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OBJECTIVE: To develop and validate a prediction model based on targeted sequencing for glucocorticoid (GC)-associated osteonecrosis of the femoral head (GA-ONFH) in GC-treated adults. METHODS: This two-centre retrospective study was conducted between July 2015 and April 2019 at Zhongshan Hospital (training set) and the Sixth People's Hospital (test set) in Shanghai, China. All patients had a history of GC therapy, with a dose exceeding 2000 mg equivalent prednisone within 6 weeks. Patients were divided into two groups according to whether they were diagnosed with GA-ONFH within 2 years after GC initiation. Blood or saliva samples were collected for targeted sequencing of 358 single nucleotide polymorphisms and genetic risk score (GRS) calculating for developing GA-ONFH prediction model. Receiver operating characteristic (ROC) curve analysis and decision curve analysis (DCA) were performed to evaluate and validate the model. RESULTS: . The training set comprised 117 patients, while the test set comprised 30 patients for external validation. Logistic regression analysis showed that GRS was significantly associated with GA-ONFH (OR 1.87, 95% CI: 1.48, 2.37). The ROC and DCA curves showed that the multivariate model considering GRS, age at GC initial, sex and underlying diseases had a discrimination with area under the ROC curve (AUC) of 0.98 (95% CI: 0.96, 1.00). This model was further externally validated using the test set with an AUC of 0.91 (95% CI: 0.81, 1.00). CONCLUSION: Our prediction model comprising GRS, age, sex and underlying diseases yields valid predictions of GA-ONFH incidence. It may facilitate effective screening and prevention strategies of GA-ONFH.
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Necrose da Cabeça do Fêmur/induzido quimicamente , Glucocorticoides/efeitos adversos , Adolescente , Adulto , Fatores Etários , Idoso , Feminino , Necrose da Cabeça do Fêmur/genética , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Análise de Sequência de DNA , Fatores Sexuais , Adulto JovemRESUMO
Circadian genes control most of the physiological functions including cell cycle. Cell proliferation is a critical factor in the differentiation of progenitor cells. However, the role of Clock gene in the regulation of cell cycle via wingless-type (Wnt) pathway and the relationship between Clock and adipogenesis are unclear. We found that the circadian locomotor output cycles kaput (Clock) regulated the proliferation and the adipogenesis of 3T3-L1 preadipocytes. We found that Clock attenuation inhibited the viability of 3T3-L1 preadipocytes in the cell counting kit 8. The expression of c-Myc and Cyclin D1 decreased dramatically in 3T3-L1 when Clock was silenced with short interfering RNA and was also decreased in fat tissue and adipose tissue-derived stem cells of Clock(Δ19) mice. Clock directly controls the expression of the components of Wnt signal transduction pathway, which was verified by serum shock, chromatin immunoprecipitation, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, IWR-1, a Wnt signal pathway inhibitor, inhibited the cell cycle promotion by CLOCK, which was detected by cell viability assay, flow cytometry, and qRT-PCR. Therefore, CLOCK transcription control of Wnt signaling promotes cell cycle progression in 3T3-L1 preadipocytes. Clock inhibited the adipogenesis on day 2 in 3T3-L1 cells via Oil Red O staining and qRT-PCR detection and probably related to cellular differentiation. These data provide evidence that the circadian gene Clock regulates the proliferation of preadipocytes and affects adipogenesis. © 2016 IUBMB Life, 68(7):557-568, 2016.
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Adipogenia/genética , Proteínas CLOCK/genética , Proliferação de Células/genética , Proteínas Proto-Oncogênicas c-myc/genética , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Diferenciação Celular/genética , Sobrevivência Celular/genética , Ciclina D1/genética , Regulação da Expressão Gênica , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
PURPOSE: Hip preservation therapy of early ONFH (Osteonecrosis of the femoral head) has emerged as one of the hot areas of research. We have optimized the procedure of traditional MFCVBG (medial femoral circumflex vascularized bone grafting) by using specialized surgical tools and used the finite element analysis to guide the implantation position of the bone flap during surgery and validate the biological mechanical stability of the modified MFCVBG. METHODS: This study was based on the data of a male patient with left hip (ARCO stage IIB, JIC type C) hormonal ONFH. Harris score (HHS), anteroposterior and lateral hip radiographs, frog position hip radiographs and SPECT/CT of femoral head flow imaging were performed postoperatively to evaluate clinical efficacy. The patient's CT data were used to establish upper femur finite element model of the normal group, osteonecrosis group and postoperative group, respectively. The force on the femoral structure of each group was analyzed under four different loads in the gait cycle of 0.5 times the body weight (0.5 G, standing on two feet), 2.75 G (standing on one foot), 4 G (walking with the middle foot on the ground) and 7 G (walking with the toe off the ground) to validate the biological mechanical stability of the modified MFCVBG, predict femoral head collapse risk, simulate of the different healing conditions of postoperative bone flap, and analyze the postoperative effect of non-ideal surgical model. RESULTS: According to the follow-up results, the bone flap and the inner wall of decompression channel healed well, no osteonecrosis progression, no local collapse or micro-fracture occurred in the femoral head, and the articular surface was intact and the necrosis was well repaired. According to the result of the finite element analysis, compared with the osteonecrosis group, the overall stress and displacement peak of the upper femur and the cortical bone stress peak of the femoral head in the postoperative group and normal group were significantly reducing; modified MFCVBG can significantly improve the biomechanical stability of necrotic femoral head and reduce the risk of femoral head collapse; there was no obvious abnormal stress distribution in the greater trochanter and intertrochanter region after the flap was removed; the bone flap of the complete removal of necrotic focus + long bone flap group was directly placed at the bottom of the decompression passage, and the bone flap cortical bone can provide substantial mechanical support; in theory, patients can try to reduce the load with crutches or walking aids and carry out appropriate flat activities to effectively promote the early postoperative recovery. CONCLUSIONS: The modified MFCVBG resulted in good efficacy, safety and feasibility. The necrotic focus should be completely removed during the operation, and the long bone flap should be placed directly under the subchondral bone. For patients with better bone healing ability, a more positive attitude can be taken to promote early postoperative weight-bearing.
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Necrose da Cabeça do Fêmur , Cabeça do Fêmur , Transplante Ósseo/métodos , Fêmur/cirurgia , Fêmur/transplante , Cabeça do Fêmur/diagnóstico por imagem , Cabeça do Fêmur/cirurgia , Necrose da Cabeça do Fêmur/diagnóstico por imagem , Necrose da Cabeça do Fêmur/cirurgia , Análise de Elementos Finitos , Humanos , Masculino , Resultado do TratamentoRESUMO
Circadian genes such as Clock, Bmal1, Cryptochrome1/2, and Period1/2/3 constitute the precise circadian system. ClockΔ19 is a commonly used mouse model harboring a circadian clock gene mutation, which lacks the EXON-19-encoded 51 amino acids. Previous reports have shown that ClockΔ19 mice have severe metabolic abnormalities. Here, we report that the mitochondria of ClockΔ19 mice exhibit excessive fission and dysfunction. We also demonstrate that CLOCK binds to the RNA-binding protein PUF60 through its EXON 19. Further, we find that PUF60 directly maintains mitochondrial homeostasis through regulating Drp1 mRNA stability, while the association with CLOCK can competitively inhibit this function. In ClockΔ19 mice, CLOCKΔ19 releases PUF60, leading to enhanced Drp1 mRNA stability and persistent mitochondrial fission. Our results reveal a direct post-transcriptional role of CLOCK in regulating mitochondrial homeostasis via Drp1 mRNA stability and that the loss of EXON 19 of CLOCK in ClockΔ19 mice leads to severe mitochondrial homeostasis disorders.
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Proteínas CLOCK , Relógios Circadianos , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Homeostase/genética , Camundongos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Estabilidade de RNARESUMO
PURPOSE: Osteoarthritis (OA) is a progressive disease characterized by pain and impaired joint functions. Engeletin is a natural compound with anti-inflammatory and antioxidant effects on other diseases, but the effect of engeletin on OA has not been evaluated. This study aimed to elucidate the protective effect of engeletin on cartilage and the underlying mechanisms. METHODS: Chondrocytes were isolated from rat knee cartilage, and TNF-α was used to simulate OA in vitro. After treatment with engeletin, the expression of extracellular matrix (ECM) components (collagen II and aggrecan) and matrix catabolic enzymes (MMP9 and MMP3) was determined by Western blotting and qPCR. Chondrocyte apoptosis was evaluated using Annexin V-FITC/PI and flow cytometry. Apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) were evaluated by Western blotting. The mitochondrial membrane potential of chondrocytes was measured with JC-1, and intracellular reactive oxygen species (ROS) levels were determined with DCFH-DA. Changes in signaling pathways (Nrf2, NF-κB and MAPK) were evaluated by Western blotting. In vivo, anterior cruciate ligament transection (ACLT) was used to induce the rat OA model, and engeletin was administered intraarticularly. The therapeutic effect of engeletin was analyzed by histopathological analysis. RESULTS: Pretreatment with engeletin alleviated TNF-α-induced inhibition of ECM components (collagen II and aggrecan) and upregulation of matrix catabolic enzymes (MMP9 and MMP3). Engeletin ameliorated chondrocyte apoptosis by inhibiting Bax expression and upregulating Bcl-2 expression. Engeletin maintained the mitochondrial membrane potential of chondrocytes and scavenged intracellular ROS by activating the Nrf2 pathway. The NF-κB and MAPK pathways were inhibited by treatment with engeletin. In vivo, ACLT-induced knee OA in rats was alleviated by intraarticular injection of engeletin. CONCLUSION: Engeletin ameliorated OA in vitro and in vivo. It may be a potential therapeutic drug for OA.
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Physiological function and metabolic regulation are the most important outputs of circadian clock controls in mammals. Mitochondrial respiration and ROS production show rhythmic activity. Mitochondrial carriers, which are responsible for mitochondrial substance transfer, are vital for mitochondrial metabolism. Clock (Circadian Locomotor Output Cycles Kaput) is the first core circadian gene identified in mammalian animals. However, whether CLOCK protein can regulate mitochondrial functions via mitochondrial carriers is unclear. Here, we showed that CLOCK can bind to the mitochondrial carrier SLC25A10. For further analysis, we established a Slc25a10-/--Hepa1-6 cell line using CRISPR/Cas9 gene-editing technology. Slc25a10-/--Hepa1-6 cells showed disordered glucose homeostasis, increased oxidative stress levels, and damaged electron transport chains. Next, using an immunoprecipitation assay, we found that amino acids 43-84 and 169-210 in SLC25A10 are key sites that respond to CLOCK binding. Finally, forced expression of wild-type SLC25A10 in Slc25a10-/--Hepa1-6 cells could compensate for the loss of SLC25A10; the decreased glucose metabolism, severe oxidative stress and damaged electron transport chain were recovered. In addition, a mutant Slc25a10 with changes in two key sites did not show a rescue effect. In conclusion, we identified a new protein-protein interaction mechanism in which CLOCK can directly regulate cell metabolism via the mitochondrial membrane transporter SLC25A10. Our study might provide some new insights into the relationship between circadian clock and mitochondrial metabolism.
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Proteínas CLOCK/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Metabolismo Energético , Proteínas Mitocondriais/metabolismo , Animais , Proteínas CLOCK/genética , Transportadores de Ácidos Dicarboxílicos/genética , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Proteínas Mitocondriais/genéticaRESUMO
Early diagnosis and prevention of glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH) continues to be a challenging problem for clinicians and researchers. However, the role of circulating biomarkers for GC-induced ONFH, which may reveal individual susceptibility and facilitate earlier diagnosis, remains to be determined. The aim of this study was to identify potential biomarkers that may predict early GC-induced ONFH. A total of 123 patients scheduled for initial systemic GC therapy were enrolled in this prospective nested case-control study. The serum concentrations of 13 potential biomarkers were measured in seven patients with GC-induced ONFH, diagnosed instantly after short-term use of GCs and in 20 controls who did not develop osteonecrosis despite similar GC therapy. Biomarkers were measured both before and after taking GCs to identify any differences in marker levels and the changes during GC therapy between two groups. Type I collagen cross-linked C-telopeptide (ß-CTX; p = 0.000) was significantly lower, high-density lipoprotein cholesterol (p = 0.092) and apolipoprotein (apo)-B/apo-A1 (p = 0.085) tended to be lower and higher, respectively, before GC treatment in osteonecrosis group. After GC therapy, ß-CTX (p = 0.014) was significantly lower and amino terminal telopeptide of procollagen type I (PINP; p = 0.068) tended to be lower in the osteonecrosis group. As secondary outcomes, we observed remarkable changes in nine potential biomarkers following short-term GC therapy in both groups. In conclusion, we found that ß-CTX, could potentially be used to predict GC-induced ONFH before GC therapy. Lower ß-CTX and PINP are promising biomarkers for the early diagnosis of GC-induced ONFH. These findings need to be confirmed in large-scale prospective studies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2348-2357, 2019.
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Biomarcadores/sangue , Necrose da Cabeça do Fêmur/sangue , Glucocorticoides/efeitos adversos , Adolescente , Adulto , Estudos de Casos e Controles , Necrose da Cabeça do Fêmur/induzido quimicamente , Glucocorticoides/administração & dosagem , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
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 ClockΔ19 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.
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Proteínas CLOCK/metabolismo , Relógios Circadianos/fisiologia , Osteogênese/fisiologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Proteínas CLOCK/genética , Feminino , Masculino , Camundongos , Camundongos Mutantes , Isomerases de Dissulfetos de Proteínas/genéticaRESUMO
Mesenchymal stem cells (MSCs) provide promising applications for clinical treatments. However, patients often take medications that affect the viability of transplanted MSCs. The aim of this study was to assess the effects and underlying mechanism of action of aspirin on the proliferation of MSCs. We showed that aspirin inhibited the growth of MSCs in a concentration- and time-dependent manner. Analysis of cell-cycle distributions showed significantly increased cell populations in the G0/G1 phase and decreased cell populations in the S phase and G2/M phase with increasing concentrations of aspirin. We further analyzed the expression of cyclins and found that the level of cyclin D1 was significantly reduced after aspirin treatment, while there was no obvious effect on the levels of cyclin A2 and cyclin E1. Because we showed that the expression of miRNA145 was significantly increased after aspirin treatment, we further transfected MSCs with an miRNA145 mimic or miRNA145 inhibitor. Transfection with the miRNA145 mimic resulted in decreased expression of cyclin D1, while transfection with miRNA145 inhibitor resulted in increased expression of cyclin D1. Transfection with miRNA145 inhibitor abolished the downregulation of cyclin D induced by aspirin. The results suggested that aspirin inhibited the proliferation of MSCs and caused cell-cycle arrest in the G0/G1 phase through downregulation of cyclin D1, which could be related to the increased expression of miRNA145.
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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 Clock∆19 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 Clock∆19 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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Proteínas CLOCK/genética , Senescência Celular/genética , Estresse do Retículo Endoplasmático/genética , Isomerases de Dissulfetos de Proteínas/genética , Animais , Feminino , Regulação da Expressão Gênica/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Isomerases de Dissulfetos de Proteínas/biossínteseRESUMO
The clinical translation of tissue engineering methods is confined by the limited external cell sources, which is hopefully to be addressed by the cell guidance approach as cytokine-induced homing and differentiation of the patients' autologous cells. Synovium-derived stem cells (SDSCs) are a potent cell source for cartilage restoration due to its intrinsic proximity and tissue-specific chondrogenic capacity. In this study, stromal cell-derived factor-1α (SDF-1α) in combination with transforming growth factor ß1 (TGF-ß1) were used to induce SDSCs migration and chondrogenesis in vitro. The migration capacity was evaluated by transwell assay and for chondrogenic evaluation, the expression of Sox9, ACAN and COL2A1 were assessed by quantitative RT-PCR while the expression of sulfated GAG and collagen II were evaluated by Alcian Blue stain and immunohistochemistry respectively. Our data showed that SDF-1α/CXC chemokine receptor 4 (CXCR4) was involved in SDSCs migration through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Exogenous TGF-ß1 enhanced SDF-1α-induced SDSCs migration in a concentration and time-dependent manner through CXCR4, evidenced as complete blockage by AMD3100, the CXCR4 antagonist and this effect was mediated by extracellular regulated protein kinases (ERK) activation. Moreover, the addition of SDF-1α augmented the TGF-ß1-induced SDSCs chondrogenesis, evidenced by the increased pellet sizes and the expressions of COL 2A1, ACAN and Sox9. This effect was related to c-Jun N-terminal kinase (JNK) activation. Collectively, these results suggest that SDF-1α and TGF-ß1 interacts with each other and synergistically enhance the SDSCs migration and chondrogenesis through MAPK pathways.
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Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.
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Proteínas CLOCK/genética , Reprogramação Celular , Relógios Circadianos/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Apoptose , Sequência de Bases , Proteínas CLOCK/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular , Proliferação de Células , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Background. The risk of atherosclerosis is elevated in abnormal lipid metabolism and circadian rhythm disorder. We investigated whether abnormal lighting condition would have influenced the circadian expression of clock genes and clock-controlled lipid metabolism-related genes in ApoE-KO mice. Methods. A mouse model of atherosclerosis with circadian clock genes expression disorder was established using ApoE-KO mice (ApoE-KO LD/DL mice) by altering exposure to light. C57 BL/6J mice (C57 mice) and ApoE-KO mice (ApoE-KO mice) exposed to normal day and night and normal diet served as control mice. According to zeitgeber time samples were acquired, to test atheromatous plaque formation, serum lipids levels and rhythmicity, clock genes, and lipid metabolism-related genes along with Sirtuin 1 (Sirt1) levels and rhythmicity. Results. Atherosclerosis plaques were formed in the aortic arch of ApoE-KO LD/DL mice. The serum lipids levels and oscillations in ApoE-KO LD/DL mice were altered, along with the levels and diurnal oscillations of circadian genes, lipid metabolism-associated genes, and Sirt1 compared with the control mice. Conclusions. Abnormal exposure to light aggravated plaque formation and exacerbated disorders of serum lipids and clock genes, lipid metabolism genes and Sirt1 levels, and circadian oscillation.