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The management and therapy of bone cancer pain (BCP) remain formidable clinical challenges. Curcumin and its analogues have been shown to have anti-inflammatory and analgesic properties. In the present study, we investigated the efficacy of curcumin analogue NL04 (NL04) in modulating inflammation in spinal dorsal horn (SDH), thereby exploring its potential to reduce central sensitization of BCP in a rat model. Differing doses of NL04 and curcumin were administered intrathecally either once (on day 12 of BCP) or over seven consecutive days (from day 6-12 of BCP). Results indicated that the ED50 for NL04 and curcumin ameliorating BCP-induced mechanical hyperalgesia is 49.08 µg/kg and 489.6 µg/kg, respectively. The analgesic effects at various doses of NL04 lasted between 4 and 8 h, with sustained administration over a week maintaining pain relief for 1-4 days, while also ameliorating locomotor gait via gait analysis and reducing depressive and anxiety-like behaviors via open-field and light-dark transition tests. The analgesic effects at various doses of curcumin lasted 4 h, with sustained administration over a week maintaining pain relief for 0-2 days. ELISA, Western blotting, qPCR, and immunofluorescence assays substantiated that intrathecal administration of NL04 on days 6-12 of BCP dose-dependently lowered spinal IL-1ß and IL-18 levels and significantly reduced the expression of IKKß genes and proteins, as well as the downstream cleavage of the trans-Golgi network (TGN). Whole-cell patch-clamp results demonstrated that NL04 inhibits potassium ion efflux in rat primary spinal neurons. Thus, NL04 exhibits significant analgesic effects in a BCP rat model by downregulating IKKß expression and inhibiting neuronal potassium ion efflux, which, in turn, suppresses the activation of NLRP3 inflammasomes and reduces IL-1ß production, potentially ameliorating pain management in BCP.
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Neoplasias Óseas , Dolor en Cáncer , Curcumina , Ratas , Animales , Dolor en Cáncer/tratamiento farmacológico , Dolor en Cáncer/metabolismo , Curcumina/farmacología , Curcumina/uso terapéutico , Curcumina/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Sensibilización del Sistema Nervioso Central , Quinasa I-kappa B/metabolismo , Dolor/tratamiento farmacológico , Neoplasias Óseas/complicaciones , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Analgésicos/farmacología , Analgésicos/uso terapéutico , Analgésicos/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Médula Espinal , Potasio/metabolismoRESUMEN
Background: Bone cancer pain (BCP) represents one of the most challenging comorbidities associated with cancer metastasis. Long non-coding RNAs (lncRNAs) have garnered attention as potential therapeutic agents in managing neuropathic pain. However, their role in the regulation of nociceptive information processing remains poorly understood. In this study, we observed a significant down-regulation of the spinal lncRNA ENSRNOG00000051325 (lncRNA51325) in a rat model of bone cancer pain. Our study sought to elucidate the potential involvement of lncRNA51325 in the development of BCP by modulating the expression of molecules associated with pain modulation. Methods: We established the BCP model by injecting Walker 256 cells into the tibial plateau of rats. We conducted tests on the pain behaviors and anxiety-like responses of rats through von-Frey test, Gait analysis, and Open Field Test. Spinal lumbar expansion was harvested for molecular biology experiments to explore the relationship between lncRNA51325 and Pumilio RNA binding family member 2 (Pum2). Results: Notably, the overexpression of lncRNA51325 effectively attenuated mechanical allodynia in rats afflicted with BCP, whereas the knockdown of lncRNA51325 induced pain behaviors and anxiety-like responses in naïve rats. Additionally, we observed a time-dependent increase in the expression of Pum2 in BCP-afflicted rats, and intrathecal injection of Pum2-siRNA alleviated hyperalgesia. Furthermore, our investigations revealed that lncRNA51325 exerts a negative modulatory effect on Pum2 expression. The overexpression of lncRNA51325 significantly suppressed Pum2 expression in BCP rats, while the knockdown of lncRNA51325 led to elevated Pum2 protein levels in the spinal cord of naïve rats. Subsequent treatment with Pum2-siRNA mitigated the downregulation of lncRNA51325-induced mechanical allodynia in naïve rats. Conclusion: Our findings indicate that lncRNA51325 plays a role in regulating bone cancer pain by inhibiting Pum2 expression, offering a promising avenue for novel treatments targeting nociceptive hypersensitivity induced by bone metastatic cancer.
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PURPOSE: The aim of this study was to investigate the success rate of surgical technique and associated risk factors based on the follow-up of patients who underwent CT-guided percutaneous radiofrequency sympathectomy (RFS) to treat primary hyperhidrosis (PH). METHODS: 370 patients who underwent RFS for PH treatment between January 2018 and April 2022 were enrolled. Patients responded to a questionnaire and telephone follow-up on the effects of treatment and their electronic medical records and imaging findings were reviewed. Logistic regression was performed to identify risk factors related to the success rate of surgical technique. RESULTS: A temperature difference ≥2 °C before and after procedure was defined as a successful surgical technique. Among the 370 patients (740 sides), 637 sides had successful RFS, and the technical success rate was 86.1 %. Immediately after procedure, 636 sides (85.9 %) were completely dry, 64 (8.7 %) were partially dry, and 40 (5.4 %) were still wet. During the longest follow-up period (54 months), 103 patients relapsed. The median hyperhidrosis disease severity scale score on both sides decreased from 4 to 1 after RFS. According to logistic regression analysis, only the pre-post pulse index was associated with the success rate of surgical technique (odds ratio, 1.14; 95 % confidence interval, 1.06-1.23; p = 0.0004). CONCLUSIONS: We observed that the immediate efficiency and success rate of surgical technique after RFS for PH treatment were relatively high, although there is a possibility of recurrence in the long term. In general, RFS is a safe and effective procedure for alleviating the symptoms of patients with hyperhidrosis.
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Hiperhidrosis , Simpatectomía , Humanos , Hiperhidrosis/cirugía , Satisfacción del Paciente , Estudios Retrospectivos , Simpatectomía/efectos adversos , Simpatectomía/métodos , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Due to its complex pathological mechanisms, bone cancer pain (BCP) has become an increasingly challenging clinical issue, there is an urgent need to identify the underlying mechanisms of BCP. In our present study, we found that decreased expression of miR-199a-3p in spinal dorsal horn (SDH) neurons contributed to BCP hypersensitivity. Intrathecal administration of miR-199a-3p agomir alleviated the initiation of tumor inoculation induced pain hypersensitivity and suppressed the expression of DNMT3A. Subsequently, luciferase assays confirmed direct binding between miR-199a-3p and Dnmt3a mRNA. AAV-DNMT3A-shRNA microinjection relieved mechanical hyperalgesia and upregulated the expression of Nrf2 levels in BCP. In naïve rats, Overexpression of DNMT3A yielded the opposite effects. Finally, increase of DNMT3A by lentiviral vector abolished miR-199a-3p-mediated alleviation hypersensitivity effects on BCP progression. Taken these together, our findings highlighted a novel contribution of miR-199a-3p to BCP and provided a fresh outlook on potential mechanism research for BCP.
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Neoplasias Óseas , Dolor en Cáncer , MicroARNs , Osteosarcoma , Ratas , Animales , Dolor en Cáncer/genética , Dolor en Cáncer/metabolismo , Regulación hacia Arriba , Dolor/metabolismo , Neoplasias Óseas/complicaciones , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Células del Asta Posterior/metabolismo , Osteosarcoma/metabolismo , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Notch signal plays an important role in regulating cell-cell interactions with the adjacent cells. However, it remains unknown whether Jagged1 (JAG-1) mediated Notch signaling regulates bone cancer pain (BCP) via the spinal cell interactions mechanism. Here, we showed that intramedullary injection of Walker 256 breast cancer cells increased the expression of JAG-1 in spinal astrocytes and knockdown of JAG-1 reduced BCP. The supplementation of exogenous JAG-1 to the spinal cord induced BCP-like behavior and promoted expression of c-Fos and hairy and enhancer of split homolog-1 (Hes-1) in the spinal cord of the naïve rats. These effects were reversed when the rats were administered intrathecal injections of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The intrathecal injection of DAPT reduced BCP and inhibited Hes-1 and c-Fos expression in the spinal cord. Furthermore, our results showed that JAG-1 up-regulated Hes-1 expression by inducing the recruitment of Notch intracellular domain (NICD) to the RBP-J/CSL-binding site located within the Hes-1 promoter sequence. Finally, the intrathecal injection of c-Fos-antisense oligonucleotides (c-Fos-ASO) and administration of sh-Hes-1 to the spinal dorsal horn also alleviated BCP. The study indicates that inhibition of the JAG-1/Notch signaling axis may be a potential strategy for the treatment of BCP.
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Neoplasias Óseas , Dolor en Cáncer , Ratas , Animales , Dolor en Cáncer/etiología , Neoplasias Óseas/complicaciones , Transducción de Señal/fisiología , Dolor , Médula EspinalRESUMEN
ABSTRACT: Bone cancer pain (BCP) is a pervasive clinical symptom which impairs the quality life. Long noncoding RNAs (lncRNAs) are enriched in the central nervous system and play indispensable roles in numerous biological processes, while its regulatory function in nociceptive information processing remains elusive. Here, we reported that functional modulatory role of ENSRNOT00000071132 (lncRNA71132) in the BCP process and sponging with miR-143 and its downstream GPR85-dependent signaling cascade. Spinal lncRNA71132 was remarkably increased in the rat model of bone cancer pain. The knockdown of spinal lncRNA71132 reverted BCP behaviors and spinal c-Fos neuronal sensitization. Overexpression of spinal lncRNA71132 in naive rat generated pain behaviors, which were accompanied by increased spinal c-Fos neuronal sensitization. Furthermore, it was found that lncRNA71132 participates in the modulation of BCP by inversely regulating the processing of miR-143-5p. In addition, an increase in expression of spinal lncRNA71132 resulted in the decrease in expression of miR-143 under the BCP state. Finally, it was found that miR-143-5p regulates pain behaviors by targeting GPR85. Overexpression of miR-143-5p in the spinal cord reverted the nociceptive behaviors triggered by BCP, accompanied by a decrease in expression of spinal GPR85 protein, but no influence on expression of gpr85 mRNA. The findings of this study indicate that lncRNA71132 works as a miRNA sponge in miR-143-5p-mediated posttranscriptional modulation of GPR85 expression in BCP. Therefore, epigenetic interventions against lncRNA71132 may potentially work as novel treatment avenues in treating nociceptive hypersensitivity triggered by bone cancer.
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Neoplasias Óseas , Dolor en Cáncer , MicroARNs , Animales , Ratas , Neoplasias Óseas/complicaciones , Neoplasias Óseas/genética , Dolor en Cáncer/genética , Dolor en Cáncer/complicaciones , MicroARNs/genética , MicroARNs/metabolismo , Dolor/metabolismo , Médula Espinal/metabolismo , Regulación hacia Arriba , ARN Largo no Codificante/genéticaRESUMEN
Chronic, inflammatory pain is an international health concern that severely diminishes individuals' quality of life. Catalpol is an iridoid glycoside derived from the roots of Rehmannia glutinosa that possesses anti-inflammatory, antioxidant, and neuroprotective properties for the treating multiple kinds of disorders. Nevertheless, catalpol's impacts on inflammatory pain and its potential methods of action are still unclear. The purpose of this investigation is to determine the mechanism of catalpol to reduce the inflammatory pain behaviors in a rat model with complete Freund's adjuvant (CFA). Catwalk, Von-Frey, and open field testing were performed for behavioral assessment. Western blot analysis and real-time quantitative PCR (RT-PCR) were employed to identify variations in molecular expression, while immunofluorescence was utilized to identify cellular localization. Catalpol effectively reduced CFA-induced mechanical allodynia and thermal hyperalgesia when injected intrathecally. Moreover, catalpol can regulate the HDAC4/PPAR-γ-signaling pathway in CFA rat spinal cord neurons. Meanwhile catalpol significantly decreased the expression of the NF-κB/NLRP3 inflammatory axis in the spinal cord of CFA rats. In addition, both in vivo and in vitro research revealed that catalpol treatment inhibited astrocyte activation and increase inflammatory factor expression. Interestingly, we also found that catalpol could alleviate peripheral pain by inhibiting tissue inflammation. Taken together, the findings declared that catalpol may inhibit inflammatory pain in CFA rats by targeting spinal cord and peripheral inflammation.
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Bone cancer pain (BCP) is a clinically intractable mixed pain, involving inflammation and neuropathic pain, and its mechanisms remain unclear. CXC chemokine receptor 1 (CXCR1, IL-8RA) and 2 (CXCR2, IL-8RB) are high-affinity receptors for interleukin 8 (IL8). According to previous studies, CXCR2 plays a crucial role in BCP between astrocytes and neurons, while the role of CXCR1 remains unclear. The objective of this study was to investigate the role of CXCR1 in BCP. We found that CXCR1 expression increased in the spinal dorsal horn. Intrathecal injection of CXCR1 siRNA effectively attenuated mechanical allodynia and pain-related behaviors in rats. It was found that CXCR1 was predominantly co-localized with neurons. Intrathecal injection of CXCR1-siRNA reduced phosphorylated JAK2/STAT3 protein levels and the NLRP3 inflammasome (NLRP3, caspase1, and IL-1ß) levels. Furthermore, in vitro cytological experiments confirmed this conclusion. The study results suggest that the spinal chemokine receptor CXCR1 activation mediates BCP through JAK2/STAT3 signaling pathway and NLRP3 inflammasome (NLRP3, caspase1, and IL-1ß).
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Neoplasias Óseas , Dolor en Cáncer , Neuralgia , Ratas , Femenino , Animales , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Dolor en Cáncer/etiología , Dolor en Cáncer/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , ARN Interferente Pequeño/metabolismo , Neoplasias Óseas/complicaciones , Neoplasias Óseas/metabolismo , Receptores de Interleucina-8B/metabolismo , Neuralgia/metabolismo , Médula Espinal/metabolismoRESUMEN
BACKGROUND: Noncoding microRNAs have emerged as critical players of gene expression in the nervous system, where they contribute to regulating nervous disease. As stated in previous research, the miR-155-5p upregulation happens in the spinal cord at the nociceptive state. It was unclear if miR-155-5p is linked to bone cancer pain (BCP). Herein, we aimed at investigating the miR-155-5p functional regulatory function in BCP process and delineating the underlying mechanism. METHODS: The miRNA-155-5p levels and cellular distribution were determined by RNA sequencing, fluorescent in situ hybridization (FISH), and quantitative real-time PCR (qPCR). Immunoblotting, qPCR, dual-luciferase reporter gene assays, immunofluorescence, recombinant overexpression adeno-associated virus, small interfering RNA, intraspinal administration, and behavioral tests were utilized for exploring the downstream signaling pathway. RESULTS: The miR-155-5p high expression in spinal neurons contributes to BCP maintenance. The miR-155-5p blockage via the intrathecal injection of miR-155-5p antagomir alleviated the pain behavior; in contrast, upregulating miR-155-5p by agomir induced pain hypersensitivity. The miR-155-5p bounds directly to TCF4 mRNA's 3' UTR. BCP significantly reduced protein expression of TCF4 versus the Sham group. The miR-155-5p inhibition relieved the spinal TCF4 protein's down-expression level, while miR-155-5p upregulation by miR-155-5p agomir intrathecal injection decreased TCF4 protein expression in naïve rats. Additionally, TCF4 overexpression in BCP rats could increase Kv1.1. Moreover, TCF4 knockdown inhibited Kv1.1 expression in BCP rats. Indeed, TCF4 and Kv1.1 were co-expressed in BCP spinal cord neurons. CONCLUSION: The study findings stated the miR-155-5p pivotal role in regulating BCP by directly targeting TCF4 in spinal neurons and suggested that miR-155-5p could be a promising target in treating BCP.
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Neoplasias Óseas , Dolor en Cáncer , MicroARNs , Regiones no Traducidas 3' , Animales , Antagomirs/metabolismo , Neoplasias Óseas/complicaciones , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Dolor en Cáncer/genética , Dolor en Cáncer/metabolismo , Hibridación Fluorescente in Situ , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Dolor/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Médula Espinal/metabolismoRESUMEN
Bone cancer pain (BCP) is a serious clinical problem that affects the quality of life of cancer patients. However, the current treatment methods for this condition are still unsatisfactory. This study investigated whether intrathecal injection of rosiglitazone modulates the noxious behaviors associated with BCP, and the possible mechanisms related to this effect were explored. We found that rosiglitazone treatment relieved bone cancer-induced mechanical hyperalgesia in a dose-dependent manner, promoted the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in spinal cord neurons, and inhibited the activation of the nuclear factor-kappa B (NF-κB)/nod-like receptor protein 3 (NLRP3) inflammatory axis induced by BCP. However, concurrent administration of the PPAR-γ antagonist GW9662 reversed these effects. The results show that rosiglitazone inhibits the NF-κB/NLRP3 inflammation axis by activating PPAR-γ in spinal neurons, thereby alleviating BCP. Therefore, the PPAR-γ/NF-κB/NLRP3 signaling pathway may be a potential target for the treatment of BCP in the future.
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Electroacupuncture (EA) is effective in various chronic pains. NF-κB and CXCL12 modulate the formation of chronic pain. Herein, we hypothesized that EA alleviates cancer-induced bone pain (CIBP) through NF-κB/CXCL12 axis in midbrain periaqueductal gray (PAG), which participates in "top-down" pain modulatory circuits. In order to filter the optimum EA frequency for CIBP treatment, 2, 100, or 2/100 Hz EA was set up. In addition, ipsilateral, contralateral, and bilateral EA groups were established to affirm the optimal EA scheme. Bilateral 2/100 Hz EA was considered as the optimal therapeutic scheme and was applied in a subsequent experiment. Western blotting along with immunofluorescence illustrated that CIBP induces a rapid and substantial increase in CXCL12 protein level and NF-κB phosphorylation in vlPAG from day 6 to day 12. Anti-CXCL12 neutralizing antibody and pAAV-U6-shRNA(CXCL12)-CMV-EGFP-WPRE in vlPAG remarkably improved the mechanical pain threshold of the hind paw in CIBP model relative to the control. EA inhibited the upregulation of pNF-κB and CXCL12 in vlPAG of CIBP. The recombinant CXCL12 and pAAV-CMV-CXCL12-EF1a-EGFP-3Xflag-WPRE reversed the abirritation of EA in the CIBP rat model. NF-κB phosphorylation mediated-CXCL12 expression contributed to CIBP allodynia, whereas EA suppressed NF-κB phosphorylation in CIBP. According to the above evidence, we conclude that bilateral 2/100 Hz EA is an optimal therapeutic scheme for CIBP. The abirritation mechanism of EA might reduce the expression of CXCL12 by inhibiting the activation of NF-κB, which might lead to the restraint of descending facilitation of CIBP.
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Electroacupuntura , Neoplasias , Animales , FN-kappa B/metabolismo , Umbral del Dolor , Sustancia Gris Periacueductal/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Due to the efficacy and tolerability of the available drugs, the current treatment for cancer-induced bone pain (CIBP) is not considered ideal, and new drugs are required for better treatment results. This study investigated whether intrathecal injection of sulforaphane (SFN) can modulates the noxious behavior associated with CIBP and enhances the analgesic effects of morphine and the possible mechanisms related to these effects were investigated. Walker256 breast cancer cells were injected into the bone marrow cavity of rats to establish the CIBP model. When CIBP rats began to exhibit painful behavior (CIBP 6 days), SFN was injected intrathecally for 7 days. The results showed that SFN alleviated the painful behavioral hypersensitivity caused by cancer, accompanied by nuclear factor, erythroid 2 like 2 (Nrf2), Haem oxygenase 1 (HO-1) activation, nuclear factor kappa B (NF-κB) inhibition and inflammation-related factors (tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-ß), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) reduction. In addition, SFN treatment inhibited the proliferation of Walker 256 cells in a dose-dependent manner, promoted mu-opioid receptor (MOR) expression in SH-SY5Y cells and enhanced the antihyperalgesic effects of morphine on CIBP rats by restoring the downregulation of MOR expression in the spinal cord. Interestingly, the antihyperalgesic effects of SFN were partially blocked by opioid receptor antagonists. This study showed that SFN combined with morphine might be a new way to treat CIBP.
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Neoplasias Óseas/complicaciones , Dolor en Cáncer/tratamiento farmacológico , Hiperalgesia/tratamiento farmacológico , Isotiocianatos/farmacología , Morfina/farmacología , Sulfóxidos/farmacología , Animales , Neoplasias Óseas/secundario , Dolor en Cáncer/etiología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Humanos , Hiperalgesia/etiología , Isotiocianatos/uso terapéutico , Morfina/uso terapéutico , Ratas , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Asta Dorsal de la Médula Espinal/metabolismo , Sulfóxidos/uso terapéuticoRESUMEN
Bone cancer pain (BCP) is a clinical pathology that urgently needs to be solved, but research on the mechanism of BCP has so far achieved limited success. Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) has been shown to be involved in pain, but its involvement in BCP and the specific mechanism have yet to be examined. This study aimed to test the hypothesis that BCP induces the transfer of Nrf2 from the cytoplasm to the nucleus and further promotes nuclear transcription to activate heme oxygenase-1 (HO-1) and inhibit the activation of nuclear factor-kappa B (NF-κB) signalling, ultimately regulating the neuroinflammatory response. Von-Frey was used for behavioural analysis in rats with BCP, whereas western blotting, real-time quantitative PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect molecular expression changes, and immunofluorescence was used to detect cellular localization. We demonstrated that BCP induced increased Nrf2 nuclear protein expression with decreased cytoplasmic protein expression in the spinal cord. Further increases in Nrf2 nuclear protein expression can alleviate hyperalgesia and activate HO-1 to inhibit the expression of NF-κB nuclear protein and inflammatory factors. Strikingly, intrathecal administration of the corresponding siRNA reversed the above effects. In addition, the results of double immune labelling revealed that Nrf2 and NF-κB were coexpressed in spinal cord neurons of rats with BCP. In summary, these findings suggest that the entry of Nrf2 into the nucleus promotes the expression of HO-1, inhibiting activation of the NF-κB signalling pathway, reducing neuroinflammation and ultimately exerting an anti-nociceptive effect.
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Neoplasias Óseas/metabolismo , Dolor en Cáncer/metabolismo , Hiperalgesia/metabolismo , Factor 2 Relacionado con NF-E2/biosíntesis , FN-kappa B/metabolismo , Médula Espinal/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Neoplasias Óseas/patología , Dolor en Cáncer/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Femenino , Hiperalgesia/patología , FN-kappa B/antagonistas & inhibidores , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Médula Espinal/patologíaRESUMEN
Background: Diisoheptyl phthalate (DIHP) is a phthalate plasticizer, which is a branched phthalate. Here, we reported the effects of gestational exposure to DIHP on testis development in male rats. Methods: Pregnant Sprague-Dawley rats were orally fed with vehicle (corn oil, control) or DIHP (10, 100, 500, and 1,000 mg/kg) from gestational day (GD) 12-21. At GD21, serum testosterone levels, the number and distribution of fetal Leydig cells, and testicular mRNA and protein levels, the incidence of multinucleated gonocytes, and focal testicular hypoplasia in the neonatal testis were measured. Results: DIHP increased the fetal Leydig cell cluster size and decreased the fetal Leydig cell size with LOAEL of 10 mg/kg. DIHP did not affect the fetal Leydig cell number. DIHP significantly lowered serum testosterone levels, down-regulated the expression of steroidogenesis-related genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and testis descent-related gene (Insl3) as well as protein levels of cholesterol side-chain cleavage enzyme (CYP11A1) and insulin-like 3 (INSL3). DIHP dose-dependently increased the percentage of multinucleated gonocytes with the low observed adverse-effect level (LOAEL) of 100 mg/kg. DIHP induced focal testicular hypoplasia. Conclusion: Gestational exposure to DIHP causes testis dysgenesis in rats.
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Perfluorododecanoic acid (PFDoA) is an endocrine-damaging compound in contaminated food and water. However, the potential role and underlying mechanism of PFDoA in Leydig cell regeneration from stem Leydig cells remain unclear. The current study aims to investigate the effect of PFDoA on the regeneration of Leydig cells in the testis of rats treated with ethylene dimethane sulfonate (EDS). PFDoA (0, 5 or 10 mg/kg/day) was gavaged to adult Sprague-Dawley male rats for 8 days, and 75 mg/kg EDS was intraperitoneally injected to eliminate Leydig cells to initiate its regeneration from day 21-56 after EDS. The serum testosterone levels in the 5 and 10 mg/kg/day PFDoA groups were significantly reduced at day 21 after EDS and the levels of serum luteinizing hormone and follicle-stimulating hormone were significantly decreased in the 10 mg/kg/day PFDoA groups at day 56 after EDS. PFDoA significantly reduced Leydig cell number and proliferation at a dose of 10 mg/kg at days 21 and 56 after EDS. PFDoA significantly down-regulated the expression of Leydig cell-specific genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1 and Cyp17a1) and their proteins at both doses at days 21 and 56 after EDS. PFDoA significantly down-regulated the gene expression of Sertoli cells (Fshr, Dhh, and Sox9) at 5 mg/kg or higher at days 21 and 56 after EDS. In addition, we found that PFDoA significantly inhibited EdU incorporation into putative stem Leydig cells and their differentiation into the Leydig cell lineage in vitro. In conclusion, short-term PFDoA exposure in adulthood delayed the regeneration of Leydig cells by preventing Leydig cells from stem cells via multiple mechanisms.
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Proliferación Celular/efectos de los fármacos , Ácidos Láuricos/toxicidad , Células Intersticiales del Testículo/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Fluorocarburos , Histonas/metabolismo , Humanos , Células Intersticiales del Testículo/citología , Masculino , Metilación , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Recuento de Espermatozoides , Células Madre/citología , Testículo/efectos de los fármacos , Testosterona/sangre , Testosterona/metabolismoRESUMEN
As a cis-acting non-depolarizing neuromuscular blocker through a nicotinic acetylcholine receptor (nAChR), cisatracurium (CAC) is widely used in anaesthesia and intensive care units. nAChR may be present on Leydig cells to mediate the action of CAC. Here, by Western blotting, immunohistochemistry and immunofluorescence, we identified that CHRNA4 (a subunit of nAChR) exists only on rat adult Leydig cells. We studied the effect of CAC on the synthesis of testosterone in rat adult Leydig cells and mouse MLTC-1 tumour cells. Rat Leydig cells and MLTC-1 cells were treated with CAC (5, 10 and 50 µmol/L) or nAChR agonists (50 µmol/L nicotine or 50 µmol/L lobeline) for 12 hours, respectively. We found that CAC significantly increased testosterone output in rat Leydig cells and mouse MLTC-1 cells at 5 µmol/L and higher concentrations. However, nicotine and lobeline inhibited testosterone synthesis. CAC increased intracellular cAMP levels, and nicotine and lobeline reversed this change in rat Leydig cells. CAC may increase testosterone synthesis in rat Leydig cells and mouse MLTC-1 cells by up-regulating the expression of Lhcgr and Star. Up-regulation of Scarb1 and Hsd3b1 expression by CAC was also observed in rat Leydig cells. In addition to cAMP signal transduction, CAC can induce ERK1/2 phosphorylation in rat Leydig cells. In conclusion, CAC binds to nAChR on Leydig cells, and activates cAMP and ERK1/2 phosphorylation, thereby up-regulating the expression of key genes and proteins in the steroidogenic cascade, resulting in increased testosterone synthesis in Leydig cells.
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Atracurio/análogos & derivados , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Receptores Nicotínicos/metabolismo , Testosterona/biosíntesis , Animales , Atracurio/farmacología , Biomarcadores , Vías Biosintéticas/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Hormona Luteinizante/metabolismo , Hormona Luteinizante/farmacología , Masculino , Ratones , Fosforilación , Ratas , Receptores Nicotínicos/genética , Esteroides/biosíntesis , Testículo/metabolismoRESUMEN
Di-n-butyl phthalate (DBP) is widely used in consumer products as a plasticizer. Here, we report a natural product taxifolin that can attenuate developmental and reproductive toxicity of DBP. Pregnant rats were daily gavaged with 500 mg/kg DBP alone or together with taxifolin (10 and 20 mg/kg) from gestational day (GD) 12-21. At GD21, sera and testes of male fetus were collected. DBP significantly lowered serum testosterone level at 500 mg/kg and taxifolin can completely reverse its action. DBP caused abnormal aggregation of fetal Leydig cells and taxifolin can reverse it. DBP down-regulated the expression of the genes of cholesterol side-chain cleavage enzyme (Cyp11a1), 17ß-hydroxysteroid dehydrogenase 3 (Hsd17b3), and insulin-like 3 (Insl3) and taxifolin can reverse its action. DBP increased malondialdehyde levels and decreased superoxide dismutase and glutathione peroxidase expression and taxifolin can reverse it. DBP increased incidence of multinucleated gonocytes and taxifolin can prevent it. Moreover, DBP lowered sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and phosphorylated AMP-activated protein kinase (pAMPK) signalling and taxifolin antagonized DBP. In conclusion, in utero exposure to DBP caused developmental/reproductive toxicity of male offspring via increasing reactive oxygen species and taxifolin is an effective food component that completely reverses DBP-mediated action.
Asunto(s)
Antioxidantes/farmacología , Dibutil Ftalato/toxicidad , Plastificantes/toxicidad , Quercetina/análogos & derivados , Testículo/efectos de los fármacos , Animales , Femenino , Masculino , Exposición Materna , Quercetina/farmacología , Ratas , Ratas Sprague-Dawley , Testículo/embriologíaRESUMEN
Male fetal Leydig cells in the testis secrete androgen and insulin-like 3, determining the sexual differentiation. The abnormal development of fetal Leydig cells could lead to the reduction of androgen and insulin-like 3, thus causing the male reproductive tract anomalies in male neonates, including cryptorchidism and hypospadias. Environmental pollutants, such as phthalic acid esters (phthalates), can perturb the development and differentiated function of Leydig cells, thereby contributing to the reproductive toxicity in the male. Here, we review the epidemiological studies in humans and experimental investigations in rodents of various phthalates. Most of phthalates disturb the expression of various genes encoded for steroidogenesis-related proteins and insulin-like 3 in fetal Leydig cells and the dose-additive effects are exerted after exposure in a mixture.
RESUMEN
Background: Propofol is a widely used anesthetic. Whether propofol inhibits androgen production by rat Leydig cells and the underlying mechanism remains unclear. The objective of the current study was to examine the effects of propofol exposure to rat primary immature Leydig cells and to define propofol-induced inhibition of steroidogenic enzymes in both rat and human testes in vitro. Methods: Immature Leydig cells were purified from 35-day-old male Sprague-Dawley rats and were exposed to propofol for 3 h. The androgen production by Leydig cells under basal, luteinizing hormone, 8bromo-cAMP, and steroid-substrate stimulated conditions and gene expression of Leydig cells after exposure to propofol were measured. Immature Leydig cells were treated with propofol for 3 h and switched to propofol-free medium for additional 3 and 9 h to test whether propofol-induced inhibition is reversible. 3H-Steroids were used to evaluate the direct action of propofol on cytochrome P450 cholesterol side chain cleavage (CYP11A1), 3ß-hydroxysteroid dehydrogenase (HSD3B), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1), and 17ß-hydroxysteroid dehydrogenase 3 (HSD17B3) activities in rat and human testes in vitro. Results: Propofol significantly lowered luteinizing hormone and 8bromo-cAMP stimulated androgen production by Leydig cells after 3-h exposure. Further investigation showed that propofol down-regulated the expression of Cyp11a1 and Cyp17a1 and their proteins at 5 and 50 µM, although it up-regulated Lhcgr expression at 50 µM. Propofol significantly suppressed phosphorylation of ERK1/2 and induced ROS production in immature Leydig cells at 5 and 50 µM. Propofol significantly induced apoptosis of immature Leydig cells at 50 µM. Propofol specifically inhibited rat and human testis HSD3B activities in vitro. The half maximal inhibitory concentrations of propofol for rat and human HSD3B enzymes were 1.011 ± 0.065 and 3.498 ± 0.067 µM, respectively. The mode of action of propofol of inhibiting HSD3B was competitive when pregnenolone was added. At 50 µM, propofol did not directly inhibit rat and human testis CYP11A1, CYP17A1, and HSD17B3 activities in vitro. Conclusion: Propofol inhibits androgen production via both directly inhibiting HSD3B activity and down-regulating Cyp11a1 and Cyp17a1 expression in Leydig cells. Suppression of steroidogenic enzymes is presumably associated with the lower production of androgen by Leydig cells after propofol treatment. However, propofol-induced inhibition on androgen production is reversible.
RESUMEN
Flurbiprofen is one of the nonsteroidal anti-inflammatory drugs. Whether flurbiprofen affects androgen biosynthesis in Leydig cells is still unknown. Immature Leydig cells (ILCs) isolated from 35-day-old male Sprague-Dawley rats were cultured with 0-100 µM flurbiprofen for 24 h and medium androgen levels and Leydig cell mRNA levels were measured. Immature Leydig cells were also incubated with 100 µM flurbiprofen for 3 h in combination with luteinizing hormone (LH), 8bromo-cAMP, 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively, and medium androgen levels were measured. The ROS generation and apoptosis rate were also investigated. The direct effects of flurbiprofen on androgen biosynthetic and metabolizing enzyme activities were measured. Flurbiprofen significantly inhibited basal, LH, and 8bromo-cAMP stimulated androgen production at 10 and 100 µM. Further study demonstrated that flurbiprofen competitively inhibited rat and human testis 3ß-hydroxysteroid dehydrogenase (HSD3B) activity with the half maximal inhibitory concentration (IC50) values of 0.95 µM for rat enzyme and 6.31 µM for human enzyme. In addition, flurbiprofen down-regulated the expression of Srd5a1 and Akr1c14 at 1, 10, and 100 µM. Flurbiprofen also down-regulated Lhcgr expression at 100 µM. Flurbiprofen at 10 and 100 µM increased ROS production and apoptosis rate of rat Leydig cells. In conclusion, flurbiprofen directly inhibits HSD3B activity and the expression levels of Srd5a1 and Akr1c14 in rat Leydig cells, thus leading to the reduction of androgen secretion.