The Mechanism Of Jianpiwan On Hepatoma

Objective: To study the experimental on Hepatoma with Jianpiwan. The aim was to find its possible mechanism, to produce the scientific evidence for further guiding.And to creat the standpoint that the foundation of Hepatoma was spleen asthenia.Methods:1 in vitro:Using the method of pharmacology of the serum with Chinese medicine drugs (PSCD), we prepared Jianpiwan-medicated blood serum. TO observed the suppression of different dose of Jianpiwan-medicated blood serum at 24 hours,48 hours,72 hours on Hepatoma cell line SMMC-7721 by using the colorimentric 3-(4,5-dimethy -lthiazol-2-yl)-2,5biphenyl tetrazolium(MTT).To detected the ratio of apoptosis of Hepatoma cell line SMMC-7721 and the cell cycle at different dose of Jianpiwan-medicated blood serum after 72 hours by using the Flow Cytometry (FCM). And observed the expression of bcl-2,bax gene protein. 2 in vivo: Sixty healthy kunming mouse were transplanted by armpit injection of H22 tumor cells (0.2ml per mice) in asepsis conditions. Then these mice were randomly divided into five groups, twelve mice in each group. The mice in five groups were treated with 5-Fluorouracil (0.2ml per mice intraperitoneal injection), Jianpiwan low dose (0.302g/ml intragastric administration), Jianpiwan middle dose (0.604g/ml intragastric administration), Jianpiwan high dose (1.208g/ml intragastric administration), Sodium Chloride (0.4ml per mice) once per day respectively. Ten days later all mice were killed and the tumor were taken and weighed. Then the ratio of tumor suppression could be calculated.At the same time the spleens were taken and weighed.The spleen index could be calculated.To detected the ratio of apoptosis and the cell cycle of the tumor cells, and observed the expression of bcl-2 and bax gene protein by using the Flow Cytometry (FCM) .To observe the morphous of the tumor, and detected the gene protein expression of bcl-2,bax gene protein by using immunohistochemistry(IH).Results1 the result in vitro1.1 The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum,meddle-medicate blood serum and high-medicate blood serum after 24 hours were 7.28%,11.44%,26.14% respectively, comparing to the negative control group there was significant difference(P<0.05). The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum,meddle-medicate blood serum and high-medicate blood serum after 48 hours were 15.67%,20.90%,33.47%respectively, comparing to the negative control group there was significant difference(P<0.05). The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum,meddil-medicate blood serum and high-medicate blood serum after 72 hours were: 17.55%,25.47%,34.29%respectively, comparing to the negative control group there was significant difference(P<0.05).The growth inhibiting depends on the dose and time. The results show that inside the certain density Jianpiwan can inhibit the growth of the tumor.1.2 The three groups of cells treated with Jianpiwan-medicated blood serum of low dose,middle dose and high dose after 72 hours were analyzed by flow cytometry. The proportion of G0/G1 period were 58.800%,62.600%,67.667% respectively. The proportion of the negative control group was 50.700%.There was significant difference comparing to the negative control group (P<0.05). The proportion of S period were 34.167%,24.767%,19.633%respectivly. The proportion of the negative control group was 35.933%. There was not significant difference comparing to the negative control group(P>0.05).The apoptosis inhibition rate of the three group were 6.985%,7.615%,8.883% respectively. The apoptosis inhibition rate of the negative control group was 5.688%. There was significant difference comparing to the negative control group (P<0.05). And the inhibition rate depends on the dose.1.3 The flourescence index (FI) of bcl-2 gene protein analyzed by flow cytometry(FCM).The results of the three group treated with Jianpiwan low-medicate blood serum,middle- medicate blood serum,high- medicate blood serum were 1.1059±0.0069,1.0779±0.0053,1.0422±0.0431.The result of the negative control group was 1.1814±0.0035.Comparing to the negative control group there was significant difference (P<0.05).And the expression of bcl-2 gene protein degraded by the dose. The flourescence index (FI) of bax gene protein treated with Jianpiwan low-medicate blood serum,middle- medicate blood serum,high- medicate blood serum were 1.086±0.0338,1.2024±0.0112,1.2561±0.0148,The result of the negative control group was 1.0392±0.0208.Comparing to the negative control group there was significant difference (P<0.05).Besides the expression of bax gene protein upgraded by the dose.2 the result in vivo2.1 The inhibition rate of the tumor treated with Jianpiwan low-dose,middle-dose and high-dose were 19.62%,39.71%,48.74% respectively. There was significant difference comparing to the negative control group错误!链接无效。And the inhibition rate depends on the dose. The inhibition rate of the chemo group was 52.76%, but it has not significant difference comparing to the Jianpiwan high-dose (P>0.01).2.2 The spleen index of the Jianpiwan low -dose,middle-dose and high -dose increased obviously. The results were 8.038±0.9318,9.778±0.8659,11.584±1.4685.The result of the chemo group was 5.278±0.4226. Comparing to the group treated with 5- Fluorouracil there was remarkable difference (P<0.01).2.3 The three groups of the tumor treated with Jianpiwan high dose,middle dose and low dose were analyzed by flow cytometry. The proportion of G0/G1 period were 66.667%,76.100%,81.633% respectivly. The propotion of the negative control group was 58.400%.There was significant difference comparing to the negative control group (P<0.05). The apoptosis inhibition rate of the three group were 22.16%,28.42%,37.94%respectivly. The apoptosis of the negative control group was 9.660%. There was significant difference comparing to the negative control group (P<0.05). The apoptosis of the chemo group was 47.71%. The apoptosis of the Jianpiwan high-dose was low than the control group (P<0.05).2.4 The flourescence index (FI) of bcl-2 gene protein analyzed by flow cytometry(FCM).The results of the there group treated with Jianpiwan low-dose,middle- dose,high- dose were 1.2049±0.0077,1.1423±0.0054,1.0859±0.0173.The result of the control group was 1.2454±0.0052. The result of the 5-FU was 1.0420±0.0682.Comparing to the negative control group there was significant difference (P<0.01).And the expression of bcl-2 gene protein degraded by the dose. The flourescence index (FI) of bax gene protein treated with Jianpiwan low-dose,middle- dose,high- dose were 1.1559±0.0121,1.2396±0.0109,1.2989±0.0119,The result of the negative control group was 1.0349±0.0190. The result of the 5-FU was 1.2611±0.0855.Comparing to the negative control group there was significant difference (P<0.05).Besides the expression of bax gene protein upgraded by the dose.2.5 Bcl-2 and bax gene protein were detected by immunohistochemistry (IH).But the results were not analyzied by statistics.According to histomorphology buffy granules appeared in cytoplasm which were treated with Jianpiwan low dose,middle dose and high dose.It indicated that bcl-2 gene protein expressed.And the expression of bcl-2 gene protein was weakening gradually. Buffy granules also appeared in cytoplasm which were treated with Jianpiwan low dose,middle dose and high dose.It indicated that bax gene protein expressed.And the expression of bax gene protein was strengthening gradually.The tendency agreed to the results analyzed by FCM.Conclusion1 Three group treated with Jianpiwan could inhibit the growth of Hepatoma comparing to the negative control group at 24huors,48hours,72hours. The growth inhibiting depends on the dose and time. The results show that inside the certain density Jianpiwan can inhibit the growth of the tumor.2 The propotion of G0/G1 period and the apoptosis ratio were increased treated with Jianpiwan.Comparing to the negative control group there was significant difference.And the apoptosis depends on the dose.The results show that the Hepatoma cells could be inhibit at G0/G1 period.3 And the expression of bcl-2 gene protein degraded by the dose. And the expression of bax gene protein upgraded by the dose.They all had significant difference compared to the negative control group.The results show that Jianpiwan could inhibit the growth of Hepatoma cells.The mechanism may be that it could degrade the expression of bcl-2 gene protein and upgrade the expression of bax gene protein.4 The spleen index treated with Jianpiwan low -dose,middle-dose and high -dose increased obviously. Comparing to the group treated with 5- Fluorouracil there was remarkable difference.It show that Jianpiwan could elevate the immunity of the tumor-bearing mice.5 Jianpiwan could invigorate the spleen.The results show that Jianpiwan could inhibit the growth of Hepatoma.To some extent the standpoint was that the foundation of Hepatoma was spleen asthenia.