Phenotypes and ATP7B gene variants in 316 children with Wilson disease / 中华儿科杂志

Objectives: To summarize the clinical phenotypes and the variation spectrum of ATP7B gene in Chinese children with Wilson's disease (WD) and to investigate their significance for early diagnosis. Methods: Retrospective analysis was performed on the clinical data of 316 children diagnosed as WD in Guangzhou Women and Children's Medical Center during the period from January 2010 to June 2021. The general situations, clinical manifestations, lab test results, imaging examinations, and ATP7B gene variant characteristics were collected. The patients were divided into asymptomatic WD group and symptomatic WD group based on the presence or absence of clinical symptoms at the time that WD diagnosis was made. The χ2 test, t test or Mann-Whitney U test were used to compare the differences between groups. Results: Among the 316 children with WD, 199 were males and 117 were females, with the age of 5.4 (4.0, 7.6) years at diagnosis; 261 cases (82.6%) were asymptomatic with the age of 4.9 (3.9, 6.4) years; whereas 55 cases (17.4%) were symptomatic with the age of 9.6 (7.3, 12.0) years. The main symptoms invloved liver, kidney, nervous system, or skin damage. Of all the patients, 95.9% (303/316) had abnormal liver function at diagnosis; 98.1% (310/316) had the serum ceruloplasmin lever lower than 200 mg/L; 97.7% (302/309) had 24-hour urine copper content exceeding 40 μg; only 7.4% (23/310) had positive corneal K-F rings, 8.2% (23/281) had abnormal MRI signals in the lenticular nucleus, and all of them had symptoms of damage in liver, kidney or nervous system. Compared with the group of symptomatic WD, asymptomatic group had higher levels of serum alanine aminotransferase and lower levels ceruloplasmin and 24-hour urine copper [(208±137) vs. (72±78) U/L, (55±47) vs. (69±48) mg/L, 103 (72, 153) vs. 492 (230, 1 432) μg; t=9.98, -1.98, Z=-4.89, all P<0.001]. Among the 314 patients completing genetic sequencing, a total of 107 mutations in ATP7B gene were detected, of which 10 are novel variants, and 3 cases (1.0%) had large heterozygous deletion (exons 10 to exon 11) in ATP7B gene. The percentage of missense mutation in asymptomatic WD children was significantly higher than that in symptomatic WD (81.5% (422/518) vs. 69.1% (76/110), χ²=8.47, P<0.05). WD patients carrying homozygous variant of c.2 333G>T had significantly low levels of ceruloplasmin than those not carrying this variant ((23±5) vs. (61±48) mg/L, t=-2.34, P<0.001). Conclusions: The elevation of serum ALT is an important clue for early diagnosis of WD in children, while serum ceruloplasmin and 24-hour urine copper content are specific markers for early diagnosis of WD. In order to confirm the diagnosis of WD, it is necessary to combine the Sanger sequencing with multiplex ligation-dependent probe amplification or other testing technologies.

Effect of Bushen Tongluo Prescription on Synaptic Plasticity of Hippocampal Neurons of Vascular Dementia Model Rats Based on PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

Objective:To explore the effect of Bushen Tongluo prescription （BSTLP） on the synaptic plasticity of hippocampal neurons in vascular dementia （VD） model rats and its mechanism. Method:SD male rats of SPF grade were selected. The rat model of VD was established by permanent bilateral ligation of the common carotid artery several times. The model rats were randomly divided into a model group， an insulin-like growth factor-1 （IGF-1， 20 μg·kg^-1） group， high-dose （3 g·kg^-1）， medium-dose （1.5 g·kg^-1）， and low-dose （0.75 g·kg^-1） BSTLP groups. A sham operation group was also set. Drugs were administered to rats by gavage once a day for four weeks. The model group and the sham operation group received the same volume of normal saline. After the last administration， all the rats were detected for spatial learning and memory by the Morris water maze. The apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase （TdT） dUTP nick-end labeling （TUNEL） assay. The changes in synaptic morphological structure and the number of dendritic spines in hippocampal neurons were detected by Golgi's method. The expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， mammalian target of rapamycin （mTOR）， synaptophysin （SYP）， and amyloid precursor protein （APP） in hippocampal neurons were detected by Western blot. Result:Compared with the sham operation group， the model group showed prolonged escape latency， lengthened swimming distance， dwindled the number of times for the platform crossing after platform removal （<italic>P</italic><0.05）， increased apoptotic cells （<italic>P</italic><0.05）， declining synaptic dendritic spines （<italic>P</italic><0.05）， down-regulated expression levels of PI3K， Akt， mTOR， and SYP proteins， and up-regulated expression level of APP protein in hippocampal neurons （<italic>P</italic><0.05）. Compared with the model group， the BSTLP groups and the IGF-1 group showed shortened escape latency and swimming distance， increased number of times for the platform crossing after platform removal （<italic>P</italic><0.05），declining apoptotic cells （<italic>P</italic><0.05）， up-regulated expression levels of PI3K， Akt， mTOR， and SYP proteins， and down-regulated expression level of APP protein in hippocampal neurons （<italic>P</italic><0.05）. Compared with the IGF-1 group， the high-dose BSTLP group showed no significant difference in the escape latency， swimming distance， the number of times for the platform crossing after platform removal， apoptotic cells， synaptic dendritic spines， and expression levels of PI3K， Akt， mTOR， SYP， and APP proteins in hippocampal neurons. However， the differences were significant in the medium-dose and low-dose BSTLP groups （<italic>P</italic><0.05）. Conclusion:BSTLP can improve the learning and memory of rats with VD. The mechanism is presumedly related to the activation of thePI3K/Akt/mTOR pathway and improvement of synaptic plasticity of hippocampal neurons.

Preparation and properties of extracellular vesicle-like nanoparticles from medicinal and edible plants / 药学学报

Extracellular vesicle-like nanoparticles (EVNs) isolated from edible plants have been shown to have multiple activities, while EVNs from medicinal plants have rarely been reported. In this paper, medicinal parts of medicinal and edible homologous fresh Curcumae Longae Rhizoma (CLR), Lilii Bulbus (LB), Polygonati Rhizoma (PR), and Gastrodiae Rhizoma (GR) are used to squeeze juice to collect EVNs. The physical and chemical properties, antioxidant capacity, and cellular uptake behavior of EVNs are determined. The results show that the particle size of EVNs from different sources ranges from 150 nm to 200 nm, and the polydispersity index (PDI) values of four EVNs are less than 0.2. Different EVNs all contain lipids, proteins, and carbohydrates, but their contents are different. The stability of EVNs is different at 4 ℃ and -80 ℃, among which the CLR-derived EVNs are most stable. Antioxidant experiments confirm that the four EVNs have different antioxidant activities while structural damage of EVNs leads to the reduced antioxidant capacity. Cellular uptake studies prove that four EVNs differ in the uptake capacity by RAW264.7 cells, which is associated with the structural interference of EVNs. The available evidence implies that the specific structure of EVNs may be necessary to their pharmacological activity and transport property.

Research progress of plant-derived vesicles and their biomedical applications / 药学学报

Plant-derived extracellular vesicles (EVs) are membranous vesicles secreted by plants, which include lipid bilayer as the basic framework and encapsulate various proteins, nucleic acid and other active substances. They play an important role in plant growth and development, tissue repair and self-defense. In recent years, extracellular vesicle-like nanoparticles (EVNs) are prepared from plant samples referring to the separation method of EVs and show unique functions. In this review, the above structures are collectively called plant-derived vesicles (PDVs). The biogenesis, separation and characterization methods, in vivo and in vitro properties of PDVs have been reviewed. The biomedical applications of PDVs as natural therapeutic agents and functional drug carriers are described, and finally some opinions on the existing problems and future prospect in this field are put forward.

mV, respectively, and the encapsulation efficiency and drug loading content were 87.9% and 0.57%. The final concentration of LM was 63.4 μg/mL. The release results showed 58.59% drug was released in 12 h. MTT results showed that the IC50 of LM@Lip on HepG2 and A549 cells was 20.16 and 15.39 μg/mL, respectively, and its in vitro antitumor was superior to that of LM. Conclusion Liposomes can increase the stability and solubility of LM. LM@Lip showed slow-release profile and significant tumor inhibition superior to LM. Key words: limonin; liposomes; thin-film dispersion method; orthogonal experiment; in vitro release; antitumor; druggability / 中草药

Objective: In view of druggability issue of limonin (LM), the liposomal preparation was developed. The liposomal formulation and preparation process were optimized, and its in vitro antitumor activity was investigated. Methods: In this study, LM was loaded in liposomes to increase its stability and solubility. Meanwhile, in vitro cytotoxicity of LM@Lip was evaluated. LM@Lip were prepared by thin-film dispersion method, and formulation selection and process optimization were operated by single factor and orthogonal experiment. Size distribution, PDI and zeta potential were measured by Malvern sizer, and the encapsulation efficiency and drug loading content were determined by HPLC. The dialysis method was used to investigate the release profile of LM@Lip. In vitro cytotoxicity against HepG2 and A549 cells were estimated by MTT method. Results: The optimized preparation conditions of liposomes were as follows: drug/lipid ratio was 1:150, cholesterol/lipid ratio was 1:9, the ultrasonic power was 120 W for 6 min (1 s interval). The average particle size, PDI and Zeta potential of optimized LM@Lip were (119.5 ± 6.2) nm, 0.318 ± 0.124, (-17.2 ± 1.3) mV, respectively, and the encapsulation efficiency and drug loading content were 87.9% and 0.57%. The final concentration of LM was 63.4 μg/mL. The release results showed 58.59% drug was released in 12 h. MTT results showed that the IC50 of LM@Lip on HepG2 and A549 cells was 20.16 and 15.39 μg/mL, respectively, and its in vitro antitumor was superior to that of LM. Conclusion: Liposomes can increase the stability and solubility of LM. LM@Lip showed slow-release profile and significant tumor inhibition superior to LM.