Background: Understanding the molecular events and mechanisms underlying development and progression of nonalcoholic steatohepatitis is essential in an attempt to formulating a specific treatment. Here, we uncover Platr4 as an oscillating and NF-κB driven lncRNA that is critical to the pathological conditions in experimental steatohepatitis Methods: RNA-sequencing of liver samples was used to identify differentially expressed lncRNAs. RNA levels were analyzed by qPCR and FISH assays. Proteins were detected by immunoblotting and ELISA. Luciferase reporter, ChIP-sequencing and ChIP assays were used to investigate transcriptional gene regulation. Protein interactions were evaluated by Co-IP experiments. The protein-RNA interactions were studied using FISH, RNA pull-down and RNA immunoprecipitation analyses Results: Cyclic expression of Platr4 is generated by the core clock component Rev-erbα via two RevRE elements (i.e., -1354/-1345 and -462/-453 bp). NF-κB transcriptionally drives Platr4 through direct binding to two κB sites (i.e., -1066/-1056 and -526/-516 bp), potentially accounting for up-regulation of Platr4 in experimental steatohepatitis. Intriguingly, Platr4 serves as a circadian repressor of Nlrp3 inflammasome pathway by inhibiting NF-κB-dependent transcription of the inflammasome components Nlrp3 and Asc. Loss of Platr4 down-regulates Nlrp3 inflammasome activity in the liver, blunts its diurnal rhythm, and sensitizes mice to experimental steatohepatitis, whereas overexpression of Platr4 ameliorates the pathological conditions in an Nlrp3 -dependent manner. Mechanistically, Platr4 prevents binding of the NF-κB/Rxrα complex to the κB sites via a physical interaction, thereby inhibiting the transactivation of Nlrp3 and Asc by NF-κB. Conclusions: Platr4 functions to inactivate Nlrp3 inflammasome via intercepting NF-κB signaling. This lncRNA might be an attractive target that can be modulated to ameliorate the pathological conditions of steatohepatitis.

While growing evidence suggests that circadian clock and obesity are intertwined, the underlying mechanism is poorly understood. Here, we investigate how circadian clock is linked to obesity. Methods: Metabolomics profiling of WAT (white adipose tissue) samples was performed to identify the metabolites altered in obese model. mRNA levels were analyzed by qPCR assays. Proteins were detected by immunoblotting, immunofluorescence and ELISA. ChIP and luciferase reporter assays were used to investigate epigenetic and transcriptional regulation. Results: Obesity causes perturbance of circadian clock in WAT in mice and humans, particularly, BMAL1 is markedly reduced. Metabolomic analysis reveals reduced glutamine and methionine in obese WAT. Glutamine metabolism contributes to production of acetyl-CoA, whereas methionine metabolism generates S-adenosyl methionine (SAM). Acetyl-CoA and SAM are the substrates for histone acetylation and methylation, respectively. Reduced glutamine and methionine in obese WAT are associated with decreased H3K27ac and H3K4me3 at Bmal1 promoter. Consistently, glutamine or methionine administration in vitro and in vivo increases H3K27ac or H3K4me3, promoting Bmal1 transcription and expression. A screen of transport and metabolic genes identifies downregulation of the uptake transporter SLC1A5 as a cause of reduced glutamine or methionine in obese WAT. Moreover, we observe impaired expression of PPAR-γ in obese WAT. PPAR-γ trans-activates Slc1a5 via direct binding to a response element in promoter. Conclusion: Impaired PPAR-γ in obesity provokes downregulation of SLC1A5 and reductions in adipocyte uptake of glutamine and methionine (two epigenetic modulators), leading to disruption of Bmal1 . Therefore, PPAR-γ integrates obesity and adipocyte clock, promoting a vicious cycle between circadian disruption and obesity development.

Rheumatoid arthritis is a systemic autoimmune disease characterized by synovial inflammation and bone destruction. Identifying drugs with time-varying efficacy and toxicity, and elucidating the mechanisms would help to improve treatment efficacy and reduce adverse effects. Here, we aimed to determine the chronoefficacy of semen strychni (SS) and tripterygium glycoside tablet (TGT) against rheumatoid arthritis in mice, and to investigate a potential role of circadian pharmacokinetics in generating chronoefficacy. SS extract and TGT suspension were prepared with ultrasonication. Effects of SS and TGT on collagen-induced arthritis (CIA) were evaluated by measuring TNF-α and IL-6 levels. SS dosed at ZT18 was more effective in protecting against CIA than drug dosed at ZT6 (i.e., lower levels of key inflammatory factors at ZT18 than at ZT6). This was accompanied by higher systemic exposure levels of strychnine and brucine (two main putative active ingredients of SS) in ZT18-treated than in ZT6-treated CIA mice. TGT dosing at ZT2 showed a better efficacy against CIA as compared to herb doing at ZT14. Consistently, ZT2 dosing generated a higher exposure of triptolide (a main putative active ingredient of TGT) as compared to ZT14 dosing in CIA mice. Moreover, strychnine, brucine, and triptolide significantly inhibited the proliferation of fibroblast-like synoviocytes, and reduced the production of TNF-α and IL-6 and the mRNAs of TNF-α, IL-6, COX-2, and iNOS, suggesting that they possessed an anti-arthritis activity. In conclusion, SS and TGT display chronoefficacy against rheumatoid arthritis in mice, that is attributed to circadian pharmacokinetics of main active ingredients. Our findings have implications for improving treatment outcomes of SS and TGT via timed delivery.

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