ObjectiveTo explore the effect of Qingre Huayu Jianpi prescription （QHJ） on colitis-associated colorectal cancer （CAC） in mice， and its related mechanism. MethodC57BL/6 mice were randomly divided into four groups including the normal， model， QHJ low-dose （QHJ-L， 10 g·kg^-1）， and QHJ high-dose （QHJ-H， 40 g·kg^-1） groups. Azoxymethane （AOM） and dextran sodium sulfate （DSS） were combined to chemically build a CAC mouse model for 14 weeks. Each drug group was given intragastrically from the 5^th week to the 14^th week， once per day. An equal volume of water was fed to the normal and model groups. The mouse survival rate， colon length， weight， and pathological alterations were assessed. The protein expressions of Wnt-3a protein signaling （Wnt3a）， β-catenin， Non-phosphor-β-catenin （Non-p-β-catenin）， and cholesterol-binding glycoproteins 133 （CD133） were detected by Western blot. The localization and expression of the cluster of differentiation （CD） 80 and CD11 antigen-like family member B （CD11b） were detected by immunohistochemistry （IHC）. The colon organoids derived from CAC mice were isolated and cultured to detect the expression of Wnt signaling pathway-related proteins. ResultThe survival rate of the CAC mice was improved by QHJ treatment and the number of colon tumors was inhibited significantly. Compared with those in the normal group， the expression levels of Wnt3a， β-catenin， Non-p-β-catenin， and CD133 in colon tissues in the model group were significantly increased （P<0.05， P<0.01）. Compared with those in the model group， the levels of Wnt3a and β-catenin in the QHJ-L group were significantly decreased （P<0.01）， and the protein levels of Wnt3a， β-catenin， Non-p-β-catenin， and CD133 in the QHJ-H group were significantly decreased （P<0.05， P<0.01）. Meanwhile， the expression level of CD11b in the model group was significantly increased compared with that in the normal group while the CD80 level was significantly decreased （P<0.05， P<0.01）. Compared with those in the model group， CD11b in QHJ-L and QHJ-H groups was significantly decreased， and CD80 was significantly increased（P<0.05， P<0.01）. The expressions of Non-p-β-catenin and CD133 in colonic organoids of CAC model mice were significantly increased， while QHJ treatment could inhibit the expressions of Non-p-β-catenin and CD133 in colonic organoids （P<0.01）. ConclusionQHJ could inhibit the inflammation-cancer development in CAC mice， the mechanism of which might be related to regulating the microenvironment and inhibiting the over-activation of Wnt signaling.

OBJECTIVE To provide reference for exploring alternative resources of Gentiana rigescens from the plants of Gentiana. METHODS The contents of four components （gentiopicroside， swertiamarin， swertioside and amarogentin） in the roots and rhizomes from 3 plants of Gentiana （G. rigescens， G. cephalantha， G. delavayi） were determined by high-performance liquid chromatography （HPLC）. The chemical compositions in the above roots and rhizomes were identified by ultra-performance liquid chromatography electrospray ionization quarter-time of flight mass spectrometry （UPLC-ESI-Q-TOF-MS）， and the differences were analyzed by principal component analysis （PCA）. RESULTS Four active components such as gentiopicroside， swertiamarin， swertioside and amarogentin were detected in the roots and rhizomes of G. rigescens and G. cephalantha， and the contents of the four components were similar in both. The contents of gentiopicroside in the root and rhizome of G. cephalantha and G.rigescens were more than four times of the limit standard of the Chinese Pharmacopoeia （Part Ⅰ） in 2020； However， only swertiamarin， swertioside and amarogentin were detected in the roots and rhizomes of G.delavayi， and the contents of swertioside and amarogentin were 34.12 and 8.81 times of those of G. rigescens， respectively. In addition， a total of 33 compounds 术。E-mail：515227235@qq.com were identified from the roots and rhizomes of 3 plants of Gentiana by UPLC-ESI-Q-TOF-MS， mainly iridoids. Additionally， G. rigescens and G. cephalantha contained xantones， G. delavayi contained flavonoids. PCA showed that there was a small difference between G. rigescens and G. cephalantha； however， there was a big difference between G. delavayi and G. rigescens. CONCLUSIONS The difference between the roots and rhizomes of G. cephalantha and G. rigescens from the same origin is small and there is substitutability； while the difference in the chemical components from roots and rhizomes between G. delavayi and G. rigescens is great and G. delavayi cannot be used as medicine instead of G. rigescens.

OBJECTIVE: To investigate the effect of microRNA-509-3p (miR-509-3p) on the apoptosis of atherosclerotic vascular endothelial cells. METHODS: Mouse aortic endothelial cells (MAECs) were divided into normal control group, oxidized low-density lipoprotein (ox-LDL) group, miR-509-3p overexpression group, miR-509-3p overexpression control group, miR-509-3p inhibitor + ox-LDL group, and miR-509-3p inhibitor control + ox-LDL group. MAEC were induced with 100 mg/L ox-LDL for 24 hours, and then transfected with miR-509-3p overexpression/inhibitor and corresponding control for 48 hours. The miR-509-3p expression in MAECs exposed to ox-LDL was detected using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Flow cytometry was used to detect the level of apoptosis, and cell counting kit (CCK-8) was used to detect the proliferation activity of MAECs. The direct gene targets of miR-509-3p were predicted using bioinformatics analyses and confirmed using a dual luciferase reporter assay. The expression of Bcl-2 mRNA and protein was detected by RT-qPCR and Western blotting, respectively. RESULTS: Compared with the normal control group, miR-509-3p was significantly upregulated in ox-LDL-stimulated MAECs (1.68±0.85 vs. 1.00±0.30, t = 2.398, P < 0.05). After transfection of MAECs with miR-509-3p overexpression, the luciferase activity of the BCL2 3'UTR WT reporter gene was significantly lower than that of miR-509-3p overexpression control group (0.83±0.06 vs. 1.00±0.07, t = 4.531, P = 0.001). The luciferase activity of the BCL2 3'-UTR mutant (MUT) reporter gene was not significantly different from that of miR-509-3p overexpression control group (0.94±0.05 vs. 1.00±0.08, t = 1.414, P = 0.188). Compared with the normal control group and miR-509-3p mimics control group, the cell proliferation activity was decreased [(0.60±0.06)% vs. (1.00±0.09)%, (0.89±0.04)%, both P < 0.01], the percentage of apoptotic cells were increased [(23.46±2.02)% vs. (7.66±1.52)%, (10.40±0.78)%, both P < 0.05], and the mRNA and protein expression of Bcl-2 were significantly downregulated (Bcl-2 mRNA: 0.52±0.13 vs. 1.00±0.36, 1.10±0.19, Bcl-2 protein: 0.42±0.07 vs. 1.00±0.11, 0.93±0.10, both P < 0.01) in miR-509-3p overexpression group. Compared with the ox-LDL group, inhibition of miR-509-3p expression could increase the proliferation activity of MAECs induced by ox-LDL [(0.64±0.35)% vs. (0.34±0.20%)%, P < 0.05], and reduce the apoptosis rate [(13.59±2.22)% vs. (29.84±5.19)%, P < 0.01], and up-regulated the expression of Bcl-2 mRNA and protein in MAECs induced by ox-LDL (Bcl-2 mRNA relative expression: 0.82±0.09 vs. 0.52±0.10, Bcl-2 protein relative expression: 0.83±0.17 vs. 0.40±0.07, both P < 0.05). CONCLUSIONS Bcl-2 was one of the target genes of miR-509-3p. miR-509-3p can reduce the proliferation activity of endothelial cells, reduce the expression of Bcl-2, and promote cell apoptosis, thereby promoting the occurrence and development of atherosclerosis. Inhibition of miR-509-3p expression may be a potential therapeutic target for atherosclerosis.
Animals ; Mice ; Humans ; Endothelial Cells ; MicroRNAs/metabolism* ; Signal Transduction ; Lipoproteins, LDL/metabolism* ; Apoptosis ; RNA, Messenger/metabolism* ; Proto-Oncogene Proteins c-bcl-2/pharmacology* ; Atherosclerosis/metabolism* ; Luciferases/pharmacology* ; Cell Proliferation ; Human Umbilical Vein Endothelial Cells

Background Diabetes is a major threat to public health across the world. Studies have shown that exposure to p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) is closely related to the occurrence of type 2 diabetes mellitus. However, the relevant molecular mechanism is not clear. Objective To investigate the effects of p,p'-DDE on H19 differentially methylated region (DMR) methylation and insulin secretion of rat insulinoma cells (INS-1 cells). Methods INS-1 cells were cultured with different concentrations (0, 3.125, 6.25, 12.5, 25, 50, and 75 µmol·L⁻¹) of p,p'-DDE for 24 h, and the viability of INS-1 cells was detected by CCK-8 method. INS-1 cells were exposed to 0, 12.5, 25, and 50 µmol·L⁻¹ p,p'-DDE for 24 h in subsequent experiments. The methylation levels of 24 CpG sites in H19 DMR were analyzed by bisulfite genomic sequencing. The expression levels of insulin-like growth factor 2 (IGF2) mRNA were detected by real-time quantitative PCR. The expression levels of IGF2 and insulin-like growth factor-1 receptor (IGF1R) proteins were detected by Western blotting. The insulin secretion function of INS-1 cells was determined by glucose-stimulatedinsulin secretion test (5 and 25 mmol·L⁻¹ glucose, respectively). Results Compared with the control group, the viability of INS-1 cells increased significantly after treatment with 12.5 µmol·L⁻¹ p,p'-DDE; however, it was significantly inhibited after treatment with 50 or 75 µmol·L⁻¹ p,p'-DDE (P<0.01); therefore, 50 µmol·L⁻¹ was chosen as the maximum concentration of exposure for subsequent experiments. The 25 µmol·L⁻¹ p,p'-DDE treatment decreased the methylation levels of CpG18 and CpG22-CpG24 sites in H19 DMR, and the 50 µmol·L⁻¹ p,p'-DDE treatment decreased the methylation levels of CpG10-CpG24 sites (P<0.05 or P<0.05). Multiple concentrations (12.5, 25, and 50 µmol·L⁻¹) of p,p'-DDE down-regulated the mRNA and protein relative expression levels of IGF2 and the protein relative expression levels of IGF1R. The transcription level of IGF2 decreased to 67.8%, 68.6%, and 62.5% of the control group, the protein level of IGF2 decreased to 73.3%, 79.5%, and 80.9% of the control group, and the protein level of IGF1R decreased to 54.8%, 25.6%, and 12.9% of the control group, respectively (P<0.01). In the high glucose context, p,p'-DDE at selected concentrations inhibited the insulin secretion levels to 85.0%, 58.6%, and 49.5% of the control group, respectively (P<0.01). Conclusion p,p'-DDE could down-regulate methylation level of H19 DMR, interfere the IGF2/IGF1R signaling pathway, and inhibit insulin secretion of islet cells.

Objective:To understand the incidence of post-traumatic stress disorder(PTSD) in fathers with premature infants and analyze the risk factors.Methods:Conveniently, the 203 fathers of premature infants in NICU of Affiliated Hospital of Jining Medical University of Shandong Province from May to August 2021 were selected as the subjects to fill in the general data questionnaire, Perinatal Post-traumatic stress disorder Questionnaire-Chinese edition (PPQ-C), Parents, Perception of Uncertainty Scale, Social Support Rating Scale, Simplified Coping Style Questionnaire. Logistic regression was used to analyze the risk factors of PTSD and establish a nomogram model. ROC curve was used to verify the discrimination ofthe model. Hosmer-Lemeshow goodness-of-fit test and Calibration Plot were used to verify the calibration.Results:PPQ-C total score of 203 fathers was 17.17 ± 8.77, 81 fathers, symptoms were positive and the incidence of PTSD was 39.90%(81/203). Logistic regression analysis showed college degree ( OR = 0.297, 95% CI 0.116 - 0.763, P<0.05), very low birth weight ( OR = 2.491, 95% CI 1.027 - 6.044, P<0.05), sense of disease uncertainty ( OR = 1.038, 95% CI 1.012 - 1.066, P<0.05), negative coping style ( OR = 1.871, 95% CI 1.127 - 3.108, P<0.05) were risk factors of PTSD in fathers with premature infants. The nomogram model was established basing on the results of the Logistic regression analysis, and the ROC curve proved (AUC = 0.751) the model having a good discrimination.The Hosmer-Lemeshow goodness-of-fit test ( P = 0.974) and the calibration plot demonstrated that the prediction values tends to coincide with the actual monitoring values. Conclusions:A higher incidence of PTSD was observed in fathers with hospitalized premature infant. It was related to education level, the infants′s birth weight, disease uncertainty and coping style. Therefore, the nurses should give information support and psychological guidance according to the individual situation of the infants′ father to reduce the incidence of PTSD.

Objective:To investigate the protective effect of liriodendrin on acute myocardial infarction in rats and to explore the related mechanisms.Methods:From January to December 2019, 30 SPF male Wistar rats with a body weight of(200±10)g were randomly divided into a sham operation group, a control group, and a liriodendringroupwith 10 rats in each group using the numerical sampling method.The liriodendron group was intragastrically administered with a liriodendrinsolution(10 ml/kg)once a day from 5 days before myocardial infarction model construction to 3 days after surgery.The control group and the sham surgery group were intragastrically administered with 10 ml/kg normal saline.After surgery, high-sensitivity troponin T levels were measured in the three groups.Cardiac function of the rats was assessed using echocardiography on the 3rd day post-surgery.Then, the rats were sacrificed, followed by hematoxylin-eosin(HE)staining and TdT-mediated dUTP nick-end labeling(TUNEL)staining of cardiac tissues and measurement of interleukin(IL)-1β and tumor necrosis factor(TNF-α)levels.Western blot and real-time polymerase chain reaction(PCR)were used to detect the expression of apoptosis-related proteins and transcriptional activity.Results:High-sensitivity troponin T levels in the liriodendrin group[(1.74±0.63)μg/L]were lower than in the myocardial infarction group[(3.54±1.60)μg/L]at 2 hours after surgery( t=2.69, P<0.05). Echocardiography showed that, compared with the myocardial infarction group, the ejection fraction was higher in the liriodendrin group, and the left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular end-diastolic volume and left ventricular end-systolic volume were lower in the liriodendrin group( P<0.05). Histological staining showed that the myocardial tissue of the control group was severely damaged, with infiltration of a large number of in flammatory cells.The number of TUNEL-positive cells in the liriodendrin group(56.66±2.414)was statistically significantly reduced, compared with in the myocardial infarction group(76.55±1.843)( t=6.55, P<0.05). The levels of IL-1β and TNF-α in the myocardial infarction group were higher than those in the liriodendrin group( P<0.05). The expression of apoptosis-related proteins in the liriodendrin group was lower( P<0.05)and the transcriptional activity of mRNA was also lower( P<0.05)than in the myocardial infarction group. Conclusions:Liriodendrin may protect cardiomyocytes after myocardial infarction in rats by inhibiting local inflammation and cell apoptosis.

Objective:To evaluate the effect of transcutaneous electrical acupoint stimulation (TEAS) at Neiguan on dexmedetomidine-induced bradycardia in patients.Methods:Sixty American Society of Anesthesiologists physical status Ⅰ or Ⅱ patients, aged 20-50 yr, weighing 48-60 kg, scheduled for elective gynecological surgery under general anesthesia, were divided into 2 groups ( n=30 each) using a random number table method: control group (group C) and TEAS group (group T). Dexmedetomidine 1 μg/kg was infused intravenously over 10 min followed by intravenous infusion 0.5 μg·kg -1·min -1 in two groups, and the patients in group T simultaneously received TEAS (frequency 2/100 Hz, disperse-dense wave, intensity 5-10 mA according to the current that could be tolerated) at bilateral Neiguan acupoints.The stimulator was only connected, and no current was given in group C. Before the infusion of dexmedetomidine (T 0) and at 10 min of dexmedetomidine infusion (T 1), mean arterial pressure (MAP) and heart rate (HR) was recorded, and electrocardiogram (ECG) was collected to calculate the PR interval, QT interval, QT interval, Tp-e interval and index of cardiac electrophysiological balance (iCEB). The development of arrhythmia was recorded. Results:Compared with the baseline value at T 0, HR was significantly decreased, and QT interval and PR interval were prolonged at T 1 in two groups, and iCEB was increased, and Tp-e interval was prolonged at T 1 in group C ( P<0.05). Compared with group C, HR was significantly increased, PR interval and Tp-e interval were shortened at T 1, and the incidence of bradycardia and atrioventricular block was increased in group T ( P<0.05). Conclusion:TEAS at Neiguan can decrease the risk of bradycardia induced by dexmedetomidine, and the mechanism may be related to shortening atrioventricular conduction time and reducing heterogeneity of ventricular repolarization in patients.

Objective:To investigate the effects of recombinant adenovirus with human vascular endothelial growth factor 165 (Ad-hVEGF 165) and recombinant adenovirus with human tissue inhibitor of metalloproteinase 1 (Ad-hTIMP-1) on rats with myocardial infarction (MI) and its mechanism. Methods:A total of 30 healthy 8-week-old male Wistar rats were randomly divided into 5 groups: sham-operated group (sham), virus control group (Ad-Track), Ad-hVEGF 165 group, Ad-hTIMP-1 group and Ad-hVEGF 165+Ad-hTIMP-1 group (hVEGF 165+hTIMP-1) ( n=6 per group). Except the sham group, all rats were ligated the left anterior descending coronary artery to induce MI model with ST-segment elevation and Q waves or T-wave inversion on electrocardiogram and local myocardial whitening. The corresponding recombinant adenovirus comprising 100 μL (1×10 10 VP/100 μL) combined with NaCl solution was injected into the myocardial infarction area at four points respectively. The sham group received no treatment. After 4 weeks, all rats were sacrificed after echocardiography was completed and heart tissues were collected. The expression of hVEGF 165 and hTIMP-1 were detected by immunohistochemistry. The mRNA expression of apoptosis-related factors were detected by real-time PCR. The protein expression of apoptosis-related factors were detected by immunohistochemistry. Differences between groups were determined by One-way analysis of variance. Multiple comparisons between groups were performed using the least significant difference t-test. Results:(1) Both heart rate (HR) (480.83±24.09) beats/min, left ventricular end-diastolic dimension (LVEDD) (6.88±0.44) mm and left ventricular end-systolic dimension (LVESD) (4.85±0.42) mm were increased in the Ad-Track group than those in the sham group (433.16±17.86) beats/min, (6.20±0.45) mm, (4.06±0.70) mm (all P<0.05), and left ventricular ejection fraction (LVEF) (62.70±3.17) % and left ventricular fractional shortening (LVFS) (29.52±1.88) % were significantly decreased in the Ad-Track group than those in the sham group (72.78±5.44)%, (29.52±1.88) % (both P<0.01). Compared with the Ad-Track group, LVEF (71.50±6.23) % and LVFS (36.17±5.27) % in the hVEGF 165-hTIMP-1 group were significantly increased (both P<0.01), and LVEDD (6.22±0.39) mm and LVESD (4.13±0.23) mm were decreased (both P<0.05). LVEF and LVFS in the hVEGF 165-hTIMP-1 group were increased significantly than those in the Ad-hVEGF 165 group (64.65±4.00) %, (30.95±2.57) % (both P<0.05). The mRNA expression of BCL2-associated X protein (Bax), cysteine aspartate specific proteinase 3 (Caspase-3) and BCL-xL/BCL-2-associated death promoter (Bad) in the hVEGF 165-hTIMP-1 group were decreased than those in the Ad-Track group ( P<0.01 or P<0.05), and B-cell lymphoma/leukemia-2 (Bcl-2) in the hVEGF 165-hTIMP-1 group were increased than those in the Ad-Track group ( P<0.01). The mRNA expression levels of Bax and Caspase-3 in the hVEGF 165-hTIMP-1 group were decreased than those in the Ad-hVEGF 165 group (both P<0.05). There was no statistically difference in the mRNA expression of Bax, Caspase-3, Bad, and Bcl-2 between the hVEGF 165-hTIMP-1 group and the sham group (all P>0.05). The protein expression of Bax and Caspase-3 in the hVEGF 165-hTIMP-1 group were significantly decreased than those in the Ad-hVEGF 165 group, the Ad-hTIMP-1 group and the Ad-Track group (all P<0.01), and the protein expression of Bcl-2 in the hVEGF 165-hTIMP-1 group was increased than those in the Ad-hVEGF 165 group, the Ad-hTIMP-1 group and the Ad-Track group (all P<0.05). There were no statistically differences in the protein expression of Bax, Caspase-3 and Bcl-2 between the hVEGF 165-hTIMP-1 group and the sham group (all P>0.05). Conclusions:Ad-hVEGF 165 and Ad-hTIMP-1 can improve cardiac contractile function of MI rats and the beneficial effects are largely attributable to inhibiting myocyte apoptosis. The combination of hVEGF 165 and hTIMP-1 may have a synergistic effect on MI.

Objective:To determine the changes in the expression of myocardial miRNA and the target genes in the rats with hypothermic ischemia-reperfusion (I/R) arrhythmia.Methods:Clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 300-400 g, were anesthetized, the chest was opened, and the heart was taken to establish an isolated heart perfusion model.Six successfully perfused isolated hearts were divided into 2 groups ( n=3 each) using a random number table method: control group (group C) and heart I/R group (IR group). The model of hypothermic global I/R injury was established by interrupting perfusion for 60-min followed by 30-min reperfusion in chloral hydrate-anesthetized rats.The arrhythmia score was recorded during reperfusion.High-throughput sequencing was used to identify the differentially expressed miRNAs in two groups.The RNAhybrid and miRanda databases were used to predict the target genes of mRNA regulated by the differentially expressed miRNAs, and the enrichment for target genes was performed by Gene Ontology and KEGG databases, and the miRNAs closely related to arrhythmia and with higher expression were selected to carry out the real-time polymerase chain reaction detection. Results:The results of high-throughput sequencing showed that there were 7 differentially expressed miRNAs (novel-miR-17, novel-miR-19, novel-miR-30, novel-miR-43, rno-miR-122-5p, novel-miR-16 and rno-miR-429) in group IR as compared with group C. There were 4 miRNAs that were closely related to arrhythmia and had higher expression: the expression of novel-miR-17, novel-miR-30 and rno-miR-122-5p was significantly up-regulated, and the expression of rno-miR-429 was down-regulated in group IR when compared with group C ( P<0.05). The miRNA-mRNA correlation analysis revealed that GJA1 gene was the target of novel-miR-17. Conclusion:Myocardial novel-miR-17 is involved in the occurrence of hypothermic I/R arrhythmia probably by acting on GJA1 gene in rats.

The most common metastatic site of breast cancer is the bone. Metastatic bone disease can alter the integrity of the bone and cause serious complications, thereby greatly reducing health-related quality of life and leading to high medical costs. Although diagnostic methods and treatments for bone metastases (BM) are improving, some patients with early breast cancer who are at high risk of BM are not diagnosed early enough, leading to delayed intervention. Moreover, whole-body scintigraphy cannot easily distinguish BM from nonmalignant bone diseases. To circumvent these issues, specific gene and protein biomarkers are being investigated for their potential to predict, diagnose, and evaluate breast cancer prognosis. In this review, we summarized the current biomarkers associated with BM in breast cancer and their role in clinical applications to assist in the diagnosis and treatment of BM in the future.