| Objective This research is based on the theory of cardiopulmonary correlation and its representative agent,Danggui Buxue Decoction in the doctrine of viscera state.the right lung of Wistar Rats was radiation-induced into lung injury,which serve as a model of Radiation-induced Lung Injury(RILI),from the perspective of Pathological morphology and Molecular Biology,To explore the protective effect and possible mechanism of Danggui Buxue Decoction which has the function of benefiting Qi,filling blood and activating blood on acute radiation-induced lung injury in Wistar Rats by adjusting NF-?B signal pathway,In order to provide new research ideas for clinical prevention and treatment of radiation lung injury from the perspective of traditional Chinese medicine(TCM).Methods Theoretical Part:Through the retrospective analysis of TCM and Western medicine on the knowledge of Radiation-induced Lung Injury,This paper explores into the source flow of cardio-pulmonary correlation theory and analyzes its typical representative agent,Danggui Buxue Decoction.60 male Wistar rats with Specific pathogen free,was randomly divided into 6 groups:Normal Control group,Model dose group,Low dose group of Danggui Buxue Decoction,Middle dose group of Danggui Buxue Decoction,High dose group of Danggui BuxueDecoction;Rats were sacrificed from the five groups at 3,7,14 and 28 day post-irradiation,After irradiation,the general conditions of the rats were observed.On the 7th,14 th and 28 th day,the pathological changes of the lung tissues which was injured by radiation was detected by hematoxylin-eosin(HE)staining and Masson staining;Histopathological changes of lung were observed by light microscope and electron microscope;The contents of tumor necrosis factor alpha(TNF-a)and endothelin-1(ET-1)in Wistar rats were collected at 3,7,14 and 28 days.The level of protein expression of angiotensin-?(ANG-?)?against angiotensin II type 1 receptor(AT1R)as well as endothelin-1(ET-1)in lung tissues were detected by immunohistochemical method,And calculate the accumulated light density of These indicators.Real-Time PCR(SYBR)absolute quantification method was used to detect and analyze the content of NF-?Bp65 mRNA ? IKB-a mRNA and ET-1mRNA.Western blot was used to detect the expression of NF-?Bp65?IKB-aand ET-1 protein in Wistar rats lung tissue which was injured by radiation.Results 1.The results of experiment one show that:according to the literature and pretest results of TCM and Western medicine,Rat model of radiated lung injury were induced by ionizing radiation in Wistar rats,which can simulate the clinical manifestation of acute radiation-induced lung injury.Not only the mortality rate is low,but also this model has fine repeatability.2.The results of experiment two show that the pathological changes of lung tissue in each group.Compared with blank control group,The results of HE staining showed that on the 7th day of HE staining of lung tissue in model group,pulmonary edema,inflammatory cell infiltration,thickening of alveolar septa,inflammatory exudation in alveoli.On the 14 th and 28 th day,pulmonary inflammatory reactiongradually decreased,and alveolar septum continued to thicken,partial alveolar septum rupture,and finally the normal structure of lung tissue was destroyed;Compared with the model group,the pathological changes of the lung tissue were similar to those of the model group,but the degree was less;Compared with hormone group,in the later period(14 to 28days),Danggui Buxue Decoction was better than hormone group.Masson staining results showed that radiation-induced pulmonary inflammation and radiation-induced pulmonary fibrosis occurred simultaneously.With the extension of time,radiation-induced pneumonia was reduced and fibrosis was gradually aggravated.From Day 7 to Day 14,The pathological changes of pulmonary lesions were mainly caused by radioactive pneumonia;From Day 14 to Day 28,The pathological changes of pulmonary lesions are mainly due to radiation pulmonary fibrosis.After the intervention of Danggui Buxue Decoction,the effect of high dose group and medium dose group of Danggui Buxue Decoction was better than that of low dose group.The results of electron microscopy showed that the three layers were complete,the gas and blood barrier was normal in contraction,the capillary was dilated,the alveolar cavity was clean,the pulmonary septum was not enlarged,the collagenous fibers were deposited,the macrophage adsorbed,the type 2 alveolar epithelial cells were less,the surface villous,Under the electron microscope,there are many Goldenite matrices,the macrophages and red blood cells also could be seen;In the model group,collagen fiber increased,mitochondrial swelling of type II alveolar epithelial cells was serious,and there were more internal active substances,less cytoplasm,severe vacuoles,more megakaryocytes and fibroblasts.The collagen fibers increased under electron microscope,and there was a model structure in the blood vessel.It may be the residue after cell disintegration.There were macrophages but a few.The mitochondria of the alveolar cell of type 2was slightly swollen.3.The results of experiment three show that:the effect of Danggui Buxue Decoction on the secretion of vasoactive substances by inflammatory factors and vascular endothelial cells.Compared with normal control group,theserum levels of TNF-a and ET-1 in model group effectively increase with statistically significant difference.Comparison with model group,After the intervention of Danggui Buxue Decoction with high and medium dose group respectively,the serum levels of TNF-a and ET-1 in high dose group and medium dose group in Danggui Buxue Decoction showed a significant downward trend(P<0.05),And the trend of change was positively correlated with the dosage of Danggui Buxue Decoction(P<0.05),Compared with the hormone group,the serum TNF-a decreased but not statistically on the 7th day,but on the 14 th and28th days(P<0.05),the serum ET-1 increased and then decreased,during the 14 th and 28 th days(P<0.05).4.The results of experiment four show that:at various points in time,The accumulated light density of ANG-??AT1R and ET-1 protein expression in blank control group was only a few basic expressions;Compared with blank control group,The cumulative light density of Ang II,AT1 R and ET-1 proteins in lung tissue of Wistar rats in model group decreased gradually,Especially in the later period(14d,28d);Comparison with model group,After the intervention of Danggui Buxue Decoction with high and medium dose group respectively,the cumulative light density of ANG-II,AT1 R and ET-1 protein expression in lung tissue of Wistar rats in medium and high dose groups gradually increased,And the trend of change was positively correlated with the dosage of Danggui Buxue Decoction(P<0.05),Compared with hormone group,the cumulative light density of ANG-II and AT1 R protein in Danggui Buxue Decoction in middle dose group and high dose groupdecreased on 28 days(P<0.05).The cumulative light density of ET-1protein in Danggui Buxue Decoction was also decreased on 28days(P<0.05).5.The results of experiment five show that:compared with the blank control group,the Level of expression of NF-?Bp65 mRNA in the model control group increased significantly(P<0.05),The protein expression of IKB-a mRNA in lung tissue of Wistar rats in model group was significantly reduced(P<0.05),The protein expression level of ET-1mRNA in lung tissue of Wistar rats in model group increased significantly(P<0.05);Comparison with model group,After the intervention of Danggui Buxue Decoctionwith high and medium dose group respectively,NF-?Bp65 mRNA transcription levels in high dose group and medium dose group in Danggui Buxue Decoction showed a significant downward trend(P<0.05),The protein transcriptional expression level of IKB-a mRNA in lung tissue was significantly increased(P<0.05),while the protein transcriptional expression level of ET-1 mRNA in lung tissue showed a significant downward trend as well(P<0.05),And the trend of change was positively correlated with the dosage of Danggui Buxue Decoction(P<0.05).6.The results of experiment six show that:compared with the blank control group,the Level of expression of NF-?Bp65 protein in the model control group increased significantly(P<0.05),The protein expression of IKB-a in lung tissue of Wistar rats in model group was significantly reduced(P<0.05),The protein expression level of ET-1 in lung tissue of Wistar rats in model group increased significantly(P<0.05);Comparison with model group,After the intervention of Danggui Buxue Decoction with high and medium dose group respectively,NF-?Bp65 mRNA transcription levels in high dose group and medium dose group in Danggui Buxue Decoction showed a significant downwardtrend(P<0.05),The protein transcriptional expression level of IKB-a in lung tissue was significantly increased(P<0.05),while the protein transcriptional expression level of ET-1 in lung tissue showed a significant downward trend as well(P<0.05),And the trend of change was positively correlated with the dosage of Danggui Buxue Decoction(P<0.05).Conclusion:Pathological mechanism of radiation-induced lung injury is a complex process that involves the interaction of various cells and molecules,a lot of literatures show that Chinese medicine compound plays an important role in the prevention and treatment of radiation-induced lung injury.On the basis of theoretical discussion and experimental results,it is shown that the classical formula of Danggui Buxue Decoction could protect against acute radiation-induced lung injury in Wistar Rats,It may have the following mechanisms:1.Danggui Buxue Decoction can reduce acute radiation lung injury in Wistar rats by lowering the levels of TNF-aand ET-1 in the serum;2.The expression of ET-1-ANG-II-AT1 R signal axis can be reduced by Danggui Buxue Decoction.3.Danggui Buxue Decoction may be used to prevent and treat radiation-induced lung injury by means of the regulation of NF-?B/ET-1/ANG-II/AT1R/signaling pathway.enzyme linked immunosorbent assay(ELISA)was used to detect the levels of TNF-aand ET-1 in serum. |
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