Protective Effect Of Shikonin On Endotoxin-induced Myocardial Injury In Mice And Its Mechanism

Background and objective:Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection,which is often associated with severe cardiac insufficiency,namely septic cardiomyopathy(SIC).Studies have shown that the inflammatory injury mediated by NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome plays a vital role in the occurrence and development of sepsis,and inhibiting the activation of the NLRP3 inflammasome may become a new therapeutic target for SIC.As a nicotinamide adenosine dinucleotide-dependent deacetylase,silent information regulator 1(SIRT1)deacetylates nuclear factor-κ B(NF-κ B)and inhibits the transcription of the NLRP3 gene.Therefore,up-regulation of SIRT1 can inhibit NLRP3-mediated inflammatory damage,which may block the occurrence and development of SIC and improve the efficacy of sepsis treatment.As a monomer component extracted from the plant comfrey,shikonin has various biological functions,such as anti-inflammatory,anti-oxidative stress,and anti-tumor activities.Studies have reported that shikonin can improve the survival rate of mice with lethal sepsis caused by cecal ligation.However,does shikonin play a protective effect on SIC?Does it exert anti-inflammatory effects by regulating SIRT1? What is its underlying molecular mechanism? Unfortunately,there have been no reports at home and abroad.In the present study,we aimed to investigate the protective effect of shikonin on endotoxin-induced myocardial injury in mice and explore its molecular mechanism.Methods:(1)A mouse model of endotoxemia was established by intraperitoneal injection of Escherichia coli lipopolysaccharide(LPS)at a 15 mg/kg body weight.Subsequently,modeled mice received intraperitoneal injection of shikonin at a dose of 8 mg/kg body weight at0.5 h before and 12 h after LPS injection,and the 72-h survival rate of mice was observed.Cardiac function was evaluated by echocardiography 18 h after the above treatment.The cardiac tissue was collected for H&E staining and immunohistochemical staining to evaluate the myocardial injury and infiltration of inflammatory cells.Moreover,the TUNEL assay was adopted to detect the apoptosis of cardiac tissue.Meanwhile,The H9C2 cell injury model was established by exposing the cells to LPS(2 μg/m L)and adenosine triphosphate(ATP)(5 m M)for 24 h,and the myocardial cell injury model was intervened by adding different concentrations of shikonin.The cell viability was detected by MTT assay,the cell injury was examined by LDH assay,and the cell apoptosis was assessed by flow cytometry.ELISA was used to detect the levels of inflammatory factors,IL-18 and IL-1?,in murine cardiac tissue homogenate and cell supernatant,and Western blotting analysis was used to detect the expression of NLRP3 inflammasome at the protein level in LPS-treated murine cardiac tissue and cardiomyocytes.(2)The H9C2 cell injury model was established by exposing the cells to LPS plus ATP as above mentioned,and Western blotting analysis was used to detect the expression of SIRT1 and the nuclear translocation of NF-κB p65 in H9C2 cells.Subsequently,in this cardiomyocyte injury model,EX527 inhibitor or si RNA was used to inhibit the activity or expression of SIRT1,and whether shikonin exerted an anti-inflammatory effect through SIRT1 was observed at the cellular level.ELISA was used to detect the levels of inflammatory factors IL-18 and IL-1 ? in the supernatant of cells,and Western blotting analysis was used to detect the levels of SIRT1,NLPR3 inflammasome-related proteins,and the nuclear translocation of NF-κB p65 in cardiomyocytes.The mouse endotoxemia model was established according to the above mentioned method and treated with shikonin and SIRT1 inhibitor EX527(Shikonin was injected intraperitoneally at a dose of 8 mg/kg body weight 0.5 h before LPS injection and 12 h after injection.Additionally,EX527 was injected intraperitoneally at a dose of 5 mg/kg body weight 0.5 h before LPS injection).TUNEL assay was used to detect the apoptosis of cardiac tissue,and the levels of inflammatory factors,IL-18 and IL-1 ?,in cardiac tissue homogenate were also determined.Besides,Western blotting analysis was adopted to detect NLRP3 inflammasome in LPS-treated cardiac tissue.and whether shikonin exerted and anti-inflammatory effect through SIRT1 was observed at the animal level.(3)The transcription factor binding sites in the promoter region of the murine SIRT1 gene were analyzed by bioinformatics technology,and it was found that the promoter region of SIRT1 contained the binding element of forkhead box protein A2(FOXA2).It was speculated that the transcription factor FOXA2 might transcriptionally regulate SIRT1 in LPS-induced cardiomyocyte injury.PCR was used to detect the expression of FOXA2 at the m RNA level in cardiomyocytes.Western blotting analysis was used to detect the expression.Immunofluorescence was used to detect the changes in nuclear translocation of FOXA2.Silencing of FOXA2 was achieved by FOXA2 si RNA,and overexpressing SIRT1 plasmid up-regulated SIRT1 expression.The levels of inflammatory factors IL-18 and IL-1? were assessed by ELISA,cell apoptosis was determined by flow cytometry,and the expressions of FOXA2,SIRT1,and NLRP3 at the protein level were assessed by Western blotting analysis.CHIP-PCR was used to detect whether FOXA2 directly bound to the SIRT1 promoter,and dual-luciferase reporter gene analysis was used to analyze whether FOXA2 up-regulated the transcriptional activity of the SIRT1 gene.Results:(1)After 18 h of intraperitoneal injection of LPS,the cardiac function of the mice was decreased,the level of serum myocardial enzyme was increased,the cardiac histopathological score was increased,the contents of inflammatory factors IL-18 and 1L-1? in cardiac tissue were increased.Inflammatory cell infiltration was enhanced,and apoptosis of cardiac tissue was elevated.However,after treatment with shikonin,the survival rate and cardiac function of mice were improved,the level of serum myocardial enzyme was decreased,the pathological score of cardiac tissue was reduced,the contents of inflammatory factors IL-18 and 1L-1? in cardiac tissue were decreased,and inflammatory cell infiltration in cardiac tissue was decreased.Besides,apoptosis of cardiac tissue was reduced.In addition,we established an H9C2 cell model stimulated by LPS and ATP.We found that 5 μ M of shikonin could significantly reduce the release of LDH in the supernatant,inhibit the release of inflammatory factors,and alleviate cell apoptosis after 24 h treatment with different concentrations of shikonin.The activation of NLRP3 inflammasome was enhanced in LPS-treated myocardial tissue and cardiomyocytes,while shikonin could inhibit the activation of such protein.(2)The expression of SIRT1 at the m RNA and protein levels and its activity were down-regulated in myocardial tissue and cardiomyocytes of LPS-treated animals.The activation of NF-κ B and NLRP3 inflammasome was enhanced,and the inflammatory response was aggravated.Shikonin could inhibit the above changes induced by LPS.In the H9C2 cell model stimulated by LPS and ATP,SIRT1 si RNA or SIRT1 inhibitor EX527 could prevent the inhibitory effect of shikonin on the activation of NF-κB and NLRP3 inflammasome in LPS-treated cardiomyocytes,thereby partially blocking its anti-inflammatory function and weakening the protective effect of shikonin on myocardial cell injury.In addition,inhibition of SIRT1 could reduce the enhanced survival rate by shikonin in endotoxin-induced mice,weaken its anti-inflammatory impact on myocardial apoptosis,and block its inhibitory effect on activation of NLRP3 inflammasome and inflammatory response.(3)In the H9C2 cell model stimulated by LPS and ATP,the expression of FOXA2 at the m RNA and protein levels and its nuclear translocation were enhanced.In contrast,shikonin could inhibit the above changes.Furthermore,FOXA2 si RNA could block the protective effect of shikonin on LPS-induced cardiomyocyte inflammation and apoptosis and weaken its inhibitory effect on activation of NLRP3 inflammasome.However,the above effects of shikonin could be restored after overexpression of the SIRT1 plasmid.Further Chip experiments found that FOXA2 could directly bind to the FOXA2-binding element region of the SIRT1 promoter,and luciferase reporter gene analysis found that shikonin could enhance the transcriptional activity of FOXA2 when binding to the SIRT1 gene.Conclusions:(1)Shikonin could reduce the inflammatory response in myocardial tissue and myocardial apoptosis,protect against endotoxin-induced myocardial injury in mice,improve cardiac function and cardiac pathology scores,and enhance the survival rate of endotoxic mice.The above protective effect of shikonin was related to its inhibitory effect on the NLRP3 inflammasome.(2)Shikonin inhibited the activation of NF-κ B and NLRP3 inflammasome,alleviating the inflammatory response of cardiomyocytes mediated by SIRT1.(3)Shikonin up-regulated and activated the transcription factor FOXA2 and promoted the expression of SIRT1,thereby exerting a protective effect on myocardial injury caused by LPS.