| Objective: To investigate the effect of Yanghe decoction on anti-tumor activity and mechanism of human lung carcinoma cell line (GLC-82) by serologic pharmacological method and provide theory basis for Yanghe decoction to popularize and apply in clinic practice. It also provides the theory basis for warm-method treating tumor of traditional Chinese medicine.Methods: According to the basic Traditional Chinese Medicine theory, the pathologic characteristic of lung carcinoma is stagnation of phlegm and turbid qi. The human lung carcinoma GLC-82 cell line was adopted as target cell. The Yanghe decoction is divided into two parts: the hot-natured part of Yanghe decoction and the non-hot-natured part of the Yanghe decoction . After incubating GLC-82 cell and Chinese drug borne rat serum at different dose with serologic pharmacological method. It was divided into 10 groups: low dose serum of the Yanghe decoction, middle dose serum of the Yanghe decoction, high dose serum of the Yanghe decoction, low dose serum of the hot-natured part of Yanghe decoction, middle dose serum of the hot natured part of Yanghe decoction, high dose serum of the hot-natured part Yanghe decoction, low dose serum of the non-hot-natured part of Yanghe decoction, middle dose serum of the non-hot-natured part of Yanghe decoction, high dose serum of the non-hot-natured part of Yanghe decoction, the control serum given saline solution. MTT assay is used to calculate the inhibitory rate. The apoptosis morphological changes were observed under the inverted microscope, light microscope. The target cells were harvested to analyze the cycles and apoptosis using flow cytometry (FCM). The protein expression of bcl-2 was checked through imunocytochemistry by S-P method.Results:1 MTT assay shows both the Yanghe decoction drug-bearing serums and the hot–natured part of the Yanghe decoction drug-bearing serums can inhibit the growth of GLC-82 cell significantly. When it was 48 hours and 72 hours, there was remarkable difference in the inhibitory rate between 10% high dose Yanghe decoction drug-bearing serum(the inhibition rate of 24h,48h,72h were -1.1%,45.2%,74.6%) and the control serum(P<0.01); there was also remarkable difference in the inhibitory rate between 10% high dose hot–natured part of the Yanghe decoction drug-bearing serums(the inhibition rate of 24h,48h,72h were -0.4%,36.4%,59.8%) and the control serum(P<0.01).A time-dependendent proliferation inhibition was demonstrated in GLC-82 cell in the two groups. Meanwhile, the optical density became smaller as the time went. There was remarkable difference in the inhibitory rate between 10% high dose drug-bearing serum of the non-hot-natured part of Yanghe decoction and the control serum (P<0.01) when it was only 72 hours. There was also a time-dependent proliferation inhibition in this group(the inhibition rate of 24h,48h,72h were-1.8%,0.8%,9.3%), but the optical density did not become smaller as the time went. The inhibitory rate of the high dose drug-bearing serums of Yanghe decoction is larger than both that of high dose drug-bearing serums of hot–natured part of the Yanghe decoction and high dose drug-bearing serums of the non-hot-natured part of Yanghe decoction when it was 48 hours or 72 hours(P<0.01). The inhibitory rate of high dose drug-bearing serums of hot–natured part of the Yanghe decoction was larger than that of high dose drug-bearing serums of the non-hot-natured part of Yanghe decoction at equal time(P<0.01). 2 When it was 48 hours cell cycle analysis of GLC-82 cell showed that there was significant difference in S phase between 10% low, middle, high dose drug-bearing serum of Yanghe decoction and the control serum by flow cytometric (P<0.01). There was also significant difference in G0/G1 phase between the middle, high dose drug-bearing serum of the Yanghe decoction and the control serum(P<0.01).There was significant difference in S phase between 10% low, middle, high dose drug-bearing serum of hot–natured part of the Yanghe decoction and the control serum(P<0.01). There was also significant in G0/G1 phase difference between 10% low, middle, high dose drug-bearing serum of the hot-natured part of the Yanghe decoction and the control serum(P<0.01). There was significant difference in S phase between 10% high dose drug-bearing serums of the non-hot-natured part of Yanghe decoction and the control serum (P<0.01). There was also significant in G0/G1 phase difference between 10% middle, high dose drug-bearing serums of the non-hot-natured part of the Yanghe decoction and the control serum(P<0.01). The proportion of S phase of middle dose drug-bearing serum of Yanghe decoction was larger than both that of middle dose drug-bearing serums of the non-hot-natured part of Yanghe decoction and that of middle dose drug-bearing serum of hot–natured part of the Yanghe decoction in equal condition (P<0.01). The proportion of G0/G1 phase of high dose drug-bearing serum of Yanghe decoction was larger than that of high dose drug-bearing serums of the non-hot-natured part of Yanghe decoction and that of high dose drug-bearing serum of hot–natured part of the Yanghe decoction (P<0.01). 3 When it was 48 hours there was significant difference between the apoptotic percentage of 10% middle, high dose drug-bearing serum of Yanghe decoction(the apoptotic were 24.64% and 28.76%) and the control serum(P<0.01). There was also significant difference between the apoptotic percentage of 10% middle, high dose drug-bearing serum of hot–natured part of the Yanghe decoction (the apoptotic were 18.57% and 23.6%) and the control serum(P<0.01). There was no significant difference between the apoptotic percentage of 10% other traditional Chinese medicine serum group and the control serum(P>0.05). There was also significant difference between the apoptotic percentage of 10% middle, high dose drug-bearing serum of hot–natured part of the Yanghe decoction and that of 10% middle, high dose drug-bearing serum of Yanghe decoction in equal condition (P<0.05). 4 With the inverted microscope,after being treated with drug-bearing serum of hot–natured part of the Yanghe decoction and Yanghe decoction, GLC-82 cell grew against the wall and then peeled off gradually, which showed the characters of cells apoptosis like chromatin condensation. With HE stain and light microscope, after being treated with drug-bearing serum of hot–natured part of the Yanghe decoction and Yanghe decoction, GLC-82 cell showed the typical apoptosis morphological changes, such as shrinkage, crescent shape concentrated chromatin and fragmentation. 5 Imunocytochemistry assay shows that the protein expression of bcl-2 was strong positive after given low, middle, high dose drug-bearing serum of the non-hot-natured part of Yanghe decoction or control serum; the protein expression of bcl-2 decreased and became weakly positive after given low, middle, high dose drug-bearing serum of the hot–natured part of the Yanghe decoction; the protein expression of bcl-2 decreased and became negative after given of low, middle, high dose drug-bearing serum of Yanghe decoction。Conclusions: 1 The Yanghe decoction could be effective in lung carcinoma which was dependent on both time and dose. The anticancer mechanisms of the both may be its effect on inducing the S phase arrest and apoptosis of Glc-82 cells. Decreasing the protein expression of bcl-2 is one of the mechanisms of the apoptosis of GLC-82 after given the Yanghe decoction. 2 The hot-natured part of the Yanghe decoction could be effective in lung carcinoma which was dependent on both time and dose. The anticancer mechanisms of the both may be its effect on inducing the S phase arrest and apoptosis of Glc-82 cells. Decreasing the protein expression of bcl-2 is one of the mechanisms of the apoptosis of GLC-82 after given the hot-natured of the Yanghe decoction. 3 Only high dose drug-bearing serum of the non-hot-part of the Yanghe decoction could be effective in lung carcinoma by inducing the S phase arrest and apoptosis of Glc-82 cells. the non-hot-part of the Yanghe decoction can not decrease the protein expression of bcl-2. 4 In inhibitory rate, the protein expression of bcl-2, inducing both the S phase arrest and apoptosis, the antineoplastic effect of Yanghe decoction is greater than both the hot–natured part of the Yanghe decoction and the non-hot-natured part of Yanghe decoction; meanwhile the antineoplastic effectiveness of the hot–natured part of the Yanghe decoction is greater than the non-hot-natured part of Yanghe decoction. So it can be concluded that the hot–natured part of the Yanghe decoction may play a significant role in the Yanghe decoction prescription; non-hot-natured part of Yanghe decoction plays a same or synergistic role in the Yanghe decoction prescription. Meanwhile, it shows the feasibility of warm-method treating tumor and support the view that cancer belongs to cold syndrome.
|
PDF Full Text Request