The Experimental And Clinical Study On TCM Staging Treatment Of Radiation-induced Lung Injury

Part 1 A clinical observation of the TCM staging treatment on radiation-induced lung injuryObjectiveTo study clinical effect and evaluate safety of radiation-induced lung injury treated by TCM staging treatment with patients confirmed of chest tumor after receiving radiation therapy that is in accordance to acute radiation pneumonitis diagnosis, using prospective randomized comparative clinical research as principle and dyspnea scores,TCM clinical symptom scores, imageological examination, and KPS as the major index.MethodsBased on multicenter, random, parallel comparison, and single-blind trial method,64 samples between July,2013 and August,2014 from clinics and ward of the Second People’s Hospical of Sichuan and clinics of Sichuan Province TCM Hospital that are in accordance to RIPI diagnostic patients are divided into two groups:32 patients are in control group and the other 32 in test group. Control group is treated with regular modern medicine while test group is treated with regular modern medicine with additional complex prescription of TCM (prescription 1 within 1-3 months and prescription 2 within 4-6 months). Both groups will be evaluated with dyspnea scores, TCM clinical symptom scores, imageological examination, KPS and post-treatment radiation induced lung injury classification at anticipated time.Results1. RTOG classification results:RIPI classification observation is carried out after 6 months of treatment for both groups. By comparing the 2nd,3rd, and 4th level of data, the result shows P<0.05 with Mann-Whitney U method, which means there is a significant difference. It can be therefore said that the test group is in a better condition than control group in patients’condition evaluation.2. dyspnea scores result:grading is in a decreasing trend within each group. For both groups, P<0.05 at post treatment time points compared with those prior to treatment, which indicates a significant difference. During 6 weeks and 3 months, P>0.05; during 3 months and 6 months, P>0.05; there is no significant difference. Therefor it can be said both groups have dyspnea scores significantly decreased after 6 weeks of treatment. However, after 3 months of treatment dyspnea scores slightly increased, but there is no statistical meaning as well as after 6 months of treatment when dyspnea scores decreased again. Comparison between the groups shows that P>0.05 after lweek,3 weeks, and 6 weeks of treatment, which means data has no significant difference. On the other hand, P<0.05 after 3 months and 6 months of treatment which means data has a significant difference. It can be said that within 6 weeks of treatment both groups have no significant progress in improving dyspnea scores, but after 3 months and 6 months of treatment test group has achieved noticeable improvement than control group.3. Clinical symptoms of TCM evaluation result:within each group, dyspnea scores is in a decreasing trend. After 1 week of treatment of test group and 1 week and 3 weeks of treatment of control group, P>0.05, which means no significant difference. The rest time points of both groups all have P<0.05, indicating a significant difference. Compared between both groups, P<0.05 for all time points except for those prior to treatment and after 1 week of treatment. It can be said after 1 week of treatment test group has a better result in improving TCM clinical symptom scores than control group.4. Iconography scores:result shows scores are in an increasing trend. For all time points within a group P<0.05, indicating a significant difference. Comparing both groups, P<0.05 for all time points except for prior to treatment, indicating a significant difference. It can be said after 1 week of treatment test group is achieving a better result in improving TCM clinical symptom scores.5. KPS evaluation:analyzed with Mann-Whitney U test, Z=-2.640, P<0.05, indicating a significant difference. It can be said test group has achieved a better result in improving quality of life and stability.Conclusion1.RIPI patients in both groups have their dyspnea significantly improved. TCM prescription 1 with additional regular modern medicine is slightly better than pure regular modern medicine in improving dyspnea remission rate. It can be said that modern medicine with additional TCM prescription 1 would greatly improve dyspnea remission rate.2. After 3 months of treatment both groups have dyspnea scoresslightly increased, while control group is suffering a more sever consequence that does not end until after 6 months of treatment. In contrast, test group has dyspnea scores decreased after 6 months. Considering it is illness related, it can be said regular modern medicine has its limitations, while with additional TCM prescription 2 is continuously improved.3. RTOG classification:both numbers of level 3 and level 4 RIPI patients of test group are lower than those of control group, indicating TCM prescription 1 is not only able to significantly help early stage of dyspnea caused by radiation-induced lung inflammation, it may also possibly delay lung fibration.4. TCM staging therapy is effective in help improving RIPI patients’clinical symptoms. With TCM prescription 1 it can greatly improve imaging performance of early stage alveolus inflammation from radiation-induced lung injury. In addition, it can possibly delay the progress of RIPI step from early stage. With TCM prescription 2 it can further delay the progress of lung fibration.5. TCM staging therapy can improve the quality of life.Part 2 experiments and study of TCM staging therapy about radiation-induced lung injury in ratsObjectiveTo observe the effect of TCM staging therapy on radiation-induced lung injury rats at pathohistology and molecular biology level and further understand its efficiency on treating radiation-induced lung injury and discuss associated mechanism, this research used linear accelerator radiation modeled C57BL/6 female rats as research objects, randomized comparison and repetitive equipoise as principle, rats’lung coefficient, lung tissue pathohistology variation, HE stained pulmonary alveolitis observation, Masson stained pulmonary fibrosis observation, and expression of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-a (TNF-a) in lung tissue as main target of research.Method45 C57BL/6 female rats are randomly evenly divided into blank group, NS group, and TCM group. Blank group is treated with fake irradiation and 100μL of NS intragastric administration per day. NS group is treated with single 16Gy irradiation and 100μL of NS intragastric administration per day. TCM group is treated with single 16Gy irradiation and 100μL of TCM intragastric administration per day, where TCM prescription 1 is used within 1-3 weeks and prescription 2 is used within 4-6 weeks. At designated time (2nd week,4th week, and 6th week) 5 rats from each group are randomly executed. Lung tissues are collected, weighed, and calculated lung coefficient variation. To evaluate effect of TCM compound staging therapy on RIPI and its mechanism, microscopies of paraffin sections of lung tissues are used to study pathohistology variation, HE stained pulmonary alveolitis observation, Masson stained pulmonary fibrosis observation and the expression of TGF-β1 nd TNF-α within immunohistochemistry stained lung tissue.Results1. Lung coefficient:comparison results between groups:groups at 2,4,6 weeks, P< 0.05, all have significant differences. Group comparison results:TCM treatment group and blank control group lung coefficient at each time point both have P> 0.05, indicating no significant difference; Saline control group lung coefficient of 2 weeks to 6 weeks, P< 0.05, indicating a significant difference, whereas there is no significant difference for the rest of time points.2. Lung tissue observation:lung tissue from blank control group has smooth surface and pink color with good elasticity. It can fully spread out; lung from NS group turned into a dark red color 2 weeks after irradiation with swelling. Color turned darker at week 4 with elasticity decreased and hardness increased. At week 6 there is an apparent shrink in lung volume with a pale surface. Irregular nodule is visible at this stage. It further hardened with an apparent decrease in elasticity. Lung tissue from TCM group has no apparent abnormal signs at week 2. At week 4 color turned darker with swelling of lung tissue. At week 6 lung tissue starts to show some shrink in volume but not as severe as NS group.3. Histopathology change under light microscope:for blank control group at week 2, 4. and 6, only very few inflammation cells are visible. Alveolar structure is complete. There is no apparent exudation observed within alveolar space. For each time point there are neither fibroblasts nor collagen observed. Formation of pulmonary fibrosis is not observed. For NS group at week 2, alveolar epithelial hyperplasia and alveolar septa expansion are observed. Hyperemia, hemorrhage, fairly amount of macrophages, and neutrophils infiltration are observed among alveolar septa. At week 4, alveolar septa expand. Inflammatory cells from alveolar septa and infiltrated alveolar space have composition changed, a few of which have collagen formed. There is no apparent change in hyperemia and hemorrhage. At week 6 alveolar septa further expand. Alveolar space destroyed. Gathering of fibroblasts and deposition of collagen are visible. A portion of alveolus have structure disappeared and alveolar space collapsed. Pulmonary fibrosis formation is observed. For TCM group, at week 2 there is minor inflammation. Both of hyperemia and neutrophils infiltration are at a lesser level than those of NS group. At week 4, hyperemia, hemorrhage, and exudation are all at a lesser level than those of NS group. A small amount of plasma cells and lymphocyte are observed. At week 6, most of neutrophils disappear, with mainly lymphocyte and other chronic inflammatory cells left. A small amount of fibroblasts and collagen deposition are observed. Its pathological process is similar to NS group, whereas the pathological scope and collagen deposition are at a lesser level as well as a decrease in alveolar space and an increase in air-bearing area. Results suggest pulmonary alveolitis of model rats mainly happened in the first 4 weeks. At week 4 gathering of fibroblasts started, with a small amount of collagen formation. This dynamic change is almost consistent with the division of acute and chronic phase of this research.4. Observation of pulmonary alveolitis with HE staining:at week 2,4, and 6, no pulmonary alveolitis is observed. On the other hand, both of NS group and TCM group have confirmed cases of pulmonary alveolitis, with the latter mainly have mild or moderate cases and the former mainly have moderate or severe cases. Results of group comparison in terms of pulmonary alveolitis score show P<0.05 at week 2,4, and 6 for all groups, indicating a significant difference. Comparison within a group shows at week 4 to week 6 for both of NS group and TCM group, P>0.05, indicating no significant difference. The rest of comparison within a group all have P<0.05, indicating a significant difference.5. Observation of pulmonary fibrosis with Masson staining:at week 2,4, and 6, no pulmonary fibrosis is observed. On the other hand, both of NS group and TCM group have confirmed cases of pulmonary fibrosis, with the latter mainly have mild or moderate cases and the former mainly have moderate or severe cases. TCM group is at a lesser level of pulmonary fibrosis than NS group. Results of group comparison in terms of pulmonary fibrosis score show P<0.05 for all groups at week 2,4, and 6, indicating a significant difference. Comparison within a group shows P<0.05, indicating a significant difference.6. Immunohistochemical microscopy results of TGF-β1:for each time point blank control group has extremely little tan particles observed at epithelial cells from bronchial mucosa of lung tissue and inflammatory cells around bronchi. Expression of TGF-β1 from NS group is extensively observed among epithelial cells from bronchial mucosa of lung tissue, alveolar epithelial cells, fibroblasts, and vascular endothelial cells at week 2 after irradiation. The expression is also fairly frequent among pulmonary interstitial macrophages and alveolar macrophages and inflammatory cells around bronchi. Expression of positive cells from NS group reaches maximum at week 6. TCM group has the same expression of positive cells as NS group but at a considerably lesser level. Overall, NS group and TCM group both have a considerably higher level of expression of positive cells than blank control group.7. Immunohistochemical quantitative results of TGF-β1:group comparison shows P<0.05 at week 2,4, and 6, indicating a significant difference. Comparison within each group shows P<0.05, indicating a significant difference.8. Immunohistochemical microscopy results of TNF-a:blank control group has an uneven expression of TNF-a in bronchial epithelia at week 2,4, and 6, with a positive expression in a small amount of alveolar macrophages in the lung parenchyma and a negative expression in vascular endothelial and alveolar epithelial cells. For NS group positive expression is observed in alveolar macrophages and type II alveolar epithelial cells at week 2 after irradiation. A large amount of positive macrophages in lung parenchyma gathered in blood vessels at the outer membrane. At week 4 the lung parenchyma is full of large amount of positive macrophages. Expression of TNF-a is extensively found in macrophages in pulmonary interstitial, bronchial mucosa epithelial cells, endothelial cells and alveolar epithelial cells. The positive expression reaches maximum at week 4. TCM group has the same expression of positive cells as NS group but at a considerably lesser level. Overall, NS group and TCM group both have a considerably higher level of expression of positive cells than blank control group.9. Immunohistochemical quantitative results of TNF-a:group comparison shows P<0.05 at week 2,4, and 6, indicating a significant difference. For blank control group, P>0.05, indicating no significant difference. For NS group and TCM group, each group has P<0.05, indicating a significant difference. In addition, both groups reaches maximum at week 4. Compared with blank group, both of NS group and TCM group have significant difference, while TCM group has a considerably higher level of expression of TNF-a than NS group.Conclusion1. TCM staging therapy can reduce loss of body weight for RIPI rats during acute or chronic period as well as decrease the elevation of lung coefficient.2. Pulmonary alveolitis of rats from NS group mainly occurred within the first 4 weeks. Fibroblasts start to assemble from the 4 week, with small amount of collagen observed, whereas large amount of collagen are observed in the 6th week. This dynamic variation is in accordance to the hypothesis of week 1-3 is the acute period of inflammation and week 4-6 is the chronic period of fibrosis.3. TCM staging therapy on RIPI rats is able to effectively make a relief to lung tissue inflammation during acute period at pathohistology level (with less lung tissue hyperemia, hemorrhage, and edema) as well as to progress and stage of lung tissue fibration during chronic period (hinders gathering of fibroblasts, deposition of collagen, damage on alveolus, and expansion of alveolar septa).4. TGF-β1 plays a dynamic role during the process of radiation-induced lung injury. At the early stage of irradiation the level of TGF-β1 goes up, with a lower rate first then a rapid growth at week 3 and 4, which reaches maximum at week 6. The origin of TGF-β1 is not inflammatory cells but rather type II alveolar epithelial cells and fibroblasts. Refers to the pathology change, at early stage there is an apparent hyperplasia of type Ⅱ alveolar epithelial cells while 3 to 4 weeks later it switches to fibroblasts.5. TCM staging therapy hinders the expression of TGF-β1 for both acute and chronic cases. In addition, referring to the pathology change, while TGF-β1 is inhibited, pulmonary fibration level has an apparent decrease as well as inflammation level. This indicates TGF-β1 is involved in both late stage fibration and inflammatory reaction.6. The expression of TNF-a starts immediately after irradiation and shows a peak at week 4. Meanwhile pathology indicates pulmonary alveolitis is at the worst case at week 4. The expression of TNF-a is positively correlated to pulmonary alveolitis within a certain range. TCM staging therapy can effectively hinder the expression of TNF-a at early stage to lessen the inflammation.7. Mechanism of TCM staging therapy on RIPI is possibly related to hindering the expression of TGF-β1 and TNF-a.