Study On The Preventive Effect And Mechanisms Of Rac1 Inhibition On Hepatic Ischemia-Reperfusion Injury In Mice

Backgrounds:As a major risk factor for post-operative complication and mortality,hepatic ischemia-reperfusion injury（IRI）not only leads to liver dysfunction and failure but also affects other remote organs,and in sever cases,causing multiple organ dysfunction syndrome（MODS）or systemic inflammatory response syndrome（SIRS）,which severely affects patient’s survival and quality of life（Qo L）.As one of the most dangerous factor during hepatic IRI,ROS is produced both during ischemia and reperfusion phases.Reducing ROS production has been proved to be an effective way for alleviating oxidative stress in various IRI animal models.According to previous studies,inhibition of Ras-related C3 botulinum toxin substrate1（Rac1）,a small guanosine triphosphatases（GTPases）could reduce ROS production and prevent oxidative stress injury in several animal models.Interestingly,previous studies suggested a positive inter-activation feedback loop between Rac1 and hypoxia inducible factor（HIF）-1α,the latter is up-regulated early during ischemia and exerts some protective effects against hypoxia.However,the positive inter-activation between Rac1and HIF-1αwould aggravate ROS production,thereby promoting IRI.Objectives:To verify the effects of Rac1 inhibition on hepatic IRI both at animal and cellular levels and to explore the interaction between Rac1 and HIF-1αduring hepatic IRI.Methods:C57BL/6 mice were randomly assigned to be intraperitoneally injected with2.5mg/kg of NSC23766（a specific Rac1 inhibitor）,50mg/kg of N-Acetyl-L-cysteine（NAC,a postive control drug）or same volume of PBS（as negative control）on alternative days for 2weeks before underwent sham or hepatic IRI surgery.Blood samples were collected by eyeball removing,and then mice were sacrificed by cervical dislocation and liver samples were collected.The content of ALT/AST in serum and liver tissues were analyzed.Hematoxylin-eosin（H&E）staining and Terminal Deoxynucleotidyl Transferase（Td T）-mediated d UTP Nick-End Labeling（TUNLE）for cell apoptosis analysis were conducted.The markers for macrophages（F4/80）and neutrophils（Ly6G）were analyzed by immunohistochemistry（IHC）analysis.And ROS level in liver tissue was detected.For mimicking hepatic IRI condition,alpha mouse liver cell line（AML-12）was put in an incubator with 95%N₂and 5%CO₂at 37℃,and was incubated with DMEM/F12 cell culture medium containing 1%fetal calf serum（FBS）,24 hours later,cells were put in normal condition（21%O₂and 5%CO₂,37℃）with normal culture medium（containing 10%FBS）changed for reoxygenation.At designed time points,cells were collected for CCK8 drug toxicity detection,mitochondrial membrane potential（MMP）detection,lactate dehydrogenase（LDH）cytotoxicity assay,flow cytometry apotosis detection and western blotting analysis of apoptosis and DNA damage-related proteins.The dose of NSC23766 used for cell experiments was 50μM.Rac1-knockdown（Rac1-SH）and negative control（Rac1-NC）AML-12 cells were constructed using lentiviral-vectors.Besides,the influence of Rac1knockdown on the m RNA levels of inflammation-related factors in AML-12 cells during IRI was detected by real-time quantitativepolymerase chain reaction（q PCR）.And the effect of Rac1 inhibition/knockdown on HIF-1αsignaling during hepatic IRI was analyzed by WB.Results:Compared with PBS,NSC 23766 treatment significantly alleviated the hepatic IRI in mice,manifesting as less vacuolation score and apoptosis cells,lower ROS and serum/liver ALT/AST levels,and fewer activation of inflammatory cells,suggesting that Rac1inhibition could effectively alleviate the hepatic IRI in this mouse model.IRI of AML-12 was also alleviated by 50μM NSC 23766 or Rac1-knockdown,manifesting as reduction in cell apoptosis,less interruption in MMP,down-regulating the apoptosis and DNA damage-related proteins up-regulated by IRI.Interestingly,Rac1 inhibition/knockdown could inhibit the up-regulation of HIF-1αinduced by ischemia-reperfusion process through reducing the phosphorylation of p21-activated kinase 1（PAK-1）.Conclusion:Our study supports a protective effect of Rac1 inhibition on hepatic IRI.Aside from the classic topics of reducing ROS production and alleviating oxidative stress,our study showed an interaction between Rac1 and HIF-1αsignaling during hepatic IRI,suggesting that there exists more sophisticated mechanisms by which Rac1 inhibition mediate its protective effects.