Investigación preclínica en animales. Pautas actuales / Preclinical animal research. Current guidelines

El uso de animales en experimentación científica está regulado por normativas diseñadas para garantizar la ética y el bienestar de los animales. A nivel mundial las regulaciones varían, pero muchos países como Argentina adoptan directrices establecidas por organizaciones internacionales como la Oficina de Protección de los Derechos de los Animales, EE.UU. (Animal Welfare Office) y el Instituto Nacional de Salud, EE.UU. (National Institute of Health). Los protocolos que se elaboran teniendo en cuenta las recomendaciones de estas entidades deben ser revisados por comités de ética para asegurarse que se cumplan con los estándares internacionales. En resumen, las pautas actuales buscan equilibrar el avance científico con la ética, asegurando que el uso de animales en investigación sea racional, humanitario y respetando el bienestar animal.(AU)

The use of animals in scientific experiments is regulated by guidelines designed to ensure ethics and welfare of animals. Regulations vary around the world, but many countries, such as Argentina, follow guidelines established by international organizations such as the US Office of Animal Welfare and the US National Institutes of Health. Protocols developed using the recommendations of these entities must be reviewed by ethics committees to ensure that they follow international standards. In summary, the current guidelines seek to strike a balance between scientific progress and ethics, ensuring that the use of animals in research is rational, humane and respects animal welfare.(AU)

Tecnologia educativa para disseminação do programa Fall TIPS Brasil / Educational technology for disseminating the Fall TIPS Brasil program

Resumo: Esta tese integra o projeto de pesquisa e desenvolvimento tecnológico "Difusão e adoção do programa Fall Tailoring Interventions for Patient Safety (Fall TIPS): engajamento de pacientes, profissionais e liderança clínica para a prevenção de quedas em ambiente hospitalar". Está conectada à linha de Pesquisa de Gerenciamento de Serviços de Saúde e Enfermagem do Programa de Pós-Graduação em Enfermagem da Universidade Federal do Paraná e objetivou construir, de maneira colaborativa, uma tecnologia educativa em formato de Curso Massivo, Aberto e Online (MOOC) sobre o programa Fall TIPS Brasil para a prevenção de quedas hospitalares, valendo-se de recursos variados, como infográficos, vídeos, podcasts e jogos interativos, destinada à disseminação da proposta entre profissionais e estudantes da área da saúde; e verificar o aprendizado estatisticamente significativo em relação ao conhecimento dos cursistas antes e após o seu acesso. Primeiramente ocorreu o processo de construção do MOOC com colaboração multidisciplinar, envolvendo docentes, estudantes de graduação e pós-graduação, profissionais de saúde e do setor de ensino a distância da universidade participante, fase de desenvolvimento tecnológico. Após, a abordagem quase experimental não randomizada e não concorrente foi realizada com 21 estudantes do último período do curso de graduação em enfermagem de uma universidade pública, tendo sido submetidos a um questionário contendo 10 questões objetivas antes e após o acesso ao MOOC. Os dados foram analisados mediante estatística descritiva e analítica. A construção do MOOC foi iniciada em 2020, com a primeira edição lançada em outubro de 2023. As etapas observadas na sua elaboração incluíram a escolha do tema, a produção do curso, a avaliação preliminar e a divulgação. O conhecimento dos cursistas após o acesso foi significativamente (p <0,001) maior, quando comparado com os resultados antes do acesso. A média das notas antes do acesso ao curso foi 7,1 e, após, foi 8,9. As evidências encontradas demonstram o sucesso da intervenção. O desenvolvimento do MOOC de maneira colaborativa, o direcionamento dos materiais para o público-alvo respeitando suas dificuldades e potencialidades permitem aderência a proposta, contribuindo para que a intervenção possibilite a ampliação do conhecimento sobre o programa Fall TIPS Brasil. Assim, destaca-se a importância de os profissionais de saúde possuírem conhecimento para poderem promover o engajamento dos pacientes e acompanhantes em prol da prevenção de quedas, sendo o MOOC um recurso promissor para essa finalidade.

Abstract: This thesis integrates the research and technological development project "Diffusion and adoption of the Fall Tailoring Interventions for Patient Safety (Fall TIPS) program: engagement of patients, professionals, and clinical leadership for the prevention of falls in the hospital environment." It is connected to the Research Line in Health Services Management and Nursing of the Graduate Program in Nursing at the Federal University of Paraná and aimed to collaboratively construct an educational technology in the form of a Massive Open Online Course (MOOC) on the Fall TIPS Brazil program for the prevention of hospital falls, utilizing various resources such as infographics, videos, podcasts, and interactive games, intended for disseminating the proposal among healthcare professionals and students; and to verify statistically significant learning regarding the knowledge of the participants before and after accessing the course. First, the process of building the MOOC occurred through multidisciplinary collaboration, involving faculty, undergraduate and graduate students, healthcare professionals, and the university's distance education sector, in the phase of technological development. Subsequently, a quasi-experimental non-randomized and non-concurrent approach was conducted with 21 final-year nursing students from a public university, who were subjected to a questionnaire containing 10 objective questions before and after accessing the MOOC. Data were analyzed using descriptive and analytical statistics. The construction of the MOOC began in 2020, with the first edition launched in October 2023. The stages observed in its development included choosing the theme, producing the course, preliminary evaluation, and dissemination. Participants' knowledge after accessing the course was significantly higher (p <0,001) compared to the results before access. The average score before accessing the course was 7,1 and after was 8,9. The evidence found demonstrates the success of the intervention. Collaborative development of the MOOC, directing materials to the target audience while respecting their difficulties and potentialities, allows adherence to the proposal, contributing to expanding knowledge about the Fall TIPS Brazil program. Thus, the importance of healthcare professionals having knowledge to promote the engagement of patients and caregivers in fall prevention is highlighted, with the MOOC being a promising resource for this purpose.

Temporal evolution of postsurgical bone repair in a rabbit model: A [99mTc]Tc-MDP scintigraphic study

Abstract Bone regeneration is crucial for repairing bone tissue following various injuries. Research techniques that enable the study of metabolic changes in bone tissue under different conditions are important for understanding bone repair and remodeling. This study used bone scintigraphy to evaluate osteogenesis secondary to osteotomy in a preclinical model of New Zealand rabbits. For this purpose, we conducted a longitudinal, prospective, case-control study in which scintigraphic variables were measured in both the right forearm (case-operated) and the left forearm (control - non-operated). The study sample consisted of 10 rabbits subjected to osteotomy, followed by a 12-week postoperative evaluation period, divided into six imaging stages at 1, 2, 3, 4, 8, and 12 weeks. We observed that the operated forearm showed significantly higher external radiation than the control side, using the pinhole collimator, denoting an increase in the biodistribution and tropism of the radiopharmaceutical to the operated forearm. Among the three evaluated time points, osteoblastic activity was highest in the second week and presented a significant decline in the 8th and 12th weeks, denoting regeneration and resolution of the surgical injury; the control forearm was also influenced by the inactivity imposed by the operated forearm. This fact was notably evidenced by the reduction in the metabolic activity of osteoblasts in the left forearm. Our study suggested that bone scintigraphy was sensitive enough to semi-quantitatively differentiate the metabolic activity of osteoblasts in the operated forearm in the three temporal landmarks evaluated in the study.

Adjuvant-induced arthritis promotes vascular hyporesponsiveness to phenylephrine through a nitric oxide-related mechanism

Arthritis has important cardiovascular repercussions. Phenylephrine-induced vasoconstriction is impaired in rat aortas in the early phase of the adjuvant-induced arthritis (AIA), around the 15th day post-induction. Therefore, the present study aimed to verify the effects of AIA on hyporesponsiveness to phenylephrine in rat aortas. AIA was induced by intradermal injection of Mycobacterium tuberculosis (3.8 mg/dL) in the right hind paw of male Wistar rats (n=27). Functional experiments in isolated aortas were carried out 15 days after AIA induction. Morphometric and stereological analyses of the aortas were also performed 36 days after the induction of AIA. AIA did not promote structural modifications in the aortas at any of the time points studied. AIA reduced phenylephrine-induced contraction in endothelium-intact aortas, but not in endothelium-denuded aortas. However, AIA did not change KCl-induced contraction in either endothelium-intact or denuded aortas. L-NAME (non-selective NOS inhibitor), 1400W (selective iNOS inhibitor), and ODQ (guanylyl cyclase inhibitor) reversed AIA-induced hyporesponsiveness to phenylephrine in intact aortas. 7-NI (selective nNOS inhibitor) increased the contraction induced by phenylephrine in aortas from AIA rats. In summary, the hyporesponsiveness to phenylephrine induced by AIA was endothelium-dependent and mediated by iNOS-derived NO through activation of the NO-guanylyl cyclase pathway.

Effect of Longmu Piyan Prescription on Oxidative Stress in Atopic Dermatitis Mice / 中国实验方剂学杂志

ObjectiveThis study observes the intervention effect of Longmu Piyan prescription on oxidative stress in BALB/c mice with atopic dermatitis （AD） induced by 2，4-dinitrochlorobenzene （DNCB） and explores its mechanism. MethodThe AD model was established using the method of DNCB sensitization on the back skin of BALB/c mice. Forty male BALB/c mice were randomly divided into a blank group， a model group， a vitamin C control group （0.5×10-3 mg·kg-1）， and a Longmu Piyan prescription group （26 g·kg-1）. Except for the blank group， other groups were sensitized with different concentrations of DNCB on the back to induce AD， and the blank group was treated with matrix coating. The gastric administration was started on the seventh day after sensitization with 2% DNCB and on the 24th day after sensitization with 0.2% DNCB continuously for 21 days. The changes in skin lesions of each group were directly observed after the experiment. Enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of interleukin （IL）-4， tumor necrosis factor （TNF）-α， immunoglobulin E （IgE）， and reactive oxygen species （ROS） in the serum of each group. The total antioxidant capacity determination kit-trace method (ABTS method) was used to measure the level of total antioxidant capacity （TAOC） in serum. The Hematoxylin eosin （HE） staining method was used to observe the pathological and morphological changes of the skin lesion site. The immunohistochemical method was used to detect the expression of thymic stromal lymphopoietin （TSLP） in the skin lesion site. Western blot was used to detect the expression of filaggrin （FLG） in the dorsal skin lesions. ResultThe results showed that compared with the blank group， the skin lesion score of the model group mice was significantly increased （P<0.01）， and HE staining showed characteristic pathological changes of AD in the skin lesion site. At the same time， the expression of TSLP in the skin lesion was significantly increased， and that of FLG was reduced （P<0.05）. The levels of TNF-α， IL-4， IgE， and ROS in serum increased， while the activity of TAOC decreased （P<0.01）. Compared with the model group， the Longmu Piyan prescription group showed a significant decrease in skin lesion scores and a significant improvement in skin lesion pathology. At the same time， the expression of TSLP decreased， and the expression of FLG increased in the skin lesions （P<0.05）. In addition， compared with the model group， the serum levels of TNF-α， IL-4， IgE， and ROS also decreased to varying degrees （P<0.05，P<0.01）， and TAOC activity increased in the Longmu Piyan prescription group （P<0.01）. ConclusionThere is a significant correlation among the degree of oxidative stress， the severity of skin lesions in AD， and the levels of inflammatory cytokines. Longmu Piyandu prescription can improve AD-like skin lesions in BALB/c mice by promoting ROS clearance， enhancing TAOC， and inhibiting oxidative stress， thus protecting the skin barrier and reducing inflammation.

Mechanism of Cuscutae Semen to Control Premature Ovarian Failure Based on Lipid and Atherosclerosis Pathways / 中国实验方剂学杂志

ObjectiveThe active ingredients， action targets， and signaling pathways of Cuscutae Semen to control premature ovarian failure were initially predicted by network pharmacology and molecular docking techniques， and an animal model of premature ovarian failure was constructed to explore the mechanism of Cuscutae Semen based on lipid and atherosclerosis signaling pathways. MethodThe effective components and corresponding targets of drugs were obtained from Traditional Chinese Medicines Systems Pharmacology Platform （TCMSP）， Swiss Target Prediction， Pharmmapper， and other databases. GeneCards database was used to collect disease-related targets. Venny2.1.0 online tool was used to screen out the intersection targets of drugs and diseases， and STRING database and Cytoscape v3.7.2 software were used to construct the network diagram of "drug-component-target" and protein-protein interaction （PPI）. The gene ontology （GO） and the Kyoto encyclopedia of genes and genomes （KEGG） enrichment analyses of the intersection targets were performed by running the R language script. The molecular docking technology was utilized to dock drug components with targets and visualize some of the docking results. The mice were randomly divided into a blank group， a model group， a Cuscutae Semen group， and an estradiol valerate group， and the ovarian premature failure model was prepared by chronic stress. The blank group and the model group were gavaged with the same amount of normal saline， and the Cuscutae Semen group was given a Cuscutae Semen decoction of 2.6 g·kg-1·d-1. The estradiol valerate group was given an estradiol valerate solution of 0.13 mg·kg-1·d-1. After four weeks， samples were collected， and hematoxylin-eosin （HE） staining was performed to observe the histopathological changes in the ovary. Serum levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E2）， Muller's tube inhibitor/anti-Muller's tube hormone （AMH）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， and low-density lipoprotein cholesterol （LDL-C） were determined by enzyme-linked immunosorbent assay （ELISA）. The expression levels of extracellular regulatory protein kinase （ERK）， nuclear transcription factor-κB p65 （NF-κB p65）， nuclear transcription factor-κB suppressor α （IκBα）， interleukin-1β （IL-1β）， IL-6， and tumor necrosis factor-α （TNF-α） were measured by Western blot. ResultA total of 171 targets of Cuscutae Semen for the prevention and treatment of premature ovarian failure were screened， mainly including tumor protein p53 （TP53）， protein kinase B1 （Akt1）， sarcoma （SRC）， tumor necrosis factor （TNF）， epidermal growth factor receptor （EGFR）， etc. KEGG pathway enrichment analysis predicts that Cuscutae Semen is mainly involved in lipid and atherosclerosis， TNF signaling pathway， and TP53 signaling pathway to control premature ovarian failure. The animal experiments show that compared with the premature ovarian failure model group， the Cuscutae Semen group can significantly upregulate AMH， E2， and HDL-C （P<0.05， P<0.01）， significantly downregulate LH， TC， and LDL-C （P<0.01）， greatly reduce IL-1β， IL-6， and TNF-α protein levels， as well as ERK， NF-κB p65， and their phosphorylation levels （P<0.01）. ConclusionCuscutae Semen can regulate hormone levels and improve ovarian function through a multi-component， multi-target， and multi-pathway approach， and the mechanism may be related to the regulation of lipid and atherosclerosis signaling pathways.

Discussion on mechanism of modified Biminkang Granules in treatment of allergic rhinitis based on network pharmacology and experimental verification / 国际中医中药杂志

Objective:To predict the molecular mechanism of Biminkang Granules in the treatment of allergic rhinitis using network pharmacological methods combined with animal experiments.Methods:Active component targets and allergic rhinitis targets were screened from TCMSP, OMIM, GeneCards, TTD, DrugBank and PharmGKB databases; R language software was used to map the intersection of drug and disease targets; Cytoscape software and String platform were used to construct intersection target PPI network and conduct network topology analysis; DAVID platform was used to perform GO enrichment and KEGG pathway analysis, and perform molecular docking verification on the main active components and key targets. 32 rats were divided into a blank group of 8 and a model group of 24 using a random number table method. Model rats were induced by ovalbumin to establish an allergic rhinitis model. 24 SD rats that were successfully modeled and were randomly divided into model group, Western medicine group, and Biminkang Granules group using a random number table method, with 8 rats in each group. The Western medicine group was gavaged with 1 mg/kg of loratadine solution, the Biminkang Granules group was gavaged with 4.1 g/kg of Biminkang Granules solution, and the blank group and model group rats were gavaged with the same volume of physiological saline once a day for 2 consecutive weeks. The symptoms of rhinitis in each group of rats for 30 minutes were observed and recorded, and the pathological changes of the rat nasal mucosa were observed using HE staining. ELISA method was used to detect the levels of IL-17 and IL-6 in rat serum, and Western blot method was used to determine the expressions of TNF and STAT3 proteins in rat tissues.Results:A total of 41 target proteins of BiMinKang Dranule in the treatment of allergic rhinitis were predicted, and TNF, STAT3 and other core target proteins were obtained by PPI network topology analysis. The biological process of GO involved drug response, inflammatory response, cytokine response, etc.KEGG enrichment is involved in Th17 cell differentiation, lipid and atherosclerosis, IL-17, toll-like receptor and other pathways. Molecular docking results indicated that the main active components had good binding activity to key target proteins.Animal experiments showed that BiMinKang Dranule could improve the inflammatory symptoms of allergic rhinitis rats, down-regulate the expression of IL-17 and IL-6 in blood, and inhibit the expression of TNF and STAT3 proteins.Conclusion:Biminkang Granules can treat allergic rhinitis through multiple active components, multiple target proteins and multiple pathways, and the mechanism may be related to the regulation of Th17 cell differentiation pathway related proteins.

Progress in Clinical Research and Experimental Study of Danggui Shaoyao Powder in the Treatment of Chronic Pelvic Inflammatory Disease / 广州中医药大学学报

Chronic pelvic inflammatory disease(CPID)is a common chronic inflammatory disease in women,and has a long course and is easy to relapse.Danggui Shaoyao Powder is from Jin Gui Yao Lve(Synopsis of the Golden Chamber),which was a commonly-used formula for the treatment of women's abdominal pain in ancient medical records.It is now often used in the treatment of CPID and has achieved satisfactory therapeutic effect.The article summarizes and analyzes the achievements in the clinical research and experimental study of Danggui Shaoyao Powder in the treatment of CPID over the past 10 years,and invesigates the clinical efficacy of Danggui Shaoyao Powder in the treatment of CPID and its therapeutic mechanism.In the field of clinical studies,Danggui Shaoyao Powder for the treatment of CPID was used by modification,or alone,or in combination with antibiotics and Chinese medicine external treatment,and its combined use was effective on significantly improving the indicators of inflammatory response and immune function,alleviating the clinical signs and symptoms such as pain,and did not increase the incidence of adverse reactions compared with the application of western medicines alone.In the field of experimental studies,Danggui Shaoyao Powder played the therapeutic role in CPID by decreasing the adhesion of endothelial cells,regulating the degradation of extracellular matrix,improving the level of inflammatory factors,and down-regulating the expression of proteins related to the nuclear factor κB(NF-κB)pathway.

Study on Optimization of spray Drying Process of Banlangen Formula Granules / 世界科学技术-中医药现代化

Objective To optimize the spray drying process of Banlangen(Isatidis Radix)formula granules based on quality by design(QbD)concept.Methods Using powder yield and the contents of uridine,adenosine,guanosine,and(R,S)-goitron as the critical quality attributes(CQAs),Plackett-Burman design was used to screen out critical process parameters(CPPs)for inlet temperature,spray pressure,liquid temperature,pump speed,and liquid relative density.The central-composite design(CCD)test was used to optimize the CPPs,which were screened.Based on the quadratic polynomial regression model,the design space of spray drying process of Banlangen(Isatidis Radix)formula granules was established,and further validated by experiments.Results Plackett-burman test results show that liquid relative density and inlet velocity are the key parameters for the study.The variance analysis results of CCD test showed that the constructed model in a good prediction ability,since the P-values of model was less than 0.01 and P-values of items lack of fit was more than 0.05.The optimized design space of CPPs was the liquid relative density 1.05-1.08,and pump speed 30%-40%.Conclusion Based on the QbD concept,the design space for the spray drying process of Banlangen(Isatidis Radix)formula granules can improve the stability of its process and help ensure the consistency of product quality.

Comparison of protocols for constructing animal models of early traumatic knee osteoarthritis / 中国组织工程研究

BACKGROUND:Current osteoarthritis modeling methods include anterior cruciate ligament transection(ACLT)and ACLT combined with medial meniscal anterior horn resection.ACLT requires excessive postoperative exercise,which is time and labor-intensive.Complete removal of anterior horn of the medial meniscus can cause collateral damage and increase variability in modeling outcomes,requiring higher surgical skills from the surgeon. OBJECTIVE:To modify and simplify the traditional method to create animal osteoarthritis model and compare osteoarthritis symptoms of different modeling methods under a low-load exercise environment. METHODS:Forty-eight Sprague-Dawley rats were randomly assigned in four groups(n=12 per group):sham operation(complete exposure of the knee cavity of the left hind limb followed by suturing the joint cavity and skin),ACLT,ACLT+anterior horn resection(removal of the anterior horn of the medial meniscus)and ACLT+anterior horn tear(anterior horn tear of the medial meniscus).At 4 weeks after modeling,the rats were euthanized and their knee specimens were collected for gross observation,X-ray and CT scans,pathological observation,and PCR detection. RESULTS AND CONCLUSION:Gross observation:Mild meniscal wear was observed in the ACLT group.In the ACLT+anterior horn tear group,severe wear of the lateral condyle articular surface,mild wear of the medial condyle articular surface,severe meniscal wear,and full wear of the medial meniscus were observed.The ACLT+resection group showed severe wear of the lateral condyle articular surface,mild wear of the medial condyle articular surface,absence of the anterior horn of the medial meniscus,and meniscus wear area＞50%.Imaging examinations showed no significant difference among the four groups.However,the anterior tibial translocation sign was observed in the three operation groups and the anterior horn of the medial meniscus was missing in the ACLT+anterior horn resection group.Histopathological section observation:Hematoxylin-eosin,toluidine blue,and Sirius red staining showed smooth joint surfaces in the sham operation group and ACLT group;cartilage damage and matrix degradation were evident in the ACLT+anterior horn tear and ACLT+anterior horn transection groups,with less cartilage damage and matrix degradation in the ACLT+anterior horn tear group.PCR results showed higher mRNA expressions of interleukin 1β,interleukin 6,interleukin 8,tumor necrosis factor α,matrix metalloproteinase 1 and matrix metalloproteinase 3 and lower mRNA expressions of aggrecan in the ACLT+anterior horn tear group and ACLT+anterior horn resection group than in the sham operation group and ACLT group(P＜0.05).The mRNA expressions of interleukin 6,matrix metalloproteinase 1,and matrix metalloproteinase 3 were higher in the ACLT + anterior horn resection group than in the ACLT +anterior horn tear group(P＜0.05).To conclude,ACLT alone is less likely to induce osteoarthritis with obvious cartilage wear.ACLT combined with anterior horn resection or tear of the medial meniscus can induce obvious symptoms of osteoarthritis and achieve similar modeling effects.

Immunomodulatory effect of astragaloside IV on T cells of experimental autoimmune encephalomyelitis mice / 中国组织工程研究

BACKGROUND:In the initial stage of multiple sclerosis,central immune cells activate and release a large number of inflammatory factors,causing white matter demyelination and even involving gray matter neurons.The equilibrium of differentiation between different subsets of CD4+ T cells plays an important role in the progression of experimental autoimmune encephalomyelitis.The previous results of the research group showed that the active ingredient astragalus glycoprotein in astragalus can regulate the immune response in experimental autoimmune encephalomyelitis mice,and whether it has a regulatory effect on the differentiation of T cell subsets has not been determined. OBJECTIVE:To explore the therapeutic effects and immune regulatory mechanisms of astragaloside IV on experimental autoimmune encephalomyelitis mice. METHODS:Female C57BL/6 mice were divided into the normal control group,experimental autoimmune encephalomyelitis disease model group,and astragaloside IV treatment group(n=8 per group).Myelin oligodendrocyte glycoprotein peptides 35-55 were used for experimental autoimmune encephalomyelitis model induction in the last two groups.On day 10 to 28 after immunization,the astragaloside IV treatment group was treated with 40 mg/kg per day astragaloside IV intragastrically.Body weight and clinical scores of mice in each group were recorded from the immunization day to the 28th day.On the 28th day after immunization,the mouse spinal cord was taken and made into frozen sections for hematoxylin-eosin staining and Lux fast blue staining to observe pathological changes in the spinal cord.Percentage of splenic T cell subsets was detected using flow cytometry.Western blot assay was used to determine the protein expression of interferon-γ,interleukin-17 and interleukin-6 in the spinal cord.Levels of interferon-γ,interleukin-17,interleukin-6 and interleukin-4 in supernatants of cultured splenocytes were determined by ELISA. RESULTS AND CONCLUSION:(1)Compared with the experimental autoimmune encephalomyelitis disease model group,astragaloside IV could reduce the degree of weight loss in experimental autoimmune encephalomyelitis mice(P<0.05),ameliorate clinical symptoms(P<0.05),inhibit the infiltration of inflammatory cells and alleviate myelin loss(P<0.01,P<0.05).(2)Compared with the experimental autoimmune encephalomyelitis disease model group,astragaloside IV could inhibit the proportion of CD4+T cell subsets expressing interferon-γ(P<0.001)and interleukin-17(P<0.001),but increase percentages of CD4+ interleukin-10+(P<0.001)and CD4+ transforming growth factor-β+(P<0.01)T cell subsets.(3)Astragaloside IV could inhibit the expression of interferon-γ(P<0.05,P<0.01),interleukin-17(P<0.05,P<0.05),and interleukin-6(P<0.05,P<0.05)in the spinal cord and spleen,and up-regulate the expression of interleukin-4(P<0.01)in spleen.(4)These findings confirm that astragaloside IV alleviates clinical symptoms in experimental autoimmune encephalomyelitis mice,which may be related to regulating the splenic T cell subsets,therefore,inhibiting the infiltration of inflammatory cells into the center and reducing the demyelination.

N6-methyladenosine related regulatory factors in osteoarthritis:bioinformatics analysis and experimental validation / 中国组织工程研究

BACKGROUND:Increasing evidence suggests that N6-methyladenosine(m6A)regulators are closely associated with osteoarthritis and are considered to be a new direction in the prevention and treatment of osteoarthritis,but their specific mechanism of action is unknown. OBJECTIVE:To conduct a bioinformatics analysis of the osteoarthritis gene microarray dataset in order to explore the role of m6A in osteoarthritis and analyze the pathogenesis of osteoarthritis. METHODS:The m6A regulators associated with osteoarthritis and their expression were first extracted from the GSE1919 dataset in the GEO database using R software,and then the results were analyzed by gene difference analysis and GO and KEGG enrichment analyses.Subsequently,the results of protein-protein interaction network topology analysis and machine learning results were intersected to obtain the m6A Hub regulators,which were validated by in vitro cellular experiments. RESULTS AND CONCLUSION:A total of 16 osteoarthritis-related m6A regulators were extracted and 11 m6A differential regulators,including ZC3H13,YTHDC1,YTHDF3 and HNRNPC,were obtained by differential analysis.GO enrichment analysis showed that osteoarthritis-related m6A differential regulators played a role in the biological processes such as mRNA transport,RNA catabolism,and regulation of insulin-like growth factor receptor signaling pathway.(3)KEGG enrichment analysis showed that the differential regulators were mainly involved in the p53,interleukin-17 and AMPK signaling pathways.The combined protein-protein interaction network topology analysis and machine learning results obtained the m6A Hub regulator-YTHDC1.(5)The results of in vitro cellular experiments showed that there was a significant difference in the expression of m6A key regulator between the control and experimental groups(P<0.05).To conclude,YTHDC1 is closely related to the development of osteoarthritis,which is expected to be a molecular target of m6A for the treatment of osteoarthritis.

Anti-inflammatory effect of Celastrol in the ocular tissues of mice with exper-imental autoimmune uveitis and its impact on microglia polarization / 眼科新进展

Objective To investigate the anti-inflammatory action of Celastrol in the ocular tissues of mice with ex-perimental autoimmune uveitis(EAU)and its effect on microglia polarization.Methods A total of 36 healthy B10.RⅢmice at 6-8 weeks of age were selected and randomly divided into the normal control group,EAU solvent control group and Celastrol intervention group,with 12 mice in each group.The interphotoreceptor retinoid-binding protein(IRBP)161-180 and Freund's complete adjuvant were mixed by thorough emulsification and injected subcutaneously into the bilateral thighs and tails of mice in the EAU solvent control group and the Celastrol intervention group with a total volume of 200 μL and 50 μg IRBP 161-180 in each mouse.On 7-14 days after immunization,mice in the Celastrol intervention group received a daily intraperitoneal injection of 0.5 mg·kg-1 Celastrol,and mice in the EAU solvent control group were injected with an equivalent dose of sterile Phosphate Buffered Saline solution.On the 14th day after immunization,the anterior segment of mice in each group was observed by slit-lamp microscope and Hematoxylin and Eosin(HE)staining of tissue sections was performed;the clinical and histopathological scores of mice in each group were obtained by reference to the Caspi grading standards;immunofluorescence staining was used to observe the activation of microglia in the eyes of mice;Western blot was used to detect the protein expression levels of inducible nitric oxide synthase(iNOS)and arginase-1(Argl)in the reti-na;quantitative real-time PCR was used to detect the relative mRNA expression of inflammatory factors in the retina,such as tumor necrosis factor(TNF)-α,interleukin(IL)-1β and IL-6.GraphPad Prism 9.0 was used for data analysis.Results On the 14th day after immunization,it was observed by the slit-lamp microscope that the anterior segment of mice in the EAU solvent control group was markedly congested with dilated iris blood vessels,corneal edema,and anterior chamber exudation;the inflammation in the anterior segment of mice in the Celastrol intervention group was markedly at-tenuated,and the iris blood vessels were seen to be mildly congested.Compared with the normal control group,the clini-cal scores of mice in the EAU solvent control group and the Celastrol intervention group were significantly elevated(both P＜0.05);the clinical scores of mice in the Celastrol intervention group were lower than those in the EAU solvent control group(P＜0.05).HE staining results showed that on the 14th day after immunization,mice in the EAU solvent control group showed severe retinal folds and detachment with diffuse infiltration of inflammatory cells,while mice in the Celastrol intervention group showed slight structural damage to the retina and a small amount of inflammatory cell infiltration.Com-pared with the normal control group,the histopathological scores of mice in the EAU solvent control group and the Celas-trol intervention group were significantly elevated(both P＜0.05);the histopathological scores of mice in the Celastrol in-tervention group were lower than those in the EAU solvent control group(P＜0.05).The intraocular Iba1+cell densities of mice in the normal control,EAU solvent control and Celastrol intervention groups were(1.00±0.12)％,(36.07± 4.57)％,and(1.83±0.36)％,respectively.Compared with the normal control group,the number of Iba1+cells in the eyes of mice in the EAU solvent control group and the Celastrol intervention group significantly increased(both P＜0.05);compared with the EAU solvent control group,the number of Iba1+cells in the eyes of mice in the Celastrol intervention group was significantly reduced(P＜0.05).Compared with the normal control group,the expression levels of iNOS and Arg1 proteins in the retinas of mice in the EAU solvent control group were significantly elevated(both P＜0.01);compared with the EAU solvent control group,the expression of iNOS protein in the retinas of mice in the Celastrol intervention group was significantly reduced(P＜0.01).Compared with the normal control group,the relative mRNA expressions of TNF-α.IL-1β,and IL-6 in the retinas of mice in the EAU solvent control group was significantly elevated(all P＜0.05);compared with the EAU solvent control group,the relative mRNA expressions of TNF-α,IL-1 β,and IL-6 in the retinas of mice in the Celastrol intervention group significantly decreased(all P＜0.05).Conclusion Celastrol inhibits Ml microglia activation and reduces the production of retinal inflammatory factors TNF-α,IL-1 β and IL-6 in EAU mice,thereby attenuating the in-flammatory reaction.

Design and finite element analysis of experimental device for simulating interface micro motion of bone trabecular prosthesis / 医疗卫生装备

Objective To design an experimental device for simulating the interface micro motion of bone trabecular prosthesis and carry out the finite element analysis.Methods The experimental device was composed of a screw-in cylinder with threads,a flexible hinge,a micro-motion rod,a trabecular prosthesis,a connecting rod and a fixation post.A model of the experimental device was constructed with SolidWorks software,and then imported into ABAQUS software to establish a finite element model.An axial displacement load was applied to the femur to analyze the effects of the position of the flexible hinge,the gap between the prosthesis and the femur and the length of the connecting rod on the micro motion.Results The interface micro motion produced by the experimental device increased with the distance of the flexible hinge from the lower end of the screwed-in cylinder;the gap between the prosthesis and the femur did not affect the interface micro motion when the gap was not lower than 20 μm;the interface micro motion rose with the length of the connecting rod.Conclusion The experimental device can accurately simulate the micro motion of different bone trabecular prosthesis interfaces,and can be used for studying the effect of the interface micro motion on osseointegration.[Chinese Medical Equipment Journal,2024,45(1):25-30]

Research on the creating scenarios method in the experimental teaching reform of food hygiene / 中华医学教育探索杂志

Objective:To explore the effect of creating scenarios method in the experimental teaching of food hygiene.Methods:A total of 170 students from two grades of preventive medicine major were selected as the research objects, and the students from grade 2016 were selected as the experimental group ( n=88) taking the creating scenarios method, and the students from grade 2015 were selected as the control group ( n=82) taking the traditional method. The scores of the two groups were compared, and the evaluation from students and teachers on the course was collected. Meanwhile, the qualitative interviews with senior students and practice teachers were taken as the reference. The teaching effects was comprehensively evaluated from the student side and the teacher side. SPSS 20.0 was used for t test, and Chi-square test. Results:The scores of thinking questions in experimental reports from experimental groups (course 1, P=0.038; course 2, P=0.043) and food hygiene paper ( P=0.034) were significantly higher than those of the control group. Eighty percent of the teachers who attended the course [8 teachers of the department (80.00%), 4 teaching supervisors (80.00%)] believed that creating scenarios method fully embodied the cultivation of professional knowledge, skills, cross-professional ability and comprehensive practical ability. The qualified rates of students' self-mastery in professional knowledge, skills, cross-professional ability and comprehensive practical ability were 100% (80 students), 100% (80 students) and 97.75% (79 students), respectively. The students' satisfaction with teaching methods reached 90% (70 students). The results of qualitative interview gave a full recognition to the application of creating scenarios method. Conclusions:The application of creating scenarios method in the experimental course of food hygiene not only improves the course results of students, but also improves their cognition of the course. In addition, it can fully display the cultivation of the core competence of students majoring in preventive medicine.

Establishment and practice of virtual simulation experimental teaching platform of Health Management / 中华医学教育探索杂志

In order to solve the problems in traditional health management experimental teaching, such as high cost, safety and difficult repeatability, a virtual simulation experimental teaching platform for health management combined with Traditional Chinese Medicine (TCM) characteristics has been constructed by using virtual simulation, multimedia, and human-computer interaction technologies, which comprises TCM constitution identification, TCM health management and health management service process. Through the combination of virtual simulation and reality situation, the platform has formed an online and offline model of experimental teaching, which has improved the innovation and practice ability of students and enhanced the teaching quality.

Mdivi-1 protects oligodendrocytes through inhibiting apoptotic signaling pathway / 中国病理生理杂志

AIM:To investigate the therapeutic effect of mitochondrial fission inhibitor-1(Mdivi-1)on experi-mental autoimmune encephalomyelitis(EAE)in mice,and to explore its mechanism.METHODS:The mice immunized with myelin oligodendrocyte glycoprotein peptide fragment 35-55(MOG35-55)were randomly divided into DMSO model group and Mdivi-1 intervention group.All mice were sacrificed on the 28th day after the first immunization.The demyelination was analyzed by Luxol fast blue staining.The protective mechanism of Mdivi-1 in the spinal cord tissue was investigated by immunofluorescence staining,TUNEL staining and the in vitro experiment with MO3.13 oligodendrocytes treated with staurosporine.The mitochondrial depolarization was detected by JC-1 staining,the cell injury was checked by LDH leakage,and the viability of MO3.13 oligodendrocytes was determined by MTT assay.RESULTS:Compared with DMSO model group,the demyelinating injury was alleviated and the proportion of apoptotic CC1+ oligodendrocytes in Mdivi-1 group was decreased.The cleaved caspase-3,caspase-9,cytochrome C and Bax protein expression levels in the spinal cord of Mdivi-1-treated mice was also attenuated.The in vitro MO3.13 cell experiments suggested that Mdivi-1 inhibited MO3.13 cell mitochondrial depolarization,attenuated the cell damage and increased the cell viability.CONCLUSION:Mdivi-1 pro-tects against the myelin injury in EAE mice,which may be related to the suppression of oligodendrocyte apoptosis.

Role of clearance of exogenous myelin antigen in experimental autoimmune encephalomyelitis / 中华微生物学和免疫学杂志

Objective:To investigate the role of the clearance of exogenous myelin antigen in experimental autoimmune encephalomyelitis (EAE).Methods:EAE was induced in C57BL/6J mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) or FITC-MOG 35-55. The concentration of exogenous myelin antigen was assessed by analyzing the proliferation of the transferred CFSE-labeled mT/mG-2D2 CD4 + T cells in spleen tissues. The release of exogenous myelin antigen from the inoculation sites was analyzed by immunohistochemistry and flow cytometry. HE staining was used to investigate the mechanism underlying the rapid clearance of exogenous myelin antigen. The role of the clearance of exogenous myelin antigen in EAE was investigated by comparative analysis of EAE induced by subcutaneous immunization in the back and footpads, and analyzing the therapeutic effect of soluble MOG 35-55. Results:The proliferation of mT/mG-2D2 CD4 + T cells in mice was enhanced on day 2 than on day 7 after immunization [(52.6±6.8)% vs (18.5±4.9)%, P<0.01]. There was no significant difference in the proliferation of mT/mG-2D2 CD4 + T cells between EAE mice (day 13) and naive mice [(4.4±1.5)% vs (2.5±1.4)%, P=0.11]. Immunohistochemistry and flow cytometry showed that MOG 35-55 was released and engulfed by CD11b + cells at the inoculation sites, and no more MOG 35-55 was released at the onset of EAE. HE staining showed that granuloma that formed surrounding the antigen emulsion during EAE development prevented antigen release from the emulsion, completely isolating the antigen from the peripheral immune system. The incidence of EAE was relatively low in mice immunized via footpads, which was related to the sustained release of MOG 35-55, but had no direct relation to CD4 + regulatory T cells. Continuous intraperitoneal injection of soluble MOG 35-55 could prevent and treat EAE. Conclusions:Exogenous myelin antigen has been completely cleared in EAE mice, and the occurrence of EAE depends on the clearance of the myelin antigen.

Excavation of Ancient Prescription of Chinese Medicinals for Whitening and Freckle-Removing(Erbai Yixin)Based on the Theory of"Lung Governs the Skin and Hair" / 中药新药与临床药理

Objective Based on the theory of"lung governs the skin and hair"in"Yellow Emperor's Inner Classic",this paper analyzes the medication rules of whitening and freckle-removing.The aim of this study is to provide reference for the clinical practice of traditional Chinese medicine(TCM)theory and the medication in TCM cosmetics.Methods"Chinese Medical Classics"was used to search the records of whitening and freckle-related drugs.The frequency,nature,flavor,meridian tropism and compatibility laws of TCM for whitening and freckle-removal were analyzed by statistics and association rules.The network pharmacology research was used to analyze the whitening and freckle-removing effects and mechanisms of high-frequency drugs.Then,the potential active ingredients were analyzed.The whitening and anti-freckle effect was verified through cytotoxicity experiments and melanin content detection.Results A total of 171 external prescriptions were selected in eligible articles,including 261 Chinese medicinals,most of which were pungent and belong to the lung meridian.The most frequently used Chinese medicinals was"Erbai Yixin"(EBYX,Angelicae Dahuricae Radix,Typhonii Rhizoma,Asari Radix et Rhizoma).Network pharmacological analysis showed that the core targets of EBYX for whitening and removing freckles are TP53,EGFR,ALB,etc.,which are mainly involved in oxygen perception and response,skin immune regulation,skin cell growth,differentiation,stress,inflammatory response,and other biological processes.Based on the results of molecular docking,biological analysis proved that the active ingredients of EBYX are chrysophanol,gallic acid and caffeic acid,which have inhibitory effects on the proliferation of melanoma cells and melanin production.Conclusion Most of the ancient prescriptions for whitening and removing freckles are pungent and belong to the lung meridian,which embodies the theory of"lung governs the skin and hair".The high-frequency drug EBYX may play a role by regulating skin redox,immunity and inflammation.The active ingredients of EBYX have an inhibitory effect on melanin formation.This study enriches the scientific connotation of TCM whitening and freckle-removing prescriptions based on the theory of"lung governs the skin and hair",realizes interdisciplinary integration and provides support for the modernization of TCM.

Network Pharmacological Analysis and Experimental Verification of the Mechanism of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma Drug Pair in the Treatment of Hypertension / 中药新药与临床药理

Objective To investigate the mechanism of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair in the treatment of hypertension based on the network pharmacology method and animal experiment verification.Methods(1)TCMSP,BATMAN and TCMIP databases were used to screen the active components and targets of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair.The hypertension-related targets were obtained by searching the Drugbank,Genecard,TTD and Disgenet databases.The intersection(common target)of the active component target and the target related to hypertension disease was taken,and the obtained intersection target was the potential target of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair for the treatment of hypertension.The active ingredients and their targets of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair were imported into Cytoscape 3.9.1 software to construct a'Chinese medicines-active ingredients-targets'network and screen key active ingredients.The protein-protein interaction(PPI)network of potential targets was constructed to screen potential core targets.The Metascape platform was used to analyze the GO function and KEGG pathway enrichment of potential targets.The key active components and potential core targets were selected for molecular docking verification.(2)Thirty male spontaneously hypertensive rats(SHR)were randomly divided into model group,western medicine group(Candesartan Cilexetil,0.72 mg·kg-1)and low-,medium-and high-dose groups of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma(2.25,4.50,9.00 g·kg-1).Another male WKY rats were selected as blank group,with 6 rats in each group,once a day for 8 weeks.The systolic blood pressure of rat tail artery was detected before administration and 2,4,6 and 8 weeks after drug intervention.The pathological changes of thoracic aorta were observed by HE staining.The protein expression levels of GRP78,CHOP and Caspase-12 in aorta abdominalis were detected by Western Blot.Results(1)A total of 83 active components of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma were obtained,and 158 potential targets(intersection targets)for the treatment of hypertension were screened out.Five key active ingredients:p-hydroxybenzoic acid,4-hydroxybenzylamine,tanshinone I,tanshinone,γ-sitosterol;6 potential core targets:IL6,TNF,CASP3,JUN,PTGS2,IL1B;GO functional enrichment analysis obtained 1 826 biological process items,89 cell component items,and 199 molecular function items.KEGG pathway enrichment analysis obtained 186 pathways,mainly involving neuroactive ligand-receptor interaction,calcium signaling pathway,inflammatory response(such as TNF and MAPK signaling pathway),vascular protection(such as HIF-1 and cAMP signaling pathway),oxidative stress(such as PI3K-Akt signaling pathway)and other signaling pathways.Tanshinone I and tanshinone had strong binding force to 6 potential core targets,and γ-sitosterol had strong binding force to IL6,CASP3,JUN,PTGS2 and IL1B.(2)Compared with the blank group,the systolic blood pressure of the model group was significantly increased(P＜0.01).The thoracic aortic endothelial injury was obvious,the endothelial cell morphology was abnormal,swelling and exfoliated cells could be seen,the intima of the tissue was disordered,the intima structure was incomplete,and the intima was thickened.The protein expressions of GRP78,CHOP and Caspase-12 in abdominal aorta were significantly increased(P＜0.01).Compared with the model group,the systolic blood pressure of the rats in the administration group was significantly decreased(P＜0.01);the injury of thoracic aorta was alleviated,and the morphology,intima structure and thickness of endothelial cells were improved to varying degrees.The protein expressions of GRP78,CHOP and Caspase-12 in abdominal aorta were significantly decreased(P＜0.01).Conclusion Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair may act on core targets such as IL6,TNF,CASP3,JUN,PTGS2,and IL1B through key active components such as p-hydroxybenzoic acid,tanshinone,and γ-sitosterol,and regulate key signaling pathways such as TNF signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,and PERK signaling pathway to improve vascular endothelial dysfunction,inhibit endoplasmic reticulum stress,and lower blood pressure.