Expression And Significance Of Sonic Hedgehog Signaling Pathway In Lipopolysaccharide-induced Acute Lung Injury Of Mice

Objective:To explore the expression and the significance of the related molecules SHH, PTC, and GLI1 in the Sonic hedgehog (SHH) signaling pathway from mice acute lung injury (ALI) induced by lipopolysaccharide (LPS) and propose clinically new ideas for the treatment of acute lung injury.Methods:The male BALB/c mice aging between 5 to 7 weeks and weighing 20-25g were selected as the experimental animals. They were divided randomly into control group (with equal amount of normal saline intraperitoneal injection), LPS-treated group (with 5mg/kg LPS intraperitoneal injection), and cyclopamine-treated group (first with 5mg/kg LPS intraperitoneal injection, and 30 minutes later 50mg/kg cyclopamine intraperitoneal injection). The mice then underwent pentobarbital sodium anesthesia via intraperitoneal injection for specimen taking 6h,12h or 24h later after being injected with different substances. When the lung tissue had undergone some general changes by visual inspection with 4% paraformaldehyde fixed, took the lung tissue section, and its pathological changes were observed in hematoxylin-eosin (HE) staining, after which its pathological injury was also graded. Take a piece of lung lobe to be dried at 60℃ for 48h to measure its wet mass/dry mass (W/D) mass ratio. Test and measure the mRNA expressions of lung tissue turner necrosis factor-alpha (TNF-a), SHH, PTC and downstream transcription factor GLI1 through quantitative real-time PCR as well as the protein expressions of lung tissue nuclear factor-κB (NF-κB), SHH and GLI1 through Western blot detecting in different treatment groups.Result:The alveolar spaces of the lung tissue could be seen clearly without obvious inflammatory cell infiltration under the light microscope for the control group, thus its pathological injury grading being low. However, the LPS-treated group could be seen with alveolar congestion and edema, obviously-thickened alveolar septa, and broad inflammatory cell infiltration in the interstitial, thus the pathological injury grading for the lung tissue in this group was much higher than that of the control group (P<0.01). For the cyclopamine-treated group, the alveolar spaces were mostly lost and the lung tissue mostly consolidated with inflammatory cell infiltration, the pathological injury grading being higher than the LPS-treated group (P<0.05). And the LPS-treated group had a larger W/D mass ratio than that of the control group at any time (P<0.01 at 6h and 12h, and P<0.05 at 24h), while that of the cyclopamine-treated group was higher than the LPS-treated group (P<0.05). The TNF-a mRNA expression of the control group was low at any time, while that of the LPS-treated group was obviously higher than it from time to time (P<0.01), and for the cyclopamine-treated group, its TNF-a mRNA expression at 12h and 24h were all larger than the LPS-treated group (P<0.05). With regard to the NF-κB expression, the control group had a lower value than that of the LPS-treated group, which was much higher than the control group(P<0.01 at 6h and 12h, and P<0.05 at 24h), while the value for the cyclopamine-treated group was still higher than that of the LPS-treated group (P<0.01).For the control group, its mRNA expressions of the SHH, PTC and GLI1 were low, and there were no obvious differences at this respect between it and the LPS-treated group at 6h (P>0.05), while at 12h and 24h big differences concerning the mRNA expressions occurred (P<0.01) and were positively correlated with time (P<0.01). For the cyclopamine-treated group, no outstanding differences occurred between it and the LPS-treated group regarding the mRNA expressions for the SHH, PTC and GLI1 at 6h (P>0.05), while there was a drop at 12h (P<0.05) compared with the LPS-treated group, and a sharp drop at 24h (SHH P<0.01, PTC P<0.05, GLI1 P<0.01). As for the protein expressions of the SHH and GLI1, the control group had a low value at any time point, but the values for the LPS-treated group saw an obvious increase at 6h,12h and 24h (SHH 6h P<0.05, P<0.01 at 12h and 24h, and GLI1 P<0.01 at any time point) and were positively correlated with time (P<0.01). The protein expressions of the SHH and GLI1 for the cyclopamine-treated group were both lower than the LPS-treated group (SHH P<0.01, GLI1 P<0.05).Conclusions:1. The mRNA expressions for the SHH, PTC, and GLI1 as well as the protein expression for the SHH and GLI1 were low in the control group, indicating the signal path was inhibited. However, the mRNA expressions for the SHH, PTC, and GLI1 at 12h and 24h as well as the protein expressions for the SHH and GLI1 at 6h,12h and 24h for the LPS-treated group were all higher than the control group, indicating an activating mechanism for the SHH signaling pathway in the LPS-induced ALI and an obvious increase of the expression quantity.2. After intervention with cyclopamine, the mRNA expressions for the SHH, PTC, and GLI1 as well as the protein expressions for the SHH and GLI1 at 12h and 24h were all lower than the LPS-treated group, and the expression for the signal path inhibited. The pathological injury grading, W/D mass ratio, TNF-a mRNA expression as well as NF-κB expression for the cyclopamine-treated group was, at this time, higher than the LPS-treated group when the SHH signaling pathway expression up-regulation was activated, and the lung tissue was more severely damaged than when the SHH signaling pathway was activated. All the indications speak to the fact that the activation of the SHH signaling pathway may alleviate lung tissue injury and assist the lung tissue recovery after it being damaged.