Circadian light/dark cycle reversal exacerbates the progression of chronic kidney disease in mice.

Circadian disruption such as shift work, jet lag, has gradually become a global health issue and is closely associated with various metabolic disorders. The influence and mechanism of circadian disruption on renal injury in chronic kidney disease (CKD) remains inadequately understood. Here, we evaluated the impact of environmental light disruption on the progression of chronic renal injury in CKD mice. By using two abnormal light exposure models to induce circadian disruption, we found that circadian disruption induced by weekly light/dark cycle reversal (LDDL) significantly exacerbated renal dysfunction, accelerated renal injury, and promoted renal fibrosis in mice with 5/6 nephrectomy and unilateral ureteral obstruction (UUO). Mechanistically, RNA-seq analysis revealed significant immune and metabolic disorder in the LDDL-conditioned CKD kidneys. Consistently, renal content of ATP was decreased and ROS production was increased in the kidney tissues of the LDDL-challenged CKD mice. Untargeted metabolomics revealed a significant buildup of lipids in the kidney affected by LDDL. Notably, the level of ß-NMN, a crucial intermediate in the NAD+ pathway, was found to be particularly reduced. Moreover, we demonstrated that both ß-NMN and melatonin administration could significantly rescue the light-disruption associated kidney dysfunction. In conclusion, environmental circadian disruption may exacerbate chronic kidney injury by facilitating inflammatory responses and disturbing metabolic homeostasis. ß-NMN and melatonin treatments may hold potential as promising approaches for preventing and treating light-disruption associated CKD.

Circadian control of ConA-induced acute liver injury and inflammatory response via Bmal1 regulation of Junb.

Background & Aims: Circadian rhythms play significant roles in immune responses, and many inflammatory processes in liver diseases are associated with malfunctioning molecular clocks. However, the significance of the circadian clock in autoimmune hepatitis (AIH), which is characterised by immune-mediated hepatocyte destruction and extensive inflammatory cytokine production, remains unclear. Methods: We tested the difference in susceptibility to the immune-mediated liver injury induced by concanavalin A (ConA) at various time points throughout a day in mice and analysed the effects of global, hepatocyte, or myeloid cell deletion of the core clock gene, Bmal1 (basic helix-loop-helix ARNT-like 1), on liver injury and inflammatory responses. Multiple molecular biology techniques and mice with macrophage-specific knockdown of Junb, a Bmal1 target gene, were used to investigate the involvement of Junb in the circadian control of ConA-induced hepatitis. Results: The susceptibility to ConA-induced liver injury is highly dependent on the timing of ConA injection. The treatment at Zeitgeber time 0 (lights on) triggers the highest mortality as well as the severest liver injury and inflammatory responses. Further study revealed that this timing effect was driven by macrophage, but not hepatocyte, Bmal1. Mechanistically, Bmal1 controls the diurnal variation of ConA-induced hepatitis by directly regulating the circadian transcription of Junb and promoting M1 macrophage activation. Inhibition of Junb in macrophages blunts the administration time-dependent effect of ConA and attenuates liver injury. Moreover, we demonstrated that Junb promotes macrophage inflammation by regulating AKT and extracellular signal-regulated kinase (ERK) signalling pathways. Conclusions: Our findings uncover a critical role of the Bmal1-Junb-AKT/ERK axis in the circadian control of ConA-induced hepatitis and provide new insights into the prevention and treatment of AIH. Impact and Implications: This study unveils a critical role of the Bmal1-Junb-AKT/ERK axis in the circadian control of ConA-induced liver injury, providing new insights into the prevention and treatment of immune-mediated hepatitis, including autoimmune hepatitis (AIH). The findings have scientific implications as they enhance our understanding of the circadian regulation of immune responses in liver diseases. Furthermore, clinically, this research offers opportunities for optimising treatment strategies in immune-mediated hepatitis by considering the timing of therapeutic interventions.

[Research progress in control strategies of biological clock disorder].

Circadian clock is an internal mechanism evolved to adapt to cyclic environmental changes, especially diurnal changes. Keeping the internal clock in synchronization with the external clock is essential for health. Mismatch of the clocks due to phase shift or disruption of molecular clocks may lead to circadian disorders, including abnormal sleep-wake cycles, as well as disrupted rhythms in hormone secretion, blood pressure, heart rate, body temperature, etc. Long-term circadian disorders are risk factors for various common critical diseases such as metabolic diseases, cardiovascular diseases, and tumor. To prevent or treat the circadian disorders, scientists have conducted extensive research on the function of circadian clocks and their roles in the development of diseases, and screened hundreds of thousands of compounds to find candidates to regulate circadian rhythms. In addition, melatonin, light therapy, exercise therapy, timing and composition of food also play a certain role in relieving associated symptoms. Here, we summarized the progress of both drug- and non-drug-based approaches to prevent and treat circadian clock disorders.

Impact of Time-Restricted Feeding on Adaptation to a 6-Hour Delay Phase Shift or a 12-Hour Phase Shift in Mice.

Nowadays, more and more people are suffering from circadian disruption. However, there is no well-accepted treatment. Recently, time-restricted feeding (TRF) was proposed as a potential non-drug intervention to alleviate jet lag in mice, especially in mice treated with a 6-h advanced phase shift. Here, we challenged C57BL/6 mice with a 6-h delay phase shift or a 12-h shift (day-night reversal) combined with 6- or 12-h TRF within the dark phase and found the beneficial effects of given TRF strategies in certain phase-shifting situations. Although behavioral fitness did not correlate well with health status, none of the TRF strategies we used deteriorated lipopolysaccharide-induced sepsis. These findings improve our understanding of the benefits of TRF for adaptation to circadian disruption.

Strain and Age Dependent Entrainable Range of Circadian Behavior in C57BL/6 and BALB/c Mice.

The mammalian circadian system has a plasticity in a certain range, rather than a strict 24-hour cycle, with considerable variations among species, strains, and ages. As the most widely used mouse strains in circadian research, C57BL/6 and BALB/c mice were well known to have different internal periods and responses to various non-24-hour light-dark cycles. However, their entrainable range of circadian behavior was not specifically studied, neither was the effect of aging. Besides, it is not well known if mice with appeared behavioral adaptation are really healthy. In the current study, we exposed C57BL/6 and BALB/c mice at 3 months and 18 months old to a series of short (T cycles < 24 h) and long (T cycles > 24 h) light-dark cycles. Wheel running activities were monitored continuously for calculation of the entrainable range and glucose homeostasis was investigated to reflect their health status. Our results showed that the range in both young and old C57BL/6 mice is between T23 and T26. By contrast, due to the strong adaptability to extreme LD cycles, the entrainable range on a circadian scale in both young and old BALB/c mice cannot be well determined. Despite the adaptation appeared at the behavioral level, glucose homeostasis revealed by glucose tolerance test and insulin tolerance test was impaired in mice upon T cycle treatment. In summary, our study explored the entrainment range in two popular mouse strains and suggested that behavioral adaptation may not well reflect their health status.

Impact of Time-Restricted Feeding to Late Night on Adaptation to a 6 h Phase Advance of the Light-Dark Cycle in Mice.

In modern society, more and more people suffer from circadian disruption, which in turn affects health. But until now, there are no widely accepted therapies for circadian disorders. Rhythmic feeding behavior is one of the most potent non-photic zeitgebers, thus it has been suggested that it was important to eat during specific periods of time (time-restricted feeding, TRF) so that feeding is aligned with environmental cues under normal light/dark conditions. Here, we challenged mice with a 6 h advanced shift, combined with various approaches to TRF, and found that food restricted to the second half of the nights after the shift facilitated adaptation. This coincided with improved resilience to sepsis. These results raise the possibility of reducing the adverse responses to jet lag by subsequent timing of food intake.

Rhein Inhibits the Migration of Ovarian Cancer Cells through Down-Regulation of Matrix Metalloproteinases.

The root of Rheum officinale BAILL as a traditional Chinese medicine, which main function is removing heat from the blood, promoting blood circulation and clearing toxins away. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is one of the most important active components in the root of Rheum officinale BAILL, which could inhibit the proliferation of tumor cells. However, the study on the mechanism of anti-cell migration capacity of Rhein on ovarian cancer is not yet clear. Here, we demonstrated that Rhein had dose-dependent effects of ovarian tumors on drugs and could inhibit the proliferations and migration of two typical ovarian cancer cell lines, A2780 and OV2008. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that the survival rate of ovarian cancer cells was significantly decreased when treated with Rhein. Rhein inhibited the proliferation of ovarian cancer cells in dose-dependent manner. Moreover, the wound healing assay and transwell assay indicated that the cell migratory potential and expression of matrix metalloproteinases were markedly inhibited by Rhein. Our findings suggested that Rhein could be a potential candidate to be developed as a drug for the prevention of ovarian cancer cell migration.

Bmal1 deletion in mice facilitates adaptation to disrupted light/dark conditions.

Recently, by utilizing conventional and tamoxifen inducible Bmal1 (Brain and muscle Arnt-like protein 1) knockout mice, we found that delaying the loss of circadian rhythms to adulthood attenuates the impact on general integrity and survival at least under 12h light/12h dark conditions (LD). To understand further the contribution of Bmal1 in post-natal life under conditions of circadian disruption, we subjected inducible knockout mice (KO) and their littermate controls (Ctrl) to forced desynchrony protocols including cycles with non-24h periods, randomized light/dark cycles, and jet lag, and monitored their locomotor activity using radiotelemetry. Under these conditions, control mice cannot be entrained, as reflected by their maintenance of circadian behavior irrespective of schedules. By contrast, KO mice displayed higher activity levels in the dark phases of most cycles. Under a 3h light/3h dark regime, Ctrls displayed higher activity levels in the dark phases of all cycles although there were still obvious circadian rhythms, suggesting that an ultradian mechanism is also involved. Insulin sensitivity was markedly reduced by disrupted light schedules as expected in Ctrls, but not in the KOs. Thus, Bmal1 deletion in adult mice facilitates adaptation to new light/dark schedules and protects from insulin resistance induced by circadian disruption.

Time-Dependent Hypotensive Effect of Aspirin in Mice.

Objective- Evening but not morning administration of low-dose aspirin has been reported to lower blood pressure in hypertensive patients. The present study was designed to determine whether this phenomenon could be replicated in mice, and if so, whether a time-dependent effect of aspirin on blood pressure was because of alteration of circadian clock function. Approach and Results- We recapitulated the protective effect of aspirin (50 µg/d for 7 days) at zeitgeber time 0 (active-to-rest transit), but not at zeitgeber time 12, on a high-salt diet-induced increase of blood pressure. However, the time of aspirin administration did not influence expression of canonical clock genes or their acetylation. We used mouse Bmal1 and Per2-luciferase reporters expressed in U2OS cells to determine the real-time effect of aspirin on circadian function but found that the oscillation of bioluminescence was unaltered. Timing of aspirin administration also failed to alter urinary prostaglandin metabolites or catecholamines, or the acetylation of its COX-1 (cyclooxygenase-1) target in platelets. Conclusions- The time-dependent hypotensive effect of aspirin in humans has been recapitulated in hypertensive mice. However, this does not seem to reflect a direct impact of aspirin on circadian clocks or on acetylation of platelet COX-1.

Formononetin, an isoflavone from Astragalus membranaceus inhibits proliferation and metastasis of ovarian cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus membranaceus which was originally described in the Shennong's Classic of Materia Medica, the earliest complete Pharmacopoeia of China written from the Warring States Period to Han Dynasty, has been widely used in Chinese medicine for > 2000 years, especially in the prescription of curing cancer. A. membranaceus has various bioactivities, such as anti-tumor, anti-viral, anti-oxidant, anti-diabetes, anti-inflammation, anti-atherosclerosis, immunomodulation, hepatoprotection, hematopoiesis, neuroprotection and so on. As an important component of A. membranaceus, whether formononetin has a close relationship with its tumor-inhibiting effect on ovarian cancer cell has been investigated. AIM OF STUDY: The present study aimed to demonstrate the anti-proliferation, anti- migration and invasion effects of formononetin on ovarian cancer cells and further explore the underlying molecular mechanisms associated with apoptosis, migration and invasion. MATERIALS AND METHODS: MTT assay was performed to detect the viability of ovarian cancer cells. DAPI staining, Annexin-V assay and assay for mitochondrial membrane potential detected the apoptosis of ovarian cancer cells treated by formononetin. The migration and invasion of ovarian cancer cells which exposed to formononetin were detected by scratch assay and transwell assay. Meanwhile, the protein-level changes of in ovarian cancer cells treated by formononetin were assessed by western blot analysis. RESULTS: MTT assays indicated that cell viability significantly decreased in ovarian cancer cells treated with formononetin. DAPI staining, Annexin-V assay and assay for mitochondrial membrane potential suggested that formononetin suppressed cells proliferation by inducing apoptosis. We detected the expression of apoptosis-related proteins in ovarian cancer cells after treatment with formononetin and found the expression of caspase 3/9 proteins and the ratio of Bax/Bcl-2 were increased in a dose-dependent manner. In addition, wound healing and transwell chamber assays showed that formononetin suppressed the migration and invasion of ovarian cancer cells. And formononetin decreased expression of MMP-2/9 proteins and phosphorylation level of ERK. CONCLUSIONS: The present results demonstrated that formononetin have potential effects on induction of apoptosis and suppression of migration and invasion.

Down-regulation of GADD45A enhances chemosensitivity in melanoma.

Melanoma is a malignant skin cancer with considerable drug resistance. Increased expression of DNA repair genes have been reported in melanoma, and this contributes to chemotherapy resistance. GADD45A is involved in DNA repair, cell cycle arrest and apoptosis in response to physiologic or environmental stresses. In this study, we investigated the role of GADD45A in chemotherapy response. Firstly, the mRNA expression of profiled DNA repair genes in cisplatin-treated melanoma cells was detected by RT2 profilerTM PCR array. We found the expression of GADD45A upregulated in a dose- and time- dependent manner. In addition, suppression of GADD45A sensitized melanoma cells to cisplatin and enhanced cisplatin-induced DNA damage. Flow cytometry revealed that downregulating GADD45A released cells from cisplatin-induced G2/M arrest and increased apoptosis. By using a MEK inhibitor, GADD45A was shown to be regulated by MAPK-ERK pathway following cisplatin treatment. Thus, the induction of GADD45A might play important roles in chemotherapy response in human melanoma cancer and could serve as a novel molecular target for melanoma therapy.

Anti-tumor effect of Scutellaria barbata D. Don extracts on ovarian cancer and its phytochemicals characterisation.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria barbata D. Don is a widely used medicinal herb in China. It possess various medicinal properties including antioxidative, anti-inflammatory and anti-cancer effects. The aim of this study was to explore whether Scutellaria barbata D. Don could inhibit the growth of ovarian cancer cells in vitro and further investigate the underlying mechanisms. MATERIALS AND METHODS: Effects of Scutellaria barbata D. Don on the viability of ovarian cancer A2780 cells were measured by MTT assay. Apoptosis was measured by cell morphologic observation through DAPI staining and Annexin V-FITC staining assay for apoptosis analysis. The migration of ovarian cancer cells which exposed to Scutellaria barbata D. Don were measured by wound healing and transwell chamber assays. The protein levels of caspase 3/9, Bcl-2 and MMP-2/9 in human ovarian cancer cells treated with Scutellaria barbata D. Don were assessed by western blotting analysis. The potential bioactive compounds were characterized by HPLC-Q-TOF-MS. RESULTS: The present study was to investigate the anticancer effects of crude extracts from Scutellaria barbata D. Don on ovarian cancer A2780 cells by MTT, DAPI staining, wound healing assay, transwell migration assay and western blotting analysis. Our study showed that Scutellaria barbata D. Don reduced the viability of A2780 cells and induced apoptosis by down-regulated Bcl-2 protein and increased Caspase 3/9 proteins. Furthermore, migration of A2780 cells were significantly inhibited by Scutellaria barbata D. Don and the underlying mechanism may be related to the decrease of MMP-2/9. The main constituents from Scutellaria barbata D. Don were identified to be thirteen flavonoids. A HPLC-Q-TOF-MS analysis of Scutellaria barbata D. Don indicated the presence of 14 flavonoids compounds, which may contribute to the anticancer activity of the Scutellaria barbata D. Don. CONCLUSIONS: Scutellaria barbata D. Don could inhibit proliferation and induce apoptosis in A2780 cells through mitochondrial pathway. Moreover, the inhibitory effect of Scutellaria barbata D. Don on the migration of ovarian cancer cells was associated with the down-regulation of MMP-2/9 expression. These findings could shed a light on the therapy of ovarian cancer.

Anti-tumor and chemosensitization effects of Cryptotanshinone extracted from Salvia miltiorrhiza Bge. on ovarian cancer cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Cryptotanshinone, a natural compound isolated from the roots of Salvia miltiorrhiza Bge. (Danshen), is a commonly used traditional Chinese medicine to treat high blood pressure in some countries. It has been shown that Cryptotanshinone induces cancer cells apoptosis and impairs cell migration and invasion. However, the antiproliferation and chemosensitization effects of Cryptotanshinone on ovarian cancer and the underlying mechanism are not fully elucidated. AIM OF STUDY: In this study, we evaluated the inhibitory effect of Cryptotanshinone on ovarian cancer cells and explored the underlying molecular mechanism. Additionally, the chemosensitization potential of Cryptotanshinone was evaluated in combination with cisplatin. MATERIALS AND METHODS: MTT assay was used for cell viability assessment of ovarian cancer A2780 cells treated with Cryptotanshinone and/ or cisplatin. Flow cytometry was used for apoptosis analysis. Wound healing and transwell assays were used for migratory and invasive potential assessment of Cryptotanshinone-treated ovarian cancer cells. Western blot was used to investigate proteins involved in the mechanisms for metastasis and apoptosis. γH2AX immunocytochemistry was used to detect DNA damage in A2780 cells exposed to Cryptotanshinone and/or cisplatin. RESULTS: Cryptotanshinone significantly induced ovarian cancer A2780 cells apoptosis by activating caspase cascade. Additionally, wound healing and transwell assays revealed that Cryptotanshinone could suppress migration and invasion of ovarian cancer cells and dramatically inhibited MMP-2 and MMP-9 expression. Furthermore, Cryptotanshinone could sensitize A2780 cells to cisplatin treatment in a dose-dependent manner. CONCLUSION: Our data confirmed the anti-tumor effect of Cryptotanshinone on ovarian cancer cells and provided new findings that Cryptotanshinone could sensitize ovarian cancer cells to chemotherapy.

Anti-tumor effects of osthole on ovarian cancer cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Cnidium monnieri (L.) Cusson is a commonly used traditional Chinese medicine to treat gynecological disease in some countries. Osthole, an active O-methylated coumadin isolated from Cnidium monnieri (L.) Cusson, has been shown to induce various beneficial biochemical effects such as anti-seizure and anti-inflammatory effects. However, the anti-tumor mechanism of osthole is not well known. AIM OF STUDY: Here, we show that osthole inhibited the proliferation and migration of two widely used ovarian cancer cell lines, A2780 and OV2008 cells, in a dose-dependent manner. The study investigated the molecular mechanisms underlying ovarian cancer cells proliferation, apoptosis, cell cycle arrest and migration triggered by osthole. MATERIALS AND METHODS: Ovarian cancer cell lines A2780, OV2008 and normal ovarian cell line IOSE80 were used as experimental model. MTT assay was employed to evaluate cell viability. Flow cytometry assays were performed to confirm apoptosis and cell cycle. We employed wound healing and transwell assays to delineate invasive and migratory potential triggered by osthole. RESULTS: MTT assays indicated that cell viability significantly decreased in ovarian cancer cells treated with osthole without effect on normal ovarian cells. Flow cytometric analysis revealed that osthole suppressed cells proliferation by promoting G2/M arrest and inducing apoptosis. The underlying mechanisms involved were regulation of the relative apoptotic protein Bcl-2, Bax and Caspase 3/9. In addition, wound healing and transwell assays revealed that the migratory potential and activity of matrix metalloproteinase MMP-2 and MMP-9 were markedly inhibited when cells were exposed to osthole. CONCLUSION: Our findings suggested that osthole has the potential to be used in novel anti-cancer therapeutic formulations for ovarian cancer treatment.