A Study On The Significance Of Autophagy In The Pathogenesis Of Perfluoroisobutylene Inhalation-Induced Acute Lung Injury

The perfluoroisobutylene comes from the manufacturing industry of the organic fluorine and is a gaseous by-product with strong poisonousness.It can penetrate the gas masks and significantly harm the human beings.The poisonousness of the perfluoroisobutylene is 10 times stronger than the phosgene,and acute lung injury will be caused with the little amount inhalation of this gaseous.Acute respiratory distress syndrome can be developed by the serious patient,which can lead to death.Perfluoroisobutylene inhalation-induced acute lung injury develops very fast.Normally,its clinical features include progressive dyspnea and intractable hypoxaemia.Currently,the exact pathogenesis of perfluoroisobutylene inhalation-induced acute lung injury is unclear,leading the ineffective treatment and protection.Based on the previous foreign and domestic research as well as the preliminary study of our research group,it is identified that perfluoroisobutylene inhalation-induced acute lung injury is an inflammatory response,of which mainly is attributed to the excessive activation of the polymorphonuclear neutrophils.However,clinically,simply using the hormones impact anti-inflammatory treatment is not significantly effective and the death rate is still very high.Autophagy is the process that eukaryotic cells use the lysosome to degrade the intracellular substances.Autophagy has double functions.On the one hand,it can degrade the excessive or degenerative macromolecule as well as the damaged organelle in the cytoplasm,providing the energy and the nutriment for the cell and protein synthesis.On the other hand,the excessive activation or under the active of autophagy can cause the adverse effects for the cells or damage them.Based on the recent research,it is noticed that autophagy is closely related to the many lung diseases,such as acute lung injury,pulmonary arterial hypertension,pulmonary infection,chronic obstructive pulmonary disease,and idiopathic pulmonary fibrosis.Since autophagy is a ?double-edged sword?,it has different function on different cells as well as on different types of lung injury.The overall purpose of this research is to investigate whether or not autophagy participates in the development of perfluoroisobutylene inhalation-induced acute lung injury,identify its change rule and explore the function of autophagy on perfluoroisobutylene inhalation-induced acute lung injury,thus providing the new concept for the treatment and protection of perfluoroisobutylene inhalation-induced acute lung injury.First,42 SPF class Wistar male rats(8 weeks old,180-200 g)were randomly divided into seven groups(n=6).According to the morbid model built previously,the six groups of infected rats were totally exposed to the dynamic PFIB-inhalation intoxication(PFIB concentration was 290 mg/m3,exposure time was 8 mins),normal group had filtered air and was under exposure for 8 min.42 rats were anesthesia to death after being exposed to the PFIB for 1 h、2 h、4 h、8 h、16 h and 24 h,and samples of lung tissues,bronchoalveolar lavage fluid are collected.By measuring the coefficient of rats(the weight rate of wet and dry lung,lung water content,lung wet body ratio and lung dry body ratio),total protein content in BALF,conducing the lung tissue pathological examination,and comparing the results with previous research to prove the successful copy of the PFIB induced ALI model.The change of autophagy was observed under the transmission electron microscopy and expressed by measuring the LC3 using western blot.The result showed that the lung coefficient and protein content in BALF increased dramatically after being intoxicated for 16 and 24 hours,pulmonary interstitial and intra-alveolar edema with exudate of PMN were seen.Autophagy could be found in normal group and type I and II alveolar epithelial cells in all groups after being intoxicated by PFIB for 1 h,2 h,4 h,and 8 h,especially obvious after 1 h.The ratio of LC3Ⅱ/ LC3Ⅰwent up rapidly after 1 h PFIB intoxication and went down subsequently to the normal level after 24 h.A conclusion could be drawn,autophagy did participate in the PFIB-induced ALI accompanied by certain growth and decline.Based on the results mentioned above,the research group decided to use the common autophagy tool medicine-RAPA and autophagy inhibitor 3-MA to intervene the autophagy level of rat cells and observed the effect on PFIB-induced ALI to further explore the role autophagy played in PFIB-induced ALI.In 3-MA autophagy inhibition experiment,48 SPF male rats(eight-week-old,180-200 g)were separated into 8 groups(n=6)in random,including a 1 h solvent control group,a 1 h drug control group,a 1h solvent exposure group,a 1 h drug exposure group,a 8 h solvent control group,a 8 h drug control group,a 8 h solvent exposure group and a 8 h drug exposure group.One intraperitoneal injection of 3-MA was given 30 mins before the exposure with the dose of 30 mg/kg bw.One intraperitoneal injection of normal saline with the same volume was given to the four solvent control groups simultaneously.The four exposure groups were completely exposed to dynamic PFIB inhalation(PFIB concentration was 290 mg/m3,exposure time was 8 mins),the four control groups inhaled filtered air for 8 mins.After exposure,8 groups of rats were anesthesia to death at 1h and 8 h,lung tissues and BALF were collected.W/D ratio,protein content in BALF,lung tissue pathological examination and the LC3 in lung tissues measured by western blot were used to conduct research.The result showed that after 1 h exposure,1)the LC3 II/LC3 I ratio of drug control group and drug exposure group were significantly lower than solvent control group and solvent exposure group based on the Western blot results,it indicated that the autophagy had been inhibited in the lung tissue;2)There was no big difference in the protein content of BALF among all groups;3)but the W/D ratio of drug exposure groups and solvent exposure group increased dramatically compared with the control groups,no obvious difference was found between the two exposure groups.Suggests there was an obvious edema of the lung tissue,but intervention autophagy had no significant effect on pulmonary edema.After 8 h exposure,1)although the LC3 II/LC3 I ratio drug control groups was lower than the corresponding solvent groups,no obvious difference was observed,3-MA played an inhibitive role at the moment.2)The W/D ratio of drug exposure group was significantly lower than solvent exposure group,but higher than drug control group,solvent exposure group was higher than solvent control group.3)From the results of protein content in BALF,it could be found that two drug exposure groups had much higher value than drug control groups,drug exposure groups had lower value than solvent exposure groups though the difference was not significant.4)The result of lung tissue pathological examination demonstrated that solvent exposure group had more severe pulmonary edema than drug exposure group.All the results above indicated the obvious inhibitive effect 3-MA had on autophagy,the protection effect is not obvious in the early PFIB inhalation induced ALI(1 h after exposure),but 3-MA in the later(8 h after exposure)has certain protective effect.In the RAPA drug intervention experiment,48 SPF male rats(eight-week-old,180-200 g)were also separated into 8 groups(n=6)randomly,including a 1 h solvent control group,a 1 h drug control group,a 1 h solvent exposure group,a 1 h drug exposure group,a 8 h solvent control group,a 8 h drug control group,a 8 h solvent exposure group and a 8 h drug exposure group.Five days before the exposure,intraperitoneal injection of RAPA was given once a day with the dose of 10 mg/kg bw,drug was given 30 mins before the exposure on the fifth day.The same drug with the same dose was given to 4 solvent control groups simultaneously.Exposure mode,sampling time and experiment method were identical to the experiment of 3-MA.The content of IL-1β、IL-6、TNF-α in BALF was determined by ELISA.Here are the results: 1)LC3 measured by Western blot showed LC3 II/LC3 I ratio of drug exposure group was significantly higher than solvent exposure group and drug control group,solvent exposure group had higher ratio than solvent control group,therefore,RAPA had started having inductive effect on autophagy,and so was the inductive effect of PFIB.2)No difference was found among the groups in terms of W/D ratio,protein content in BALF,pathological examination and ELISA after 1h exposure,thus indicating there was no change in lung tissue.Suggest lung tissue injury was not obvious at this time.3)The result from Western blot after 8 h exposure showed that LC3 II/LC3 I ratio of the two drug control groups were much higher than the two solvent groups,thus RAPA had great inductive effect on autophagy at the time of 8 h.4)The W/D ratio of drug exposure group was significantly lower than solvent exposure group after 8 h exposure.Protein content in BALF of drug exposure group was much lower than solvent exposure group at the same time.The result of pathological examination displayed that drug exposure group had slighter pulmonary edema than solvent exposure group,with less PMN exudation.5)After PFIB 8 h exposure,the content of IL-1β、IL-6、TNF-α in BALF increased in different degrees,and IL-1β was the most significant.The inflammatory factor content of drug exposure group was significantly lower than solvent exposure group after 8 h exposure,IL-1β and TNF-α were the most obvious.All the results above demonstrated that drug exposure group had slighter ALI than solvent exposure group,indicating the protective effect RAPA had on lung tissue.To sum up the results above : 1)Autophagy did participate in the PFIB-induced ALI accompanied by certain growth and decline.At early stage(1 h),autophagy was active in type I and II alveolar epithelial cells,and lowered to basic level in 24 h.2)The auxo-action of autophagy on PFIB induced ALI.Both RAPA and 3-MA had protective effect on the lung tissue in PFIB-induced ALI.For 3-MA,the auxo-action of autophagy on PFIB induced ALI and the inhibition of autophagy from 3-MA lead to its protective effect.RAPA has the function of anti-inflammatory.Since its anti-inflammatory function has stronger power in protecting the lung tissue than the injury caused by the induced autophagy,therefore,it can significantly reduced the PFIB inhalation-induced ALI.In conclusion,all the results from the research provided reference to the prevention and treatment of PFIB-induced ALI.