The circadian clock plays a critical role in regulating various physiological processes, including gene expression, metabolic regulation, immune response, and the sleep-wake cycle in living organisms. Post-translational modifications (PTMs) are crucial regulatory mechanisms to maintain the precise oscillation of the circadian clock. By modulating the stability, activity, cell localization and protein-protein interactions of core clock proteins, PTMs enable these proteins to respond dynamically to environmental and intracellular changes, thereby sustaining the periodic oscillations of the circadian clock. Different types of PTMs exert their effects through distincting molecular mechanisms, collectively ensuring the proper function of the circadian system. This review systematically summarized several major types of PTMs, including phosphorylation, acetylation, ubiquitination, SUMOylation and oxidative modification, and overviewed their roles in regulating the core clock proteins and the associated pathways, with the goals of providing a theoretical foundation for the deeper understanding of clock mechanisms and the treatment of diseases associated with circadian disruption.
Protein Processing, Post-Translational/physiology* ; Circadian Rhythm/physiology* ; Humans ; Animals ; CLOCK Proteins/physiology* ; Circadian Clocks/physiology* ; Phosphorylation ; Acetylation ; Ubiquitination ; Sumoylation

This study aims to explore the protective effect of Chaihu Jia Longgu Muli Decoction on rats with heart failure after myocardial infarction, and to clarify its possible mechanisms, providing a new basis for basic research on the mechanism of classic Chinese medicinal formula-mediated inflammatory response in preventing and treating heart failure induced by apoptosis after myocardial infarction. A heart failure model after myocardial infarction was established in rats by coronary artery ligation. The rats were divided into sham group, model group, and low, medium, and high-dose groups of Chaihu Jia Longgu Muli Decoction, with 10 rats in each group. The low-dose, medium-dose, and high-dose groups of Chaihu Jia Longgu Muli Decoction were given 6.3, 12.6, and 25.2 g·kg~(-1) doses by gavage, respectively. The sham group and model group were given an equal volume of distilled water by gavage once daily for four consecutive weeks. Cardiac function was assessed using color Doppler echocardiography. Myocardial pathology was detected by hematoxylin-eosin(HE) staining, apoptosis was measured by TUNEL assay, and mitophagy was observed by transmission electron microscopy. The levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-1β, and N-terminal pro-B-type natriuretic peptide(NT-proBNP) in serum were detected by enzyme-linked immunosorbent assay(ELISA). The expression of apoptosis-related proteins B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), and cleaved caspase-3 was detected by Western blot. Additionally, the expression of phosphorylated nuclear transcription factor-κB(NF-κB) p65(p-NF-κB p65)(upstream) and nuclear factor kappa B inhibitor alpha(IκBα)(downstream) in the NF-κB signaling pathway was assessed by Western blot. The results showed that compared with the sham group, left ventricular ejection fraction(LVEF) and left ventricular short axis shortening(LVFS) in the model group were significantly reduced, while left ventricular end diastolic diameter(LVEDD) and left ventricular end systolic diameter(LVESD) increased significantly. Myocardial tissue damage was severe, with widened intercellular spaces and disorganized cell arrangement. The apoptosis rate was increased, and mitochondria were enlarged with increased vacuoles. Levels of TNF-α, IL-1β, and NT-proBNP were elevated, indicating an obvious inflammatory response. The expression of pro-apoptotic factors Bax and cleaved caspase-3 increased, while the anti-apoptotic factor Bcl-2 decreased. The expression of p-NF-κB p65 was upregulated, and the expression of IκBα was downregulated. In contrast, the Chaihu Jia Longgu Muli Decoction groups showed significantly improved of LVEF, LVFS and decreased LVEDD, LVESD compared to the model group. Myocardial tissue damage was alleviated, and intercellular spaces were reduced. The apoptosis rate decreased, mitochondrial volume decreased, and the levels of TNF-α, IL-1β, and NT-proBNP were lower. The expression of pro-apoptotic factors Bax and cleaved caspase-3 decreased, while the expression of the anti-apoptotic factor Bcl-2 increased. Additionally, the expression of p-NF-κB p65 decreased, while IκBα expression increased. In summary, this experimental study shows that Chaihu Jia Longgu Muli Decoction can reduce the inflammatory response and apoptosis rate in rats with heart failure after myocardial infarction, which may be related to the regulation of the IκBα/NF-κB signaling pathway.
Animals ; Apoptosis/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Myocardial Infarction/physiopathology* ; Male ; NF-kappa B/genetics* ; Heart Failure/etiology* ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; NF-KappaB Inhibitor alpha/genetics* ; Humans ; Tumor Necrosis Factor-alpha/genetics*

A high performance liquid chromatography (HPLC) method utilizing correction factors was established for the quantitative detection of related substances in flumazenil. Separation was achieved using an Agilent Pursuit XRs C18 column (250 mm × 4.6 mm, 5 μm) with an isocratic elution of dilute phosphoric acid, methanol, and tetrahydrofuran as the mobile phases. Correction factors calculated from a standard curve method were applied to determine the impurity content. The quantification of impurities in flumazenil was conducted using both external standard and correction factor methods, followed by validation and comparison of the two. For the identification of degradation products, a forced degradation approach was employed to prepare a flumazenil degradation solution, and the resulting impurities were confirmed by LC-MS analysis. The separation of flumazenil and its impurities was found to be efficient. The limits of quantification for impurities A, B, D, and E were established at 0.169 9, 0.314 7, 0.143 9, and 0.270 8 ng, respectively, with the limits of detection at 0.055 8, 0.096 9, 0.048 8, and 0.089 0 ng. These impurities demonstrated a strong linear relationship across the concentration ranges of 0.034 9-7.847 0, 0.038 7-8.710 7, 0.034 6-7.794 1, and 0.032 4-7.292 8 µg·mL^-1, respectively (n = 7). The method achieved average recoveries between 98.25% and 99.42%, with an RSD of less than 2.0% (n = 9), indicating high accuracy. The external standard and correction factor methods were used to determine the related substances in flumazenil, and the results of the two methods were consistent. The established HPLC method is characterized by its high accuracy, sensitivity, and repeatability, and is suitable for determining related substances in flumazenil.

In recent years,the prevalence of carbapenem-resistant Klebsiella pneumoniae(CRKP)infection has become a global public health issue.Ceftazidime/avibactam(CAZ/AVI)has been approved as a novel antimicrobial agent for the treatment of healthcare-associated pneumonia/ventilator-associated pneumonia,bloodstream infection,infection after kidney transplantation,and severe infection combined with liver cirrhosis.However,the use of CAZ/AVI has also led to the emergence of drug-resistant strains.The major mechanisms of drug-resistance include over-expression of blaKPC gene,mutation of β-lactamase and amino acids at key sites,changes in cell permeability caused by loss of membrane porin,and over-expression of efflux pump.This article reviews the research progress of CAZ/AVI in the treatment of CRKP infection,providing reference for clinical diagnosis and treatment.

Aim To investigate the effect of discoidin domain receptor 1(DDR1)on high glucose induced endothelial cell dysfunction and the underlying mecha-nism.Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and divided in-to the control group and high glucose induction group(HG).HUVECs were treated with 33 mmol·L-1 D-glucose for 48 hours to construct endothelial dysfunc-tion.Pyroptosis was detected using propidium iodide staining(PI);lactate dehydrogenase(LDH)and IL-1β,IL-18 levels were determined using enzyme linked immunosorbent assay(ELISA);the expression of DDR1 and NF-κB/NLRP3 signaling pathway proteins and pyroptosis related proteinses were detected using Western blot.Subsequently,the experiment was divid-ed into the control group,HG group,HG+DDR1 NC group,and HG+DDR1 siRNA group.The effect of high glucose on the proliferation and migration of HU-VECs was observed after transfection with DDR1 siR-NA for 24 hours;ELISA was used to detect the endo-thelial nitric oxide synthase(eNOS),vascular cell ad-hesion molecule-1(VCAM-1),intercellular adhesion molecule-1(ICAM-1),as well as LDH,IL-1β,IL-18 levels;PI was employed to detect pyroptosis;Western blot was applied to detect DDR1 and NF-κB/NLRP3 signaling pathway proteins and pyroptosis related pro-teins.Results Compared with the control group,HG group decreased eNOS content,increased VCAM-1 and ICAM-1 contents,decreased cell viability and migration ability,and significantly increased the expressions of DDR1,p-NF-κB,NLRP3 and pyroptosis related pro-teins.The levels of LDH,IL-1β,IL-18 and the rate of pyroptosis significantly increased(P＜0.05).Com-pared with HG group,DDR1 siRNA could promote the secretion of eNOS,decrease the levels of VCAM-1,ICAM-1,LDH,IL-1β and IL-1 8,increase cell viability and migration ability,reduce the expression of p-NF-κB,NLRP3 and pyroptosis related proteins,and inhibit high glucose-induced pyroptosis of HUVECs(P＜0.05).Conclusions Gene silencing DDR1 can im-prove vascular endothelial cell dysfunction induced by high glucose,and the mechanism is related to the inhi-bition of NF-κB/NLRP3 signaling pathway mediated pyroptosis.

Background: and Purpose The relationships among interleukin (IL)-10 levels, anxiety, and cognitive status after stroke remain controversial. We aimed to determine the associations of serum IL-10 levels with poststroke anxiety (PSA) and poststroke cognitive impairment (PSCI). Methods: We recruited 350 patients with stroke, of whom only 151 completed a 1-month follow-up assessment. The Mini Mental State Examination (MMSE) and Hamilton Anxiety Scale (HAMA) were used to assess the cognitive status and anxiety, respectively. Serum IL-10 levels were measured within 24 hours of admission. Results: IL-10 levels were significantly lower in the PSA group than in the non-PSA group, and they were negatively associated with HAMA scores (r=-0.371, p<0.001). After adjusting for all potential confounders, IL-10 levels remained an independent predictor of PSA (odds ratio=0.471, 95% confidence interval=0.237–0.936, p=0.032). IL-10 levels were strongly correlated with behavior during interviews, psychic anxiety, and somatic anxiety. Patients without PSCI had higher IL-10 levels were higher in non-PSCI patients than in PSCI patients, and they were positively associated with MMSE scores in the bivariate correlation analysis (r=0.169, p=0.038), and also with memory capacity, naming ability, and copying capacity.However, IL-10 did not predict PSCI in the univariable or multivariable logistic regression. Conclusions Low IL-10 levels were associated with increased risks of PSA and PSCI at a 1-month follow-up after stroke. Serum IL-10 levels may therefore be helpful in predicting PSA.

Objective:To explore the establishment and application of ovarian cancer organoids.Methods:Fresh ovarian tumor tissues, obtaining from patients underwent surgery in the First Affiliated Hospital of Nanjing Medical University between October 2021 and March 2022, were collected, enzymatic degraded, digested, and embedded into matrigel to establish organoids. A total of 32 ovarian cancer samples were collected. Hematoxylin eosin (HE) staining and immunofluorescence (IF) procedure were used to verify the morphological structure of organoids and their expression of molecular markers. 3D cyto-live or dead assay was used to detecte the live or dead cells in organoids. Carboplatin with a concentration ranging from 5 to 80 μmol/L (5, 10, 20, 40, 80 μmol/L) was added to organoids to calculate the 50% inhibitory concentration (IC 50) in different organoids. Results:(1) Organoids from a total of 32 patients were established, of which 18 cases could be passaged stably in the long term in vitro, while 14 could be passaged in the short time. The average amplification time of long-term passage in vitro was over 3 months, and the longest reached 9 months. (2) In HE staining, significant nuclei atypia and local micropapillary structures were observed in organoids. IF staining revealed that ovarian cancer organoids expressed molecular markers similar to primary tumor tissues, such as Pan cytokeratin (Pan-CK), p53, paired box gene 8 (PAX8), and Wilms tumor gene 1 (WT1). (3) In 3D cyto-live or dead assay, a large number of apoptotic cells were observed inside and around the organoids after added carboplatin. The sensitivity to carboplatin varied in 18 organoids could amplify in the long term, with an average IC 50 of (29.5±15.8) μmol/L. Moreover, IC 50 values of 4 organoids derived from patients received neoadjuvant chemotherapy were much higher than the 14 organoids which did not received neoadjuvant chemotherapy [(48.7±11.3) μmol/L vs (24.0±12.1) μmol/L; t=3.429, P=0.022]. Conclusions:Organoids recapitulate ovarian cancers in vitro and could be stably passaged. Organoids derived from patients received neoadjuvant chemotherapy have higher resistance to carboplatin.

OBJECTIVE: To examine the anti-inflammatory effect of grape seed extract (GSE) in animal and cellular models and explore its mechanism of action. METHODS: This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17 and the inflammatory factors such as tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), IL-6, IL-12, IL-17 and interferon-γ (IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and Western blot, respectively. RESULTS: GSE reduced the secretion of TNF-α, IL-1 β and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 β, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days (P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors (P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4 (TLR4) and Rho-associated kinase (ROCKII, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors (P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45⁺CD11b⁺ and CD45⁺CD4⁺ cells, and weakened the differentiation of Th1 and Th17 (P<0.05). Moreover, it reduced the secretion of inflammatory factors (P<0.01), and prevented the activation of microglia (P<0.05). CONCLUSION GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.
Mice ; Animals ; Encephalomyelitis, Autoimmune, Experimental/pathology* ; Grape Seed Extract/therapeutic use* ; Interleukin-17 ; Interleukin-1beta ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Th1 Cells ; Mice, Inbred C57BL ; Interferon-gamma/therapeutic use* ; Th17 Cells/metabolism* ; Interleukin-12/therapeutic use* ; Cytokines/metabolism*

Metastasis and resistance are main causes to affect the outcome of the current anticancer therapies. Heat shock protein 90 (Hsp90) as an ATP-dependent molecular chaperone takes important role in the tumor metastasis and resistance. Targeting Hsp90 and downregulating its expression show promising in inhibiting tumor metastasis and resistance. In this study, a redox-responsive dual-drug nanocarrier was constructed for the effective delivery of a commonly used chemotherapeutic drug PTX, and a COA-modified 4-arm PEG polymer (4PSC) was synthesized. COA, an active component in oleanolic acid that exerts strong antitumor activity by downregulating Hsp90 expression, was used as a structural and functional element to endow 4PSC with redox responsiveness and Hsp90 inhibitory activity. Our results showed that 4PSC/PTX nanomicelles efficiently delivered PTX and COA to tumor locations without inducing systemic toxicity. By blocking the Hsp90 signaling pathway, 4PSC significantly enhanced the antitumor effect of PTX, inhibiting tumor proliferation and invasiveness as well as chemotherapy-induced resistance in vitro. Remarkable results were further confirmed in vivo with two preclinical tumor models. These findings demonstrate that the COA-modified 4PSC drug delivery nanosystem provides a potential platform for enhancing the efficacy of chemotherapies.