Study For Emulsified Isoflurane Post-conditioning In Protection Against Lipopolysaccharide-induced Acute Lung Injury In Rats

Partone:Theeffectanddose-responserelationshipof8%emulsifiedisofluranepost-conditioningonLPS-inducedacutelunginjuryinratsObjective To investigate the protective effect of8%emulsified isoflurane (EI)post-conditioning on LPS-induced acute lung injury(ALI) in rats and the dose-effectrelationship.Methods Forty-eight healthy male SD rats,200~250g，were randomly divided into6groups(n=8, each group): control group(C); LPS group(L); low, middle and high dose ofEI post-conditioning group(L-EI, M-EI, H-EI) and the solvent of lipid emulsion group(LE).ALI model in rat was made by tail intravenous injection of LPS(10mg/kg), while the samevolume of saline was injected intravenously into C group. After about two hours,3.5,7.0,10.5ml/kg of8%EI were administered intraperitoneally into L-EI, M-EI and H-EI groupsrespectively, LE group was injected intraperitoneally with10.5ml/kg lipid emulsion, while10.5ml/kg of NS was administered intraperitoneally into C group and LE group. Afterabout6hours, arterial blood gas(ABG) was measured for oxygenation index(OI). Thebronchoalveolar lavage fluid (BALF) and blood samples were taken for the measurementof IL-6and TNF-α by ELISA. The ratio of wet to dry weight of middle lobe of right lungwas measured and the pathological changes of lower lobe of right lung was evaluated.Results Light microscopic examination of lung tissue: the lung in control groupshowed intact tissue structure, clarity alveolar space, without alveolar septal edema,inflammatory cell infiltration, and other special inflammatory performance; While the lungin LPS group displayed severe structural damage, massive inflammatory cell infiltration,alveolar septa with serious edema, hemorrhage, some alveolar collapse, etc; The lung tissue structure was heavily damaged in LE group, with more inflammatory cell infiltration,hemorrhage, alveolar septal edema, some alveolar collapse, etc; Compared with LE group,the improvement was not obvious in lung tissue structure of L-EI group; The lung tissue inM-EI group showed moderate damage, alveolar collapse, inflammatory cell infiltration,alveolar septa moderate edema with hemorrhage; The lung tissue structure is relativelyintact in H-EI group, a small amount of hemorrhage with mild inflammatory cellinfiltration, and the alveolar septa was slightly widened. The lung tissues showed severepulmonary inflammation in L, LE and L-EI groups, moderate pulmonary inflammation inM-EI group and mild pulmonary inflammation in H-EI group. Compared with C group, thelevels of TNF-α and IL-6in serum and BALF, the ratio of W/D and pathomorphologicalintegration of lung increased significantly in the other groups(P<0.05). The levels ofTNF-α and IL-6in serum and BALF, the ratio of W/D and pathomorphological integrationof lung in M-EI group and H-EI group were lower than that in L, LE and L-EIgroups(P<0.05), but no difference was observed among L, LE and L-EI groups(P>0.05),while the difference was significant between M-EI group and H-EI group(P<0.05). Nosignificant difference was observed when compared the baseline of OI with each othergroups(P>0.05); In addition to the C group, compared with the baseline of the same group,the OI significantly decreased at2hours and8hours after the injection of LPS(P<0.05),while2hours after the injection of LPS, the OI was lower than300mmHg suggested asuccessful model of ALI; Compared with the control group, the OI of the other groupsdecreased significantly after the injection of LPS(P<0.05); But8hours after the injectionof LPS, the OI was higher in M-EI group and H-EI group than in L, LE and L-EI groups(P<0.05), and the OI was higher in H-EI group than in M-EI group(P<0.05), while the OIwas higher in M-EI group than in L-EI group(P<0.05); But there was no significantdifference among L, LE and L-EI groups(P>0.05); However compared with2hours afterthe injection of LPS, the OI decreased significantly in L group about8hours after theinjection of LPS(P<0.05), while increased significantly in H-EI group(P<0.05).Conclusion1. Two hours after the injection of LPS(10mg/kg), the model of ALI could be copied.2. Emulsified isoflurane post-conditioning showed protective effect againstLPS-induced ALI in a dose-response manner. Part two: The effects of post-conditioning with8%emulsifiedisoflurane on Toll-like receptor4expression in the lung tissue of ratswith acute lung injuryObjective To investigate the effect of post-conditioning with8%emulsifiedisoflurane on TLR4expression and downstream signal to explore the possible mechanismof post-conditioning with emulsified isoflurane on LPS-induced acute lung injury in rats.Methods Thirty-two healthy male SD rats,200~250g，were randomly divided into4groups(n=8, each group): control group(C), LPS group(L), EI post-conditioning group(EI)and the solvent of lipid emulsion group(LE). L, EI and LE groups were intravenouslyadministered with LPS(10mg/kg), while the same volume of saline was injectedintravenously into C group. After about two hours,10.5ml/kg of8%EI was administeredintraperitoneally into EI group, LE group was injected intraperitoneally with10.5ml/kglipid emulsion and10.5ml/kg of NS was administered intraperitoneally into C group andLE group. Then after about6hours, ABG was measured for OI. The blood samples andBALF were taken for the measurement of TNF-α and IL-6by ELISA. The ratio of wet todry weight of middle lobe of right lung was measured and the pathological changes oflower lobe of right lung was evaluated. The relative protein level of TLR4and the relativemRNA expression of TLR4and NF-κB of the upper lobe of right lung were determined bywestern blot and RT-PCR correspondingly.Results Compared with control group, the levels of TNF-α and IL-6in serum andBALF, and W/D ratio of right middle lobe of lung and pathomorphological integration ofright lower lobe of lung increased significantly in the other groups(P<0.05). Comparedwith L group or LE group, the levels of TNF-α and IL-6in serum and BALF, the ratio ofW/D and pathomorphological integration of lung decreased significantly in EIgroup(P<0.05); While there was no significant difference between L group and LEgroup(P>0.05). No significant difference was observed when compared the baseline of OIwith each other groups(P>0.05); In addition to the C group, compared with the baseline ofthe same group, the OI significantly decreased at2hours and8hours after the injection ofLPS(P<0.05), and2hours after the injection of LPS, the OI was lower than300mmHg suggested a successful model of ALI; And8hours after the injection of LPS, the OI wasobviously higher in EI group than in L and LE groups(P<0.05), but no significantdifference was observed between L group and LE group (P>0.05); Compared with2hoursafter the injection of LPS, the OI decreased significantly in L group about8hours after theinjection of LPS, while increased significantly in EI group(P<0.05).Conclusion Post-conditioning with EI can inhibit the expression of TLR4andactivation of NF-κB. And the secretion of inflammatory factor also can be decreased byemulsified isoflurane. Therefore the down-regulation of TLR4and its downstream signaltransduction may be one of the mechanism through which emulsified isofluranepost-conditioning can alleviate LPS-induced acute lung injury in rats.