The Effect Of Sini Ruangan Recipe On P53 And CyclinD 1 Factors Of Hepatic Stellate Cells

Objective:This research chooses an effective treatment--Sini Ruanggan Prescription,which derives from Dr.Zhao Guorong 's clinical experience as a study object.After taking drugs intervention toward Human hepatic stellate cell,observing the morphological variations of cells and the adjustive transformation of P53 and Cyclin D 1 factors in an attempt to proving the mechanism and effect of this effective treatment in terms of anti-Liver Fibrosis,thus further seeking modern biological theoretical support for the overwhelmingly curative effect of traditional Chinese medicine.Methos:1.Fetching LX-2 cells,adjusting its concentration to 1 × 105 · ml-1,and then inoculating them in 96-hole plates at the ratio of 100 ?l per hole.A positive control group(Fuzheng Huayu tablets),an intervention group(Sini Ruanggan Prescription),and a blank group are set up.Installing 6 complex holes in each group,and enabling the drug to take effect for 24 h.After that,we put these hole plates into a 37-degree carbon dioxide incubator to incubate for 90 min,measure the absorption value(OD value)of each pole by using a microplate reader,and calculate the survival rate of cell.Selecting concentrations of Sini Ruanggan Prescription--1mg/ml,2mg/ml,4mg/ml,under which the survival rate of the intervention group are close to that of the positive control group(Fuzheng dispersing blood stasis Tablet)under a 50% inhibition rate.2.Treating LX-2 cells with the drug for 24 hours and then observing them under an inverted microscope to record changes in cell morphology.3.Taking the logarithmic growth cells,adjusting the cell suspension with cell concentration of 1×105 cells/ml after the trypsin digested,inoculating the cells in a6-well plate with 2 ml of cell suspension per well.The cells are intervened with Sini Ruanggan Prescription with three concentrations of 1 mg/ml,2 mg/ml,4 mg/ml and Fuzheng Huayu tablets with a concentration of 0.3mg/ml.Culturing in a 37-degree carbon dioxide incubator for 24 hours and collecting cells that may have fallen off.After trypsinization again,it is incubated in a 37 ° C carbon dioxide incubator for 1 min.Centrifuging the supernatant and pipette the cells,staining with Annexin V-FITC/PI apoptosis kit: discarding the supernatant of the centrifuged cells,resuspending the cells by adding 500 ?l of Binding Buffer working solution,and adding 5 ?l of Annexin V-FITC and 10 ?l of PI.Gently mixing and incubating for 5min at room temperature in the dark.Apoptosis analysis is performed using a Cellometer K2 dual fluorescent cell analyzer.4.The expression of Cyclin D1 and P53 is detected by immunofluorescence and Western Blot.Results:1.After the intervention of Sini Ruanggan Prescription,the proliferation of the LX-2cells is inhibited.Its survival rate has decreased significantly,which was positively correlated with the concentration.2.Morphological changes: As the medicine's concentration of intervention increases,the LX-2 cells separate,shed,and clump together.3.The apoptosis rate of LX-2 cells increases obviously after 24 hours of intervention.There are positive correlation between the concentration and apoptosis rate.4.There was no evident difference in the expression of Cyclin D1 between the immunofluorescence assay and Western Blout after the detection of relative gray value(P>0.05).The expression of P53 in each group was different and the difference has statistically significant(P<0.05).Compared with the blank group,the expression of support group,2mg/ml group and 4mg/ml group increase apparently.Conclusion:Sini Ruanggan Prescription can inhibit the proliferation of LX-2 cells in vitro and lead to the apoptosis of LX-2 cells;It can distinctly up-regulate the expression of P53 protein.Having known that Sini soft liver and Fuzheng Huayu tablets have little difference in curative effects,so we speculate that they may play a similar role and have the same anti-fibrosis effect.