Expression Of Circadian Clock Genes In Basic Pharmacology And Toxicology Research

Objective: Hepatocellular carcinoma(HCC)is the major threat to human health,and disruption of circadian clock genes is implicated in hepatocarcinogenesis.This study examined the dysregulation of metallothioneins and circadian genes in achieved human HCC(n = 24),peri-HCC tissues(n = 24)as compared with normal human livers(n = 36).Methods: All the human liver samples were provided by Oriental Liver Translation Center(Beijing,China)as previously reported.The human liver samples included 36 normal donor trimmed livers,24 paired HCC and Peri-HCC tissue from individual paired patients.The liver samples were immediately frozen in liquid nitrogen and stored at-80℃ for RNA analysis.Total RNA was extracted and reverse transcribed.Real-time RT-q PCR was performed to determine the expression of genes of interest.Results: The results demonstrated the downregulation of MT-1,MT-2,and MFT-1in human HCC as compared with Peri-HCC and normal tissues.MTs are a biomarker for HCC and have typical circadian rhythms;the expression of major circadian clock genes was also determined.HCC produced a dramatic decrease in the expression of core clock genes,Clock and Bmal1,and decreased the expression of the clock feedback control genes Per1,Per2,Cry1,Cry2.On the other hand,the expression of clock target genes Nr1d1 and Dbp was upregulated as compared with Peri-HCC and normal livers.Peri-HCC also had mild alterations in these gene expressions.Conclusion: The present study clearly demonstrated the dysregulation of MTs and circadian clock genes in human HCC,which could provide the information of targeting MT and circadian clock genes in HCC management.Objective: Manganese(Mn)neurotoxicity displays non-motor dysfunction and motor impairment like Parkinson’s disease(PD),and is called as Manganism.Circadian disruption is a non-motor symptom found in PD and Manganism.Clock genes are essential to drive and maintain circadian rhythm,but little is known about Mn exposure on circadian clock genes expression.Both the brain and liver are targets of Mn,we hypothesize that repeated Mn administration could affect clock gene expression in the hypothalamus and liver.Methods: Male Sprague-Dawley rats were intraperitoneally injected Mn2+ 1 mg and5 mg/kg as Mn Cl2·4H2O,every other day for 30 days.Mn neurotoxicity was evaluated the loss of dopaminergic neurons in the substantia nigra,and activated microglia in the brain via immunohistochemistry.The expression of circadian clock genes was determined via real-time RT-q PCR.Results: Repeated Mn administration dose-dependently decreased the number of dopaminergic neurons in the substantia nigra,and activated microglia in the brain.Expressions of circadian core genes Bmal1,Clock,Npas2,and clock feedback gene Cry1,Per1 and Per2 in the hypothalamus and liver were decreased after exposure to Mn in a dose-dependent manner,while expressions of clock-targeted genes Nr1d1 and Dbp were increased.Conclusion: Repeated Mn administration produced dysregulation of circadian clock gene expressions in both the brain and liver.Objective: LPS-induced chronic neurogenic inflammation aggravates the neurotoxicity of rotenone and successfully simulates the model of Parkinson’s disease.Circadian clock disorders are associated with many neurodegenerative diseases.In this study,we investigated the expression of circadian clock genes and proteins in Parkinson model rats.Methods: Male SD rats(180 g)were injected LPS(5 mg/kg,ip x 1)to produce chronic,low-grade neuroinflammation.Approximately 200 days after LPS,low dose of rotenone(0.5 mg/kg,sc)was administered 5 times per week for 20 times.Behavioral tests(rotarod,open field and Y maze)were performed 7 days after the end of rotenone administration.Subsequently,brain regions(cortex,hippocampus,and midbrain)were collected for analysis.Mitochondrial activity,Dopamine neuron loss,and microglia activation were evaluated.The expression of circadian clock genes and proteins was examined via RT-q PCR and Western blot.Results: Chronic LPS plus rotenone challenge produced PD like symptoms including reduced motor activity(rotarod and open field),loss of dopamine neurons in substantia nigra(THr staining),increased neuroinflammation(Iba1 staining),and reduced mitochondria complex-1 activity.The expressions of clock core gene Bmal1,Clock,Npas2 were decreased in the LPS group,rotenone group and LPS plus rotenone group.LPS plus rotenone produced more pronounced effects(60%decreases).Circadian feedback control genes Per1 and Per2 were also decreased.The clock target genes Nr1d1 and Dbp were also decreased,although to a lesser extent.Consistent with the transcript levels,circadian clock proteins BMAL1,CLOCK,NR1D1,and DBP were decreased,especially in the LPS plus rotenone group.Conclusion: The expression of circadian clock genes and proteins was decreased in Parkinson’s disease model rats induced by LPS plus rotenone,implying circadiandisruption could contribute to PD symptoms.Objective: The circadian clock is involved in drug metabolism,efficacy and toxicity.Drugs could in turn affect the biological clock as a mechanism of their actions.Zuotai is an essential component of many popμLar Tibetan medicines for sedation,tranquil and “detoxification,” and is mainly composed of metacinnabar(β-Hg S).The pharmacological and/or toxicological basis of its action is unknown.This study aimed to examine the effect of Zuotai on biological clock gene expression in the liver of mice.Methods: Mice were orally given Zuotai(10 mg/kg,1.5-fold of clinical dose)daily for 7 days,and livers were collected every 4 h during the 24 h period.Total RNA was extracted and subjected to real-time RT-q PCR analysis of circadian clock gene expression.Results: Zuotai decreased the oscillation amplitude of the clock core gene(Clock,Npas2,Bmal1)at 10:00.For the clock feedback negative control genes,Zuotai had no effect on the oscillation of the clock gene(Cry1,Per1–3).For the clock-driven target genes,Zuotai increased the oscillation amplitude of Dbp,decreased Nfil3 at10:00,but had no effect on Tef;Zuotai increased the expression of Nr1d1 at 18:00,but had little influence on Nr1d2 and RORa.Conclusion: The Tibetan medicine Zuotai could influence the expression of clock genes,which could contribute to pharmacological and/or toxicological effects of Zuotai.