Polysaccharide extracts of Astragalus membranaceus and Atractylodes macrocephala promote intestinal epithelial cell migration by activating the polyamine-mediated K channel / 中国天然药物

Astragalus membranaceus (Radix Astragali, RA) and Atractylodes macrocephala (Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control media or media containing spermidine (5 μmol·L, SPD), alpha-difluoromethylornithine (2.5 mmol·L, DFMO), 4-Aminopyridine (40 μmol·L, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca ([Ca]) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca] and accelerated migration of IEC-6 cells, compared with the controls (P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca], but also restored IEC-6 cell migration to control level (P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased (P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0 : 14.1 : 0.3 : 19.9 : 181.3 : 6.3 in RA and 1.0 : 4.3 : 0.1 : 5.7 : 2.8 : 2.2 in RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.

The experimental model of intestinal epithelial cell growth established by real time cell analysis / 中国药理学通报

Aim To observe the effect of epidermal growth factor (EGF) on cell growth under different culture conditions using the real-time cell analyzer and EGF as a tool medicine to promote cell growth,and to provide reference for establishing pharmacokinetic model of IEC-6 cell growth (proliferation).Methods IEC-6 cell was inoculated on E-Plate 16 plate at a density of 1 × 104 cell/well and cultured in DMEM with 10％ serum for 24 h,then replaced by serum-free DMEM culture (serum starvation)for 20 h,then the effects of different culture conditions on cell growth as well as EGF efficacy were observed.Results ① When the serum concentration was 10％,the cell growth index of EGF group(1,10,100 μg · L-1) after drug administration 24 h,48 h and 72 h was P ＞ 0.05 compared with the blank group,suggesting that 10％ serum culture could not reflect the efficacy of EGF.② When the serum concentration was 0％,EGF (1,10 μg · L-1) improved cell growth inhibition caused by serum-free cultivation,but could not recover it to normal level (the EGF group after drug administration 24 h,48 h and 72 h was P ＜0.01 compared with 5％ serum),which suggested that serum-free culture could not reflect the EGF efficacy.③ 0％,0.5％,1％ serum had different effects on cell growth,of which 0.5％ serum could neither have obvious inhibition on cell growth,nor reflect the EGF effect due to promoting cell growth for a long time.④When the serum concentration was 0.5 ％,the cell growth index of EGF groups after drug administration 24h,48h and 72h was P ＜0.01 compared with the blank group,suggesting that 0.5％ serum culture could better reflect EGF efficacy.⑤The efficacy of EGF (10 μg· L-1) in promoting cell growth was confirmed by repeated validation of 0.5 ％ serum.Condusions A reference scheme of the IEC-6 cell growth (proliferative) pharmacological experimental model is established in the real-time cell analyzer:cells are cultured in DMEM with 10％ serum for 24h,then in serum-free DMEM (serum starvation) for 20h,then after the adding of reagent,cells are cultured in DMEM with 0.5％ serum for 48-72 h to observe its effect on cell growth (proliferation).

Polysaccharide extracts of Astragalus membranaceus and Atractylodes macrocephala promote intestinal epithelial cell migration by activating the polyamine-mediated K channel / 中国天然药物

Astragalus membranaceus (Radix Astragali, RA) and Atractylodes macrocephala (Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control media or media containing spermidine (5 μmol·L, SPD), alpha-difluoromethylornithine (2.5 mmol·L, DFMO), 4-Aminopyridine (40 μmol·L, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca ([Ca]) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca] and accelerated migration of IEC-6 cells, compared with the controls (P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca], but also restored IEC-6 cell migration to control level (P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased (P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0 : 14.1 : 0.3 : 19.9 : 181.3 : 6.3 in RA and 1.0 : 4.3 : 0.1 : 5.7 : 2.8 : 2.2 in RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.

Effect of flavonoid from Glycyrrhizae Radix on small intestine epithelium IEC-6 cell migration and intracellular polyamine content / 中草药

Objective: To observe the effect of flavonoid from Glycyrrhizae Radix (FGR) on the migration of small intestinal epithelial cells (IEC-6) and intracellular polyamines content. Methods: The cell migration model was constructed by scratch method. Under the intervention of α-difluoromethylornithine (DFMO), the specific inhibitor of polyamine synthesis, or 4-aminopyridine (4-AP), the specific inhibitor of potassium channel, the effect of FGR on cell migration was observed. The intracellular polyamine content being cultured for 12 or 24 h in normal and DFMO intervention condition was detected by precolumn derivatization-HPLC. Results: FGR could reverse the inhibitory effects of DFMO and 4-AP on cell migration, increase the intracellular content of polyamines after 12 or 24 h treatment, and reserve the decrease of polyamine content caused by DFMO. Conclusion: FGR could affect the polyamine-dependent signaling pathway, which may be related to the mechanisms for FGR promoting cell migration.

Study on ribosomal protein gene expression in patients with ulcerative colitis of Pi-asthenic syndrome and Pi-asthenic syndrome / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study differential expression profiles of ribosomal protein (RP) genes in healthy subjects and ulcerative colitis (UC) patients of Pi-asthenic syndrome (PAS) and of dampness-heat syndrome (DHS), thus providing experimental bases for " Pi as the source of qi and blood" theory from the view of protein synthesis.</p><p><b>METHODS</b>RP genes arrays were made. The mucous membrane of colon was detected in four UC patients of PAS (UC-PAS), four UC patients of DHS (UC-DHS), and four healthy subjects (N), and data analyzed using BRB-TOOL Software Package (3.9). Bioinformatics analyses were conducted in differential genes.</p><p><b>RESULTS</b>Low-density RP gene chips were successfully produced, including 77 RP genes and two RP like genes (RPL26-like1 and RPL7-like1). There were twelve differential genes between UC (PAS+DHS) and N, all of which were down-regulated genes. There were nineteen differential genes between UC-DHS and N, all of which showed down-regulating tendency. There were three differential genes between UC-PAS and N, all of which were down-regulated genes. There were six differential genes between UC-PAS and UC-DHS, all of which were up-regulated genes. Cluster analysis showed that normal and UC samples of this chip can be classified according to gene expression profiles, and UC-PAS and UC-DHS can be classified by clustering. Various differential genes had a common transcription regulatory factor.</p><p><b>CONCLUSIONS</b>RP genes arrays were successfully produced. RP gene expressions were down-regulated in UC-PAS and UC-DHS. Corresponding gene expression profiles were shown in N, UC-PAS and UC-DHS.</p>

Effect and mechanism of reserpine for changing salivary protein secretion in Pi-deficient rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of reserpine (RSP) for changing salivary protein secretion in Pi-deficient rats and to explore its possible mechanism.</p><p><b>METHODS</b>Twenty rats allocated in the RSP group were given subcutaneous injection of RSP [0.4 mg/(kg x d)] for 9 successive days, while the other 20 rats in the control group were injected with same volume of saline instead. On the 10th day, ten rats randomly selected from each group were subjected for extracting saliva to detect salivary amylase activity (sAA) before and after an acid stimulation; and drawing blood from the orbital vein to measure the contents of vasoactive intestinal peptide (VIP) and cyclic adenosine monophosphate (cAMP). Then they were sacrificed and their parotids were taken out for pathological examination with HE staining, as well as for VIP and cAMP measuring, and zymogen granules counting under a transmission electron microscope. The remainder animals were stopped injecting and normally fed to 40 days, then subjected to be detected as above-mentioned.</p><p><b>RESULTS</b>Food intake and body weight reduction were more significantly in the RSP group than in the control group. On the 10th day, the ratio of sAA before/after stimulation in the RSP group was 0.39 +/- 0.18, significantly lower than that in the control group (0.80 +/- 0.21, P < 0.01), but it was restored rapidly, reaching the normal range on the 25th day, on the 40th day, it became significantly different to the level on the 10th day (P < 0.05) and approached the level in the control group (P > 0.05). No significant pathological change of parotid was found in both groups; but the number of zymogen granules in the RSP group was remarkably more than that in the control group (41.4 +/- 4.9 vs 34.6 +/- 5.2, P < 0.01). Serum level of VIP in the RSP group was significantly less while that of cAMP was higher than that in the control group (22.5 +/- 13.1 mg/L vs 38.5 +/- 14.1 mg/L, and 125.8 +/- 15.5 micromol/L vs 105.3 +/- 16.7 micromol/L, both P < 0.05), but no inter-group difference was found in parotid tissue contents of both VIP and cAMP. All the indices detected became equivalent in the two groups on the 40th day.</p><p><b>CONCLUSION</b>The reduction of salivary protein in Pi-deficient rats induced by RSP may be related to the regulatory pathway of VIP and cAMP.</p>

Bioinformatics research on chronic superficial gastritis of Pi-deficiency syndrome by gene arrays / 中国结合医学杂志

<p><b>OBJECTIVE</b>To determine the bioinformatical characteristics of differential gene expression in patients with chronic superficial gastritis (CSG) with the Pi-deficiency syndrome (PDS) and those of the non-Pi-deficiency syndrome (non-PDS), i.e. patients of CSG with Pi-Wei dampnese-heat syndrome and healthy persons.</p><p><b>METHODS</b>With the BRB-Array Tools software package, original data collection and bioinformatic: analysis of gene arrays were conducted in 6 CSG patients of PDS (CSG-PDS), 6 CSG patients of non-PDS (CSG-nPDS), and 6 healthy volunteers (Normal).</p><p><b>RESULTS</b>Compared with non-PDS, the gene expressions: in PDS with regards to protein synthesis, energy metabolism, immune reaction and ionic transport tended to be down-regulated, while those concerning secretion, cytoskeleton and ubiquitinization were up-regulated dominantly.</p><p><b>CONCLUSIONS</b>The two kinds of samples, CSG-PDS/Normal and CSG-PDS/CSG-nPDS, have their respective gene expression profiles with different characteristics. Gene expression profile has certain referential significance in syndrome classification.</p>