Chronoeffect Studies On Tripterygium Wilfordii And Coptidis Rhizoma Basing On Circadian Rhythms In Pharmacokinetics And Diseases

Objective:Tripterygium wilfordii is a woody vine belonging to Tripterygium wilfordii Hook.f..It has strong anti-inflammatory and immunomodulatory effects and is known as"Chinese herbal hormone".It is often used in clinical treatment of rheumatic diseases.Coptidis rhizoma is a perennial herb of Coptis chinensis Franch（the Ranunculaceae family）.It has strong anti-inflammatory and anti-diarrhea effects.It is often used to treat chronic colitis and other diseases.Our previous study found that the toxicity of Tripterygium wilfordii or Coptidis rhizoma was time-dependent,but whether the efficacy of Tripterygium wilfordii or Coptidis rhizoma was time-dependent is still unclear.Therefore,this study will investigate whether the efficacy of Tripterygium wilfordii or Coptidis rhizoma is time-dependent,and further explore whether the time-dependent efficacy of Tripterygium wilfordii or Coptidis rhizoma is related to the time-difference in pharmacokinetics（time-pharmacokinetics）and disease rhythms.Methods:1.Based on C57BL/6 mice,the mouse model of rheumatoid arthritis was established by collagenic induction.At six time points（ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22）,model mice were given suspension of Tripterygium wilfordii polyglycosides tablets（TGT）.The inflammatory factors related to rheumatoid arthritis were detected by ELISA method,the expression of inflammatory factors at the m RNA level was analyzed by RT-q PCR technology,and the joint injury was characterized by H&E staining to explore whether the efficacy of Tripterygium wilfordii was time-dependent.2.The rheumatoid arthritis mice were used as experimental objects,and the inflammatory factors were detected at six time points by ELISA method,and the severity of rheumatoid arthritis in mice at different time points was compared.Model mice were given suspension of TGT at the time points of the most severe rheumatoid arthritis（ZT2）and the least severe rheumatoid arthritis（ZT14）.The inflammatory factors related to rheumatoid arthritis were detected by ELISA method to explore whether the temporal efficacy of TGT was related to disease rhythm.3.Mice with rheumatoid arthritis were treated with TGT suspension at different time points,and the main absorbed components of Tripterygium Wilfordii in the blood were detected by UPLC-QTOF/MS technology,and then the drug-time curve was fitted by using PKsolver software.The pharmacokinetic parameters of Tripterygium wilfordii polyglycosides tablets were obtained.4.Primary fibroblast synovial cells were extracted from mice with rheumatoid arthritis and used to evaluate the anti-inflammatory effect of triptolide（the main active ingredient of TGT）and explore the therapeutic effect of triptolide on rheumatoid arthritis.5.The mice model of chronic colitis was established by using sodium glucan sulfate in wild-type mice.Body weight,disease activity index and colon length were evaluated during modeling,and the severity of disease was measured.Mice with chronic colitis were given different doses of Coptidis rhizoma（CR）extract,and the degree of colitis in mice was characterized by observing disease activity index,colon length,histopathology and detecting the expression of related disease factors.The therapeutic effect of CR on chronic colitis at different doses were assessed in mice.6.Chronic colitis model mice were used as experimental objects.Mice with chronic colitis were given CR extract at different time points.The expression levels of inflammatory factors and the content of malondialdehyde/myeloperoxidase in colon tissues at different time points were detected.The m RNA expression levels of Nlrp3,IL-1β,IL-6,IL-18 and Ccl2 were analyzed by RT-q PCR,so as to explore whether CR had time-dependent efficacy.7.Chronic colitis model mice were used as experimental objects.The expression levels of inflammatory factors and myeloperoxidase content in colon tissues of model mice were compared at six time points to explore the severity of chronic colitis in mice at different time points.At the time points of the most severe chronic colitis（ZT2）and the least severe chronic colitis（ZT10）,CR extract was administered to model mice.The expression levels of inflammatory factors and the content of myeloperoxidase in colon tissues at different time points were detected to explore whether the temporal effects of berberine were correlated with the severity of chronic colitis.After ZT2 and ZT10 were administered to model mice,the main absorbed components of CR in the blood were detected by UPLC-QTOF/MS.8.The mouse model of chronic colitis was established with Rev-erbα^-/-mice,which were used as experimental objects.The levels of inflammatory factors and the content of malondialdehyde/myeloperoxidase in model mice were detected after CR extract was administered,and the correlation between the time-dependent efficacy of CR and the clock controlling gene Rev-erbαwas analyzed.Results:1.The efficacy of Tripterygium wilfordii against rheumatoid arthritis is dosing time-dependent.Compared with ZT14,ZT2 administration of TGT showed better efficacy against rheumatoid arthritis in mice.Specifically,the levels of inflammatory cytokines TNF-αand IL-6 were lower in model mice treated at ZT2 compared with those treated at ZT14.In line with this,m RNA expression of Tnf-α,IL-6,Cox-2 and i NOS in model mice for ZT2administration.It was lower than when ZT14 was administered.In addition,H&E staining analysis showed that the degree of joint injury during ZT2 administration was greater than that during ZT14 administration The lightness of the medicine.It is noteworthy that Tripterygium wilfordii has no significant hepatotoxicity at therapeutic doses.2.The chronoefficacy of Tripterygium wilfordii is associated with time-dependent severity of rheumatoid arthritisLevels of inflammatory factors TNF-αand IL-6 in model mice peaked at daytime（ZT2）and reached a trough at night（ZT14）,suggesting that there was a significant circadian rhythm in the severity of rheumatoid arthritis in mice.For ZT2 and ZT14 administration,TGT had anti-inflammatory effects,but the anti-inflammatory effects produced by daytime administration（ZT2）were better than those produced by night administration（ZT14）.Specifically,the relative levels（percent change）of the inflammatory cytokines TNF-αand IL-6 decreased in model mice treated at ZT2 were higher compared with those treated at ZT14.3.Chronopharmacokinetics is one factor responsible for the chronoefficacy of Tripterygium wilfordii and the main active ingredient（triptolide）has an anti-inflammatory effect in vitroPlasma concentrations and area under the curve（systemic exposure）of triptolide in rheumatoid arthritis mice after ZT2 administration were higher and had a longer half-life compared with ZT14 administration.Triptolide inhibited the abnormal proliferation of primary fibroblast synovial cells,inhibited the levels of inflammatory factors TNF-αand IL-6,and decreased the m RNA expression of Tnf-α,IL-6,Cox-2 and i NOS.4.Coptidis rhizoma has a therapeutic effect on chronic colitis,and the therapeutic effect is dosing time-dependentCompared with the solvent group,the drug group had a lower disease activity index,longer colon length,and lower levels of MOA,MPO,IL-1β,and IL-6.The therapeutic effect of CR at ZT10 was significantly better than that at ZT2.Specifically,protein levels of MOA,MPO,IL-1βand IL-6 were lower in model mice after ZT10 administration compared with ZT2 administration.Similarly,the expression of Nlrp3,IL-1β,IL-6,IL-18,and Ccl2 m RNA was lower in model mice treated at ZT10 than at ZT2.5.The severity of chronic colitis in mice was dosing time-dependent,and the chronoefficacy of Coptidis rhizoma was related to the disease rhythms,but not to the pharmacokineticsWe found that the levels of MPO,IL-1β,and IL-6 peaked in the daytime（ZT2）.The results showed that the severity of chronic colitis in mice was dosing time-dependent.CR produced anti-inflammatory effect after ZT2 and ZT10 administration.CR produced a better anti-inflammatory effect in the evening（ZT10）than in the daytime（ZT2）.Specifically,the relative levels（percent change）of MPO,IL-1β,and IL-6 decreased in chronic colitis mice treated at ZT10 were higher compared with those treated at ZT2.There was no significant difference in the area under the curve（systematic exposure）and colon distribution of berberine at the two dosing time points,indicating that the chronoefficacy of coptidis was independent of pharmacokinetics.6.The time-dependence of Coptidis rhizoma was related to the clock controlling gene Rev-erbαThere was no time difference in the therapeutic effect of CR on chronic colitis in Rev-erbα^-/-mice.Specifically,in Rev-erbα^-/-mice with chronic colitis,there was no significant difference in the therapeutic effect produced by evening administration（ZT10）compared with daytime administration（ZT2）.Conclusion:The efficacy of Tripterygium wilfordii was dosing time-dependent,which was related to time-dependent pharmacokinetics and disease rhythms.The effect of Coptidis rhizoma was time-dependent and correlated with disease rhythms（regulated by the clock gene Rev-erbα）,but not with pharmacokinetics.This thesis work may provide a scientific basis for chronotherapy application for Tripterygium wilfordii and Coptidis rhizoma.