| Part 1 Clinical Efficacy Study of Yin Nourishing,Heat Clearing and Stasis Removing Prescription with Thoracic Radiotherapy on the Regulation of Th1/Th2 Immune Imbalance and Radiation PneumonitisObjective:To discuss the clinical efficacy of Yin nourishing,heat clearing and stasis removing prescription with thoracic radiotherapy on the regulation of Th1/Th2 immune imbalance and radiation pneumonitis.Methods: A total of 136 patients receiving thoracic radiotherapy in our hospital from July 2014 to July 2016 were randomly divided into treatment group(n=67)and control group(n=69)by hierarchical number table.Patients in the treatment group were given Yin nourishing,heat clearing and stasis removing prescription on the basis of radiotherapy or concurrent radiochemotherapy,one dose/day.The prescription was decocted with water twice according to the routine,and 200 m L of the drug solution was taken orally in divided twice.It was taken from the beginning of radiotherapy to one month after radiotherapy.Those in the control group received single radiation therapy or concurrent radiochemotherapy.A total of 3 m L venous blood was taken on an empty stomach at the beginning,during and after radiotherapy,respectively.Enzyme-linked immunosorbent assay was used to detect the contents of interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-4(IL-4)and interleukin-6(IL-6)in the serum.Moreover,the incidence of acute radiation pneumonitis was evaluated using Radiation Therapy Oncology Group criteria for acute radiation pneumonitis,and the quality of life and clinical symptoms of patients were assessed using TCM clinical symptom scoring table(self-made).Results: The incidence of acute radiation pneumonitis was 13.43%(9/67)in the treatment group and 28.98%(20/69)in the control group;the comparison between the two groups had statistical difference(p<0.05).IFN-γ and TNF-α in the treatment group had no obvious changes during and after radiotherapy,while they were increased notably in the control group;there were statistical differences in the comparison between the two groups(p<0.01).IL-4 and IL-6 in the treatment group showed a decreasing trend during and after radiotherapy,while in the control group,IL-4 presented downtrend,and IL-6 showed an upward trend;the comparison between the two groups had statistical differences(p<0.01).The ratio of IFN-γ/IL-4 in the control group had significant change during and after radiotherapy;the comparison between the treatment group and the control group was statistically significant(p<0.01).The scores of TCM clinical symptoms in the treatment group were remarkably reduced after radiotherapy,and the improvement of the scores in the treatment group was superior to that in the control group,which showed statistical significance(p<0.05).Conclusion: Yin nourishing,heat clearing and stasis removing prescription can reduce the incidence of radiation pneumonitis,regulate Th1/Th2 immune balance through regulating the expressions of Th1 and Th2 cytokines and improve the immune imbalance status of radiation-induced lung injury.Part 2 Basic Research on Protective Effects of Yin Nourishing,Heat Clearing and Stasis Removing Prescription on Radiation-induced Lung InjuryExperiment 1: Effects of Yin Nourishing,Heat Clearing and Stasis Removing Prescription on the Cell Count in Alveolar Lavage Fluid and Pulmonary Pathological Morphology of the Rats with Radiation-induced Lung InjuryObjective: To preliminarily discuss the protective effects of Yin nourishing,heat clearing and stasis removing prescription on the rats with radiation-induced lung injury(RILI)through the establishment of reliable and stable rat models of RILI and the effective evaluation of the models.Methods: A total of 162 female Wistar rats were randomly divided into 6 groups by random number table,including blank control group(Control Group),model control group(Model Group),TCM group/Yin nourishing,heat clearing and stasis removing prescription group(Decoction Group),TCM-intervention group/Yin nourishing,heat clearing and stasis removing prescription-intervention group(Intervention Group),pyrrolidine dithiocarbamate group(PDTC Group)and hormone group(Prednisone Group).Except for the Control Group,other 5 groups received a single 6MV X-ray irradiation on the right lung of the rats at 20 Gy to establish rat models of RILI.Decoction Group: A total of 9.7g/kg/d of crude drug solution was given on the first day after model establishment.Intervention Group: A total of 9.7g/kg/d of crude drug solution was given in the first week before model establishment.Prednisone Group: A total of 1.25mg/kg/d of prednisone solution was given in Week 1-2,0.625mg/kg/d in Week 3-4 and 0.312mg/kg/d in Week 5-12.PDTC Group: PDTC(10mg/kg)was given by subcutaneous injection on the first day after model establishment.Control Group and Model Group: Physiological saline was given.The general condition of the rats was observed after irradiation.In the 1st,4th,8th and 12 th week,the rats were sacrificed to observe the pathological sections of their lungs so as to calculate the lung coefficients and the cell count in the bronchoalveolar lavage fluid(BALF).Results:(1)General condition In the Control Group,the fur,movement,response,food intake and other conditions of the rats were relatively normal,and no rats died during the observation period.The rats in the Model Group developed lassitude,reduced appetite,curled back,upside-down bur and withered fair without luster and reacted a little slowly from the 2nd week.Since the 8th week,some rats in the Model Group were observed with tarnished fur,obvious polypnea,slow movement and slowly increased body weight.A total of 4 rats died during the observation period in the Model Group.Around Week 3,a few rats in the Decoction Group,Intervention Group and Prednisone Group started to experience decreased appetite,slow movement,curled back and sparse fur,and since Week 7,their appetite,fur color and movement had different degrees of improvement.In the Decoction Group and Intervention Group,there was a rat died in Week 5.In the Prednisone Group,there was a rat died in Week 6 and Week 12,respectively.The mental state,fur color and appetite of the rats in the PDTC Group were poorer than those of the rats in the hormone group.Moreover,in the PDTC Group,there was a rat died in Week 1 and 2 rats died in Week 6.(2)Lung coefficient The lung coefficients of the rats in the Model Group were obviously higher than those of the rats in the Control Group in Week 1,4,8 and 12(p<0.01).In comparison with those in Model Group,the lung coefficients in the Decoction Group,Intervention Group,PDTC Group and Prednisone Group were decreased in varying degrees in Week 1,4,8 and 12(p<0.05).In comparison with those in Prednisone Group,the lung coefficients in Decoction Group and Intervention Group were reduced in the same degree in Week 1,4 and 8(p>0.05).In Week 12,the lung coefficients in Decoction Group and Intervention Group were lower than that in Prednisone Group(p<0.05).The lung coefficient in PDTC Group was higher than those in Decoction Group,Intervention Group and Prednisone Group,which was more obvious in Week 8 and 12(p<0.05).(3)Pathomorphology Compared with Control Group,the rats in Model Group were observed with a little amount of inflammatory cell infiltration in their lung tissues in Week 1 after irradiation,obvious edema,inflammatory cell infiltration around the bronchia,destruction of alveolar structure and partial alveolar atrophy in Week 4,reduction of inflammatory cells,partial alveolar collapse and atelectasis,widening or destruction of alveolar septum and the formation of a few fusiform fibroblasts in Week 8 and alveolar ectasia and more significant alveolar collapse with fibrosis in Week 12.As the time went on,the degrees of alveolitis and pulmonary fibrosis in Decoction Group and Intervention Group were lighter than those in Model Group.In Week 8 and 12,alveolar atrophy and intramural fibroblasts in Decoction Group and Intervention Group presented varying degrees of reduction compared with those in Prednisone Group.In Week 1 and 4,the pathological changes of lung tissues in the rats in Decoction Group had no significant differences with those in the rats in Intervention Group.In Week 8,the degree of inflammatory cell infiltration in Decoction Group was slightly worse than that in Intervention Group.(4)Count of cells in BALF Compared with that in Control Group,total number of cells in alveolar lavage fluid in Model Group was obviously increased at each time point,which was significant in Week 4,8,and 12(p<0.01),and those in Decoction Group,Intervention Group,Prednisone Group and PDTC Group were also elevated at each time point,while they were lower than that in Model Group at the same time point(p<0.05)with the obvious reduction in PDTC Group(p<0.01).In Week 1,8 and 12,there were obvious differences in total cell number between Decoction Group and Prednisone Group or between Intervention Group and Prednisone Group(p>0.05).In addition,there was no significant difference in total cell number between Decoction and Intervention Group at each observation time point(p>0.05).Conclusion: Rat models of RILI can be successfully established using a single 6MV X-ray irradiation on the right lung of the rats at 20 Gy.Meanwhile,the pathological changes of lung tissues in the rats undergo acute inflammation period,hyperplasia period and fibrosis period,providing the experimental basis for studying the mechanism of drugs in prevention and treatment of RILI as well as screening drugs for prevention and treatment in the future.Yin nourishing,heat clearing and stasis removing prescription can alleviate the pathological changes of RILI and delay the pathological process of RILI.Moreover,the prescription helps to improve general condition of the rats,including appetite,fur color,movement and weight.However,the intervention with Yin nourishing,heat clearing and stasis removing prescription 1 week in advance is not superior to synchronous intervention in terms of pathological morphological changes.PDTC can significantly alleviate inflammatory cell infiltration in BALF after RILI.The mechanism of RILI may be related to the nuclear factor-kappa B signaling pathway.Experiment 2 Effects of Yin Nourishing,Heat Clearing and Stasis Removing Prescription on Th1/Th2 in the Serum and Lung Tissues of Rats with Radiation-Induced Lung InjuryObjective: To study the immunomodulatory effects of Yin nourishing,heat clearing and stasis removing prescription on Th1/Th2 imbalance in rats with radiation-induced lung injury(RILI).Methods: A total of 162 female Wistar rats were randomly divided into 6 groups by random number table,including blank control group(Control Group),model control group(Model Group),TCM group/Yin nourishing,heat clearing and stasis removing prescription group(Decoction Group),TCM-intervention group/Yin nourishing,heat clearing and stasis removing prescription-intervention group(Intervention Group),pyrrolidine dithiocarbamate group(PDTC Group)and hormone group(Prednisone Group).Except for the Control Group,other 5 groups received a single 6MV X-ray irradiation on the right lung of the rats at 20 Gy to establish rat models of RILI.Decoction Group: A total of 9.7g/kg/d of crude drug solution was given on the first day after model establishment.Intervention Group: A total of 9.7g/kg/d of crude drug solution was given in the first week before model establishment.Prednisone Group: A total of 1.25mg/kg/d of prednisone solution was given in Week 1-2,0.625mg/kg/d in Week 3-4 and 0.312mg/kg/d in Week 5-12.PDTC Group: PDTC(10mg/kg)was given by subcutaneous injection on the first day after model establishment.Control Group and Model Group: Physiological saline was given.In the 1st,4th,8th and 12 th week,the rats were sacrificed,and the levels of tumor necrosis factor-α(TNF-α)and interleukin-1α(IL-1α)of Th1 cytokines and interleukin-4(IL-4),interleukin-6(IL-6),interleukin-10(IL-10)and transforming growth factor-β(TGF-β)of Th2 cytokines in the serum of the rats were detected using enzyme-linked immunosorbent assay,and the levels of TNF-α and IL-1α of Th1 cytokines and messenger RNA expressions of IL-4,IL-6,IL-10 and TGF-β of Th2 cytokines in the lung tissues of the rats were detected using reverse transcription-polymerase chain reaction method.Results:(1)Change of Th1/Th2 cytokines in the serum of the rats: Compared with those in Control Group,the levels of TNF-α,IL-1α,IL-6 and TGF-β in each irradiation group were obviously increased in Week 1,4,8 and 12(p<0.01);the level of IL-4 in the serum had no significant change in Week 1 and 4(p>0.05),but was elevated obviously in Week 8 and 12(p<0.01);the level of IL-10 in the serum was notably reduced in Week 1 and 4(p<0.01),but was gradually increased in Week 8 and 12(p<0.01).The levels of TNF-α,IL-1α and TGF-β in the serum of the rats in Decoction Group and Intervention Group were obviously lower than those in the serum of the rats in Model Group(p<0.05).In Decoction Group,there was no statistically significant difference in the level of IL-6 in the serum in Week 1(p>0.05).In Week 4,8 and 12,the level of IL-6 in the serum of the rats in Decoction Group was remarkably lower than that in Model Group(p<0.01).The level of IL-6 in Intervention Group was notably lower than those in Model Group in Week 1,4,8 and 12(p<0.01).In Week 1 and 4,there were no obvious changes in the level of IL-4 in the serum between Decoction Group and Model Group as well as between Intervention Group and Model Group(p>0.05).In Week 8 and 12,the levels of IL-4 in the serum of the rats in Decoction Group and Intervention Group were obviously lower than that of the rats in Model Group(p<0.01).The levels of IL-10 in the serum of the rats in Decoction Group and Intervention Group were significantly higher than that of the rats in Model Group in Week 1 and 4(p<0.05),but obviously lower than that in Model Group in Week 8 and 12(p<0.01).Compared with those in Decoction Group,the levels of TNF-α,IL-1α,IL-6,TGF-β and IL-4 in the serum of the rats in Intervention Group were remarkably decreased in Week 1,4,8 and 12 (p>0.05).The level of IL-10 in the serum of the rats in Intervention Group was notably higher than that in Decoction Group in Week 1(p<0.05),while in Week 4,8 and 12,there were no significant differences in the levels of IL-10 between the two groups(p>0.05).Compared with those in Prednisone Group,the levels of TNF-α,IL-1α and TGF-β in the serum of the rats in Decoction Group and Intervention Group had no obvious differences in Week 1 and 4(p>0.05),while they were lower than those in Prednisone Group in Week 8 and 12(p<0.05).The levels of IL-6 in the serum of the rats in Decoction Group and Intervention Group presented no obvious differences with those of Prednisone Group in Week 1,4 and 8(p>0.05),but they were significantly lower than those in Prednisone Group in Week 8 and 12(p<0.05).The levels of IL-6 in the serum of the rats in Decoction Group and Intervention Group had no obvious differences with those in Prednisone Group in Week 1,4 and 8(p>0.05),but it was significantly lower than that in Prednisone Group in Week 12(p<0.05).In Week 1,4,8 and 12,the levels of IL-10 in the serum of the rats in Decoction Group and Intervention Group were all higher than those in Prednisone Group,but the level of IL-10 in the serum of the rats only showed significant difference in Week 8(p<0.05).(2)Protein expression of Th1/Th2 cytokines in lung tissues of the rats: In Week 1,4,8 and 12,mRNA expressions of TNF-α,IL-1α and IL-6 in Model Group were obviously higher than those in Control Group(p<0.01),and reached the peak value in Week 8.In Week 1,4,8 and 12,mRNA expressions of TGF-β in Model Group were significantly higher than those in Control Group(p<0.01),and were gradually increased over time.In Week 1 and 4,mRNA expressions of IL-4 in Model Group were slightly higher than those in Control Group(p>0.05),and obviously higher than those in Control Group in Week 8 and 12(p<0.01).The mRNA expression of IL-10 in Model Group began to decrease from Week 1,and was decreased to the minimum value in Week 4,and started to increase gradually in Week 8.The comparison of mRNA expression of IL-10 between Model Group and Control Group was statistically significant(p<0.01).In Week 1,4,8 and 12,mRNA expressions of TNF-α,IL-1α,IL-6 mRNA and TGF-β in Decoction Group and Intervention Group were obviously lower than those in Model Group(p<0.05).In Week 1 and 4,mRNA expressions of IL-4 and IL-10 in Decoction Group and Intervention Group were significantly higher than those in Model Group(p<0.05),and notably lower than those in Model Group in Week 8 and 12(p<0.05).There were no significant differences in mRNA expressions of TNF-α,IL-6,TGF-β,IL-4 and IL-10 between Decoction Group and Intervention Group in Week 1,4,8 and 12(p>0.05).In Week 1,8 and 12,mRNA expressions of IL-1 in Intervention Group were lower than those in Decoction,which suggested no statistical differences(p>0.05).In Week 4,mRNA expression in Intervention Group was obviously lower than that in Decoction Group,which showed statistical difference(p<0.05).MRNA expressions of TNF-α in Decoction Group and Intervention Group were higher than those in Prednisone Group in Week 1 and 4,showing statistical differences(p<0.05),and lower than those in Prednisone Group in Week 8 and 12,suggesting statistical differences(p<0.05).MRNA expressions of IL-1 in Decoction Group and Intervention Group were higher than that in Prednisone Group in Week 1,showing statistical differences(p<0.05).There were no statistical differences in mRNA expressions of IL-1 between Decoction Group and Prednisone Group as well as between Intervention Group and Prednisone Group in Week 4,8 and 12(p>0.05).MRNA expressions of IL-4 in Decoction Group and Intervention Group were lower than that in Prednisone Group in Week 1,which had statistical differences(p<0.05),higher than those in Prednisone Group in Week 4 and 8,presenting no statistical differences(p>0.05),and significantly higher than that in Prednisone Group in Week 12, showing statistical difference(p<0.05).MRNA expressions of IL-6 in Decoction Group and Intervention Group were higher than those in Prednisone Group in Week 1 and 4,suggesting statistical significance(p<0.05),and were significantly lower than that in Prednisone Group in Week 12,showing statistical significance(p<0.05).MRNA expressions of IL-19 in Decoction Group and Intervention Group were equivalent to those in Prednisone Group in Week 1,4 and 8,having no statistical differences(p>0.05),and were obviously higher than that in Prednisone Group in Week 12,showing statistical difference(p<0.05).MRNA expressions of TGF-β in Decoction Group and Intervention Group were lower than those in Prednisone Group in Week 8 and 12,and were significantly lower than that in hormone group in Week 12,suggesting statistical differences(p<0.05).Conclusion: Th1/Th2 immune imbalance is one of the important factors affecting the occurrence and development of RILI.The regulation of the imbalance of Th1/Th2 function by regulating the levels of TNF-α,IL-1α,IL-4,IL-6,IL-10 and TGF-β is one of the mechanisms of Yin nourishing,heat clearing and stasis removing prescription in preventing and treating RILI.Experiment 3 Effects of Yin Nourishing,Heat Clearing and Stasis Removing Prescription on TLR4/NF-κB Signaling Pathway Molecules of Lung Tissues in Rats with Radiation-Induced Lung InjuryObjective: To explore whether Yin nourishing,heat clearing and stasis removing prescription plays its role in prevention and treatment of radiation-induced lung injury(RILI)through the toll-like receptor 4/nuclear factor-kappa B(TLR4/NF-κB)signal transduction mechanism.Methods: A total of 162 female Wistar rats were randomly divided into 6 groups by random number table,including blank control group(Control Group),model control group(Model Group),TCM group/Yin nourishing,heat clearing and stasis removing prescription group(Decoction Group),TCM-intervention group/Yin nourishing,heat clearing and stasis removing prescription-intervention group(Intervention Group),pyrrolidine dithiocarbamate group(PDTC Group)and hormone group(Prednisone Group).Except for the Control Group,other 5 groups received a single 6MV X-ray irradiation on the right lung of the rats at 20 Gy to establish rat models of RILI.Decoction Group: A total of 9.7g/kg/d of crude drug solution was given on the first day after model establishment.Intervention Group: A total of 9.7g/kg/d of crude drug solution was given in the first week before model establishment.Prednisone Group: A total of 1.25mg/kg/d of prednisone solution was given in Week 1-2,0.625mg/kg/d in Week 3-4 and 0.312mg/kg/d in Week 5-12.PDTC Group: PDTC(10mg/kg)was given by subcutaneous injection on the first day after model establishment.Control Group and Model Group: Physiological saline was given.In Week 1,4,8 and 12,the rats were sacrificed,and the expressions of TLR4,NF-κB P65,inhibitor of nuclear factor kappa-B kinase-α(IKKα)and IKKβ in lung tissues of the rats were detected using Western blot assay.Results: The expressions of TLR4,NF-κB P65,IKKα and IKKβ in each irradiation group were obviously higher than those in Control Group,which was statistical significant(p<0.01).In Week 1,4,8 and 12,the expressions of TLR4,NF-κB P65,IKKα and IKKβ in Decoction Group and Intervention Group were obviously lower than those in Model Group(p<0.01).In Week 1,4,8 and 12,there were no obvious differences in the expressions of TLR4,NF-κB P65,IKKα and IKKβ in the lung tissues of the rats between Decoction Group and Intervention Group(p>0.05).The expressions of IKKβ in the lung tissues of the rats in Decoction Group and Intervention Group were significantly higher than that in Prednisone Group in Week 8(p<0.05),but obviously lower than those in Prednisone Group in Week 1,4 and 12(p<0.05).The expressions of IKKα in Decoction Group and Intervention Group were obviously higher than that in Prednisone Group in Week 1(p<0.05),but significantly lower than that in Prednisone Group in Week 12(p<0.05).The expressions of NF-κB p65 in Decoction Group and Intervention Group were notably higher than those in Prednisone Group in Week 1 and 4(p<0.05),but there was no statistical significance in Week 8 and 12(p>0.05).The expressions of TLR4 in Decoction Group and Intervention Group were higher than those in Prednisone Group in Week 1,4 and 8(p<0.05),and there was no obvious difference with Prednisone Group in Week 12(p>0.05).The expressions of NF-κB P65,IKKα and IKKβ in lung tissues of rats in PDTC Group were lower than those in Decoction Group,Intervention Group and Prednisone Group(p<0.01).Conclusion: TLR4/NF-κB signaling pathway plays an important role in the occurrence and development of RILI.Yin nourishing,heat clearing and stasis removing prescription can reduce the expressions of TLR4,NF-κB P65,IKKα and IKKβ in lung tissues of the rats with RILI.Moreover,the aforementioned indexes are obviously positively correlated with the severity of RILI,which may be related to the role of Yin nourishing,heat clearing and stasis removing prescription in regulating the signaling molecules in the TLR4/NF-κB signal transduction pathway. |
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