Pkcα/ho-1 Signaling Pathway Attenuates Lps-attacked Injury Of Rat Type Ⅱ Alveolar Epithelial Cells By Mediating Mitochondrial Fusion

BackgroundSepsis is a life-threatening organ dysfunction caused by imbalance of body response to infectious factors,which seriously affects the prognosis of patients and threatens their lives.Sepsis has the complicated pathogenesis,severe injury to tissues and organs,difficult treatment and poor prognosis.Acute lung injury is the most common complication of sepsis,various factors(trauma,shock,sepsis,inhalation of irritant gas,aspiration of gastric reflux,etc.)can cause injury of alveolar epithelial cells and capillary endothelium,causes lung interstitial and alveoli diffusely dropsy,may cause the organism hypoxia finally,further induces other organic function failure.TypeⅡalveolar epithelial cells are progenitor cells of alveolar epithelium and have the functions of proliferation,differentiation,immunity,lung water transport,synthesis and secretion of surfactant,etc.They are essential to maintain the stability of the pulmonary environment in acute lung injury and acute respiratory distress syndrome.Mitochondria are highly dynamic organelles that maintain dynamic equilibrium by constantly fusion and fission.By merging mitochondria,damaged mitochondria return to normal and apoptosis is inhibited.Heme oxygenase-1(HO-1),an endogenous stress protein,plays an important role in lipopolysaccharide(LPS)-attacked acute lung injury in rats.Previous studies in our group have shown that activation of the PKCα/HO-1 signaling pathway promotes mitochondrial fusion and reduces LPS-attacked alveolar macrophage injury in rats.However,the effect of PKCα/HO-1 signal pathway on LPS-attacked mitochondrial fusion in rat typeⅡalveolar epithelial cells is still unclear.The aim of this study is to investigate the effect of mitochondrial fusion in typeⅡalveolar epithelial cells and its relationship with PKCα/HO-1 signaling pathway.ObjectiveTo evaluate the effects of mitochondrial fusion in rat typeⅡalveolar epithelial cells damage stimulated by LPS and the relationship with PKCα/HO-1 signal pathway.MethodsThe cultured rat typeⅡalveolar epithelial cells were seeded in 96-well plate at a density of 2×10~5/ml and were randomly divided into 7 groups using a random number table:control group(group C),LPS group(group L),LPS+PKCαinhibitor Go6976group(group LG),LPS+PKCαagonist PMA group(group LP),LPS+DMSO group(group LD),Go6976 group(group G)and PMA group(group P).Group LG,group LP and group LD were respectively pretreated with Go6976 5μmol/L,PMA100nmol/L and 0.1%DMSO for 30 min,then group L,group LG,group LP and group LD were given lipopolysaccharide(LPS)10μg/mL to establish the model of rat typeⅡalveolar epithelial cells damage,group G,group P and group C were respectively given with Go6976 5μmol/L,PMA 100nmol/L and the same amount of PBS as control groups.The cells were collected after 24 h of incubation for measurement of malondialdehyde(MDA)and reactive oxygen species(ROS)contents,superoxide dismutase(SOD)activity and expression of PKCα,HO-1,mitochondrial fusion related proteins 1 and 2(Mfn1,Mfn2),optic atrophy 1(OPA1)and mRNA(by fluorescent quantitative polymerase chain reaction and Western blot).ResultsCompared with group C,MDA and ROS were increased,the SOD activity was decreased,the expression of PKCα,HO-1 protein and mRNA were up-regulated,and the expression of Mfn1,Mfn2,OPA1 protein and mRNA were down-regulated in L,LG,LP and LD groups(P<0.05),there was no significant difference among the above parameters in G and P groups(P>0.05).Compared with group L,MDA and ROS contents were increased,the SOD activity was decreased,and the expression of PKCα,HO-1,Mfn1,Mfn2,OPA1 protein and mRNA were down-regulated in group LG,the above parameters are reversed in group LP(P<0.05),there was no significant difference among the above parameters in group LD(P>0.05).ConclusionPKCα/HO-1 signaling pathway attenuates LPS-attacked injury of rat typeⅡalveolar epithelial cells by mediating mitochondrial fusion.