Vitexin Attenuated Smoke Inhalation Induced Acute Lung Injury In Rats And Research On Its Associated Mechanisms

Objective: To establish the SI-ALI rats model,and explore the role of oxidative stress in SI-ALI.To expole the protective effect of vitexin in SI-ALI rat,and study its possible molecular mechanisms.Methods: 1 Establishment and evaluation of the SI-ALI rats model and detection of oxidative stress indexes SD male rats（8 weeks old）were randomly divided into the control group and the smoke inhalation group.According to the different time points after smoke inhalation,the smoke inhalation group was randomly divided into 4 subgroups.The subgroups included the 1 h,6 h,12 h and 24 h times points（n=10）.The SI-ALI rats model was established by using self-designed smoking generator.Rats were sacrificed at different time points after smoke inhalation.Blood and lung samples were harvested for subsequent experiment.Then,the arterial blood gas,W/D ratio,MDA and SOD levels of lung tissue and the histological evaluation of lung tissues were detected.2 The protective effect of vitexin on SI-ALI in rats SD male rats（8 weeks old）were randomly divided into the control group,smoke+DMSO group and smoke+vitexin group（n=12）.Before smoke inhalation,vitexin was intraperitoneal injected into rats at a dose of 3 mg/（kg*d）for 5 days in the smoke+vitexin group,and the equal volume of DMSO was intraperitoneal injected into rats in the smoke + DMSO group.Any treatment was not given to the control group rats.Then the SI-ALI rats model was established as described above.After establishment of the rat model,the arterial blood gas analysis was detected at 1 h time point.The rats were sacrificed at 12 h time point.Lung tissue and BALF samples were collected.The lung W/D ratio,protein concentration of BALF,and lung histological changes were analyzed.Additionally,to evaluate the effect of vitexin on the outcome of SI-ALI,another 18 rats were divided into 3 groups（6 rats/group）and given the same treatments as described above.Survival rate recorded for 72 h,and Kaplan-Meier curves were depicted.3 Vitexin attenuated rats from SI-ALI by inhibition oxidative stress and inflammatory response SD male rats（8 weeks old）were randomly divided into 7 groups.Including the control group,smoke group,smoke+vitexin group,smoke+LY333531 group,smoke+PMA group,smoke+vitexin+LY333531 group and smoke+vitexin+ PMA group（n=6）.Different pretreatments（dose: vitexin,3mg/kg*d LY333531,1mg/kg*d and PMA,1mg/kg*d）were administrated for 5 days before smoke inhalation.Then,the SI-ALI model was established as described above.The rats were sacrificed at 12 h time point.Lung tissue and plasma samples were collected.The MDA,LPO,SOD and GSH-PX levels of lung tissue and plasma,IL-1β,IL-6 and TNF-α levels of lung tissue,expression of PKC β,p66 Shc and phosphorylated p66 Shc,and the histological evaluation of lung tissues were detected.Results: 1 Establishment and evaluation of the SI-ALI rats model and detection of oxidative stress indexes（1）The arterial blood gas analysis: Compared with the control group,PaO₂ was significantly decreased（P<0.05）in each group after smoke inhalation.PaCO₂ was increased at the 1h time points group（P<0.05）,and p H was decreased at the 1h time points group and 24 h times group（P<0.05）.（2）The W/D ratio: The W/D was increased significantly（P<0.05）in each group after smoke inhalation.（3）The Oxidative stress indexes: The MDA levels of lung tissue were increased significantly（P<0.05）in each group after smoke inhalation.However,the SOD levels were decreased significantly（P<0.05）.（4）Lung pathology: After smoke inhalation,the lung tissue showed different degrees of alveolar hyperemia,alveolar septal thickening and inflammatory cell infiltration in each group.2 The protective effect of vitexin on SI-ALI in rats（1）The survival rate: Compared with the control group,the survival rate was significantly decreased（P<0.05）in the smoke+DMSO group.In the smoke+DMSO group,the survival rate of rats was 33.3%,and in the smoke+vitexin group,the survival rate was 66.7%;（2）The arterial blood gas analysis: Compared with the control group,PaO₂ and oxygenation index were significantly decreased（P<0.05）in the smoke+DMSO group.Compared with the smoke+DMSO group,PaO₂ and oxygenation index were increased（P<0.05）in the smoke+vitexin group.（3）The W/D ratio: Compared with the control group,The W/D ratio was significantly increased（P<0.05）in the smoke+DMSO group.Compared with the smoke+DMSO group,The W/D ratio was decreased（P<0.05）in the smoke+vitexin group.（4）The protein concentration of BALF: Compared with the control group,the protein concentration of BALF was significantly increased（P<0.05）in the smoke+DMSO group.Compared with the smoke+DMSO group,the protein concentration of BALF was decreased（P<0.05）in the smoke+vitexin group.（5）Lung pathology: Compared with the control group,the lung tissue showed the alveolar septal thickening,inflammatory cell infiltration,the lung injury score was significantly increased（P<0.05）in the smoke+DMSO group.Compared with the smoke+DMSO group,the lung injury score was decreased（P<0.05）in the smoke+vitexin group.3 Vitexin attenuated rats from SI-ALI by inhibition oxidative stress and inflammatory response（1）The oxidative stress indexes of lung tissue: Compared with the control group,the MDA levels of lung tissue were significantly increased（P<0.05）in the smoke group,smoke+PMA group and smoke+vitexin+PMA group,and the SOD levels were significantly decreased（P<0.05）.The LPO levels of lung tissue were significantly increased（P<0.05）in each group after smoke inhalation,and the GSH-PX levels were decreased（P<0.05）.Compared with the smoke group,the MDA and LPO levels of lung tissue were decreased（P<0.05）in the smoke+vitexin group,smoke+LY333531 group and smoke+vitexin+LY333531 group,and the SOD levels were increased（P<0.05）.The GSH-PX levels of lung tissue were increased（P<0.05）in the smoke+vitexin group,smoke+ vitexin+LY333531 group.（2）The oxidative stress indexes of plasma: Compared with the control group,the MDA levels of plasma were significantly increased（P<0.05）in the smoke group,smoke+PMA group and smoke+vitexin+PMA group,and the SOD levels were significantly decreased（P<0.05）.The LPO levels of plasma were significantly increased in each group（P<0.05）after smoke inhalation.The GSH-PX levels of plasma were significantly decreased（P<0.05）after smoke inhalation except in the smoke+vitexin+LY333531 group.Compared with the smoke group,the MDA and LPO levels of plasma were decreased in the smoke+vitexin group,smoke+LY333531 group and smoke+vitexin+LY333531 group（P<0.05）,the SOD and GSH-PX levels were increased（P<0.05）.（3）The inflammation index of lung tissue: Compared with the control group,the IL-1β,IL-6 and TNF-α levels of lung tissue were significantly increased（P<0.05）in the smoke group,smoke+LY333531 group,smoke+PMA group and smoke+vitexin+PMA group.Compared with the smoke group,the IL-6 and TNF-α levels were decreased in the smoke+vitexin group and smoke+vitexin+LY333531 group（P<0.05）.（4）The result of western blot: Compared with the control group,the expressions of PKC β,p66 Shc and p-p66 shc of lung tissue was increased in the smoke group,smoke+PMA group and smoke+vitexin+PMA group.Compared with the smoke group,the expressions of PKC β,p66 Shc and p-p66 shc of lung tissue was decreased in the smoke+vitexin group,smoke+LY333531 group and smoke+vitexin+LY333531 group.（5）Lung pathology: Compared with the control group,the lung tissues showed different degrees of injury in each group after smoke inhalation.Among them,in the smoke group and smoke+PMA group,the alveolar septal thickening,inflammatory cell infiltration and alveolar hyperemia was the most serious,and the lung injury score was significantly increased（P<0.05）.Compared with the smoke group,the alveolar septal thickening,inflammatory cell infiltration and alveolar hyperemia was mitigated,and the lung injury score was decreased in the smoke+LY333531 group and smoke+vitexin+LY333531 group（P<0.05）.Conclusion 1 The SI-ALI rats model was successfully established,and oxidative stress participated in the development of SI-ALI.2 Vitexin could protect rats from SI-ALI by inhibition oxidative stress and inflammatory response 3 The protective mechanism of vitexin might be partially associated with I the inhibition of PKCβ/p66 Shc pathway.