ABSTRACT
Aim To investigate the effect of Sijunzi Decoction on mRNA and protein expression related to growth and cell cycle in polyamine/HuR signaling pathway during small intestinal epithelial cell (IEC-6) proliferation, and to explore its mechanism on intestinal mucosal injury repair. Methods Sijunzi Decoction-containing serum (SJZD) was prepared from SD rats, the expression of HuR protein in cytoplasm and nucleus was analyzed by immunofluorescence and Western blot, the mRNA level of activating transcription factor-2 (A T F - 2), JunD and cyclin dependent kinase 4 (CDK4) were determined by real-time fluorescent quantitative PCR (RT-PCR), Western blot was used to detect protein level of HuR, ATF-2, JunD and CDK4, and flow cytometry was applied to analyse cell cycle distribution. Results Compared with the control group, the mRNA and protein expression of ATF-2 and JunD decreased, while the expression of Cdk4 mRNA and protein increased in SZJD group, and the proportion of G
ABSTRACT
OBJECTIVE： To compare low-polarity volatile constituents in supercritical CO2 extract from the roots and stem of Ilex asprella and its effects on the proliferation of IEC-6 in vitro， and to provide reference for making full use of wild resources of I. asprella and expanding its medicinal parts. METHODS： The low-polarity volatile constituents were extracted from the root and stem of I. asprella with supercritical fluid CO2 extraction（SFE-CO2）. The chemical constituents were analyzed by GC-MS. IEC-6 cells were treated with different concentrations of supercritical CO2 extracts （0， 1， 5， 10， 20， 40， 60， 80， 100 μg/mL） from roots or stems of I. asprella. MTT assay was used to detect the relative viability， and cell proliferation curve was drawn and EC50 of each extract were calculated. RESULTS： Sixty-two and forty-six low-polarity volatile constituents were identified from supercritical CO2 extract in the roots and stem of I. asprella with GC-MS； there were 24 common constituents totally， mainly including pelargonic acid（14.18％ and 6.14％），octanoic acid（10.59％ and 4.35％），hexanoic acid（8.63％ and 10.86％），paeonol（7.79％ and 6.00％），2-methyl-3-phenyl-propanal（6.3％ and 0.58％），acetic acid（1.72％ and 33.77％） in root and stem， respectively. The results of cell culture in vitro showed that when the concentration of supercritical CO2 extract from the roots and stems of I. asprella was lower （≤60 μg/mL）， it could significantly promote the proliferation of IEC-6 cells and their EC50 were 16.35， 20.20 μg/mL， respectively； when the concentration of the extract was higher （≥80 μg/mL）， it showed cytotoxicity and inhibited the proliferation of IEC-6 cells. CONCLUSIONS： There are similar species of volatile constituents in roots and stems of I. asprella and similar in vitro bioactivity of the supercritical CO2 extracts to IEC-6 cells. The short-chain fatty acids may be the active ingredient to promote cell proliferation， while paeonol may be the cytotoxic active ingredient.
ABSTRACT
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).
ABSTRACT
Aim To investigate the protective effect of RTP1,one of the polysaccharide isolated from Rheum tanguticum,on H2O2 induced apoptosis in IEC-6 cells and its possible mechanism.Methods H2O2(100 mmol?L~-1)was used to induce IEC-6 cell apoptosis.Different doses(10,30,100 mg?L~-1)of RTP1 were administrated before H2O2 was added into IEC-6 cell culture.Cell viability was observed by MTT assay.Reactive oxygen species were measured with laser scanning confocal microscopy(LSCM).DNA content and percentage of apoptosis were assayed by DNA agarose gel electrophoresis,acridine orange staining and flow cytometry.The activation of Caspase-3 was detected with Western blot analysis.Results Following treatment with H2O2 for 2 h,H2O2 induced a significant decrease in cell viability,while DNA ladder was observed and apoptosis percentage was as high as 31.3%.Accumulation of intracellular ROS and increase in Caspase-3 activity were also detected.Pretreatment with RTP1 for 24 h exhibited cytoprotective effects in a dose-dependent manner.RTP1 obviously enhanced cell viability,reduced formation of DNA ladder and significantly reduced the number of cells labeled with Annexin V.The percentage of apoptosis/necrosis cells was markedly decreased to 24.4% and 21.5%,respectively.LSCM showed that RTP1 attenuated the accumulation of ROS.The significant decrease in Caspase-3 activity was detected.Conclusion RTP1 has cytoprotective capacity to antagonize H2O2-induced IEC-6 cell apoptosis and injury,and this effect may be related to decrease ROS and inhibit Caspase-3 activity
ABSTRACT
Object To observe the effects of Astragali Injection (Hi) and extracts from Atractylodes macrocephala Koidz. (B1, B4, B9, B13) on the migration of rat small intestinal crypt like cells line (IEC 6). Methods IEC 6 cells were inoculated in 6 well microplates and injured in 72 h, and added with Hi, B1, B4, B9, B13. Then D PBS to negative control in the same amount and EGF to positived control, the cell migration was observed in 24, 48 and 72 h after treatment. Results B4?B9?B13 and EGF significantly promoted the migration of wounded IEC 6 cells (P
ABSTRACT
Objective To study the invasion and proliferation in IEC-6 cells of Toxoplasma gondii RH strain tachyzoites in vitro.Methods T.gondii tachyzoites of RH strain were co-cultured with IEC-6 cells in vitro,the process of cell adhesion,invasion and proliferation by tachyzoites was observed consecutively with inverted microscope.At 5 min,10 min,20 min,30 min,1,2,4,6,12,24 and 48 h after co-culture,the tachyzoite invasion to IEC-6 and intra-cellular proliferation were observed with Giemsa-Wright's staining,respectively.The invasive rate of tachyzoites to IEC-6 was counted.Results T.gondii tachyzoites invaded the IEC-6 cells 5 min after culture,thenceforth the invasive rate increased gradually.The invasive rate was about 55.0% at the first hour after culture with 1-5 tachyzoites in one cell.In the second hour after culture,the rate reached highest with 81.8 % and there were many pseudocysts emerging.At the same time,tachyzoites invaded the cell nucleus and proliferated in the nucleus.At the 4th hour after culture,the invasive rate began to decrease(80.8?9.2)%,the pseudocysts began to break and tachyzoites were released to cluster.The clustering tachyzoites increased significantly at the 6th hour.At the 12th hour the clustering tachyzoites decreased and most tachyzoites were free,the number of complete cells decreased obviously.There were only a few cells and pseudocysts left at the 24th hour,and a great quantity of free tachyzoites existed out of the IEC-6 cells.There were plenty of mobile tachyzoites while none of IEC-6 cells existed after 48 h culture.Conclusion IEC-6 cell may be the suitable target cell of Toxoplasma gondii tachyzoite.The tachyzoites can invade the IEC-6 cells quickly in vitro and proliferate in the plasma and nucleus with a reproductive cycle of about 6 to 12 hrs.