Maackiain Plays A Protective Role In Sepsis By Activating AMPK/Nrf2/HO-1 Pathway

Sepsis is a life-threatening syndrome caused by infection-triggered aberrant immune responses,leading to host tissue and organ injury.Despite advances in medical interventions,the mortality rate for septic shock remains high.The pathophysiological mechanism of sepsis-induced organ dysfunction is complex,involving endothelial dysfunction,immune system disorders and other aspects.Oxidative stress is caused by the imbalance between antioxidation and prooxidation of the body,which is involved in the pathological process of many diseases.Recent studies highlight the role of oxidative stress in the occurrence and development of sepsis,providing a potential therapeutic target for preventing sepsis-associated organ injury.Natural compounds have attracted increasing attention due to their pharmacological properties and are widely used to treat a variety of diseases.As one of the important components of Sophora flavescens,a traditional Chinese herbal medicine,Maackiain has been shown to have antioxidant,antibacterial,and anti-tumor effects,but its effect on sepsis is still unclear.In this study,we found that Maackiain exerted a protective role in a cecal ligation and puncture(CLP)-induced murine model of sepsis and we demonstrated the corresponding targets and pathways.(1)Protective effect of Maackiain on organs in septic mice.In this part of the study,the sepsis mice model was established by CLP method,and the mice were intraperitoneally injected with different doses of Maackiain(0mg/kg,2.5mg/kg,5mg/kg).After treatment,the survival of the septic mice was monitored and the clinical scoring was completed.The damage degree of major organs and tissues was evaluated by hematoxylin-eosin staining.The results showed that Maackiain significantly reduced the mortality rate of septic mice(P < 0.05)and improved the prognosis in a dose-dependent manner.According to the clinical manifestations,the clinical scores of septic mice were scored.The results showed that the clinical scores of septic mice were significantly improved after pretreatment with Maackiain in a dose-dependent manner(P < 0.05).The lung,liver,spleen,and kidney of septic mice were stained with hematoxylin-eosin,and the pathological changes such as inflammatory cell exudation and cell necrosis in septic mice were significantly reduced in the Maackiain treatment group compared with those in the group without Maackiain treatment,and the organ damage in septic mice was significantly reduced by treatment with Maackiain.In order to clarify the reason of the antagonism of Maackiain against septic injury,we first excluded the antibiotic-like bactericidal ability of Maackiain by the antibacterial experiment.Then,serum levels of cytokines IL-1,IL-6,and TNF-α and oxidative stress-related markers GSH,TEAC,and MDA in septicmice were measured using ELISA.The results showed that Maackiain significantly reduced the levels of IL-1,IL-6 and TNF-α in septic mice,and could significantly inhhibit oxidative stress in septic mice(P < 0.05).Maackiain had obvious anti-inflammatory and anti-oxidative effects.These results suggest that Maackiain plays a protective role in sepsis-induced organ damage.(2)Effects of Maackiain on LPS-induced inflammatory response and oxidative stress in macrophages.Before LPS stimulation,RAW264.7 cells were pretreated with different concentrations of Maackiain(0 ng/m L,50 ng/m L and 100 ng/m L).The levels of cytokines IL-1,IL-6 and TNF-α in the supernatant and the levels of oxidative stress-related indicators GSH,TEAC and MDA were detected by ELISA.The nuclear translocation of NFκB was evaluated by immunofluorescence technique,and the mitochondrial membrane potential of RAW264.7 cells was detected.The results showed that Maackiain significantly inhibited the LPS-induced increase of cytokines IL-1,IL-6 and TNF-α in RAW264.7 cells(P < 0.05),and also inhibited the activation of NFκB.It also significantly antagonized LPS-induced decreases in GSH and TEAC levels and increases in MDA levels(P < 0.05),and significantly reversed LPS-induced mitochondrial membrane potential damage in the cells.These results suggest that Maackiain also has anti-inflammatory and anti-oxidative effects in vitro.(3)Study on the mechanism of protective effect of Maackiain in sepsis.The above experiments have proved the anti-inflammatory and antioxidative effects of Maackiain both in vivo and in vitro.In this part of the study,we further explored the specific mechanism of anti-inflammatory and antioxidative effects of Maackiain.The results showed that Maackiain could activate the AMPK/Nrf2/HO-1 pathway,and by the use of AMPK or Nrf2 inhibitors,the anti-inflammatory and anti-oxidative effects of Maackiain,as well as the improvement of prognosis and reduction of organ damage were offset in septic mice or LPS-induced RAW264.7 cells.In addition,by molecular docking analysis,we found that Maackiain directly interacted with AMPK to activate AMPK.In summary,the present study demonstrated that the anti-inflammatory and anti-oxidative effects of Maackiain in sepsis were mediated through activation of AMPK/Nrf2/NO pathway.Therefore,Maackiain has the potential to treat sepsis and provides a new option for the treatment of sepsis.