Objective: To investigate the clinical significance of the monocyte count/high-density lipoprotein cholesterol ratio(MHR)in evaluating imperfect ST-segment resolution in elderly patients with acute ST-elevation myocardial infarction(STEMI)after percutaneous coronary intervention(PCI). Methods: This was a retrospective cohort study.A total of 274 elderly patients with STEMI underwent PCI in our hospital from December 2015 to December 2018 were enrolled.Based on the extent of the ST-segment resolution of the postoperative electrocardiogram, patients were divided into an imperfect ST-segment resolution group(observation group, n=79)and a favorable ST-segment resolution group(control group, n=195). General clinical data were compared between the two groups, and logistic regression equation was used to analyze the association of MHR with ST-segment resolution.Receiver operating characteristic(ROC)curve was performed to assess the predictive value of MHR for imperfect ST-segment resolution. Results: Compared with patients in the control group, patients in the observation group were associated with a significantly higher proportion of anterior wall myocardial infarction and heart failure(≥Killip 2), A longer duration of chest pain to balloon expansion, higher levels of creatine kinase isoenzyme, N-terminal pro-brain natriuretic peptide, hypersensitive C-reactive protein, blood sugar, blood uric acid, fibrinogen, triglyceride and mononuclear cell count, and lower levels of high density lipoprotein cholesterol and lymphocyte count(all P<0.05). Meanwhile, there was a significant difference in MHR between the observation group and the control group [(0.75±0.22)vs.(0.48±0.19), t=9.831, P=0.001]. Multivariate Logistic regression analysis showed that MHR was an independent risk factor for imperfect ST-segment resolution(OR=1.950, 95%CI: 1.646-5.430, P=0.003)and ROC curve showed the threshold value of MHR at 0.67, the area under the curve at 0.867, the sensitivity at 79.72%, and the specificity at 79.61%. Conclusions MHR may be an independent risk factor and a good predictive index for imperfect ST-segment resolution in elderly patients with STEMI after PCI.

Objective To investigate the neuroprotective effect and mechanism of liraglutide on diabetic rats. Methods 24 healthy male SPF Goto-Kakizaki (GK) rats with random blood glucose greater than 11.1 mmol/L were selected as the experimental group, and randomly divided into diabetes mellitus group ( n＝12) and liraglutide group (n＝12). Ten healthy male SPF Wistar rats with the same age and weight as GK rats were selected as normal control group. After adaptively feeded for 2 weeks, the liraglutide group was given liraglutide (400 μg·kg－1·d－1, subcutaneous injection), while the control group and diabetes mellitus group were given the same volume of saline, and continued to be administered for 8 weeks. After 10 weeks, data and biochemical indicators were recorded. Effects of liraglutide on learning and memory in diabetes mellitus rats were detected by Morris water maze test. HE staining observed the hippocampal neurons morphology. Western blotting method detected the expression of p- IκB kinase (IKK) β, p-NF-κB, NF-κB, Klotho, and PRX2 in hippocampus. Results Morris water maze test showed that liraglutide can improve the spatial learning and memory ability of diabetes mellitus rats. HE staining showed that liraglutide significantly reduced the pathological damage of hippocampal neurons of diabetes mellitus rats. Western blotting showed that liraglutide inhibited NF-κB signaling pathway in hippocampus of diabetes mellitus rats. The expression of Klotho protein in hippocampus of diabetes mellitus group was significantly lower than that of control group, while the expression of PRX2 protein was higher than control group (t＝8.298,－7.398,all P<0.01). The expression of Klotho and PRX2 protein in hippocampus of liraglutide group were higher than diabetes mellitus group (t＝－13.059, 14.113, all P<0.01). The expression of Klotho protein of liraglutide group was similar to that of control group ( t ＝ －1. 137, P>0. 05 ). The expression of PRX2 protein was significantly higher than control group (t＝－28.055, P<0.01). Conclusions Liraglutide may enhance the expression of antioxidant stress protein including Klotho and PRX2, by inhibiting NF-κB signaling pathway in hippocampus of diabetes mellitus rats, reduced oxidative stress and improved the injury of hippocampal neuronal in diabetes mellitus rats, which seems to play a neuroprotective effect, to prevent and delay the occurrence of diabetic encephalopathy.

Purpose To investigate the effect of autophagy intervention on ferroptosis and drug resistance of colorectal canc-er cells and its molecular mechanism.Methods The human colorectal cancer cell lines HCT-8,COLO205,HCT-116,SW620,and SW480 were cultured.HCT-116 cells with moder-ate expression of LC3 were screened,and the expression differ-ences of LC3,p62,Keap1,Nrf2,GPX4 proteins,Fe2+,GSH,and MDA between them and OXA-resistant HCT-116/OXA cell lines were detected.The expression levels of LC3,p62,Keap1,Nrf2,GPX4,Fe2+,GSH and MDA were assessed in HCT-116/OXA cells through the intervention of autophagy and ferroptosis intervention agent combined with oxaliplatin.The proliferative activity and sensitivity to oxaliplatin in each group were detected by CCK-8 assay.Cell growth and invasion ability of each group were detected by plate cloning and Trans well assay.Results LC3,p62 and GPX4 expression levels of HCT-116 cells in the 5 groups were moderate.Compared with HCT-116 cells,HCT-116/OXA was less sensitive to oxaliplatin,and the proteins of p62,Nrf2 and GPX4 were highly expressed,LC3 and Keap1 were lowly expressed,and the expression of Fe2+,GSH and MDA were increased(P＜0.05).The levels of LC3,Keap1 protein,Fe2+and MDA in Rapa and Rapa+Fer-1 groups were higher than those in Fer-1 and control groups,while p62,Nrf2,GPX4 and GSH levels were lower.The expressions of GPX4 pro-tein and GSH in Rapa+Fer-1 group were lower than those in Rapa group(P＜0.05).In the autophagy inhibitor group,LC3,p62,Nrf2,GPX4 and GSH were highly expressed in the CQ and CQ+Erastin groups compared with the control and Eras-tin groups,while Keap1 protein,Fe2+and MDA were low.The levels of GPX4 protein and GSH in Erastin group were lower than those in the other three groups,and the levels of Fe2+and MDA were higher than those in the other three groups(P＜0.05).The combination of autophagy activator OXA showed that Rapa intervention group had higher chemical sensitivity to OXA,less number of migrating cells and lower cell proliferation activity than the other three groups.The sensitivity of Rapa+Fer-1 group to oxaliplatin was lower than that of Rapa group,but higher than that of Fer-1 group and control group(P＜0.05).There was no significant difference between Fer-1 group and con-trol group(P＜0.05).Compared with the control group,the cell activity,migration capacity and clonogenesis capacity of Erastin,CQ+Erastin and CQ groups were decreased when auto-phagy inhibitor was combined with OXA,and the Erastin group was the lowest,while the CQ+Erastin group was higher than the Erastin group,and lower than the CQ group(P＜0.05).Con-clusion In colorectal cancer,autophagy is involved in the regu-lation of ferroptosis,and intervention in autophagy can regulate ferroptosis in colorectal cancer cells through the p62-Keap1/Nrf2-GPX4 pathway,thereby reversing oxaliplatin resistance.

Endometriosis is a common chronic gynecological disease with endometrial cell implantation outside the uterus.Angiogenesis is a major pathophysiology in endometriosis.Our previous studies have demon-strated that the prodrug of epigallocatechin gallate(ProEGCG)exhibits superior anti-endometriotic and anti-angiogenic effects compared to epigallocatechin gallate(EGCG).However,their direct binding targets and underlying mechanisms for the differential effects remain unknown.In this study,we demonstrated that oral ProEGCG can be effective in preventing and treating endometriosis.Additionally,1D and 2D Proteome Integral Solubility Alteration assay-based chemical proteomics identified metadherin(MTDH)and PX domain containing serine/threonine kinase-like(PXK)as novel binding targets of EGCG and ProEGCG,respectively.Computational simulation and BioLayer interferometry were used to confirm their binding affinity.Our results showed that MTDH-EGCG inhibited protein kinase B(Akt)-mediated angiogenesis,while PXK-ProEGCG inhibited epidermal growth factor(EGF)-mediated angiogenesis via the EGF/hypoxia-inducible factor(HIF-1a)/vascular endothelial growth factor(VEGF)pathway.In vitro and in vivo knockdown assays and microvascular network imaging further confirmed the involvement of these signaling pathways.Moreover,our study demonstrated that ProEGCG has superior therapeutic effects than EGCG by targeting distinct signal transduction pathways and may act as a novel anti-angiogenic therapy for endometriosis.

Objective:To investigate the expression characteristics of phosphorylated enhancer of Zeste homolog 2(EZH2)in head and neck squamous cell carcinoma(HNSCC)and their effect on chemosensitivity.Methods:Fifty-three patients with HNSCC treated between January 2018 and March 2021 in Tianjin Medical University Cancer Institute&Hospital were selected.Expression levels of p-EZH2S21,p-STAT3Y705,HIF-1α,and Ki-67 in HNSCC tissues were analyzed by immunohistochemical staining.Western blot was used to detect p-EZH2S21 expression in HNSCC tissues and cell lines.HNSCC cell lines stably transfected with wild type(EZH2-WT)or S21 mutant EZH2(EZH2-S21A)were construc-ted.CCK8 and colony formation assays were performed to detect the effect of EZH2 and S21 phosphorylation on HNSCC cell proliferation and their sensitivity to cisplatin(DDP)and 5-fluorouracil(5-FU).Results:Elevated levels of p-EZH2S21 were observed in HNSCC tissues and positively correlated with p-STAT3Y705(P<0.05)and HIF-1α(P<0.01)levels.p-EZH2S21 level correlated positively with lymph node metastasis(P<0.000 5),T(P<0.05),N(P<0.000 1)and AJCC stages(P<0.05).In vitro experiments confirmed that EZH2 expression promoted cell prolifer-ation and attenuated chemosensitivity of HNSCC cells.Inhibition of p-EZH2S21 restored HNSCC cell sensitivity to DDP and 5-FU.Conclusions:p-EZH2S21 plays an important role in tumor progression in HNSCC,and phosphorylation at S21 is an important way for EZH2 to affect HNSCC cell proliferation and chemotherapy sensitivity.

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.
Animals ; Apoptosis ; Base Sequence ; CLOCK Proteins ; genetics ; metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; Cellular Reprogramming ; Circadian Clocks ; genetics ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Hepatocyte Nuclear Factor 3-beta ; genetics ; metabolism ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; SOXB1 Transcription Factors ; genetics ; metabolism

OBJECTIVETo investigate the effect of PD0332991 (C24H29N7O2) on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells (HSC) in mice.

METHODSThe self renewal ability of HSCs was measured by cobblestone forming cell assay (CAFC). The colony-forming cell (CFC) assay was used to quantify the changes of numbers and functions of HPC after the treatment of the compound. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were analyzed by flow cytometry.

RESULTSThere were obvious changes in cell cycle regulation between control and PD0332991 groups. HSCs in G1 phase increased significantly from 76.3% to 89.5% after treatment of PD0332991 (P<0.05) while cells in S, G2 and M phase reduced from 20.5% to 7.3% (P<0.05). HPCs in G1 phase also increased from 74% to 87.4% after treatment of PD0332991 (P<0.05) while cells in S, G2 and M phase reduced from 25.54% to 11.6% (P<0.05). The apoptotic fractions between control and PD0332991 groups had no statistical difference (P>0.05). After cultured with PD0332991, the expression levels of cell cycle genes CDK1, CyclinA2, CyclinF, p18, p19 and p27 decreased by 58.77%, 66.35%, 56.33%, 62.18%, 32.28% and 36.53% respectively, while expression of CDK7 increased by 27.27% (P<0.05). No visible expression difference was observed in apoptosis and self-renew related genes. After treatment of PD0332991, the self-renewal ability of HSC decreased significantly. There were almost no CFCs in PD0332991 group in CAFC assay. Similarly, the frequency of CFCs was dramatically lower in PD0332991 group.

CONCLUSIONThese results suggested that PD0332991 affected HSC/HPC from mice mainly through inhibiting the cell cycle rather than apoptosis. It also suggested that CDK4/6 might play a key role in the regulation of HSC/HPC.

Animals ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Piperazines ; pharmacology ; Pyridines ; pharmacology

Mesp family proteins comprise two members named mesodermal posterior 1 (Mesp1) and mesodermal posterior 2 (Mesp2). Both Mesp1 and Mesp2 are transcription factors and they share an almost identical basic helix-loop-helix motif. They have been shown to play critical regulating roles in mammalian heart and somite development. Mesp1 sits in the core of the complicated regulatory network for generation of cardiovascular progenitors while Mesp2 is central for somitogenesis. Here we summarize the similarities and differences in their molecular functions during mammalian early mesodermal development and discuss possible future research directions for further study of the functions of Mesp1 and Mesp2. A comprehensive knowledge of molecular functions of Mesp family proteins will eventually help us better understand mammalian heart development and somitogenesis as well as improve the production of specific cell types from pluripotent stem cells for future regenerative therapies.
Amino Acid Sequence ; Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Cell Differentiation ; genetics ; Gene Expression Regulation, Developmental ; Mesoderm ; embryology ; metabolism ; Mice, Knockout ; Molecular Sequence Data ; Pluripotent Stem Cells ; metabolism ; Sequence Homology, Amino Acid

Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans.

Cardiomyopathy, Dilated/metabolism* ; Humans ; Microfilament Proteins/metabolism* ; Models, Cardiovascular ; Mutation ; Myocytes, Cardiac ; Pluripotent Stem Cells/metabolism* ; Transcription Factors/metabolism* ; Troponin T/metabolism*