Luteolin Alleviates LPS-induced Myocardial Injury In Rats Through Activating Nrf2 Pathway And Inhibiting NLRP3 Inflammasome

Background:Sepsis is a life-threatening disease caused by severe systemic inflammation caused by infection.Sepsis myocardial injury(SIMI)is a common complication of sepsis.20-60% of sepsis patients suffer from sepsis-related myocardial injury and cardiac dysfunction,and sepsis patients with myocardial damage have a much higher mortality rate than patients without myocardial damage with a 2-3-fold increase in mortality.However,the treatment of this fatal septic myocardial injury is limited,and supportive care is almost the only option in most cases.Therefore,there is an imperative need to promote basic research on the pathogenesis and therapeutic targets of septic myocardial injury to improve its treatment.Various mechanisms are involved in the pathological process of myocardial injury and cardiac dysfunction induced by sepsis,and inflammation is the initiating factor of sepsis,and excessive inflammation is crucial for the initiation and progression of myocardial injury.Pyroptosis is a type of programmed death associated with inflammation.The main mechanism of pyroptosis is the formation and activation of the classic inflammasome Nod-like receptor protein 3(NLRP3)inflammasome,which leads to the processing and activation of caspase-1,cleaving the precursor IL-1β and precursor IL-18 into mature activated forms,and GSDMD,eventually inducing pyroptosis and cell loss.The NLRP3 inflammasome plays a crucial part in the cardiac damage brought on by sepsis.Many factors can activate NLRP3 inflammasome,including reactive oxygen species(ROS),and Nrf2(nuclear factor E2 related factor)is a key regulator of antioxidant stress response and a key factor of cell protection.Therefore,regulating the interaction between Nrf2 and NLRP3 inflammasome may have vital therapeutic potential in the treatment of myocardial injury in sepsis.In recent years,more and more studies have suggested that natural products such as flavonoids,alkaloids and terpenoids have multi-effect biological activities such as anti-inflammatory and antioxidant with few side effects,providing a good therapeutic prospect for a variety of tissue and organ injuries.Luteolin,a flavonoid found in vegetables and fruits,have been reported that it has anti-inflammatory,antioxidant,antinerve damage,anti-cancer and other effects.According to a previous study,luteolin can protect myocardial damage in diabetic cardiomyopathy by regulating Nrf2-mediated oxidative stress as well as NF-κB mediated inflammatory responses.In the LPSinduced macrophage inflammation model,luteolin inhibited ROS production and NLRP3 inflammasome activation through Nrf2 activation and NF-κB inactivation,thereby preventing THP-1 macrophage pyroptosis.However,there are few studies on the effect of luteolin on myocardial injury in sepsis,and whether luteolin can play a protective role against myocardial pyroptosis in sepsis is still unknown.Therefore,to investigate the role and specific molecular mechanisms of luteolin in sepsis induced myocardial injury,we conducted the following in vivo and in vitro studies.Methods:1.The SD rat model of sepsis was established.Rats in the LPS sepsis model was divided into Control group,drug control group(LUT group),model group(LPS group)and luteolin pretreatment group(LUT+LPS group).By intraperitoneal injection of LPS(10 mg/kg)for 24 hours,the acute rat model of sepsis was established.LUT 15 mg/kg was intraperitoneally injected 30 minutes before LPS treatment.The rat heart was examined by ultrasound before dosing and after modeling.The heart tissue was collected.Hematoxylin-Eosin Stain(HE)was used to observe the histological morphology,and NLRP3 immunohistochemistry and Nrf2 immunofluorescence staining were used to detect the expression levels of NLRP3 and Nrf2 in heart tissue.2.Construct the myocardial cell inflammation model induced by LPS.The cell model was constructed at the H9C2 cell level and divided into Control group,model group(LPS group),luteolin pretreatment group(LUT+LPS group)and inhibitor ML385 group(ML385+LUT+ LPS group).In addition,NAC(ROS inhibitor)was used as a positive control,to detect whether the inhibition of luteolin on the activation of NLRP3 inflammasome depended on ROS,and the Nrf2 gene was knocked down by si RNA technology to verify the effect of luteolin on the activation of Nrf2 signal and the inhibition of NLRP3 inflammasome.The myocardial cell inflammation model was constructed by stimulation with LPS(10 μg/ml)for 24 h.LUT was added to the luteolin pretreatment group 30 minutes before LPS,and Nrf2 inhibitor ML385 was added to the inhibitor group 30 minutes before LUT.3.Serum markers of myocardial injury LDH and CK-MB,inflammatory factors IL-1β and IL-18,and oxidative stress markers SOD and MDA,were evaluated by kit and enzyme-linked immunoassay(ELISA).The ROS production level was detected by DCFH staining;the level of pyroptosis was detected by PI staining.4.Western Blot and q PCR were used to analyze the protein and m RNA expression levels of Nrf2/HO-1 pathway,NLRP3 inflammasome and pyroptosis-related genes.And nuclear/cytoplasmic protein extraction kit was used to extract protein and detect the change of Nrf2 protein expression in the nucleus/cytoplasm.The distribution and abundance of Nrf2 were observed by cellular immunofluorescence to detect nuclear translocation.Results:1.Protective effect of luteolin on myocardial injury in rats with LPS-induced sepsisWe constructed the myocardial injury model of rats with sepsis induced by LPS.In LPS group,compared to Control group,rats had decreased systolic function of the heart(70.5 ± 6.716% vs 81.6 ± 4.98%,P=0.0172)with disordered and irregular myocardial cells.And the release of LDH,CK-MB,MDA,IL-1β and IL-18 increased,and the activity of SOD decreased in LPS group.And the relative expression of NLRP3,ASC,caspase-1,GSDMD,IL-1β and IL-18 associated with NLRP3 inflammasome was significantly up-regulated,compared to Control group.But pretreatment with luteolin mitigated the above changes,especially improving systolic function of the heart(87.17± 6.242% vs 70.5 ± 6.716%,P=0.0003).And the expression of Nrf2 in the nucleus of the LPS group was up-regulated,while the expression of Nrf2 in the nucleus of the LUT+LPS group was more obvious than that of LPS group.The immunofluorescence of Nrf2 cardiac tissue also showed that the abundance of Nrf2 was significantly increased after luteolin pretreatment.These results demonstrated that NLRP3 inflammasome activation and myocardial pyroptosis were increased in the myocardium of LPS-induced sepsis rats,while luteolin pretreatment could improve the LPS-induced cardiac dysfunction and inhibit NLRP3 inflammasome activation and myocardial pyroptosis.Moreover,luteolin pretreatment promoted Nrf2 nuclear translocation and increased the expression of Nrf2 and its target gene HO-1,that is,activated Nrf2 pathway.2.Study on mechanism by which luteolin alleviated H9C2 cardiomyocyte injury induced by LPSWe constructed the H9C2 myocardial inflammatory injury model induced by LPS.In H9C2 cells of LPS group,LDH activity was increased compared with control group,SOD activity was decreased and ROS production was increased.The relative expression levels of NLRP3 inflammasomes and pyroptosis-related molecules were upregulated in the LPS group.But luteolin pretreatment in LUT+LPS group downregulated these changes,which was similar to NAC+LPS group.But in ML385+LUT+LPS group,compared with luteolin pretreatment group,the expressions of inflammasome-related molecules and pyroptosis-related molecules were up-regulated,and the protein expressions of NLRP3,caspase-1 and GSDMD in si Nrf2 group were also up-regulated.Cell pyroptosis with disrupted integrity of cell membrane,detected by LDH and PI staining,was significantly lightened by luteolin pretreatment.And its effect was reduced by ML385.The results suggested that luteolin can inhibit LPSinduced activation of NLRP3 inflammasome and pyroptosis in H9C2 cells by inhibiting ROS production,which was weakened by Nrf2 pathway inhibitor ML385 or Nrf2 knockdown.And in H9C2 cells of LPS group,the relative m RNA and protein expression of Nrf2 pathway-associated molecules were similar,but the expression significantly increased in group with luteolin pretreatment.However,the expression of Nrf2 pathway-related molecules in ML385 pretreatment group was decreased,and the expression level in si Nrf2 group was also decreased.Moreover,the expression of Nrf2 protein in the nucleus in LUT+LPS group was more obvious,in which the effect of luteolin was inhibited by ML385.And Nrf2 immunofluorescence also verified the above changes.In summary,the results suggested that luteolin pretreatment could promote Nrf2 nuclear translocation and increase the expression of Nrf2 and its target gene HO-1,that is,activate the Nrf2 pathway,and then inhibit the activation of NLRP3 inflammasome and alleviate cell pyroptosis.Conclusion:1.This study confirmed that myocardial pyroptosis is involved in myocardial damage in LPS-induced sepsis.2.This study showed that luteolin could alleviate myocardial dysfunction induced by LPS-induced sepsis through anti-pyroptosis effects.3.It was found that luteolin could inhibit the activation of NLRP3 inflammasome and inhibit myocardial pyroptosis by activating Nrf2 signal.