Therapeutic Effect And Mechanism Of Alstonia-leaf Alkaloids On Radiation Pneumonitis

[Objectives]Radiation pneumonitis is a common and early complication of radiotherapy for thoracic neoplasms,which can influence patients’ quality of life.Alstonia-leaf,as a traditional Chinese medicine with Yunnan characteristics,has functions of eliminating phlegm and relieving cough.However,no research about alstonia-leaf on radiation pneumonitis is reported in the literature.The aim of this study is to observe the efficacy and mechanism of alstonia-leaf on radiation-induced lung injury by Radiotion pneumonitis-associated inflammatory mediators.[Methodology]1.Cellular Level1)Cells are divided into Radiotherapy Group and Alstonia-leaf Group.Comparative study of inflammation-related cytokines(TNF-α,TGF,IL-2,IL-4,IL-6,IL-10,NF-κB)were performed.a)Human alveolar epithelial cell line BEAS-2B was inoculated into 96-well plates,and different concentrations of alstonia-leaf were applied to the cell line.The toxicity of alstonia-leaf in human alveolar epithelial cell line BEAS-2B was detected by MTT assay.IC50s after 24h,48h and 72h were calculated seperatedly.b)We utilized CBA flow cytometry and ELISA to detect the change of cytokines(TNF-α,TGF,IL-2,IL-4,IL-6,IL-10 and NF-κB).2)The expression of EMT-related(Vimentin,Snail)mRNAs and proteins between Radiotherapy Group and Alstonia-leaf Group was compared by Real-time PCR and Western blotting.2.Animal Level1)Animal model construction and grouping SD rats were used to replicate the model of radiation-induced pneumonitis.According to the reference and the previous experimental basis,the dose was set at 6Mv X Ray 16Gy.2)After successful construction of the animal model,SD rats are divided into negative control group,positive control group,radiotherapy alone group,low dose group(10mg),medium dose group(20mg)and high dose group(40mg).CBA flow cytometry was used to compare the serum changes of TNF in the 10th,13rd,18th and 21st day respectively.ELISA was done to compare that of IL-2、IL-4、IL-6、NF-κB separatedly.3)The expression level of EMT-related molecular markers(E-cadherin,N-cadherin,Vimentin,Snail)was examined by immunohistochemistry(IHC).[Results]1.Cellular Experiment1)MTT experiments confirmed that alstonia-leaf’s inhibition against BEAS-2B cell line gradually increase.The effect is time-dose dependent.2)The levels of IL-2、IL-4、IL-6、IL-10 and TNF-α were decreased in the supernatant of BEAS-2B cells treated with alstonia-leaf after radiotherapy(p<0.05).That of IL-6 showed no significant changes after the treatment of alstonia-leaf.3)Real-time PCR results showed the expression of N-cadherin and E-cadherin:That in radiotherapy group outweighed that in radiotherapy group with alstonia-leaf;The expression of Snail and Vimentin mRNA in radiotherapy group was inferior to that in radiotherapy group with alstonia-leaf.Western Blotting results showed that the relative expression levels of Vimentin and Snail mRNA in radiotherapy group was lower than that in radiotherapy group with alstonia-leaf.2.Animal Experiment1)The level of proinflammatory cytokines(IL-2,NF-κB and TNF-α)in alstonia-leaf treatment group(10mg,20mg and 40mg)showed time-dependent decreased,but the drug-dose-dependent effect of the serum cytokine was not observed.After radiotherapy,the serum level of anti-inflammatory factor(IL-4)in dexamethasone-positive control group and radiotherapy group was the same as that of IL-2,NF-κB and TNF-α.The level of anti-inflammatory factor(IL-4)in alstonia-leaf treatment group(10mg,20mg and 40mg)gradually increased with time,but the serum level of IL-4 did not show a significant dose-dependent effect.2)Immunohistochemistry results showed that alstonia-leaf could reduce the expression of E-cadherin and N-Cadherin protein and increase the expression of Snail and Vimentin protein.[Conclusions]1.The Alstonia-leaf had a therapeutic effect on radiation pneumonitis in rats,which could alleviate pulmonary inflammation in patients with radiation pneumonitis,so as to maintain the balance of pulmonary homeostasis;2.The Alstonia-leaf achieved the effect of treating radiation pneumonitis by reducing the serum levels of pro-inflammatory mediators and increasing serum levels of anti-inflammatory mediators;3.The Alstonia-leaf could not treat early radiation-induced lung disease(radioactive pneumonia)through EMT pathway.