ObjectiveTo provide a new idea for exploring the molecular genetic approach to the pathogenesis of schizophrenia via construction of microRNA-messenger RNA （miRNA-mRNA） regulatory network in schizophrenia. MethodsThe microarray datasets of GSE54578 miRNA expression profiles in peripheral blood and GSE145554 mRNA expression in the anterior cingulate in postmortem brain of schizophrenic subjects were downloaded from Gene Expression Omnibus （GEO） database since July 2021. The GEO2R was used to identify the differentially expressed miRNAs and mRNAs， screen the miRNA with target differentially expressed mRNA， and predict their potential upstream transcription factors. The overlapping genes from the mRNA targeted by the differentially expressed miRNA and the mRNA differentially expressed in GSE145554 dataset were collected. Then the biological features of hub genes were analyzed via Gene Ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis， and the protein-protein interaction （PPI） network and miRNA-mRNA regulatory network of hub genes were constructed. ResultsA total of 8 up-regulated differentially expressed miRNAs with targeted mRNA were screened out in GSE54578 datasets regarding schizophrenia， which involved in the regulation of 10 transcription factors， 247 down-regulated differentially expressed mRNAs were screened out in GSE145554 datasets， and 17 overlapping mRNAs were obtained. GO analysis showed that the target mRNAs were mainly involved in astrocyte differentiation and development. KEGG pathway enrichment analysis showed that the target mRNAs were mainly involved in Rap1 and Ras signaling pathways. PPI network analysis showed that the mRNAs （KRAS and CD28） might be key genes in schizophrenia. ConclusionThe integrated bioinformatics analysis based on GEO database can identify potential susceptibility genes in schizophrenia， and it also contributes to the construction of miRNA-mRNA regulatory network in schizophrenia.

Aim To investigate the neuroprotective effects of osthole(Ost)on the primary cultured cortical neurons transfected with APP595/596 gene and its underlying mechanism.Methods Neonatal mouse cortical neurons were transfected with APP595/596 gene to establish AD cell models for the further study.Then,the cell viability was detected by CCK-8 assay,and the leakage of lactate dehydrogenase(LDH)was assayed by LDH kit to evaluate the injury degree.Transferase-mediated nick end labeling(TUNEL)was used to evaluate the cell apoptosis.The expression of β-amyloid peptide(Aβ)and β-site APP cleaving enzyme 1(BACE1)was measured by immunofluorescence,while the miRNA-107 was measured by RT-PCR.Results Compared to model group,Ost could significantly improve the neurons viability,decrease the LDH release and prevent the apoptosis.Ost also inhibited the expression of Aβ and BACE1 at protein level,while enhanced the expression of miRNA-107 at gene level.Conclusion Ost plays a neuroprotective role in neurons transfected with APP595/596 gene in part through up-regulating miRNA-107.

OBJECTIVE：To study the effects and mechanism of panaxadiol （PD） on Tau protein phosphorylation in the SH-SY5Y cells transfected with APP gene（APP-SH-SY5Y）. METHODS ：The target of PD and non-receptor tyrosine kinases Fyn was verified by molecular docking. SH-SY 5Y cells were cultured in vitro ，and the APP-SH-SY 5Y cell models and green fluorescent （GFP）-SH-SY5Y cell model （control cell ）was constructed. The expression of Aβ1-42 was detected so as to verify the success of APP-SH-SY5Y cell model. Taking GFP-SH-SY 5Y cells as control ，the effects of 5，10，20，30，40 μmol/L PD and 125，250， 500，1 000，2 000 nmol/L PP 2（Fyn inhibitor ，positive control ）on the survival rate of APP-SH-SY 5Y cells were detected by CCK-8 assay after treated for 24 h，so as to confirm the optimal concentration. The concentration of Ca 2 + ，the ratio fophosphorylated Tau protein （p-Tau）/Tau，phosphorylatedn Src（p-Src）/Fyn and phosphorylated glutamate receptor 2B（p-GluN2B）/ GluN2B were detected in APP-SH-SY 5Y cells after trated with the optimal concentration of PD and PP 2 for 24 h. RESULTS ：The results of molecular simulation docking showed that PD could target Fyn protein. Compared with GFP-SH-SY 5Y cells ，the protein expression of Aβ1-42 in APP-SH-SY 5Y cell were increased significantly （P＜0.01）. The optimal concentration of PD and PP 2 were 20 μmol/L and 500 nmol/L. The 20 μmol/L PD and 500 nmol/L PP 2 could increase the survival rate of the cells and reduced the concentration of Ca 2+，the ratio of p-Tau/Tau ，p-Src/Fyn，and p-GluN 2B/GluN2B. CONCLUSIONS：PD can reduce the the phosphorylation of Tau protein through inhibiting Fyn/GluN 2B signaling pathway.

OBJECTIVE To study the neuroprotective effects of Shenzao jianna o oral liquid （SZJN）on Alzheimer ’s disease （AD）model mice and its mechanism. METHODS The mice were randomly divided into sham operation group ，model group ， Donepezil hydrochloride tablet group （0.65 mg/kg），SZJN low-dose ，medium-dose and high-dose groups （0.3，1.5 and 7.5 g/kg， calculated by crude drug quantity ），with 12 mice in each group ，half male and half female. Each group was given relevant medicine（intragastric administration of water at constant volume in sham operation group and model group ），twice a day ，for consecutive 28 d. On the 15th day of administration ，intracerebroventricular injection of β-amyloid 1-42（Aβ1-42）combined with intraperitoneal injection of scopolamine hydrobromide were used to induce AD model. Morris water maze was used to detect the learning and memory ability of mice. HE staining and Nissl staining were used to evaluate the pathological changes of brain tissue in mice. The levels of MDA and SOD in brain tissue of mice were detected. The phosphorylation level of cyclic adenosine monophosphate response element binding protein （CREB） and expression of brain-derived neurotrophic factor （BDNF） in hippocampal tissues were detected by Western blot. RESULTS Compared with sham operation group ，the escape latency of the model group was significantly prolonged ，and the number of crossing the platform and the percentage of residence time in the target quadrant were significantly reduced （P＜0.01）. The level of SOD in brain tissue ，the phosphorylation level of CREB and the expression level of BDNF in hippocampus decreased significantly （P＜0.01），while the level of MDA increased significantly （P＜ 0.01）. In hippocampal CA 1 area and cortical tissue ，nerve cells showed significantly decreased number ，the disordered arrangement and large gap ；the shape of nucleus was irregular and deeply stained ，and Nissl body was blurred ，loosely arranged and the number decreased. Compared with model group ，the escape latency of mice in each dose group of SZJN was significantly shortened ，and the times of crossing the platform and the percentage of residence time in the target quadrant were significantly jing- increased（P＜0.01）. Above indexes of brain tissue in mice were reversed sig nificantly in SZJN high-dose group （P＜0.01），and pathological damage of brain tiss ue was improved. CONCLUSIONS SZJN can significantly improve the learning and memory ability of AD model mice ，and alleviate the pathological injury and oxidative stress of brain tissue ，which may be related to the activation of CREB/BDNF signaling pathway.

OBJECTIVE: To investigate the effect of osthole on the expression of amyloid precursor protein (APP) in Alzheimer's disease (AD) cell model and its mechanism. METHODS: The SH-SY5Y cell with over expression of APP was established by transfection by liposome 2000. The cells were treated with different concentrations of osthole, and the cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The differentially expressed miRNAs with and without osthole treatment were detected by miRNA array, and the target genes binding to the differentially expressed miRNAs were identified and verified by databases and Cytoscape. After the inhibitor of the differentially expressed miRNA was transduced into cells, the changes of APP and amyloid β (Aβ) protein were determined by immunofluorescence cytochemistry, and the mRNA expression of APP was determined by RT-PCR. RESULTS: The AD cell model with over expression of APP was established successfully. The results of MTT and LDH assay showed that osthole had a protective effect on cells and alleviated cell damage. miR-101a-3p was identified as the differentially expressed miRNA, which was binding to the 3'-UTR of APP. Compared with APP group, the expression of APP and Aβ protein and APP mRNA increased in the miR-101a-3p inhibitor group (all <0.01), while the expression of APP and Aβ protein and APP mRNA decreased in the cells with osthole treatment (all <0.01). CONCLUSIONS Osthole inhibits the expression of APP by up-regulating miR-101a-3p in AD cell model.
Alzheimer Disease ; Amyloid beta-Peptides ; Amyloid beta-Protein Precursor ; genetics ; Cell Line ; Coumarins ; pharmacology ; Gene Expression Regulation ; drug effects ; genetics ; Humans ; MicroRNAs ; genetics ; metabolism

OBJECTIVE: To explore the mechanisms of large-conductance calcium-activated potassium channel (BKCa) involved in inflammatory response in sepsis. METHODS: The serum levels of BKCa were measured by enzyme-linked immunosorbent assay (ELISA) in patients with sepsis (28 cases), patients with common infection (25 cases) and healthy people (25 cases). The relationship between levels of BKCa and acute physiology and chronic health evaluation II (APACHE II) were analyzed. Cultured RAW 264.7 cells were stimulated by lipopolysaccharide (LPS). In some experiments, a cell model of sepsis was constructed using Nigericin as the second stimulus signal. The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS (0, 50, 100, 1 000 μg/L) were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RAW 264.7 cells were transfected with small interfering RNA of BKCa (siRNA-BKCa), and the levels of caspase-1 precursor (pro-caspase-1), interleukin-1β precursor (pro-IL-1β) in cell, and the levels of caspase-1 p20, IL-1β p17 of cell culture medium, and NOD-like receptor protein 3 (NLRP3), nuclear factor-κB (NF-κB) were measured by Western blotting. The apoptosis were detected by staining with propidium iodide (PI), the release rate of lactate dehydrogenase (LDH) were measured, and the expression of apoptotic protein Gasdermin D (GSDMD) was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis. RESULTS: The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples (ng/L: 165.2±25.9 vs. 102.5±25.9, 98.8±20.0, both P < 0.05). In addition, the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHE II score (r = 0.453, P = 0.013). LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner. The mRNA and protein expressions of BKCa in the cells stimulated by 1 000 μg/L LPS were significantly higher than that in the blank group (0 μg/L) [BKCa mRNA (2^-ΔΔCt): 3.00±0.36 vs. 1.00±0.16, BKCa/β-actin: 1.30±0.16 vs. 0.37±0.09, both P < 0.05]. Compared with the control group, the ratios of caspase-1 p20/pro-caspase-1 and IL-1β p17/pro-IL-1β in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1β p17/pro-IL-1β: 0.77±0.12 vs. 0.23±0.12, both P < 0.05), however, transfection of siRNA-BKCa induced the decrease both of them (caspase-1 p20/pro-capase-1: 0.23±0.12 vs. 0.83±0.12, IL-1β p17/pro-IL-1β: 0.13±0.05 vs. 0.77±0.12, both P < 0.05). Compared with the control group, the number of apoptotic cells, LDH release rate and GSDMD expression in the model group were significantly increased [LDH release rate: (30.60±8.40)% vs. (15.20±7.10)%, GSDMD-N/GSDMD-FL: 2.10±0.16 vs. 1.00±0.16, both P < 0.05], however, transfection of siRNA-BKCa induced the decrease both of them [LDH release rate: (15.60±7.30)% vs. (30.60±8.40)%, GSDMD-N/GSDMD-FL: 1.13±0.17 vs. 2.10±0.16, both P < 0.05]. The mRNA and protein expressions of NLRP3 in sepsis cells were significantly higher than those in the control group [NLRP3 mRNA (2^-ΔΔCt): 2.06±0.17 vs. 1.00±0.24, NLRP3/GAPDH: 0.46±0.05 vs. 0.15±0.04, both P < 0.05]. However, the expression of NLRP3 after siRNA-BKCa transfection was significantly lower than that in model group [NLRP3 mRNA (2^-ΔΔCt): 1.57±0.09 vs. 2.06±0.17, NLRP3/GAPDH: 0.19±0.02 vs. 0.46±0.05, both P < 0.05]. Compared with the control group, the NF-κB p65 nuclear transfer of sepsis cell were significantly increased (NF-κB p65/Histone: 0.73±0.12 vs. 0.23±0.09, P < 0.05). However, the NF-κB p65 expression in the nucleus were decreased after siRNA-BKCa transfection (NF-κB p65/Histone: 0.20±0.03 vs. 0.73±0.12, P < 0.05). CONCLUSIONS BKCa is involved in the pathogenesis of sepsis, and its possible mechanism is to activate NF-κB/NLRP3/caspase-1 signaling pathway to induce inflammatory factor production and cell death.
Humans ; Histones ; Caspase 1 ; Large-Conductance Calcium-Activated Potassium Channels ; Lipopolysaccharides ; NF-kappa B ; NLR Family, Pyrin Domain-Containing 3 Protein ; L-Lactate Dehydrogenase ; Sepsis ; RNA, Small Interfering ; Caspases

Captopril can have nephrotoxic effects, which are largely attributed to accumulated renin and "escaped" angiotensin II (Ang II). Here we test whether angiotensin converting enzyme-1 (ACE1) inhibition damages kidneys via alteration of renal afferent arteriolar responses to Ang II and inflammatory signaling. C57Bl/6 mice were given vehicle or captopril (60 mg/kg per day) for four weeks. Hypertension was obtained by minipump supplying Ang II (400 ng/kg per min) during the second 2 weeks. We assessed kidney histology by periodic acid-Schiff (PAS) and Masson staining, glomerular filtration rate (GFR) by FITC-labeled inulin clearance, and responses to Ang II assessed in afferent arterioles in vitro. Moreover, arteriolar H₂O₂ and catalase, plasma renin were assayed by commercial kits, and mRNAs of renin receptor, transforming growth factor-β (TGF-β) and cyclooxygenase-2 (COX-2) in the renal cortex, mRNAs of angiotensin receptor-1 (AT₁R) and AT₂R in the preglomerular arterioles were detected by RT-qPCR. The results showed that, compared to vehicle, mice given captopril showed lowered blood pressure, reduced GFR, increased plasma renin, renal interstitial fibrosis and tubular epithelial vacuolar degeneration, increased expression of mRNAs of renal TGF-β and COX-2, decreased production of H₂O₂ and increased catalase activity in preglomerular arterioles and enhanced afferent arteriolar Ang II contractions. The latter were blunted by incubation with H₂O₂. The mRNAs of renal microvascular AT₁R and AT₂R remained unaffected by captopril. Ang II-infused mice showed increased blood pressure and reduced afferent arteriolar Ang II responses. Administration of captopril to the Ang II-infused mice normalized blood pressure, but not arteriolar Ang II responses. We conclude that inhibition of ACE1 enhances renal microvascular reactivity to Ang II and may enhance important inflammatory pathways.
Angiotensin II/pharmacology* ; Animals ; Arterioles/metabolism* ; Captopril/pharmacology* ; Hydrogen Peroxide/pharmacology* ; Kidney ; Mice