ABSTRACT
ObjectiveTo study the mechanism of astragaloside Ⅳ （AS Ⅳ） on db/db mice with type 2 diabetes mellitus （T2DM） and non-alcoholic fatty liver disease （NAFLD） based on network pharmacology and experimental validation. MethodA total of 24 db/db mice were randomly divided into four groups： model group， metformin group， and low-dose and high-dose AS Ⅳ groups. Six C57 mice were used as the blank group. The low-dose and high-dose AS Ⅳ groups were given AS Ⅳ of 0.015 and 0.030 g·kg-1 by gavage， and the metformin group was given 0.067 g·kg-1 by gavage. The blank and model groups were given equal volumes of distilled water by gavage. After intragastric administration， fasting blood glucose （FBG） was detected， and an oral glucose tolerance test was performed. Serum lipid level and liver histopathology were detected. The target and enrichment pathway of AS Ⅳ for treating T2DM and NAFLD were predicted by network pharmacology， and the main enrichment pathway was verified by molecular biology techniques. The protein expressions of AMPK， p-AMPK， sterol regulatory element-binding protein-1 （SREBP-1）， and fatty acid synthetase （FAS） in liver tissue were detected by Western blot. ResultCompared with the blank group， the levels of body mass， liver weight coefficient， fasting blood glucose， serum total cholesterol， triglyceride， and low-density lipoprotein cholesterol in mice treated with AS Ⅳ were decreased （P<0.05， P<0.01）. The pathology of liver tissue showed significant improvement in lipid accumulation， and imaging results showed that the degree of fatty liver was reduced after AS Ⅳ therapy. Network pharmacological prediction results showed that vascular endothelial growth factor α （VEGFA）， galactoagglutinin 3 （LGALS3）， serine/threonine kinase B2 （Akt2）， RHO-associated coiled-coil protein kinase 1 （ROCK1）， serine/threonine kinase B1 （Akt1）， signaling and transcriptional activator protein （STAT3）， and messtimal epidermal transformation factor （MET） were key targets in "drug-disease" network. The results from the Kyoto encyclopedia of genes and genomes （KEGG） enrichment showed that the AMP-dependent protein kinase （AMPK） signaling pathway was strongly associated with T2DM and NAFLD. Western blot results showed that compared with the blank group， the expression levels of p-AMPK/AMPK in the model group were significantly down-regulated， while those of SREBP-1 and FAS proteins were significantly up-regulated （P<0.01）. Compared with the model group， the expression levels of p-AMPK/AMPK in the metformin group and high-dose AS Ⅳ group were significantly up-regulated， while those of SREBP-1 and FAS proteins were significantly down-regulated （P<0.05， P<0.01）. ConclusionAS Ⅳ regulates the expression of lipid proteins by activating the AMPK signaling pathway， thereby improving lipid metabolism.
ABSTRACT
BACKGROUND Leishmaniases encompass a spectrum of neglected diseases caused by parasites of the genus Leishmania, grouped in two forms: tegumentary and visceral leishmaniasis. OBJECTIVES In this study, we propose Friend Virus B NIH Jackson (FVB/NJ) mouse strain as a new experimental model of infection with Leishmania (Leishmania) amazonensis, the second most prevalent agent of tegumentary leishmaniasis in Brazil. METHODS AND FINDINGS We performed in vitro infections of FVB/NJ macrophages and compared them with BALB/c macrophages, showing that BALB/c cells have higher infection percentages and a higher number of amastigotes/cell. Phagocytosis assays indicated that BALB/c and FVB/NJ macrophages have similar capacity to uptake parasites after 5 min incubations. We also investigated promastigotes' resistance to sera from FVB/NJ and BALB/c and observed no difference between the two sera, even though FVB/NJ has a deficiency in complement components. Finally, we subcutaneously infected FVB/NJ and BALB/c mice with 2 × 106 parasites expressing luciferase. Analysis of lesion development for 12 weeks showed that FVB/NJ and BALB/c mice have similar lesion profiles and parasite burdens. MAIN CONCLUSIONS This work characterises for the first time the FVB/NJ mouse as a new model for tegumentary leishmaniasis caused by Leishmania (L.) amazonensis.
ABSTRACT
Introducción: Tropaeolum tuberosum, conocido comomashua, es un tubérculo andino que tiene un valor tanto económico como nutritivo para las poblaciones de pocos recursos. Se cree que afecta la fertilidad masculina, porque los hombres andinos lo relacionan con impotencia y disminución de la capacidad fecundante. Estudios hechos en ratas que se alimentaron con mashua demostraron que hubo un 45% de decrecimiento de la tasa testosterona/dihidrotestosterona. El efecto de esta planta en la reproducción está relacionada a su contenido de isotiocianatos, compuestos que se unen covalentemente a las proteínas, las cuales pueden estar directa o indirectamente involucradas en el proceso espermatogénico. El propósito de esta investigación fue evaluar el efecto del extracto acuoso de la mashua sobre la espermatogénesis y la fisiología reproductiva de ratones. Métodos: Se evaluaron los parámetros morfofuncionales in vivo de espermios de ratones (espermatograma) y se cuantificó la expresión de: Cytochrome P450 17α-hydroxylase/17,20-lyase, proteína reguladora de esteroidogénesis aguda, ciclina y protamina, relacionados a la espermatogénesis. Resultados: A los 7, 14 y 21 días de dosificación, se vio afectado el conteo de espermatozoides, así como su motilidad progresiva (MP); por otra parte, se observó un retardo en la maduración de los mismos. En cuanto a la expresión génica, no se encontró diferencias significativas entre la expresión de los dos genes estudiados (cytochrome P450 17α-hydroxylase/17,20-lyase, ciclina). Conclusión: El efecto de la mashua no se da a nivel de la expresión de los genes involucrados en la espermatogénesis, sino a nivel de sus funciones como proteína.
Introduction: Tropaeolum tuberosum, known as "mashua" is an Andean tuber that holds both economic and nutritional value for low-income populations. It is believed that it affects male fertility because Andean men associate it with impotence and decreased fertility. Studies conducted on rats fed with "mashua" showed that there was a 45% decrease in the testosterone/dihydrotestosterone ratio. The effect of this plant on reproduction is related to its content of isothiocyanates, compounds that covalently bind to proteins, which may be directly or indirectly involved in the spermatogenic process. The purpose of this research was to evaluate the effect of the aqueous extract of "mashua" on spermatogenesis and reproductive physiology of mice. Methods: In vivo morphofunctional parameters of mouse sperm (spermatogram) were evaluated and the expression of Cytochrome P450 17α-hydroxylase/17,20-lyase, acute steroidogenesis regulatory protein, cyclin, and protamine related to spermatogenesis was quantified. Results: The results indicated that at 7, 14 and 21 days of dosing, the sperm count was affected, as well as their progressive motility (PM), on the other hand, a delay in their maturation was observed. Regarding gene expression, no significant differences were found between the expression of the two genes studied (Cytochrome P450 17α-hydroxylase/17,20-lyase, Cyclin). Conclusion: The effect of "mashua" does not occur at the level of gene expression involved in spermatogenesis, but at the level of its functions as a protein.
ABSTRACT
Psoriasis is a chronic, mild and common inflammatory skin condition. Still an ideal treatment for psoriasis, effective, safe, convenient, and economical is not available. In this scenario, the search for suitable alternative treatments with minimal side effects is necessary. Plants can be effective and alternative in this regard. Therefore, this article discusses the leaves of the plants Thespesia populnea (Malvaceae) that are traditionally used in the treatment of psoriasis. The present study aimed to assess anti-psoriatic activity. The dried leaves of the plants were subjected to soxhlation with 95% ethanol and phytochemical studies were performed. The anti-psoriatic activity was evaluated by the Mouse-Tail model. It is a relatively sensitive and reproducible morphometric method that allows quantitative evaluation of the effects of anti-psoriatics through epidermal differentiation. Extracts were applied topically at a dose of 500mg/kg over 14 days and at the end, the animals were sacrificed, longitudinal histological sections were made of the tail skin and the degree of orthokeratosis was determined. It was significantly (P <0.05) increased by the ethanolic extract of Thespesia populnea (52.86±2.86) compared to the control (17.30±4.09). In relative epidermal thickness, the ethanolic extract of Thespesia populnea (92.68±8.8) showed a significant difference (P <0.05) compared to the control (100±10.7). The data obtained suggest that the selected plant has anti-psoriatic activity and confirms its traditional use in the treatment of psoriasis.
ABSTRACT
SUMMARY OBJECTIVE: The expression of cytotoxic T lymphocyte-associated antigen 4, E-cadherin, and CD44 in the area of tumor budding was investigated in breast carcinomas in our study. METHODS: Tumor budding was counted at the invasive margins in 179 breast carcinomas. To understand the microenvironment of tumor budding, we examined the expression status of the immune checkpoint molecules such as cytotoxic T lymphocyte-associated antigen 4, E-cadherin, and CD44. RESULTS: Tumors were separated into low (≤5) and high tumor budding groups (>5) based on the median budding number. Lymphovascular, perineural invasion, and the number of metastatic lymph nodes were significantly higher in high-grade budding tumors (p=0.001, p<0.001, and p=0.019, respectively). Tumor-infiltrating lymphocytes were significantly higher in tumors without tumor buddings (p<0.001). When the number of budding increases by one unit, overall survival decreases by 1.07 times (p=0.013). Also, it increases the risk of progression by 1.06 times (p=0.048). In high tumor budding groups, the cytotoxic T lymphocyte-associated antigen 4 staining percentage of lymphocytes was significantly higher (p=0.026). With each increase in the number of buds, an increase in the percentage of cytotoxic T lymphocyte-associated antigen 4 staining was seen in lymphocytes in the microenvironment of TB (p=0.034). CONCLUSION: Tumor budding could predict poor prognosis in breast carcinomas, and anti-cytotoxic T lymphocyte-associated antigen 4 immunotherapies may be beneficial in patients with high tumor budding tumors.
ABSTRACT
Objective:To investigate the effects of hydroxysafflor yellow A (HSYA) on depressive-like behavior and expression of type A γ-aminobutyric acid receptor(GABAAR)in hippocampus of chronic restraint stress model mice.Methods:The SPF grade male C57BL/6C mice were divided into Control group, HSYA group, Model group, Model + HSYA group and Model + fluoxetine group according to random number table method, with 12 mice in each group.Mice model of depression was established by chronic restraint stress.Mice in HSYA group and Model+ HSYA group were intraperitoneally injected with HSYA(20 mg/kg), mice in Model+ fluoxetine group were injected intraperitoneally with fluoxetine (10 mg/kg), and mice in Control group and Model group administered with 0.9% sodium chloride solution intraperitoneally once a day for 14 days.Then, the forced swimming test (FST) and tail suspension test (TST) were performed to evaluate the depressive-like behavior of mice, and the protein expression levels of different subtypes of GABAAR in the hippocampus of mice were determined by Western blot.SPSS 19.0 and GraphPad Prism 8.0 software were used for data statistical analysis and mapping.One-way ANOVA was used for comparison among groups, and Tukey-HSD test was used for further pairwise comparison.Results:(1) In the behavioral tests, there were significant differences in swimming immobility time of FST and tail suspension immobility time of TST among the five groups ( F=21.59, 20.81, both P<0.05). The swimming immobility time ((143.91±9.97) s) and tail suspension immobility time (( 107.00±6.54) s) in Model group were higher than those in Control group ((52.92±6.70) s, ( 43.50±5.96) s, both P<0.05). There were no significant difference in swimming immobility time and tail suspension immobility time between Model+ HSYA group ((26.17±7.69)s, ( 20.17±7.89)s) and Model+ fluoxetine group ((61.60±16.22)s, (34.14±10.74)s)(both P>0.05), but the swimming immobility time and tail suspension immobility time in these two groups were lower than those in Model group (both P<0.05). (2) The Western blot results showed that there were significant differences in the expression of GABAARβ1 and GABAARβ2 protein in hippocampus among the four groups ( F=12.21, 11.40, both P<0.05). The expression levels of GABAARβ1(45.60±10.76) and GABAARβ2 (46.27±4.82) protein in hippocampus of Model group were lower than those in Control group ((100.00±3.44), (100.00±3.26), both P<0.05). Compared to Model group, the expression of GABAARβ1 (79.91±5.00) and GABAARβ2 (79.08±5.53) protein in hippocampus of Model+ HSYA group were higher (both P<0.05). In addition, the expression of GABAARα1 and GABAARγ1 proteins in hippocampus were not significantly different among the four groups( F=0.23, 0.10, both P>0.05). Conclusion:HSYA can effectively alleviate depressive-like behavior in depression model mice, which may be related with the upregulation of GABAARβ1 and GABAARβ2 of hippocampus tissue.
ABSTRACT
Objective:To evaluate the cerebral infarct volume and the nerve fiber connectivity between cortical and neurogenesis-related regions in the mouse model of reperfusion after middle cerebral artery occlusion (MCAO) by 11.7 Tesla(11.7 T) magnetic resonance imaging (MRI).Methods:MCAO models were established in SPF grade adult male C57BL/6 mice using the suture-occluded method.MRI scans were performed at 3 days before and 1 day after modeling.Infarct volumes were calculated, and nerve fiber tracking was performed on specific brain regions to analyze the nerve fiber number and the parameters of fractional anisotropy(FA), mean diffusivity(MD), axial diffusivity (AD)and radial diffusivity(RD). SPSS 26.0 was used for statistical analysis, and paired t test was used to compare the data before and after modeling. Results:(1) After MCAO-induced ischemia, the infarct volume was up to (35.11±17.57)mm 3, and the FA value of the infarct area was significantly reduced compared with that of before modeling( t=4.73, P<0.01). (2) At the anterior-posterior(AP): + 1.2 mm section, the results of fiber tracking showed that compared with before modeling, the number of fiber bundles originating from the dorsal horn of the lateral sub-ventricle zone(SVZ)to the cortex reduced ((92 584.20±14 751.00) vs (59 815.60±6 752.46), t=4.87, P<0.01), and the number of fiber bundles projected to the infarcted area reduced ((107 671.40±10 497.57) vs (61 658.60±10 178.21), t=6.43, P<0.01). FA, AD, MD, and RD values were all decreased in different degrees( t=3.38-6.43, all P<0.05). (3) At the AP: -3.8 mm section, the number of fiber bundles originating from the dorsal horn of the SVZ to the cortex decreased (after modeling(96 944.00±18 331.09), before modeling(58 767.80±16 445.25), t=2.99, P<0.05), and the values of FA, AD, MD and RD decreased after ischemia ( t=7.30, 5.05, 6.74, 4.13, all P<0.05). Conclusion:The ultra-high field strength of 11.7 T MRI can accurately detect the following results that the number of nerve fiber bundles from the SVZ to the cortex or infarct area are both significantly reduced, and diffusion tensor parameters are consistently changed in mice after 1 day of ischemia-reperfusion.
ABSTRACT
Objective:To investigate the effect of early intervention with electroacupuncture (EA) on the gut microbiota in a mouse model of post-traumatic stress disorder(PTSD).Methods:Totally 32 C57BL/6 mice were randomly assigned to the following 4 groups ( n=8 for each group): Control group, EA group, PTSD group and PTSD+ EA group.After 7 days acclimation, mice in the PTSD group and PTSD+ EA group were subjected to modified single prolonged stress (mSPS). Mice in the EA group and PTSD+ EA group received EA (2/15 Hz, 1 mA, dilatational wave, 30 min/d) on "Baihui" for 7 days. Mice in the Control group and PTSD group received false stimulation (stimulated the same acupiont without electricity) for 7 days. Seven days after the last stimulation, elevated plus maze test and fear conditioning test were conducted to observe the effect of EA on PTSD-like behavior of mice. At the same time, feces of the mice were collected for gut microbiota detection by 16S rRNA sequencing.SPSS 19.0 was used for statistical analysis.One-way ANOVA was used for multiple group comparison and Bonferrani test was done for further pairwise comparision. Results:(1) There were statistically differences in the open arm activity time of the elevated plus maze test and the immobility time in contextual and cued fear conditioning test among the four groups ( F=6.93, 5.26, 14.51, all P<0.01). In the elevated plus maze test, mice in PTSD group ((60.17±15.52) s) showed significant less time in the open arms than mice in Control group((96.37±14.62) s) and PTSD+ EA group ((86.89±15.02) s) (both P<0.05). In the fear conditioning test, mice in PTSD group ((121.99±29.67) s, (130.82±29.11) s) showed significant increased immobility time both in contextual and cued fear conditioning tests than mice in Control group((74.50±26.65) s, (39.50±23.52) s) and PTSD+ EA group ((76.77±22.60) s, (102.17±3.39) s)(both P<0.05). (2) There were no significant differences among the four groups in the alpha diversity of gut microbiota ( F=0.79-2.45, all P>0.05). (3)Correlation analysis showed that 13 gut microbiotas were negatively correlated with the immobility time in contextual fear conditioning test, 2 gut microbiotas were positively correlated with it; 7 gut microbiotas were negatively correlated with the immobility time in cued fear conditioning test, 1 gut microbiota was positively correlated with it; 3 gut microbiotas were positively correlated with time spent in open arms of elevated plus maze test. Conclusion:Early intervention with EA can improve anxiety-fear like behaviors and gut microflora disorder in PTSD model mice.
ABSTRACT
Objective:To investigate the icariin on cognitive function and astrocytic pyroptosis in hemorrhagic shock resuscitation model mice.Methods:Forty-eight SPF grade C57BL/6 mice (male) were randomly divided into four groups ( n=12 in each group): Sham operation control group (Group C), hemorrhagic shock and resuscitation group (Group H), hemorrhagic shock and resuscitation plus icariin group (Group HI) and hemorrhagic shock resuscitation plus icariin and SSK1 group (Group HIS, SSK1 was a phosphorylation agonist of mitogen-activated protein kinase p38(p38MAPK). The mice in Group H, HI and HIS were subjected to hemorrhagic shock and resuscitation model by bleeding and retransfusion via left femoral vein; the mice in Group HI and HIS were administered with icariin (10 mg/kg) intragastrically for 7 days; the mice in Group C and H were administered with the same amount of normal saline containing dimethyl sulfoxide(DMSO). The mice in Group HIS were administered with SSK1 (0.5 mg/kg) intraperitoneally, but the mice in Group C, H and HI were only administered with the same amount of normal saline containing DMSO.At 15 days after resuscitation, novel objective recognition test and fear conditioning test were used to assess cognitive dysfunction of mice.Microtubule-associated protein 2(MAP2), a specific marker protein of neurons reflecting astrocytic pyroptosis in the hippocampus of mice, were detected by immunofluorescence assay so as to assess neuronal injury and astrocytic pyroptosis.The levels of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the hippocampus were evaluated by Western blot.SPSS 21.0 software was used for data analysis, multiple samples among groups were compared by one-way ANOVA, and SNK- q test was used for further pairwise comparison. Results:The results of new object recognition test showed that the difference of new object recognition index among the four groups was statistically significant ( F=50.75, P<0.05). The new object recognition indexes in H group(22.7±6.9), HI group(40.1±7.0) and HIS group (22.5±7.5) were significantly lower than that in C group (58.5±11.2). The index in HI group was higher than that in H group, while the index in HIS group was lower than that in HI group (all P<0.05). The results of the fear conditioning test showed that there was a statistically significant difference in the percentage of freezing time among the four groups of mice ( F=60.54, P<0.05). And the percentage of freezing time in H group((21.8±5.0)%), HI group ((38.4±7.4) %)and HIS group((21.3±4.2)%)were lower than that in C group((49.1±7.0)%), which in HI group was higher than that in H group ( P<0.05)and which in HIS group was lower than that in HI group(all P<0.05). The results of immunofluorescence showed that there were significant decreases of MAP2 intensity ((35.3±9.3)%, (63.3±6.1)%, (28.7±10.3)%) but increases of pyroptotic astrocytes ((24.5±4.2)%, (9.3±1.5)%, (22.1±3.3)%) in the H, HI and HIS groups compared with those of C group ((106.7±19.7) %, (3.4±2.0)%). There was an increase of MAP2 intensity but a decrease of pyroptotic astrocytes in the HI group compared with those in H group, and there was a decrease of MAP2 intensity but an increase of pyroptotic astrocytes in the HIS group compared with those of HI group (all P<0.05). The Western blot results showed that there were significant increases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the H, HI and HIS groups compared with C group, there were decreases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the HI group compared with H group, and there were increases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the HIS group compared with those in HI group (all P<0.05). Conclusion:Icariin alleviates hemorrhage shock and resuscitation-induced cognitive dysfunction and astrocytic pyroptosis in mice, and the mechanism may be associated with inhibition of phosphorylated p38MAPK.
ABSTRACT
Objective:To investigate the therapeutic effect of the combination of mouse nerve growth factor and edaravone in the treatment of carbon monoxide poisoning and its effect on patients′ cognitive function, lactic acid clearance rate, and related indicators of oxygen free radicals.Methods:A selection of 158 patients with carbon monoxide poisoning in the Huxi Hospital Affilliated Jining Medical College from May 2017 to June 2020 were divided into study group (80 cases) and control group (78 cases) according to the treatment plan. Both groups were given conventional treatment. On this basis, the control group was given edaravone, and the study group was given mouse nerve growth factor combined with edaravone, both of which were treated for 2 weeks. The clinical efficacy of the two groups was compared with those before treatment and 1 week and 2 weeks after treatment. Neurological impairment score (NIHSS), disease severity score (APACHE Ⅱ), cognitive function score (MMSE), serum inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP)], oxygen free radical related indicators [lipid peroxide (LPO), superoxide dismutase (SOD), gluten Glutathione peroxidase (GSH-PX), malondialdehyde (MDA)] levels, blood lactic acid levels before treatment and lactic acid clearance rates after 12 h, 24 h, 72 h treatment, and statistics of adverse reactions and 30-day mortality.Results:The total effective rate of the study group was higher than that of the control group after 2 weeks of treatment [95.00% (76/80) vs. 78.21% (61/78)] ( P<0.05); NIHSS and APACHEⅡ scores of the study group after 1 week and 2 weeks of treatment Lower than the control group: (6.08 ± 1.15) points vs. (8.94 ± 1.71) points, (4.58 ± 0.74) points vs. (6.32 ± 0.93) points and (6.79 ± 1.03) points vs. (8.02 ± 1.47) points, (5.94 ± 1.47) points vs. (7.25 ± 0.94) points, the MMSE score was higher than that of the control group: (22.09 ± 4.35) points vs. (19.34 ± 5.32) points, (26.05 ± 2.37) points vs. (22.47 ± 4.64) points ( P<0.05) After 1 and 2 weeks of treatment, the serum TNF-α, IL-6, CRP, LPO and MDA levels in the study group were lower than those in the control group: (22.62 ± 4.12) ng/L vs. (29.43 ± 4.68) ng/L and (18.21 ± 2.09) ng/L vs. (24.37 ± 3.16) ng/L, (39.67 ± 4.35) ng/L vs. (52.14 ± 5.48) ng/L and (34.83 ± 3.75) ng/L vs. (41.07 ± 4.09) ng/L, (12.63 ± 1.85) mg/L vs. (17.02 ± 2.47) mg/L and (8.27 ± 1.16) mg/L vs. (11.05 ± 1.62) mg/L, (11.06 ± 1.28) μmol/L vs. (15.97 ± 1.85) μmol/L and (8.24 ± 1.12) μmol/L vs. (12.97 ± 1.40) μmol/L, (7.15 ± 1.16) μmol/L vs. (9.02 ± 1.47) μmol/L and (6.12 ± 0.96) μmol/L vs. (7.84 ± 1.25) μmol/L, the levels of SOD and GSH-PX were higher than those in the control group ( P<0.05); the lactate clearance rate in the study group was higher than that in the control group after 12, 24 and 72 h of treatment: (18.49 ± 3.63)% vs. (14.62 ± 2.95)%, (23.19 ± 4.20)% vs. (17.42 ± 3.57)%, (29.86 ± 6.37)% vs. (25.38 ± 5.21)% ( P<0.05); the incidence of adverse reactions in the study group during treatment Compared with the control group, there was no significant difference ( P>0.05); there was no significant difference in the 30-day mortality between the study group and the control group ( P>0.05). Conclusions:The combination of mouse nerve growth factor and edaravone in the treatment of carbon monoxide poisoning can reduce the severity of disease and neurological deficits, improve cognitive function and lactate clearance rate, reduce inflammation and oxidative stress, improve efficacy, and have good safety.
ABSTRACT
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.
ABSTRACT
ObjectiveTo investigate whether phosphodiesterase （PDE） 5 inhibitors sildenafil （SIL） or LW1646 prevented renal interstitial fibrosis induced by unilateral ureteral obstruction （UUO）. MethodsMale C57BL/6 mice were randomly divided into four groups （n =6）， namely the Sham group， 7UUO group， 7UUO+SIL group and 7UUO+LW1646 group. Sildenafil （SIL） or LW1646， or vehicle was administered 1 hour before surgery， and the mice were continuously treated once daily （i. g.， 50 mg/kg） for 7 days. The obstructed kidneys were harvested on day 7. Hematoxylin-eosin （HE） and Masson’s staining was used to examine renal histology. Immunoblotting and RT-qPCR were used to detect the expression levels of protein and mRNA for fibrosis， apoptosis， endoplasmic reticulum （ER） stress， autophagy， and pro-fibrotic factors. Human proximal tubule epithelial cells （HK-2） were treated with TGF-β1 for 48 hours or tunicamycin for 24 hours， respectively， to evaluate whether cyclic guanosine monophosphate （cGMP） or PDE5 inhibitors prevents ER stress and pro-fibrotic responses. ResultsAt the 7th days after UUO， the body weight of the mice showed a significant decrease （P< 0.000 1） compared with that in the sham group. The obstructed kidneys showed a significant tubular dilation and interstitial inflammation. The levels of protein and mRNA expression in apoptosis， ER stress， autophagy-related protein and pro-fibrotic factors were also markedly increased in UUO mice （P <0.05）. In contrast， SIL or LW1646 treatment was associated with attenuated tubular dilation， infiltration of inflammatory cells and collagen content in the obstructed kidney of the mice. The protein and mRNA expression levels of renal TGF-β1 were markedly decreased， and the protein expression levels of apoptosis， endoplasmic reticulum stress， and autophagy markers were also significantly downregulated by PDE5 inhibitors. In HK-2 cells， TGF-β1 induced increased expression levels of fibronectin and BiP， which was at least partially reversed by cGMP， a product of PDE inhibition. Additionally， PDE5 inhibitors were found to modulate aberrant levels of autophagy and apoptosis. ConclusionIn conclusion， PDE5 inhibitors， in particular， LW1646， can alleviate the progression of fibrosis by improving ER stress， apoptosis and autophagy as well as downregulating protein and mRNA expression of TGF-β1.
ABSTRACT
@#Objective To investigate the effect of cold stimulation on the phenotype of alveolar macrophages(MH-S cells) in mice. Methods MH-S cells were cultured at 37 ℃ for 24 h,and cold stimulated at 36,34 and 32 ℃ for 0,0. 5,1,3,6,9 and 12 h respectively. The mRNA transcription levels of interleukin-1β(IL-1β) and interleukin-10(IL-10) genes in MH-S cells were detected by qRT-PCR. MH-S cells were cultured at 37 ℃ for 24 h,and cold stimulated at 34 ℃ for 0. 5 h,which were detected for the mRNA transcription levels of tumor necrosis factor-α(TNF-α),inducible nitric oxide synthase(iNOS)and Arginase1(Arg1)genes by qRT-PCR(MH-S cells with 0 h cold stimulation as control),detected for the expression of iNOS and Arg1 by immunofluorescence assay(MH-S cells cultured at 37 ℃ for 0. 5 h as negative control)and detected for the expression levels of iNOS,TNF-α and nuclear factor-kappa B(NF-κB)by Western blot(MH-S cells cultured at 37 ℃ for 0. 5 h as negative control). Results The mRNA transcription levels of IL-1β and IL-10 genes in MH-S cells were the highest when the cells were cultured at 34 ℃ for 0. 5 h,therefore,the cold stimulation model of MH-S cells was established under this condition. Compared with the cells cultured for 0 h,the mRNA transcription levels of iNOS,TNF-α and Arg1genes in MH-S cells cultured at 34 ℃ for 0. 5 h increased significantly(t = 3. 733,12. 190 and 6. 793,respectively,each P < 0. 05). Compared with the negative control group,the fluorescence expression intensity of iNOS and Arg1 in MH-S cells in the stimulation group increased,especially iNOS,the expression levels of iNOS and TNF-α proteins increased with no significant difference(t = 0. 675 and 1. 514,respectively,each P > 0. 05),and the expression level of NF-κB increased significantly(t = 3. 092,P < 0. 05). Conclusion Cold stimulation at 34 ℃ for 0. 5 h can increase the expression of inflammatory factors such as IL-1β,IL-10,TNF-α,iNOS,Agr1 and NF-κB in MH-S cells,activate NF-κB signaling pathway in MH-S cells,induce the expression of inflammatory proteins and promote cell activation.
ABSTRACT
Objective:To construct a C57BL/6 mouse model of simulating transurethral thulium laser vaporization prostatectomy.Methods:Twelve male C57BL/6 mice were selected to undergo transvesical vaporization resection of the urothelium covering the urethra of the prostate using thulium laser. The urethral tissue of the prostate was retrieved on the 1st, 3rd, 5th, and 7th days after the surgery. HE staining was used to observe the process of re-epithelialization of the urethral wound of the prostate. Immunohistochemical (IHC) staining was used to detect whether the re-epithelialized cells of the urethral wound of the prostate expressed urothelin Ⅲ (UPⅢ).Results:On the first day after surgery, HE staining showed complete destruction to the urothelium covering the urethra of the prostate, with a large amount of coagulative necrotic tissue on the wound surface, and IHC staining showed no expression of UPⅢ on the wound surface. On the 3rd day after surgery, HE staining showed that there were still no regenerated epithelial cells on the wound surface, with coagulation necrosis tissue significantly reduced, and the urethral cavity was clearly visible. And IHC staining showed no expression of UPⅢ on the wound surface. On the 5th day after surgery, HE staining showed 1-2 layers of regenerated epithelial cells lacking cell polarity on the wound surface, and IHC staining showed that the regenerated epithelial cells expressed UPⅢ. On the 7th day after surgery, HE staining showed 4-6 layers of polar regenerated epithelial cells on the wound surface, and IHC staining showed the multiple layers of regenerated epithelial cells expressing UPⅢ.Conclusions:Based on the simulation of transurethral thulium laser vaporization resection of the prostate, the thulium laser and ultra micro endoscope system were used to vaporize the urothelium covering the urethra of the prostate, and the process of urethral re-epithelialization of the prostate can be observed after surgery. The establishment of the C57BL/6 mouse model simulating thulium laser vaporization prostatectomy provides a new research platform for studying the mechanism of wound repair after prostatectomy.
ABSTRACT
Objective:To evaluate the effects of high mobility group protein box 1 (HMGB1) on clinical prognosis of esophagus squamous cell carcinoma (ESCC) patients treated with chemoradiotherapy and the radiosensitivity of xenograft in nude mice.Methods:A total of 90 endoscopic biopsy specimens were obtained from ESCC patients treated with chemoradiotherapy. The expression level of HMGB1 was determined by immunohistochemical staining. High expression level was defined when staining was observed on ≥50% of the tumor cells. All patients were divided into the high expression group ( n=48) and low expression group ( n=42), and their survival information was retrospectively analyzed. Cell transfection was performed with the plasmid carrying human HMGB1-shRNA to knockdown HMGB1 expression in ECA109 cells and xenograft mouse models were established. The tumor volume and mass were calculated after irradiation with a dose of 15 Gy. The cell apoptosis in xenograft tissues were detected. Survival analysis was performed using Kaplan-Meier method. Univariate prognostic analysis was conducted by log-rank test. Intergroup comparison was performed by analysis of variance (ANOVA). Results:The expression level of HMGB1 was significantly associated with gross tumor volume, longest diameter of tumor, T staging and distant metastasis ( χ2=9.663, 5.625, 4.068, 7.146, all P<0.05). In the low expression group, the overall survival (OS) ( χ2=4.826, P=0.028), progression-free survival (PFS) ( χ2=4.390, P=0.036) were longer compared with that in the high expression group. Further analysis of HMGB1-high expression patients showed that the radiation dose and the combination of chemoradiotherapy did not significantly affect the OS or PFS of ESCC patients. We observed that knockdown of HMGB1 slowed the growth rate of xenograft, decreased the tumor volume and increased the apoptosis rate after irradiation. Conclusions:ESCC patients with high expression level of HMGB1 obtain poor prognosis after chemoradiotherapy, which can be enhanced by increasing the sensitivity to radiotherapy and chemotherapy. HMGB1 knockdown can effectively increase the radiosensitivity of xenograft in ESCC nude mice.
ABSTRACT
Objective To investigate the dynamic expression of cluster of differentiation 47 (CD47) and its ligands signaling regulatory protein α (SIRPα) and thrombospondin-1 (TSP-1) in mice infected with Toxoplasma gondii in the second and third trimesters.. Methods C57BL/6J mice (6 to 8 weeks old) were used for modeling T. gondii infection in the first trimester, and the pregnant mice were randomly divided into the normal control and infection groups, of 10 mice in each group. Pregnant mice in the infection group were intraperitoneally injected with 150 T. gondii tachyzoites on gestational day (Gd) 6.5, while pregnant mice in the normal control group were intraperitoneally injected with the same volume of physiological saline at the same time. The uterine and placental specimens were collected from all pregnant mice on Gd12.5 and Gd18.5, and the pregnant outcomes were recorded. The pathological damages of mouse uterine and placental specimens were observed using hematoxylin-eosin (HE) staining on Gd12.5 and Gd18.5. The relative expression of CD47, SIRPα, TSP-1, surface antigen 1 (SAG1), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-13 mRNA was quantified in mouse uterine and placental specimens using real-time fluorescence quantitative PCR (qPCR) assay, and the CD47, SIRPα, TSP-1 expression was determined in mouse uterine and placental specimens using immunohistochemical staining. Results As compared with those in the normal control group, the pregnant mice in the infection group showed back arching, bristling, trembling and listlessness during pregnancy, and several mice presented virginal bleeding and abortion. Pathological examinations showed inflammatory cell infiltration, congestion and necrosis in uterine and placental specimens of pregnant mice in the infection group, a higher abortion rate of pregnant mice was seen in the infection group than in the normal control group on Gd12.5 (χ2 = 20.405, P < 0.001) and Gd18.5 (χ2 = 28.644, P < 0.001). qPCR assay showed significant differences in the expression of CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 genes in mouse placental specimens between the normal control and infection groups on Gd12.5 and Gd18.5 [F′ (F) = 37.511, 29.337, 97.343, 53.755, 67.188, 21.145, 8.658 and 13.930, all P values < 0.001]. Higher CD47, SIRPα and TSP-1 gene expression was quantified in mouse placental specimens in the infection group than in the normal control group on Gd12.5 (all P values < 0.01), and lower CD47, SIRPα and TSP-1 gene expression was quantified in the infection group than in the normal control group on Gd18.5 (all P values < 0.001), while higher SAG1 gene expression was detected in placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01). In addition, higher INF-γ and IL-2 expression and lower IL-4 and IL-13 expression was detected in mouse placental specimens in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001), and there were significant differences in the CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 gene expression in uterine specimens of pregnant mice between the normal control and infection groups on Gd12.5 and Gd18.5 [H(F′ and F) = 14.951, 25.977, 18.711, 48.595, 39.318, 14.248 and 15.468, all P values < 0.01], and higher CD47 and TSP-1 expression was detected in mouse uterine specimens in the infection group than in the control group on Gd12.5 and Gd18.5 (all P values < 0.01); however, no significant difference was found in the SIRPα expression (P > 0.05). Higher SAG1 expression was detected in uterine specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01), and higher INF-γ and IL-2 gene expression and lower IL-4 and IL-13 gene expression was found in the placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001). Spearman correlation analysis showed that the CD47 gene expression correlated positively with IFN-γ (rs = 0.735, P < 0.05) and IL-2 (rs = 0.655, P < 0.05) and negatively with IL-4 (rs = −0.689, P < 0.05) and IL-13 expression (rs = −0.795, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd12.5, and the CD47 gene expression correlated negatively with IFN-γ (rs = −0.745, P < 0.05) and IL-2 expression (rs = −0.816, P < 0.05) and positively with IL-4 (rs = 0.704, P < 0.05) and IL-13 (rs = 0.802, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd18.5. Immunohistochemical staining showed mild CD47, SIRPα and TSP-1 expression in uterine and placental specimens of pregnant mice in the normal control group on Gd12.5 and Gd18.5, strong CD47, SIRPα and TSP-1 expression in the placental specimens of pregnant mice in the infection group on Gd12.5 and strong CD47 and TSP-1 expression in the uterine specimens of pregnant mice in the infection group on Gd12.5. Conclusions T. gondii infection in the first trimester may cause abnormal expression of CD47 and its ligands SIRPα and TSP-1 in the maternal-fetal interface of pregnant mice in the second and third trimesters, which may be associated with the immune escape of T. gondii at the maternal-fetal interface.
ABSTRACT
@#Objective To develop and verify a plaque method for detection of infectious titer of tick-borne encephalitis virus(TBEV)strain(PHKT strain for short)adapted to primary hamster kidney(PHK)cells.Methods PHK cells were infected with TBEV,a primary mouse brain adaption strain,and passed consecutively for 12 passages.The titer of PHKT was detected by plaque method(Monolayer BHK-21 cells were infected with PHKT of various passages at different dilution ratios,and the plaque number was calculated by neutral red staining)and challenge titration in mouse brain(Mice were challenged with PHKT of various passages at different dilution ratios through brain cavity,0.03 mL for each,observed continuously for 14 days,and calculated for the median lethal dose(LD50)by Reed-Muench method)respectively,and the correlation between the results of two methods was analyzed.The developed plaque method for the detection of TBEV titer was verified for specificity,repeatability and intermediate precision.Results The plaque titer of PHKT virus was up to8.9 lgPFU/mL;The correlation between the results of plaque method and mouse brain challenge titration method was good(r = 0.92);The specificity of plaque method for detecting infectious titer of PHKT virus was good,and the coefficients of variation(CVs)of repeatability and intermediate precision were both less than 5%.Conclusion A plaque method for detecting infectious titer of PHKT virus was developed,which may be used as an alternative method for challenge titration in mouse brain.
ABSTRACT
Objective@# To explore the preventive effect of nicotinamide (NAM) on cleft palate induced by all-trans retinoic acid (RA), to provide research evidence for the prevention of cleft palate. @*Methods @#The mouse cleft palate model was induced by intragastric administration of 70 mg/kg all-trans retinoic acid at embryonic day 10.5 (E10.5) in the control group. The mouse cleft palate model was treated by caudal vein injection of 20 mg/kg NAM at E8.5 to E13.5 in the experimental group (1). The cleft palate model was treated by caudal vein injection of 40 mg/kg NAM at E8.5-E13.5 in the experimental group (2). The cleft palate of fetal rats was observed by laparotomy on E16.5 and statistically analyzed. Annexin V-FITC/PI double staining was used to detect the apoptosis of mouse embryonic palatal mesenchyme (MEPM) cells treated with RA 1 μmol/L (RA 1 group), NAM 200 μmol/L (NAM 200 group), and both NAM 200 μmol/L and RA 1 μmol/L (NAM 200+RA 1 group) for 24 hours by flow cytometry and the apoptosis rate in groups were compared. Culture without RA or NAM was used as a control. @*Results @# The cleft palate rate in the control group was 98%. The cleft palate rate in experimental group (1) was 87%. There was no significant difference between groups (P>0.05). The cleft palate rate in the experimental group (2) was 63%, compared with the control group, there was a significant difference (P<0.01). The cell apoptosis rate was 16.53%±2.89% in the CONTROL group. The cell apoptosis rate was 22.9%±1.85% in the RA 1 group, which was a significant increase compared with the CONTROL group (P<0.01). The apoptotic rate of the NAM 200 group was 9.23%±1.39%, which was a significant decrease compared with NA 1 group (P<0.01). The apoptosis rate of the NAM 200+RA 1 group was 14.9%±7.67%, which was a significant decrease compared with the RA 1 group (P<0.01).@*Conclusion@#NAM can prevent cleft palate. 40 mg/kg nicotinamide during pregnancy is an effective concentration for the prevention of RA-induced cleft palate. The mechanism by which NAM prevents cleft palate may be that NAM inhibits RA-induced apoptosis of MEPM cells.
ABSTRACT
Background Benzo[a]pyrene (BaP) has neurotoxicity, which can induce the loss of hippocampal neurons in humans and animals and lead to spatial learning and memory dysfunction, but its mechanism is still unclear. Objective To observe the ferroptosis of mouse hippocampal neuron HT22 cells induced by 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene (BPDE), an active metabolite of BaP, and to explore its potential mechanism, so as to provide a basis for the study of BaP neurotoxicity mechanism. Method Mouse hippocampal neuron HT22 cells were selected and divided into four groups: solvent control group and low, medium, and high concentration BPDE exposure groups (0.25, 0.50, and 0.75 μmol·L−1). Cell survival was detected by CCK8 method. Cell morphology and ultrastructure were observed under light and electron microscopes. The levels of reactive oxygen species (ROS) and Fe2+ were detected by fluorescence probe method. Iron, 4-hydroxynonenoic acid (4-HNE), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-PX) levels were detected with commercial kits. The expression levels of acyl-CoA synthase long chain family member 4 (ACSL4), cyclooxygenase 2 (COX2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were detected by Western blotting. After interventions with ferroptosis inhibitors 20 μmol·L−1 deferoxamine (DFO) and 10 μmol·L−1 ethyl 3-amino-4-cyclohexylaminobenzoate (Fer-1), the cell survival rate of each BPDE exposure group and the changes of the ferroptosis characteristic indicators and protein expression levels were observed. Results With the increase of BPDE concentration, the survival rate of HT22 cells decreased gradually, and the survival rate of each BPDE group was significantly lower than that of the solvent control group (P<0.01). Under light microscope, the number of cells in the high concentration BPDE group was significantly reduced, and atrophic cells and reduced synapses were recorded. Under electron microscope, the HT22 cells in the high concentration BPDE group showed mitochondrial shrinkage, decreased crista, and increased mitochondrial membrane density. Compared with the solvent control group, the levels of intracellular lipid ROS, Fe2+, 4-HNE, and MDA significantly increased in the high concentration group (P<0.01), the GSH and GSH-PX levels were significantly decreased (P<0.01), the protein expression levels of ASCL4 and COX2 were significantly increased (P<0.01), and the protein expression levels of SCL7A11 and GPX4 were significantly decreased (P<0.01). The ferroptosis inhibitors DFO and Fer-1 significantly reversed the cell survival rate (P<0.01), the ferroptosis characteristic indicators (ROS, Fe2+, 4-HNE, MDA, GSH, and GSH-PX levels) (P<0.01), and the expression levels of ferroptosis-related proteins (ACSL4, COX2, SLC7A11, and GPX4) (P<0.01) in the high concentration BPDE group. Conclusion BPDE can induce ferroptosis in mouse hippocampal neuron HT22 cells, which may be related to the inhibition of SLC7A11/GSH/GPX4 axis and the induction of iron metabolism disorder.
ABSTRACT
The mammalian carboxylesterase 1 (Ces1/CES1) family comprises several enzymes that hydrolyze many xenobiotic chemicals and endogenous lipids. To investigate the pharmacological and physiological roles of Ces1/CES1, we generated Ces1 cluster knockout (Ces1 -/- ) mice, and a hepatic human CES1 transgenic model in the Ces1 -/- background (TgCES1). Ces1 -/- mice displayed profoundly decreased conversion of the anticancer prodrug irinotecan to SN-38 in plasma and tissues. TgCES1 mice exhibited enhanced metabolism of irinotecan to SN-38 in liver and kidney. Ces1 and hCES1 activity increased irinotecan toxicity, likely by enhancing the formation of pharmacodynamically active SN-38. Ces1 -/- mice also showed markedly increased capecitabine plasma exposure, which was moderately decreased in TgCES1 mice. Ces1 -/- mice were overweight with increased adipose tissue, white adipose tissue inflammation (in males), a higher lipid load in brown adipose tissue, and impaired blood glucose tolerance (in males). These phenotypes were mostly reversed in TgCES1 mice. TgCES1 mice displayed increased triglyceride secretion from liver to plasma, together with higher triglyceride levels in the male liver. These results indicate that the carboxylesterase 1 family plays essential roles in drug and lipid metabolism and detoxification. Ces1 -/- and TgCES1 mice will provide excellent tools for further study of the in vivo functions of Ces1/CES1 enzymes.