Effect of total polysaccharides of Sijunzi decoction on the proliferation of intestinal epithelial cell line IEC-6 of rats / 中国组织工程研究

ABSTRACT

BACKGROUND: In a series of recent studies it was demonstrated thatpolysaccharides play important roles in many physiologic and pathologicprocessions, such as infection, inflammation, inter-cell adherence and sig nal conduction, immune identification, cell proliferation and differentiation, as well as maintenance of cell structure and function. But the protectiveeffect of plant polysaccharides on gastrointestinal mucosa needs further re search. OBJECTIVE: To observe the effect of the total polysaccharides of SijunziDecoction (SJZD) (TPSJ) in different concentrations on the proliferation ofrat intestinal epithelial cell line IEC-6. DESIGN: Observational controlled trial. SETTING: Central Laboratory, Guangdong Hospital of Traditional ChineseMedicine. MATERIALS ①Cell line The IEC-6 of normal rats (Catalog No. RL 1592) was purchased from American Type Culture Collection (ATCC). IEC6 cells were originated mainly from intestinal crypt cells. ②Reagents anddrugs DMEM medium, bovine insulin, gentamicin, fetal bovine serum (FBS) and DPBS were purchased from GIBCO Ltd. Cell proliferation kit(MTT) was purchased from Roche Ltd. Indomethacin was purchased fromSigma Company. SJZD was composed of Dangshen (Codonopsis pilosula),Baizhu (Atractylodes macrocephala), Fuling (Poria cocos) and Gancao (Glycyrrhizae uralensis), and these four drugs were in same ratio as Pharmacopoeia. The four herbs were boiled in water, extracted twice for 8 hours.Extract was combined, decompressed, concentrated, centrifugated with high speed to take out insoluble substance, put in glass paper to receive reverse lotic water dialysis for 2 hours. The final decoction was concentrated by heating followed by extraction with 80% ethanol. After overnight precipitation at room temperature and combination of sedimen, the total polysaccharide was obtained by deproteinating with the Sevag method.METHODS: ①The IEC-6 cell line was maintained in T-150 flasks with DMEM culture solution, and then put in CO2 incubator at 37 ℃, at saturated humidity, cultured at 0.05 volume fraction CO2, after being taken out from dry ice and defrosted rapidly in water-bath at 37 ℃. Flasks were incubated at 37 ℃ in 5% CO2· Stock cells were subcultured at a dilution of17 every 5-7 days and the medium was changed once every 2 days. The cells in passage 15-20 were used for testing. ②IEC-6 cells were inoculated at a density of 1×l04 cells/well in 96-well plates. Cultured were supplemented with TPSJ in a final concentrations ranging from 50, 100 and 200 mg/L after 6 hours, which was 3 TPSJ groups. One plate would be taken out for the examination of cell proliferation using MTT assay everyday. The cells that not administrated by any intervention were used as normal control group and cell proliferation was assayed using MTF at corresponding time points. ③IEC-6 cells were inoculated at a density of1×104 cells/well in 96-well plates, and then cultured in the DMEM supplemented with no serum from the following day for 24 hours. For the examiation of mucosal restitution, indomethacin at concentration of 40 mmol/L was employed to induce IEC-6 cells injured, which was indomethacin group. The three concentration of TPSJ was 50, 100 and 200 mg/L, respectively, which was 50,100,200 mg/L TPSJ groups. After drug action for 20 hours, the proliferation of cells was measured using MTT according to the manufacturer's instructions. IEC-cells without any intervention were used in the normal control group. Cell proliferation was determined with TT method at corresponding time points.MAIN OUTCOME MEASURES: MTT assay was used to examine the effects of TPSJ on IEC-6 cell proliferation in different times. MTT assay was used to detect the effect of TPSJ on IEC-6 cell proliferation inhibited by indomethacin.RESULTS: TPSJ could accelerate IEC-6 cells growth at different doses and in different time. After the cells were treated by 40 mmol/L indomethacin for 24 hours, the absorbance (A) of IEC-6 cells apparently declined compared with that in the normal control group (0.17±0.02,0.31±0.03; P ＜ 0.01). The A of IEC-6 cells treated by TPSJ in 100 mg/L group was apparently higher compared with indomechacin group (0.25±0.04, 0.17±0.02; P ＜ 0.01). The A of IEC-6 cells treated by TPSJ did not restored to the normal level, but there was no insignificant difference compared with normal group (P ＞ 0.05).CONCLUSION: TPSJ can accelerate the proliferation of IEC-6 cells. TPSJ can exert regulatory function both in intestinal mucosa absorption and immunity by affecting intestinal epithelial cells.