Oral administration of resveratrol is able to ameliorate the progression of diabetic nephropathy (DN); however, its mechanisms of action remain unclear. Recent evidence suggested that the gut microbiota is involved in the metabolism therapeutics. In the current study, we sought to determine whether the anti-DN effects of resveratrol are mediated through modulation of the gut microbiota using the genetic db/db mouse model of DN. We demonstrate that resveratrol treatment of db/db mice relieves a series of clinical indicators of DN. We then show that resveratrol improves intestinal barrier function and ameliorates intestinal permeability and inflammation. The composition of the gut microbiome was significantly altered in db/db mice compared to control db/m mice. Dysbiosis in db/db mice characterized by low abundance levels of Bacteroides, Alistipes, Rikenella, Odoribacter, Parabacteroides, and Alloprevotella genera were reversed by resveratrol treatment, suggesting a potential role for the microbiome in DN progression. Furthermore, fecal microbiota transplantation, derived from healthy resveratrol-treated db/m mice, was sufficient to antagonize the renal dysfunction, rebalance the gut microbiome and improve intestinal permeability and inflammation in recipient db/db mice. These results indicate that resveratrol-mediated changes in the gut microbiome may play an important role in the mechanism of action of resveratrol, which provides supporting evidence for the gut-kidney axis in DN.

The expression patterns of clock-controlled genes (ccgs) are regulated by circadian rhythm, which is a major regulatory and physiological mechanism tied to the solar day. Disruptions in circadian rhythm contribute to the development of cardiovascular diseases, cancer, metabolic syndromes, and aging. It has been reported that bone remodeling is also regulated by circadian rhythm. However, the molecular mechanism by which the circadian gene Clock regulates bone remodeling has yet to be elucidated. Here, we show that Clock mutant mice exhibit a significant reduction in bone density as well as increased apoptosis. Protein disulfide isomerase family A member 3 (PDIA3) is a 1,25-dihydroxy-vitamin D3 [1α,25(OH)2D3] receptor that can regulate bone formation and apoptosis. Using luciferase and ChIP assays, we confirmed that Pdia3 is a ccg. Clock activates Pdia3 transcription by binding the E-box promoter, and transcription is decreased in Clock mutant mice. Forced expression of Pdia3 or of Clock completely rescues the osteogenic disorders found in the mutant background and inhibits apoptosis in vivo and in vitro. Furthermore, ablation of PDIA3 via RNA interference completely blocks the compensatory effect of forced expression of Clock in osteoblasts. Our results demonstrate that the core circadian gene Clock regulates bone formation via transcriptional control of 1,2,5(OH)2D3 receptor PDIA3. © 2016 American Society for Bone and Mineral Research.

Objectives: To examine the effect of the circadian gene Clock on posttranscriptional function and proinflammatory mechanisms in osteoarthritis (OA). Methods: The cartilage from Clock mutant mice was assessed using histology, (OA) score, and real-time polymerase chain reaction (PCR) quantification of key pro-inflammatory genes. Nuclear factor kappalight-chain-enhancer of activated B cells (NFkB) translocation, posttranslational state and expression levels during day and night conditions were assessed using immunoblot and IP. The regulation of transcription by Clock in cartilage tissue was assessed by using chromatin immunoprecipitation (ChIP) and luciferase assays. Total acetylation level and pattern over 24 h were quantified using immunoblot and real-time PCR. Finally, the effects of exogenous Clock nanoparticle treatment were quantified by histology and immunoblot. Results: The Clock mutation significantly promoted the degradation of cartilage and the expression of the key pro-inflammatory mediators, IL-1b, IL-6 and MCP-1. The Clock mutation significantly promoted NFkB nuclear translocation. The circadian protein CLOCK positively regulates NFkB at the transcriptional level by binding the E-box domain. The Clock mutation significantly inhibited the total lysine acetylation level in cartilage and inhibited NFkB acetylation at the Lys310 residue but promoted phosphorylation at the Ser276 residue. The forced expression of Clock in vivo inhibited NFkB activation by increasing acetylation and decreasing phosphorylation levels and by decreasing cartilage damage and inflammation. Conclusions: This study demonstrates the mutation of Clock promotes inflammatory activity by mediating the posttranscriptional regulation of NFkB in OA pathogenesis.

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Accumulated evidence indicates that circadian genes regulate cell damage and senescence in most mammals. Endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) regulate longevity in many organisms. However, the specific mechanisms of the relationship between the circadian clock and the two stress processes in organisms are poorly understood. Here, we show that Clock-mediated Pdia3 expression is required to sustain reactive oxidative reagents and ER stress. First, ER stress and ROS are strongly activated in the liver tissue of ClockΔ19 mutant mice, which exhibit a significant aging phenotype. Next, transcription of Pdia3 is mediated by the circadian gene Clock, but this process is affected by the ClockΔ19 mutant due to the low affinity of the E-box motif in the promoter. Finally, ablation of Pdia3 with siRNA causes ER stress with sustained phosphorylation of PERK and eIF1α, resulting in exaggerated up-regulation of UPR target genes and increased apoptosis as well as ROS. Moreover, the combined effects result in an imbalance of cell homeostasis and ultimately lead to cell damage and senescence. Taken together, this study identified the circadian gene Clock as a regulator of ER stress and senescence, which will provide a reference for the clinical prevention of aging.

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Adiposity is a worldwide health threat that needs to be prevented. Circadian gene Clock (circadian locomotor output cycles kaput) is closely correlated to adiposity; for example, weight gain, adipocytes size expansion, and serum lipid level rise in Clock mice compared to C57BL/6J mice. However, the precise role of Clock during adipogenic differentiation is unknown. Herein, the circadian gene Clock is shown to regulate adipogenesis mediated by GILZ. Clock-mediated attenuation and upregulation influenced lipid synthesis and affected the levels of adipogenic transcriptional factors, C/EBP-β, C/EBP-α, PPAR-γ, and FABP4, both in vivo and in vitro (primary adipose-derived stromal cells and 3T3-L1 cells). Chromatin immunoprecipitation (ChIP) assay, reporter gene assay, and serum shock assay found that Clock transcriptionally regulated the glucocorticoid-induced leucine zipper (GILZ). Furthermore, GILZ attenuation could relieve the inhibitory effect of Clock on lipid synthesis and GILZ overexpression also reduced the promotion role of Clock attenuation in adipogenesis suggesting that Clock inhibits adipogenic differentiation of preadipocytes via GILZ. The current results demonstrate how circadian genes are likely to regulate adiposity, affecting the adipogenic differentiation process, as well as, increasing the fat cells number. Therefore, this study may provide novel insights into the underlying mechanism explaining the correlation between Clock mutation and adiposity.

In addition to the role of programmed cell death ligand 1 (PD-L1) in facilitating tumour cells escape from immune surveillance, it is considered as a crucial effector in transducing intrinsic signals to promote tumour development. Our previous study has pointed out that PD-L1 promotes non-small cell lung cancer (NSCLC) cell proliferation, but the mechanism remains elusive. Here we first demonstrated that PD-L1 expression levels were positively correlated with p-MerTK levels in patient samples and NSCLC cell lines. In addition, PD-L1 knockdown led to the reduced phosphorylation level of MerTK in vitro. We next showed that PD-L1 regulated NSCLC cell proliferation via Gas6/MerTK signaling pathway in vitro and in vivo. To investigate the underlying mechanism, we unexpectedly found that PD-L1 translocated into the nucleus of cancer cells which was facilitated through the binding of Karyopherin β1 (KPNB1). Nuclear PD-L1 (nPD-L1), coupled with transcription factor Sp1, regulated the synthesis of Gas6 mRNA and promoted Gas6 secretion to activate MerTK signaling pathway. Taken together, our results shed light on the novel role of nPD-L1 in NSCLC cell proliferation and reveal a new molecular mechanism underlying nPD-L1-mediated Gas6/MerTK signaling activation. All above findings provide the possible combinational implications for PD-L1 targeted immunotherapy in the clinic.