Differential Expression Of MicroRNAs In Rats With Smoke Inhalation Injury

ObjectiveWe established an advanced rat model of Smoke inhalation injury(SII)based on the previous works,and explored the dynamics changes of pathophysiology and inflammatory factors during 28 days post SII.We also measured the different expressions of miRNAs in bronchoalveolar lavage fluid(BALF)between SII and normal control rats,and evaluated potential roles of miRNAs and target genes in SII.Methods42 male wistar rats were randomly divided into 7 groups: normal control group(n = 6)and smoke inhalation injury 6 groups(n = 36).The rats were sampled after being injured 1,2,3,7,14 and 28 days(n = 6),and the pathological changes of the lung tissue were observed by HE staining and Masson trichrome staining.The degree of pulmonary edema was evaluated by measuring wet and dry weight ratio(W/D ratio)of lung tissue.The interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and Interleukin-10(IL-10)in plasma and bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay(ELISA).The different expressions of miRNAs in bronchoalveolar lavage fluid(BALF)between SII and normal control rats were preliminarily screened by Affymetrix miRNA 4.0 microarray technology and then qRT-PCR validation assay confirmed the potential roles of miRNAs in SII.Finally,we preliminary forecasted potential roles of miRNAs and target genes in SII.ResultsThe histopathology of lung tissue showed that the first 3 days after the injury was in the acute phase.The main change was airway lesion,with the ciliated structure of airway epithelial cells damaged,airway local secretions increased,the alveolar stroma thickening,alveolar rupture and fusion;At 7 days after the injury,the hisopathological changes mainly included local pulmonary interstitial lesions,alveolar fusion,occasional inflammatory cell infiltration;At 28 days postinjury,large lamellar interstitial lesions were founded,which indicated local lung fibrosis,Collagen fiber deposition.The wet and dry weight ratios of lung tissue in 1 day and 2 days injury groups were higher than that in normal controls(P <0.05).In contrast,W/D ratios of lung tissue in 7 days,14 days and 28 days was significantly lower than those in one day group(P <0.05).Compared with the normal control group,plasma IL-6 increased significantly after 1 day of injury,then decreased,14 days after injury showed the second peak(P <0.05).IL-6 in alveolar lavage fluid injury did not change significantly,and it slightly decreased after 28 days of injury(P <0.05).The level of TNF-α in plasma was significantly increased after 1 day of injury,and the content of TNF-α in 28 days group was significantly lower than that in normal control group(P <0.05).The content of TNF-α in alveolar lavage fluid was significantly increased after injury,and there were two peaks at 2 and 14 days after injury(P <0.05).Plasma content of IL-10 in the second day after injury significantly increased,then decreased,the seventh day after the injury began to continue to increase until 28 days(P < 0.05).The change of BALF IL-10 was similar to TNF-α,there were two peaks appeared in post 2 days and 14 days after the injury(P < 0.05).Compared with the normal control group,there were 23 upregulated miRNAs(Fold Change≥2.0)and 2 downregulated miRNAs in BALF of SII group at 1 day post-injury.From top 12 upregulated miRNAs,9 miRNAs that have same sequences with human were selected for subsequent validation by qRT-PCR.It confirmed that the changes of miR-34c-5p,miR-92b-3p,miR-205,miR-34b-3p,miR-92a-3p,let-7b-5p,let-7c-5p in BALF were consistent with the conclusion of the miRNAs microarray(P < 0.05).Through the prediction of target gene software and preliminary study in vitro,it was founded that the increase of rno-miR-34c-5p promoted a lower expression of target gene ntn-1(P < 0.05).ConclusionsWe showed the dynamic changes of pathologic changes and inflammatory factors in rats with SII,and the differential expression of miRNAs in BALF of SII rats was screened by miRNAs microarray,which suggesting that these differential miRNAs may serve as SII markers for injury diagnosis and potential therapeutic targets.