The Anti-inflammatory Effect And Mechanism Of Active Components Of Rabdosia Japonica Var.glaucocalyx Via Targeting NLRP3 Inflammasome Activation

Rabdosia japonica var.glaucocalyx has the bitter,sweet and cold characteristic,which has the effect of relieving heat and diuresis,promoting blood circulation and dispersing blood stasis,detoxification and detumescence.As a traditional herbal medicine,it has been reported to possess marked anti-inflammatory properties and is widely used in the treatment of various inflammatory diseases,such as hepatitis,gastritis and so on.At present,it is known that Glaucocalyxin A(GLA),an ent-kauranoid diterpenoid component,is the main anti-inflammatory active component in Rabdosia japonica var.glaucocalyx.But the anti-inflammatory mechanism of GLA remains unclear.Inflammasomes are a group of protein complexes assembled by cytoplasmic pattern recognition receptor(Pattern Recognition Receptors,PRRs).It is an important part of human innate immune system and plays a key role in the process of the occurrence and development of many inflammation-related diseases.NLRP3inflammasome is the most widely studied type of inflammasomes at present.Targeted regulation of NLRP3 inflammasome pathway is also the molecular mechanism of the role of a variety of anti-inflammatory drugs,but whether GLA achieves anti-inflammatory effect by regulating NLRP3 inflammasome pathway has not been reported.Objective This paper aims to explore the regulatory effect and mechanism of GLA on NLRP3 inflammasome pathway from the aspects of molecule,cell and whole animal.The anti-inflammatory effect and mechanism of GLA,the active component of Camellia oleifera,were studied by using mouse septic shock model and macrophage inflammation model in vitro,so as to expand herb's clinical application of in the prevention and treatment of inflammatory diseases and to provide a new scientific basis for the development and utilization of Rabdosia japonica var.glaucocalyx's resources.Methods To establish the activation model of NLRP3 inflammasome,bone marrow-derived macrophages(BMDMs)were primed with lipopolysaccharide(LPS),and then stimulated by nigericin.LPS-primed BMDMs were pretreated with GLA,and then given nigericin or other stimuli to observe the effect of GLA on the activation of NLRP3 inflammasome.Caspase-Glo~?1 Inflammasome Assay and ELISAs were used to detect the activity of caspase-1,the secretion of interleukin(IL)-1?and tumor necrosis factor-?(TNF-?)in the culture supernatant.Lactate dehydrogenase(LDH)assay kit was used to quantitatively detect cell viability,and TUNEL detection kit was used to directly detect apoptosis caused by activation of NLRP3 inflammasome.Western blotting was used to detect the production of mature IL-1?and caspase-1 in the supernatant and the expression of NLRP3 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain(ASC),pro-caspase-1 and pro-IL-1?in the cell lysate.In addition,BMDMs were also used to further study the effects of GLA on poly(dA:dT)-stimulated the absent in melanoma-2(AIM2)and Salmonella-stimulated NOD-like receptor family caspase-specific protease activation recruitment domain 4(NLRC4)inflammasome.The expression of NLRP3 and pro-IL-1?protein in cell lysate was detected by Western blotting to explain the effect of GLA on NLRP3 inflammasome and TLR4 signaling pathway.The degree of mitochondrial damage induced by nigericin was detected by immunofluorescence to explore the inhibitory mechanism of GLA on NLRP3 inflammasome activation.At the same time,the oligomerization of ASC in the supernatant and the expression of ASC in the cell lysate were further detected by Western blotting on the basis of the activation of NLRP3inflammasome so as to clarify the mechanism.In the in vivo experiment of septic shock model in mice induced by intraperitoneal injection of LPS,the effect of GLA on the NLRP3 inflammasome activation was further verified.Results1.GLA could inhibit the activation of NLRP3 inflammasome and cell pyrogenesis by inhibiting pro-caspase-1 cleavage and blocking caspase-1 p20 cutting pro-IL-1?.It also inhibited the activation of NLRC4 inflammasome induced by Salmonella,but had no effect on the activation of AIM2 inflammasome induced by poly(dA:dT).2.GLA could inhibit NLRP3 and NLRC4 inflammasomes activation via blocking ASC oligomerization.3.GLA significantly reduced the mortality caused by LPS-induced bacterial infection by inhibiting NLRP3 inflammasome-dependent inflammatory factors.Conclusion Glaucocalyxin A,active component of Rabdosia japonica var.glaucocalyx,could suppress the BMDMs macrophage activated by NLRP3inflammasome and ameliorate LPS-induced septic shock and inflammation.Therefore,the paper clarified the anti-inflammatory effect and mechanism.It provides a natural active compound with development prospect for the treatment of inflammation-related diseases mediated by NLRP3 inflammasome,and also provides a reference basis for the development,utilization and quality control of Chinese herbal medicine resources of Rabdosia.