Protective Effects And Possible Mechanisms Of Colchicine On Lipopolysaccharide(LPS)-induced Myocardial Injury

Part I.Establishment of a model of lipopolysaccharide-induced myocardial injury in mice and the protective effect of colchicine on lipopolysaccharide-induced myocardial injury in miceBackground and objectives:Myocarditis refers to the myocardial inflammation caused by various etiologies.Its clinical manifestations are diverse and patients without suitable treatments are highly susceptible to malignant arrhythmias,cardiogenic shock and even cardiac arrest.Colchicine has a variety of anti-inflammatory properties and offers an effective treatment for a variety of inflammatory diseases.In this section,we will establish a model of lipopolysaccharide(LPS)-induced myocardial injury in mice and to investigate the effects of colchicine on LPS-induced myocardial injury in mice.Methods:The experiments were carried out with male C57BL/6 mice(6-8 weeks old).Mice were randomly divided into three groups:control group,LPS group and colchicine(Col)+LPS group.We established a model of LPS-induced myocardial injury by intraperitoneal injection of 10mg/kg LPS in mice.Mice in LPS group were injected with 10mg/kg LPS intraperitoneally.While mice in the Col+LPS group were pretreated with colchicine 0.4mg/kg for 0.5 hours and then got the LPS injection.An equal volume of normal saline was intraperitoneally injected in mice of the control group.12 hours later,we observed the general condition of each group,evaluated heart function with echocardiography,detected serum LDH and CKMB levels in peripheral blood,collected heart tissues for sectioning and H&E staining,used Western Blot to detect the protein level of NLRP3,cleaved Caspase-1(p20),GSDMD-Nterm,IL-1β,cleaved Caspase-11,and detected the mRNA levels of ASC,IL-1β and NLRP3 using qPCR.Results:(1)Compared with the control group,the general condition of the mice worsened significantly in the LPS group,with reduced food consumption,diminished activity levels,and increased purulent secretions around the eyes.While the general condition of mice in the Col+LPS group improved when compared with the LPS group.(2)Compared with the control group,EF and FS of mice in the LPS group decreased significantly(both P<0.01),and EF and FS of mice in the Col+LPS group increased compared with the LPS group(P<0.01).(3)Compared with the control group,LDH and CKMB levels in the serum of mice in the LPS group were significantly increased(both P<0.01),and those indexes of mice in the Col+LPS group were decreased compared with the LPS group(both P<0.01).(4)Compared with the control group,H&E staining of myocardial tissue of mice in the LPS group showed significant myocardial damage.Myocardial edema,myocardial fiber disarray,accompanied with large amount of inflammatory cells infiltration were observed in the LPS group.While cell damage and inflammatory cell infiltration reduced in the Col+LPS group compared with the LPS group.(5)Compared with the control group,the protein expression of NLRP3,cleaved caspase-1(p20),GSDMD-Nterm,IL-1β,and cleaved caspase-11 were significantly increased in the LPS group(all P<0.01).Protein expression of the above proteins was decreased in the Col+LPS group compared with the LPS group(all P<0.05).(6)Compared with the control group,the mRNA expression levels of ASC,IL-1β and NLRP3 in cardiomyocytes were significantly increased in the LPS groups(all P<0.01).While they decreased in the Col+LPS group compared with the LPS group(all P<0.01).Conclusions:In this section,we successfully established a model of LPS-induced myocardial injury in mice.Pretreatment with colchicine could in part improve the cardiac function and reduce myocardial injury caused by LPS in mice.Part II.Investigation of the protective effects of colchicine on lipopolysaccharide-induced H9c2 cells injury and possible mechanismsBackground and Objectives:Several studies revealed that myocardial injury due to LPS may be associated with pyroptosis of cardiomyocytes.Reduction of pyroptosis may possibly reduce myocardial injury due to LPS.Recent studies about other diseases demonstrated that colchicine can inhibit the activation of NLRP3/caspase-1/ASC inflammasome and thus reduce pyroptosis of cardiomyocytes.But it is unclear whether it can reduce cardiomyocytes pyroptosis due to LPS.In this section,we propose to investigate the mechanisms by which colchicine attenuates LPS-induced myocardial injury.Methods:H9c2 cells were divided into 5 groups and treated with colchicine at 1nM,5nM,10nM,50nM and 100nM for 12 hours,followed by the cell viability test to investigate the highest concentration of colchicine that did not damage H9c2 cells.Then H9c2 cells were divided into three groups:the control group,the LPS group and the Col+LPS group.Cells in the LPS group were treated with LPS 10μg/mL,and cells in the Col+LPS group was pretreated with colchicine at the optimum concentration for 0.5 hours and then treated with LPS 10μg/mL.An equal volume of PBS was added in the control group at the same time.After 12 hours,Calcein AM/PI staining was performed to compare the cell mortality rate of each group,and the LDH level in the supernatant of each cell culture medium was measured.We used Western Blot to detect the protein level of NLRP3,cleaved Caspase-1(p20),GSDMD-Nterm,IL-1β and cleaved Caspase-11.qPCR was used to detect the mRNA levels of NLRP3 and GSDMD.Results:(1)Compared with the control group,H9c2 cells with 1nM,5nM and 10nM colchicine treated for 12 hours showed no significant difference on cell viability(P>0.05),while cell viability decreased after 50nM and 100nM colchicine intervention for 12 hours(P<0.01).(2)GSDMD-Nterm protein expression decreased in the 1nM and 5nM colchicine pretreatment groups compared to the LPS group(P<0.05),and 5nM colchicine pretreatment group showed a more pronounced decrease.We used 5nM colchicine to intervene cells in subsequent experiments.(3)Calcein AM/PI staining indicated a significant increase in cell mortality in the LPS group compared to the control group,and a decrease in the Col+LPS group compared with the LPS group.(4)LDH levels in the supernatant of cell culture medium were significantly increased in the LPS group compared with the control group(P<0.01),and decreased in the Col+LPS group compared with the LPS group(P<0.05).(5)The protein expression of NLRP3,cleaved caspase-1(p20),GSDMD-Nterm,IL-1βand cleaved caspase-11 increased in the LPS group compared with the control group(P<0.01),while the levels of related proteins were decreased in the Col+LPS group compared with the LPS group(P<0.01).(6)The mRNA expression of NLRP3 and GSDMD increased in the LPS group compared with the control group(P<0.01),while decreased in the colchicine+LPS group when compared with the LPS group(P<0.05).Conclusions:The protective effects of colchicine on LPS-induced H9c2 myocardial cell injury may be attributed to the inhibition of caspase-1-dependent and caspase-11dependent pyroptosis pathways by colchicine,suggesting that the protective effects of colchicine on LPS-induced myocardial injury may be related to the reduction of pyroptosis.