNF-α in the knee joints were also reduced by DC32 in papain-induced OA mice. In conclusion, DC32 inhibited the inflammatory response in osteoarthritic synovium through regulating Nrf2/NF-κB pathway and attenuated OA. In this way, DC32 may be a potential agent in the treatment of OA.
Assuntos
Antirreumáticos/uso terapêutico , Artemisininas/uso terapêutico , Inflamação/tratamento farmacológico , Osteoartrite/tratamento farmacológico , Membrana Sinovial/imunologia , Sinoviócitos/fisiologia , Animais , Movimento Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Norverapamil, the N-demethylated derivative of verapamil, is a novel promising leading compound for attenuating multidrug resistance with less side effects compared with verapamil. However, the efficient synthetic method for norverapamil is absent. In this study, an innovative biotechnological method based on enzymatic catalysis was presented for the high-efficient production of norverapamil. CYP105D1, a cytochrome P450 from Streptomyces griseus ATCC 13273, was identified to carry out a one-step specific N-demethylation of verapamil along with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) as the redox partner. Docking calculations rationalized the specific N-demethylation observed in experiment and identified important amino acid residues for verapamil binding. Furthermore, a CYP105D1-based whole-cell system in E. coli BL21(DE3) was established and optimized for highly efficient N-demethylation of verapamil. The bioconversion rate of verapamil by the whole cell system came up to 60.16% within 24 hours under the optimized conditions. These results demonstrated the high potential of CYP105D1-based biocatalytic system for norverapamil production.