Study On The Mechanism Of Electroacupuncture Promoting TFEB Translocation Into The Nucleus To Improve Pulmonary Inflammation In COPD Rats

Objective:In this study,We established COPD rats models to study the effect of electroacupuncture"Zusanli"（ST36）and"Feishu"（BL13）on pulmonary autophagy and pulmonary inflammation of COPD.To further investigate the the role of TFEB-mediated autophagy in COPD pulmonary inflammation,we used cigarette smoke extract（CSE）to induce NR8383 cells（rat alveolar macrophages）to build a cell inflammatory model.Methods:1.The group of cells and detecting methods:NR8383 cells in logarithmic growth phase were treated with CSE at final concentrations of 5%,10%,20%,and 40%for 24h,48h,and 72h,respectively.The CCK-8 kit was used to detect the effect of CSE on cell viability.Then,the cells were cultured with 5%,10%and 20%CSE for 24 hours,respectively.Based on the western blot,the ratio of autophagosome marker protein LC3II/LC3I and the level of P62,an autophagy substrate degradation protein,were detected.The quantity of autophagosomes was detected by transmission electron microscopy（TEM）.Based on the results of CCK-8 and Western blot,20%CSE,24h were selected as the concentration and time of CSE intervention in subsequent experiments,respectively.To investigate the relationship between autophagy and inflammation,we used chloroquine（CQ）-an autophagy inhibitor,and rapamycin（RAPA）,an autophagy activator,to pretreat cells respectively.To investigate the relationship between TFEB-mediated autophagy and CSE-induced inflammation,we used fisetin,a kind of TFEB-activator,to induce cells.The expressions of tumor necrosis factor（TNF）-α,interleukin（IL）-1βand IL-6 in the supernatant were detected by ELISA.The levels of P62 protein、TFEB nucleoplasmic protein and the ratio of LC3II/LC3I protein were detected by western blot.The relative protein levels of LAMP1 and the nucleoplasmic distribution of TFEB were detected by immunofluorescence.2.Animal and grouping:According to the random principle,forty-eight rats of adult Sprague-Dawley（SD）healthy males were falled into normal,normal+EA,COPD,COPD+CQ,COPD+EA,COPD+CQ+EA group,with eight rats in each group.The rats in the normal group didn’t accept any treatment,and the rats in the normal+EA group received EA treatment for 30 minutes daily for 14 days;the rats were exposed to cigarette smoke for 12 weeks to establish COPD models;in the COPD+EA group,the rats were given EA treatment for 14 days,with 30 mins/d;and the rats in COPD+CQ group were intraperitoneally injected with autophagy inhibitor CQ（50mg/kg）30 min before the last EA treatment,for 4 days.The rats in the COPD+CQ+EA group were intraperitoneally injected with CQ（50mg/kg）30min before EA treatment.3.Model establishment and appraisal:after adaptive feeding,the rats were put into a1m³smoking box,1 hour for each time,twice a day,with an interval of 8 hours between the two smokes for 12 weeks.COPD model was evaluated as following:lung function significantly decreased,pathological manifestations of lung inflammation were significant,and the levels of inflammatory cytokines in broncho-alveolar lavage fluid（BALF）evidently increased.4.Electro-acupuncture treatment:COPD rats were given EA treatment at bilateral"Feishu"（BL13）and"Zusanli"（ST36）.The parameters of EA stimulation are following:density wave,frequency 4Hz/20Hz,lasting 0.5ms,intensity 1～3m A.The rats were treated with EA once a day for 30min each time for 14 days.5.Index detection:After treatment,the lung function and the pathological changes of lung tissue were tested.The levels of cytokines TNF-α,IL-1βand IL-6 in BALF were detected by ELISA.Autophagosomes in lung of rats were observed by transmission electron microscopy.The expression of P62 protein,LAMP1 protein,TFEB nucleoplasmic protein and the ratio of autophagic microtubule-associated protein light chain 3II/I（LC3II/LC3I）in lung of rats were detected by Western blot.Immunohistochemistry was used to observe the immunopositive expression of TFEB in lung of rats.Results:1.The viability of NR8383 cells was inhibited by CSE in a concentration-dependent manner（P<0.05,P<0.01）.Comparing to the 24h group,the cell viability of 48h group and 72h group was decreased（P<0.05,P<0.01）;there was no significant difference between 48h group and 72h group（P<0.05）.And after 24h of CSE stimulation,the cell survival rate was in a good level.therefore,24h was selected as the CSE intervention time in the following experiment.2.The results of western blot showed that the expression of autophagy substrate degradation protein P62 and the ratio of LC3II/I protein positively dependent on CSE concentration（P<0.05,P<0.01,P<0.001）.There were rare autophagosomes in the control group,while autophagosomes in CSE group were clearly visible and abundant.Compared with the control group,the fluorescence intensity of LAMP1 protein on the lysosomal surface of NR8383 cells in the CSE group was significantly decreased（P<0.01）;the red fluorescence intensity of Lyso-Tracker Red significantly decreased（P<0.01）;the expressions of cytokines in supernate obviously increased（P<0.001）.In addition,comparing to the control group,in the CQ group,the level of P62 protein and the ratio of LC3II/I protein obviously increased（P<0.01）;the contents of inflammatory cytokines were significantly increased（P<0.001）.Rapamycin,a common autophagy activator,partially reversed CSE-induced secretion of TNF-α,IL-1βand IL-6 in NR8383 cells（P<0.01）.3.Compared with the control group,the nuclear localization of TFEB protein in the CSE group significantly decreased（P<0.01,P<0.001）.Pretreating NR8383 cells with TFEB inducer fisetin.As results showed,compared with the CSE group,the expression of P62 protein and the ratio of LC3II/I protein in the CSE+fisetin group were decreased（P<0.05）;the fluorescence intensity of LAMP1 on lysosomal surface was upregulated（P<0.05）;the fluorescence intensity of Lyso-tracker Red significantly increased（P<0.05）;and the contents of TNF-α,IL-1βand IL-6 in supernate obviously decreased（P<0.001）.4.After 12 weeks of cigarette smoke expose,compared with the normal group,forced vital capacity（FVC）,forced expiratory volume in 0.1s(FEV_0.1)and 0.3 s(FEV_0.3),FEV_0.1/FVC and FEV_0.3/FVC were decreased in COPD rats（P<0.001）;the alveolar septum of the lung tissue of rats was thickened and the infiltration of inflammatory cells was obvious.the contents of TNF-α,IL-1βand IL-6 in BALF were significantly increased（P<0.001）;Autophagosomes were abundant in lung of COPD rats;the immunopositive expression of TFEB in lung was significantly decreased（P<0.001）;the expression level of autophagy-related protein P62 and the ratio of LC3II/I were significantly increased,while the expression of LAMP1 protein and TFEB nucleoplasmic ratio were significantly decreased（P<0.001）.5.Compared with the COPD group,the indexes of lung function in the COPD+EA group were significantly improved（P<0.001）;the alveolar wall was slightly thickened,and the inflammatory infiltration of lung tissue was alleviated;the contents of TNF-α,IL-1βand IL-6 in BALF were significantly decreased（P<0.001）;there were rare autophagosomes in lung;the immunopositive expression of TFEB in lung significantly increased（P<0.05）;the protein expression level of P62 and the ratio of LC3II/LC3I significantly decreased（P<0.05,P<0.001）,while the protein expression level of LAMP1 and the nucleoplasmic ratio of TFEB were significantly increased（P<0.01）.6.Compared with the COPD group,in the COPD+CQ group,the rats were injected with autophagy inhibitor CQ intraperitoneally for the last four days,the indexes of pulmonary function in COPD+CQ group were significantly decreased（P<0.001）;alveolar wall were thickened,alveolar cavity were enlarged,and inflammatory cell infiltration was obvious;the levels of inflammatory cytokines in BALF were increased（P<0.05）;the level of P62 protein and the ratio of LC3II/LC3I protein were increased（P<0.001）,while LAMP1 protein expression was decreased（P<0.05）.7.Comparing to the COPD+EA group,the above indexes of the COPD+CQ+EA group were obviously reversed（P<0.05,P<0.01,P<0.001）.Conclusion:1.CSE induced impaired autophagy by inhibiting TFEB from entering into the nucleus in NR8383 cells,which led to increased secretions of inflammatory factors.2.The lung histopathological changes and pulmonary function decline of COPD model rats established by cigarette smoking alone were consistent with the characteristics of COPD patients.The COPD model rats were successfully prepared in this study.3.EA can significantly improve pulmonary function and reduce pulmonary inflammation in COPD rats,which suggest that EA had a good therapeutic effect on COPD.4.Cigarette smoke can lead to the impairement of autophagy and induce pulmonary inflammation in COPD rats,which may be related to the inhibition of TFEB entry into the nucleus.5.Electro-acupuncture can alleviate the impaired autophagy in the lung of COPD rats,thus alleviate lung inflammation,and improve lung function,which may be related to the promotion of TFEB entering into the nucleus.