| Radiation therapy is a very imporinntcancer treatment, but at the same time it brings a lot of side effects to cancer patients that impact the treatment efficacy, and radiation-induced lung fibrosis is one of the common side effects Radiation-induced lung injury (RILI) is caused by radiation in most lung, chest,head and neck cancer patients.Radiation pneumonitis (RP) is an early manifestation of RTLI,and then develops as radiation-induced lung fibrosis (RPF). Radiation-induced lung fibrosis seriously affects the patient’s quality of life and survival time. So far, Western medicine still has no effective treatment to RILI, Yet TCM can reduce the incidence of radiation-induced lung injury in some degree, as well as all eviate symptoms, improve quality of life in cancerpatients.1. PurposeLung fibroblasts are the effector cells of Radiation-induced pulmonary fibrosis. Based on pre-expcrimental studies, the metha of serum pharmacology was used in the study to observe the effects of Yang Yin Qing Fei (YYQF) formula on human embryo lung fibroblasts after Co60radiation.And further confirm whether YYQF can inhibit cell proliferation and promote apoptosis, change the cell growth cycle, reduce the expression of TGF-β1and α-actin expression. Clarifying the effect of YYQF on RILI, we can provide experimental basis for further development and application of new drugsand promote TCM in the treatment of RILI.2. MethasThe cells were divided into four groups:traditional Chinese medicine (TCM) group, hormone (H) group, TCM+II groupandcontrol group, control group. Wistar rats in four groups were administrated with gavagc, and their serum were collected, repacked and saved. Human embryo lung fibroblasts were radiated by Co60-ray, and then were treated with different drug-containing serum.First,human embryo lung fibroblasts were treated by di fferent Co60radiation doses (respectively3Gy5Gy,8Gy),and then eell prol iferation rate was detected by MTT to choose the strongest radiation dose on cell proliferation as the radiation dose infollowing experiments.Lung fibroblasts were treated under the best radiation dose, and then were treated with drug-containing serum in different group, cell prol iferations were detected at24,48and72hours respectively after radiation. The inhibition rates of lung fibroblasts were calculated.After treated with radiation and drug-containing serum, apoptosis and cell cycle of lung fibroblast were detected by flow cytometry to analysis the change in apoptosis and cell cycle by different drug-containing scrum intervention.TGF-β1plays a very important role in the development of pulmonary fibrosis. TGF-β1in different groups of24、48、72hours were detected by human TGF-β1ELISA Kit, and to analysis the drug effect on TGF-β1secreted by lung fibroblasts.α-actin is a muscle-specific expression of fibroblast and myofibroblast also plays a very important role in the occurrence and development of lung fibrosis. Treated cells were detected by high content screening methas to observethe changes of α-actin, to analysis the effect on α-actin protein expression of different drugs.3. ResultsAfter treated with different doses (0,3,5,8Gy) of radiation, the avalue of each group showed a significant difference (P<0.05)compared with control group, there were also differences (P<0.05) between each experimental group. With the increase of radiation dose, the survival rate and proliferationrate of the cells also increased. So8Gy was determinate as radiation dose in following experiments.Lung fibroblasts after radiated by8Gy Co60were cultivated respectively in B, Y, Q, I drug containing serum. Proliferation of cells was assayed by MTT. There was a difference between B group and drug groups (group Y, Q, andI)(P <0.05), and differences were also showed between group Y, Q, andI (P<0.05).Cell apoptosis were analyzed by Flow cytometryof in24hours. Each experimental group (Y, Q, I group) and controlgroup (B) were different (P<0.05), and there are differences between each experimental group (groupY,Q and I),(P<0.05), TCM group was most effective. In48hours there was difference between group Y, Q, I and B (P<0.05), the group Y, while there was no difference between group Y, Qand I (P>0.05), but this time point cell apoptosis was significantly less than the24-hour rate of apoptosis. At this point in time of72hours, Y, and Q, the I group and B group are different (P<0.05); there are differences between the Y group, Q group, I group (P<0.05). Rach cell cycle was analyzedby flow cytometry, and inhour24, control group (B), each cycle is more evenly distributed in G0/G1proportion is slightly larger; each drug group (Y, Q Group I), cell cycle arrest in G0/G1phase and G2/M phase; B group and drug group (Y, Q, I group), a significant difference (P<0.05) was showed in the S phase, and each drug group in S phase was significantly lower than the control group; there are significant differences between B group and drug group (P<0.05)in G2/M phase, and drug group was significantly higher than the control group in G2/M phase. The same trendwas showed in48, and72hours.RLISA kitwas used to detect the expression of TGF-β1, there was signi ficant difference between group B, Y, Q,I and K before treated with radiation (P<0.05);no difference between group B and Y at24hours (P>0.05), and difference was showed between the remaining groups (P<0.05);no difference between group B and Q at48hours (P>0.05), and difference was showed between the remaining groups (P<0.05);no difference between group B and I,between group Y and Q at72hours (P>0.05), and difference was showed between the remaining groups (P <0.05).High content drug screening system was used to detect the expression of α-actin (α-SMA). group B group and drug group (Y, Q, I group) are significantly different (P<0.05); Y、Q were with the group I have significant differences (P<0.05), and Y, Q between the two groups was not statistically significant (P>0.05).4. ConclusionThe proliferationof lung fibroblasts significantly accelerate after treated with radiation, in the0-8Gy irradiation range, this trend is positively correlated with radiation dose.Cell proliferation and survivalrate also increased with the increase of radiation dose.TCM plus hormone group (I group) showed the most cells suppression rate. The cell suppresscapacity of Q group is larger than the Y group. Within48hours, the rate of drug inhibition of cell went upward; peaked in the48hour time point drug inhibition of celland went down after that.Each drug group can promote radiation lung fibroblasts apoptosis. Tn the24-hour time point, the Chinese medicine group (Y group) showed a stronger ability to promote cell apoptosis than the other two groups (group Q and I), and in this time point, each drug group showed the most prominent ability to promote cell apoptosisEach drug groups can probably block G0/G1phase and G2/M phase, and reduce S phase cell cycle change; there by they can reducecell proliferation and promote apoptosis.ELlSA was used to detect lung fibroblast secretion of TGF-β1. In24hours TCM+hormone group showed the strongest inhibition ability to cell secretion of TGF-β1, while TCM group showed the weakest. In48hours, Chinese medicine inhibits the secretion of TGF-β1in lung fibroblasts strongest. In72hours, the inhibition ability of TCM+hormone group decreased, both TCM and hormones can inhibit cell secretion of TGF-β1.High content technology analysis of α-actin expression showed that each drug group could reduce the expression of α-actin, TCM+hormone group inhibit the most α-actin expression, and there was no difference between TCM group and hormone group. |
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