Effect And Mechanism Of ROS-Mediated Activation Of The NLRP3 In Hepatic Alveolar Echinococcosis

Objective:The immune response plays an important role in resisting disease by acting as a stress mechanism to protect the body from harmful stimuli.The NLRP3 inflammasome plays an important role in Inflammation has received widely attention.The NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasome is a significant component of the innate immune system that plays a vital role in the development of various parasitic diseases.However,its role in hepatic alveolar echinococcosis(HAE)remains unclear.Although the association of NLRP3 inflammasome with other inflammatory factors and the specific signaling pathways mediated in parasitic diseases are still in the early stages of study.Alveolar echinococcosis is a common liver disease and parasitic disease in Qinghai Province.However,investigating the role and mechanism of NLRP3 inflammasome in parasitic diseases can provide ideas for us to find better interventions and treatments.Methods:1.Collect 60 patients with hepatic alveolar echinococcosis who underwent surgery in the Affiliated Hospital of Qinghai University from December 2019 to December2020,and conduct HE staining on the focus tissue.Use immunohistochemistry and Western blot to detect NLRP3,caspase-1,IL-18,IL-1β in the marginal zone of the focus and the corresponding normal liver tissue Detect the expression of NLRP3 and analyze the correlation between NLRP3 expression and clinical characteristics of patients with hepatic alveolar echinococcosis.2.Establish a rat model of Echinococcus multilocularis(E.m)infection,obtain the inflammatory marginal zone around the lesion and corresponding normal liver tissue of rats infected with E.multilocularis.Use flow cytometry to detect the expression of reactive oxygen species(ROS)in the marginal zone of the lesion and corresponding normal liver tissue of rats infected with E.multilocularis,and analyze the relationship between ROS and the relative expression level of NLRP3.Utilizing PBN(N-term Butyl-α-Phenylnitrone inhibits the production of ROS in vivo experiments,and detects the efficiency of PBN at three different concentrations(20,50,and 100 mg/kg/d)in inhibiting the infection of Echinococcus multilocularis in rats,establishing appropriate concentrations.Subsequently,30 rats infected with Echinococcus multilocularis were randomly divided into three groups,with 10 rats in each group.PBN group,saline group,and normal culture group were established to observe the effect of ROS inhibitors on rats infected with Echinococcus multilocularis.3.In vitro experiments,in the transfer model,normal rat hepatocytes(BRL)were placed on the upper layer,Echinococcus multilocularis and rat Kupffer cells were placed on the lower layer.Normal rat hepatocytes,rat Kupffer cells and Echinococcus multilocularis were cultured in high glucose medium containing 10% Fetal bovine serum(FBS).In a co culture system,NAC(N-Acetyl-L-Cysteine)was selected as the ROS scavenger.Cells were treated with NAC at concentrations of 5,10,and 20 m M,and the expression of ROS in Kupffer cells was detected at 24,48,and 72 hours to determine the appropriate concentration.Establish a control group to detect the expression levels of NLRP3,caspase-1,and ROS in Kupffer cells at 24,48,and 72 hours,while also measuring IL-1β in a co-culture system And IL-18 expression.Results:1.By collecting NLRP3,caspase-1,IL-18,and IL-1 from the edge zone of lesions and corresponding normal liver tissues of 60 patients with hepatic alveolar echinococcosis The expression of NLRP3,caspase-1,and IL-1β in the inflammatory marginal zone of patients with hepatic alveolar echinococcosis was detected The expression of NLRP3—caspase-1—IL-1β is significantly increased compared to the corresponding normal liver tissue,indicating that NLRP3—caspase-1—IL-1β The response pathway is activated in the inflammatory marginal zone of patients with hepatic alveolar echinococcosis.2.Clinical pathological analysis shows that the expression of NLRP3 in the inflammatory marginal zone of patients with hepatic alveolar echinococcosis is related to the patient’s jaundice symptoms and Child-Pugh grading,but not to the patient’s age,ethnicity,gender,tumor markers,and size of the primary lesion.The activation of NLRP3 inflammasomes in hepatic alveolar echinococcosis is associated with the grading of liver function in patients with hepatic alveolar echinococcosis,indicating that the activation of NLRP3 inflammasomes directly affects liver function,However,its lack of correlation with serological indicators reveals its early concealment,and we cannot analyze symptoms through routine blood testing indicators.3.By establishing a rat model infected with Echinococcus multilocularis,the inflammatory marginal zone around the lesion and corresponding normal liver tissue were obtained.Flow cytometry was used to detect the expression of ROS in the marginal zone of the lesion and corresponding normal liver tissue of rats infected with Echinococcus multilocularis.The relationship between the relative expression of ROS and NLRP3 was analyzed,and the results showed a positive correlation between the expression of ROS and the relative expression of NLRP3.4.Inhibiting the expression of ROS can effectively inhibit NLRP3—caspase-1—IL-1β in the established rat model of multilocular echinococcus infection Expression,accompanied by NLRP3—caspase-1—IL-1β The pathway was inhibited,and the inflammation of the marginal zone of the lesion in rats infected with Echinococcus multilocularis was alleviated.5.In the Transwell model,the apoptosis of normal liver cells in rats was detected at three time periods of 24 hours,48 hours,and 72 hours.The results showed that there was no statistical difference in the apoptosis rate of liver cells between the NAC treatment group and the co culture group at 24 hours(P>0.05),but at 48 hours(P<0.01)and 72 hours(P<0.05),the apoptosis rate of liver cells in the NAC treatment group was significantly lower than that in the normal co-culture group,indicating that with the downregulation of ROS expression,ROS—NLRP3—caspase-1—IL-1β The inhibition of the response pathway reduces the apoptosis rate of liver cells,and inhibiting the production of ROS can alleviate liver cell apoptosis.Conclusion:1.ROS—NLRP3—caspase-1—IL-1β response pathway is activated in HAE.2.Inhibiting the expression of ROS in HAE rats can inhibit the activation of ROS-mediated NLRP3 inflammasome thereby inhibiting the activation of ROS—NLRP3—caspase-1—IL-1β response pathway.