Abstract
Dysregulation of the circadian clock machinery is a critical mechanism in the pathogenesis of fibrosis. This study aimed to investigate whether the antifibrotic effect of melatonin is associated with attenuation of circadian clock pathway disturbances in mice treated with carbon tetrachloride (CCl4) and in human hepatic stellate cells line LX2. Mice received CCl4 5 μL/g body weight i.p. twice a week for 4 or 6 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p., beginning 2 weeks after the start of CCl4 administration. Treatment with CCl4 resulted in fibrosis evidenced by the staining of α-smooth muscle actin (α-SMA) positive cells and a significant decrease of peroxisome proliferator-activated receptor (PPARα) expression. CCl4 led to a lower expression of brain and muscle Arnt-like protein 1 (BMAL1), circadian locomotor output cycles kaput (CLOCK), period 1–3 (PER1, 2, and 3), cryptochrome 1 and 2 (CRY1 and 2) and the retinoic acid receptor-related orphan receptor (RORα). The expression of the nuclear receptor REV-ERBα showed a significant increase. Melatonin significantly prevented all these changes. We also found that melatonin (100 or 500 μM) potentiated the inhibitory effect of REV-ERB ligand SR9009 on α-SMA and collagen1 expression and increased the expression of PPARα in LX2 cells. Analysis of circadian clock machinery revealed that melatonin or SR9009 exposure upregulated BMAL1, CLOCK, PER2, CRY1, and RORα expression, with a higher effect of combined treatment. Findings from this study give new insight into molecular pathways accounting for the protective effect of melatonin in liver fibrosis.
Highlights
Hepatic fibrosis is a common scarring response to all forms of chronic liver injury (Pellicoro et al, 2014), which can result in elevated extracellular matrix protein production by activated hepatic stellate cells (HSCs) (Lan et al, 2015)
To corroborate previous results about the protective effect of melatonin in the CCl4 mice model of liver fibrosis, the expression of α-smooth muscle actin (α-SMA), the main gene related to fibrogenesis in the liver, FIGURE 2 | Effect of CCl4 and treatment with melatonin on the expression of circadian clock genes brain and muscle Arnt-like protein 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK). (A) mRNA levels of BMAL1 and CLOCK were analyzed by real-time PCR assay and normalized against β-Actin. (B) Representative Western blot photographs and densitometric quantification of BMAL1 and CLOCK
We further investigated if circadian clock restoration induced by melatonin in the CCl4 model of fibrosis occurred in human HSCs by analyzing expression of the different clock genes in LX2 cells
Summary
Hepatic fibrosis is a common scarring response to all forms of chronic liver injury (Pellicoro et al, 2014), which can result in elevated extracellular matrix protein production by activated HSCs (Lan et al, 2015). A number of studies have shown that alterations of hepatic clock genes result in altered organ function and injury, and circadian clocks may represent relevant targets for the development of new therapeutic approaches in liver fibrosis (Zhou et al, 2016). CLOCK and BMAL1 proteins dimerize and activate the transcription of target genes PERs and CRYs (Ko and Takahashi, 2006). ROR activates whereas REV-ERB represses the expression of BMAL1, reinforcing stability and robust rhythmicity of the internal clock systems (Curtis et al, 2014)
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