| Objective1. To observe the effects of dexmedetomidine hydrochloride pre-conditioning on acute lung injury and the inflammatory factors induced by intestinal ischemia-reperfusion(I/R) in rat.2. To explore whether a2-adrenoreceptor play a role in dexmedetomidine hydrochloride pre-conditioning on acute lung injury induced by intestinal I/R, providing a new laboratorial and theoretical basis for the clinical application.3. To investigate the role of TLR4/MyD88/NF-κB and PI3K/Akt signaling pathways in the dexmedetomidine hydrochloride pre-conditioning reducing acute lung injury induced by intestinal I/R. In order to study the molecular biological mechanisms of the effects of dexmedetomidine hydrochloride on acute lung injury.Methods1. To study the effects of dexmedetomidine hydrochloride pre-conditioning on acute lung injury induced by intestinal I/R. We detect the changes of arterial blood gas analysis, wet/dry(W/D) ratio of the lung, pathologic examination, and the concentration of inflammatory factors in bronchoalveolar lavage fluid(BALF) using different doses of dexmedetomidine hydrochloride preconditioning on an intestinal I/R model in vivo.48healthy male Sprague-Dawley ratswere randomly divided into four groups, including sham group(Sham), intestinal ischemia-reperfusion(IR) group,2.5ug/(kg-h) dexmedetomidine hydrochloride pre-conditioning(DL) group and5ug/(kg-h) dexmedetomidine hydrochloride pre-conditioning(DH) group. Rats of the sham group underwent only a laparotomy without clamping the anterior mesenteric artery. In the IR group, after continuous infusion of0.9%NaCl for1h via the caudal vein, the anterior mesenteric artery was clamped with a noninvasive micro vascular clip for1h and then reperfusion for2h. In dexmedetomidine pre-conditioning groups(including the DL and DH group), dexmedetomidine hydrochloride was continuously infused at the rate of2.5ug/(kg-h) or5ug/(kg-h) for1h via the caudal vein before intestinal I/R. During the whole operation,0.9%saline was administered at1.5ml/h via caudal vein for fluid maintainance. At the end of each experiment,0.5ml of blood was drawn from the left ventricular for arterial blood gas analysis. Then all the rats were sacrificed by bleeding out through the right ventricular. Six rats of each group were operated bronchoalveolar lavage for determining the concentrations of TNF-α and IL-6in BALF. The middle lobe of right lung of the other six rats were tied and removed for wet/dry ratio analysis, and the rest parts of the lung were perfused with4% formaldehyde and then removed for HE staining.2. To explore the role of a2-adrenoreceptor in the effects of dexmedetomidine hydrochloride pre-conditioning on acute lung injury induced by intestinal I/R.48healthy male rats were randomly divided into4groups, including IR group, DH group, yohimbine hydrochloride+dexmedetomidine hydrochloride pre-conditioning(YD) group, yohimbine hydrochloride pre-conditioning(Y) group. In the IR group, after continuous infusion of0.9%NaCl for1h via the caudal vein, the anterior mesenteric artery was clamped with a noninvasive microvascular clip for1h and then reperfusion for2h. In the DH group, dexmedetomidine hydrochloride was continuously infused at the rate of5ug/(kg-h) for1h via the caudal vein before intestinal I/R. In the Y group, yohimbine hydrochloride(1mg/kg,>15min) was given before the continuous infusion of0.9%NaCl at the rate of1.5ml/h for1h, then rats in this group underwent intestinal I/R. In the YD group, yohimbine hydrochloride(1mg/kg,>15min) was given before the continuous infusion of dexmedetomidine hydrochloride at the rate of5ug/(kg-h) for1h via the caudal vein, then rats were subjected to intestinal I/R. At the end of each experiment,0.5ml of blood was drawn from the left ventricular for arterial blood gas analysis and then all rats were sacrificed by bleeding out through the right ventricular. The test items and techniques were as same as the first part. 3. To investigate the role of TLR4/MyD88and PI3K/Akt signaling pathways in dexmedetomidine hydrochloride pre-conditioning reducing acute lung injury induced by intestinal I/R.36healthy male SD rats were randomly divided into6groups, including Sham group, IR group, DL group, DH group, YD group and Y group. The dosage of dexmedetomidine hydrochloride was chosen5ug/(kg-h). The reagent and the model building were as same as the above two parts. At the end of each experiment, all rats were sacrificed by bleeding out through the right ventricular, and then the lung were removed. The expression of TLR4mRNA and MyD88mRNA in the lung tissues were detected by real-time polymerase chain reaction (RT-PCR), and the levels of phosphorylation of IκBα and Akt were detected by western blotting.Resuts1. There were no statistical differences among Sham, IR, DL, DH groups in arterial blood gas results(P>0.05). However, the pathological examination of the lung tissue showed different results. In the sham group, the pulmonary structure was almost normal, there were little exudation in the alveolar space, no congestion or bleeding in the capillary. In the IR group, the alveolar walls were thick along with alveolar edema, hemorrhage and a lot of inflammatory cells infiltrating into the pulmonary interstitial, which indicated that the lungs were severely damaged. Compared with IR group, the lung injury in DL and DH groups were significantly reduced, the exudation in the alveolar space was decreased, only a small part of the alveolar septa was slightly thickened and a small amount of inflammatory cells were infiltrated. The tendency of pathological scoring results was in accordance with the change of the pathological results. The pathological scoring of lung injury in DL and DH groups were lower than that in the IR group(P<0.05). The lung W/D ratio of DL and DH groups were lower than that of the IR group respectively(P<0.01), so did the concentrations of TNF-a and IL-6in BALF. When compared DH group with DL group, the pathological scoring, the lung W/D ratio and the concentrations of TNF-a and IL-6in BALF of DH group were lower than those of DL group(P<0.05).2. There were no statistical differences among IR, DH, YD, Y groups in arterial blood gas results(P>0.05). The lung W/D ratio of DH and YD groups were lower than that of the IR group(P<0.01), but the W/D ratio of YD group was still higher than that of the DH group(P<0.01); there was no difference between the Y group and IR group(P>0.05). The lung tissue pathological results showed that the lungs were damaged severely in IR and Y groups, but the lung injury in the DH group was improved; the lung was middle damaged in the YD group but significantly improved compared with that in the IR group. The tendency of pathological scoring was in line with the HE staining results. The pathological scoring was lower in DH and YD groups than those in IR and Y group(P<0.05), but there was no statistical difference between Y and IR group(P>0.05); but the pathological scoring of YD group was higher than that of the DH group statistically(P<0.01). The concentrations of TNF-α and IL-6in BALF of DH and YD groups reduced compared with the IR group(P<0.01), but there was no statistical difference between Y and IR groups(P>0.05); the concentrations of TNF-α and IL-6in BALF of DH group was lower than those of YD group statistically(P<0.01).3. The expression of TLR4mRNA and MyD88mRNA was increased in the IR group compared with the sham group according to the RT-PCR detection(P<0.05). The phosphorylation of IκcBα in lung tissue also increased according to western blotting results in the IR group, but the phosphorylation of Akt in the IR group has no statistical difference(P>0.05). Compared with the IR group, the expression of TLR4mRNA, MyD88mRNA, and the phosphorylation of IκBα in lung tissue were decreased(P<0.05), but the phosphorylation of Akt increased in DH and YD groups statistically(P<0.05). Compared with the YD group, the expression of TLR4mRNA, MyD88mRNA and the phosphorylation of IκBα in lung tissue were inhibited further in DH group(P<0.05), but the phosphorylation of Akt increased in DH group statistically(P<0.05).The expression of TLR4, MyD88mRNA, the phosphorylation of IκBα and Akt in lung tissue of Y group were not statistically different compared with that of the IR group(P>0.05).Conclusions1. Dexmedetomidine hydrochloride pre-conditioning reduced the acute lung injury and inflammation induced by intestinal I/R through a dose-dependent manner.2. Dexmedetomidine hydrochloride was capable of attenuating the lung injury and inflammation induced by intestinal I/R in rats via both a2-adrenoreceptor dependent and independent mechanisms.3. TLR4/MyD88/NF-κB and PI3K/Akt signaling pathways played role in the protective effect of dexmedetomidine hydrochloride pre-conditioning on lung injury induced by intestinal I/R and the effect may be partly depended on the α2-adrenoreceptor-mediated mechanism. |
PDF Full Text Request