Diclofenac Disrupts the Circadian Clock and through Complex Cross-Talks Aggravates Immune-Mediated Liver Injury-A Repeated Dose Study in Minipigs for 28 Days.

Diclofenac effectively reduces pain and inflammation; however, its use is associated with hepato- and nephrotoxicity. To delineate mechanisms of injury, we investigated a clinically relevant (3 mg/kg) and high-dose (15 mg/kg) in minipigs for 4 weeks. Initially, serum biochemistries and blood-smears indicated an inflammatory response but returned to normal after 4 weeks of treatment. Notwithstanding, histopathology revealed drug-induced hepatitis, marked glycogen depletion, necrosis and steatosis. Strikingly, the genomic study revealed diclofenac to desynchronize the liver clock with manifest inductions of its components CLOCK, NPAS2 and BMAL1. The > 4-fold induced CRY1 expression underscored an activated core-loop, and the dose dependent > 60% reduction in PER2mRNA repressed the negative feedback loop; however, it exacerbated hepatotoxicity. Bioinformatics enabled the construction of gene-regulatory networks, and we linked the disruption of the liver-clock to impaired glycogenesis, lipid metabolism and the control of immune responses, as shown by the 3-, 6- and 8-fold induced expression of pro-inflammatory CXCL2, lysozyme and ß-defensin. Additionally, diclofenac treatment caused adrenocortical hypertrophy and thymic atrophy, and we evidenced induced glucocorticoid receptor (GR) activity by immunohistochemistry. Given that REV-ERB connects the circadian clock with hepatic GR, its > 80% repression alleviated immune responses as manifested by repressed expressions of CXCL9(90%), CCL8(60%) and RSAD2(70%). Together, we propose a circuitry, whereby diclofenac desynchronizes the liver clock in the control of the hepatic metabolism and immune response.

Assessing the safety of an Ephedrae Herba aqueous extract in rats: A repeat dose toxicity study.

Ephedrae Herba (EH) has been used in Asian traditional herbal medicine to cure bronchial asthma, cold, flu, chills, fever, headache, nasal congestion, and cough. In this study, we evaluated the subchronic toxicity of an Ephedrae Herba aqueous extract (EHAE) in male and female F344 rats. The EHAE was administered orally daily at doses of 0, 125, 250, 500, and 1000 mg/kg bw/day for 13 weeks. Toxicological assessment was performed to determine mortality, clinical signs, and changes in body weight, food consumption, ophthalmological, urinary, hematological, and serum biochemical parameters, macroscopic and microscopic evaluations, and organ weights. We found that oral administration of EHAE to F344 rats for 13 weeks resulted in histopathological changes in the kidneys and salivary glands. In the kidneys, increased incidence and severity of tubular basophilia were observed in females administered 1000 mg/kg bw/day of the extract. In the salivary glands, acinar cell hypertrophy was observed in males administered 500 mg/kg bw/day and in both sexes administered 1000 mg/kg bw/day of the extract. All test article-treated groups of males and females administered ≥250 mg/kg bw/day showed increased absolute and relative salivary gland weights. Therefore, the NOAEL (No Observed Adverse Effect Level) was determined as 125 mg/kg bw/day for both sexes of rats under the present experimental conditions.

A novel missense mutation in the mouse hairless gene causes irreversible hair loss: genetic and molecular analyses of Hr m1Enu.

A novel autosomal recessive mutant was produced using N-ethyl-N-nitrosourea mutagenesis. The characteristics of the mutant mice included progressive irreversible hair loss within a month of birth, wrinkled skin, and long curved nails. Linkage analysis revealed that the causative gene is linked to D14Mit193 on chromosome 14. Sequence analysis of the complete cDNA of the candidate gene, hairless (Hr), identified a homozygous G-to-T transition at nucleotide 3572, leading to the substitution of glycine by tryptophan, designated Gly960Trp. This missense mutation occurs in the vicinity of repression domain 3 of the hairless protein (HR). This allele was named Hr(m1Enu). The relative amounts of Hr mRNA and HR protein determined by real-time PCR and Western blot analyses, respectively, were slightly elevated in the mutant mice. Quantitative real-time PCR analysis revealed the increased expression of Kc1 and Vdr in the mutant mice, whereas the expression of Nrs1 and Krtap16-6 was decreased. These results suggest that the Gly960Trp substitution in HR protein in Hr(m1Enu) mice may alter the function of HR as a transcriptional corepressor.