| BackgroundFrom the Chinese medicine, we know that the spleen is the source of generating QI and blood, the foundation of acquired constitution. Only the spleen has prosperity function ,our body can active and well, resist the exogenous evil . Such as 《lingshu-wulongjinyebie》 said:"the spleen acting as the protector of the body". The intestinal tract has the most wide areas which contact with the outsides. Many toxin and causative agent like cytokine can enter blood circulation through destructed gut barrier. It can cause the local lesion and lead to the diseases of the whole body. So it has very important sense to investigate the influence of the yiqijianpi Chinese medicine effecting the gut barrier.The gut barrier contain the mechanical barrier, biological barrier,, chemical barrier and immune barrier. The intestinal mechanical barrier are composed of intestinal epithelial cells and the tight junction between them et al. Intestinal epithelial cells are made up by many kinds of cells with different functions, are the main basis of organization structure of the the intestinal mechanical barrier.Between the intestinal epithelial cells there has a special membrane structure — tight junction which local the tip between epithelial cells at the lateral membrane. The tight junction play standing in the breach role to resist the many toxins hand immunogen. It is the important structure in maintaining the membrane permeability. Under the physiological circumstances, only 2μm size of ion or micromolecule can be allowed to transit the tight junction in order to communicate the internal environment and external environment. The tight junction regulate the passage about the ion, water molecule, macromolecule even cancer cell. So the tight junction is closely linked with oedema, diarrhea, blood transfer et al. Any reason make the change of the member of the tight junction , or injury directly the tight junction, it can cause the raise of intestinal permeability to the intestinal contents. Subsequently, thegerms and toxins enter the body, evoke a series of changes of pathological and physiological.Yiqijianpi Chinese medicine are main medicine for curing spleen deficiency. The classics prescription Decoction of Four Mild Drugs , buzhongyiqi tang, huangqijianzhongtang , senlinbaizhu san, baizhu huangqi tang and so on are all consisted mainly by the Atractylodes macrocephala. Astragalus mongholicus, Glycyrrhiza uralensis, Codonopsis pilosula et al. Yiqijianpi Chinese medicine can influence mechanical barrier, biological barrier, chemical barrier and immune barrier. It can be reflect through accelerating epithelium regeneration, getting rid of the toxin, reducing inflammation cytokines, reducing germ shift, regulating alteration of intestinal flora . promoting intestinal secreting SIgA, regulating blood circulation and so on.Modern pharmacology research presume Yiqijianpi Chinese medicine can enhance and regulate function of digestive system through enhancing barrier function, promoting mucosa protection factor secretion. Yiqijianpi Chinese medicine was mainly used to cure the patients with spleen deficiency. However, spleen deficiency syndrome reflect general synthetic pathologic state based on digestive system dysfunction. The patients and animals of spleen deficiency have abnormal appearance about intestinal epithelial cells and the tight junction. So one of the mechanism of action of the yiqi jianpi Chinese medicine treating the spleen deficiency maybe is to protect and recovery the intestinal mucosa.we have purchased the intestinal epithelial cell line(IEC-6) in 2000, an in vitro cell model, used it as the cellular experimental modal to evaluate the effect of Chinese herbs on gastrointestinal mucosal repairation. The result indicate that Astragalus injection (the main component is Astragalus saponin) can promote IEC-6 cell differentiation and migration, and secreting IL-6. The complex polysaccharide AMPS-II from Atractylodes macrocephala can promote IEC-6 cell differentiation through changing the IEC-6 cell morphology, and the extent is positive correlation with the dose and time. At the same times, The complex polysaccharide AMPS-II can promote IEC-6 cell migration through activating orinithine decarboxylase, promote villin protein expression. Glycyrrhiza uralensis total flavonoids can promote IEC-6 cell proliferation significantly.In the recent years,we study the pharmacology of the the complex prescription of Atractylodes macrocephala and Astragalus mongholicus composed by the utility sites of atractylodes macrocephala, Astragalus mongholicus, Glycyrrhiza uralensis.The complex prescription of Atractylodes macrocephala and Astragalus mongholicus origined from ^Suwen bingji qiyibaomingji-juanzhong-xielilun-dishijiu)) of Liuwansu—the one of the four notable doctor in the JinYuan period . In this contribution it was described : baizhu huangqi tang;drunk the prodrug. Although the dysentery has been cured,but it wassuitable for taking this medicine.......baizhu one uncia., Astragalus mongholicus seven qian,Glycyrrhiza uralensis three qian. On the basis of the cell and animal experiment results of our study, a new complex prescription of Atractylodes macrocephala and Astragalus mongholicus was made up by the complex polysaccharide AMPS-II from Atractylodes macrocephala, Astragalus mongholicusinjection -. Glycyrrhiza uralensis total flavonoids through the homogeneous design. Subsequently, the animal experiment results indicated the complex prescription has therapeutical effect on animal ulcerative colitis, it can reduce mucosa inflammatory extent % promote the reparation and reconstruction of the injured mucosa.In this research the purpose is to observe the Caco-2 cell proliferation and expression of ZO-1 protein -. the permeability and tight junction of Caco-2 cell monolayer more carefully in order to find out the effect of yiqi jianpi Chinese medicine on intestinal epithelium barrier, explore potential material basis of "yiqi jianpi" for the yiqi jianpi Chinese medicine. And provide some thinking about looking for the effective regular pattern of "yiqi jianpi" on yiqi jianpi Chinese medicine.Methods and Result1. Observation the growth curve of Caco-2 cell through the MTT methodMethod: Cells were seeded into 96-well plate;The cells were maintained at 37*C in an atmosphere of 5 % CO2 and 95% relative humidity;taking 4 well, using MTT detection. The growth curve was drawn through the lateral axis of time, longitudinal axis of optical density value.Result: The Caco-2 cell display a proliferation state from 1st to 7th day. From 8th, the cell number was descended. It indicated there has density inhibition. So the experiments of cell proliferation, cell differentiation and expression of protein were done suitably from 1st to 7th.2. Observation of the Caco-2 cellmonolayer growingMethod: Caco-2 cells, the colon carcinoma cell line were obtained from Shanghai institute of Biochemistry and Cell Biology. Caco-2 cell were routinely cultured with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum at 37"C with 5% CO2. Cells were sub-cultured with 2ml trypsin (0.25%) for every 3 days.Cells were seeded into polycarbonate membrane at a density of 5><105 cells/cm2.At the same time, the cell were seeded into 6-well plates. Phenol red exclusion was used as a measure of monolayer integrity. The medium in the Transwell insert chamber was 0.15mg/ml phenol red containing culture medium. The Transwell insert was later placed in the lower chamber and diffusion of phenol red was allowed to removed the culture medium in the lower and upper chamber of 500 ul at 1 -. 3-. 4-. 5<* 7-. 9% 13d. A 560 nm were determined . At the same time , the cell junction was observed under the transmission electron microscope.Result: After the cell was culture on the polycarbonate for 3d, the conspicuous microvilli can be seen under the transmission electron microscope. And the content of phenol red in the lower of Transwell Bicameral Chambers was decreased significantly from 3rd to 4th day. After 13d, the conspicuous microvilli and cell junction were seen.. 3.PharmacoIogical effects of the complex prescription of Atractylodes macrocephala andAstragalus mongholicus on Caco-2 cell3.1 Observation the Caco-2 cell proliferation effected by the complex prescription of Atractylodes macrocephala and Astragalus mongholicus through MTTMethod: Cells were seeded into 6-well plates. The cells were maintained at 37*C in an atmosphere of 5 % CO2 and 95% relative humidity .After 48 hours, 2% DMEM 0.1ml and 2%DMEM containing different density(2000 mg/L . 1000 mg/L ? ......)of the complex prescription ofAtractylodes macrocephala and Astragalus mongholicus 0.1ml was added. After 24 hours, assessment of cell proliferation was measured in terms of optical absorbance (OD) per well by a semi-automated tetrazolium-based colorimetric assay using MTT. Briefly, 20uL 5g-L"' MTT solution was added to a different well, further incubated at 37°C for an additional 4h, 120uL supernatants were then removed from the well and 150uL of DMSO was added to each well and the plates were mixed thoroughly for 15min to dissolve the dark blue crystals of formazan. The blank assay (containing culture medium alone with no cells) was subtracted from the other readings. Relative percentage of viability was determined by the OD value at 490nm with Bio-Rad EIA reader.Result : The Toxic Concentration (TC50)of the complex prescription of Atractylodes macrocephala and Astragalus mongholicus on Caco-2 cell according to the OD value is 14680mg/L. The experiment concentration of the complex prescription of Atractylodes macrocephala and Astragalus mongholicus 500 mg/L, 250 mg/L, 125 mg/L, 62.5mg/L, 30.25mg/L were choosed. Under the inverted microscope, the poison cell can not adherence and fall off. It indicated the 125 mg/L, 62.5mg/L, 30.25 mg/L of the complex prescription of Atractylodes macrocephala and Astragalus mongholicus can promote Caco-2 cell proliferation to some extent.3.2 Observation of the Caco-2 cell proliferation effected by the complex prescription of Atractylodes macrocephala and Astragalus mongholicus through 3H methodMethod: Assessment of cell proliferation was measured using 3H-TDR. Briefly;cells were plated on 24-wells plates. After 24 hours, the complex prescription of Atractylodes macrocephala and Astragalus mongholicus with different density(500 mg/L, 250 mg/L, 125 mg/L, 62.5 mg/L, 30.25mg/L) was added in. And then cells were cultured for 12 hours.Tritiated thymidine ([3H]-TDR, 5ulCi/well) was added in. After 12 hours.pulsed cells were harvested by an automated cell harvester and incorporation of 3H-TdR was evaluated by p scintillation counting.Result: The 125 mg/L-. 62.5mg/L, 30.25 mg/L of the complex prescription of Atractylodes macrocephala and Astragalus mongholicus can promote Caco-2 cell proliferation to some extent.3.3 Observation the permeability of the Caco-2 cell monolayer effected by the complex prescription of Atractylodes macrocephala and Astragalus mongholicus through phenol red excreting experimentMethod:Cells were seeded into polycarbonate membrane at a density of 5x105 cells/cm2.At thesame time, the cell were seeded into 6-well microplates. 80% confluent cultures of cells were obtained after 2 days. Phenol red exclusion experiment was used as a measure of monolayer integrity. The medium in the Transwell insert chamber was 0.15mg/ml phenol red containing culture medium and 500 mg/L. 250 mg/L. 125 mg/L. 62.5 mg/L. 30.25mg/L the complex prescription of Atractylodes macrocephala and Astragalus mongholicus. The Transwell insert was later placed in the lower chamber and diffusion of phenol red was allowed to removed the culture medium in the lower chamber of 500 1 at 4. 10> 16. 22. 28 h. A 560 nm were determined.Result: The Caco-2 cell monolayer permeability was raised. At 4, 10. 16. 22. 28 h, the optical density value had been elevated 117.8%. 15.5%. 11.2%. 9.2%. 8.2% respectively. Among these, it was raised obviously after the drug additiomed for 4 hours.3.4 Observation the expression of the ZO-1 effected by the complex prescription of Atractylodes macrocephala and Astragalus mongholicus through immumofluorescenceMethod:cells were plated on cover glass at the concentration of 5><105/ml. 80% confluent cultures of cells were obtained after 2 days, the complex prescription of Atractylodes macrocephala and Astragalus mongholicus with different density (lOOOmg/L , 500 mg/Li 250mg/L, 125 mg/L, 62.5 mg/L , 30.25mg/L) was added. After 24 hours, fixing 15min with 95% alcohol (or cold acetone), blocking with bovine serum albumin .adding the antibody for ZO-1 and the antibody labeled with FITC, mounting, at last, the cover glass was observation with laser confocal microscopy.Result: The fluorescence labeled ZO-1 can be seen near the cytomembrane. The fluorescence of 62.5mg/L group was the most bright. The fluorescence of other group were a little bright compared with the control group. It indicate the 62.5mg/L of the complex prescription of Atractylodes macrocephala and Astragalus can affect the expression of the tight junction associated protein zo-1 on Caco-2 cell.3.5 Observation the expression of the ZO-1 effected by the complex prescription of Atractylodes macrocephala and Astragalus mongholicus through Western BlotMethod: Cells were seeded into 6-well plates. 80% confluent cultures of cells were obtained after 2 days, the complex prescription of Atractylodes macrocephala and Astragalus mongholicus with different density (1000 mg/L, 500 mg/L, 250 mg/L, 125 mg/L, 62.5 mg/L, 30.25mg/L) was added. After 24 hours ,the fluid containing Tris-Hcl (PH 7.5). Nad . EDTA. EGTA . Na3VO4. NP-40. sodium deoxycholate. SDS. Na3N. (PMSF) Aprotinin lysed the cell;The quantitation of the protein with the Bradford method;The electrophoresis was carried out with 8% polyacrylamide gel;After trarsmembrane. immunoblot. visualization and fixation, the result was analyzed by the gel imaging system.Result: From the gray scale on the films, the group of 62.5mg/L has deep color than the control group. It indicate initially the 62.5mg/L of the complex prescription of Atractylodes macrocephala and Astragalus can affect the expression of zo-1 on Caco-2 cell.4. Pharmacological effects of the Atractylodes macrocephala AMPS-II onCaco-2 cell4.1. Observation the permeability and cell junction of the Caco-2 cell monolayer effected by theAtractylodes macrocephala AMPS-II through phenol red excret experimentMethod: Cells were seeded into polycarbonate membrane.At the same time, the cell were seeded into 6-well microplates.. 80% confluent cultures of cells were obtained after 2 days. Phenol red exclusion was used as a measure of monolayer integrity. The medium in the Transwell insert chamber was 0.15mg/ml phenol red containing culture medium and 500mg/L AMPS-II. The Transwell insert was later placed in the lower chamber and diffusion of phenol red was allowed to removed the culture medium in the lower chamber of 500 1 at 37 C for 4, 10> 16^ 22, 28 h. A 560 nm were determined. Caco-2 cells grown in Transwell chambers were examined by transmission electron microscopy using standard procedures. Briefly, cells were fixed overnight with 3% glutaryldehyde followed by postfixation postfixation in 1% OsO4 and embedding in Araldite. Ultrathin sections were then cut from suitable areas, stained with uranyl acetate and lead citrate, and examined in a H-600 electron microscope.Result: The 500mg/L AMPS-II can deseased significantly the Caco-2 cell monolayer permeability(p<0.01). At 4, 10> 16, 22, 28 h, the optical density value had been decreased 40.5% , 50.5%, 47.7%, 49.5%, 53.9% respectively. The structure like the tight junction was observed under the transmission electron microscope.4.2 Observation the expression of the ZO-1 effected by the Atractylodes macrocephala AMPS-II through immumofluorescenceMethod: cells were plated on cover glass. 80% confluent cultures of cells were obtained after 2 days, the 500mg/L AMPS-II was added. After 24 hours, fixing 15min with 95% alcohol (or cold acetone), blocking with bovine serum albumin .adding the antibody for ZO-1 and the antibody labeled with FITC, mounting, at last, the cover glass was observation with laser confocal microscopy.Result: The fluorescence labeled ZO-1 can be seen near the cytomembrane. But the fluorescence of AMPS- II 500mg/L group has no significant difference compared with the control group. It indicate the 500mg/L of AMPS-II can not affect the expression of the tight junction associated protein zo-1 on Caco-2 cell.4.3 Observation the expression of the ZO-1 effected by the Atractylodes macrocephala AMPS-II through Western BlotMethod: Cells were seeded into 6-well plates. 80% confluent cultures of cells were obtained after 2 days, the 500mg/L AMPS-II was added. After 24 hours ,the fluid containing Tris-Hcl (PH 7.5) > Nacl > EDTA, EGTA > Na3VO4, NP-40, sodium deoxycholate, SDS, Na3N, (PMSF) Aprotinin lysed the cell;The quantitation of the protein with the Bradford method;The electrophoresis was carried out with 8% polyacrylamide gel;After trarsmembrane, immunoblot, visualization and fixation, the result wasanalyzed by the gel imaging system.Result: From the gray scale on the films, the 500mg/L AMPS-II has has no significant difference compared with the control group. It indicate the 500mg/L of AMPS- II can not affect the expression of the tight junction associated protein zo-1 on Caco-2 cell.5. Pharmacological effects of the Astragalus mongholicus injection on Caco-2 cell 5.1. Observation the permeability and cell junction of the Caco-2 cell monolayer effected by the Astragalus mongholicus injection through phenol red excret experimentMethod: Cells were seeded intopolycarbonate membrane.At the same time, the cell were seeded into 6-well microplates. 80% confluent cultures of cells were obtained after 2 days. Phenol red exclusion was used as a measure of monolayer integrity. The medium in the Transwell insert chamber was 0.15mg/ml phenol red containing culture medium and 250mg/L Astragalus mongholicus injection. The Transwell insert was later placed in the lower chamber and diffusion of phenol red was allowed to removed the culture medium in the lower chamber of 500 P 1 at 37°C for 4, 10% 16, 22> 28 h. A 560 nm were determined . Caco-2 cells grown in Transwell chambers were examined by transmission electron microscopy using standard procedures. Briefly, cells were fixed overnight with 3% glutaryldehyde followed by postfixation postfixation in 1% OsO4 and embedding in Araldite. Ultrathin sections were then cut from suitable areas, stained with uranyl acetate and lead citrate, and examined in a H-600 electron microscope.Result: The 250mg/L Astragalus mongholicus injection can deseased significantly the Caco-2 cell monolayer permeability(p<0.01). At 4,10> 16,22-.28 h, the optical density value had been decreased 92.0 % > 69.5 %, 43.6 %, 49.5 %, 53.9 % respectively. The desmosome and microvilli was observed under the transmission electron microscope.5.2 Observation the expression of the ZO-1 effected by the Astragalus mongholicus injection through immumofluorescenceMethod: cells were plated on cover glass. 80% confluent cultures of cells were obtained after 2 days, the 250mg/L the Astragalus mongholicus injection was added. After 24 hours, fixing 15min with 95% alcohol (or cold acetone), blocking with bovine serum albumin .adding the antibody for ZO-1 and the antibody labeled with FITC, mounting, at last , the cover glass was observation with laser confocal microscopy.Result:The fluorescence labeled ZO-1 can be seen near the cytomembrane. But the fluorescence of Astragalus mongholicus injection 250mg/L group has no significant difference compared with the control group. It indicate the 250mg/L of Astragalus mongholicus injection can not affect the expression of the tight junction associated protein zo-1 on Caco-2 cell.5.3 Observation the expression of the ZO-1 effected by the Astragalus mongholicus injection through Western BlotMethod: Cells were seeded into 6-well microplates. 80% confluent cultures of cells were obtained after 2 days, the 250mg/L Astragalus mongholicus injection was added. After 24 hours ,the fluid containing Tris-Hcl (pH 7.5^ Nacl ^ EDTA, EGTA , Na3VO4, NP-4(h sodium deoxycholate, SDS. Na3N> (PMSF) Aprotinin lysed the cell;The quantitation of the protein with the Bradford method;The electrophoresis was carried out with 8% polyacrylamide gel;After trarsmembrane> immunoblot% visualization and fixation, the result was analyzed by the gel imaging system.Result: From the gray scale on the films, the 250mg/L Astragalus mongholicus injection has has no significant difference compared with the control group. It indicate the 250mg/L of Astragalus mongholicus injection can not affect the expression of the tight junction associated protein zo-1 on Caco-2 cell.ConclusionsThe complex prescription of Atractylodes macrocephala and Astragalus mongholicus has the therapia effect for ulcerative colitis of animal, such as relieving the extent of inflammation > promoting reparation of injured mucosa. In this investigation, the findings indicated The complex prescription of Atractylodes macrocephala and Astragalus mongholicus has a trend to promote the Caco-2 cell proliferation and the expression of the ZO-1 protein. Maybe these are the effective target and material basis for treating ulcerative colitis of The complex prescription of Atractylodes macrocephala and Astragalus mongholicus.At the same time, the ministerial drug Atractylodes macrocephala-, the ministerial drug Astragalus mongholicus of The complex prescription of Atractylodes macrocephala and Astragalus mongholicus have been investigated. The result manifested the Atractylodes macrocephala AMPS— II > Astragalus mongholicus injection (the main component is Astragalus mongholicus saponin) can promote the formation of tight junction of the Caco-2 cell monolayer.In the future.the effective target of the The complex prescription of Atractylodes macrocephala and Astragalus mongholicus should be studied deeply.The compatibility research of the Atractylodes macrocephala AMPS—IK Astragalus mongholicus saponhu Glycyrrhiza uralensis flavone or the Glycyrrhiza uralensis flavone itsel should be investigated.
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