Research On Action Mechanism Of The Method Of Tonifying Qi And Detoxication On Rat Hepatic Stem Cell Line WB-F344

objectivesStudy the effects of NHS (Bezoar, American ginseng) drug serum with the function of tonifying qi and detoxication on rat hepatic stem cell line WB-F344proliferation, apoptosis and expression of cell factor and its receptor in the process of differentiation, to explore the mechanism of the method of tonifying qi and detoxication on hepatic stem cell,and explain the function principle of the method of tonifying qi and detoxication in treating chronic liver from the level of molecular biology,which of fered new ideas for the further study on the treating methods and also provided theoretical and experimental basises for clinical application.Methods1. Preparation of drug serum:In vitro cultivated calculus bovis, American ginseng compatibility group with traditional decoction containing serum gastric perfusion of animal production (referred to the decoction group); on the basis of modern pharmacology research, extraction of ginseng with anti fatigue effects of American ginseng saponins, total saponins, extraction of cultured calculus bovis with protecting liver cells, restoring liver function and lidan autigo role of bilirubin, cholic acid and deoxycholic acid,which were made NHS capsules, after the capsules’ powder taken dilution using gavage animal production of medicated serum (referred to the component group). The decoction and component groups in the experiment were control groups. The rats were divided into the blank control group, the decoction group and the component group, with16rats in each group. Each group given the corresponding drugs orally for7days (1/d), the concentration:containing bezoar3.6g/L and American ginseng21.6g/L, according to the ammount of1mL every rat weight100g, and the blank group given an equal volume of saline, all for a week. Drawing blood from the rats’carotid arteries after2hours of the last administration, placing them at room temperature,3000rpm, centrifugal15min, and collecting the serum, inactivated30min at56℃water bath, then filtrated and sterilized in the super-clean bench with injection filter (0.22um microporous membrane filtration), saved-20℃in many vials,were made the necessary concentration of serum in the culture liquid when needed.2. Culture of WB-F344cells:Using50mL culture bottles, WB-F344cells were vaccinated in the high glucose medium DMEM containing10%FBS,2mmol/L glutamine,100U/mL penicillin and streptomycin, whose density were2×104/mL, and cultured in5%CO2,37℃culture box. Cell culture fluid renewed one time for2-3days,and cells were passed from genengration to genenration one time for3-5days,1:2after digested by0.25%trypsin.3.Effects of the method of tonifying qi and detoxication on WB-F344proliferation:Logarithmic growth phase cells digested by trypsin, whose density were1×105/mL, vaccinated in96cell culture plates, cultured for24hours under the condition of37℃、5%CO2, were added5%,10%,20%drug serums of the component and decoction groups, the blank control group with the same concentration gradient in the normal rat serum, the normal control group added equal amount of culture medium, the normal culture medium. After every group serum had effected the cells for24hours, MTT method was used to detect the cell proliferation, taking the best proliferation of drug serum concentration, all experiments follwed made this concentration.4. Effects of the method of tonifying qi and detoxication on the expression of WB-F344HGFR, EGF, EGFR, TGF β1R and TGF β2R:All serums were divided into the normal control group (10%FBS), the blank control group (10%normal rat serum), the component group(10%component serum), the decoction group (10%decoction serum). After the serums had effected on the cells for24hours, lysing them, extracting the cells’total proteins, Western blotting was used to detect the expression of WB-F344HGFR, EGF, EGFR, TGF β1R and TGF β2R.5. Effects of the method of tonifying qi and detoxication on the expression of WB-F344AFPmRNA and ALBmRNA:Cells taken by5x103in each hole, inoculated in the cell culture plates, and cultured by the differentiation system(10%FBS, HGF50ng/mL, EGF20ng/mL,high glucose DMEM, Insulin lg/mL, Dexl μmol/L) for8hours, were added10%drug serums of the component and decoction groups, with normal and blank serum control holes. In0,7,10,14,21days the cultured cells were collected, then total RNA extracted. RT-PCR method was used to detect the mRNA expression of WB-F344hepatic stem cell surface marker AFP and liver cell surface marker ALB, and the morphological changes of the cells were observed after stimulated by the drug serum.6. Effects of the method of tonifying qi and detoxication on WB-F344apoptosis:When the density a justed to1×106/mL, the cells were placed in the culture plates for6hours at37℃, and added40ng/mL TGF-β after adhered. All serums were still divided into the normal control group (10%FBS), the blank control group (10%normal rat serum), the component group (10%component serum)and the decoction group (10%decoction serum). After the serums had effected the cells for24hours, Annexinv-FITC and PI staining,the flow cytometry was used to deter and analyze datas.Results1.Effects of the method of tonifying qi and detoxication on WB-F344proliferation:10%,20%of NHS drug serum of the component and decoction groups could promote WB-F344cell proliferation, compared to the normal and blank control groups, OD value increased significantly (P<0.01).10%was the optimal concentration gradient of cell proliferation. Each group serum showed no toxic effect on the cells,which growed well under microscope.2. Effects of the method of tonifying qi and detoxication on the expression of WB-F344HGFR, EGF, EGFR, TGF β1R and TGF β2R:After intervened by10%NHS drug serum of the component and decoction groups, the expression of WB-F344HGFR, EGF, EGFR, TGFβ1R and TGF β2R were significantly increased, compared to the blank and normal control groups(P<0.01); and partial indexes ofthe component group better than the decoction group (P<0.05).3. Effects of the method of tonifying qi and detoxication on the expression of WB-F344AFPmRNA and ALBmRNA:After intervened by10%NHS drug serum of the component and decoction groups, the mRNA expression of WB-F344hepatic stem cell surface markers AFP gradual ly decreased (P<0.01), while liver cell surface markers ALB increased gradual ly along with the t ime (P<0.01); and the component group better than the decoction group (P<0.05).4.Effects of the method of tonifying qi and detoxication on WB-F344apoptosis:After intervened by10%NHS drug serum of the component and decoction groups, WB-F344cell apoptosis rate was significantly decreased compared to the blank and normal control groups(P<0.01); and the component group better than the decoction group (P<0.05).Conelusions1.The method of tonifying qi and detoxication could promote liver stem cell WB-F344proliferation, no obvious toxicity on the cells.2. The method of tonifying qi and detoxication could promote the upregulation of the expression of WB-F344HGFR, EGF, EGFR, TGF β1R and TGFβ2R of liver stem cell WB-F344.3. The method of tonifying qi and detoxication could induce the differentiation of hepatic stem cell to liver cell characteristic and direction through cytokine systems.4. The method of tonifying qi and detoxication could ihbit apoptosis of hepatic stem cells WB-F344.5.The method of tonifying qi and detoxication selected by the chinese medicine compound with different dosages, effets are also different, NHS capsule better than NHS decoction in the experiment.