Mechanistic Studies On Circadian Anti-colitis Effects Of Berberine

Objective:Berberine,a pharmacologically active compound,isolated from the traditional Chinese medicine Coptis chinensis,is frequently used to treat arthritis,colitis and other inflammatory diseases.Chronic colitis is the chronic irritation or inflammation of colon.It is characterized with a long onset time and high rate of recurrence,and is a threat of human health.REV-ERBα is the core component of the circadian clock and has been identified as a potential therapeutic target for inflammatory diseases including colitis.However,it is unclear whether REV-ERBα plays a role in the pharmacological effect of berberine in treating chronic colitis.Therefore,the objective of this dissertationare to evaluate the chronoefficacy of berberine in the treatment of chronic colitis,and to clarify the contribution of the circadian clock protein REV-ERBα,in mediating the chronopharmacodynamic effect of berberine.Methods: 1.A mouse model of chronic colitis was established by oral administration of dextran sodium sulfate.The severity of chronic colitis was estimated by measuring the changes in body weight,disease activity index,colon length and the contents of malondialdehyde and myeloperoxidase of mice with chronic colitis,as compared with those of vehicle-treated mice.The colon tissues collected at different time points(ZT2,ZT6,ZT10,ZT14,ZT18,ZT22)were determined by using real-time PCR and Western blot analysis,and the expressions of inflammatory factors(Nlrp3,IL-1β,IL-6,IL-18 and Ccl2)were analyzed.2.The REV-ERBα knockout mouse model was verified by PCR phenotype and qPCR,and primary cells derived from bone marrow were extracted.In vitro anti-inflammatory effects of berberine were investigated in LPS-treated bone marrow-derived macrophage,and the expression levels of inflammatory factors,malondialdehyde and myeloperoxidase levels were detected by qPCR,Western blotting and ELISA,respectively.The key role of REV-ERBα in the suppression of inflammation response by berberine was clarified.3.To investigate the circadian pharmacological effects of berberine,berberine were orally administrated to mice with chronic colitis at ZT2 and/or ZT10.The expression levels of inflammatory factors and the contents of malondialdehyde content and myeloperoxidase in colon tissues of vehicle-and berberine-treated mice were detected by qPCR and ELISA Pharmacokinetic experiments were conducted at ZT2 and ZT10 to clarify the contribution of pharmacokinetics in the chronopharmacodynamics of berberine.4.To elucidate the regulatory effect of berberine on the circadian protein REV-ERBα,the expression levels and transcriptional activities of Bmal and Nlrp3(two REV-ERBα targeted genes)were measured by using qPCR,western blot and luciferase reporter assays.Furthermore,the interaction between berberine and the ligand-binding domain(LBD)of REV-ERBα protein was evaluated using the mammalian one-hybrid GAL4-LBD assay.Results: 1.The severity of chronic colitis in mice is time-dependent.The weight of mice in the chronic colitis model group decreased significantly,and the disease activity index score was significantly increased,and the length of the colon was significantly shortened compared with the vehicle mice,proving that the mouse chronic colitis model was successfully constructed.The colon tissues of chronic colitis mice were collected at different time points(ZT2,ZT6,ZT10,ZT14,ZT18,ZT22),and the inflammatory factors(Nlrp3,IL-1β,IL-6,IL-18 and Ccl2)were analyzed.The trend of m RNA and protein expression levels and the trend of malondialdehyde content and myeloperoxidase content,which are two other indicators of the severity of colon inflammation.We found that the ratio of the highest point to the lowest point of the expression curve was at least 5 times,and the largest more than 30 times.Chronic colitis disease in mice have severe rhythm fluctuations,and it is concluded that the severity of chronic colitis disease is time-dependent.2.The anti-inflammatory effect of berberine depends on the circadian clock protein REV-ERBα.Berberine was orally administered to the model mice,and the DAI,colon length,MDA,and MPO,the m RNA and protein expression of inflammatory factors were analyzed.Berberine has anti-inflammatory effects in view of animal levels,and the dose of 100 mg/kg has the most significant effect,up to 40%,compared with the non-administered group.In addition,at the cellular level of BMDM and THP-1,berberine has anti-inflammatory effects,also.The effect is most obvious at a concentration of 5 μM,even better than the positive control SR9009.Primary cells derived from bone marrow of REV-ERBα knockout mice were extracted for berberine treatment,and the anti-inflammatory effect of berberine was confirmed to be lost by qPCR and Western blot experiments.The above research shows that the anti-inflammatory effect of berberine depends on the circadian clock protein REV-ERBα.3.Berberine has a time-varying anti-inflammatory effect.Berberine was used to treat in mice with chronic colitis at ZT2 and ZT10,and the expression of inflammatory factors and MPO content were detected.Research data shows that the treatment effect of ZT10 is much better than ZT2 at the same dose,and the therapeutic effect of the index IL-6 is as much as 2.5 times.In addition,the study found that the exposure of berberine at two different circadian time points ZT2 and ZT10 did not have a significant difference.It is obvious that pharmacokinetics did not have contribution in the chronopharmacodynamics of berberine.4.Berberine activates the circadian clock protein REV-ERBα.The luciferase reporter assay of the Bmal1 and Nlrp3 promoter segments showed that berberine could significantly inhibit the transcriptional activities of the two targeted genes of REV-ERBα,Bmal1 and Nlrp3.Gal4-cotransfection experiments showed that berberine could significantly enhance the transcriptional repression activity of REV-ERBα.In addition,qPCR and western blotting results displayed that berberine could reduce the m RNA and protein expression levels of Bmal1 and Nlrp3 in a concentration-dependent manner.The above data indicate that berberine has an agonistic effect on the circadian protein REV-ERBα.Conclusion: 1.Clarified that the severity of chronic colitis disease is time-dependent.2.It was discovered that the anti-inflammatory effect of berberine depends on the circadian clock protein REV-ERBα.3.Clarified the time-dependence of anti-inflammatory effects of berberine.4.Clarified that the time-dependence of berberine effects through agonistic circadian protein REV-ERBα.