ABSTRACT
Pancreatic stellate cell (PSC) activation is a major cause of chronic pancreatitis and pancreatic cancer, yet the mechanisms by which PSCs switch from quiescent to activated state are poorly studied. In this study, we identified JUN, a key transcription factor that maintains the quiescent state of PSCs, by integrating single-cell sequencing data from multiple pancreatic tissues and using WGCNA and SCENIC algorithms, and demonstrated that the expression and activity of JUN is a major regulator of the quiescent state of PSCs through cellular experiments and multiple pancreatic-related disease bulk RNAseq data. This study explores the main mechanism of PSC activation and provides a theoretical basis for the treatment of multiple pancreatic injury-related diseases caused by PSCs.
Subject(s)
Pancreatic Neoplasms , Pancreatitis, Chronic , Humans , Pancreatic Stellate Cells/metabolism , Transcription Factors/metabolism , Pancreatic Neoplasms/genetics , Pancreatitis, Chronic/metabolism , Pancreas/metabolism , Cells, CulturedABSTRACT
BACKGROUND: Circulating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown. METHODS: Cord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated ß-galactosidase (SA-ß-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot. RESULTS: B2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK. CONCLUSION: BK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.
Subject(s)
Bradykinin/pharmacology , Cellular Senescence/drug effects , Endothelial Progenitor Cells/drug effects , Receptor, Bradykinin B2/metabolism , Case-Control Studies , Cells, Cultured , DNA, Mitochondrial/genetics , Diabetes, Gestational , Dinoprost/analogs & derivatives , Dinoprost/blood , Endothelial Progenitor Cells/cytology , Female , Fetal Blood , Gene Dosage , Glucose/pharmacology , Humans , Infant, Newborn , Nitric Oxide/blood , Nitric Oxide Synthase Type III/metabolism , Pregnancy , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Telomerase , TelomereABSTRACT
OBJECTIVE: To observe the effect of acupuncture therapy on 14-3-3, Bcl-2 and Bax expression levels in the cerebral cortex in neonatal rats with hypoxic-ischemic brain damage(HIBD). METHODS: Timed pregnant Sprague-Dawley rat dams were delivered either vaginally (normal group), or by C-section (sham-operation group) or by C-section with 5 min of global anoxia (anoxia group), with 8 rats in each group. The rat pups of the anoxia group were randomly divided into model group and acupuncture group (n =8). Acupuncture stimulation of "Naosanzhen" "Niesanzhen" and "Zhisanzhen" acupoints was given begin- ning from the 14th day after birth, once daily for 7 consecutive days. All rat pups were killed by decapitation on day 21 after birth, and then 14-3-3, Bcl-2 and Bax immunoactivity (expression) in the cerebral cortex were detected by immunohistochemistry. RESULTS: In comparison with the normal group, the expression level of cerebral cortical 14-3-3 was significantly decreased, and that of Bax remarkably increased in the model group (P<0. 01, P<0. 05). Compared to the model group, cortical 14-3-3 and Bcl-2 expression levels were markedly up-regulated in the acupuncture group (P<0.01, P<0.05). Compared to the normal group, cortical 14-3-3 expression level was obviously lower, but Bax expression level significantly higher in the sham-operation group (P<0. 05, P<0. 01). No significant differences were found between the model and normal groups in the expression levels of Bcl-2, and between the acupuncture and model groups in the expression levels of Bax (P>0. 05). CONCLUSION: Acupuncture intervention can increase the expression of 14-3-3 and Bcl-2 in the cerebral cortex in HIBD rats.
