Eugenol and its liposome-based nano carrier reduce anxiety by inhibiting glyoxylase-1 expression in mice / Eugenol e seu nanocarreador à base de lipossoma reduzem a ansiedade ao inibir a expressão de glioxalase 1 em camundongos

Abstract The most common form of psycho-social dysfunction is anxiety with depression being related closely without any age bar. They are present with combined state of sadness, confusion, stress, fear etc. Glyoxalase system contains enzyme named glyoxalase 1 (GLO1).It is a metabolic pathway which detoxifies alpha-oxo-aldehydes, particularly methylglyoxal (MG). Methylglyoxal is mainly made by the breakdown of the glycolytic intermediates, glyceraldehyde-3-phosphates and dihydroxyacetone phosphate. Glyoxylase-1 expression is also related with anxiety behavior. A casual role or GLO-1 in anxiety behavior by using viral vectors for over expression in the anterior cingulate cortex was found and it was found that local GLO-1 over expression increased anxiety behavior. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of GLO-1 was analyzed by real time RT-PCR. Moreover, the GLO-1 protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating GLO-1 protein expression in mice.

Resumo A forma mais comum de disfunção psicossocial é a ansiedade intimamente relacionada com a depressão, sem qualquer barreira de idade. Elas estão presentes em um estado combinado de tristeza, confusão, estresse, medo etc. O sistema de glioxalase contém uma enzima chamada glioxalase 1 (GLO1). É uma via metabólica que desintoxica alfa-oxo-aldeídos, particularmente metilglioxal (MG). O metilglioxal é produzido principalmente pela quebra dos intermediários glicolíticos, gliceraldeído-3-fosfatos e fosfato de diidroxiacetona. A expressão da glioxalase 1 também está relacionada ao comportamento de ansiedade. Um papel casual ou GLO1 no comportamento de ansiedade usando vetores virais para superexpressão no córtex cingulado anterior foi encontrado e descobriu-se que a superexpressão local de GLO1 aumentava o comportamento de ansiedade. O presente estudo trata do mecanismo molecular da atividade protetora do eugenol contra o transtorno ansiolítico. Um estudo pré-clínico em animais foi realizado em 42 camundongos BALB / c. Os animais foram submetidos ao estresse por meio do modelo de contenção convencional. A expressão de mRNA de GLO1 foi analisada por RT-PCR em tempo real. Além disso, a expressão da proteína GLO1 também foi examinada por imuno-histoquímica em todo o cérebro e a densidade média foi calculada. Verificou-se que as expressões de mRNA e proteínas estavam aumentadas em animais que receberam ansiedade em comparação com o controle normal. Considerando que as expressões foram diminuídas nos animais tratados com eugenol e seus nanocarreadores baseados em lipossomas de forma dependente da dose. No entanto, os resultados foram melhores em animais tratados com nanocarreadores em comparação com o composto sozinho. Conclui-se que o eugenol e seus nanocarreadores baseados em lipossomas exercem atividade ansiolítica por regulação negativa da expressão da proteína GLO1 em camundongos.

BALB/c and C57BL/6 mouse strains influence gastric function outcomes with administration of cisplatin and dexamethasone

Abstract Our aim was to evaluate the effects of cisplatin and dexamethasone alone and combined on gastric contractility and histomorphometry of BALB/c and C57BL/6 mice. BALB/c and C57BL/6 male mice (8-week-old) were randomly separated into: Control; Cisplatin (7.5 mg/Kg); Dexamethasone (2.0 mg/Kg); and Dexamethasone plus Cisplatin (2.0 mg/Kg of dexamethasone 1-hour prior to 7.5 mg/Kg of cisplatin). Drugs were administered intraperitoneally for three days. Body weight and food intake were evaluated on 2nd day. Alternating Current Biosusceptometry technique was employed to measure gastric contractions on 3rd day. Afterward, mice were killed for gastric histomorphometric analysis. Cisplatin decreased food intake and caused bradygastria in BALB/c mice; however, the amplitude of gastric contractions decreased in both BALB/c and C57BL/6. Dexamethasone and cisplatin combined restored the gastric frequency and food intake only in BALB/c, but drug combination reduced the gastric amplitude of contractions in both strains. Dexamethasone alone increased gastric mucosa thickness in C57BL/6 and decreased muscular thickness in BALB/c. In conclusion, the mouse strains presented differences in acute effects of cisplatin and dexamethasone alone and combined on gastric function. This reinforces the importance of choosing the appropriate mouse strain for studying the acute effects of drugs on the gastrointestinal tract.

Efecto del estrés crónico y de la masticación sobre los niveles séricos de interleuquina-6 e interferón-gama / Effect of chronic stress and chewing on serum interleukin-6 and interferon-gamma levels

Introducción: el estrés crónico afecta el equilibrio inmunológico alterando los niveles séricos de interleuquina-6 (IL-6) e interferón gama (INF-γ), dicha alteración afecta al sistema nervioso y al comportamiento humano. La masticación adecuada disminuiría dichos efectos. El objetivo del estudio fue determinar el efecto del estrés crónico y de la masticación sobre los niveles séricos de IL-6 e INF-γ. Métodos: experimento donde se emplearon 64 ratones Balb/c de 8 semanas de edad. Se dividieron en 4 tratamientos: Grupo NE: Masticación normal + estrés, Grupo N: masticación normal sin estrés, Grupo DE: Masticación deficiente + estrés, Grupo D: masticación deficiente sin estrés. Mediante test de ELISA se midió IL-6 e IFN-γ alfinal de la 4ta y de la 8va semana de tratamiento. Resultados: tanto la IL-6 como el IFN-γ fueron mayores en el grupo DE (p<0,05) al final de la 4ta semana. Al evaluarlos al término de la 8va semana se observó que en el grupo NE se incrementó la IL-6 respecto al resto de grupos (p<0,0001), y en el grupo DE fue donde se encontró mayor cantidad de IFN-γ (p<0,0001). Conclusión: el estrés crónico y la masticación deficiente incrementan los niveles séricos de IL-6 e IFN-γ. En cambio, la adecuada masticación disminuye el nivel de tales citoquinas al final de la cuarta semana de tratamiento.

Introduction: chronic stress affects the immune balance by altering the serum levels of interleukin-6 (IL-6) and gamma interferon (INF-γ), this alteration affects the nervous system and human behavior. Appropriate chewing would lessen these effects. The aim of this study was to determine the effect of chewing and chronic stress over serum levels of IL-6 and INF-γ. Methods: experiment in which 64 Balb/C mice of 8 weeks of age were used, they were divided into 4 treatments: Group NE: Normal chewing + stress, Group N: normal chewing without stress, Group DE: Chewing poor + stress, Group D: poor chewing without stress. IL-6 and IFN-γ were measured by ELISA after 4 and 8 weeks of treatment. Results: both IL-6 and IFN-γ were higher in the DE group (p < 0,05) at the end of fourth week of treatment. When evaluating the animals at the end of the eighth week of treatment, it was observed that in the NE group, the IL-6 was increased with respect to the rest (p < 0,0001) and the DE group showed more IFN-γ (p < 0,0001). Conclusion: stress and poor chewing increase serum IL-6 and IFN-γ. In contrast, appropriate chewing decreases the effects of stress on the increase of such cytokines at the end of the fourth week of treatment in animals.

Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Prokaryotic expression of the GapC protein of Streptococcus uberis and prediction, identification of its B-cell epitopes / 生物工程学报

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide 266AANDSYGYTEDPIVSSD282 reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.

Effect of fire needling on imiquimod induced psoriasis-like lesion and STAT3 pathway in mice / 中国针灸

OBJECTIVE@#To observe the effect of fire needling on psoriasis-like lesion and the signal transducer and activator of transcription 3 (STAT3) pathway in mice and compare the therapeutic effect between different interventions of fire needling therapy (surrounding technique of fire needling, fire needling at "Dazhui" [GV 14] and "Zusanli" [ST 36]).@*METHODS@#Thirty male BALB/c mice were randomized into a blank group, a model group, a dexamthasone group, a surrounding technique group and an acupoint group, 6 mice in each one. Except the blank group, the mice in the rest groups were established as psoriasis-like lesion model by topical application with imiquimod cream, once daily, consecutively for 8 days. From day 4 to day 8, in the dexamthasone group, gastric infusion with 0.2 mL dexamthasone was administered, once daily. On day 4, 6 and 8, in the surrounding technique group, fire needling was exerted around the skin lesion; and fire needling was applied to "Dazhui" (GV 14) and "Zusanli" (ST 36) in the acupoint group, once a day. The changes in skin lesion on the dorsal parts of mice were observed in each group to score the psoriasis area and severity index (PASI). Using HE staining, the dermal morphological changes and epidermal thickness were observed in the mice of each group. The positive expression of proliferating cell-associated antigen Ki-67 was determined by immunofluorescence. Immunohistochemistry method was used to determine the expressions of , and T cells of skin tissue in each group. Using real-time PCR, the expressions of interleukin (IL)-17, IL-22, tumor necrosis factor α(TNF-α) mRNA were determined. Western blot method was adopted to determine the protein expressions of STAT3 and p-STAT3 in skin tissue in each group.@*RESULTS@#Compared with the blank group, the scores of each item and the total scores of PASI, as well as the epidermal thickness were all increased in the mice of the model group (P<0.01). Except for the erythema scores of the dexamethasone group and the surrounding technique group, the scores of each item and the total scores of PASI, as well as the epidermal thickness were all decreased in each intervention group as compared with the model group (P<0.01). The infiltration scores and the total scores in the dexamethasone group and the acupoint group were lower than those in the surrounding technique group respectively (P<0.01, P<0.05). In comparison with the blank group, Ki-67 positive cell numbers and the numbers of , and T cells in skin tissue were increased in the mice of the model group (P<0.01). Ki-67 positive cell numbers and the numbers of , and T cells were reduced in each intervention group as compared with the model group (P<0.01), and the numbers of and T cells in the acupoint group were less than the surrounding technique group (P<0.01). Compared with the blank group, the mRNA expressions of IL-17, IL-22 and TNF-α and the ratio of p-STAT3 to STAT3 were all increased in the model group (P<0.01). The mRNA expressions of IL-17, IL-22 and TNF-α and the ratio of p-STAT3 to STAT3 were all decreased in each intervention group as compared with the model group (P<0.01, P<0.05). The mRNA expressions of IL-17, IL-22 and TNF-α in the acupoint group, as well as mRNA expression of IL-17 in the surrounding technique group were all lower than the dexamethasone group (P<0.01), while, the mRNA expression of IL-22 in the acupoint group was lower than the surrounding technique group (P<0.01).@*CONCLUSION@#Fire needling therapy improves skin lesion severity in imiquimod induced psoriasis-like lesion of the mice, which is probably related to the inhibition of STAT3 pathway activation and the decrease of Th17 inflammatory factors expression. The systemic regulation of fire needling at "Dazhui" (GV 14) and "Zusanli" (ST 36) is superior to the local treatment.

Anti-inflammatory Effects of Ampelopsis Japonica Root on Contact Dermatitis in Mice / 中国结合医学杂志

OBJECTIVE@#To investigate the anti-inflammatory potential of Ampelopsis japonica on contact dermatitis (CD).@*METHODS@#A total of 38 Balb/c mice were divided into 5 groups by using a random number table: normal mice (n=6), CD model mice (n=8), CD mice treated with 3 or 30 mg/kg of the ethanol extract of A. japonica (EEAJ, n=8) and 7.5 mg/kg dexamethasone treated CD mice (DEX, n=8). CD was induced using topical application of 1-fluoro-2,4-dinitrofluorobenzene in mice. EEAJ and DEX were topically applied to the shaved skin of each mouse for 6 days, and the effects of EEAJ and DEX on skin lesions and color, histopathological abnormalities such as epidermal hyperplasia and immune cell infiltration, and tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) production were investigated. The effects on changes in body weights and spleen/body weight ratio were also investigated.@*RESULTS@#EEAJ at 30 mg/kg significantly prevented scaling, erythema and enlargement of skin weight compared to using carbon dioxide. EEAJ also prevented epithelial hyperplasia and immune cell infiltrations induced by repeated application of DNFB (P<0.01). In addition, EEAJ significantly lowered levels of TNF-α, IL-6 and MCP-1 (P<0.05 or P<0.01). The anti-inflammatory effects of EEAJ were similar to those of DEX.@*CONCLUSION@#A. japonica may be a new therapeutic agent with the potential to reduce or replace corticosteroids and its mechanisms are closely related to regulation of TNF-α production.

Acupoint Autohemotherapy Attenuates Atopic Dermatitis Lesions by Regulating Th1/Th2 Balance in DNCB-Induced BALB/c Mice / 中国结合医学杂志

OBJECTIVE@#To evaluate the therapeutic effects of acupoint autohemotherapy (A-AHT) on 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD) in mice focusing on regulating T helper 1/T helper 2 (Th1/Th2) immune responses.@*METHODS@#Thirty BALB/c mice were divided into 5 groups by a random number table, including normal control (NC), AD model (AD), A-AHT, sham A-AHT (sA-AHT), and acupoint injection of normal saline (A-NS) groups, 6 mice per group. Mice were challenged by DNCB for the establishment of experimental AD model. On the 8th day, except for the NC and AD groups, the mice in the other groups received management once every other day for a total of 28 days. For the A-AHT and sA-AHT groups, 0.05 mL of autologous whole blood (AWB) was injected into bilateral Zusanli (ST 36) and Quchi (LI 11) and sham-acupoints (5 mm lateral to ST 36 and LI 11), respectively. The A-NS group was administrated with 0.05 mL of normal saline by acupoint injection into ST 36 and LI 11. Dermatitis severity for dorsal skin of mice was determined using the Severity Scoring of Atopic Dermatitis (SCORAD) every week. The total immunoglobulin E (IgE), interleukin-4 (IL-4), and interferon-gamma (IFN-γ) cytokine levels in serum were examined by enzyme-linked immunosorbent assay (ELISA). Spleen Th1/Th2 expression were analyzed via flow cytometry and immunohistochemical assay was used to detect T-box expressed in T cell (T-bet) and GATA-binding protein 3 (GATA3) expressions in skin lesions of mice.@*RESULTS@#Compared with the AD group, both A-AHT and sA-AHT reduced the SCORAD index and serum IgE level (P<0.05 or P<0.01); A-AHT, sA-AHT and A-NS down-regulated serum IL-4 level and upregulated IFN-γ/IL-4 ratio (P<0.05 or P<0.01); A-AHT regulated the Th1/Th2 shift specifically and increased the related transcription factors such as T-bet expression and T-bet/GATA3 ratio (P<0.05).@*CONCLUSION@#A-AHT showed significant effectiveness on the AD model mice, through regulating Th1/Th2 immune responses.

Inhibitory Effect of Interference with PD-L1 Expression on B-cell lymphoma in Mice / 中国实验血液学杂志

OBJECTIVE@#To investigate the potential inhibitory effect of interference with PD-L1 on B cell lymphoma in mice.@*METHODS@#Three shRNA vectors for mouse CD274 (PD-L1) were constructed and transiently transfected into 293T cells. RT-qPCR was used to validate the interference efficiency of CD274. The shRNA vector that interfere efficiently with CD274 expression was packaged by using lentivirus packaging system to generate shRNA lentivirus, and then transfected into A20 lymphoma cell line. The methyl thiazol terazolium (MTT) assay was used to detect proliferation after 48 h culture of CD274-sh A20 cells. Meanwhile, BALB/c mice were hypodermically infected with CD274-sh A20 cells. Infected mice were observed daily and assessed to visualize tumor by in vivo fluorescence imaging.@*RESULTS@#The proliferation rate of CD274-sh A20 cells in vitro was significantly lower than that of A20 cells (P<0.05). The tumor size detected by in vivo fluorescence imaging showed a significant reduce in tumor bearing mice with CD274-sh compared with other tumor bearing mice. And the weight and size of tumor in CD274-sh group were also significantly reduced compared with other group (P<0.05). Moreover, the survival time of tumor bearing mice in CD274-sh group was longer than that of the PD-L1 high expression group.@*CONCLUSION@#PD-L1 plays an important role in the incidence and the progression of lymphoma, and the shRNA-based PD-L1 knockdown can inhibit cell proliferation of A20 cells and partly suppress tumor growth.

Phytochemical characterization and biological activities of Plectranthus barbatus Andrews / Caracterização fitoquímica e atividades biológicas de Plectranthus barbatus Andrews

Plectranthus barbatus Andrews (Lamiaceae) is widely distributed in the world and has a range of popular therapeutic indications. This work aimed to evaluate the phytochemical characterization of two leaf extracts of P. barbatus, and their antimicrobial, antineoplastic and immunomodulatory potential. After collection, herborization and obtainment of the P. barbatus aqueous extract (PBA) and acetone:water 7:3 P. barbatus organic extract (PBO), the phytochemical characterization was performed by high-performance liquid chromatography (HPLC). The antimicrobial activity was performed to determine the minimum inhibitory concentration (MIC) against eight bacterial strains using the microdilution test and the fungus Trichophyton rubrum by disc diffusion assay and microdilution test. Cytotoxicity was assessed by MTT and trypan blue methods in normal peripheral blood mononuclear cells (PBMCs) at concentrations ranged between 0.1 to 100 µg.mL-1 and in neoplastic cell lines Toledo, K562, DU-145 and PANC-1 at 1, 10 and 100 µg.mL-1 . Immunomodulatory activity, was evaluated by sandwich ELISA of proinflammatory cytokines at BALB/c mice splenocytes cultures supernatant. Both extracts presented flavonoids, cinnamic derivatives, steroids and ellagic acid. PBO showed bacteriostatic activity against Acinetobacter baumannii (MIC = 250 µg.mL-1) clinical isolate and PBA fungistatic activity against Trichophyton rubrum (MIC = 800 µg.mL-1). The extracts did not exhibit toxicity to PBMCs and neoplastic cells (IC50 > 100 µg.mL-1). Additionally, PBO at 100 µg.mL-1 significantly inhibited IFN-γ and IL-17A cytokines (p = 0.03). Plectranthus barbatus is a potential candidate for therapeutic use due to its low toxicity in healthy human cells and exhibits biological activities of medical interest as bacteriostatic, fungistatic and immunomodulatory.

Plectranthus barbatus Andrews (Lamiaceae) é amplamente distribuída no mundo e com uma série de indicações terapêuticas populares. Este trabalho teve como objetivo avaliar a caracterização fitoquímica de dois extratos da folha de P. barbatus e seu potencial antimicrobiano, antineoplásico e imunomodulador. Após coleta, herborização e obtenção do extrato aquoso (PBA) e acetona: água 7: 3 (orgânico) (PBO) de P. barbatus, a caracterização fitoquímica foi realizada por cromatografia líquida de alta eficiência (CLAE). A atividade antimicrobiana foi realizada para determinar a concentração inibitória mínima (CIM) contra oito cepas bacterianas usando o teste de microdiluição e o fungo Trichophyton rubrum por ensaio de difusão em disco e teste de microdiluição. A citotoxicidade foi avaliada por métodos MTT e azul de tripan em células normais mononucleares do sangue periférico (CMSP) em concentrações variadas entre 0,1 a 100 µg.mL-1 e nas linhagens celulares neoplásicas Toledo, K562, DU-145 e PANC-1 em 1, 10 e 100 µg.mL-1 . A atividade imunomoduladora foi avaliada por ELISA sanduíche de citocinas pró-inflamatórias em sobrenadante de culturas de esplenócitos de camundongos BALB/c. Ambos os extratos apresentaram flavonoides, derivados cinâmicos, esteróides e ácido elágico. O PBO mostrou atividade bacteriostática contra Acinetobacter baumannii (CIM = 250 µg.mL-1) e atividade fungistática do PBA contra Trichophyton rubrum (CIM = 800 µg.mL-1). Os extratos não apresentaram toxicidade para CMSP e células neoplásicas (IC50 > 100 µg.mL-1). Além disso, o PBO a 100 µg.mL-1 inibiu significativamente as citocinas IFN-γ e IL-17A (p = 0,03). Plectranthus barbatus é um candidato potencial para uso terapêutico devido à sua baixa toxicidade em células humanas saudáveis e exibe atividade de interesse médico como bacteriostática, fungistática e imunomoduladora.

Experimental study of dopamine ameliorating the inflammatory damage of olfactory bulb in mice with allergic rhinitis / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the effects of dopamine on olfactory function and inflammatory injury of olfactory bulb in mice with allergic rhinitis (AR). Methods: AR mouse model was established by using ovalbumin (OVA), and the mice were divided into two groups: olfactory dysfunction (OD) group and without OD group through buried food pellet test (BFPT). The OD mice were randomly divided into 2 groups, and OVA combined with dopamine (3, 6, 9 and 12 days, respectively) or OVA combined with an equal amount of PBS (the same treatment time) was administered nasally. The olfactory function of mice was evaluated by BFPT. The number of eosinophils and goblet cells in the nasal mucosa were detected by HE and PAS staining. Western blotting, immunohistochemistry or immunofluorescence were used to detect the expression of olfactory marker protein (OMP) in olfactory epithelium, the important rate-limiting enzyme tyrosine hydroxylase (TH) of dopamine, and the marker proteins glial fibrillary acidic protein (GFAP) and CD11b of glial cell in the olfactory bulb. TUNEL staining was used to detect the damage of the olfactory bulb. SPSS 26.0 software was used for statistical analysis. Results: AR mice with OD had AR pathological characteristics. Compared with AR mice without OD, the expression of OMP in olfactory epithelium of AR mice with OD was reduced (F=26.09, P<0.05), the expression of GFAP and CD11b in the olfactory bulb was increased (F value was 38.95 and 71.71, respectively, both P<0.05), and the expression of TH in the olfactory bulb was decreased (F=77.00, P<0.05). Nasal administration of dopamine could shorten the time of food globule detection in mice to a certain extent, down-regulate the expression of GFAP and CD11b in the olfactory bulb (F value was 6.55 and 46.11, respectively, both P<0.05), and reduce the number of apoptotic cells in the olfactory bulb (F=25.64, P<0.05). But dopamine had no significant effect on the number of eosinophils and goblet cells in nasal mucosa (F value was 36.26 and 19.38, respectively, both P>0.05), and had no significant effect on the expression of OMP in the olfactory epithelium (F=55.27, P>0.05). Conclusion: Dopamine can improve olfactory function in mice with AR to a certain extent, possibly because of inhibiting the activation of glial cells in olfactory bulb and reducing the apoptotic injury of olfactory bulb cells.

The role of TNF-α/TNFR1 inhibiting autophagy of M1-type Kupffer cells in liver injury in trichloroethylene-sensitized mice / 中华劳动卫生职业病杂志

Objective: To detect the expression levels of M1-type polarization and autophagy-related indicators in the liver of trichloroethylene (TCE) -sensitized mice, and to explore the role of liver tumor necrosis factor-α (TNF-α) and tumor necrosis factor receptor 1 (TNFR1) in regulating M1-type Kupffer cells autophagy in liver injury in TCE-sensitized mice. Methods: In November 2019, according to simple random grouping, 45 SPF grade BALB/c female mice (6-8 weeks old) were divided into 4 groups: blank control group (n=5) , solvent control group (n=5) , TCE treatment group (n=18) , TCE+R7050 (inhibitor) treatment group (n=17) . Transdermally sensitized mice, 24 h after the last challenge, the mice were divided into TCE sensitized group and TCE non-sensitized group according to the skin reaction score. The livers of mice were harvested, and the pathological changes of the livers were observed under light and electron microscopes. Western blotting was used to detect the expressions of TNF-α, TNFR1 and autophagy-related indexes. The expression of inducible nitric oxide synthase (iNOS) , a marker of M1-type Kupffer cells, was detected by immunohistochemistry, and the occurrence of autophagy in M1-type Kupffer cells was detected by immunofluorescence double-labeling method. Results: The sensitization rate of TCE treatment group was 38.9% (7/18) , and TCE+R7050 treatment group was 35.3% (6/17) , with no significant difference between the two groups (P=1.000) . Compared with the blank control group, mice in the TCE sensitized group had abnormal liver ocytes, obvious liver injury, reduced mitochondria and broken endoplasmic reticulum. Western blotting results showed that the expressions of TNF-α and TNFR1 protein in the liver of the mice in the TCE sensitized group increased, the expression of iNOS protein in M1-type Kupffer cells increased, and the expressions of autophagic microtubule-associated protein 1 light-chain 3 (LC3B) and Beclin1 protein were decreased (P<0.05) . The results of immunohistochemistry showed that iNOS was not significantly expressed in the blank control group and solvent control group, and a small amount of expression was found in the TCE non-sensitized group, the positive staining area was obvious in TCE sensitized group, and the expression of iNOS was significantly increased (P<0.05) . Immunofluorescence results showed that the iNOS protein levels in the blank control group, solvent control group and TCE non-sensitized group were lower, and only partially colocalized with P62; the colocalization of iNOS with P62 in the TCE sensitized group was significantly increased. Conclusion: TNF-α/TNFR1 signaling pathway may promote liver injury in TCE-sensitized mice by inhibiting autophagy of M1-type Kupffer cells.

The role of ROS/TXNIP/NLRP3 pathway in the skin injury of trichloroethylene sensitized mice / 中华劳动卫生职业病杂志

Objective: To explore the mechanism of reactive oxygen species/thioredoxin-interacting protein/nucleotide-binding oligomerization domain-like receptor 3 (ROS/TXNIP/NLRP3) pathway in the skin injury of trichloroethylene (TCE) sensitized mice. Methods: In August 2020, 40 female BALB/c mice were randomly divided into control group (n=5) , solvent control group (n=5) , TCE treatment group (n=15) and TCE+(2-(2, 2, 6, 6-Tetrameyhylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito TEMPO) treatment group (n=15) . The TCE sensitization model was established. Mice in the TCE treatment group and TCE+Mito TEMPO treatment group were divided into the sensitized positive group and the sensitized negative group according to the skin erythema and edema reactions on the back of the mice 24 h after the last stimulation. The mice were sacrificed 72 h after the last stimulation, the back skin of the mice was taken, and the skin lesions were observed. Immunohistochemistry (IHC) was used to detect the expression level of NLRP3, and the Western Blot was performed to detect the expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) , cysteinyl aspartate specific proteinase 1 (Caspase 1) , Interleukin-1β (IL-1β) and TXNIP proteins in the skin of the mice, the reactive oxygen species (ROS) kit was used to detect the level of intracellular ROS in the back skin tissue. Results: The sensitization rates of TCE treatment group and TCE+Mito TEMPO treatment group were 40.0% (6/15) and 33.3% (5/15) , respectively, and there was no significant difference between the two groups (P>0.05) . The back skin of the mice in the TCE sensitized positive group was thickened and infiltrated by a large number of inflammatory cells. The number of mitochondria in the epidermis cells was significantly reduced, the mitochondrial crest disappeared and vacuolar degeneration occurred. TCE+Mito TEMPO sensitized positive group had less damage, more mitochondria and relatively normal cell structure. Compared with the solvent control group and corresponding sensitized negative groups, the expression levels of NLRP3, ASC, Caspase 1, IL-1β, TXNIP proteins and the content of ROS in the TCE sensitized positive group and TCE+Mito TEMPO sensitized positive group were significantly increased (P<0.05) . Compared with TCE sensitized positive group, the expression levels of NLRP3, ASC, Caspase 1, IL-1β, TXNIP proteins and the content of ROS in the TCE+Mito TEMPO sensitized positive group were significantly decreased (P<0.05) . Conclusion: ROS/TXNIP/NLRP3 pathway was activated and then encouraged the release of IL-1β, finally aggravated the TCE-induced skin injury.

An injectable hydrogel/staple fiber composite for sustained release of CA4P and doxorubicin for combined chemotherapy of xenografted breast tumor in mice / 南方医科大学学报

OBJECTIVE@#To prepare an injectable hydrogel/staple fiber composite loaded with combretastain A-4 disodium phosphate (CA4P) and doxorubicin (DOX) and evaluate its antitumor efficacy via intratumoral injection.@*METHODS@#DOX-loaded PELA staple fibers (FDOX) were prepared using electro-spinning and cryo-cutting, and the drug distribution on the surface of the fibers was observed using a fluorescence microscope, and the encapsulation efficiency and loading capacity of FDOX were determined with a fluorospectro photometer. The fibers were then dispersed in CA4P-loaded PLGA-PEG-PLGA tri-block polymer solution at room temperature to obtain the hydrogel/staple fiber composite (GCA4P/FDOX). The thermo-sensitivity of this composite was determined by a test tube inverting method. An ultraviolet spectrophotometer and a fluorospectrophotometer were used to detect the release profile of CA4P and DOX, respectively. We observed in vivo gel formation of the composite after subcutaneous injection in mice. The in vitro cytotoxicity of GCA4P/FDOX composite in MCF-7 and 4T1 cells was assessed using cell Counting Kit-8 (CCK-8) reagent. In a mouse model bearing breast tumor 4T1 cell xenograft, we evaluated the antitumor efficacy of the composite by monitoring tumor growth within 30 days after intratumoral injection of the composite. HE staining, immunohistochemistry for Ki67 and immunofluorescence (TUNEL) assay were used for pathological examination of the tumor tissues 21 days after the treatments.@*RESULTS@#The average length of FDOX was 4.0±1.3 μm, and its drug loading capacity was (2.69±0.35)% with an encapsulation efficiency of (89.70±0.12)%. DOX was well distributed on the surface of the fibers. When the temperature increased to 37 ℃, the composite rapidly solidified to form a gel in vitro. Drug release behavior test showed that CA4P was completely released from the composite in 5 days and 87% of DOX was released in 30 days. After subcutaneous injection, the composite solidified rapidly without degradation at 24 h after injection. After incubation with GCA4P/FDOX for 72 h, only 30.6% of MCF-7 cells and 28.9% of 4T1 cells were viable. In the tumor-bearing mice, the tumor volume was 771.9±76.9 mm3 in GCA4P/FDOX treatment group at 30 days. Pathological examination revealed obvious necrosis of the tumor tissues and tumor cell apoptosis induced by intratumoral injection of G4A4P/FDOX.@*CONCLUSION@#As an efficient dual drug delivery system, this hydrogel/staple fiber composite provides a new strategy for local combined chemotherapy of solid tumors.

Pharmacodynamic of cilostazol for anti-altitude hypoxia / 中南大学学报(医学版)

OBJECTIVES@#The plateau environment is characterized by low oxygen partial pressure, leading to the reduction of oxygen carrying capacity in alveoli and the reduction of available oxygen in tissues, and thus causing tissue damage. Cilostazol is a phosphodiesterase III inhibitor that has been reported to increase the oxygen release of hemoglobin (Hb) in tissues. This study aims to explore the anti-hypoxic activity of cilostazol and its anti-hypoxic effect.@*METHODS@#A total of 40 male BALB/C mice were randomly divided into a low-dose cilostazol (6.5 mg/kg) group, a medium-dose (13 mg/kg) group, a high-dose (26 mg/kg) group, and a control group. The atmospheric airtight hypoxia experiment was used to investigate the anti-hypoxic activity of cilostazol and to screen the optimal dosage. Twenty-four male Wistar rats were randomly divided into a normoxia control group, a hypoxia model group, an acetazolamide (22.33 mg/kg) group, and a cilostazol (9 mg/kg) group. After 3 days of hypoxia in the 4 010 m high altitude, blood from the abdominal aorta was collected to determine blood gas indicators, the levels of IL-6 and TNF-α in plasma were determined by enzyme-linked immunosorbent assay, and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutataione (GSH) were measured. The degree of pathological damage for rat tissues was observed with HE staining.@*RESULTS@#Compared with the control group, the survival time of mice in the low, medium, and high dose group of cilostazol was significantly prolonged, and the survival time of mice in the medium dose group was the longest, with an extension rate at 29.34%, so the medium dose was the best dose. Compared with the hypoxia model group, the P50 (oxygen partial pressure at Hb oxygen saturation of 50%) value of rats in the cilostazol group was significantly increased by 1.03%; Hb and Hct were significantly reduced by 8.46% and 8.43%, and the levels of IL-6 and TNF-α in plasma were reduced by 50.65% and 30.77%. The MDA contents in heart, brain, lung, liver, and kidney tissues were reduced by 37.12%, 29.55%, 25.00%, 39.34%, and 21.47%, respectively. The SOD activities were increased by 94.93%, 9.14%, 9.42%, 13.29%, and 20.80%, respectively. The GSH contents were increased by 95.24%, 28.62%, 28.57%, 20.80%, and 44.00%, respectively. The results of HE staining showed that compared with the hypoxia model group, cilostazol significantly improved the damage of heart, lung, and kidney tissues in rats after hypoxia.@*CONCLUSIONS@#Cilostazol can significantly improve the oxidative stress and inflammatory reaction caused by rapid altitude hypoxia, and it has a significant protective effect on tissue damage caused by hypoxia, suggesting that it has obvious anti-hypoxic activity.

Establishment of a Mouse Model of Acquired Aplastic Anemia Mediated by Cyclophosphamide Combined with Cyclosporine / 中国实验血液学杂志

OBJECTIVE@#To establish a stable mouse model of acquired aplastic anemia.@*METHODS@#Female BALB/C mice aged 6 months were intraperitoneally injected with cyclophosphamide and cyclosporine for 14 days. The number of peripheral blood cells, the concentration of hemoglobin, the number of bone marrow nucleated cells, bone marrow smear, bone marrow pathological sections and other indexes were observed.@*RESULTS@#In BALB/C mice injected intraperitoneally with cyclophosphamide and cyclosporine, the number of peripheral blood cells and the concentration of hemoglobin were significantly decreased, especially the white blood cells and platelets. Bone marrow smear showed a significant decrease in the number of nucleated cells and bone marrow hyperplasia. Bone marrow pathology showed decreased hematopoietic cells and increased non-hematopoietic cells such as adipocytes.@*CONCLUSION@#The mouse model with intraperitoneal injection of cyclophosphamide and cyclosporine can meet the diagnostic criteria of acquired aplastic anemia, which can be used as a mouse model for the study of the pathogenesis and treatment of acquired aplastic anemia.

Ershiwuwei Shanhu Pills regulate Akt/mTOR/GSK-3β signaling pathway to alleviate Alzheimer's disease mice / 中国中药杂志

The present study investigated the mechanism of the Tibetan patent medicine Ershiwuwei Shanhu Pills(ESP) in alleviating Alzheimer's disease in mice via Akt/mTOR/GSK-3β signaling pathway. BALB/c mice were randomly assigned into a blank control group, a model group, low(200 mg·kg~(-1)), medium(400 mg·kg~(-1)) and high(800 mg·kg~(-1)) dose groups of ESP, and donepezil hydrochloride group. Except the blank control group, the other groups were given 20 mg·kg~(-1) aluminum chloride by gavage and 120 mg·kg~(-1) D-galactose by intraperitoneal injection for 56 days to establish Alzheimer's disease model. Morris water maze was used to detect the learning and memory ability of mice. The level of p-tau protein in mouse hippocampus and the levels of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in hippocampus and serum were detected. Hematoxylin-eosin staining and Nissl staining were performed for the pathological observation of whole brain in mice. TdT-mediated dUTP nick-end labeling(TUNEL) staining was employed for the observation of apoptosis in mouse cortex. Western blot was adopted to detect the protein levels of p-mTOR, p-Akt, and GSK-3β in the hippocampus. Compared with the model group, the ESP groups showcased alleviated pathological damage of the whole brain, decreased TUNEL positive cells, reduced level of p-tau protein in hippocampus, and risen SOD, CAT, and T-AOC levels and declined MDA level in hippocampus and serum. Furthermore, the ESP groups had up-regulated protein levels of p-mTOR and p-Akt while down-regulated protein level of GSK-3β in hippocampus. Therefore, ESP can alleviate the learning and memory decline and oxidative damage in mice with Alzheimer's disease induced by D-galactose combined with aluminum chloride, which may be related to Akt/mTOR/GSK-3β signaling pathway.

Effect of Sishen Pills and its split prescriptions on Tfr/Tfh9/Tfh17 cells in colitis mice / 中国中药杂志

This study aims to investigate the regulatory effect of Sishen Pills(SSP) and its split prescriptions Ershen Pills(EP) and Wuweizi Powder(WP) on T follicular helper(Tfh) cell subset in the dextran sodium sulfate(DSS)-induced colitis mice and the mechanism. A total of 60 male SPF BALB/c mice were used, 10 of which were randomly selected as the normal group. The rest 50 were induced with 3% DSS solution for colitis modeling. After modeling, they were randomized into 5 groups: model group, SSP group, EP group, WP group, and mesalazine group. Body mass, colon mass, colon mass index, colon length, and unit colon mass index in each group were observed. After hematoxylin-eosin(HE) staining, the pathological injury of colon tissue was scored. The expression levels of molecules related to the STAT/SOCS signaling pathway in colon tissues were analyzed by Western blot. Differentiation levels of Tfh cells such as CD4~+CXCR5~+IL-9~+(Tfh9), CD4~+CXCR5~+IL-17~+(Tfh17), and CD4~+CXCR5~+Foxp3~+(Tfr) in peripheral blood of mice were detected by flow cytometry. The results showed each treatment group demonstrated significant increase in body mass and colon length, decrease in colon mass, colon mass index, unit colon mass index, and histopathological score(P<0.05, P<0.01), reduction of the expression of p-STAT3, STAT3, p-STAT6, and STAT6(P<0.05, P<0.01), rise of the expression of SOCS1 and SOCS3(P<0.05, P<0.01), decrease of Tfh9 and Tfh17 cells, and increase of Tfr cells(P<0.05, P<0.01) compared with the model group. These results indicated that SSP and the split EP and WP may alleviate ulcerative colitis by inhibiting the activation of STAT/SOCS signaling pathway and regulating the balance of Tfr/Tfh9/Tfh17 cells.

Effect of Astragali Radix-Curcumae Rhizoma compatibility combined with 5-fluorouracil on Th17/Treg balance and tumor-related mRNA and protein expression in orthotopic xenograft model mice of CT26.WT colorectal carcinoma / 中国中药杂志

Astragali Radix-Curcumae Rhizoma(AR-CR) is a combination commonly used in the clinical treatment of tumors. Based on the T helper 17(Th17)/regulatory T cell(Treg) balance, the present study explored the possible mechanism of AR-CR combined with 5-fluorouracil(5-FU) on the tumor growth of orthotopic xenograft model mice of colorectal carcinoma. Ninety male BALB/c mice were randomly divided into nine groups, i.e., a blank group, a model group, a 5-FU group, high-, medium-, and low-dose AR-CR(2∶1) groups, and high-, medium-, and low-dose AR-CR+5-FU groups, with 10 mice in each group. The orthotopic xenograft model of CT26.WT colorectal carcinoma was induced in mice except those in the blank group. Twenty-four hours after the ope-ration, mice in the blank group and the model group received normal saline by gavage(10 mL·kg~(-1), once per day), and those in the 5-FU group received 5-FU by intraperitoneal injection(25 mg·kg~(-1), once every other day). Mice in the AR-CR groups received AR and CR decoctions by gavage(12, 6, and 3 g·kg~(-1), once a day) and those in the combination groups received AR and CR decoctions and 5-FU(doses and administration methods were the same as above). After intervention for three weeks, all mice were sacrificed and tumor tissues were collected. The tumor mass was weighed and the average tumor weight was calculated. The changing trend of Th17/Treg(%) in the CD4~+T lymphocytes of the spleen tissues of the mice in each group was detected. The mRNA expression in the blood and protein expression in the tumor tissues of transforming growth factor-β(TGF-β), tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), Smad4, N-cadherin, matrix metalloproteinase-7(MMP-7) were detected. The experimental results revealed that compared with the model group, the groups with drug intervention showed reduced tumor mass(P<0.01), decreased CD4~+IL-17~+ in the spleen tissues to varying degrees(P<0.001), and increased proportion of CD4~+Foxp3~+(P<0.001 or P<0.05), indicating that Th17/Treg maintained dynamic balance, and the effect of the combination groups was predominant. Additionally, the mRNA expression in the blood and protein expression in the tumor tissues of TGF-β, TNF-α, IFN-γ, Smad4, N-cadherin, and MMP-7 declined to varying degrees in a dose-dependent manner(P<0.01 or P<0.001). The AR-CR combined with 5-FU can inhibit the tumor growth of orthotopic xenograft model mice of CT26.WT colorectal carcinoma. The mechanism may be related to maintenance of Th17/Treg dynamic balance in the body and down-regulation of TGF-β, TNF-α, IFN-γ, Smad4, N-cadherin, and MMP-7 expression.

A monoclonal antibody-based icELISA for puerarin / 中国中药杂志

Puerarin was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) by periodate oxidation to serve as the immunogen and coating antigen, respectively. BALB/c mice were immunized with puerarin-BSA according to the routine immunization procedure, and the titer and specificity of serum were detected after three immunization. After booster immunization, mouse spleen lymphocytes were fused with mouse myeloma cells, and 24 hybridoma cell lines of the monoclonal antibodies against puerarin were screened by monoclonal antibody screening technique. Ascites was prepared and purified. The cross-reactivity of monoclonal antibody(mAb) M1 with 4'-methoxy puerarin, daidzin, puerarin-6″-O-xyloside, daidzein, mirificin, 3'-methoxy puerarin, and 3'-hydroxy puerarin was 239.84%, 112.18%, 67.89%, 58.28%, 22.37%, 0.40%, and 0.20%, respectively, and those with other analogs such as baicalein and baicalin were all less than 0.10%. The IC_(50) and the working range of the indirect competitive enzyme-linked immunosorbent assay(icELISA) for puerarin were 44.80 ng·mL~(-1) and 8.20-292.30 ng·mL~(-1), respectively. The average recovery was 91.95%-98.20% with an RSD in the range of 0.70%-2.60%. The content of puerarin in different Puerariae Lobatae Radix samples was determined with icELISA and validated by UPLC-MS. The correlation between data obtained from icELISA and UPLC-MS was 0.999 0, indicating that icELISA is suitable for the rapid detection of puerarin in Puerariae Lobatae Radix samples.