The Study Of The Alterations Of Some Inflammatiory Mediators And The Protective Effect And Possible Mechanism Of Edaravone In The Lipopolysaccharide-induced Acute Lung Injured Rats

ObjectivesTo explore the changes of oxidative stress, inflammatiory cytokinesand the signal pathway of NF-κB in acute lung injury(ALI) throughobserving the alterations of pathology in lung tissues, the expressions ofrelative inflammatiory mediators and NF-κB in thelipopolysaccharide(LPS)-induced acute lung injured rats. To determinethe the possible protective effect and mechanism of edaravone bymeasurements of the pathological alterations in lung tissues, the changesof relative inflammatiory mediators and the level of NF-κB with theintervention of edaravone.Methods72 healthy male Wistar rats were randomly divided into saline (NS)control group (NS group, n=24), model group (LPS group, n=24) andedaravone therapy group (ED+LPS group, n=24). Every group wasfurther grouped by different time-dependent subgroups (2h, 6h and 12h, 8 rats per subgroup). In NS group, the same volume of NS was injectedthrough tail vein, and in LPS group, 10 mg/kg LPS was injected throughtail vein to reproduce the ALI rats models while in ED+LPS group, therats were injected with edaravone (3 mg/kg) just after the injection ofLPS. The general conditions of the rats were observed at the set timepoint. Then the rats were sacrificed and the tissues samples includinglung and blood tissues were collected and stored for relative parametersdetection. The alterations of PaO₂, the wet/dry (W/D) weight ratio of thelung tissues were detected. The gross change of the rats lungs, HEstaining of the lung tissue samples, and the pathological rating weredetermined. The levels of the oxidative injuries parameters such as MPO,MDA, iNOS and antioxidative parameters such as SOD in the lungtissues homogenate were assayed. The levels of TNF-αand IL-10 ofserum were detected by ELISA. The expression of NF-κBp65 of the lungtissues were observed by immunohistochemistry methods and comparedby semiquantitive analysis.Results1. The general conditions observation: the rats of the NS groupbreathed a little more quickly and recovered soon. No symptoms likecyanosis, abnormal excreta were observed. The rats acted agilely to avoidcatching. The rats of LPS group had series of symptoms such as hairerection, accelerated breath frequency, less activity, refusal of drink, watery stool, incapability of escaping while being caught whichdisplayed the most seriously at the time point of 6 h after the injection ofLPS, even with the appearance of cyanosis. 2 rats died for the dyspneicrespiration .While in the ED+LPS group, except for some individual ratshad slight cyanosis, the symptoms of most rats were much slighter thanthat of the rats of LPS group. The rats were more active, drank the waterfreely, had mushy stool and could escape rapidly while facing catching.2. The alterations of PaO₂: the PaO₂ level of the rats in the NS groupfluctuated at about 104.5 mmHg. Compared with NS group, the level ofPaO₂ of the rats of LPS group decreased obviously at all the set timepoint (P＜0.01) and showed its lowest level at time point of 6h after theinjection of LPS. Compared with LPS group, the level of PaO₂ of the ratsof ED+LPS group increased with statistical significance. However, it wasstill lower than that of NS group.3. The alterations of W/D weight ratio of lung tissues: the W/D weightratio of lung tissues of the rats in the NS group was about 3.88.Compared with NS group, the W/D weight ratio of lung tissues in theLPS group increased obviously at all the set time point (P＜0.01) andreached its highest ratio at the time point of 6 h after the injection of LPS.Compared with LPS group, the level of W/D weight ratio of lung tissuesin the ED+LPS group decreased (P＜0.01). However, it was still higherthan that in the NS group (P＜0.01). 4. Pathological changes: by gross specimen observation, in the NSgroup, the lungs of the rats appeared equably pinky white and smoothly.No petechia and swelling was observed. By microscopy, the interalveolarseptum had no thickening. The structure of alveoli was normal and thealveolar space was clear with the filtration of few inflammatory cells.The ALI pathological score was the lowest. In the LPS group, the lungsof the rats appeared to be swelling and congestion. The petechia could befound in the severely injured rats. By microscopy, in the LPS group, theobiviously thickened interalveolar septum, the infiltration ofinflammatory cells and the leakage of erythrocyte could be found.Especially, in the severely injured rats, numbers of alveolar spacecollapsed and pulmonary hyaline membrane formed in alveolar space.Such changes listed above appeared most severely at the time point of 6h after the injection of LPS. The ALI pathological score of the rats of theLPS group was higher than that of NS group (P＜0.01). In ED+LPS group,the according pathological changes were slighter and the ALIpathological score decreased obviously at all the set time point (P＜0.01).However, the ALI pathological score of the ED+LPS group was stillhigher than that of NS group (P＜0.01).5. The alterations of levels of the oxidative and antioxidativeparameters in the lung tissues homogenate: compared with NS group, thelevel of MPO,MDA and iNOS in the LPS group increased at all the set time point (P＜0.01) and the peak level of such parameters appeared at thetime point of 6 h. The level of SOD in the LPS group was lower than thatinthe NS group at all the set time point (P＜0.01). When treated withedaravone, the level of MPO,MDA and iNOS decreased but was stillhigher than that of NS group (P＜0.01) and the level of SOD increasedwith statistically significance (P＜0.01) but was lower than than that ofNS group (P＜0.01).6. The alterations of inflammatory cytokines of the serum: comparedwith NS group, the level of TNF-αin the LPS group increased at all theset time point. The peak level appeared at the time point of 2 h and withthe time last, the level decreased and reached the lowest at the time pointof 12 h but still higher than that in NS group. The therapy of edaravonecould decrease the level of TNF-α. As to IL-10, when the rats wereinjected with LPS, it increased with the time lasting (P＜0.01). However,the intervention of edaravone had no effect on the level of IL-10.7. The expression of NF-κBp65: in the NS group, a few NF-κBp65positive immunologic reactive products scattered in the lung tissue slice.The positive immunologic reactive products located mostly in thecytoplasm. However, in the LPS group, the increasing quantity ofNF-κBp65 positive immunologic reactive products appeared and variedwith the different set time point of LPS stimuli. At the time point of 6h,the numbers of the positive immunologic reactive products reached maximum. The positive dyeing located mostly in the nuclei suggestingthe activation of NF-κB. The IOD of the expression of NF-κBp65 washigher in LPS group than that in NS group at all the set time point(P＜0.01). As to the ED+LPS group, the positively reactive products werealso mostly located in nuclei with decreased intensity. The IOD of theexpression of NF-κBp65 of all the set time point was lower than that ofthe LPS group (P＜0.01), but still higher than that of the NS group(P＜0.01), suggesting the expression inhibition of NF-κB induced by LPSin the lungs tissues.Conclusions1. The ALl rat model can be successfully duplicated by injection ofLPS in vein.2. The injection of LPS can increase the oxidative parameters such asMPO,MDA and iNOS, decrease the antioxidative parameters like SOD;It can also improve the level of serum inflammatory cytokine TNF-αandcause the relative shortage of the level of anti-inflammatory cytokineIL-10. The application of LPS also results the activation of the NF-κBsignal pathway in the lung.2. Edaravone has the protective effect on ALl. The mechanisms of theeffect are due to its ability of clearing the free radicals and inhibiting theactivation of NF-κB. Edaravone can inhibit the accumulation ofinflammatory cells in the lung and the oxidative stress, decrease the level of MPO,MDA and iNOS and increase the activity of SOD; On the otherhand, it can suppress the release of the proinflammatory cytokine TNF-αinduced by LPS and had no effect on the level of anti-inflammatorycytokine IL-10 induced by LPS.