Experimental Study Of Qingfei Yiyin Recipe On Regulation Of AMPK-mediated Autophagy In Prevention And Treatment Of Radiation Pneumonitis

1 BackgroundRadiation therapy is one of the commonly used treatments for malignant tumors of the chest.The lungs are moderately sensitive to radiation.After receiving radiation therapy,it will inevitably cause certain damage to normal lung tissue,including early radiation pneumonitis and advanced radiation lungs.Fibrosis.The main clinical manifestations of radiation pneumonitis are:cough,sputum,chest pain,fever,fatigue and so on.At present,the pathogenesis of radiation pneumonitis is still unclear.There are various hypotheses,such as target cell theory,cytokine cascade theory,and free radical theory.Radioactive pneumonia is commonly used in the treatment of glucocorticoids,ACEIs,antioxidants,other drug therapies,and stem cell therapies.In recent years,some scholars have confirmed that autophagy as a self-protective mechanism of cells can reduce the damage of radiation to normal tissues,so autophagy may play a role in radiation pneumonitis.Chinese medicine believes that radiation is a hot and poisonous evil.The pathogenesis of this disease lies in heat poisoning,yin deficiency,blood stasis,qi deficiency,etc.,often appearing together.The clinical experience of the instructor for many years is that the early stage of the disease is mainly caused by heat-induced yin and yin.The principle of clearing heat and nourishing yin should be used as the treatment principle.The clinical experience of "Qingfei Yiyin Recipe" is added and subtracted to prevent and treat radiation pneumonitis.Clinical efficacy.In this study.rats with radiation pneumonitis were studied,and the mechanism of the prevention and treatment of radiation pneumonitis by the instructor's experience party "Qingfei Yiyin Decoction" was discussed around the AMPK-mediated autophagy pathway.2 ObjectivesTo observe the effect of Chinese medicine "Qingfei Yiyin Recipe" on the pathological morphology,lung coefficient,expression of inflammatory factors in alveolar lavage fluid,expression of autophagosome and autophagy-related protein in rats with radiation pneumonitis Fang is in the prevention and treatment of radiation pneumonitis,providing a theoretical basis for guiding clinical medication.3 Methods3.1 Establishment of a rat model of radiation pneumonitisThe rats were anesthetized with 10%chloral hydrate at a dose of 3 ml/kg,lying flat,limbs fixed on a self-made wooden frame,fully exposed to the chest,and 6 Gy with a single full chest irradiation with a 6MV-X line accelerator.3.5cm × 4.0cm(up to two armpits,down to the sternum xiphoid,the rest is shielded with 10cm thick lead brick),the source target distance is 100cm,the dose rate is 250cGy/min.In the blank group,the lungs were irradiated with OGy under the same conditions,and the rats in the experimental group were irradiated with normal dose.3.2 Animal administration methodEach group of rats was started to be administered one day after the irradiation,once a day for 28 days.The dose was weighed three times a week,and the dose was adjusted according to the body weight,and administered at 1 ml/100 g.Blank group:normal saline,1 time/day gavage;model group:normal saline,1 time/day gavage;Chinese medicine group:107 g/kg/d crude drug solution,1 time/day gavage;hormone group:1-1.25 mg/kg/d was given for 2 weeks,0.625 mg/kg/d prednisolone acetate solution was administered intragastrically for the third week,and normal saline was administered once per day for the fourth week;Chinese medicine plus hormone group:1-2 weeks to Chinese medicine+hormone liquid(10.7g crude drug+1.25mg prednisone/kg/d),1 time/day gavage;3 weeks from the Chinese medicine+hormone liquid(10.7g crude drug+0.625mg prednisone/Kg/d),1 time/day gavage;4 weeks to Chinese medicine(10.7.g/kg/d crude drug solution),1 time/day gavage.3.3 Pathological sections of lung tissue and ELISA were used to detect the expression levels of inflammatory factors such as IL-6,TNF-? and TGF-? in alveolar lavage fluid.3.4 The formation of autophagosomes in lung tissue of the rats in each group was observed by electron microscopy.3.5 The ratio of autophagy-related protein LC3II to LC3I,the expression of P62 protein and the expression of AMPK pathway protein were detected by Western-Blot assay.4 results 4.1 Pathological observation of lung tissue in each groupThe rats were sacrificed by exsanguination 4 weeks after irradiation,and the lung tissues were taken out.After HE staining,the alveolar wall structure of the rats in the blank group was intact,the thickness of smooth muscle was normal and no pathological changes such as inflammatory cell infiltration were observed.The rats in the irradiation group showed different degrees of inflammation.Sexual changes,including thickening of the alveolar wall,narrowing of the alveolar space,obvious inflammatory cell infiltration and exudation,capillary pathological changes such as capillary congestion.4.2 Detection of inflammatory factorsRats were sacrificed by blood transfusion 4 weeks after irradiation,and the whole lung lavage was performed by bronchoalveolar lavage.The lavage fluid was collected,the supernatant was centrifuged,and the ELISA kit was used for detection.The result was IL-6 in the irradiated group.The expression levels of TNF-a and TGF-? were higher than those in the blank group,and the combination group was superior to the traditional Chinese medicine alone or the hormone alone group in reducing the expression level of inflammatory factors.4.3 Electron microscopic observation of autophagosome formationAfter 14 days of irradiation,the cells were fixed by electron microscopy.The formation of autophagosomes in the lung tissue of each group was observed by electron microscopy.Under the same field of view,the number of autophagosomes in the combination group was more than that in the combination group.Other groups.4.4 Detection of autophagy-related proteins and AMPK pathwayAfter 14 days of irradiation,the ratio of LC3II to LC3I,the expression of P62 protein and the expression of AMPK protein were detected by Western-Blot method.The results showed that the degree of autophagy in the lung tissue of irradiated rats was inhibited.Yiyin Recipe,prednisolone acetate and the combination of the two can increase the degree of autophagy and activate the AMPK pathway.The effect of the combination group is better than that of the traditional Chinese medicine or the hormone a one.5 Research conclusionsTraditional Chinese medicine Qingfei Yiyin Recipe can alleviate the degree of inflammation in rats with radiation pneumonitis.The mechanism of its action may be achieved by activating the AMPK pathway and enhancing the degree of autophagy in rat lung tissue.