Berberine Contributes To Alleviation Of Ventilator Induced Lung Injury Via AMPK-SIRT1-NF?B Pathway

Objective:Mechanical ventilation(MV)plays a core role in treatment of ICU patients,especially for those enduring ALI/ARDS.Nonetheless,MV can also attribute to aggravation of lung injury as a side effect of ventilation induced lung injury(VILI).Previous studies have found that AMP-activated protein kinase,which known as AMPK,gains such property as significant alleviation of mouse acute lung injury.Meanwhile,beneficial effect of AMPK in regulating COPD has been uncovered in some recent findings.However,as an AMPK activator,berberine as a traditional C hinese medicine has not yet been identified as a eligible medicine in alleviating VILI at present.O ur research is to determine whether berberine could bring benefits in regulation of VILI and test the hypothesis that berberine could play an anti-inflammatory role via AMPK-SIRT1-NF? B pathway in a mouse VILI model.Methods:1.40 C57BL/6 mouse were used and were divided randomly into 5 experimental groups.Compare group(C): 4h of free breath without any additional treatment.Low tidal volume ventilation group(LV)which mouse accepted a 4h MV,VT=7ml/kg.High tidal volume ventilation group which mouse accepted a 4h MV,VT=20ml/kg.A berberine pre-treatment group which mouse accepted berberine ahead of conducting HV,namely HBV.Mouse in this group were pre-treated with berberine 30mg/kg ip.48 h,24h respectively ahead of HV was conducted.And AHBV group which is totally the same of HBV group except for a pre-treatment of an AMPK inhibitor Compound C 100mg/kg,ip.24 h ahead of MV,2.VILI in mouse model: mouse were kept a food-free,water-available condition 12 h before MV.After being weighed,mouse was anesthetised with a 0.75% pentobarbital sodium interpreperitoneally and fixed on an auto-temperature-controling experimental pad in abdominal position as soon as a surgical anethesia phase were reached.And after sterized,trachea was found after a incision of 7mm in the central neck skin.A 22 G Casing swabs vein needle sets was inserted into the trachea and fixed.Adjust the mechanical setting board and conduct the MV.Mo use breath spontaneously in group C.VT=7ml/kg with Respiratory rate(RR)100/min in LV;VT= 20ml/kg with RR=140/min in HV;HBV: pre-treatment of berberine 30mg/kg i.p.48 h,24h respectively ahead of MV+HV;AHBV: HBV+ pre-treatment of AMPK inhibitor,compo und C 100mg/kg i.p.24 h ahead of MV.MV or spontaneously breath for 4h in each group,the whole process are inhaled air ventilation.Temperature was detected constantly and maintained at 37?;maintained anesthesia and added 0.2 ml saline solution i.p.per hour to maintain fluid balance in mice and reduce fluid loss caused by breathe.3.Lung sample collection and processing: briefly,completely expose thorasis and observe pulmonary pathological changes,expose each porta pulmonis followed with ligation of right lung and extraction of the upper lobe,fixed and embeded for H-E staining and observation of morphological changes.Conduct immunohistorchemical(IHC)and observe AMPK,SIRT1 and NF-? B presentation in lung.The right middle lobe was used to peform W/D ratio test via a 60 oC oven baked for 72 h,each sample weighed before and after this process respectively.Extract the posterior lobe of the right lung and put into liquid nitrogen immediately,keep at-80?.Western-Blot analysis is conducted for detection of AMPK,NF-? B and SIRT1 presentation.4? 1ml saline is used to peform brocheoalveolar lavage(BALF)in left lung,3 times respectively,and centrifuged at 2000r/m,5min,4oC,supernatant is collected for determination of inflammatory factor TNF-? and IL-1?.4.Mouse lung tissue AMPK,NF-? B,SIRT1 levels were detected via Western Blot analysis so as to determine whether berberine could up-regulate AMPK level in a mouse VILI model,and meanwhile to determine its anti-inflammatory effect via AMPK/SIRT1/NF-? B pathway.This hypothesis is further verified by usage of AMPK inhibitor which was proposed to play an converse role.5.IHC approach to detect lung AMPK,NF-? B,SIRT1 positive rate and further determine alleviation of lung morphological changes were associated with regulated protein expression.Results:1.Morphological findings suggest berberine could alleviate mouse VILI significantlyCompared to HV(P<0.05),and contrast with C and LV,mice in HBV group show alleviation in inflammatory infiltration,alveolar edema,Pulmonary interstitial thickening,etc.While,AHBV show a converse morphological change compared to HBV(P<0.05),which is similar to HV group.2.BALF inflammatory factor level sharply reduced after berberine pre-treatment BALF was collected immediately after MV.Each experimental group TNF-? and IL-1?concertrations were measured via ELISA.Results indicated that levels of both inflammatory factors were down regulated significantly compared to HV and AHBV(P<0.05 respectively).3.W/D ratio is lower in HBV compared to HV and AHBV.Right middle lobe of mouse lung was obtained and weighed after MV,baked in a 60? oven for 72 h and weighed for dry value.Results showed that W/D ratio was reduced in HBV group compared to HV and AHBV(P<0.05,respectively),which indicate that pulmonary edema was alleviated in MV via berberine pre-treatment.4.ICH result suggested that AMPK and SIRT1 level were up-regulated significantly while the NF-? B level was down-regulated in berberine per-treatment group.AHBV showed a converse outcome.5.Western Blot findings indicates that AMPK and SIRT levels in HBV group are highly presented while NF-? B presents less compared to HV and AHBV.(P<0.05,respectively).AMPK was down-regulated in AHBV group,the same trend was also observed in SIRT level while NF-? B presentation suggested its activation.Conclusion:(1)Berberine alleviates VILI via activation of AMPK in mouse lung.(2)AMPK-SIRT1-NF ? B serves as the pathway of alleviation of VILI via berberine pre-treatment in a mouse model.