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Objective:To investigate the expression of adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation in corneal epithelial cells and the effects of fungus on AMPK phosphorylation and interleukin-6 (IL-6) production in corneal epithelial cells.Methods:The human immortalized corneal epithelial cell line was selected.The safe concentration range of AMPK agonist 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (100, 300, 500, 1 000 μmol/L) and inhibitor Compound C (10.0, 12.5, 15.0, 17.5, 20.0 μmol/L) on corneal epithelial cells was screened by multi-function real-time unlabeled cell analyzer.Corneal epithelial cells without any treatment were used as the normal control group, and those co-cultured with spores were used as the spore control group.Corneal epithelial cells co-cultured with spores were treated with AICAR and Compound C for 4 hours in the AICAR group and Compound C group, respectively.The expression of phosphorylated AMPK (p-AMPK) and AMPK in corneal epithelial cells was detected by Western blot, and the concentration of IL-6 in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA).Results:After treatment with different concentrations of AICAR for different periods, there was no statistical significance in the cell index of corneal epithelial cells (all at P>0.05). The cell index of corneal epithelial cells was increased with 10.0 μmol/L and 12.5 μmol/L Compound C treatment compared with that of the normal control group.The expression levels of p-AMPK were 0.67±0.15, 2.57±0.12, 3.67±0.58 and 1.50±0.50, respectively, in the normal control group, spore control group, AICAR group and Compound C group, showing a statistically significant difference among them ( F=32.820, P<0.001). The expression level of p-AMPK was significantly higher in the spore control group compared with the normal control group ( P<0.001). The expression level of p-AMPK in the AICAR group was higher than that in the spore control group, and the expression level of p-AMPK in the Compound C group was lower than that in the spore control group, and the differences were statistically significant (both at P=0.010). There was no significant difference in the relative expression level of AMPK among the four groups ( F=0.120, P=0.950). The expression levels of IL-6 concentration in the normal control group, spore control group, AICAR group and Compound C group were (107.81±17.15), (156.32±9.94), (167.96±14.16) and (127.42±19.75)pg/ml, respectively, showing a statistically significant difference among them ( F=15.210, P<0.001). The IL-6 concentration of the spore control group was higher than that of the normal control group, and the difference was statistically significant ( P<0.001). The IL-6 concentration of the AICAR group was higher than that of the spore control group, but the difference was not statistically significant ( P=0.260). The IL-6 concentration of the Compound C group was lower than that of the spore control group, and the difference was statistically significant ( P=0.010). Conclusions:In corneal epithelial cells, AMPK phosphorylation is found, which is enhanced after fungal spores stimulation, and the secretion of IL-6 increases.
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ObjectiveTo explore the therapeutic effect and possible mechanism of Banxia Xiexintang and its disassembled prescriptions in regulating the flora disorder induced by mixed antibiotics in young rats. MethodSeventy male BALB/C young rats were randomly assigned into 7 groups： blank group， model group， Bifidobacterium tetralogy viable tablets （0.68 g·kg-1） group， Banxia Xiexintang （9.1 g·kg-1） group， Xinkai （3.19 g·kg-1） group， Kujiang （1.82 g·kg-1） group， and Ganbu （4.1 g·kg-1） group， with 10 rats in each group. Except the blank group， the other groups were given mixed antibiotics by gavage to induce intestinal flora disorder. After 14 days， the rats in different drug groups were administrated with corresponding drugs by gavage， and those in the blank group and model group with the same amount of normal saline once a day for 14 days. After that， fecal samples were collected aseptically for 16S rDNA sequencing of intestinal flora， and lipopolysaccharide （LPS， 10 mg·kg-1） was injected intraperitoneally to induce inflammatory reaction. The tissue morphology of colonic mucosa was observed via hematoxylin-eosin （HE） staining， and the macrophage infiltration of colonic mucosa was observed via toluidine blue staining and immunohistochemistry. The expression of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， tumor necrosis factor-α （TNF-α）， and interleukin-10 （IL-10） mRNA were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the modeling changed the intestinal flora structure of the young rats （P<0.01）， damaged the colonic mucosa， reduced the macrophage infiltration， and down-regulated the mRNA levels of IL-1β， IL-6， IL-8， TNF-α， and IL-10 （P<0.01）. Compared with the model group， bifidobacterium quadruple viable tablets， Banxia Xiexintang and its disassembled prescriptions increased the diversity of intestinal flora and the relative abundance of beneficial bacteria such as Bacteroidetes and Firmicutes （P<0.01）. At the same time， they ameliorated colonic mucosal injury （P<0.05， P<0.01）， increased macrophage infiltration （P<0.05， P<0.01）， and up-regulated the mRNA levels of IL-6， IL-8， and TNF-α （P<0.01）. The mRNA level of IL-1β was up-regulated in Bifidobacterium tetralogy viable tablets， Banxia Xiexintang， Kujiang， and Ganbu groups （P<0.01）， and that of IL-10 was up-regulated in Bifidobacterium tetralogy viable tablets， Banxia Xiexintang， Xinkai， and Ganbu groups （P<0.01）. ConclusionBanxia Xiexintang and the disassembled prescriptions can adjust the intestinal flora of young rats exposed to antibiotics and protect the immune barrier of colonic mucosa after intestinal flora disorder. In particularly， the whole prescription of Banxia Xiexintang demonstrates the best performance.
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OBJECTIVES@#To analyze the differentially expressed genes (DEGs) with radiation-induced rat lung injury, and to reveal the protective mechanism for mild hypothermia in the radiation-induced lung injury in rats at the transcriptome level.@*METHODS@#A total of 10 male SD rats aged 6-8 weeks were randomly divided into 2 groups to establish a rat model of radiation-induced lung injury, and one group was treated with mild hypothermia. RNA was extracted from left lung tissue of each group, and sequenced by BGISEQ-500 platform. Significance analysis of DEGs was carried out by edgeR software. Gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to analyze the gene function. Then 5 key DEGs were verified by real-time reverse transcription PCR (real-time RT-PCR).@*RESULTS@#There were 2 790 DEGs (false discovery rate<0.001, |log@*CONCLUSIONS@#The DEGs and pathways related to mild hypothermia protection against radiation-induced lung injury in rats are obtained, which provides an experimental basis for the protection of mild hypothermia against radiation-induced lung injury.
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Animals , Male , Rats , Gene Expression Profiling , Hypothermia , Lung Injury , RNA-Seq , Rats, Sprague-Dawley , TranscriptomeABSTRACT
Objective: To investigate the reversal effect of astragaloside IV on multidrug resistance of MDA-MB-231/DOX in breast cancer cells. Methods: The cytotoxicity of astragaloside IV and sensitivity or drug resistance of breast cancer cells to doxorubicin (DOX) before and after treatment were determined by MTT assay. Liposome co-delivery system containing doxorubicin and astragaloside IV (LPs-DOX/AS) was constructed by ethanol injection-ammonium sulfate gradient method. The reversal effect of LPs-DOX/AS on multidrug resistance of breast cancer cells was determined by MTT method. The effect of LPs-DOX/AS on apoptosis was determined by flow cytometry. Results: Astragaloside IV had no significant cytotoxicity to breast cancer cells in the experimental concentration range. After combined with astragaloside IV, the IC50 values of DOX on MDA-MB-231 and MDA-MB-231/DOX cells decreased (P < 0.05), and the intervention effect on drug-resistant cells was more significant (P < 0.01). Compared with free DOX/AS-IV, the IC50 values of LPs-DOX/AS-IV on both breast cancer cells decreased (P < 0.05), and the effect on drug-resistant strains was more significant (P < 0.01). The apoptosis rate of drug-resistant strains treated with LPs-DOX/AS-IV was also significantly higher than that of free drug group (P < 0.05). Conclusion: Astragaloside IV has reversal effect on multidrug resistance of human breast cancer cell MDA-MB-231 to doxorubicin. The combination of astragaloside IV and doxorubicin and its liposome co-delivery system can effectively reverse or sensitize multidrug resistance in breast cancer.
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Objective:To study the antimicrobial activity of diacerein on common pathogens of the ocular surface in vitro.Methods:Pathogens were collected from patients with ocular surface infections in Henan Eye Hospital, including Gram-positive cocci and bacilli, Gram-negative bacilli, filamentous fungi, and Candida.The antimicrobial activity of diacerein was determined by the K-B agar diffusion method, and its minimum inhibitory concentration (MIC) was determined by the micro-liquid method.Levofloxacin and voriconazole were used as the control of antibacterial and antifungal drug, respectirely.Results:Diacerein showed antibacterial activity against 42 strains of Gram-positive cocci and 10 strains of Gram-positive bacilli, its inhibition zone diameters for Staphylococcus epidermidis, S.aureus, S.intermedius and Gram-positive Corynebacterium were not significantly different from those of levofloxacin (all at P>0.05). Its MIC range of diacetate against Staphylococcus epidermidis, S. aureus, S. intermedius and other Staphylococci was 1-32 μg/ml, and its respective MIC 90 was 16, 8, 16, and 32 μg/ml.Diacerein had no bacteriostatic effect on 23 strains of Gram-negative bacilli, 10 strains of filamentous fungi and 3 strains of candida. Conclusions:Diacerein has antibacterial effects against Gram-positive Staphylococcus and Corynebacterium isolated from the ocular surface, but shows no antimicrobial activity against Gram-negative bacilli and fungi.Diacerein offers a new drug option and method for the treatment of bacterial keratitis.
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Serum samples were tested for IgG antibodies using indirect immunofluorescence assays. We then analyzed associated risk factors. Serum samples were considered positive when reactive at a dilution of more than 1:320. Differences between groups and risk factors associated with exposure were statistically analyzed using Chi-square tests and the generalized linear model. 122 of 1,260 samples (9.68%) were positive for infection. The infection rate ranged from 0% to 30.43% and differed significantly among age groups ( < 0.01); infection rate in the 50-59 years group was significantly higher than that in other age groups. The seroprevalence of varied significantly among sites within the four provinces, and the infection rate of field workers was significantly higher than that of urban workers.
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Objective:In the study of urine metabolomics of rats,necessary antiseptic measures should be taken for collection of urine samples,the effect of several antiseptic measures on the endogenous metabolites in urine was studied. Method:The urine samples of rats were collected on ice,sodium azide was added,and both of them were used together to prevent corrosion.Differences of antiseptic measures were analyzed by nuclear magnetic resonance (NMR) metabolomics. Result:The results of NMR metabolomics showed that sodium azide+ice group and ice group had many overlaps,but they clearly separated with the control group and sodium azide group;sodium azide group and the control group had a small part overlap,but there was a tendency of separation.The antiseptic effect of sodium azide+ice group and ice group was similar;compared with control group,valine,betaine and hippuricacid in these two groups increased,but the alanine and 2-ketoglutaric acid decreased. Conclusion:In the study of rat urine metabolomics,low temperature antiseptic measures must be taken when urine samples are collected,and the addition of sodium azide can improve the antiseptic effect slightly under protective conditions.
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Objective@#To study the effects of platelets on macrophages phagocytosis and inhibition of fungi.@*Methods@#Macrophages were cultured, Fusarium Pythium spores were extracted and platelets were isolated from blood of mouse.Simple spore group, spore+ macrophage group and mixed platelet group were set, and were inoculated with fungal spore, equal proportion spore+ macrophage and platelet+ spore+ macrophage, respectively.The prepared plate was placed on a spinning disk laser scanning confocal microscope at 1 hour, 2, 3 and 4 hours after culture, five visual fields were randomly selected at the corresponding time points for photography.The phagocytic rate, phagocytic index and fungal spore germination rate were calculated.Fungal hyphae length of each group at 4, 6 and 8 hours after culture were calculated.The single macrophage group, spore+ macrophage group and mixed platelet group were set and the cytotoxicity was measured by real-time cell analyzer.The breeding and use of mice was in according with the ARVO statement.This study was approved by the Experimental Animal Ethics Committee of Henan Eye Institute (HNEECA-2017-04).@*Results@#The phagocytic rate and phagocytic index of macrophages in mixed platelet group at 1 hour, 2, 3 and 4 hours after culture were significantly higher than those in spore+ macrophage group at corresponding time point (all at P<0.05). There were significant differences in spore germination rate at 1 hour, 2, 3 and 4 hours after culture among different groups (H=60.05, 37.89, 55.15, 60.52; all at P<0.001). The spore germination rates of spore+ macrophage group at 1 hour, 2, 3 and 4 hours after culture were lower than those of simple spore group, while the spore germination rates of mixed platelet group at 1 hour and 3, 4 hours after culture were lower than that of spore+ macrophage group, the differences were statistically significant (all at P<0.01). There were significant differences in fungal hyphae length at 4, 6 and 8 hours after culture among the three groups (H=13.76, 43.57, 60.87; all at P≤0.001). The fungal hyphae lengths of spore+ macrophage group at 4, 6 and 8 hours after culture were lower than those of simple spore group, and the fungal hyphae lengths of mixed platelet group at 6 and 8 hours after culture were lower than those of spore+ macrophage group at the corresponding time point.The differences were statistically significant (all at P<0.05). There was no significant difference in cell index between 0 hour, 6, 12, 18 and 24 hours after culture (F=0.02, 1.08, 1.61, 1.58, 2.52; all at P>0.05). There were significant differences in cell index among different groups at 30, 36, 42 and 48 hours after culture (F=10.81, 8.08, 5.61, 5.72; all at P<0.05). The cell indexes in spore+ macrophage group at different time points were significantly lower than those in simple macrophage group (all at P<0.01).@*Conclusions@#Platelets can promote the phagocytosis and inhibition of macrophages on fungi, and platelets may have antagonistic effect on fungal cytotoxicity.
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Objective To investigate the protective effect of hydrogen-rich water on rat cognitive dysfunction induced by ionizing radiation.Methods A total of 20 SD rats were randomly divided into four groups with the ramdom number table method: control group(C),hydrogen-rich water group(HRW),irradiation group(IR)and hydrogen-rich water intervention group(HRW+IR),with 5 rats in each group.The spatial memory ability of rats was tested by a morris water maze.The expression of apoptosis-related genes was detected by real-time fluorescence quantitative PCR.The changes of glutathione(GSH),8-hydroxydeoxy guanosine(8-OHdG)and malondialdehyde(MDA)and SOD were also measured.Results The escape latency(F＝6.003,P＜0.05)and the swimming distances(F＝3.850,P＜0.05)of rats in four different groups had statistically significant differences.Compared with the IR group,the escape latency of the HRW+IR group was significantly decreased at 3,4,5 d after irradiation(P＜0.05),and the swimming distance of this group became much longer(P＜0.05).The levels of GSH,8-OHdG and MDA in these four groups had statistically significant differences(F＝6.450,5.033,4.113,P＜0.05).Compared with IR group,the concentration of GSH was increased(P＜0.05),but MDA and 8-OHdG decreased(P＜0.05)in the brain tissue of HRW+IR group,and the expressions of caspase-3,caspase-9 and bax genes were reduced(t＝2.956,3.087,5.246,P＜0.05),while the expression of bcl-2 gene was enhanced(t ＝-3.640,P ＜0.05)in the HRW+IR group.Conclusions Hydrogen-rich water attenuates the oxidative damage of ionizing radiation by neutralizing oxyhydrogen free radicals and thus protects brain from radiation damage.
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Objective This study was to assess the efficacy of corneal collagen cross-linking treatment on fungal keratitis.Methods Eighty SPF male C57BL/6 mice aged 6-8 weeks were selected for the experiment.Fusarium solani infected model was established on the left eyes of all 80 mice.Forty mice were distributed randomly into sham operation group,model control group,scraped epithelium group and corneal collagen cross-linking (CXL)group (treated with epithelium scraped and CXL).Three days after modeling,the levels of the corncal disease sevcrity were scored by slit lamp microscopy.The fungal activity was confirmed by plate counts.The left 40 mice were divided randomly into sham operation group,model control group,scraped epithelium group and CXL group (treated with epithelium scraped and CXL).In 1 day and 2,3,4,5,6,7,14 days after modeling,the corneas were examined under the slit lamp microscope.The corneal pathological examination of each group were conducted with hematoxylin and eosin staining at postoperative 14 days.The animal feeding and use was in accordance with the standards set by the ARVO,and the experiment was approved by the Ethic Committee for Experimental Animal of Henan Eye Institute.Results The colony-forming units (CFUs) of fungal solutions in culture significantly decreased with CXL treatment (F =11.97,P =0.00).The Pearson correlation analysis of CFU and clinical scores in CXL group showed that inflammatory cells infiltration was positively correlated with corneal disease severity (r =0.723,P =0.043).Corneal inflammatory score was significantly lower in the CXL group in various time points,with a significant differences among the groups and time points (Fgroup =34.44,P=0.00;Ftime =17.49,P=0.00).Corneal lesion and the depth of ulceration in scraped epithelium group and CXL group were remarkably lower than that in the model control group (all at P ＜ 0.05).Histopathology revealed that the degree of corneal collagen fibers destruction and the ratio of inflammatory cells infiltration in scraped epithelium group (59.33％) and CXL group (11.29％) were much lower than that in the model control group (73.65％).Conclusions CXL can inhibit the fungal activity effectively in the cornea of mice,and reduce the fungal induced keratitis reaction.
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Objective To compare the removal efficiency of γδT cells between cornea and ear skin and develop an alternative method for dynamic monitoring of γδT cells in mouse cornea in vivo using 2-photon laser scanning microscopy.Methods The γδT cells in mouse ear skin were monitored before and after antibody neutralization,and the mice corneas were excised and stained for counting γδT cells at 6 h,12 h,24 h after antibody neutralization by using 2-photon laser scanning microscopy,followed by comparison of the removal efficiency of γδT cells between the cornea and ear skin.Results The γδT cells in normal mouse cornea were often distributed in the limbal epithelium and superficial stromal layer.The irregular morphology of γδT cells in the epithelial layer was often accompanied by protuberances,while the stromal γδT cells were mostly round or oval and the number of cells was approximately 27 ± 4.After antibody neutralization,the number of γδT cells in the cornea of mice gradually decreased,and the number of cells at 6 h,12 h and 24 h was significantly lower than that of before depletion (P =0.03,0.00,0.00),and the removal efficiencies were 48％,78％,and 96％,respectively.The γδT cells in ear skin of the normal mice were ellipse or stellate with cell processes and they were located in epidermal layer,and the cell number was about 60 ± 9.After antibody neutralization,the number of γδT cells were significantly reduced at 6 h,12 h and 24 h compared with before depletion (P =0.000,0.000,0.000) and the removal efficiency were 43％,72％ and 95％,respectively.Conclusion The number of γδT cells in the cornea and ear skin is gradually decreased after antibody neutralization,and their removal efficiency is consistent with time.Therefore,monitoring the γδT cells in the mouse ear skin is an ideal alternative to dynamically monitoring the changes in the number of γδT cells in the cornea in vivo.
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Long non-coding RNA ( LncRNA) is a class of transcript (>200 nucleotides) that do not encode proteins. It plays an important role in epigenetic regulation and gene expression at transcriptional or post transcriptional level. The abnormal expression of LncRNA may lead to various pathological processes. Diabetic retinopathy ( DR ) is a multifactorial disease. Recent studies have shown that many specific expressions of LncRNAs are closely related to the genesis of DR. In this review, we summarized the recent advances in the function of LncRNA, the regulatory mechanisms of LncRNA involved in the development of DR, and the related therapies.
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Objective To observe the effects of mild hypothermia induced by pentobarbital sodium on hematology in male BALB/C mice.Method Healthy male BALB/C mice were divided randomly into two groups:control group ( C) and mild hypothermia group(M).The body temperature of the mild hypothermia group was maintained between 28℃ to 30℃( anal temperature ) for 4 hours induced by pentobarbital sodium injected intraperitoneally , then recover unaffected . Anal temperature, coagulation, electrolytes, and blood cell indexes were examined in 2, 24, 72 hours after treated by mild hypothermia;Control group was given equal volume of saline volume at constant temperature .Results The body temperature and coagulation in mild hypothermia group showed no significant difference compared with the control group ( P﹥0.05),but the concentration of K +and Na +in mild hypothermia group were higher than control group (P﹤0.01), the number of WBC in mild hypothermia group was lower than control group ( P﹤0.01或P﹤0.05 ) , and the RBC、HGB、MCH、MCHC in mild hypothermia group were lower than control group transiently (P﹤0.01或P﹤0.05).Conclusion Mild hypothermia induced by pentobarbital sodium affects some of hematological values in mice considerably .
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Background and purpose:The chemokine,CXCL12 and its receptor,CXCR4,have recently been shown to play an important role in the metastasis of several kinds of carcinoma. It also has been demonstrated that VEGF regulates both the expression of CXCR4 and invasiveness in cancer cell. Our aim was to study the expression of CXCR4,VEGF in osteosarcoma and the correlation between these two factors and distant metastasis. Methods: The immunohistochemical staining SP method was used to detect the expression of CXCR4 and VEGF in 56 cases of osteosarcoma. We analyzed the correlation between the expression of CXCR4 and VEGF,and the correlation between the expression of CXCR4,VEGF and clinical stage,the level of ALP. The patients were followed up for 2 years. Results:There was signifi cant correlation between the expression of CXCR4 and VEGF in 56 cases(r=5.678,P=0.02). Univariate analysis showed a signifi cant correlation between the expression of CXCR4,VEGF and clinical tumor stage(P=0.026),and no correlation between the expression of these two factors and age,sex and serum ALP level. 31 cases had metastasis in two years in a total of 56 follow-up cases,and the expression of CXCR4 and VEGF was associate with metastasis(P=0.018 and P=0.022,respectively). Conclusion:VEGF can upregulate tumor angiogenesis and promote tumor metastasis to specific organ by increasing expression of CXCR4.The increasing expression of CXCR4 and VEGF is useful to predict metastasis and prognosis of osteosarcoma.
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At the beginning of the 1980s, a concept of viable but non-culturable(VBNC) was suggested. VBNC is a survival strategy adopted by microorganisms when they are exposed to environmental stress. This article try to make a summary of research of the conditions of VBNC formation, recovery of culturability and methods of VBNC cells detection. In addition, introduces the first growth factor of microorganisms-Rpf.
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This project is targeted on exploring some improving approaches to isolate and culture the microorganisms which are difficult to be isolated and cultured through the conventional ways. The results showed that betaine, sodium pyruvate, SOD and catalase are helpful for increasing the total number and variety of isolated strains. A kind of combined method was also used to isolate the micro-colony which can not be seen by naked eyes on the plates. Totally 52 Actinomycetes and 103 bacteria and 17 fungi were obtained from 4 soil samples using the above methods. 4. 325% microorganisms were obtained as positive strains to inhibit the growth of some kinds of test bacteria, which is higher than the percent using generally isolated ones. These microbial natural products may remain an important resource for the drug discovery.