RESUMO
Objective:To observe the effect of Jianpi Xiaoai prescription on long non-coding RNA Hox transcript antisense intergenic RNA （lncRNA HOTAIR）/Janus kinase 2 （JAK2） /signal transducer and activator of transcription 3 （STAT3） signaling pathway and to explore the potential mechanism of Jianpi Xiaoai prescription in suppressing the metastasis of colon cancer. Method:The expression of lncRNA HOTAIR in different cells was analyzed. Following the treatment of HCT116 cells with 10%，15%，and 20% Jianpi Xiaoai prescription -containing serum， the invasive ability of Jianpi Xiaoai prescription on HCT116 cells was assessed by transwell assay. The mRNA expression levels of lncRNA HOTAIR，JAK2，and STAT3 were measured by Real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of JAK2， phosphorylated STAT3 （p-STAT3） and STAT3 by Western blot. Result:The highest expression of lncRNA HOTAIR was detected in HCT116 cells. Compared with the blank group， each Jianpi Xiaoai prescription group exhibited a decreased number of invasive cells （<italic>P</italic><0.05， <italic>P</italic><0.01）. The relative JAK2 mRNA expression in the middle-dose Jianpi Xiaoai prescription group was down-regulated （<italic>P</italic><0.05）， and the relative lncRNA HOTAIR mRNA expression in the middle- and high-dose Jianpi Xiaoai prescription groups and the relative JAK2 mRNA expression in the high-dose Jianpi Xiaoai prescription group were remarkably down-regulated （<italic>P</italic><0.01）. Compared with the blank group，the relative p-STAT3 protein expression was down-regulated in the middle-dose Jianpi Xiaoai prescription group （<italic>P</italic><0.05）， and the relative JAK2 protein expression in the middle- and high-dose Jianpi Xiaoai prescription groups and the relative p-STAT3 protein expression in the high-dose Jianpi Xiaoai prescription group were remarkably down-regulated （<italic>P</italic><0.01）. Conclusion:Jianpi Xiaoai prescription effectively inhibits the metastasis of colon cancer cells， which may be related to the inhibition of lncRNA HOTAIR/JAK2/STAT3 signaling pathway.
RESUMO
Objective:To observe the effect of Jianpi Xiaoai prescription on the activation of normal human embryonic lung fibroblasts （HFL1） into tumor-associated fibroblasts （CAFs） induced by human colon cancer cells （HCT116） derived exosomes. Method:SD rats were gavaged with 13.1 g·kg-1 of Jianpi Xiaoai prescription to prepare drug-containing serum， and HCT116 cell exosomes-containing 10% exosomes-free serum and 20% Jianpi Xiaoai prescription drug serum were isolated by ultra-high speed centrifugation. The particle size distribution of exosomes were detected by Nanoparticle tracking analyzer （Zetaview）， and the exosomes' marker proteins apoptotic transfer gene 2 interaction protein X （Alix）， heat shock protein 70 （HSP70）， and tumor-susceptibility gene 101 （TSG101） were identified by Western blot， and the uptake of exosomes labeled with cell membrane staining kit （PKH67） by HFL1 was observed by fluorescence microscope. HFL1 cells were divided into six groups： the blank group， the transforming growth factor-β1 （TGF-β1） group， the TGF-β1 combined with HCT116 exosomes of 2 mg·L-1 group， the TGF-β1 combined with HCT116 exosomes of 4 mg·L-1 group， the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 2 mg·L-1 group， and the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 4 mg·L-1 group， and all groups were cultivated for 48 h. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to determine the protein and mRNA expressions of α-smooth muscle actin （α-SMA）. Result:The particle size distribution detected by Zetaview was mainly between 50-100 nm， and the exosomes were verified based on the expressions of marker proteins Alix， HSP70 and TSG101. After co-incubation of HFL1 cells with exosomes， a large number of exosomes were absorbed by HFL1 cells under fluorescence microscope. Compared with the blank control group， the protein and mRNA expressions of α-SMA in the TGF-β1 group and TGF-β1 combined with HCT116 exosome groups were increased （P<0.01）. Compared with the TGF-β1 combined with HCT116 exosome groups， the protein and mRNA expressions of α-SMA were decreased in the TGF-β1 combined with Jianpi Xiaoai prescription exosome groups （P<0.01）. Conclusion:Human colon cancer cell exosomes combined with TGF-β1 can induce the activation of HFL1 into CAFs， and Jianpi Xiaoai prescription can reduce the activation of HFL1 by affecting the expressions of α-SMA， thus antagonizing the lung metastasis of colon cancer.
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Objective:To observe the effect of polyphyllin Ⅰ on the expressions of forkhead box Q1（FOXQ1）and epithelial-mesenchymal transition (EMT)-related factors, in order to explore the possible mechanism of polyphyllin Ⅰ in inhibiting the metastasis of colon cancer. Method:After the treatment with 1.25，2.50 μmol·L-1 polyphyllin Ⅰ on HCT116 cells, Western blot and Real-time PCR were used to detect the expressions of FOXQ1，E-cadherin，Vimentin protein and mRNA. Result:Compared with the blank group, relative expressions of FOXQ1 protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased, while relative expression of E-cadherin mRNA was increased, the differences were not statistically significant, and relative expressions of Vimentin protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased (P<0.05)， and relative expression of E-cadherin protein in low-concentration polyphyllin Ⅰ group was increased (P<0.01). Compared with the blank group, relative expressions of FOXQ1, Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased，while relative expressions of E-cadherin protein and mRNA were increased (P<0.05, P<0.01). Compared with low-concentration polyphyllin Ⅰ group, relative expressions of Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased, but the difference was not statistically significant, relative expressions of E-cadherin protein and mRNA in high-concentration polyphyllin Ⅰ group were increased, whereas relative expressions of FOXQ1 protein and mRNA were decreased (P<0.05, P<0.01). Conclusion:Mechanism of polyphyllin Ⅰ inhibiting the metastasis of colon cancer may be related to the decrease of FOXQ1 and Vimentin expressions, and the up-regulation of E-cadherin.
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<p><b>OBJECTIVE</b>To develop an HPLC method for determination of the four effective components (genistin; rutin; quercetin; genistein) in Huaijiao pill.</p><p><b>METHOD</b>The chromatographic separation was performed on a Shim-packODS (4.6 mm x 150 mm, 5.0 microm) eluted with a mobile phase of MeOH-H2O-HAc (40:60:0.25). The detection wavelength was set at 256 nm and column temperature was set at 30 degrees C .</p><p><b>RESULT</b>Nice linear relation between the peak area and injected amount exists when the amount is within 0.059-0.352 microg for genistin, within 0.435-2.610 microg for rutin, within 0.020-0.121 microg for quercetin and within 0.053-0.319 microg for genistein. The correlation coefficient of each component is 0.999 6, 0.998 2, 0.998 9 and 0.999 9 respectively. The average recoveries of the four components are from 98.7% to 100.2%. The RSD of each group are 1.21%, 1.36%, 0.47% and 1.54% (n = 5).</p><p><b>CONCLUSION</b>The method was accurate, repeatable and suitable to determine four effective components in Huaijiao pill. It can be use for quality control of Huaijiao pill.</p>