The Interaction Mechanism Study On Anti-inflammatory Effect Of Two Components In Glycyrrhiza Uralensis

BackgroundGlycyrrhiza uralensis（glycyrrhiza）is a commonly used clinical herbal medicine with the effects of tonifying the spleen,benefiting Qi,clearing heat and detoxifying toxins.Modern research has shown that glycyrrhiza has significant anti-inflammatory effects and can be used to treat a variety of inflammatory diseases and has been developed into a variety of preparations.Glycyrrhiza has three main types of active components:triterpenoids,flavonoids,and polysaccharides.To date,studies by several teams,including our subject group,have demonstrated that all three classes of components have strong anti-inflammatory effects.Chinese medicines are characterized by multi-component and multi-target synergistic effects;however,the multi-component synergistic mechanism of anti-inflammatory action of glycyrrhiza is still unclear,which hinders further knowledge of the anti-inflammatory properties of glycyrrhiza and affects the development of new relevant preparations of it.In this paper,we intend to investigate the combined anti-inflammatory effects of the two main active components of glycyrrhiza and explore the mechanisms of their mutual effects,in order to provide a reference for the development and application of glycyrrhiza-related preparations.ObjectiveThe aim of this study was to explore the anti-inflammatory effects of multiple active components of glycyrrhiza and to investigate the reciprocal effects and mechanisms of the combination of components for the treatment of inflammation-related diseases.Methods1.Based on mouse bone marrow-derived macrophages（BMDMs）,LPS-induced NLRP3 inflammasome activation model and H₂O₂-induced oxidative stress model were constructed,and active components regulating inflammatory pathways in glycyrrhiza were screened.Western bolt was used to analyze the expression of Caspase-1 p20,and enzyme-linked immunosorbent assay（ELISA）was used to detect the release of interleukin-1β（IL-1β）,a downstream indicator of inflammasome activation.ROS release and SOD activity of oxidative stress related indexes were determined by flow cytometry and nitrotetrazolium Blue chloride（NBT）method.2.Based on C57BL/6 mice,an LPS-induced mouse sepsis model was constructed to evaluate the therapeutic effects of the active ingredients of glycyrrhiza alone and in combination on septicemic mice,using the survival rate,the level of peritoneal exudate immune cells and the content of serum inflammatory factors as indicators,and the survival rate was counted by continuous observation of the death of mice within 72 h.The percentage of macrophages and neutrophils in the peritoneal lavage fluid was detected by flow cytometry,and the content of serum IL-1βand TNF-αwas determined by ELISA.The ratio of macrophages and neutrophils was measured by flow cytometry,and the levels of serum IL-1βand TNF-αwere determined by ELISA.In addition,we evaluated the anti-septic effect of the composition combination with glycyrrhiza extract.3.Based on C57BL/6 mice,the MCD-induced mouse NASH model was established,and the therapeutic effects of single and combination of active ingredients on NASH model mice were comprehensively evaluated based on the pathological changes of liver tissue,the expression levels of serum liver function evaluation indexes and inflammatory factors,and the expression levels of liver inflammatory factors and fibrosis indicators.The contents of AST,ALT and TNF-αin serum were determined by Lai’s method and ELISA,the m RNA expression levels of IL-1β,TNF-αandα-SMA in liver tissue were detected by QRT-PCR,and the pathological changes of liver were analyzed by hematoxylin-eosin staining（H&E）staining.Results1.In the NLRP3 inflammasome activation model,echinatin significantly inhibited the protein expression of Caspase-1 p20 and the release of inflammatory factor IL-1β;In the H₂O₂-induced oxidative stress model,all components of glycyrrhiza could reduce ROS production,among which the triterpene saponins component glycyrrhizin had the most significant inhibitory effect and significantly increased SOD activity.Based on this,we screened out the important anti-inflammatory ingredients-echinatin and glycyrrhizin in glycyrrhiza,and took them as the research objects for the next experiment.2.In the LPS-induced sepsis model in mice,glycyrrhizin group（high dose）,echinatin group（high and low dose）and combined glycyrrhizin-echinatin group all significantly increased the survival rate of mice,and the best effect was observed in the combined group;all the administered groups significantly reduced the proportion of macrophages and neutrophils in the exuding cells and decreased the serum levels of IL-1βand TNF-αIn addition,the combination group showed a better effect on the level of inflammatory factors released in the peritoneal cavity compared with the glycyrrhiza extract group.3.In MCD-induced mice NASH model,the treatment groups can significantly reduce the levels of ALT,AST,IL-1βand TNF-αin serum,significantly reduce the expression of IL-1β,TNF-αandα-SMA m RNA in liver tissue,and also alleviate the pathological damage and steatosis of liver cells to varying degrees.All the animal indexes in the combined group showed a stronger inhibitory effect.ConclusionIn this study,the in vitro NLRP3 inflammatory vesicle activation model and oxidative stress model were used to screen the main anti-inflammatory and antioxidant components of glycyrrhiza-echinatin and glycyrrhizin,and the therapeutic effects of the combination of the two components were evaluated using sepsis model and NASH model.It was demonstrated that glycyrrhiza multi-component can play a synergistic anti-inflammatory role through different targets:the activation of NLRP3 inflammatory vesicles is regulated by stimulation,which can release a large amount of inflammatory factors to mediate inflammation,and the blockage of this pathway can significantly inhibit the occurrence and development of inflammation;the body produces excessive free radicals during oxidative stress,which can lead to nucleic acid damage and cellular dysfunction,and the inhibition of oxidative stress can effectively repair the cells and tissues damaged by inflammation The inhibition of oxidative stress can effectively repair the cells and tissues damaged by inflammation and reduce the susceptibility of the body to inflammation.The present study confirmed the objectivity of the multi-targeted synergistic anti-inflammatory effect of glycyrrhiza,and discovered the mechanism of the anti-inflammatory interaction effect of the two main active ingredients,which further deepened the knowledge of the anti-inflammatory effect of glycyrrhiza and provided a reference basis for the development of component herbal medicines based on the multi-targeted anti-inflammatory effect of glycyrrhiza.