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OBJECTIVE： To study the repair effect of artificial Isaria cicadae on intestinal mucosal injury induced by 5-fluorouracil （5-FU） in rats. METHODS： Forty SD rats were randomly divided into normal group （normal saline）， model group （normal saline）， artificial I. cicadae high-dose， medium-dose and low-dose groups （3.5， 1.75， 0.875 g/kg）， with 8 rats in each group. Except for normal group， other groups were given intraperitoneal injection of 5-FU （0.25 g/10 mL） 30 mg/kg， once a day， for consecutive 5 days. At the same time， each group was given relevant medicine/normal saline intragastrically， once a day， for consecutive 8 d. After medication， body weight of rat was determined in each group. HE staining was used to observe the pathological change of small intestine. The levels of biobarrier-related factor [endotoxin （ET）， D-lactic acid （D-LA）]， immune barrier related factors （TNF-α， IFN-γ， sIgA， IL-15， G-CSF in serum and MPO， MDA in small intestine） and the levels of mechanical barrier related factors （connexin ZO-1 and Claudin-1） were detected. RESULTS： Compared with normal group， body weight of rats in model group was decreased significantly （P＜0.01）. Intestinal villus exfoliated obviously， the crypt structure was scattered， a large number of inflammatory cells gathered， and intestinal mucosa was seriously damaged. Serum levels of ET and D-LA， the levels of TNF-a， IFN-γ， MPO and G-CSF in serum， MDA level in small intestine were increased significantly （P＜0.01）. Serum levels of sIgA and IL-15 as well as the expressions of ZO-1 and Claudin-1 in small intestine were decreased significantly （P＜0.05 or P＜0.01）. Compared with model group， body weight of rats in artificial I. cicadae high-dose group was increased significantly （P＜0.01）. The pathological changes of the small intestine of rats in each administration group were improved to varying degrees. The intestine morphology of artificial I. cicadae high-dose and medium-dose groups was close to that of the normal group. The levels of and ET， D-LA， TNF-α， IFN-γ， MPO， G-CSF in serum and the level of MDA in intestinal were decreased significantly （P＜0.01）. Serum levels of sIgA and IL-15 in administration groups as well as the expressions of ZO-1 and Claudin-1 in intestinal tissue were increased significantly （P＜0.01）. CONCLUSIONS： Artificial I. cicadae can repair intestinal mucosal damage caused by 5-FU in respects of mechanical barrier， immune barrier， biological barrier.
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OBJECTIVE:To establish a method for simultaneous determination of fatty acids in Isaria cicadae,Cordyceps militaris and C.sinensis,and to compare the difference of the contents of fatty acids among above medicinal herbs.METHODS:GC-MS method was adopted.Chromatographic condition:the determination was performed on TG-5MS gas phase capillary column with carrier gas of nitrogen at the flow rate of 1.2 mL/min.The inlet temperature was 290 ℃ by splitlesssampling;valve opening time was 1 min,and volume of sample was 1 μL.Mass spectrometry condition:the ion source is an electrospray ionization source.The temperatures of ion source and transmission line were 280 ℃,the initial temperature of the chromatographic column was 80 ℃ (gradient elution),ionization voltage was 70 eV.Solvent delay time was 5 min,and scan mass range was m/z 30-400.RESULTS:The linear ranges of tetradecanoic acid,pentadecanoic acid,palmitic acid,palmitoleic acid,heptadecanoic acid,heptadecenoic acid,docosahexaenoic acid,methyl oleate,linoleic acid,arachidonic acid,eicosenoic acid,diolefinic acid,eicosatrienoic acid,heneicosanoic acid,behenic acid,tricosanoic acid,lignoceric acid were 1.400-44.520 μg/mL(r=0.999 8),2.091-93.721 μg/mL(r=0.999 7),3.146-85.856 μg/mL(r=0.998 2),1.664-61.444 g/rnL(r=0.998 7),1.773-64.983 g/mL(r=0.999 5),1.781-68.421 μg/ mL (r=0.999 7),1.706-55.606 μg/mL (r=0.999 8),1.439-47.989 μg/mL (r=0.999 6),1.738-66.908 μg/mL (r=0.999 6),2.086-94.206 μg/mL(r=0.999 5),1.356-44.966 μg/mL(r=0.999 4),1.444-56.814 μg/mL(r=0.999 7),1.375-52.335 μg/mL(r =0.999 8),1.512-60.312 μg/mL(r=0.999 5),1.450-59.760 μg/mL(r=0.999 7),1.427-58.757 μg/mL(r=0.999 1),1.269-58.109 μg/ mL(r=0.999 3),respectively.The limit of quantitation was no more than 1 764.71 μg/mL,and the limit of detection was no more than 529.42 μg/mL.RSDs of precision,stability and reproducibility tests were all lower than 2.0％.The recoveries were 84.87％-108.93％ (RSD ranged 0.19％-2.23％,n=6).There were 17 fatty acids in C.militaris,16 fatty acids in Ⅰ.cicadae and 16 fatty acids in C.sinensis.The contents of unsaturated fatty acids in above medicinal herbs were higher than that of saturated fatty acids.The content of fatty acids in artificial cultivated Ⅰ.cicadae was mostly higher than other medicinal herbs.COCLUSIONS:The method is simple,accurate,stable and reproducible.It can be used for simultaneous determination of fatty acids in I.cicadae,C.militaris and C.sinensis.Above 3 kinds of medicinal materials.From the perspective of fatty acid content,the quality of artificial cultivated I.cicadae is best.
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@#To establish a simple and sensitive LC-MS/MS method for simultaneous determination of the concentration for L-tryptophan(L-Try)and L-kynurenine(L-Kyn)in rat plasma. The changesin the process of the liver tumors formation may provide a basis for the diagnosis of liver cancer. 3-Nitro-L-tyrosine(3-NT)were added as the internal standard for the determination of two active substances and the chromatographic analysis was performed on a RRHD Eclipse Plus C18 column(3. 0 mm×100 mm, 1. 8 μm). The mobile phase was composed of water and acetonitrile(containing 0. 1%formic acid)(90 ∶10)at a flow rate of 0. 25 mL/min, and the injection volume is 5 μL. Detection and quantification were performed by mass spectrometry in multiple reaction monitoring mode with m/z 205. 12→146. 10(L-Try), m/z 209. 09 →146. 10(L-Kyn), m/z 227. 09→181. 10(3-NT), respectively. The results show that thelinear ranges were 9. 670-9 670 ng/mL for L-Try, and 9. 973-9973 ng/mL for L-Kyn(r2≥0. 9990). The limit of quantitation were 9. 670 ng/mL for L-Try, and 9. 973 ng/mL for L-Kyn, respectively. The intra- and inter-day precisions were all less than15%; the recoveries ofthe two analytes were more than 81. 17% and severe matrix effect was not observed. The ratio of L-Try/L-Kyn determined by LC-MS/MS in rat plasma showed an overall downward trend, which could used effectively for the drug metabolism studies and researches on the action mechanism of medicine on liver cancer. A rapid, simple, sensitive and specific LC-MS/MS method has been successfully developed and could also be used effectively for the drug metabolism studies and researches on the action mechanism of medicine on liver cancer.
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Background and purpose:MicroRNA (miRNA) belongs to a class of 19 to 30 nucleotide-long, endogenous noncoding RNA expressed in eukaryotes and predominantly inhibits gene expression at the post-transcriptional level. The miRNAs play critical roles in cell proliferation and differentiation, apoptosis, metabolism, and immune regulation. This study aimed to detect the expression of miR-216a-5p in lung cancer tissues and lung cancer cell lines, and to discuss the effects of miR-216a-5p on the invasion ability of lung cancer cells and the mechanism.Methods:Quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-216a-5p in lung cancer tissues of 55 cases and 7 lung cancer cell lines. Three lung cancer cell lines of A549, 95D and H460 were transiently transfected by miR-216a-5p, and Transwell was used to detect the effects of miR-216a-5p on the invasion of lung cancer cell lines. The dual luciferase reporter plasmids containing the miR-216a-5p candidate target gene and the gene of matrix metalloproteinase 16 (MMP16) were predicted and constructed. qRT-PCR and Western blot were used to detect the changes in mRNA and protein levels of target geneMMP16 by miR-216a-5p. The interference of MMP16 by siRNA and up-regulation miR-216a-5p by transfection were compared on the invasion of lung cancer cells.Results:The miR-216a-5p expression levels were all signiifcantly reduced in 90.91% (50 of 55 patients) tumor tissues compared with corresponding adjacent normal lung tissues (P<0.05). The miR-216a-5p expression levels were only 7.00%-32.00%in 7 lung cancer cells compared with the control group (P<0.05). Up-regulation of the expression of miR-216a-5p inhibited the invasion of lung cancer cells; interference of MMP16 by siRNA, as well as up-regulating miR-216a-5p by transfection, inhibited the expression of MMP16 in lung cancer leading to inhibition of the invasion of lung cancer cells. Conclusion:miR-216a-5p can be a candidate marker in clinical diagnosis and it can inhibit the invasion of lung cancer cells by down-regulating the expression of MMP16.
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Objective:To investigate the expression level of pyruvate kinase M2 (PKM2) in tissues of non-small cell lung cancer (NSCLC) patients and the correlation between PKM2 expression level and radiation sensitivity. Methods:A total of 45 NSCLC pa-tients were chosen and treated with radiotherapy for two months after surgery. The patients were classified into four groups based on the curative effect. The mRNA expression levels of PKM2 in tumor and the homologous paraneoplastic tissues of NSCLC patients were de-tected prior to radiotherapy using real time-polymerase chain reaction (RT-PCR), and the protein expressions of PKM2 in the tumor and paraneoplastic tissues of NSCLC patients were detected with Western blot analysis and immunohistochemical method. The mRNA and protein expression levels of PKM2 in the tumor tissues of different groups were detected with RT-PCR and Western blot assays. Re-sults:The effective rate of radiotherapy for 2 months is 44.8%in NSCLC patients. The expression level of PKM2 is significantly high-er in tumor tissues than in homologous paraneoplastic tissues of NSCLC patients and is negatively correlated with the curative effect of radiotherapy. Conclusion:The expression level of PKM2 is significantly higher in tumor tissues than in the paraneoplastic tissues of NSCLC patients. Patients with lower PKM2 expression level are more sensitive to radiotherapy.