| Objective: On the basis of previous experimental research,the protective effect of AS-IV,a quality control component of Gan Du Qing,on the lung tissue structure of PM2.5-induced acute lung injury,the mechanism of AS-IV protection effect was further explored.Method: The animals were randomly divided into five groups(n = 7 per group)as follows:(1)normal saline(NS,rats received normal saline by intratracheal instillation [i.t.] and normal saline containing 1% dimethyl sulfoxide [DMSO] by intraperitoneal injection [i.p.]);(2)fine particulate matter(PM2.5,rats received PM2.5 at 7.5 mg/kg by i.t.and NS containing 1% DMSO by i.p.);(3)high dose AS-IV(ASH,rats received PM2.5 at 7.5 mg/kg by i.t.and AS-IV at 100 mg/kg by i.p.);(4)low dose AS-IV(ASL,rats received PM2.5 at 7.5 mg/kg by i.t.and AS-IV at 50 mg/kg by i.p.);and(5)fasudil(fasudil,rats received PM2.5 at 7.5 mg/kg by i.t.and fasudil at 10 mg/kg by i.p.).The volume for all intratracheal instillations was 1.5 m L/kg,and the volume of all intraperitoneal injections was 10 m L/kg.AS-IV was dissolved in NS containing 1% DMSO.For the ASH/ASL and fasudil groups,AS-IV and fasudil were administered at the indicated concentrations once a day within three days as previously described.The model was then established on the third day and the fifth day.That is,thirty minutes after the prophylactic administration on the third day,the rats in each group were anesthetized with 0.4% pentobarbital sodium solution(40 mg/kg,i.p.).The actue lung injury model was established by fully exposing the rat trachea,puncturing the cricothyroid membrane with a micro-syringe,and slowly infusing the PM2.5 suspension(7.5 mg/kg,drip volume calculated at 1.5 m L/kg).The second intratracheal instillation was administered on the fifth day.Twenty-four hours after the second PM2.5 inducement,the rats were anesthetized with 0.4% sodium pentobarbital solution and sacrificed by blood loss from the right femoral artery.Lung edema was assessed by the W/D weight ratio.Pulmonary histopathology and Pathological score was assess the lung injure.ICAM-1、IL-1β 、MPO were detect by kit to estimate the level of inflammation.Protein level in BALF detect by BCA kit to observe protein exudation.Total cell count and PMN count in BALF appraise the exudation level of lung injury.The alveolar epithelial type Ⅱ was observed by transmission electron microscope.Rho A,ROCK1 and ROCK2 were locate of in lung tissue by immunohistochemistry,and detection of Rho A,ROCK1,ROCK2,NF-κB,p-NF-κB,IκB,P-IκB,E-cadherin in lung tissue by Western blot.The experimental results were analyzed by SPSS 22.0,and mapped by graphpad prism 8.0.Results:(1)Effect of AS-IV on PM2.5-induced pulmonary edema: The lung W/D ratio and total lung water content in the PM2.5 group was higher than that in the NS group(P < 0.001);Remaining groups also reduced the W/D ratio and total lung water content compared to the PM2.5 stimulation(P < 0.001).(2)Total protein content and cell exudation in lung tissue: In the total protein exudation detected by the BCA method,compared with the NS group,the total protein content in the alveolar levage fluid of the model group was significantly increased(P < 0.001);compared with the model group,the total protein content in the alveolar levage fluid of the rats in remaining groups was significantly reduced(P < 0.001).In cell count and Wright-Gimsa staining of neutrophils,compared with the NS group,the cell count and the proportion of neutrophils in the alveolar levage fluid of the PM2.5 group were significantly increased(P < 0.001);compared with the PM2.5 group,Cell counts and neutrophil percentages in the alveolar levage fluid of rats in ASH group and fasudil group decreased significantly(P < 0.001),while cell counts in the BALF of rats in ASL group decreased(P < 0.01).(3)Effect of AS-IV on the morphology of lungs after PM2.5-induced acute lung injury: There were no histopathological changes in lungs of rats from the NS,ASH,ASL,and fasudil groups;alveolar structures were intact,capillaries were not enlarged,and only a small number of inflammatory cells were observed in the alveolar septum(the number of inflammatory cells was significant in the ASL group).Compared with NS,PM2.5 damaged alveolar structures,caused extensive edema and protein buildup,induced inflammatory cell infiltration,promoted fibroblast proliferation,induced pulmonary capillary congestion,and caused bleeding.In the pathological score,compared with the NS group,the total pathological scores of the rats in the PM2.5 group were significantly increased(P < 0.001),and the scores were increased in hemorrhage(P < 0.01),alveolar congestion(P < 0.05),alveolar wall thickening,or clear membrane scores(P < 0.05);compared with the PM2.5 group,ASH significantly reduced total pathological score(P < 0.001)and hemorrhage score(P < 0.01);ASL and fasudil could reduce total pathological score(P < 0.01).(4)Effect of AS-IV on PM2.5-induced inflammatory level in acute lung injury: Compared with the NS group,the levels of IL-1β(P < 0.01),MPO activity(P < 0.001),and ICAM-1(P < 0.001)activity in the PM2.5 group were significantly increased;compared with the PM2.5 group,the levels of IL-1β and ICAM-1 in the remaining groups were significantly reduced(P < 0.001),and MPO activity in the ASH/ASL groups was significantly reduced(P < 0.001),while MPO activity in the fasudil group was reduced(P < 0.01).(5)Ultrastructure of alveolar type Ⅱ epithelial cells: In the NS group,the volume of lamellar bodies of alveolar type Ⅱ epithelial cells was normal,and the alveolar structure was basically normal.In the PM2.5 group,alveolar type Ⅱ epithelial cell lamellar bodies increased in volume and irregularly,emptying was obviously vacuolated,and the alveolar structure was disordered.The structure of alveolar type Ⅱ epithelial cells in the remaining groups was significantly improved compared with the model group,and the alveolar structure was basically normal.(6)Effect of AS-IV on the Rho A signaling pathway in PM2.5-induced acute lung injury: Compared with the NS group,the Rho A/ROCK1 protein activity in the lung tissue of the PM2.5 group was significantly increased(P < 0.001),the ROCK2 protein activity was increased(P < 0.05),and the E-cadherin protein activity was significantly reduced(P < 0.001).Compared with the PM2.5 group,the Rho A/ROCK1 protein activity was significantly increased in the remaining groups(P < 0.001);fasudil significantly inhibited the ROCK2 protein activity(P < 0.001),and ASH could inhibit the ROCK2 protein activity(P < 0.05);E-cadherin protein activity in the ASH group was deeply increased(P <0.05),and E-cadherin protein activity was significantly increased in the ASL and fasudil group(P < 0.01).(7)Effect of AS-IV on the NF-κB/IκB signaling pathway in PM2.5-induced lung injury: Compared with the NS group,the p-IκB/IκB、p-NF-κB/NF-κB ratios in the lung tissue of the PM2.5 group were significantly increased(P < 0.001);compared with the PM2.5 group,the p-IκB/IκB、p-NF-κB/NF-κB ratio decreased significantly(P < 0.001).Conclusion:(1)Astragaloside IV can protect PM2.5-induced acute lung injury by inhibiting the inflammation,edema and protein exudation.(2)Astragaloside IV can alleviate the inflammatory reaction and protect the structure and function of lung tissue.(3)Astragaloside IV can protect lungs from damage caused by PM2.5 by inhibiting the NF-κB signaling pathway through Rho A and ROCK. |
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