| ObjectivesChronic hepatitis B(CHB)is a serious health problem worldwide,and prolonged hepatitis B can progress to end-stage liver disease such as cirrhosis and primary hepatocellular carcinoma.Magnesium isoglycyrrhizate(MgIG),a single 18α isomer of glycyrrhetinic acid,has been widely recognized as a class of anti-inflammatory and enzyme-lowering drugs for clinical application in the treatment of CHB,but the molecular mechanisms underlying the anti-inflammatory and hepatoprotective effects of MgIG in the treatment of CHB have not yet been investigated.Therefore,this study was conducted to verify the mechanism of action of MgIG injection in the treatment of CHB by combining network pharmacology with experiments to provide new ideas for the diagnosis and treatment of CHB.Methods Part Ⅰ Predicting the potential mechanism of glycyrrhizic acid in the treatment of CHB1.Based on the Swiss Target prediction database,the main targets of glycyrrhizic acid were screened;2.Gene Cards,OMIM,pharm GKB and drug library databases were used to screen CHB-related targets;3.Venn analysis was performed on the screened drugs and disease targets to obtain cross-targets.Cyto Scape 3.8.0 software was used to build a "glycyrrhizic acid-active ingredient-therapeutic target" network;4.Use STRING 11.0 database to construct PPI protein interaction network;5.Screen out the core targets through topological analysis;6.Core targets were analyzed using GO and KEGG.Part ⅡDifferential expression of NLRP3 inflammatory vesicles and their downstream factors in patients with different HBV infection status1.Serum samples of 280 patients were collected,divided into three groups: low re Plication phase(n=100),immune tolerance phase(n=90),and active phase(n=90),and the expression levels of IL-1β and IL-18 were detected by Elisa and the expression levels between different groups were compared;2.Whole blood samples of 150 patients were collected,divided into three groups: low replication phase(n=50),immune tolerance phase(n=50),and active phase(n=50),density gradient centrifugation to isolate perpheral blood mononuclear cells of patients,real-time PCR to detect NLRP3 and Caspase-1 mRNA expression levels in PBMCs,Western blot to detect NLRP3 in PBMCs,Caspase-1 protein expression levels and comparing the differences in expression levels between different groups;3.Collect the pathological tissues of 121 patients,divide them into mild group and severe group according to the degree of liver inflammation,immunohistochemistry to detect the expression of NLRP3 and Caspase-1 in liver tissue,and compare the difference in expression levels between different groups.Part Ⅲ Dynamically observe whether magnesium isoglycyrrhizinate injection improves liver inflammation in CHB patients by inhibiting the expression level of NLRP3 inflammasome and its downstream factors1.This part included 20 CHB patients,who retained peripheral blood samples after a clear diagnosis within 1 week of hospitalization,and retained peripheral blood samples again after 2 weeks of treatment.2.Density gradient gradient centrifugation to separate patients’ PBMCs and detect NLRP3,Caspase-1,IL-1β and IL-18 mRNA expression levels in PBMCs by real-time PCR,and dynamically observe the changes in the expression level of NLRP3 inflammosomes and their downstream factors in PBMCs after 2 weeks of injection of magnesium isoglycyrrhizinate injection.Results Part Ⅰ Potential mechanism of action of glycyrrhizic acid in the treatment of CHB1.In this study,a total of 22 active targets of glycyrrhizic acid and 9188 targets related to CHB were screened,and 19 potential targets of glycyrrhizic acid for the treatment of CHB were identified.2.Target genes such as NR3C1,JUN,STAT3,BCL2L1,AR,IL2,CYP2D6,HTR2 C of glycyrrhizic acid play an important role in the biological network of glycyrrhizic acid in the intervention of CHB.3.GO enrichment results showed that glycyrrhizic acid intervened in CHB and mainly involved 64 biological processes,14 cellular components,and 24 molecular functions.4.The KEGG pathway enrichment results showed that the highest scoring potential inflammatory pathway was the NOD-like receptor pathway.Part Ⅱ Differential expression of NLRP3 inflammatory vesicles and their downstream factors in patients with different HBV infection status1.There were significant statistical differences in gender,age,HBV DNA load,ALT and AST in patients in the low replication group,immune tolerance group and active group(P<0.001).2.The serum levels of IL-1β and IL-18 in patients with HBV infection in the active phase group were significantly higher than those in the low replication group(P<0.001)and the immune tolerance group(P<0.05),and the serum levels of IL-1β and IL-18 in patients with HBV infection in the immune tolerance group were significantly higher than those in the low replication group(P<0.05).3.The expression of NLRP3 and Caspase-1 mRNA in PBMCs of HBV-infected patients in the active phase group was significantly higher than that in the low replication group(P<0.001)and the immune tolerance group(P<0.05).4.The expression of NLRP3 and Caspase-1 mRNA in PBMCs in HBV infected patients was positively correlated with ALT,AST and HBV DNA levels(P<0.01);Serum IL-1β and IL-18 levels were positively correlated with ALT,AST and HBV DNA levels(P<0.01).5.The protein expression levels of NLRP3 and Caspase-1 in PBMCs in patients with active HBV infection were significantly higher than those in PBMCs in patients with low replication and immune tolerance.6.The positive rate of NLRP3 and Caspase-1 in the pathological tissues of CHB patients in the severe liver inflammation group was significantly higher than that in the mild liver inflammation group(P<0.001).The positive rates of NLRP3 and Caspase-1 in the pathological tissues of CHB patients in the severe liver fibrosis group were significantly higher than those in the mild liver fibrosis group(P<0.001).Part Ⅲ Dynamic observation of the expression level of third NLRP3 inflammasome and its downstream factors in PBMCs in CHB patients before and after treatment with magnesium isoglycyrrhizinate injection1.The ALT and AST levels of patients before treatment with magnesium isoglycyrrhizinate injection were significantly higher than those in patients after treatment(P<0.01).2.The expression levels of NLRP3,Caspase-1,IL-1β and IL-18 in PBMCs in patients after treatment with magnesium isoglycyrrhizinate injection were significantly lower than those before treatment(P<0.05).Conclusions1.Licorice acid may play a role in the treatment of CHB by regulating core targets such as NR3C1,JUN,STAT3,etc.,and by affecting the NOD-like receptor signaling pathway.2.NLRP3,Caspase-1,IL-1β and IL-18 levels are correlated with different HBV infection status,which can reflect the degree of liver inflammation and liver fibrosis to a certain extent.3.Magnesium isoglycyrrhizinate injection for the treatment of CHB patients and its role in improving liver inflammation is related to reducing the expression levels of NLRP3,Caspase-1,IL-1β and IL-18 in NOD-like pathways. |
PDF Full Text Request