The Effect Of μ/m-calpain On Regulating Respiratory Inflammation Related To Cigarette Smoke

Objective:Cigarette smoke(CS)is the most important risk factor in the development of chronic obstructive pulmonary disease whose incidence has substantially increased in recent decades but short of effective treatments.The mechanisms responsible for CS-induced acute and chronic pulmonary inflammation remain unclear.The effect of calpains and their inhibitor calpeptin on the inflammatory respiratory diseases associated with CS remains to be reported.This research is to study the expression of μ-calpain and m-calpain in CS-induced lungs from mice and cigarette smoke extract(CSE)-stimulated BEAS-2B cells.The present study furtherly investigates the effect of calpeptin on attenuating cigarette smoke induced respiratory inflammation and its potential mechanisms of action.Methods:The BALB/c male mice were exposed to the smoke of a cigarette once time with one or five cigarettes per day,five days per week,for a total of thirty or ninety days in a smoking apparatus as whole body exposure.The passive smoking mice with five cigarettes per day,five days per week,for a total of thirty days,were treated with 0.2 ml calpeptin(0.2 mg/ml)intraperitoneally once time,three times per week for a total of sixty days.Morphological changes in lungs were observed under a transmission electron microscope.Hematoxylin and eosin(H&E)staining was also performed using H&E staining kit.The expression of m-calpain were determined using indirect immunofluorescence staining under a laser scanning confocal microscopy.Western blot was conducted to assess the expression of m-calpain,μ-calpain and IκBα.BEAS-2B cells were added with CSE at 1%,2%,4%,8%,16%,32%,64%for 24 h.The IC50 value of CSE which inhibited cell proliferation was calculated by CCK-8 assay.The IC50 value of CSE-stimulated BEAS-2B cells were treated with calpeptin at 1nM、5nM、25nM、50nM、75nM、100nM for 24 h.The concentration of calpeptin,which inhibited the level of expression of m-calpain mostly via western blot,was used to treat the IC50 value of CSE-stimulated BEAS-2B cells.The BEAS-2B cells across groups were observed under an inverted microscope.The expression of m-calpain,μ-calpain and IκBα across groups were determined using western blot.Results:1.When the mice were exposed to the smoke of five cigarettes a day for ninety days,pulmonary vascular endothelial cells,type II alveolar epithelial cells,mitochondria and endoplasmic reticulum presented swelling.Mitochondria and endoplasmic reticulum became vacuolated while vague structure occurred in mitochondria.Infiltration of neutrophils(P<0.001),eosinophils(P<0.001)and macrophages(P<0.01)were visible within the interstitial and alveolar spaces with fibrinous exudation and proliferation.Bronchial epithelial hyperplasia and collapse of alveolar lumen were also visible.The pulmonary vascular lumen showed more hemangiectasia,hyperemia and hemorrhage.The expression of m-calpain(P<0.001),μ-calpain(P<0.01)and IκBα(P<0.01)were significantly increased in CS-induced lungs from mice.2.CSE inhibited proliferation of BEAS-2B cells in a dose-dependent manner.The IC50 value of CSE which inhibited cell proliferation was 18.02%.The expression of m-calpain,μ-calpain and IκBα were significantly increased in BEAS-2B cells stimulated by CSE at 18.02%(P<0.05).3.Swelling of pulmonary vascular endothelial cells,type Ⅱ alveolar epithelial cells(EP Ⅱ),mitochondria and endoplasmic reticulum in CS-induced lungs were markedly reduced by calpeptin.Additionally,calpeptin histologically attenuated infiltration of neutrophils(P<0.001),eosinophils(P<0.001)and macrophages(P<0.01)within the interstitial and alveolar spaces in CS-exposed lungs from mice.Meanwhile the fibrinous exudation and proliferation had been absorbed.Bronchial epithelial hyperplasia and collapse of alveolar lumen were invisible while autophagosome and autophagic vacuoles were seen.The concentration of calpeptin which inhibited m-calpain mostly was 50 nM.The number of floating cells was significantly decreased and the number of adherent cells was increased in CSE-stimulated cells group treated with calpeptin at 50nM.In addition,the markedly increased expression of m-calpain,μ-calpain and IKBa in CS-induced lungs and CSE-stimulated BEAS-2B cells were simultaneously decreased by calpeptin(P<0.05).Conclusions:1.The level of expression of μ-calpain、m-calpain and IκBα was increased in CS-induced lungs,contributing to pulmonary inflammation.The level of IKBa was upregulated in a time-dependent manner.2.The level of μ-calpain、m-calpain and IκBκ expression was elevated in CSE-stimulated BEAS-2B cells,leading to inhibition and inflammation in proliferated cells.3.These data indicated that calpeptin attenuated cigarette smoke-induced pulmonary inflammation through suppressing μ-calpain,m-calpain and IKBa in vivo and in vitro.