Protective Effect Of Astragaloside IV On Lipopolysaccharide-induced Myocardial Damage Via The ROS/NLRP3 Pathway

ObjectiveTo observe the myocardial injury in mice induced by lipopolysaccharide(LPS),and to study the protective effects of astragaloside IV(As IV)on the anti-inflammatory and anti-oxidative stress in mouse myocardial tissue.This experiment will further study and explore the relationship between ROS /NLRP3 pathway and myocardial injury,provide new ideas to reduce inflammation and improve myocardial injury,and also provide an experimental basis for the protective effect of As IV on myocardial tissue.MethodsIn vivo experiments: 60 C57 BL / 6J mice,provided by the Experimental Animal Center of Jinzhou Medical University,were randomly divided into 4groups,15 in each group.They are:(1)blank control group,(2)LPS model group,(3)low-dose group(As IV 40 mg / kg)and(4)high-dose group(As IV 80 mg / kg).As IV was dissolved with 0.05% sodium carboxymethyl cellulose(CMC-Na)and administered by gavage.The low-dose group and the high-dose group were continuously given As IV for 14 days,while the blank group and the model group were given intragastrically with the same volume of CMC-Na solution for 14 days.On the fifteenth day,except for the blank control group,the other three groups were given LPS(10mg / kg)to induce inflammation.In vitro experiments: HL-1 cells were incubated in complete medium(90%DMEM + 10% FBS)containing 10% fetal bovine serum,100 IU/ml streptomycin and 100 IU/ml penicillin at 37 °C and 5% CO2.The HL-1 cells were periodically exchanged and passaged at 80%-90% confluence until enough cells were grown for the experiments.The cells were randomly divided into 5 groups:(1)blank control group,(2)LPS model group,(3)LPS + low-dose As IV group(40ng / μL),(4)LPS + high-dose As IV group(80 ng / μL),and(5)inhibitor group(MCC950,10 ng / μL).As IV was dissolved in DMSO to a final concentration of no more than 0.1%,and the NLRP3 inhibitor MCC950 was dissolved in complete medium.Groups 3,4,and 5 were pretreated with As IV(40 ng / μL),As IV(80 ng / μ L),and MCC950(10 ng / μ L),respectively,for 24 hours,followed by treatment with LPS(1 ng / μ L)for 1 hour.Group 2 received treatment with only LPS(1 ng / μL)for 1 hour.Group 1 has no operation.Methods for measuring indicators:(1)Observe the morphological changes of heart tissue by HE staining;(2)Use the Mitochondrial Membrane Potential Fluorescent Probe(JC-1)kit to detect the degree of mitochondrial membrane damage;(3)Reactive oxygen(ROS)sunlight probe(DHE)kit was used to detect ROS content;(4)Western Blot method was used to detect NLRP3,ASC,Caspase-1,IL-1β,IL-18 and TNF-α protein expression(5)Elisa kit was used to detect the expression levels of IL-1β,IL-18 and TNF-α in serum and cell supernatant.ResultsCompared with the blank group,in the tissue section of the model group:Cell aggregation,increased inflammatory cell infiltration,and severe deformation of myocardial tissue fibers;Cell mitochondrial membrane was severely damaged(P <0.05);The ROS content in tissues and cells was significantly increased(P <0.05);Increased expression of NLRP3,ASC,Caspase-1,IL-1β,IL-18 and TNF-α proteins in tissue and cell supernatants(P<0.05);The levels of IL-1β,IL-18 and TNF-α in serum and cell supernatants were significantly increased(P <0.05).Compared with the model group,astragaloside IV can reduce the cell aggregation and inflammatory cell infiltration in tissue sections,and restore the myocardial tissue fiber morphology to normal;Attenuated mitochondrial membrane damage of cells(P <0.05);Decrease ROS content in tissues and cells(P <0.05);Reduce the expression of NLRP3,ASC,Caspase-1,IL-1β,IL-18 and TNF-α protein in tissue and cell supernatants(P <0.05);Decreased the expression levels of IL-1β,IL-18 and TNF-α protein in serum and cell supernatants(P <0.05).Conclusion(1)As IV can significantly protect LPS-induced myocardial damage in mice on anti-inflammatory and anti-oxidative stress.(2)As IV can reduce excessive release of ROS,reduce mitochondrial membrane damage,inhibit the expression of NLRP-3 constituent monomers,reduce the synthesis of NLRP-3 inflammatory bodies,and reduce the inflammatory cytokines IL-1β,IL-18 and TNF-α expression.