| Objective The aim of this study was to investigate the impact of nebulized inhaled lysophosphatidic acid(LPA)on early pulmonary fibrosis in acute respiratory distress syndrome(ARDS).Methods 1.Clinical study: Eight patients were assigned to the ARDS group,while another eight were included in the healthy control group(NC group).Peripheral blood samples were collected on the first day of hospitalization for the ARDS group and during the physical examination for the NC group.Enzyme-linked immunosorbent assay(ELISA)was performed to measure the levels of peripheral blood inflammatory factors,including IL-6(interleukin-6),TNF-α(tumor necrosis factor-alpha),IL-8(interleukin-8),as well as fibrosis factors α-SMA(alpha-smooth muscle actin)and TGF-β(transforming growth factor-beta).2.Animal study: The animals were divided into the following groups:a.Control group: The rats received a tail vein injection of an equal amount of phosphate-buffered saline(PBS)with lipopolysaccharide(LPS).b.LPA group:The rats received a tail vein injection of PBS followed by nebulized inhalation of LPA at a concentration of 1μM after half an hour.c.Ki16425 group: The rats received a tail vein injection of PBS followed by nebulized inhalation of Ki16425(an LPA receptor inhibitor)after half an hour.d.LPS group: The rats received a tail vein injection of LPS at a dosage of 5mg/kg.e.LPS+LPA group: The rats received a tail vein injection of LPS,followed by nebulized inhalation of LPA at a concentration of 1μM after half an hour.f.LPS+LPA+Ki16425 group:LPS was administered via tail vein injection,followed by nebulized inhalation of a mixture of LPA and Ki16425 at a 1:1 ratio after 30 minutes.Each group consisted of 12 rats,and lung tissues and bronchoalveolar lavage fluid(BALF)were collected at two time points: day 1(d1)and day 4(d4)after establishing the model.The wet-to-dry weight ratio(W/D)of the lung tissue was calculated,and the pathological changes in the lung tissue of each group were observed under HE staining.Immunohistochemical analysis of α-SMA was performed,and the content of IL-6,IL-1α(interleukin-1α),TNF-α,GM-CSF(granulocyte-macrophage colony-stimulating factor),INF-γ(interferon-γ),and TGF-β in rat BALF was determined using ELISA.Results Clinical study: The levels of inflammatory cytokines(IL-6,TNF-α,and IL-8)in the serum of the ARDS group were significantly higher than those in the NC group(P < 0.05).The levels of fibrotic factors(α-SMA and TGF-β)in the serum of the ARDS group were also significantly higher than those in the NC group(P < 0.05).Animal experiment: Lung wet-todry weight ratio(W/D)results: On both d1 and d4,there were no significant differences in lung W/D between the Ki16425 group,LPA group,and Control group(P > 0.05).The LPS group showed a significant increase in lung W/D compared to the Control group on both d1 and d4(P < 0.05),with a more pronounced increase on d4.The LPS+LPA group exhibited a significant decrease in lung W/D compared to the LPS group on both d1 and d4(P < 0.05),with a more pronounced decrease on d4.The LPS+LPA+Ki16425 group showed no significant difference in lung W/D compared to the LPS+LPA group on both d1 and d4(P > 0.05).However,there was a downward trend compared to the LPS group,although it was not statistically significant(P > 0.05).HE results: The lung alveolar structure in the Control group was intact,with no evidence of alveolar cavity or interstitial edema,and no infiltration of inflammatory cells.There were no significant pathological changes observed on both d1 and d4.Compared to the Control group,the LPA group showed no significant changes on both d1 and d4.The inflammatory pathology scores of lung tissue showed no significant difference(P > 0.05).In the Ki16425 group on both d1 and d4,partial thickening of the alveolar septa and mild infiltration of inflammatory cells were observed.The inflammatory pathology score of lung tissue was significantly higher than that of the Control group(P < 0.05).In the LPS group,on d1,there was thickening of the alveolar septa,abundant infiltration of red blood cells and inflammatory cells,and interstitial edema.On d4,the inflammatory response was more severe,and the inflammatory pathology score of lung tissue was significantly higher than that of the Control group(P < 0.05).In the LPS+LPA group,on both d1 and d4,there was mild thickening of the alveolar septa,mild infiltration of inflammatory cells,mild congestion of alveoli,and mild thickening of alveolar septa with a reduced degree of infiltration of inflammatory cells compared to the LPS group.Additionally,the inflammatory pathology score of lung tissue was significantly decreased compared to the LPS group(P < 0.05).In the LPS+LPA+Ki16425 group,there were no significant changes in HE staining on both d1 and d4,and the inflammatory pathology score of lung tissue showed no significant difference(P > 0.05)compared to the LPS+LPA group.However,the degree of lung injury was reduced compared to the LPS group,and the inflammatory pathology score was significantly decreased(P < 0.05).The results of the detection of pro-inflammatory factors in the bronchoalveolar lavage fluid(BALF)of each group of rats showed the following: On both d1 and d4,there were no significant differences in the levels of IL-6,IL-1α,TNF-α,GM-CSF,and INF-γ between the LPA group and the Ki16425 group compared to the Control group(P > 0.05).The levels of IL-6,IL-1α,TNF-α,GM-CSF,and INF-γ in the LPS group were significantly increased compared to the Control group(P <0.05).The levels of IL-1α,TNF-α,GM-CSF,and INF-γ in the LPS+LPA group were significantly decreased compared to the LPS group(P < 0.05),while there was no significant difference in the IL-6 level(P > 0.05).On both d1 and d4,compared to the LPS+LPA group,the level of IL-6 decreased significantly in the LPS+LPA+Ki16425 group(P < 0.05),while there were no significant differences in the levels of IL-1α,TNF-α,GM-CSF,and INF-γ(P >0.05).However,compared to the LPS group,the levels of IL-6,IL-1α,TNF-α,GM-CSF,and INF-γ were significantly decreased in the LPS+LPA+Ki16425 group(P < 0.05).The results of TGF-β detection in the bronchoalveolar lavage fluid(BALF)of each group of rats showed the following: In the Control group,a small amount of TGF-β expression was observed in the BALF on both d1 and d4.There were no significant differences in the levels of TGF-β between the LPA group and the Ki16425 group compared to the Control group on both d1 and d4.The levels of TGF-β in the LPS group were significantly increased compared to the Control group on both d1 and d4(P < 0.05).Compared to the LPS group,the levels of TGF-β in the LPS+LPA group were significantly decreased on both d1 and d4(P < 0.05).On d1,there was a trend of increased TGF-β levels in the LPS+LPA+Ki16425 group compared to the LPS+LPA group,but it was not statistically significant(P > 0.05).On d4,the levels of TGF-β in the LPS+LPA+Ki16425 group were higher than those in the LPS+LPA group(P < 0.05).On both d1 and d4,there were no significant differences in the levels of TGF-β between the LPS+LPA+Ki16425 group and the LPS group(P>0.05).Immunohistochemical staining for α-SMA: In the Control group,on both d1 and d4,there was slight α-SMA deposition observed in the smooth muscle cells of the bronchi and the walls of blood vessels,with no deposition observed in other areas.In the LPA group,there were no significant changes observed in lung tissue compared to the Control group.In the Ki16425 group,on d1,a small amount of α-SMA deposition was observed in the blood vessel walls,which was increased compared to the Control group,and on d4,there was a significant increase in α-SMA deposition.In the LPS group,a large amount of α-SMA deposition was observed on d1,and the deposition was more pronounced on d4.In comparison to the LPS group,in the LPS+LPA group,there was no significant change in α-SMA deposition on d1,but a significant decrease was observed on d4.In the LPS+LPA+Ki16425 group,compared to the LPS+LPA group,there was no significant change in α-SMA deposition on d1,but a significant increase was observed on d4.The results of optical density(OD)calculation using Image-J 1.8 image analysis software were consistent with the observations mentioned above.Conclusion 1.In the early stage of ARDS,the expression of inflammatory and fibrotic factors is increased.2.Inhalation of 1μM LPA via nebulization significantly reduces the elevated levels of pro-inflammatory cytokines and fibrotic markers induced by LPS in ARDS model rats,indicating that inhalation of LPA can inhibit the inflammatory response and pulmonary fibrosis process in ARDS. |
