| OBJETIVE: To study the state of Thl/Th2 in human lung cancer cells and Peripheral blood mononuclear cell (PBMC) from lung cancer patients before and after the culture with Astragalus(AG) and Tetramethylpyrazine(TMPz), as well as to observe the variation of Thl/Th2 cytokines expression in PBMC from lung cancer patients after administration of AG and TMPz, investigating the effect of AG and TMPz on lung cancer.METHODS: There were three parts in this study: human lung cancer cells, PBMC from lung cancer patients (in vitro), and PBMC from lung cancer patients (in vivo). 1. human lung cancer cells : The experiment system included squamous cell carcinoma, adenocarcinoma and small cell lung cancer(SCLC). Each kind of these cells was divided into pre-control group, control group, AG group, TMPz group and AG+TMPz group. In the later three groups, there were three concentrations of medicines including AG group (5%, 10%, 20%), TMPz group (lug/ml, 100μg/ml, 1000μg/ml) and AG+TMPz group (a correlative combination of the former three concentrations respectively). After being cultured for 48h, cells were collected in the later four groups. The expression of Th1/Th2 cytokines was detected by reverse transcription polymerase chain reaction(RT-PCR) considering IFN- γ and IL-2 as Th1 cytokines, IL-4, IL-6 and IL-10 as Th2 cytokines. 2. PBMC from lung cancer patients (in vitro): PBMC from 23 lung cancer patients and 19 healthy controls were isolated with Ficoll-hypaque gradient method, which were divided into the following five groups: pre-culture group, control group, AG group, TMPz group and AG+TMPzgroup. The concentrations of medicines in the later three groups were: AG group (10%, 20%), TMPz group (lOOug/ml, lOOOug/ml) and AG+TMPz group (10%AG+100ug/ml TMPz, 20%AG+1000^g/ml TMPz). The m-RNA expression of Thl/Th2 cytokines in PBMC before and after culture was detected by RT-PCR, the secretions of Thl/Th2 cytokines in plasma and culture supematants were determined by ELISA (Enzyme-linked immunoadsordent assay). 3. PBMC from lung cancer patients (in vivo): 37 lung cancer patients were randomly divided into two groups: 19 cases in AG group, and 18 cases in TMPz group. 10 healthy volunteers were involved as normal control group. The former two groups were respectively treated with 40ml AG injection+NS 100ml IV drip qd x 7 days, or TMPz 800mg+5%GDW IV drip qd x 7 days. Venous blood was obtained from patients before and after administration of the drugs. PBMCs were isolated with Ficoll-hypaque gradient method, which were divided into two parts: culture group (cultured with PHA for 48h) and pre-culture group (cells were reserved immediately without culture). The expression of Thl/Th2 cytokines was detected by RT-PCR considering IFN- Y and IL-2 as Thl cytokines, IL-4, IL-6 and IL-10 as Th2 cytokines.RESULTS: 1. human lung cancer cells : (1) None of the human lung cancer cells evaluated express IFN- Y and IL-2 in pre-control group, whereas all Th2 type cytokines were detected except that IL-4 and IL-10 were present respectively in squamous cell carcinoma and SCLC. (2) Expression of control group was as same as that of pre-control group. (3) 10% AG can prevent all human lung cancer cells evaluated from expression IL-4, inducing SCLC to expression IFN- Y and IL-2. (4) TMPZ dose-dependently can induce squamous cell carcinoma and SCLC to express IFN- Y, even SCLC to express IL-2. Meantime, expression of IL-4 can be inhibited. (5) 10%AG+100|0.g/ml TMPz can induce all human lung cancer cells evaluated to express IFN- Y strongly, and SCLC to express IL-2 simultaneously. Meantime, IL-4 can be inhibited. 2. PBMC from lung cancer patients (in vitro): The expression and secretion of IFN- Y and IL-2 in PBMCs from lung cancer patients were lower thanthat of the normal controls, while those of IL-4, IL-6 and IL-10 were significantly higher (P<0.05). After the culture, the expression and secretion of Thl/Th2 cytokines were changed obviously, which was reflected by a significant enhancement in IL-2 (P<0.05) paralleled by a substantial decrease in IL-6(P<0.05). The expression and secretion of IFN- Y also increased showing statistical difference in lOOOug/ml TMPz group and 10%AG+100ug/ml TMPz group(P<0.05). IL-4 and IL-10 decreased showing statistical differences in 20%AG group, lOOug/ml TMPz group and 20%AG+1000ug/ml TMPz group(P<0.05). 3. PBMC from lung cancer patient (in vivo): The positive rates of mRNA expression of IL-4, IL-6 and IL-10 were significantly higher in PBMC from lung cancer patients than that of the normal controls (P<0.05). The expression of IFN- Y and IL-2 were lower, but which were no of significance(P>0.05). After treatment with AG, the expression of IL-2 in PBMC from lung cancer patients significantly increased (P<0.05), and those of IL-4, IL-6 and IL-10 significantly decreased (P<0.05). In TMPz group, the expression of Thl/Th2 cytokines in PBMCs from lung cancer patients were also changed obviously with decreased expression of IL-6, IL-10 (PO.05) and increased expression of IFN- Y and IL-2 (PO.05).Astragalus is a kind of Chinese traditional medicine. In recent years the immunity enhancing properties of astragalus have drawn more and more attention. Our study about human lung cancer cells showed that 10% AG can prevent all human lung cancer cells evaluated from expression IL-4, meantime, this dose of AG can also induce SCLC to expression IFN- Y and IL-2, showing a reversing effect on Th2 predominant state. The effect present in 5%AG group, increased in 10%AG group, while declined in 20%AG group. The results indicated that AG has a reversing effect on Th2 predominant state of human lung cancer cells, which is related to the pathology of lung cancer cells and the dose of the medicine. SCLC is the most sensitive one and 10%AG is the most efficient dose. Our study about PBMC in vitro showed that the expression and secretion of IL-4, IL-6 and IL-10 in PBMC wereinhibited significantly after being cultured with AG. The inhibitory effect on IL-10 correlated with AG dose positively. Study about PBMC in vivo showed that expression of IL-4, IL-6 and IL-10 decreased obviously after patients treated with AG, and IL-2 increased obviously. In conclusion, our study showed that AG could not only reverse Th2 predominant state in lung cancer cells, but also enhance body immunity to tumor, interpreting the anti-tumor mechanism of AG from a new point of view.TMPz is now mainly used for therapy of cardiovascular and interstitial lung diseases. Recent years its potential use for treatment of cancer has been reported occasionally. Our study about human lung cancer cells showed that TMPz could reverse Th2 predominant state of all human lung cancer cells evaluated. Similar to AG, the effect of TMPz is also related to the pathology of lung cancer cells and the dose of the medicine. SCLC is the most sensitive one too. Compared with AG, the effect of TMPz on lung cancer cells has the following characteristics: (1) The reversing effect of TMPz on SCLC is stronger than that of AG; (2) TMPz can convert Th2 predominant state of squamous cell carcinoma and adenocarcinoma, which are less sensitive to AG; (3) The correlation between the effect and the pathology of lung cancer cells and the dose of the medicine is more obvious. Study about PBMC showed that the expression of IL-6 and IL-10 decreased after patients treated with TMPz, meantime, expression of IFN- Y and IL-2 increased. It should be pointed out that TMPz could dose-dependently induce not only human lung cancer cells but also PBMCs from lung cancer patients to express IFN- y . IFN- Y has an important function of anti-tumor and immunity toning including promoting tumor cell to express MHC- II antigen, promoting the differentiation of T and B-lymphocyte , stimulating CTL to maturate as well as B-lymphocyte to secrete antibodies, etc. Furthermore, IFN- Y play an important role in Thl/Th2 cell polarization , in cooperation with IL-12, promoting Thl cell proliferation. So, the effect of TMPz on IFN- Y inducing is of important significance. Our study showed that TMPz could reverse Th2 predominant state of human lung cancer cell lines and PBMCs from lung cancer... |
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