| ?Objetive?In this study,H22 malignant hepatic ascites model was selected as the study object to observe the effect of Sanwubai Powder on the survival rate,apoptosis,cell cycle,and Bcl-2 protein expression of H22 cells in malignant ascites of mice,and then to observe the effects of Sanwubai The effect of interventional treatment of malignant ascites in mouse hepatoma H22 cells was investigated and its mechanism of action was preliminarily explored..?Methods?1.To build H22 mouse model of ascites tumor: 0.1 mL hepatoma H22 ascites tumor cell suspension(containing 1x106 H22 ascites tumor cells)was injected intraperitoneally into the right lower abdomen of mice,and the general condition and weight gain of the mice were observed.Abdominal enlargement is a successful model when the general condition is significantly worsened,body weight is significantly increased,and the abdomen is significantly enlarged.2.Sanwubai Powder intervened in the study of malignant ascites:60 KM mice were randomly divided into 5 groups after 48 hours after the model was established: the model group,the positive group,the high-dose Sanwubai Powder group,and the three substances.Baisan middle dose group,Sanwubaisan low dose group,12 in each group.The experimental mice started drug intervention 48 hours after the model was established.Seven days after continuous drug intervention,the tumors were weighed after the last administration,and were sacrificed after cervical dislocation.The ascites were taken for measurement of ascites,and the amount of ascitic fluid was measured and H22 was counted by the method of counting with trypan blue.Number of liver cancer cells.The mouse body weight change curve and the ascites formation box plot were plotted to calculate the survival rate of H22 hepatoma cells.3.The effect of Sanwubaisan on malignant ascites was related to apoptosis,cellcycle and bcl-2 protein expression.The ascitic fluid of mice was measured by FCM for cell cycle and apoptosis,and the expression of bcl-2 protein was detected by WB.?Results?1.It is successful that building animal model with a rate of 100%.After the model group was inoculated with H22 cells for 3-4 days,the mouse's abdomen gradually increased.The experimenter gently shakes the mouse's abdomen with his fingers,and the sensory subtle fluctuation can be felt.This indicates that the model group has malignant ascites.Changes in body weight in mice: From the 4th day onwards,there was a significant increase in body weight in mice.However,in the blank group,there was no significant change in the abdomen and the body weight showed a slowly increasing trend.2.Compared with the model group,the survival time of the mice in the positive group and the high dose of Sanwubaisan significantly prolonged,the difference was extremely significant(P <0.01);the survival time of the mice in the medium dose group was prolonged,the difference was significant(P <0.05).The survival time of low-dose mice with Sanwubai Powder was significantly lower than that of the positive group,and the difference was extremely significant(P<0.01).Using the trypan blue dye exclusion method,the number of living tumor cells and the total number of cells in the ascitic fluid of each group of mice was measured to obtain the survival rate of the tumor cells in the ascitic fluid.The ascitic tumor cells in the model group,the positive group,the high-dose Sanbubai Powder group,the middle-dose group,and the low-dose group were(54.49±3.81)%,(31.98±1.62)%,(43.18±3.06)%,respectively.(43.21±1.91)%,(48.73±2.08)%.Compared with the model group,the survival rate of tumor cells in the ascitic fluid of the mice in the positive group,the high-dose Sanbubai Powder group,and the middle-dose group was significantly lower(P<0.01).3.The percentage of ascites H22 cells in the G0/G1 phase of mice in the high-,middle-,and low-dose groups of Sanwubai Powder was(48.76±1.07),(43.11±1.15),(34.84±1.83),which was significantly higher than that of the model group(26.90±1.43),the difference was extremely significant(P<0.01).The percentages of ascites H22 cells in S phase of high-dose Sanwubai Powder groups were(47.33±1.23),which was higher than that of the model group(54.48±2.11).The difference was significant(P<0.05);the percentage of ascites H22 cells in the G2/M phase in the high-,middle-,and low-dose groups of Sanwubai powder was(15.03±0.85),(6.23±0.72),(12.39±1.33).),significantly lower than the model group(18.62 ± 0.71),the difference was extremely significant(P <0.01).The results suggest that the effect of Sanwubai Powder on H22 cells in ascites of mice is mainly to arrest H22 cell cycle in G0/G1 and S phases,thereby reducing the proportion of cells in G2/M phase.Apoptosis:The early apoptotic rates of the model group,the positive group,and the Sanwubai Powder high,middle,and low dose groups were:(0.60±0.14),(13.47±0.77),(1.35±0.09),(1.17±0.12),(0.93±0.05);The late apoptotic rates of the model group,the positive group,and the high-,middle-,and low-dose Sanwubai powder group were(0.66±0.06),(7.11±0.51),(1.95±0.30),(1.87,respectively).±0.32),(1.33 ± 0.39).Early apoptotic rate: Compared with the model group,the positive group had a significant difference(P<0.01),and there was a significant difference in the high-and middle-dose group of Sanwubai Powder(P<0.05).(P>0.05);Compared with the positive group,the high-,middle-,and low-dose groups of Sanwubai powder were all extremely significant(P<0.01).The late apoptotic rate: Compared with the model group,the positive group showed a significant difference(P<0.01),and there was a significant difference between the high-and middle-dose groups of Sanwubai Powder(P<0.05).There was no significant difference in the low-dose Sanwubai Powder group.(P>0.05);Compared with the positive group,the high-,middle-,and low-dose groups of Sanwubai powder were all extremely significant(P<0.01).The relative expression level of Bcl-2 protein: The relative expression level of Bcl-2 protein in ascites H22 cells of the positive group was(0.63±0.02),compared with the model group(2.57±0.08),the difference was extremely significant(P<0.01);The relative expression levels of Bcl-2 protein in mice ascitic fluid H22 cells in Hubai Powder high-and medium-dose group were(2.33±0.08),(2.38±0.05),and the model group(2.57±0.08)had significant differences(P<0.05).There was a significantdifference between the three groups in the dosage of Sanwubai Powder compared with the positive group(P<0.01).?Conclusions?1.Sanwubaisan can effectively interfere with malignant ascites2.Sanwubai Powder intervenes in the malignant ascites of H22 mice,and its mechanism may be that Sanwubai Powder blocks the cell cycle of H22 in the S phase,thereby reducing the mitogenic active G2/M phase cells and further reducing the neoplastic cells.3.Sanwubai Powder has cytotoxic effects on H22 cells and can induce apoptosis of H22 cells.4.The effect of Sanwubai Powder on the apoptosis of H22 cells may be related to the down-regulation of Bcl-2 protein by inhibiting the apoptosis of H22 cells. |
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