| Objective:To analyze the possible material basis and mechanism of Yinchenhao Decoction in regulating NLRP3（Nucleotide oligomerization domain-like receptors containing pyrin domain 3,NLRP3）inflammasome,and to explore the possible efficacy and mechanism of Yinchenhao Decoction and its main components in preventing and treating NASH（Nonalcoholic steatohepatitis,NASH）by regulating NLRP3 inflammasome.Methods:1.Yinchenhao Decoction improved NASH by regulating inflammasome:（1）Mouse BMDMs（Bone marrow-derived macrophages,BMDMs）were pretreated with Yinchenhao Decoction and drugs derived from Yinchenhao Decoction,and then stimulated with LPS（Lipopolysaccharide,LPS）,LPS+nigericin,LPS+poly dAdT and LPS+MDP respectively,the expression of IL-1β（interleukin-1 beta,IL-1β）and TNF-α（Tumor necrosis factor-alpha,TNF-α）were detected.（2）Mouse BMDMs were pretreated with Yinchenhao Decoction and drugs derived from Yinchenhao Decoction,and then stimulated with LPS+cholesterol,the expression of IL-1β and TNF-α were detected;（3）C57 mice were fed with MCD（Methionine and choline deficiency,MCD）diet and intervened with Yinchenhao Decoction and drugs derived from Yinchenhao Decoction as well as inflammasome blockers,liver histopathology（H&E staining）,liver function ALT（Alanine aminotransferase,ALT）and AST（Aspartate aminotransferase,AST）as well as serum inflammatory factors IL-1β and TNF-α were detected.2.The main components of Yinchenhao Decoction improved NASH by regulating inflammasome:2.1 To screen the active components of Yinchenhao Decoction in regulating NLRP3 inflammasome:（1）Mouse BMDMs were pretreated with the main components of Yinchenhao Decoction,and then stimulated with LPS+nigericin,the expression of IL-1β and TNF-α were detected;（2）Mouse BMDMs were pretreated with Rhubarb anthraquinones and then stimulated with LPS+ATP（Adenosine triphosphate,ATP）,LPS+alum（Aluminum potassium sulfate,Alum）,LPS+SiO2（Silicon dioxide,SiO2）and LPS+CPPD（Calcium pyrophosphate,CPPD）,the expression of IL-1β and TNF-αwere detected;（3）Mouse BMDMs were pretreated with Rhubarb anthraquinones and then stimulated with LPS+nigericin and LPS+ATP,The splicing form of pro-caspase-1 was detected;（4）Mouse BMDMs were pretreated with Rhubarb anthraquinones and then stimulated with LPS+nigericin,ASC（Apoptosis-associated speck-like protein containing CARD,ASC）fluorescent spots were detected;（5）Mouse BMDMs were pretreated with different concentrations of Rhubarb anthraquinones and then treated with LPS+nigericin,the expression of IL-1β,TNF-α,LDH（Lactate dehydrogenase,LDH）and the cleavage of pro-caspase-1 were detected.2.2 Mechanism of Rhubarb anthraquinones in regulating NLRP3 inflammasome:2.2.1 Whether Rhubarb anthraquinones regulated NLRP3 transcription through TLR4（Toll like receptor 4,TLR4）signaling pathway:（1）Mouse BMDMs were pretreated with Rhubarb anthraquinones and then stimulated with LPS and E.coli OMVs（Escherichia coli outer membrane vesicles,E.coli OMVs）respectively,the expression of IL-1β and TNF-α were detected;（2）Mouse BMDMs were pretreated with Rhubarb anthraquinones at different concentrations and then stimulated with LPS,the mRNA expression of NLRP3,IL-1β and TNF-α as well as the protein expression of NLRP3,pro-caspase-1,ASC and pro-IL-1β were detected;（3）Mouse BMDMs were pretreated with Rhubarb anthraquinones and stimulated with LPS,the phosphorylation of p65,p38,ERK and JNK were detected at different time points.2.2.2 Whether Rhubarb anthraquinones regulate NLRP3 transcription through AHR（Aromatic hydrocarbon receptor,AHR）signaling pathway:（1）Mouse BMDMs were pretreated with Rhubarb anthraquinones and the expression of CYP1A1 mRNA was detected;（2）Mouse BMDMs were pretreated with Rhubarb anthraquinones and then stimulated with LPS,the expression of CYP1A1 mRNA was detected;（3）Mouse BMDMs were pretreated with different concentrations of FICZ,ITE and L-kynurenine,and then stimulated with LPS,the mRNA expression of CYP1A1,NLRP3,IL-1β and TNF-α were detected;（4）Mouse BMDMs were pretreated with Rhubarb anthraquinones,FICZ（6-formylindolo-[3,2-b]-carbazole,FICZ）,ITE（2-（1’H-indole-3’-carbonyl）-thiazole-4-carboxylic acid methyl ester,ITE）and L-kynurenine,and then stimulated with LPS+nigericin,the expression of IL-1β,TNF-α and the cleavage of pro-caspase-1 were detected.2.2.3 Whether Rhubarb anthraquinones inhibited the assembly of NLRP3 protein:（1）The cell culture medium was replaced with new culture medium after mouse BMDMs stimulated by LPS,then mouse BMDMs were pretreated with Rhubarb anthraquinones and stimulated by nigericin,the expression of IL-1β,the cleavage of pro-caspase-1,ASC fluorescent spots and the binding of NLRP3 protein to ASC protein were detected;（2）Mouse BMDMs were pretreated with Rhubarb anthraquinones and then stimulated with LPS or LPS+nigericin respectively,the expression of H2O2（Hydrogen peroxide,H2O2）was detected.2.3 Rhubarb Anthraquinones improved NASH by regulating NLRP3 inflammasome:（1）Mose BMDMs were pretreated with Rhubarb Anthraquinones and stimulated with LPS+cholesterol,IL-1β and TNF-α were detected;（2）C57 mice were fed with MCD diet and intervened with Rhubarb,Rhubarb anthraquinones and inflammasome blockers,liver histopathology（H&E staining）,liver function ALT and AST as well as serum inflammatory factors IL-1β and TNF-α were detected.Results:1.Yinchenhao Decoction improved NASH by regulating inflammasome:（1）Yinchenhao Decoction and the drugs from Yinchenhao Decoction inhibited the expression of IL-1β in mouse BMDMs induced by LPS,LPS+nigericin,LPS+poly dAdT and LPS+MDP（P<0.001,P<0.001,P<0.001and P<0.001）,but have no effect on the expression of TNF-α.（2）Yinchenhao Decoction and the drugs from Yinchenhao Decoction inhibited the expression of IL-1β（P<0.001）in mouse BMDMs induced by LPS+cholesterol,but have no effect on the expression of TNF-α;（3）Yinchenhao Decoction,the drugs from Yinchenhao Decoction and inflammasome blockers improved the liver histopathology（H&E staining）,liver function ALT and AST（P<0.001 and P<0.001）as well as serum inflammatory factors IL-1β and TNF-α（P<0.001 and P<0.001）in MCD diet-induced NASH in mice.2.The main components of Yinchenhao Decoction improved NASH by regulating inflammasome:2.1 To screen the active components of Yinchenhao Decoction in regulating NLRP3 inflammasome:（1）Rhubarb anthraquinones inhibited the expression of IL-1β induced by LPS+nigericin in mouse BMDMs（P<0.001）,but had no effect on the expression of TNF-α;（2）Rhubarb anthraquinones inhibited the expression of IL-1β induced by LPS+ATP,LPS+alum,LPS+SiO2 and LPS+CPPD in mouse BMDMs（P<0.001,P<0.001,P<0.001 and P<0.001）,but had no effect on the expression of TNF-α;（3）Rhubarb Anthraquinones inhibited the cleavage of pro-caspase-1 induced by LPS+nigericin and LPS+ATP in mouse BMDMs.（4）Rhubarb anthraquinones inhibited the formation of ASC fluorescent spots induced by LPS+nigricin in mouse BMDMs（P<0.001）;（5）Rhubarb anthraquinones inhibited the expression of IL-1β（P<0.001）,LDH（P<0.001）and the cleavage of pro-caspase-1 induced by LPS+nigricin in a dose-dependent manner,but had no effect on the expression of TNF-α.2.2 Mechanism of Rhubarb anthraquinones in regulating NLRP3 inflammasome:2.2.1 Whether Rhubarb anthraquinones regulated NLRP3 transcription through TLR4 signaling pathway:（1）Rhubarb anthraquinones inhibited the expression of IL-1β in mouse BMDMs induced by LPS and E.coli OMVs（P<0.001 and P<0.001）,but had no effect on the expression of TNF-α;（2）Rhubarb Anthraquinones inhibited the expression of NLRP3 and IL-1β in both mRNA and protein level in a dose-dependent manner（P<0.001 and P<0.001 as well as P<0.001 and P<0.001）,but had no effect on the mRNA expression of TNF-α as well as the protein expression of pro-caspase-1 and ASC;（3）Rhein,dicerein,emodin,aloe emodin and 1,8-dihydroxyanthraquinone had no effect on the phosphorylation of p65,p38,ERK and JNK in mouse BMDMs stimulated by LPS.2.2.2 Whether Rhubarb anthraquinones regulate NLRP3 transcription through AHR signaling pathway:（1）Rhubarb anthraquinones promoted the gene expression of CYP1A1 in mouse BMDMs in a dose-dependent manner（P<0.001,P<0.001,P<0.001,P<0.001 and P<0.001）;（2）Rhubarb anthraquinones promoted the gene expression of CYP1A1 in mouse LPS-stimulated BMDMs in a dose-dependent manner（P<0.001）;（3）FICZ,ITE and L-kynurenine inhibited the gene expression of NLRP3 and IL-1β（P<0.001 and P<0.001）as well as the protein expression of NLRP3（P<0.001）in a dose-dependent manner,but promoted the gene expression of CYP1A1 and TNF-α（P<0.001 and P<0.007）;（4）Rhubarb anthraquinones,FICZ,ITE and L-kynurenine inhibited the expression of LPS+nigericin inhibited the expression of IL-1β（P<0.001）and the cleavage of pro-caspase-1 induced by LPS+nigericin in mouse BMDMs,but had no effect on the expression of TNF-α.2.2.3 Whether Rhubarb anthraquinones inhibited the assembly of NLRP3 protein:（1）Rhubarb anthraquinones inhibit the expression of IL-1β and cleavage of pro-caspase-1 in a dose-dependent manner,and also inhibited the formation of ASC protein fluorescent spots（P<0.001）and the combination between NLRP3 protein and ASC protein in mouse BMDMs induced by nigericin;（2）Rhubarb anthraquinones dose dependently promoted the expression of H2O2 in LPS and LPS+nigericin stimulated mouse BMDMs（P<0.001）.2.3 Rhubarb Anthraquinones improved NASH by regulating NLRP3 inflammasome:（1）Rhubarb anthraquinones inhibited the expression of IL-1β in mouse BMDMs induced by LPS+cholesterol（P<0.001）,but have no effect on the expression of TNF-α;（2）Rhubarb,Rhubarb Anthraquinones and inflammasome blockers improved liver histopathology（H&E staining）,liver function ALT and AST（P<0.001 and P<0.001）as well as serum inflammatory factors IL-1β and TNF-α（P<0.001 and P<0.001）in MCD diet induced NASH in mice.Conclusion:1.Yinchenhao Decoction improved NASH by regulating inflammasome;2.The main component of Yinchenhao Decoction to regulate NLRP3 inflammasome is Rhubarb Anthraquinones;3.Rhubarb anthraquinones improved NASH by regulating NLRP3 inflammasome.The mechanism Rhubarb anthraquinones regulated NLRP3 inflammasome may be related to its role in regulating the gene transcription and protein assembly of NLRP3. |
PDF Full Text Request