Objective : To investigate the brain microvascular tissue block culture method for extracting primary rat brain microvascular endothelial cells and identify its effect. Methods : Brain tissue from 4-week-old Sprague Dawley rats was screened, pre-digested and solidified to obtain brain microvascular segments. These segments were subsequently placed in a CO2incubator for primary culture. The target cells were identified by cell morphology and immunocytochemical staining for factor Ⅷ-related antigen. Results : After a 48-hour culture periodin vitro, the short spindle cells crawled out from around the brain microvascular segments. After 72 hours, island-like cell culsters formed. After 96 hours the clusters fused and the cells formed a typical monolayer, cobble stone-like, and mosaic arrangement. Factor Ⅷ-related antigen immunocytochemical staining showed that the cytoplasm of the cells appeared brown-red, indicating positive expression; DAB stained the nucleus, showing blue-dark. Conclusion The brain microvascular tissue block culture method can isolate and culture primary rat brain microvascular endothelial cells.

Objective To observe the effect of Kaixin Powder on neuroinflammation in APP/PS1 mice by regulating WDFY1/TLR4/NF-κB signaling pathway;To explore its mechanism of intervening in Alzheimer disease(AD).Methods APP/PS1 transgenic mice were randomly divided into model group,donepezil hydrochloride group(0.66 mg/kg),and Kaixin Powder low-,medium-and high-dosage groups(1.625,3.25,6.5 g/kg),C57BL/6J mice were set as blank control group,with 8 mice in each group,and corresponding drug intervention was given to medicaction group for 24 weeks.Morris water maze,Y maze and novel object recognition experiments were conducted to assess the cognitive function and learning and memory abilities of mice,immunohistochemical staining was used to detect the deposition of β-amyloid protein(Aβ)in hippocampus,the morphology and Nissl bodies of hippocampal CA1 neurons were observed using HE staining and Nissl staining,ELISA was used to detect the serum contents of interleukin(IL)-6,IL-17,IL-1β and tumor necrosis factor-α(TNF-α),Western blot was used to detect the protein expression of calcium-binding adapter molecule 1(Iba1),glial fibrillary acidic protein(GFAP),WDFY1,Toll like receptor 4(TLR4),Toll like receptor associated molecule(TRAM),TIR domain adapter protein(TRIF),NF-κB p65 and p-NF-κB p65 in hippocampal tissue,RT-qPCR was used to detect the mRNA expression of WDFY1,TLR4,TRAM,TRIF and NF-κB p65 in hippocampal tissue.Results Compared with the blank control group,the model group had significantly prolonged escape latency,reduced platform crossings,decreased autonomous reaction alternation rate and relative recognition index(P<0.05,P<0.01),with increased deposition of Aβ in hippocampal tissue(P<0.01),damaged morphological structure of neurons,reduced number of neurons and Nissl bodies,the serum contents of IL-6,IL-17,IL-1β and TNF-α significantly increased,the expression of Iba1,GFAP,WDFY1,TLR4,TRAM,TRIF,p-NF-κB p65 protein and WDFY1,TLR4,TRAM,TRIF mRNA in hippocampal tissue significantly increased(P<0.01).Compared with the model group,Kaixin Powder groups and donepezil hydrochloride group had significantly shortened escape latency and increased platform crossings,autonomous reaction alternation rate and relative recognition index(P<0.05,P<0.01),hippocampal Aβ deposition reduced in Kaixin Powder medium-,high-dosage groups and donepezil hydrochloride group,the morphological structure of neurons recovered,the number of neurons and Nissl bodies increased,the serum contents of IL-6,IL-17,IL-1β and TNF-α significantly decreased(P<0.05,P<0.01),and the protein expression of Iba1,GFAP,WDFY1,TLR4,TRAM,TRIF,p-NF-κB p65 and the mRNA expressions of WDFY1,TLR4,TRAM and TRIF in hippocampal tissue significantly decreased(P<0.05,P<0.01).Conclusion Kaixin Powder can improve cognitive function and learning and memory abilities in AD model mice,alleviate hippocampal neuron damage and Aβ deposition,inhibit the activation of microglia and astrocytes,and thereby reduce serum inflammatory cytokine release.Its mechanism may be related to regulating the WDFY1/TLR4/NF-κB signaling pathway to inhibit neuroinflammation.

Targeted protein degradation via nanoparticle-based universal strategy modifies nanoparticles with antibodies and ingeniously utilizes its cellular transport characteristics. This strategy achieved targeted degradation of extracellular proteins without complex design.Image 1.

Objective To explore the value of the ratio of soluble FmS-like tyrosine kinase-1(sFlt-1)to placental growth factor(PlGF)in predicting vitamin D deficiency in pregnant women with preeclampsia.Methods A total of 740 singleton pregnant women who underwent prenatal examination in Qijiang Health Center for Maternal and Child Care from January to November 2023 and were able to complete follow-up were selected as the research subjects.They were divided into the normal group(n=44)and the deficiency group(n=696)according to the vitamin D[1,25-(OH)2D3]level.Information such as age,BMI,pregnancy histo-ry,family history and medical history of each group was collected.5 mL of peripheral venous blood was drawn after fasting for 6-8 hours at 12-14 weeks of pregnancy to detect the levels of sFlt-1,PlGF and vitamin D in peripheral blood,and evaluated the predictive value of sFlt-1/PlGF for vitamin D deficiency in preeclampsia.Results There were no statistically significant differences in general conditions such as height,age,BMI,edu-cational level and occupation between the two groups(P>0.05).There were statistically significant differ-ences in the prenatal weight,vitamin D level,sFlt-1/PlGF and neonatal birth weight between the two groups(P<0.05).The results of univariate analysis showed that the prenatal weight,sFlt-1/PlGF,and neonatal birth weight were risk factors for vitamin D deficiency in patients with PE.The results of multivariate analysis showed that sFlt-1/PlGF and neonatal birth weight were factors for predicting vitamin D deficiency in patients with PE(P<0.05).All these variables are used to establish the nomogram prediction model.The receiver op-erating characteristic(ROC)curve showed that area under the curve(AUC)of the training group and the val-idation group was 0.66(95%CI:0.56-0.77)and 0.63(95%CI:0.49-0.76),respectively.Conclusion In pregnant women with preeclampsia,elevated sFlt-1/PlGF may be an early warning indicator for predicting vi-tamin D deficiency.

Objective:To establish a simple and efficient method for in vitro extraction,culture and identification of brain microvascular endothelial cells(BMECs)of rabbits.Method:The cerebral cortex of 3-week-old rabbits was cut up,sieved and digested with type II collagenase to obtain pure brain microvascular segments for primary culture and identification.Results:The cultured target cells grew in a typical"cobblestone-like"arrangement.Immunohistochemi-cal staining for von Willebrand factor(vWF)related antigen showed that the cytoplasm was presented as brownish red and the expression was positive.Conclusion:The physical sieving method combined with the chemical enzyme diges-tion method can simply and efficiently culture primary rabbit BMECs with strong activity and high purity.

Objective:To establish a simple and efficient method for in vitro extraction,culture and identification of brain microvascular endothelial cells(BMECs)of rabbits.Method:The cerebral cortex of 3-week-old rabbits was cut up,sieved and digested with type II collagenase to obtain pure brain microvascular segments for primary culture and identification.Results:The cultured target cells grew in a typical"cobblestone-like"arrangement.Immunohistochemi-cal staining for von Willebrand factor(vWF)related antigen showed that the cytoplasm was presented as brownish red and the expression was positive.Conclusion:The physical sieving method combined with the chemical enzyme diges-tion method can simply and efficiently culture primary rabbit BMECs with strong activity and high purity.

Objective To observe the effect of Kaixin Powder on neuroinflammation in APP/PS1 mice by regulating WDFY1/TLR4/NF-κB signaling pathway;To explore its mechanism of intervening in Alzheimer disease(AD).Methods APP/PS1 transgenic mice were randomly divided into model group,donepezil hydrochloride group(0.66 mg/kg),and Kaixin Powder low-,medium-and high-dosage groups(1.625,3.25,6.5 g/kg),C57BL/6J mice were set as blank control group,with 8 mice in each group,and corresponding drug intervention was given to medicaction group for 24 weeks.Morris water maze,Y maze and novel object recognition experiments were conducted to assess the cognitive function and learning and memory abilities of mice,immunohistochemical staining was used to detect the deposition of β-amyloid protein(Aβ)in hippocampus,the morphology and Nissl bodies of hippocampal CA1 neurons were observed using HE staining and Nissl staining,ELISA was used to detect the serum contents of interleukin(IL)-6,IL-17,IL-1β and tumor necrosis factor-α(TNF-α),Western blot was used to detect the protein expression of calcium-binding adapter molecule 1(Iba1),glial fibrillary acidic protein(GFAP),WDFY1,Toll like receptor 4(TLR4),Toll like receptor associated molecule(TRAM),TIR domain adapter protein(TRIF),NF-κB p65 and p-NF-κB p65 in hippocampal tissue,RT-qPCR was used to detect the mRNA expression of WDFY1,TLR4,TRAM,TRIF and NF-κB p65 in hippocampal tissue.Results Compared with the blank control group,the model group had significantly prolonged escape latency,reduced platform crossings,decreased autonomous reaction alternation rate and relative recognition index(P<0.05,P<0.01),with increased deposition of Aβ in hippocampal tissue(P<0.01),damaged morphological structure of neurons,reduced number of neurons and Nissl bodies,the serum contents of IL-6,IL-17,IL-1β and TNF-α significantly increased,the expression of Iba1,GFAP,WDFY1,TLR4,TRAM,TRIF,p-NF-κB p65 protein and WDFY1,TLR4,TRAM,TRIF mRNA in hippocampal tissue significantly increased(P<0.01).Compared with the model group,Kaixin Powder groups and donepezil hydrochloride group had significantly shortened escape latency and increased platform crossings,autonomous reaction alternation rate and relative recognition index(P<0.05,P<0.01),hippocampal Aβ deposition reduced in Kaixin Powder medium-,high-dosage groups and donepezil hydrochloride group,the morphological structure of neurons recovered,the number of neurons and Nissl bodies increased,the serum contents of IL-6,IL-17,IL-1β and TNF-α significantly decreased(P<0.05,P<0.01),and the protein expression of Iba1,GFAP,WDFY1,TLR4,TRAM,TRIF,p-NF-κB p65 and the mRNA expressions of WDFY1,TLR4,TRAM and TRIF in hippocampal tissue significantly decreased(P<0.05,P<0.01).Conclusion Kaixin Powder can improve cognitive function and learning and memory abilities in AD model mice,alleviate hippocampal neuron damage and Aβ deposition,inhibit the activation of microglia and astrocytes,and thereby reduce serum inflammatory cytokine release.Its mechanism may be related to regulating the WDFY1/TLR4/NF-κB signaling pathway to inhibit neuroinflammation.

Objective:To extract rat brain microvascular endothelial cells(BMECs)using thin-layer cell culture method.Methods:The brain cortex of four-week-old SD rats was obtained,which was chopped,sieved,and digested with type II collagenase to obtain microvascular segments.The amount of culture medium was strictly controlled,and it was inoculated into culture flasks for primary culture.The target cells were morphologically observed by using an invert-ed phase contrast microscope,and the factor Ⅷ related antigen(FⅧRag)was identified by using immunocytochemi-cal staining.Results:The target cells cultured in the inverted phase contrast microscope showed typical endothelial-like monolayer cobble stone-like mosaic growth;FⅧRag positive cells accounted for more than 99％of the total cells.Conclusion:Thin-layer cell culture method can successfully isolate and cultivate high-purity rat BMECs.

ObjectiveTo investigate the Alzheimer-associated neurofilament protein （AD7c-NTP） in urine of middle-aged and elderly people and its correlation between common metabolites. MethodsA total of 1 150 middle-aged and elderly people who did their physical exmanination in the health examination center of the Sichuan Science City Hospital and the Third Hopital of Mianyang were recruited from March 2017 to March 2020. The level of urine AD7c-NTP were measured by enzyme-linked immunosorbent assay （ELISA）， and common metabolites in blood were measured by biochemical analyzer. Based on urine AD7c-NTP level ≤1.5 ng/mL， the objects was divided into normal group （n=956） and elevated group （n=194）. Thier demographic data and blood biochemical indicators were collected. ResultsThe urine AD7c-NTP level in middle-aged and elderly people was 0.60（0.30~1.20） ng/mL. The urine AD7c-NTP level was higher in women than that in men ［1.04（0.40~1.30） ng/mL vs. 0.84（0.30~1.00） ng/mL， Z=4.202， P˂0.01］. And the urine AD7c-NTP level was lower in the normal group than that in the elevated group ［0.50（0.30~0.90） ng/mL vs. 2.10（1.70~2.10） ng/mL， Z=22.035， P˂0.01］. The results of the univariate comparison showed that， the differences between the two groups in age （Z=6.545）， fasting glucose （Z=3.506）， blood uric acid （Z=2.574）， urea nitrogen （Z=2.891）， creatinine （Z=2.243）， total bilirubin （Z=3.936）， glutathione （Z=0.969）， total cholesterol （t=3.956） and low density lipoprotein （Z=-5.678） were were statistically significant （P˂0.05 or 0.01）. Spearman correlation analysis showed that， the urine AD7c-NTP level was positively correlated with age and the levels of urea nitrogen， glucose， total cholesterol and low density lipoprotein （r=0.177， 0.178， 0.171， 0.109， 0.149， P˂0.01）， and negatively correlated with the level of total bilirubin （r=-0.172， P˂0.01）. Conclusionthe urine AD7c-NTP level in middle-aged and elderly females was signifitcantly higher than in middle-aged and elderly males.The urine AD7c-NTP level of middle-aged and elderly people was positively correlated with age， urea nitrogen， glucose， total cholesterol and low density lipoprotein， and negatively correlated with total bilirubin.

Background The current oil production determines oil workers’ occupational noise exposure. Without effective protection, noise will affect various aspects of worker’s body functions, including acting on the adrenal cortex system and resulting in renal function damage. Objective To evaluate the associations of noise exposure and its cumulative exposure level with renal function impairment of oil workers. Methods Oil workers from a collective medical examination in a hospital were selected as the study subjects. In accordance with the national standard Measurement of Physical Agents in the Workplace Part 8: Noise (GBZ/T 189.8—2007), noise exposure was measured three times at the oil workers' work site, and their average value was calculated to obtain the cumulative noise exposure (CNE). A questionnaire survey was conducted to collect general information such as socio-demographic characteristics, family history, lifestyles, and occupational history. All blood biochemical indicators were measured in the fasting state. Renal function impairment was judged based on the glomerular filtration rate. The relationship between CNE and renal function was analyzed using receiver operating characteristic curve (ROC) for workers with noise exposure. Results A total of 2 917 subjects were included in the study and their prevalence of renal function impairment was 14.2%. The univariate analysis results suggested statistically significant differences in the prevalence of renal function impairment among the oil workers grouped by having hypertension or not, gender, age, marital status, marital status, smoking, and alcohol consumption (P＜0.05); the prevalence of renal impairment was significantly higher in those with abnormal values of uric acid, total cholesterol, triglycerides, high-density lipoprotein, and fasting glucose than in those with normal values (P＜0.05); the oil workers with noise exposure [n=1565, 53.7%, equivalent sound level ≥80 dB(A)] showed a higher prevalence of renal function impairment than those without (P＜0.05). The results of multiple logistic regression analysis showed that being female (OR=2.811, 95%CI: 1.960-4.030), age at 31 years and above (OR_31-40=3.502, 95%CI: 1.402-8.751; OR_41-50=4.255, 95%CI: 1.759-10.291; OR_≥51=7.179, 95%CI: 2.864-17.996), showing abnormal uric acid (OR=5.932, 95%CI: 4.486-7.843), having hypertension (OR=1.593, 95%CI: 1.230-2.063), alcohol consumption (OR=2.648, 95%CI: 1.346-5.212), and smoking (OR=1.816, 95%CI: 1.133-2.911) had higher risks of developing renal function impairment; besides, those exposed to noise had 1.351 times (95%CI: 1.073-1.702) higher risks of developing renal function impairment than non-exposed individuals. Noise-exposed oil workers in the renal impairment group had higher noise exposure intensity and CNE compared to the noise-exposed oil workers in the normal renal function group (P＜0.05), and the workers had an increased risk of renal function impairment when the CNE was >95.85 dB(A)·year versus CNE ≤ 95.85 dB(A)·year (OR=2.583, 95%CI: 1.956-3.411). Conclusion Exposure to noise, higher noise exposure intensity, and higher level of CNE may be associated with developing renal function impairment in oil workers. Oil workers with CNE above 95.85 dB(A)·year are at an increased risk of renal impairment.