Establishment and observation of a mouse model of cytokine release syndrome induced by recombinant mouse IFN-γ adenovirus / 中国肿瘤生物治疗杂志

@#目的：通过向C57Bl/6J小鼠腹腔注射IFN-γ腺病毒（Ad-mIFN-γ）建立细胞因子释放综合征（CRS）的动物模型。方法：构建Ad-mIFN-γ及对照Ad-lacZ腺病毒载体，分别以MOI=100体外转染小鼠腹腔巨噬细胞，流式细胞术检测其对细胞mIFN-γ分泌的影响。将40只雌性C57Bl/6J小鼠按随机数字表法分为对照组、载体对照组、病毒低、中、高剂量组（每组8只），分别向各组小鼠腹腔注射PBS（200 μL）、Ad-lacZ（2×107 PFU/只）、Ad-mIFN-γ（5×106 PFU/只）、Ad-mIFN-γ（1.5×107 PFU/只）和Ad-mIFN-γ（2×107 PFU /只）。每日观测小鼠的体质量及生存情况；第3天时采用流式细胞术检测小鼠外周血和脾内单核细胞（CD11b+）、巨噬细胞（CD11b+/CD86+）比例，免疫荧光染色法检测脾内CD11b+的单核细胞比例；第9天时采用流式细胞术检测小鼠血清中细胞因子的分泌水平；第14天，采用颈椎脱臼法处死小鼠，H-E染色法观察小鼠肝、脾、肺和肾的病理和组织学变化。结果： Ad-mIFN-γ体外感染小鼠腹腔巨噬细胞，在第3天检测到巨噬细胞分泌mIFN-γ达到峰值（118.34±2.90）pg/mL，并在一周内持续高分泌mIFN-γ，Ad-lacZ对照组IFN-γ分泌水平较低后，第3天时为（0.17±0.08）pg/mL。小鼠腹腔注射Ad-mIFN-γ后，在14 d内病毒低、中剂量组无小鼠死亡，病毒高剂量组小鼠体质量持续减轻（P<0.001）；第3天，病毒高剂量组小鼠外周血和脾组织内单核细胞、巨噬细胞比例较对照组和中剂量组均显著增加（P<0.05或P<0.01）；第9天，病毒低、中、高剂量组小鼠血清中mIFN-γ、IL-6、单核细胞趋化蛋白-1（MCP-1）、IL-1、TNF-α等细胞因子的水平均显著升高（P<0.001）；10 d内病毒高剂量组小鼠死亡率达100%。组织病理检测可见病毒高剂量组小鼠的肝、脾、肺、肾组织有明显损伤。结论： Ad-mIFN-γ体外感染小鼠原代腹腔巨噬细胞后，可以快速分泌mIFN-γ；腹腔注射高剂量（2×107 PFU/只）Ad-mIFN-γ导致小鼠出现CRS典型表现，可作为CAR-T细胞治疗诱发CRS的动物模型。

Comparison of 17β-estradiol against retinal light damage in BALB/c or C57BL/6 mice / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the neuroprotective effect of 17β-estradiol(E2)on retina light damage in BALB/c and C57BL/6 mice and provide experimental data for the successful construction of a research model for E2 against retinal light damage.METHODS:Totally 40～45 adult female BALB/c or C57BL/6 mice were divided into six groups, 6 for each group: normal control, ovariectomized control, ovariectomized light(mice were stimulated with continuous white light at 10000 lx for 4, 8, 12, 16, and 24h after 14d of ovariectomy), intravitreal administration sham operation, saline and E2 pre-treatment groups(2μL saline or 10-5mol/L E2 were intravitreal injected respectively after 14d of ovariectomy operation and 24h of dark adaptation). The morphological and functional changes of the retina were detected by paraffin section HE staining, TUNEL staining and electroretinogram.RESULTS:In the ovariectomized light group, the thickness of the inner/outer nuclear layer decreased significantly from the 4h stimulation of 10000 lx white light group. Intravitreal administration of E2 significantly inhibited the apoptosis of retinal cells in the two strains of mice(P&#x003C;0.01)and the decrease of amplitudes of a- and b-waves in max-ERG of C57BL/6 mice(P&#x003C;0.05).CONCLUSION:The light loss sensitivity of two strains of mice was different under the same light stimulation. E2 had a protective effect on both morphology and function of the retina in BALB/c mice, and had a significant protective effect on retina function in C57BL/6 mice.

Effects of modified Zhujing Pill on mitochondrial dynamics related protein in mice with form deprivation myopia / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the effects of modified Zhujing Pill on the mitochondrion structure and dynamin-related protein of retinal pigment epithelial cells(RPEs)in mice with form deprivation myopia.METHODS: 3-week-old C57BL/6J mice were randomly divided into control group, model group and Chinese medicine group, with 10 mice in each group. Myopia model of the right eye of mice was established by means of form deprivation in model and Chinese medicine groups. After 4wk, the Chinese medicine group were given intragastric administration of modified Zhujing Pill suspension 0.546g/(kg·d)(0.2mL/d)for 4wk, and same amount of saline was given to mice in other groups at the same time of modeling. The axial length and diopter of the right eye of the mouse were measured before and after the experiment by A-ultrasound and a strip retinoscope respectively. At the end of the experiment, the mitochondrial ultrastructure of RPEs was observed by transmission electron microscope. Western blot, and real-time fluorescent quantitative PCR(q-PCR)were used to detect quantitative and gene expression of mitofusin 1(MFN1), optic atrophy 1(OPA1), and dynamin-related protein 1(DRP1)in retinal tissues respectively.RESULTS: At the beginning of the experiment, there was no statistically significant difference in axial length and diopter of the right eye of the mouse in control, model and Chinese medicine groups(P&#x003E;0.05). At the end of the experiment, compared with the control group, the mice in the model group and the Chinese medicine group had lower diopter and continuously prolonged axial length(all P&#x003C;0.05), while the mice in the Chinese medicine group had significantly shorter axial length and higher diopter than the model group(all P&#x003C;0.05). Western blot and q-PCR results showed that the relative expression of MFN1 and OPA1 decreased and DRP1 increased in both the model group and the Chinese medicine group compared with the control group(all P&#x003C;0.05), and the relative expression of MFN1 and OPA1 increased in the Chinese medicine group compared with the model group(all P&#x003C;0.05). The electron microscopic results showed that the mitochondria in the right retina of the mice were only mildly swollen in the Chinese medicine group, while the mitochondria in the model group were obviously swollen and disordered and empty.CONCLUSION: Modified Zhujing Pill could protect the retinal mitochondria by regulating the key proteins of mitochondrial dynamics(MFN1, OPA1, and DRP1), and it has a protective effect on the retina of axial myopic mice.

Establishment of a RILD model in C57BL/6J mice treated with precise radiotherapy / 中国辐射卫生

Objective To provide a reliable and stable animal model for investigating the molecular pathogenesis of radiation-induced liver disease (RILD). Methods Ninety C57BL/6J mice were divided into control, 20 Gy, 25 Gy, 30 Gy and 35 Gy radiation groups. The mice were executed at 4 weeks after radiation and the levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in the liver serum were measured. HE staining was performed on the pathological liver tissues. Masson staining was performed at 36 weeks after radiation. Results Compared with the control group, the fatality rate was higher in the 30 and 35 Gy radiation groups, and the body weight significantly decreased in the 20 and 25 Gy radiation groups. Compared with the control group, alanine aminotransferase significantly increased in mice exposed to 20 Gy, while aspartate aminotransferase and alanine aminotransferase increased in mice exposed to 25 Gy. No significant changes were observed in the livers of the mice in the 20 and 25 Gy radiation groups, but pathological examination showed liver damage induced by both 20 and 25 Gy radiation. Conclusion A stable and reliable mouse model of RILD was constructed for treatment with linear accelerator. The mouse model of RILD constructed for stereotactic body radiation therapy using linear accelerator has significant research implications for the exploration of RILD.

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.

Eficacia de la artemisina en modelos murinos de leishmaniasis cutánea experimental causada por Leishmania amazonensis / Artemisine efficacy in murine models of experimental cutaneous leishmaniasis caused by Leishmania amazonensis

Los limitados tratamientos disponibles para enfrentar la leishmaniasis requieren el desarrollo de investigaciones para buscar nuevos agentes terapéuticos. Una estrategia recomendada es el reposicionamiento farmacológico, en el que la artemisina figura como un posible candidato. El objetivo de este estudio es evaluar las potencialidades de la artemisina en dos modelos murinos de leishmaniasis cutánea experimental. Para ello, se emplearon ratones BALB/c (susceptibles) y C57BL/6 (resistentes) infectados con Leishmania amazonensis. El tratamiento se realizó por vía oral o intralesional con cinco dosis de artemisina a 30 mg/kg cada 4 días. Se determinó el comportamiento del peso, la evolución del tamaño de la lesión y la carga parasitaria. En ambos modelos animales se observó que el tratamiento con artemisina (oral e intralesional) disminuyó el tamaño de la lesión y la carga parasitaria con respecto a los grupos infectados sin tratamiento (p 0,05). Los ratones C57BL/6 tratados por vía oral fueron los únicos capaces de controlar las lesiones hasta el final del experimento. Se demuestra la eficacia in vivo de la artemisina en dos modelos de leishmaniasis cutánea inducida por L. amazonensis y se destaca la administración por vía oral en el control de la enfermedad. Se sugiere el futuro desarrollo de este fármaco para el tratamiento de la leishmaniasis cutánea.

The limited treatments available for leishmaniasis require the development of research for new therapeutic agents. One recommended strategy is the pharmacological repositioning, where artemisinin stands out as a possible candidate. The aim of this study is to evaluate the potential of artemisinin in two murine models of experimental cutaneous leishmaniasis. For this purpose, BALB/c (susceptible) and C57BL/6 (resistant) mice infected with Leishmania amazonensis were used. Oral or intralesional treatment was performed with five doses of artemisinin at 30 mg/kg every four days. Weight behavior, evolution of lesion size, and parasitic load were determined. In both animal models it was observed that treatment with artemisinin (oral and intralesional) decreased lesion size and parasitic load with respect to the untreated infected groups (p 0.05). Orally treated C57BL/6 mice were the only ones able to control lesions until the end of the experiment. The in vivo efficacy of artemisinin in two models of cutaneous leishmaniasis induced by L. amazonensis is demonstrated and oral administration is highlighted in the control of the disease. Further development of this drug for the treatment of cutaneous leishmaniasis is suggested.

Resistance of C57BL/6 Mice to Different Artemisinin-sensitive Strains of PbK173 / 中国实验方剂学杂志

ObjectiveThe tolerance of C57BL/6 mice to artemisinin-sensitive and -resistant strains of Plasmodium berghei （Pb） K173 and the differences in blood parameters， spleen coefficient and spleen structure during infection were compared to explore whether the artemisinin resistance of Pb would aggravate malaria infection. MethodPbK173 artemisinin-sensitive and -resistant strains were tested in parallel. C57BL/6 mice were randomly divided into 1 control group， 4 artemisinin-sensitive strain groups and 4 artemisinin-resistant strain groups by body weight. Each infection group was simultaneously inoculated （ip） with 1×107 infected red blood cells （iRBCs） of sensitive/resistant strain. For the mice in the survival test group， the body weight was recorded every day post infection， and the tail vein blood smear was collected to calculate the Pb infection rate. In the other infection groups， peripheral blood and spleen were collected on 2， 5 and 9 d after infection. Peripheral blood parameters， spleen coefficient， pathological section of spleen and spleen cells were detected in each group. ResultOn 1-3 d after infection， the infection rate of the resistant strain （0.4±0.0， 0.8±0.1， 1.9±0.4）% was always higher than that of the sensitive strain （0.2±0.1， 0.4±0.1， 1.1±0.3）% （P<0.01）. From the 4th d of infection， the infection rate of the two groups gradually approached. The survival period of the sensitive strain group （20.5±1.2） d was shorter than that of the resistant strain group （23.3±1.4） d （P<0.01）. On the 9th d， the white blood cell count of the sensitive strain group （16.2±1.1）×109 cells/L was higher than that of the resistant strain group （10.6±1.8）×109 cells/L （P<0.01）. Flow cytometry analysis of spleen cells showed that the sensitive strain group （3.6±0.4） demonstrated a higher CD4+/CD8+ value than the resistant strain group （2.3±0.2） on the 9th d （P<0.01）. The spleen of C57BL/6 infected mice was gradually enlarged during infection， and on the 9th d， the resistant strain group （3.1±0.1）% showed a higher spleen coefficient than the sensitive strain group （2.7±0.2）% （P<0.01）. In the early stage of C57BL/6 infected mice， the red pulp of spleen was hyperemic and swollen. On the 9th d， the marginal area of the spleen disappeared and the structure of the red and white pulp was destroyed. ConclusionWithout drug treatment， the protective immune responses of peripheral blood and spleen of C57BL/6 mice were more sensitive to PbK173 artemisinin-sensitive strain. The artemisinin-resistant strain of PbK173 bred with mouse-to-mouse blood transmission and increased artemisinin dose exhibited shortened growth period and reduced toxicity.

Effect of gender difference on antigen induced Sjogren's syndrome model in mice / 中国药理学通报

Aim To study the effect of gender differences in C57BL / 6J mice on antigen induced Sjogren's syndrome(SS)model. Methods The submandibular gland protein of C57BL/6J female and male mice was extracted and mixed with the same amount of Freund's complete adjuvant(FCA)for the first three times, the antigen concentration was adjusted to 2.5 g·L-1, mixed with Freund's incomplete adjuvant(FIA)for the fourth time, and the same-sex mouse antigen was injected into the back of mice for a total of four times to induce the mouse SS model. The mouse SS model was induced by multi-point intradermal injection of antigen on the back of mice for four times,the body weight of female and male mice was measured every week, the general condition was observed, the saliva volume of mice was measured at the sixth week of modeling. After the mice were sacrificed, the pathological changes of submandibular gland and the changes of T and B lymphocyte subsets in spleen were detected, and the differences in SS model preparation between female and male mice were compared. Results The SS model of male and female mice was successfully established, and there was no significant difference in general condition, saliva volume, submandibular gland pathology, plasma cells and memory B cells between male and female SS mice. The success rate of SS model was 75% in female mice and 60% in male mice. Compared with normal mice of the same sex, the weight loss of female SS mice was earlier and more obvious than that of male SS mice; the submandibular gland index of male mice was significantly higher than that of female mice. Compared with normal mice of the same sex, the proportion of Th17 and Treg cells in spleen of female SS mice was more statistically significant than that of male SS mice. Conclusions The success rate of SS modeling in female mice is higher than that in male mice. Compared with male SS mice, female SS mice have more significant SS like manifestations and pathological manifestations, which can provide a reference basis for the selection of gender when establishing SS model.

Effects of Total Flavonoids from Chamomile on Lipid Metabolism of Hyperlipidemia Model Mice and Its Mechanism / 中国药房

OBJECTIVE：To st udy t he effects of total flavonoids from chamomile on lipid metabolism of hyperlipidemia model mice and its potential mechanism. METHODS ：Thirty male C 57BL/6J-ApoE－/－ mice were randomly divided into model group ， positive control group（fenofibrate 30 mg/kg）and chamomile total flavonoids low-dose ，medium-dose and high-dose groups （88， 176，352 mg/kg），with 6 mice in each group. In addition ，6 male C 57BL/6J mice were used as normal control group. Mice in normal control group were fed with ordinary diet ，and mice in other groups were fed with high-fat diet for 8 weeks to replicate hyperlipidemia model. At the time of making model ，administration groups were given relevant liquid （using 1％ sodium carboxymethyl cellulose as solvent ）；normal control group and model group were given 1％ sodium carboxymethyl cellulose intragastrically，200 mL per gavage ，once a day ，for consecutive 8 weeks. The body weight of mice in each group was weighed before medication and 8 weeks after medication. The serum contents of total cholesterol （TC），triacylglycerol（TG），low-density lipoprotein cholesterol （LDL-C），high-density lipoprotein cholesterol （HDL-C），aspartate aminotransferase （AST）and alanine aminotransferase （ALT） in mice were detected after last administration ；the contents of superoxide dismutase （SOD） and malondialdehyde（MDA）as well as the protein expressions of peroxisome proliferator-activated receptor α（PPARα），carnitine palmityl transferase 1A（CPT1A）and peroxase acyl-CoA oxidase 1（ACOX1）in liver tissue were determined. The pathological changes i n liver tissue were observed. RESULTS：Compared w ith before medication ，the body weight of each group showed an increasing trend after 8 weeks of medication. Compared with normal control group ，body weight ，the contents of TC ，TG， LDL-C，AST and ALT in serum and MDA content in live r lan- tissue of mice in model group were significantly increased wei516@sina.com after 8 weeks of medication （P＜0.05 or P＜0.01）. The ·2706· China Pharmacy content of HDL-C in serum and the cont ent of SOD in liver tissue ，as well as the protein expressions of PPARα，CPT1A and ACOX1 were significantly decreased （P＜0.05 or P＜0.01），and the structure of liver tissue was disorganized ，with circular fat vacuoles of different sizes and lipid droplets of different sizes in the cytoplasm. Compared with model group ，body weight （except for chamomile total flavonoids low-dose group ）of mice ，serum contents of TC ，TG，LDL-C，AST and ALT ，content of MDA in liver tissue （except for chamomile total flavonoids low-dose and medium-dose groups ）were significantly decreased （P＜0.05 or P＜ 0.01）. Serum content of HDL-C ，content of SOD in liver tissue ，protein expressions of PPARα，CPT1A（except for chamomile total flavonoids low-dose and medium-dose groups ） and ACOX 1 were significantly increased （P＜0.05 or P＜0.01）；liver tissue structure was clear ，and liver fat vacuoles were improved to varying degrees ，and less lipid droplets. The improvement effect of the above indexes was the best in the chamomile total flavonoids high-dose group. CONCLUSIONS ：Chamomile total flavonoids can prevent the occurrence of hyperlipidemia in C57BL/6J-ApoE －/－ mice，the mechanism of which may be associated with up-regulation of PPARα expression，the improvement of liver injury and oxidant stress injury.

Effect of modified Zhujing pill on retinal thickness and apoptosis in form deprivation myopia mice / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effect of modified Zhujing pill on retinal thickness and apoptosis in form deprivation myopia(FDM)mice.<p>METHODS: Totally 72 C57BL/6J mice aged 3-week-old were randomly divided into control group 1, model group 1, intervention group 1, control group 2, model group 2 and intervention group 2, with 12 mice in each group. The first three groups were tested for 3wk and the last three groups were tested for 6wk, except for the groups of control 1 and control 2, all the mice used translucent goggles to cover their right eyes for form deprivation. The mice of intervention 1 and intervention 2 were respectively given intragastric administration modified Zhujing pill suspension 0.546g/(kg·d)(0.1mL/d)for 3wk at the beginning and after 3wk of the experiment. Same amount of saline was given to mice in other groups at the same time of modeling. The eye axis was measured before and after the experiment. At the end of the experiment, the eye of mice was taken for HE staining to observe the thickness changes of each layer of retina. Immunohistochemistry(IHC)and western blotting(WB)were used to detect Bcl-2 and Caspase3 expression of protein.<p>RESULTS: At the end of the experiment, the axis of model group 1 was significantly higher than that of control group 1(<i>P</i><0.01), the axis of intervention group 1 was significantly lower than that of model group 1(<i>P</i><0.01), and the axis of model group 2 was significantly higher than that of control group 2(<i>P</i><0.01), the eye axis of intervention group 2 was significantly lower than that of model group 2(<i>P</i><0.01); HE staining showed that the thickness of NFL and ONL of model group 1 was significantly thinner than that of control group 1(<i>P</i><0.01). The thickness of INL of model group 1 was significantly thinner than that of control group 1(<i>P</i><0.05), and the thickness of NFL, INL and ONL of intervention group 1 was significantly higher than that of model group 1(<i>P</i><0.05); The thickness of NFL, INL and ONL model group 2 was significantly thinner than that of control group 1(<i>P</i><0.01); IHC testing showed that the average optical density of Bcl-2 protein in model group 1 was significantly lower than that of control group 1(<i>P</i><0.05), which in intervention group 1 was significantly higher than that of model group 1(<i>P</i><0.01), and which in the average optical density of Bcl-2 protein of model group 2 was significantly lower than that of control group 2(<i>P</i><0.01), which in intervention group 2 was significantly higher than that of model group 2(<i>P</i><0.01); Caspase 3 protein average optical density of model group 1 was significantly higher than that of control group 1(<i>P</i><0.01), which in intervention group 1 was significantly lower than that of model group 1(<i>P</i><0.01), which in model group 2 was significantly higher than that of control group 2(<i>P</i><0.05), which in intervention group 2 was significantly lower than that of model group 2(<i>P</i><0.01); WB test proved that the relative expression level of Bcl-2 protein in model group 1 was significantly lower than that of control group 1(<i>P</i><0.01), which in intervention group 1 was significantly higher than that of model group 1(<i>P</i><0.01), and which in model group 2 was significantly lower than that of control group 2(<i>P</i><0.01), which in intervention group 2 was significantly higher than that of model group 2(<i>P</i><0.01); The relative expression level of Caspase3 protein in model group 1 was significantly higher than that of control group 1(<i>P</i><0.01), which in intervention group 1 was significantly lower than that of model group 1(<i>P</i><0.01), the intervention group 2 was significantly lower than that of model group 2. <p>CONCLUSION: The results show that the modified Zhujing pill can interfere with the pathological changes of retinal thickness thinning in the process of myopia and formed myopia mice by regulating the expression of apoptosis-related proteins Bcl-2 and Caspase3, and alleviating the apoptosis of retinal cells in myopia formation and myopia mice.

One extraction tool for in vitro-in vivo extrapolation?SPME-based metabolomics of in vitro 2D,3D,and in vivo mouse melanoma models / 药物分析学报

Solid phase microextraction(SPME)in combination with high-resolution mass spectrometry was employed for the determination of metabolomic profile of mouse melanoma growth within in vitro 2D,in vitro 3D,and in vivo models.Such multi-model approach had never been investigated before.Due to the low-invasiveness of SPME,it was possible to perform time-course analysis,which allowed building time profile of biochemical reactions in the studied material.Such approach does not require the multiplication of samples as subsequent analyses are performed from the very same cell culture or from the same individual.SPME already reduces the number of animals required for experiment;therefore,it is with good concordance with the 3Rs rule(replacement,reduction,and refinement).Among tested models,the largest number of compounds was found within the in vitro 2D cell culture model,while in vivo and in vitro 3D models had the lowest number of detected compounds.These results may be connected with a higher metabolic rate,as well as lower integrity of the in vitro 2D model compared to the in vitro 3D model resulting in a lower number of compounds released into medium in the latter model.In terms of in vitro-in vivo extrapolation,the in vitro 2D model performed more similar to in vivo model compared to in vitro 3D model;however,it might have been due to the fact that only compounds secreted to medium were investigated.Thus,in further experiments to obtain full metabolome infor-mation,the intraspheroidal assessment or spheroid dissociation would be necessary.

Immunomodulatory effect of human adipose derived mesenchymal stem cells on skin transplantation between different mouse strains / 中国组织工程研究

BACKGROUND: Skin transplantation is one of the most effective methods for treating large-area burns. How to effectively suppress the immune rejection after allogeneic skin transplantation is a problem that needs to be solved urgently. OBJECTIVE: To investigate the effect of human adipose derived mesenchymal stem cells (hADSCs) on the immunoregulation of skin grafts in different strains of mice. METHODS: Isolated hADSCs were cultured to the 3rd generation. Sixty ICR neonatal mice, 2-4 days of age, were randomly divided into four groups (n=15). The skin tissues of ICR neonatal mice were transplanted into adult C57BL/6 mice to establish a different strain of mouse skin graft immune rejection model. PBS and low dose (5×104), medium dose (10×104), high dose (20×104) hADSCs were injected into the model mice through tail vein, and the survival time of transplanted skin in each group was recorded. On the 7th day after operation, five mice from each group were randomly selected to remove their spleen and serum, and the expression of immune factors interleukin-10, tumor necrosis factor-α and interferon-γ were detected by RT-PCR and ELISA respectively. The transplanted part of the skin was taken to make pathological sections for observing the infiltration of lymphocytes. RESULTS AND CONCLUSION: Compared with the PBS group, the survival time of the skin was prolonged in the low dose hADSCs group; however, there was no significant difference between the two groups (P > 0.05). Compared with the PBS and low dose hADSCs groups, the survival time of the skin was significantly increased in the medium and high dose groups (P 0.05). Compared with the PBS group, the relative expression of tumor necrosis factor-α and interferon-γ in the spleen and serum was significantly decreased in the low, medium and high dose hADSCs groups (P < 0.05), whereas the level of interleukin-10 was significantly elevated in the medium and high dose hADSCs groups (P < 0.05). To conclude, the appropriate dose of hADSCs can significantly prolong the survival time of transplanted skin between different strains of mice, by regulating the expression of related immune factors in the recipient mice.

Lotus leaf flavonoids induce apoptosis of human lung cancer A549 cells through the ROS/p38 MAPK pathway

BACKGROUND: Leaves of the natural plant lotus are used in traditional Chinese medicine and tea production. They are rich in flavonoids. METHODS: In this study, lotus leaf flavonoids (LLF) were applied to human lung cancer A549 cells and human small cell lung cancer cells H446 in vitro to verify the effect of LLF on apoptosis in these cells through the ROS/p38 MAPK pathway. RESULTS: LLF had no toxic effect on normal cells at concentrations up to 500 µg/mL, but could significantly inhibit the proliferation of A549 cells and H446 cells. Flow cytometry showed that LLF could induce growth in A549 cells. We also found that LLF could increase ROS and MDA levels, and decrease SOD activity in A549 cells. Furthermore, qRT-PCR and western blot analyses showed that LLF could upregulate the expression of p38 MAPK (p-p38 MAPK), caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and Bax and downregulate the expression of Cu/Zn SOD, CAT, Nrf2, NQO1, HO-1, and Bcl-2 in A549 cells. Results of HPLC showed that LLF mainly contain five active substances: kaemp-feritrin, hyperoside, astragalin, phloridzin, and quercetin. The apoptosis-inducing effect of LLF on A549 cells came from these naturally active compounds. CONCLUSIONS: We have shown in this study that LLF is a bioactive substance that can induce apoptosis in A549 cells in vitro, and merits further research and development.

Enhancement of cellular activity in hyperglycemic mice dermal wounds dressed with chitosan-alginate membranes

The use of specially designed wound dressings could be an important alternative to facilitate the healing process of wounds in the hyperglycemic state. Biocompatible dressings combining chitosan and alginate can speed up wound healing by modulating the inflammatory phase, stimulating fibroblast proliferation, and aiding in remodeling phases. However, this biomaterial has not yet been explored in chronic and acute lesions of diabetic patients. The aim of this study was to evaluate the effect of topical treatment with a chitosan-alginate membrane on acute skin wounds of hyperglycemic mice. Diabetes mellitus was induced by streptozotocin (60 mg · kg-1 · day-1 for 5 days, intraperitoneally) and the cutaneous wound was performed by removing the epidermis using a surgical punch. The results showed that after 10 days of treatment the chitosan and alginate membrane (CAM) group exhibited better organization of collagen fibers. High concentrations of interleukin (IL)-1α, IL-1β, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor-alpha (TNF-α) were detected in the first and second days of treatment. G-CSF and TNF-α level decreased after 5 days, as well as the concentrations of TNF-α and IL-10 compared with the control group (CG). In this study, the inflammatory phase of cutaneous lesions of hyperglycemic mice was modulated by the use of CAM, mostly regarding the cytokines IL-1α, IL-1β, TNF-α, G-CSF, and IL-10, resulting in better collagen III deposition. However, further studies are needed to better understand the healing stages associated with CAM use.

Co-transplantation of adipose mesenchymal stem cells and endothelial progenitor cells in ulcerative colitis mice / 中国组织工程研究

BACKGROUND: Mesenchymal stem cell transplantation has been proved to be effective for ulcerative colitis, while the effect of endothelial progenitor cells in ulcerative colitis treatment is still unknown. They are both promising cells for tissue engineering, which can be used for cell transplantation. OBJECTIVE: To explore whether co-transplantation of endothelial progenitor cells and adipose mesenchymal stem cells can relieve ulcerative colitis in a mouse model. METHODS: C57BL/6J mice were randomly divided into six groups (n=24 per group): co-transplantation group, mesenchymal stem cell transplantation group, endothelial progenitor cell transplantation group, glucocorticoid treatment group, transplantation control group and normal control group. Murine ulcerative colitis model was established in all groups except for the normal control group. At 7 and 10 days after modeling, transplantation groups were respectively injected via tail vein with adipose mesenchymal stem cells and/or endothelial progenitor cells, glucocorticoid or PBS. Mice were sacrificed at 12 days after modeling. Colon length, disease activity index, histological score and the serum level of tumor necrosis factor-α were compared between groups. RESULTS AND CONCLUSION: Treatment with glucocorticoid was significantly effective for ulcerative colitis relative to the transplantation control group (P 0.05). Co-transplantation of adipose mesenchymal stem cells and endothelial progenitor cells was better than the other treatments, which significantly improved the shortening of the colon, disease activity index, histological score, and serum level of tumor necrosis factor-α.

Efectos de la corriente eléctrica de baja intensidad en ratones sanos / Effects of low-intensity electric current on healthy mice

Introducción: El conocimiento de los efectos de la terapia electroquímica, en los organismos puede ser importante para el tratamiento de diferentes enfermedades. Objetiv:o Evaluar los efectos de la corriente eléctrica directa de baja intensidad en ratones sanos. Métodos: 80 ratones C57BL/6/Cenp sanos (40 machos y 40 hembras) fueron distribuidos aleatoriamente en ocho grupos experimentales (cuatro grupos controles y cuatro grupos tratados). La corriente eléctrica directa (10 mA durante 5 min) o tensión constante (10 V durante 5 min) fue aplicada a los ratones. Fueron realizadas observaciones clínicas, macroscópicas, microscópicas y mediciones de los parámetros hematológicos y bioquímicos de la sangre. Resultados: Se indujeron cambios reversibles en los parámetros hematológicos, bioquímicos de la sangre e histológicos estudiados en ratones C57BL/6/Cenp machos y hembras cuando se usó 10 mA o 10 V y diferentes arreglos de electrodos, siendo menos notable para el modo de tensión constante. Conclusiones: Las alteraciones inducidas en los ratones C57BL/6/Cenp sanos por la corriente eléctrica directa de baja intensidad son reversibles y el proceso inflamatorio sistémico es dominado por los linfocitos(AU)

Introduction: Knowledge about the effects of electrochemotherapy on organisms may be important for the treatment of various diseases. Objective: Evaluate the effects of low-intensity direct electric current on healthy mice. Methods: Eighty healthy C57BL/6/Cenp mice (40 male and 40 female) were randomly distributed in eight experimental groups (four control and four treated). Direct electric current (10 mA during 5 min) or constant voltage (10 V during 5 min) was applied to the mice. Clinical, macroscopic and microscopic observation was performed, and measurements were taken of hematological and biochemical parameters of the blood. Results: Reversible changes were induced in hematological, biochemical and histological parameters of the blood of male and female C57BL/6/Cenp mice when 10 mA or 10 V and various electrode arrays were used. These changes were less noticeable in the constant voltage mode. Conclusions: The alterations induced by low-intensity direct electric current in healthy C57BL/6/Cenp mice are reversible. The systemic inflammatory process is dominated by the lymphocytes(AU)

RT-PCR detection of cytokine expression in gastric mucosae of mice with Helicobacter heilmannii infection / 中华微生物学和免疫学杂志

Objective To compare the changes in IFN-γ, IL-4, IL-10 and IL-1β expression at mRNA level in gastric mucosae of BALB/c, C57BL/6 and nude mice at different stages of Helicobacter heilmannii ( H. heilmannii) infection, and to investigate the types of induced immune responses. Methods Each kind of mice was randomly divided into two groups: infection ( n=30 ) and control ( n=6 ) groups. Those in the infection groups were intragastrically inoculated with H. heilmannii strains to establish long-term stable mouse infection models. Gastric mucosa tissues were collected at weeks 4, 8, 12, 24 and 36 and ana-lyzed by semi-quantitative RT-PCT to detect the expression of IFN-γ, IL-4, IL-10 and IL-1βat mRNA lev-el. Results In the early stage of infection (weeks 4-12), INF-γ, IL-4, IL-10, and IL-1β expression at mRNA level in the gastric mucosae of BALB/c and C57BL/6 mice were significantly increased compared with those of the control groups (P<0. 05). IL-4, IL-10 and IFN-γexpression peaked at weeks 8-12, while IL-1βexpression reached the peak at week 4. After 12 weeks, IFN-γexpression at mRNA level in BALB/c mice was significantly decreased, but showed no significant change in C57BL/6 mice. IL-4 expression at mRNA level in C57BL/6 mice at the late stage of infection (week 36) was lower than that in the correspond-ing control group (P<0. 05). Expression of IFN-γ, IL-4, IL-10 and IL-1β at mRNA level in nude mice were all higher than those in the control group, and there were significant differences in IL-1βand IL-4 ex-pression between groups (P<0. 05). Conclusions Expression of cytokines in H. heilmannii-infected mice increased over time. IFN-γ-mediated Th1 immune responses were the predominant immunity induced by H. heilmannii infection. Immune responses to H. heilmannii infection varied with the kinds of mice. C57BL/6 mice showed mainly Th1 cell immune responses, while Th1/Th2 mixed immune responses were induced in BALB/c mice.

Comparison of responsiveness to cancer development and anti-cancer drug in three different C57BL/6N stocks / 한국실험동물학회지

In our efforts to understand the systemic features of tumors, the importance of animal models is increasing due to the recent growth in the development of immunotherapy and targeted therapies. This has resulted in increased attention towards tumor animal models using C57BL/6N, which are mainly used in immunological studies. In this study, the C57BL/6NKorl stock and two other commercial stocks (C57BL/6NA and C57BL/N6B) are evaluated by comparing the occurrence of tumors using the syngeneic model; furthermore, we compare the response to anti-cancer drugs in the syngeneic model by evaluating survival, growth of tumors, proliferation and molecular biology analysis. In the syngeneic model using LLC (Lewis lung carcinoma) cells, the survival of mice and growth of the tumor showed a better response in the C57BL/6NKorl stock, and was dependent on the cell concentration of the dosing tumor, as compared to the other C57BL/6N stocks. However, the Ki-67 staining showed only little difference in cell proliferation within the tumor tissue each mouse stocks. Comparing the sensitivity to anti-cancer drug by examining changes in growth, volume and weight revealed that cisplatin treatment for tumor-bearing C57BL/6NKorl was more dependent on concentration. The Ki-67 staining, however, showed no difference among the C57BL/6N stocks after cisplatin treatment. The expressions of p27 and p53 tumor suppressor proteins, caspase-3 and Bax showed dose-dependent increase after exposure to cisplatin, whereas the expression of Bcl-2 was reduced in a dose-dependent manner. Furthermore, the expressions of MMP-2 and VEGF involved in metastasis, as well as inflammatory genes IL-1β, IL-6 and IL-10, showed dose-dependent decrease in tumor tissue after cisplatin exposure. Differences observed among the C57BL/6N stocks were not significant. Taken together, our studies reveal that C57BL/6NKorl has the potential of being a useful biological resource established in Korea, as it does not differ from the two commercially available C57BL/6N stocks when considering response to tumor generation and sensitivity to anti-cancer drugs using the syngeneic tumor model.

Effect of qingfei-yangyin-huoxue recipe on radiation-induced pulmonary injury via lncrna nanci-nkx2.1 pathway / 中国药学杂志

OBJECTIVE: To discuss the effect of Qingfei-yangyin-huoxue recipe on radiation-induced pulmonary injury by regulating the lncRNA NANCI-NKX2.1 pathway. METHODS: The C57BL/6 mice were randomly divided into normal control group (A group, n=16), drug only group (B group, n=16), model group (C group, n=16) and drug model group (D group, n=16). After exposure to Qingfei-yangyin-huoxue recipe for 10 w, the pathological change of lung tissue was examined by H&E and Masson staining. The expressions of lncRNA NANCI and NKX2.1 mRNA in lung tissues were detected by qRT-PCR. And the NKX2.1 protein expressions were detected by Western blot. RESULTS: The mean animal weight in D groups was less than A and B group, but more than C group after treatment of 7 d(P<0.05). There were marked interstitial edema and inflammatory cells, fibrocytes accumulation in C group but not in A and B groups by H&E and Masson stain. The alveolitis and fibrosis changes in D group were better than C group. And the mean radiation-induced pulmonary injury score in D group was (3.875±1.746), which was less than C group, but more than A and B groups (P<0.05). The expression of lncRNA NANCI and NKX2.1in D group was higher than C group, but lower than A and B groups (P<0.05). Besides, the radiation-induced pulmonary injury score was negative related with lncRNA NANCI and NKX2.1 (r=-0.510, -0.786, P<0.05). CONCLUSION: There are significant evidences that Qingfei-yangyin-huoxue recipe could protect radiation-induced pulmonary injury by up-regulation lncRNA NANCI-NKX2.1 pathway.

Effect of cold exposure on browning of white adipose tissues in mice fed with high fat diet / 中国药理学通报

Aim: To observe the effect of cold exposure on browning of white adipose tissues in mice fed with high fat diet. Methods: Male 8-week-old C57BL/6J mice were randomly divided into HFD + 5 °C group and HFD + RT group, after 8 weeks of high-fat diet. Male 8-week-old C57BL/6J mice were randomly divided into normal + 5 °C group and normal + RT group, after 8 weeks of normal diet. Each group of mice were intervened for 2 h at different temperatures in the same time period for 8 weeks. Parameters, including body weight, food intake, inguinal white adipose tissue (iWAT) mass, brown adipose tissue (BAT) mass, blood glucose, blood lipids, leptin, adiponectin, adipose tissue pathology and in situ expression of uncoupling protein 1(UCP1) and prohibitin protein (PHB) in iWAT and BAT. Results: Compared with HFD + RT group, cold exposure not only significantly reduced body weight, blood glucose, iWAT weight/body weight ratio, TC, TG, LDL-C and leptin, but also increased food intake and BAT weight in high-fat diet mice. HE staining showed that iWAT and BAT cells became smaller and had multiple compartments. The iWAT had a browning trend. Immunohistochemistry showed that UCP1 and PHB protein in iWAT and BAT significantly increased. Conclusions: Cold exposure can counteract the weight gain caused by a high-fat diet, which may be related to the activation of brown adipose tissue and the induction of browning of white adipose tissues, increasing heat production and reducing white fat accumulation.