The molecular mechanism of verbascoside(OC1) in promoting acetylcholine(ACh) release in the pathogenesis of Alzheimer's disease(AD) was studied. Adrenal pheochromocytoma cells(PC12) of rats induced by β-amyloid protein(1-42)(Aβ_(1-42)) were used as AD models in vitro and were divided into control group, model group(Aβ_(1-42) 10 μmol·L~(-1)), OC1 treatment group(2 and 10 μg·mL~(-1)). The effect of OC1 on phosphorylated proteins in AD models was analyzed by whole protein phosphorylation quantitative omics, and the selectivity of OC1 for calcium channel subtypes was virtually screened in combination with computer-aided drug design. The fluorescence probe Fluo-3/AM was used to detect Ca~(2+) concentration in cells. Western blot analysis was performed to detect the effects of OC1 on the expression of phosphorylated calmodulin-dependent protein kinase Ⅱ(p-CaMKⅡ, Thr286) and synaptic vesicle-related proteins, and UPLC/Q Exactive MS was used to detect the effects of OC1 on ACh release in AD models. The effects of OC1 on acetylcholine esterase(AChE) activity in AD models were detected. The results showed that the differentially modified proteins in the model group and the OC1 treatment group were related to calcium channel activation at three levels: GO classification, KEGG pathway, and protein domain. The results of molecular docking revealed the dominant role of L-type calcium channels. Fluo-3/AM fluorescence intensity decreased under the presence of Ca~(2+) chelating agent ethylene glycol tetraacetic acid(EGTA), L-type calcium channel blocker verapamil, and N-type calcium channel blocker conotoxin, and the effect of verapamil was stronger than that of conotoxin. This confirmed that OC1 promoted extracellular Ca~(2+) influx mainly through its interaction with L-type calcium channel protein. In addition, proteomic analysis and Western blot results showed that the expression of p-CaMKⅡ and downstream vesicle-related proteins was up-regulated after OC1 treatment, indicating that OC1 acted on vesicle-related proteins by activating CaMKⅡ and participated in synaptic remodeling and transmitter release, thus affecting learning and memory. OC1 also decreased the activity of AChE and prolonged the action time of ACh in synaptic gaps.
Animals ; Rats ; Glucosides/administration & dosage* ; Acetylcholine/metabolism* ; Alzheimer Disease/genetics* ; PC12 Cells ; Phenols/chemistry* ; Neurotransmitter Agents/metabolism* ; Drugs, Chinese Herbal ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics* ; Humans ; Phosphorylation/drug effects* ; Calcium/metabolism* ; Polyphenols

Objective:To explore the osteogenic activity of in vitro cultured preosteoblasts treated by magnesium loaded hydrogel core-shell microspheres.Methods:A microfluidics chip device was designed and made to fabricate magnesium loaded hydrogel core-shell microspheres to precisely control the stable release of magnesium ions in vitro,the mechanism of controllable magnesium ion release in the promotion of osteogenic activity of MC3T3-E1 preosteoblasts was systematically investigated.Results:The magnesium loaded hydro-gel microspheres(PLGA/MgO-alginate microspheres)prepared by the microfluidics chip exhibited to be monodisperse with special core-shell structure and could control the stable release of magnesium ions at a concentration of 50 ppm in vitro.The in vitro study showed that it significantly enhanced the activity and proliferation ability of MC3T3-E1 preosteoblasts,promoted their osteogenic differ-entiation and mineralization ability,and increased ALP activity.Conclusion:Magnesium loaded hydrogel core-shell microspheres can promote the osteogenic activity of MC3T3-E1 osteoblasts in vitro.

Aim To investigate the effect of quercetin on the aging model of bone marrow mesenchymal stem cells established under microgravity. Methods Using 3D gyroscope, a aging model of bone marrow mesenchymal stem cells was constructed, and after receiving quercetin and microgravity treatment, the anti-aging effect of the quercetin was evaluated by detecting related proteins and oxidation indexes. Results Compared to the control group, the expressions of age-related proteins p21, pi6, p53 and RB in the microgravity group significantly increased, while the expressions of cyclin D1 and lamin B1 significantly decreased, with statistical significance (P<0.05). In the microgravity group, mitochondrial membrane potential significantly decreased (P<0.05), ROS accumulation significantly increased (P <0.05), SOD content significantly decreased and MDA content significantly increased (P<0.05). Compared to the microgravity group, the expressions of age-related proteins p21, pi6, p53 and RB in the quercetin group significantly decreased, while the expressions of cyclin D1 and lamin B1 significantly increased, with statistical significance (P<0.05). In the quercetin group, mitochondrial membrane potential significantly increased (P<0.05), ROS accumulation significantly decreased (P<0.05), SOD content significantly increased and MDA content significantly decreased (P<0.05). Conclusions Quercetin can resist oxidation, protect mitochondrial function and normal cell cycle, thus delaying the aging of bone marrow mesenchymal stem cells induced by microgravity.

ObjectiveTemporal heterogeneity in lung cancer presents as fluctuations in the biological characteristics, genomic mutations, proliferation rates, and chemotherapeutic responses of tumor cells over time, posing a significant barrier to effective treatment. The complexity of this temporal variance, coupled with the spatial diversity of lung cancer, presents formidable challenges for research. This article will pave the way for new avenues in lung cancer research, aiding in a deeper understanding of the temporal heterogeneity of lung cancer, thereby enhancing the cure rate for lung cancer. MethodsRaman spectroscopy emerges as a powerful tool for real-time surveillance of biomolecular composition changes in lung cancer at the cellular scale, thus shedding light on the disease’s temporal heterogeneity. In our investigation, we harnessed Raman spectroscopic microscopy alongside multivariate statistical analysis to scrutinize the biomolecular alterations in human lung epithelial cells across various timeframes after benzo(a)pyrene exposure. ResultsOur findings indicated a temporal reduction in nucleic acids, lipids, proteins, and carotenoids, coinciding with a rise in glucose concentration. These patterns suggest that benzo(a)pyrene induces structural damage to the genetic material, accelerates lipid peroxidation, disrupts protein metabolism, curtails carotenoid production, and alters glucose metabolic pathways. Employing Raman spectroscopy enabled us to monitor the biomolecular dynamics within lung cancer cells in a real-time, non-invasive, and non-destructive manner, facilitating the elucidation of pivotal molecular features. ConclusionThis research enhances the comprehension of lung cancer progression and supports the development of personalized therapeutic approaches, which may improve the clinical outcomes for patients.

Objective To compare the effects of 20 mg qd and 10 mg bidadministration of iprrazole enteric-coated tablets on the control of gastric acid in healthy subjects.Methods A randomized,single-center,parallel controlled trial was designed to include 8 healthy subjects.Randomly divided into 2 groups,20 mg qd administration group:20 mg enteric-coated tablets of iprrazole in the morning;10 mg bid administration group:10 mg enteric-coated tablets of iprrazole in the morning and 10 mg in the evening.The pH values in the stomach of the subjects before and 24 h after administration were monitored by pH meter.The plasma concentration of iprazole after administration was determined by HPLC-MS/MS.The main pharmacokinetic parameters were calculated by Phoenix WinNonlin(V8.0)software.Results The PK parameters of iprrazole enteric-coated tablets and reference preparations in fasting group were as follows:The Cmax of 20 mg qd group and 10 mg bid group were(595.75±131.15)and(283.50±96.98)ng·mL-1;AUC0-t were(5 531.94±784.35)and(4 686.67±898.23)h·ng·mL-1;AUC0-∞ were(6 003.19±538.59)and(7 361.48±1 816.77)h·ng·mL-1,respectively.The mean time percentage of gastric pH＞3 after 20 mg qd and 10 mg bid were 82.64％and 61.92％,and the median gastric pH within 24 h were 6.25±1.49 and 3.53±2.05,respectively.The mean gastric pH values within 24 h were 5.71±1.36 and 4.23±1.45,respectively.The correlation analysis of pharmacokinetic/pharmacodynamics showed that there was no significant correlation between the peak concentration of drug in plasma and the inhibitory effect of acid.Conclusion Compared with the 20 mg qd group and the 10 mg bid group,the acid inhibition effect is better,the administration times are less,and the safety of the two administration regimes is good.

Objective To evaluate the anesthetic and postoperative analgesic effects of ultrasound-guided erector spinae plane block(ESPB)with dexmedetomidine combined with ropivacaine in percutaneous endoscopic lumbar discectomy(PELD).Method Patients underwent endoscopic discectomy were randomly divided into local infiltrative anesthesia group(group C),ropivacaine group(group R)and dexmedetomidine combined with ropivacaine group(group DR).Group C received local infiltration anesthesia with 0.375％ropivacaine 20 mL;in group R,the anesthesiologist used 0.375％ropivacaine 20 mL to perform bilateral ESPB.In DR group,0.375％ropivacaine+1 μg·kg-1 dexmedetomidine mixed solution 20 mL was used for bilateral ESPB.The changes of mean arterial pressure(MAP)and heart rate(HR)at 3 min before and after insertion of the channel were observed.The visual analogue scale(VAS)were recorded before operation(T0),at the time of needle positioning(T1),working channel placement(T2),annulus fibrosus operation(T3),at the end of operation(T4),at 4 h(T5),8 h(T6),12 h(T7)and 24 h(T8)after operation were observed.The dosage of intravenous analgesics,vasoactive drugs,and the occurrence of intraoperative adverse drug reactions were observed.Results There were 30 patients in group C,29 patients in group R and 30 patients in group DR.At 3 min after insertion into the channel,HR of group C,group R and group DR were(83.23±5.61),(78.18±4.71)and(77.31±5.13)beat·min-1;MAP were(85.97±3.89),(76.13±4.70)and(74.21±3.12)mmHg;with statistically significant difference(all P＜0.05).The VAS of group C,group R and group DR at T2were(5.80±0.85),(2.38±0.68)and(1.73±0.95)points;at T3 were(4.43±0.57),(2.55±0.57)and(1.63±0.56)points;at T7 were(4.66±0.66),(3.55±0.63)and(3.03±0.66)points;at T8 were(5.53±0.68),(4.07±0.46)and(3.23±0.57)points,all with significant difference(all P＜0.05).The number of additional cases of sufentanil in group C was 17 cases,and in group R was 5 cases,and in group DR was 3 cases,the number of additional cases of sufentanil in group R and DR was significantly less than that in group C(all P＜0.05).There was no significant difference in the incidence of adverse drug reactions among the three groups(all P＞0.05).Conclusion ESPB with dexmedetomidine combined with ropivacaine can significantly reduce intraoperative and postoperative pain,reduce intraoperative opioid use,and has no obvious adverse reactions in patients undergoing PELD.

To construct a high-quality Panax ginseng cDNA library, transcription factors binding to the P. ginseng PgD14 gene promoter were screened by yeast one-hybrid, and proteins interacting with the P. ginseng Pgpht2-1 gene-encoded protein were screened by yeast two-hybrid. In this study, root tissues of P. ginseng were used as materials. Gateway technology was used to construct the P. ginseng yeast one-hybrid library, and duplex-specific nuclease(DSN) homogenization technology was used to construct the P. ginseng yeast two-hybrid library. The pAbAi-PgD14-Pro961 vector was used as bait to screen candidate transcription factors that might bind to the PgD14 gene promoter from the yeast one-hybrid library, and the pGBKT7-Pgpht2-1 vector was used as bait to screen candidate proteins that might interact with the Pgpht2-1 gene-encoded protein from the yeast two-hybrid library. The yeast one-hybrid library had a size of 1.20×10~7 CFU, a recombination rate of 100%, and an average inserted fragment length of more than 1 000 bp. The yeast two-hybrid library had a size of 1.832×10~5 CFU, a recombination rate of 100%, and an average inserted fragment length of about 1 000 bp. The recombinant vectors pAbAi-PgD14-Pro961 and pGBKT7-Pgpht2-1 were transformed into Y1HGold and AH109 strains, respectively, and interacting proteins were screened by yeast one-hybrid and yeast two-hybrid. As a result, 54 transcription factors that could bind to the PgD14 gene promoter of P. ginseng and 42 proteins that may interact with the protein encoded by the Pgpht2-1 gene were identified. This study successfully constructed the P. ginseng yeast one-hybrid and yeast two-hybrid cDNA libraries, laying a foundation for subsequent studies on the functions of the P. ginseng PgD14, Pgpht2-1, and other genes.
Panax/metabolism* ; Two-Hybrid System Techniques ; Plant Proteins/metabolism* ; Gene Library ; Plant Roots/chemistry* ; Protein Binding ; Transcription Factors/metabolism* ; Promoter Regions, Genetic

Humans ; Angioplasty, Balloon/adverse effects* ; Antithrombin III/metabolism* ; Chronic Disease ; Pulmonary Artery ; Pulmonary Embolism/etiology* ; Thromboembolism/therapy*

Objectives: To construct a nomogram for prediction of intrahepatic cholangiocarcinoma (ICC) lymph node metastasis based on inflammation-related markers,and to conduct its clinical verification. Methods: Clinical and pathological data of 858 ICC patients who underwent radical resection were retrospectively collected at 10 domestic tertiary hospitals in China from January 2010 to December 2018. Among the 508 patients who underwent lymph node dissection,207 cases had complete variable clinical data for constructing the nomogram,including 84 males,123 females,109 patients≥60 years old,98 patients<60 years old and 69 patients were pathologically diagnosed with positive lymph nodes after surgery. Receiver operating characteristic curve was drawn to calculate the accuracy of preoperative imaging examinations to determine lymph node status,and the difference in overall survival time was compared by Log-rank test. Partial regression squares and statistically significant preoperative variables were screened by backward stepwise regression analysis. R software was applied to construct a nomogram,clinical decision curve and clinical influence curve,and Bootstrap method was used for internal verification. Moreover,retrospectively collecting clinical information of 107 ICC patients with intraoperative lymph node dissection admitted to 9 tertiary hospitals in China from January 2019 to June 2021 was for external verification to verify the accuracy of the nomogram. 80 patients with complete clinical data but without lymph node dissection were divided into lymph node metastasis high-risk group and low-risk group according to the score of the nomogram among the 858 patients. Log-rank test was used to compare the overall survival of patients with or without lymph node metastasis diagnosed by pathology. Results: The area under the curve of preoperative imaging examinations for lymph node status assessment of 440 patients was 0.615,with a false negative rate of 62.8% (113/180) and a false positive rate of 14.2% (37/260). The median survival time of 207 patients used to construct a nomogram with positive or negative postoperative pathological lymph node metastases was 18.5 months and 27.1 months,respectively (P<0.05). Five variables related to lymph node metastasis were screened out by backward stepwise regression analysis,which were combined calculi,neutrophil/lymphocyte ratio,albumin,liver capsule invasion and systemic immune inflammation index,according to which a nomogram was constructed with concordance index(C-index) of 0.737 (95%CI: 0.667 to 0.806). The C-index of external verification was 0.674 (95%CI:0.569 to 0.779). The calibration prediction curve was in good agreement with the reference curve. The results of the clinical decision curve showed that when the risk threshold of high lymph node metastasis in the nomogram was set to about 0.32,the maximum net benefit could be obtained by 0.11,and the cost/benefit ratio was 1∶2. The results of clinical influence curve showed that when the risk threshold of high lymph node metastasis in the nomogram was set to about 0.6,the probability of correctly predicting lymph node metastasis could reach more than 90%. There was no significant difference in overall survival time between patients with high/low risk of lymph node metastasis assessed by the nomogram and those with pathologically confirmed lymph node metastasis or without lymph node metastasis (Log-rank test:P=0.082 and 0.510,respectively). Conclusion: The prediction accuracy of preoperative nomogram for ICC lymph node metastasis based on inflammation-related markers is satisfactory,which can be used as a supplementary method for preoperative diagnosis of lymph node metastasis and is helpful for clinicians to make personalized decision of lymph node dissection for patients with ICC.

To compare the pancreatic proteomics and autophagy between Rehmanniae Radix-and Rehmanniae Radix Praeparata-treated mice with type 2 diabetes mellitus(T2DM). The T2DM mouse model was established by high-fat diet coupled with streptozotocin(STZ, intraperitoneal injection, 100 mg·kg~(-1), once a day for three consecutive days). The mice were then randomly assigned into a control group, low-(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix groups, low-(150 mg·kg~(-1)) and high-dose(300 mg·kg~(-1)) catalpol groups, low-(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix Praeparata groups, low-(150 mg·kg~(-1)) and high-dose(300 mg·kg~(-1)) 5-hydroxymethyl furfuraldehyde(5-HMF) groups, and a metformin(250 mg·kg~(-1)) group. In addition, a normal group was also set and each group included 8 mice. The pancreas was collected after four weeks of administration and proteomics tools were employed to study the effects of Rehmanniae Radix and Rehmanniae Radix Praeparata on protein expression in the pancreas of T2DM mice. The expression levels of proteins involved in autophagy, inflammation, and oxidative stress response in the pancreatic tissues of T2DM mice were determined by western blotting, immunohistochemical assay, and transmission electron microscopy. The results showed that the differential proteins between the model group and Rehmanniae Radix/Rehmanniae Radix Prae-parata group were enriched in 7 KEGG pathways, such as autophagy-animal, which indicated that the 7 pathways may be associated with T2DM. Compared with the control group, drug administration significantly up-regulated the expression levels of beclin1 and phosphorylated mammalian target of rapamycin(p-mTOR)/mTOR and down-regulated those of the inflammation indicators, Toll-like receptor-4(TLR4) and Nod-like receptor protein 3(NLRP3), in the pancreas of T2DM mice, and Rehmanniae Radix showed better performance. In addition, the expression levels of inducible nitric oxide synthase(iNOS), nuclear factor erythroid 2-related factor 2(Nrf2), and heine oxygenase-1(HO-1) in the pancreas of T2DM mice were down-regulated after drug administration, and Rehmanniae Radix Praeparata demonstrated better performance. The results indicate that both Rehmanniae Radix and Rehmanniae Radix Praeparata can alleviate the inflammatory symptoms, reduce oxidative stress response, and increase the autophagy level in the pancreas of T2DM mice, while they exert the effect on different autophagy pathways.
Mice ; Animals ; Diabetes Mellitus, Type 2/genetics* ; Streptozocin/pharmacology* ; Diet, High-Fat/adverse effects* ; Proteomics ; Inflammation ; TOR Serine-Threonine Kinases ; Autophagy ; Mammals