Effect of antioxidant and Pro-oxidant on bone mineralization and remodelling

Osteoporosis is characterized by abnormal bone with low bone mass and degradation of skeleton microarchitecture, thus leading to bone fragility and risk of fracture. Oxidative stress induces an imbalance in osteoblast and osteoclast activity that leads to bone degradation, consequently resulting in osteoporotic phenotype. In addition, oxidative stress is a primary cause of secondary osteoporosis caused by specific medications. Understanding the role of oxidative stress in the development of primary and secondary osteoporosis could lead to further research towards preventive and therapeutic measures to combat this significant cause of mortality and morbidity worldwide. Antioxidant supplementation has improved bone mineral density and lowered the risk of fragility fractures. While it is not experimentally evident if the antioxidant activity is the cause of this alteration. Hence, this work aimed to counteract/rescue specific medication-induced secondary osteoporosis by supplementing antioxidants on in vitro and in vivo models. In addition, to provide insights into the molecular mechanisms on medication-induced bone impairment, the transcriptome of murine osteoblasts treated with antioxidants (Resveratrol and MitoTEMPO) and pro-oxidants (Doxorubicin) alone or in combination was analyzed. RNA-Seq data revealed that osteocrin and p53 are the responsible players on doxorubicin-induced bone impairment and its reversal by resveratrol. We further studied the effect of antioxidants and pro-oxidant on osteoclast differentiation, where we found out that doxorubicin also increased osteoclast differentiation. Based on our in vitro results, we confirm the effect of Resveratrol, MitoTEMPO on Doxorubicin-induced bone impairment with the zebrafish (osteocytic bone) and seabream (non-osteocytic bone) models. Our data indicate that regular supplementation of antioxidants effectively improves overall growth, mineralization and counteracts pro-oxidant induced bone pathologies on both models. In conclusion, this work proposes that the negative effect of the specific medication (i.e., Doxorubicin) can be reversed by regular supplementation of antioxidants (i.e., Resveratrol and MitoTEMPO). Furthermore, this work also proposes osteocrin as a key responsible factor for doxorubicin-induced bone impairment, and its reversal. Osteocrin can be further exploited as a communicator molecule for crosstalk between bone and other tissues.A osteoporose é uma doença caracterizada por um osso anormal com baixa massa óssea e pela degradação da microarquitetura do esqueleto, conduzindo assim à fragilidade óssea e a um elevado risco de fraturas. Existem várias causas descritas para a origem desta condição, sendo a mais comum a osteoporose primária ou senil, provocada pelo envelhecimento e que afeta principalmente as mulheres pós-menopausa, mas também os homens idosos. Representa cerca de 95% dos casos em mulheres e 80% em homens. A principal causa da osteoporose primaria é a diminuição dos níveis de estrogénio, particularmente a rápida redução que ocorre nas mulheres durante a menopausa, embora esta diminuição também ocorra nos homens com mais de 50 anos. A deficiência de estrogênio leva à rápida perda óssea. A osteoporose secundária representa menos de 5% dos casos em mulheres e cerca de 20% em homens e é causada por diversos fatores, como doenças, distúrbios hormonais, medicação, como corticosteroides ou fármacos quimioterápicos, ou pelo consumo de álcool e drogas. Entre os mecanismos que levam ao aparecimento da osteoporose secundária, associada a tratamentos terapêuticos com medicamentos específicos, está o stress oxidativo, que induz um desequilíbrio na atividade osteoblástica e osteoclástica que leva à degradação óssea, resultando consequentemente no fenótipo osteoporótico. Compreender o papel do stress oxidativo no desenvolvimento da osteoporose primária e secundária poderia levar a uma investigação mais aprofundada no sentido de medidas preventivas e terapêuticas para combater esta causa significativa de mortalidade e morbilidade em todo o mundo. Alguns estudos mostraram que a suplementação com antioxidantes levou a melhorias na densidade mineral óssea e diminuiu o risco de fraturas de fragilidade. Embora não existam evidências experimentais para provar que a atividade antioxidante foi a causa desta alteração. Por isso, o objetivo deste trabalho visou contrariar ou recuperar a osteoporose secundária induzida por medicação específica, através da suplementação com antioxidantes, utilizando para tal a modelos in vitro e in vivo. Adicionalmente, para procurar identificar quais os mecanismos moleculares sobre a deficiência óssea induzida pela medicação, foi realizada uma análise ao transcritoma de células osteoblásticas de ratinho (MC3T3-E1) tratadas com antioxidantes (Resveratrol; RES e MitoTEMPO; MT) ou com pró-oxidantes (Doxorrubicina; DOX) sozinhos ou em combinações entre estes. Os dados da sequenciação do ARN revelaram que a osteocrina e o p53 são as moléculas intervenientes responsáveis pela deficiência óssea induzida pela DOX e estão também envolvidas na sua reversão destes efeitos pelo co-tratamento com RES. Estudamos ainda a diferenciação de células de ratinho RAW 264.7 na linhagem osteoclástica, de forma a determinar os efeitos e investigar os mecanismos da diferenciação osteoclástica induzida pelo pró-oxidante DOX. Foi utilizado o ativador de Sirt 1 resveratrol (RES) para neutralizar os efeitos induzidos pela DOX. As células RAW 264.7 foram diferenciadas em osteoclastos sob co-tratamento com DOX e RES. Foi observado que a DOX aumentou a diferenciação de osteoclastos in vitro, ao passo que o tratamento com RES inibiu a diferenciação osteoclástica induzida pelo DOX, mas tal não se verificou com MT. O tratamento com RES reduziu a expressão do marcador de fusão osteoclástica Oc-stamp e dos marcadores de diferenciação Rank, Trap, Ctsk e Nfatc1. Inversamente, o tratamento com RES induziu a sobre expressão dos genes antioxidantes Sod 1 e Nrf 2, enquanto DOX reduziu significativamente a expressão FoxM1, resultando em stress oxidativo. A utilização da linha transgénica de peixe zebra repórter para catepsina K (Tg[ctsk:DsRed]) permitiu verificar o aumento da expressão in vivo de ctsk significativamente o sinal de ctsk, enquanto o co-tratamento RES resultou numa redução significativa do número de células positivas. Adicionalmente foi observado que a exposição a RES conseguiu reverter efeitos negativos da DOX no desenvolvimento do trato intestinal, com redução da mucosite, e nos efeitos sobre o comportamento locomotor e padrão de locomoção que foram significativamente afetados no tratamento com DOX. Com base nos resultados in vitro, foi estudado o efeito do RES e MT na prevenção de malformações ósseas induzida pela DOX, utilizando os modelos de peixe-zebra (osso osteocítico) e dourada (osso anosteocítico). Estes dois modelos foram submetidos a tratamentos nutricionais com dietas suplementadas em DOX, RES e MT durante o crescimento larvar. Os nossos dados indicam que a suplementação regular com antioxidantes melhorou efetivamente o crescimento global, a mineralização das estruturas esqueléticas e contrariou a incidência e a gravidade das malformações ósseas induzidas por pro-oxidantes em ambos os modelos. Observou-se ainda uma redução da expressão de genes por ação da DOX, como os marcadores de osteoblastos (osteocalcina e osterix/sp7) e de genes antioxidantes, incluindo catalase, glutatião peroxidase 1, superóxido dismutase 1, e hsp90, sugerindo que a formação de espécies reativas de oxigénio (ROS) é central para a perda óssea induzida por DOX. A expressão dos genes antioxidantes foi significativamente aumentada pelo RES sozinho ou em tratamento combinado. Uma vez mais foi observada uma melhoria do desenvolvimento intestinal, com aumento do tamanho dos villi intestinais e redução da inflamação, tanto em peixe zebra como em dourada. Em conclusão, este trabalho propõe que o efeito negativo da medicação específica (DOX) possa ser revertido por suplementação regular com antioxidantes (RES e MT). Além disso, este trabalho propõe também a osteocrina como fator responsável pela deficiência óssea induzida pelo DOX, e a sua inversão. A osteocrina pode ser ainda mais explorada como molécula comunicadora para a conversa cruzada entre o osso e outros tecidos.This work was made possible with the financial support of the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766347- BioMedAqu, and by the Portuguese Foundation for Science and Technology (FCT) through the projects UIDB/04326/2020, UIDP/04326/2020 and LA/P/0101/2020

Regular supplementation with antioxidants rescues Doxorubicin-Induced Bone deformities and mineralization delay in Zebrafish

Osteoporosis is characterized by an abnormal bone structure with low bone mass and degradation of microarchitecture. Oxidative stress induces imbalances in osteoblast and osteoclast activity, leading to bone degradation, a primary cause of secondary osteoporosis. Doxorubicin (DOX) is a widely used chemotherapy drug for treating cancer, known to induce secondary osteoporosis. The mechanism underlying DOX-induced bone loss is still not fully understood, but one of the relevant mechanisms is through a massive accumulation of reactive oxygen and nitrogen species (i.e., ROS and NOS) leading to oxidative stress. We investigated the effects of antioxidants Resveratrol and MitoTEMPO on DOX-induced bone impairment using the zebrafish model. DOX was shown to increase mortality, promote skeletal deformities, induce alterations on intestinal villi, impair growth and mineralization and significantly downregulate osteoblast differentiation markers osteocalcin 2 and osterix/sp7. Lipid peroxidation was significantly increased in DOX-supplemented groups as compared to control and antioxidants, suggesting ROS formation as one of the key factors for DOX-induced bone loss. Furthermore, DOX affected mineral contents, suggesting an altered mineral metabolism. However, upon supplementation with antioxidants, DOX-induced effects on mineral content were rescued. Our data show that supplementation with antioxidants effectively improves the overall growth and mineralization in zebrafish and counteracts DOX-induced bone anomalies.info:eu-repo/semantics/publishedVersio

Reversal of doxorubicin-Induced bone loss and mineralization by supplementation of Resveratrol and MitoTEMPO in the early development of Sparus aurata

Doxorubicin is a widely used chemotherapeutic drug known to induce bone loss. The mechanism behind doxorubicin-mediated bone loss is unclear, but oxidative stress has been suggested as a potential cause. Antioxidants that can counteract the toxic effect of doxorubicin on the bone would be helpful for the prevention of secondary osteoporosis. We used resveratrol, a natural antioxidant, and MitoTEMPO, a mitochondria-targeted antioxidant, to counteract doxorubicin-induced bone loss and mineralization on Sparus aurata larvae. Doxorubicin supplemented Microdiets increased bone deformities, decreased mineralization, and lipid peroxidation, whereas Resveratrol and MitoTEMPO supplemented microdiets improved mineralization, decreased bone deformities, and reversed the effects of doxorubicin in vivo and in vitro, using osteoblastic VSa13 cells. Partial Least-Squares Discriminant Analysis highlighted differences between groups on the distribution of skeletal anomalies and mineralization of skeleton elements. Calcium and Phosphorus content was negatively affected in the doxorubicin supplemented group. Doxorubicin reduced the mRNA expression of antioxidant genes, including catalase, glutathione peroxidase 1, superoxide dismutase 1, and hsp90 suggesting that ROS are central for Doxorubicin-induced bone loss. The mRNA expression of antioxidant genes was significantly increased on resveratrol alone or combined treatment. The length of intestinal villi was increased in response to antioxidants and reduced on doxorubicin. Antioxidant supplements effectively prevent bone deformities and mineralization defects, increase antioxidant response and reverse doxorubicin-induced effects on bone anomalies, mineralization, and oxidative stress. A combined treatment of doxorubicin and antioxidants was beneficial in fish larvae and showed the potential for use in preventing Doxorubicin-induced bone impairment.info:eu-repo/semantics/publishedVersio

Infectious sources of Histoplasmosis and molecular techniques for its identification

Histoplasmosis, a fungal infection caused by Histoplasma capsulatum (H. capsulatum), acquired from contaminated soil with droppings of chicken or birds and found to be distributed in many parts of the world. The prevalence of histoplasmosis has not well studied in Nepal. The common symptoms of acute and epidemic histoplasmosis include high fever, cough, and asthenia and weight loss. Most of the infections associated with histoplasmosis are asymptomatic. People with compromised immune systems such as HIV/AIDS (PLWHA), cancer, and organ transplant recipients are at risk of developing this disease. In this review, we have summarised the current status of histoplasmosis in Nepal and molecular techniques available for its identification. To date, the significant outbreak is not reported in Nepal, but the risk of infection for the vulnerable population cannot be undermined. Appropriate preventive measures and treatment on time can reduce the burden of this fungal disease. Further, this review is also focused on molecular identification of H. capsulatum. Hence, careful considerations by concerned stakeholders for national surveillance programs and the treatment of patients on time after proper diagnosis is highly recommended

Procurement process and shortages of essential medicines in public health facilities: A qualitative study from Nepal

Ensuring access to essential medicines remains a formidable challenge in Nepal. The specific reasons for the shortage of essential medicines within Nepal have not been extensively investigated. This study addresses challenges associated with access to essential medicines, procurement process difficulties, and functionality of inventory management systems at different levels of public health facilities. Fifty-nine semi-structured in-depth interviews were conducted with health managers and service providers at provincial and local levels in six randomly selected districts of Bagmati province, Nepal. Interviews were audiotaped and transcribed verbatim, and the results were analyzed using the inductive approach and were later mapped within the four domains of “Procurement of essential medicines”. The major barriers for the effective management of essential medicines included delays in the procurement process, primarily locally, leading to frequent stock-out of essential drugs, particularly at the health post level. Additionally, challenges arise from storage problems, mainly due to insufficient storage space and the need to manage additional comorbidities related to COVID-19. Other identified challenges encompass the absence of training on logistics management information systems, a lack of information technology resources in primary health facilities, inadequate qualified human resources to operate the IT system, and insufficient power backup. Moreover, unrealistic demand estimation from the service points, inadequate transportation costs, and manual inventory management systems further contributed to the complex landscape of challenges. This study identified procurement delays as the primary cause of essential medicine shortages in Bagmati Province, Nepal. We recommend implementing comprehensive procurement guidelines, collaborative training, and dedicated budgets to address this issue. Improving the procurement and inventory management process in low-resource settings requires a well-trained workforce, suitable storage spaces, and enhanced coordinated administrative tiers within health facilities at different levels to ensure the year-round availability of essential medicines in these settings

What is “Opioid Stewardship”?:An Overview of Current Definitions and Proposal for a Universally Acceptable Definition

Introduction: Opioid stewardship has been widely used to promote rational use, monitoring and discontinuation of opioid therapy; however, its definition and scope of practice remain unclear. Objective: To synthesize definitions of opioid stewardship proposed by clinical practice guidelines and professional societies, and to offer a proposal for a universally acceptable definition. Methods: Systematic literature searches were performed (earliest records to May 2022) in six databases (MEDLINE, EMBASE, APA PsycINFO, Scopus, and CENTRAL) and grey sources guidelines development bodies and professional societies through Google. The conventional but widely applied content analysis and word frequencies were used to analyze the definitions and scope of practice. Results: After removing duplicates, 449 articles were retrieved (439 databases and registers and 11 from other sources), 19 of which included a definition of “opioids stewardship”. A total of 12 themes was identified in the definitions, including 1) improvement or appropriateness of prescribing opioids use, 2) mitigation of risk from opioids, 3) monitoring opioid use, 4) evaluation of opioid use, 5) judicious opioid use, 6) appropriateness of opioid disposal, 7) identification and treatment of opioid use disorder, 8) reduction in mortality associated with opioid overdoses, 9) appropriate procurement practices, 10) appropriate storage, 11) promoting better communications between patients and prescribers including education provision and 12) patient-centered decision-making. Conclusion: Opioid stewardship is inconsistently defined across professional and research literature. While there is a greater focus on appropriateness and need for improvement of prescribing and monitoring of opioid use, the importance of communications between patients and prescribers, and patient involvement in both prescribing and deprescribing decision-making remains sparse. A comprehensive definition has been proposed as part of the work. There is a need to develop and validate the proposed definition and scope of practice to promote rationale for opioid prescribing, use and attainment of favourable outcomes through international consensus involving practitioners, researchers, and patients

Case Report: Levetiracetam causing acute liver failure complicating post-operative management in a neurosurgical patient [version 1; peer review: 2 approved]

Background: Herein we report a rare case of acute liver failure due to levetiracetam, which has been considered to have an excellent safety profile with minimal hepatic side effects. Case presentation: A 55-year-old male patient presenting with sudden onset dizziness, slurring of speech and headache was operated for posterior fossa cerebellar hematoma. His post-surgical period was complicated by development of icterus with elevation of liver enzymes. After ruling out common inciting factors, it was decided to stop levetiracetam which was given prophylactically for preventing seizures owing to presence of external ventricular drain. From the next day patient had dramatic improvement in liver functions and sensorium. Conclusions: We would like to highlight this side effect that is potentially life threatening, though rare, of levetiracetam, which is very commonly used in today’s practice and fast superseding all other time-tested antiepileptics

Procurement process and shortages of essential medicines in public health facilities: A qualitative study from Nepal

Ensuring access to essential medicines remains a formidable challenge in Nepal. The specific reasons for the shortage of essential medicines within Nepal have not been extensively investigated. This study addresses challenges associated with access to essential medicines, procurement process difficulties, and functionality of inventory management systems at different levels of public health facilities. Fifty-nine semi-structured in-depth interviews were conducted with health managers and service providers at provincial and local levels in six randomly selected districts of Bagmati province, Nepal. Interviews were audiotaped and transcribed verbatim, and the results were analyzed using the inductive approach and were later mapped within the four domains of “Procurement of essential medicines”. The major barriers for the effective management of essential medicines included delays in the procurement process, primarily locally, leading to frequent stock-out of essential drugs, particularly at the health post level. Additionally, challenges arise from storage problems, mainly due to insufficient storage space and the need to manage additional comorbidities related to COVID-19. Other identified challenges encompass the absence of training on logistics management information systems, a lack of information technology resources in primary health facilities, inadequate qualified human resources to operate the IT system, and insufficient power backup. Moreover, unrealistic demand estimation from the service points, inadequate transportation costs, and manual inventory management systems further contributed to the complex landscape of challenges. This study identified procurement delays as the primary cause of essential medicine shortages in Bagmati Province, Nepal. We recommend implementing comprehensive procurement guidelines, collaborative training, and dedicated budgets to address this issue. Improving the procurement and inventory management process in low-resource settings requires a well-trained workforce, suitable storage spaces, and enhanced coordinated administrative tiers within health facilities at different levels to ensure the year-round availability of essential medicines in these settings