Research On Anti-inflammatory Active Components Of Salvia Miltiorrhiza And Related Mechanism Based On NLRP3 Inflammasome

Objective:The study was designed the NLRP3 inflammasome pathway as the target,along with exploring the active components of Danshen targeting the NLRP3inflammasome,revealing its mechanism and targets.The aim of the study to clarify the basis and specific mechanism of anti-inflammatory substances of Danshen,and further evaluate its protective effects of the active components on NLRP3 inflammasome-mediated diseases such as lipopolysaccharide-induced endotoxemia syndrome in mice and methionine-and choline-deficient-diet-induced nonalcoholic steatohepatitis（NASH）.It is expected to explore the possibility and safety of Danshen as a therapeutic drug for NLRP3 inflammasome-related diseases,and provide a scientific basis for the development of anti-inflammatory drugs.Methods:A cell model of NLRP3 inflammasome activation was constructed,and the active ingredients cryptotanshinone and tanshinone Ⅰ in Danshen that inhibit NLRP3inflammasome were screened out.Cryptotanshinone and tanshinone Ⅰ anti-inflammatory effects via NLRP3 inflammasome were studied using a combination of in vitro and in vivo experiments.Mouse bone marrow-derived macrophages BMDMs and human macrophages THP-1 were used in vitro experiments.The effect of cryptotanshinone and tanshinone Ⅰ were evaluated using canonical and noncanonical NLRP3 inflammasome,NLRC4 inflammasome and AIM2 inflammasome activation models.It was planned to analyze the effects of cryptotanshinone and tanshinone Ⅰ on its activation from multiple nodes of NLRP3 inflammasome activation,and gradually identify the ways in which cryptotanshinone and tanshinone Ⅰ affect the activation of NLRP3 inflammasomes.The effects of the components of Danshen on the biological activity of BMDMs were determined by CCK8;caspase-1 p20,IL-1βp17 in the cell supernatant and pro-caspase-1、pro-IL-1β、NLRP3 and ASC protein expression in the lysate were detected by western blotting;caspase-1 p20 inflammatory cytokine in the cell supernatant was detected by Caspase-Glo（?）1 Inflammasome Assay;The content of IL-1βand TNF-a in the cell supernatant were determined by ELISA;The level of lactate dehydrogenase（LDH）release,was used to detect cell viability by a LDH kit;The cross-linked oligomerized ASC polymer under the action of DSS（Disuccinimidyl sulfate）were analyzed by western blotting;Intracellular K⁺was measured by inductively coupled plasma mass spectrometry.ATP-induced Ca²⁺flux was measured using the FLIPRT Tetra system.Production of mitochondrial reactive oxygen species（mtROS）in cells was assayed by flow cytometry.To construct a C57BL/6J mouse model of LPS-induced sepsis based on NLRP3 inflammasome:firstly observe the effects of cryptotanshinone and tanshinone Ⅰ on the survival rate of mice.Then observe the general state of the mice after the intervention of cryptotanshinone and tanshinone Ⅰ,the main components of Danshen.The levels of IL-1βand TNF-αin serum and peritoneal lavage fluid were determined by ELISA.The proportion of neutrophils and mono-macrophages in the peritoneal lavage fluid were assayed by flow cytometry.MCD-diet-induced NASH mouse model was constructed to observe the effects of cryptotanshinone and tanshinone Ⅰ on the general state and body weight of mice,too.The ratio of immune cells Th17 and Treg cells in the whole blood were measured by flow cytometry.The level of IL-17A in serum was determined by ELISA.Further,the histopathology observation of liver were performed.The level of ALT and AST in serum were measured by kit.In addition,the expressions ofα-SMA、Col1a1、IL-1β、NLRP3 mRNA in liver were detected by qRT-PCR,and the expression of pro-caspase-1 and caspase-1 p20 protein in liver were determined by western blotting.Results:A plate-based bioluminescence assay for high-throughput screening of inflammasome modulators revealed that various components of Danshen could inhibit the activation of NLRP3 inflammasomes.Comprehensive consideration was given to selecting cryptotanshinone and tanshinone Ⅰ for the next study.Using nigericin as the NLRP3 inflammasome activator,cryptotanshinone and tanshinone Ⅰ could inhibit caspase-1 p20 activation and mature IL-1βp17 secretion in the supernatant of BMDMs cells,but hardly affect on the expression of pro-IL-1β、NLRP3、ASC and pro-caspase-1 protein in the cell lysate.Further investigation found that both cryptotanshinone and tanshinone Ⅰ could inhibit the expressions of caspase-1 p20 and IL-1βp17 protein in the cell supernatant（P<0.05）induced by nigericin,ATP,SiO₂,poly（I:C）,and intracellular transfection of LPS,but not affect those induced by poly（dA:dT）and salmonella.Above results showed that cryptotanshinone and tanshinone Ⅰ can inhibit the activation of canonical NLRP3 inflammasomes and the activation of noncanonical NLRP3 inflammasomes induced by intracellular transfection of LPS,not affect NLRC4and AIM2 inflammasome,indicating that cryptotanshinone and tanshinone Ⅰ can specifically inhibit activity of NLRP3 inflammasome.The activation of NLRP3inflammasome requires two signals,a priming signal and an activation signal.Adding cryptotanshinone before or after LPS has no effect on the production of TNF-αand the expression of pro-IL-1βand NLRP3 caused by LPS pretreatment signal,suggesting that cryptotanshinone specifically affects NLRP3 inflammation activation signal,without affecting the NF-κB signaling pathway within the current dose range.The difference is that adding tanshinone Ⅰ before or after LPS can down-regulate the expression of pro-IL-1β、TNF-αcaused by LPS pretreatment signals,suggesting that tanshinone Ⅰ has an inhibitory effect on the NF-κB signaling pathway.Both cryptotanshinone and tanshinone Ⅰ can inhibit the ASC oligomerization of the canonical and noncanonical NLRP3 inflammasomes,and have no effect on the ASC oligomerization of other NLRC4 and AIM2 inflammasomes,indicating that both cryptotanshinone and tanshinone Ⅰ have an effect on NLRP3 inflammasome assembly.The results showed that both cryptotanshinone and tanshinone Ⅰ had no effect on K⁺efflux during the activation of NLRP3 inflammasome.Cryptotanshinone can reduce the intracellular calcium ion concentration during NLRP3 inflammasome activation process,and the effect will increase with increasing dose.Cryptotanshinone can also reduce the production of intracellular mtROS during the activation of inflammasomes.Cryptotanshinone and tanshinone Ⅰ have inhibitory effects on the NLRP3inflammasome at the overall level of animals.In the LPS-induced septic shock mouse model experiment,both cryptotanshinone and tanshinone Ⅰ reduced the mortality of LPS-induced septic shock mice.In the endotoxemia syndrome C57BL/6J mice model experiment,both cryptotanshinone and tanshinone Ⅰ reduced the increase of IL-1βin serum and peritoneal lavage fluid induced by LPS,but had no significant effect on the protein expression of TNF-α.In addition,cryptotanshinone reduced the ratio of inflammatory cells F4/80 and neutrophils in the peritoneal lavage fluid.In the MCD-diet-induced NASH mouse model experiment,cryptotanshinone and tanshinone Ⅰ can improve the mental state and activity of the model mice,significantly reduced fatty vacuoles and inflammatory cell infiltration in liver;reducing ALT and AST levels in serum（P<0.01）.It shows that cryptotanshinone and tanshinone Ⅰ can improve liver injury.Compared with the model group,the cryptotanshinone intervention group down-regulated the expression ofα-SMA、Col1a1、IL-1βand NLRP3 mRNA in liver（P<0.01）,and decreased the expression level of caspase-1 p20 protein in liver,reducing the content of IL-17A and the proportion of Th17 immune cells in serum significantly（P<0.01）,but there was no significant difference in the proportion of Treg regulatory cells.Conclusions:Both cryptotanshinone and tanshinone Ⅰ can specifically inhibit the activation of NLRP3 inflammasome.Cryptotanshinone exerts anti-inflammatory effects by inhibiting the assembly of inflammasomes,the flow of calcium ions and the production of mitochondrial reactive oxygen species during the activation of NLRP3inflammasomes.Tanshinone Ⅰ exerts an anti-inflammatory effect by inhibiting the assembly of NLRP3 inflammasomes.Both cryptotanshinone and tanshinone Ⅰ can play protective effects on sepsis mice and MCD-diet-induced NASH mice through their anti-inflammatory activity.The results of the study can provide a more solid scientific basis for the clinical application of Danshen.