Protective Effect And Mechanisms Of Sivelestat On Lipopolysaccharide-induced Acute Lung Injury

ObjectiveTo investigate the protective effect of sivelestat(SIV)on lipopolysaccharide-induced acute lung injury(ALI).And to investigate whether sivelestat attenuates lipopolysaccharide-induced acute lung injury by inhibiting the downregulation of ACE2/Ang-(1-7)/Mas receptor axis expression.MethodsIn vivo,90 healthy male Sprague-Dawley rats,weighing 220 ± 10 g,were randomized into 5 groups: the control group(CON,n=18);LPS model group(LPS,n=18);LPS+5mg/kg dexamethasone group(LPS+DEX,n=18);LPS+10mg/kg sivelestat group(LPS+10mg/kg SIV,n=18)and LPS+30mg/kg sivelestat group(LPS+30mg/kg SIV,n=18).Rats in the corresponding group were pretreated with sivelestat(10,30mg/kg)or dexamethasone(5mg/kg)intraperitoneally for 30 minutes before being anesthetized.Subsequently,an acute lung injury model was established: Except for the CON group,rats were given an intratracheal instillation of LPS(5mg/kg)toward the lung.Meanwhile,rats in the CON group were intratracheally dripped with identical volumes of normal saline.The general condition of rats was observed.Rats were sacrificed at three different time points(3,6,and 12 hours)following LPS or saline injection.Each group had six rats at each time point.Then,rat serum was prepared and the expression levels of angiotensin-(1-7),TNF-α and IL-6 were measured by enzyme-linked immunosorbent assay(ELISA).The right middle lobe of the rat lung was taken for the determination of the lung wet/dry weight ratio(W/D).Left lung tissues of rats were taken for pathological observation and lung injury scoring.Lung tissue homogenates were prepared and the m RNA expression of angiotensin-converting enzyme 2(ACE2)was detected by real-time PCR.Western Blot and immunohistochemical staining(IHC)were used to detect ACE2 protein expression.The effect of the LPS+DEX group was comparable to that of the LPS+10mg/kg SIV group,and the indexes of the LPS+30mg/kg SIV group were significantly higher than those of the LPS+10mg/kg SIV group(P<0.05).In vitro,RAW264.7 cells were maintained in RAW264.7 cell-specific medium.Logarithmic growth cells were inoculated into 6-well plates and cultured in a serum-free medium at 80-90 percent fusion.The cells were divided into the following groups:(1)the control group(CON);(2)the LPS model group(LPS);(3)LPS+0.51 m M dexamethasone;(4)LPS+10-8M sivelestat;(5)LPS+10-6M sivelestat;(6)LPS+0.51 m M dexamethasone+A779;(7)LPS+10-8M sivelestat+A779;(8)LPS+10-6M sivelestat+A779;(9)LPS+0.51 m M dexamethasone+DX600;(10)LPS+10-8M sivelestat+DX600;(11)LPS+10-6M sivelestat+DX600.Groups(4),(7),and(10)were administrated with 10-8M sivelestat,groups(5),(8),and(11)with 10-6M sivelestat,and groups(3),(6)and(9)with 0.51 m M dexamethasone for 1 hour at the same time.Subsequently,groups(6),(7),and(8)were co-incubated with A779(an antagonist of Ang-(1-7))with a final concentration of 10-5M for 1 hour.Simultaneously,groups(9),(10),and(11)were co-incubated with DX600(a specific inhibitor of ACE2)with a final concentration of 100 n M.Next,the LPS-induced acute lung injury model in vitro was established: All groups except the CON group were treated with 1μg/m L LPS for 3,6,and 12 hours.Cell culture supernatants and cells were extracted respectively at three-time points.The expression levels of ACE2 protein were determined by Western Blot and the concentrations of TNF-α,IL-6 and Ang-(1-7)in the cell culture supernatant were measured by ELISA.The statistical analyses were performed using the Graph Pad Prism software,version 9.0.A one-way ANOVA was used to analyze differences across groups,and P<0.05 was considered statistically significant.ResultsThe results of in vivo experiments showed that: Rats in the LPS group showed rapid respiration,impaired mobility,depression and persistent worsening.Compared with the CON group,rats in the LPS group had severe alveolar structural damage,thickened alveolar septa,and significantly higher lung injury scores(P<0.05).Rats pretreated with sivelestat or dexamethasone showed significantly less lung injury than in the LPS group,with the most significant improvement in the LPS+30mg/kg SIV group,and lung injury scores were also remarkably lower at all time points(P<0.05).W/D ratios,TNF-α and IL-6 levels in serum exposed to LPS for 3,6,and 12 hours were notably higher than in the CON group.Sivelestat or dexamethasone considerably decreased the W/D values and the release of TNF-α and IL-6 induced by LPS(P<0.05).ACE2 m RNA,ACE2 protein and Ang-(1-7)levels in rats exposed to LPS for 3,6,and 12 hours were markedly lower than in the CON group.Sivelestat or dexamethasone considerably increased the above indicators induced by LPS(P<0.05),and the elevation was more significant in the LPS+30mg/kg SIV group(P<0.05).The results of in vitro experiments showed that: The levels of TNF-α and IL-6 in the supernatants of RAW264.7 cells exposed to LPS for 3,6,and 12 hours were substantially higher than in the CON group(P<0.05).LPS-induced TNF-α and IL-6 secretions were significantly inhibited by sivelestat or dexamethasone(P<0.05).The expression of ACE2 and Ang-(1-7)in RAW264.7cell after 3,6,and 12 hours of LPS exposure was substantially lower than in the CON group(P<0.05).Sivelestat or dexamethasone pretreatment of RAW264.7cells significantly increased the ACE2 and Ang-(1-7)levels(P<0.05).However,administration of A779 or DX600,to RAW264.7 cells in the antagonist stimulation groups,significantly increased inflammatory cytokine levels and decreased ACE2 and Ang-(1-7)levels,reaching nearly the same level as the LPS group(P<0.05).Conclusions1.Sivelestat has a protective effect on LPS-induced ALI in rats in a dose-dependent manner.2.Sivelestat inhibits the increased secretion of TNF-α and IL-6 in cells induced by LPS.3.Sivelestat can reduce the inflammatory response by upregulating the expression levels of ACE2 and Ang-(1-7),thus exerting a pulmonary protective effect.