The Effects Of Lycorine On Myocardial Injury Induced By Sepsis And Its Mechanism

Research background:Sepsis is defined as multi-organ failure resulting from a severe infectious reaction caused by pathogens,with severe effects on the heart.Cardiac dysfunction caused by sepsis is nearly correlated with the patient’s prognosis.However,some nonspecific treatment strategies commonly used in clinical practice are not ideal in improving cardiac function and mortality rate of septic patients.Hence,it is necessitated to find new and effective specific therapeutic drugs for patients with sepsis to provide better treatment options.AMP-activated protein kinase（AMPK）is a heterotrimer containing different subunits,which is involved in multiple processes such as energy sensing and metabolic regulation.Since the biological function of AMPK in cells is extremely related to the pathomechanism of sepsis-induced myocardial injury,it has been considered as a key target for the treatment of myocardial injury in sepsis.Lycorine（LYC）is a major effective monomer component isolated from bulbs of Lycoris radiata,which belongs to isoquinoline alkaloids in structure.LYC can bind to threonine at the172 site of AMPK domain and activate AMPK,thereby improving peripheral nerve function in diabetic patients.However,there are currently no studies on the application of LYC in sepsis myocardial injury,nor on the important role of AMPK in this process.Research objectives:Based on the above research background,we will put forward the following two research purposes:（1）To investigate the effect of LYC on myocardial injury induced by sepsis;（2）To explore whether LYC exerts the effect of anti-septic myocardial injury by affecting AMPK pathway.Research methods:1.Effect of LYC on CLP-induced myocardial injury in miceBALB/c male mice were stochastically divided according to body weight into:（1）Sham group,model group（CLP group）and LYC pre-protective group（LYC+CLP group）,with dimethyl sulfoxide（DMSO）,DMSO,5mg/kg LYC administered intraperitoneally 5d,3d and 1d before surgery,severally;（2）Sham group,CLP group and LYC post-protective group（CLP+LYC group）,with DMSO,DMSO,5mg/kg LYC administered intraperitoneally 30min and 4h after operation,severally.Sham mice were sutured directly after opening abdominal cavity.All remaining groups of mice replicated the sepsis-induced myocardial injury model by cecal ligation and perforation（CLP）.At 8 h after CLP modeling,the changes in cardiac function,anal temperature,blood biochemical and blood routine parameters of each group of mice were compared by using ultrasonic instrument,anal temperature detector,blood biochemical detector and blood routine detector.The changes of m RNA content in myocardial tissue of each group of mice were compared by q RT-PCR.The changes of protein expression in myocardial tissue of mice in each group were compared by Western blot.And the changes of myocardial tissue pathology of each group of mice were analyzed by histopathological staining.2.Effect of AMPK on the anti-sepsis myocardial injury effect of LYCC57BL/6J wild type（WT）male mice and AMPK knockout(AMPK^+/-)male mice were selected as the LYC+CLP WT group and LYC+CLP AMPK^+/-group,severally.And both groups were administered 5 mg/kg LYC by intraperitoneal injection 5 d,3 d and 1d before surgery.In addition,BALB/c male mice were randomly divided into Sham group,CLP group,LYC+CLP group,LYC+CC+CLP（compound C,CC）group and CC+CLP group according to body weight.DMSO,DMSO,5 mg/kg LYC,5 mg/kg LYC,2.5 mg/kg CC and 2.5 mg/kg CC were intraperitoneal injected 5 days,3 days and1 day before operation,severally.All mice except the Sham group were constructed by CLP surgery as a model of septic myocardial injury.At 8 h after CLP modeling,the changes in cardiac function,anal temperature,blood biochemical and blood routine parameters of mice in each group were compared by using ultrasonic instrument,anal temperature detector,blood biochemical detector and blood routine detector.The changes of protein expression in myocardial tissue of each group of mice were compared by Western blot.Research results:1.LYC can resist myocardial injury induced by CLP in mice（1）Compared with CLP group,the survival rate of LYC+CLP group increased（P<0.05）;anal temperature increased significantly（P<0.001）;sepsis score decreased significantly（P<0.05）;CK,AST,LDH and BUN levels decreased significantly,while ALB levels increased significantly（P<0.05）;WBC,GRA,LYM,MID and PLT in peripheral blood increased significantly,and the number of RBC decreased significantly（P<0.05）;cardiac function indexes such as CO,SV and LVESV increased significantly,while LVPWs and LVPWd decreased significantly（P<0.05）;the m RNA levels of pro-inflammatory cytokines NLRP3,TNF-α,Caspase-1,IL-6 and IL-1βin myocardial tissue increased significantly（P<0.05）;the expression levels of AMPK related proteins such as p-AMPK/AMPK,p-ACC/ACC and PPARγ,mitochondrial related proteins such as NRF1,UCP2 and TFAM,oxidative stress related proteins such as Nrf2,and endoplasmic reticulum stress related proteins ATF6,CHOP and GRP78 in myocardium up-regulated significantly,and the expression levels of some endoplasmic reticulum stress related proteins p-PERK/PERK down-regulated significantly（P<0.05）;the contents of oxidative stress markers NOX2、8-OHd G and ROS in myocardial tissue and the levels of pro-inflammatory cytokines such as TNF-αand Ly6G decreased significantly;inflammatory cell infiltration decreased;myocardial collagen deposition decreased,cardiac fibrosis improved significantly.（2）In addition,compared with the CLP group,the rectal temperature of mice in the CLP+LYC group increased significantly（P<0.001）;Sepsis score decreased significantly（P<0.01）;BUN level in serum decreased significantly（P<0.05）;the number of WBC,GRA and MID in peripheral blood increased significantly,and the number of RBC decreased significantly（P<0.05）;the cardiac function indexes such as CO,SV and LVEDV increased significantly,while the cardiac function indexes such as LVPWd and LVPWs decreased significantly（P<0.05）.2.LYC exerts an anti-CLP mouse myocardial injury effect by activating the AMPK pathway（1）Compared with LYC+CLP WT group,the rectal temperature of LYC+CLP AMPK^+/-mice decreased significantly（P<0.0001）;sepsis score increased significantly（P<0.01）;LDH,BUN and AST levels in serum increased significantly（P<0.05）;the number of WBC,GRA and LYM in peripheral blood decreased significantly,and the number of RBC increased significantly（P<0.05）;the cardiac function indexes such as CO,SV and LVEDV decreased significantly,while the cardiac function indexes such as LVPWd and LVPWs increased significantly（P<0.05）;the expression levels of AMPK related proteins such as AMPK,ACC and PPARγ,mitochondrial related proteins such as UCP2,NRF1 and TFAM,oxidative stress related proteins such as Nrf-2 and HO-1 in myocardium down-regulated significantly,and the expression levels of some endoplasmic reticulum stress related proteins GRP78,p-PERK/PERK and CHOP up-regulated significantly（P<0.05）.（2）In addition,compared with the LYC+CLP group,the rectal temperature of mice in the LYC+CC+CLP group decreased significantly（P<0.001）;Sepsis score increased significantly（P<0.05）;ALB level in serum decreased significantly,and BUN level increased significantly（P<0.05）;The number of WBC,GRA,LYM,MID and PLT in peripheral blood decreased significantly（P<0.05）;cardiac function indexes such as CO,SV,LVESV and LVEDV decreased significantly,and cardiac function indexes such as LVPWd and LVPWs increased significantly（P<0.05）.Research conclusions:1.LYC has a clear protective effect against myocardial injury in sepsis.2.LYC can activate AMPK pathway,thereby restoring mitochondrial function,regulating endoplasmic reticulum stress and inhibiting oxidative stress,thus playing a protective role against myocardial injury in sepsis.