OBJECTIVES: To investigate the molecular epidemiology of children with phenylketonuria (PKU) in Gansu, China, providing foundational data for intervention strategies. METHODS: A retrospective analysis was conducted on 1 159 PKU families who attended Gansu Provincial Maternity and Child Care Hospital from January 2012 to December 2024. Sanger sequencing, multiplex ligation-dependent probe amplification, whole exome sequencing, and deep intronic variant analysis were used to analyze the PAH gene. RESULTS: For the 1 159 children with PKU, 2 295 variants were identified in 2 318 alleles, resulting in a detection rate of 99.01%. The detection rates were 100% (914/914) in 457 classic PKU families, 99.45% (907/912) in 456 mild PKU families, and 96.34% (474/492) in 246 mild hyperphenylalaninemia families. The 2 295 variants detected comprised 208 distinct mutation types, among which c.728G>A (14.95%, 343/2 295) had the highest frequency, followed by c.611A>G (4.88%, 112/2 295) and c.721C>T (4.79%, 110/2 295). The cumulative frequency of the top 23 hotspot variants reached 70.28% (1 613/2 295), and most variant alleles were detected in exon 7 (29.19%, 670/2 295). CONCLUSIONS Deep intronic variant analysis of the PAH gene can improve the genetic diagnostic rate of PKU. The development of targeted detection kits for PAH hotspot variants may enable precision screening programs and enhance preventive strategies for PKU.
Humans ; Phenylketonurias/epidemiology* ; Female ; Male ; Retrospective Studies ; Phenylalanine Hydroxylase/genetics* ; Mutation ; Child, Preschool ; China/epidemiology* ; Child ; Infant

Objective To investigate the protective effects of secretomes released by three-dimensional cultured mesenchymal stem cells(MSCs)on neurons subjected to seawater immersion(SW)and stretch injury(SI),and to provide new insights into neuronal repair following SW combined with traumatic brain injury(TBI).Methods MSCs were cultured using the hanging drop method,and the conditioned medium(CM)containing MSCs secretomes was collected.A cellular model combining SW with SI was established using mouse hippocampal neuronal cells(HT22 cells).HT22 cells were randomly assigned to five groups:control,SI,SI+SW,SI+CM,and SI+SW+CM groups.Cell viability was assessed using the CCK-8 assay,apoptosis rate was measured by flow cytometry,cell migration ability was evaluated by scratch assay,and the expression levels of apoptosis-related proteins Bcl-2 and Bcl-2-associated protein(Bax),and ferroptosis-related proteins long-chain acyl-CoA synthetase 4(ACSL4)and cyclooxygenase-2(COX-2)were detected by Western blotting.Results Immersion in 15%seawater for 12 h significantly decreased HT22 cell viability(P<0.05).The CCK-8 assay indicated that cell viability in both the SI and SI+SW groups was significantly lower than that in control group after 12 h of treatment(P<0.05).Treatment with CM containing MSCs secretomes significantly increased cell viability in SI+CM group compared to SI group(P<0.0001),and in SI+SW+CM group compared to SI+SW group(P<0.001).Flow cytometry results revealed that the apoptosis rate in SI and SI+SW groups was significantly higher than that in control group(P<0.05 or P<0.001),while in SI+CM group was lower than that in SI group(P<0.05),and in SI+SW+CM group was lower than that in SI+SW group(P<0.05).Western blotting showed that compared to control group,SI and SI+SW groups exhibited reduced Bcl-2 expression level(P<0.01 or P<0.0001)and increased expression levels of Bax,ACSL4,and COX-2(P<0.01 or P<0.0001).Compared to SI group,the SI+CM group displayed increased Bcl-2 expression level(P<0.05)and decreased expression levels of Bax,ACSL4,and COX-2(P<0.05).Compared to SI+SW group,SI+SW+CM group exhibited increased Bcl-2 expression level(P<0.01)and decreased expression levels of Bax,ACSL4,and COX-2(P<0.01 or P<0.001).Scratch assay results demonstrated that at both 12 h and 24 h,the cell migration rate in SI and SI+SW groups was significantly lower than that in control group(P<0.01 or P<0.0001),while the migration rate in SI+CM group was significantly higher than that in SI group(P<0.0001 or P<0.01),and the migration rate in SI+SW+CM group was significantly higher than that in SI+SW group(P<0.0001).Conclusion Secretomes derived from MSCs cultured using the hanging drop method can alleviate neuronal damage caused by SW and TBI,potentially offering a therapeutic approach for SW combined with TBI.

Objective To investigate the clinical characteristics of patients with clinical and subclinical Cushing's syndrome caused by primary bilateral macronodular adrenal hyperplasia(PBMAH).Methods A retrospective analysis was performed on the clinical data of 198 patients with Cushing's syndrome caused by PBMAH diagnosed in the First Medical Center of Chinese PLA General Hospital from January 2004 to October 2024.According to clinical manifestations,the patients were classified into clinical type Cushing's syndrome(n=61)and subclinical type Cushing's syndrome(n=137),and the clinical characteristics of the two types were compared.Results The mean age at diagnosis of patients with PBMAH-induced Cushing's syndrome was(53.5±10.4)years,including 118 males and 80 females,with a male-to-female ratio of 1.475:1.Compared with the subclinical type,the clinical type had a higher proportion of females,higher levels of serum cortisol,24-hour urine free cortisol(24 h UFC),and inhibited serum cortisol after low-dose dexamethasone suppression.Additionally,the clinical type had lower plasma ACTH,larger adrenal nodules and a higher risk of surgery(P<0.05)compared with those in subclinical type.The incidences of hypertension,dyslipidemia,obesity,diabetes mellitus,hypokalemia,vitamin D deficiency,osteoporosis,coronary heart disease,and cerebrovascular disease in patients with Cushing's syndrome caused by PBMAH were 87.9%,50.5%,37.1%,36.9%,27.8%,25.9%,18.7%,18.7%and 12.1%,respectively.Among them,compared with subclinical type patients,clinical type patients had higher incidence of hypokalaemia,vitamin D deficiency and osteoporosis(P<0.05),while there were no statistically significant differences in the incidences of other comorbidities between the two types(P>0.05).The results of postoperative follow-up for PBMAH patients showed that the short-term biochemical remission rate of unilateral total adrenalectomy was 41.5%(22/53)and the long-term biochemical remission rate was 32.0%(8/25).The short-term biochemical remission rate of unilateral partial(or nodular)adrenalectomy was 52.9%(9/17),and the long-term biochemical remission rate was 14.3%(1/7).All patients who underwent unilateral total adrenalectomy plus contralateral partial resection developed adrenal insufficiency(3/3),and 1 patient(1/3)relapsed 3.4 years after surgery.Conclusion Clinical and subclinical types of Cushing's syndrome caused by PBMAH have their distinct clinical characteristics.Surgery is an effective treatment for PBMAH,but a certain proportion of patients fail to achieve biochemical remission after non-bilateral total adrenalectomy.

Objective To observe the effects of total saponins of Trillium tschonoskii Maxim(TST)on vascular cognitive impairment(VCI),neurovascular units(NVUs),and neural circuit integrity in rats.Methods Forty-eight male Sprague-Dawley(SD)rats were randomly divided into sham-operated group,model group,TST group(intragastric administration,100 mg/kg),and donepezil group(intragastric administration,0.45 mg/kg),and then subjected to ischemic stroke by 2-VO method(bilateral common carotid artery ligation)or sham surgery.After 28 days of intragastric administration,Mirros water maze test was performed to evaluate the spatial learning and memory abilities of rats in each group.HE and Nissl staining were used to observe the pathological changes of brain tissue in rats.The expression of synuclein(SYN)in rat hippocampus was observed by immunohistochemical staining.Changes in dendritic spines in rat's hippocampal neurons were observed by Golgi staining.Western blotting was used to detect the expression levels of IL-1β,IL-10,vascular endothelial growth factor A(VEGFA),postsynaptic density protein 95(PSD95),and growth associated protein 43(GAP43)in rat's hippocampus in each group.Results In Mirros water maze test,rats in model group showed significant prolonged escape latency(P＜0.05),and a significant reduction in the number of crossing platforms and the percentage of activity time in the target quadrant(P＜0.05)than those in sham-operated group;while rats in TST group and donepezil group showed significant shortened escape latency(P＜0.01),and significant increase of the number of times of crossing platforms and the percentage of activity time in the target quadrant(P＜0.05)than those in model group.Compared of sham-operated group,model group showed a decrease in the expression of SYN and the number of neurons,Nissl bodies,and dendritic spines in the CA1 region of the hippocampus(P＜0.05).Compared with model group,TST group and donepezil group showed an increase in the expression of SYN and the number of neurons,Nissl bodies,and dendritic spines in the CA1 region of the hippocampus(P＜0.05).Western blotting showed a significant increase in the expression of IL-1β and VEGF(P＜0.05),and a decrease in the expression of IL-10,PSD95,and GAP43(P＜0.01)in rat's hippocampus of model group than those in sham-operated group.Compared with model group,TST group and donepezil group showed a significant decrease in the expression of IL-1β(P＜0.05),and an increase in the expression of VEGFA,IL-10,and GAP43(P＜0.05).Conclusions TST could alleviate cognitive impairment through promoting synaptic plasticity and neurovascular unit remodeling in 2-VO model rats,suggesting its significance as a potential drug for apoplexy.

Aim This study aims to investigate the therapeutic effect and underlying mechanism of Todda-lia asiatica in the treatment of ischemic stroke(IS),utilizing network pharmacology,molecular docking technology,and animal experiments.Methods To screen the chemical components of Toddalia asiatica and its targets related to IS,a database was utilized.A protein-protein interaction(PPI)network was con-structed,followed by KEGG pathway enrichment anal-ysis.Molecular docking was performed to investigate the interaction between the components and target pro-teins.Finally,the effects of the drug on the PI3K/AKT/mTOR pathway and autophagy were validated through animal experiments.We established a middle cerebral artery occlusion(MCAO)rat model and di-vided the rats into the model group,Donepezil hydro-chloride group,Toddalia asiatica group,and sham op-eration group randomly.Observed the pathological changes in neurons of the rat hippocampal and cortical regions induced by the drug,performed immunohisto-chemical analysis to detect and localize mTOR expres-sion,and used Western blot to assess the expression levels of PI3K,p-PI3K,AKT,p-AKT,mTOR,as well as autophagy markers(LC3-Ⅱ and p62).Re-sults A total of 22 active ingredients from Toddalia asiatica,including AKT1 and MAPK3,were identified through screening.Additionally,194 signaling path-ways,such as PI3K/AKT and MAPK,were analyzed.The active compounds in Toddalia asiatica demonstra-ted stable binding affinity with targets associated with ischemic stroke.The results of the animal experiment indicated that,compared to the sham-operated group,the neuronal distribution in the hippocampal and corti-cal regions of the model group rats became sparser and more disorganized.There was a decrease in the number of Nissl bodies and cytoplasmic vacuolization.The ex-pression of mTOR-positive cells in the hippocampal and cortical regions was reduced.Additionally,the ex-pression levels of p-PI3K,p-AKT,mTOR,and p62 in the rat hippocampal tissue decreased(P＜0.05,P＜0.01),while the expression of LC3-Ⅱ increased(P＜0.01).Compared with the model group,the rats in the Toddalia asiatica and the Donepezil hydrochloride groups effectively improved the aforementioned indica-tors in rats.Conclusions Network pharmacology a-nalysis has revealed the promising potential of Toddalia asiatica in treating ischemic stroke,attributed to its di-verse components,targets,and pathways.The animal experiment showed that Toddalia asiatica can protect the neuronal structure in the hippocampal and cortical regions,which may be related to the inhibition of ex-cessive autophagy mediated by the PI3 K/AKT/mTOR pathway.

Aim To examine the neuroprotective im-pacts of total saponins from Trillium tschonoskii maxim(TST)on cerebral ischemia-reperfusion injury(CIRI)in rats and delve into the mechanisms of ferroptosis.Methods The CIRI model was prepared by dividing male SD rats into the model group,TST(0.1 g·kg-1)group,Donepezil hydrochloride(0.45 mg·kg-1)group,and sham group.The cognitive functions of rats in each group were assessed through the Morris water maze test,the changes in neurological function were evaluated using the Zea-Longa method,the infarct area was observed via TTC staining,and the pathologi-cal alterations in brain tissue were analysed using HE and Nissl staining.To further investigate the underly-ing mechanism,the mitochondrial structural changes were examined using transmission electron microscopy,and the levels of GSH-PX,MDA,and SOD were ana-lyzed.Additionally,the expressions of GPX4 and Nrf2 proteins were evaluated through immunohistochemistry and immunofluorescence.Furthermore,the protein lev-els of Keap1/Nrf2/HO-1 and Nrf2/SLC7A11/GPX4 pathways in rats were examined using Western blot-ting.Results The rats in the model group displayed diminished learning and memory capabilities in com-parison to those in the sham group,as well as a signifi-cantly increased cerebral infarction area and higher neurological function scores(P＜0.01),significantly increased cerebral infarct area,disordered and loosely arranged neurons,and reduced Nissl bodies.Addition-ally,mitochondria showed typical signs of ferroptosis.Changes related to ferroptosis included decreased activ-ities of SOD and GSH-PX(P＜0.01)and increased MDA levels(P＜0.01).The expression of GPX4 and Nrf2-positive cells was significantly reduced,along with decreased fluorescence intensity of GPX4.Further-more,the protein expression of Keap1,Nrf2,HO-1,GPX4,SLC7A11 in the hippocampus decreased(P＜0.05,P＜0.01).Following the administration of TST,these effects showed improvement.Conclusions TST has neuroprotective effects,enhancing learning and memory abilities while reducing oxidative stress levels.The mechanism may involve the inhibition of ferroptosis through the Keap-1/Nrf2/HO-1 and Nrf2/SLC7 A11/GPX4 pathways.

Objective To explore the relationship between sperm DNA fragmentation index(DFI)and sperm quality,as well as in vitro fertilization-embryo transfer(IVF-ET)preganancy outcomes in middle-aged men.Methods A total of 180 semen specimens from males aged over 38 years who received comventional IVF-ET treatment were collected and divided into two groups(＜30%and≥30%)based on the DIF threshold.Main measures result including:normal semen parameters,hormone levels,DFI,fertilization rate,good quality embryo rate and pregnancy rate.Results DFI was closely related to male follicle-stimulating hormone(FSH),sperm motility,morphology,and the sperm vitality decreased with increasing DFI level(P＜0.05).When DFI≥30%,the rate of high-quality embryos decreased(P＜0.05),but there was no significant difference in the pregnancy rate(P＞0.05).Conclusion DFI can serve as an important indicator for routine analysis of semen in middle-aged men.DFI affects the rate of high-quality embryos,but it is not related to the clinical pregnancy outcome of assistant reproduction technology(ART).

OBJECTIVE To evaluate the clinical effectiveness and safety of domestic generic and imported original clopidogrel for antiplatelet therapy in patients with acute coronary syndrome （ACS）. METHODS The clinical data of ACS patients in Nanjing Drum Tower Hospital of China Pharmaceutical University from January 2020 to June 2021 were retrospectively collected by using electronic medical record system， and the patients were divided into original drug group （321 cases） and generic drug group （328 cases） according to the drug use. Both groups were given dual antiplatelet therapy with clopidogrel and aspirin. The effectiveness and safety outcomes of the two groups were followed up for 12 months and compared， the related influential factors were analyzed. RESULTS Major adverse cardiovascular events （MACE） occurred in 16 and 22 patients in original drug group and generic drug group respectively， including nonfatal myocardial infarction （4 and 5 cases）， stroke （2 and 4 cases）， revascularization （8 and 3 cases）， cardiovascular related death （2 and 4 cases）， and all-cause death （4 and 6 cases）. There were 12 and 7 patients with major bleeding events， 38 and 29 patients with minor bleeding events， and 33 and 21 patients with non-bleeding adverse events. There was no statistically significant difference in the cumulative incidence of related events （P values of Log-Rank tests were all greater than 0.05）. Cox regression analysis showed that the use of generic clopidogrel did not increase the risk of MACE and major bleeding events in ACS patients [hazard ratio of 1.305 and 0.416， 95% confidence interval of （0.678， 2.512） and （0.155， 1.117）， respectively， P＞0.05]， and the combination of proton pump inhibitors （PPI） could reduce the risk of major bleeding events [hazard ratio of 0.196， 95% confidence interval of （0.063， 0.611）， P＜0.05]. CONCLUSIONS Compared with imported original drug， domestic generic clopidogrel has similar clinical effectiveness and good safety. Combined use of PPI may be a beneficial factor to reduce the occurrence of major bleeding events in patients.

AIM:Observation of neuroprotective effects of Toddalia asiatica(TA)on cerebral ischemia-reper-fusion injury(CIRI)in rats by investigating the effects and mechanisms of drugs on the polarization of microglia M1/M2 subtype and the Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB)path-way.METHODS:The modified thread occlusion method was used to establish a rat model of CIRI,and the rats were ran-domly divided into the model group,Toddalia asiatica(1.08 g/kg)group,donepezil hydrochloride(0.45 mg/kg)group,and sham group,with 16 rats in each group.Based on the assessment of neurofunctional changes in each group of rats,HE and Nissl staining were used to observe the pathological changes in brain tissue.TUNEL staining was performed to de-tect neuronal apoptosis,immunohistochemistry was used to detect the expression of M1 microglia marker ionized calcium-binding adapter molecule 1(Iba1),M2 microglia marker arginase 1(Arg1),and TLR4 localization.Western blot was used to detect the expression of microglia polarization proteins and proteins related to TLR4/MyD88/NF-κB pathway in the hippocampus region.RESULTS:Compared with sham group,the model group rats had higher neurological function scores(P＜0.01),and neuronal arrangement in the hippocampus and cortex was loose and disordered,Nissl bodies de-creased,and neuronal apoptosis increased.The numbers of M1 microglia marker Iba1-,M2 microglia marker Arg1-,and TLR4-positive cells were significantly increased.In addition,the protein levels of TLR4,MyD88,p-NF-κB p65,NF-κB p65,p-NF-κB inhibitory factor(p-IκB),Iba1,interleukin-6(IL-6),and Arg1 in the hippocampus were elevated(P＜0.05),while IL-4 and IL-10 expression were decreased(P＜0.01).Compared with model group,the Toddalia asiatica group and Donepezil hydrochloride group showed increased protein expression of Arg1,IL-4 and IL-10(P＜0.05),while the other indicators were decreased(P＜0.05,P＜0.01).CONCLUSION:Toddalia asiatica possesses neuroprotective effects on CIRI rats,which may be attributed to its ability to regulate M1/M2 polarization and inhibit the TLR4/MyD88/NF-κB-mediated inflammatory pathway.

Objective: To compare the effects and safety of dydrogesterone (DG) and medroxyprogesterone acetate (MPA) on the treatment in patients with endometrial hyperplasia without atypia (EH). Methods: This was a single-center, open-label, prospective non-inferior randomized controlled phase Ⅲ trial. From February 2019 to November 2021, patients with EH admitted to the Obstetrics and Gynecology Hospital of Fudan University were recruited. Enrolled patients were stratified according to the pathological types of simple hyperplasia (SH) or complex hyperplasia (CH), and were randomised to receive MPA or DG. Untill May 14, 2022, the median follow-up time after complete response (CR) was 9.3 months (1.1-17.2 months). The primary endpoint was the 6-month CR rate (6m-CR rate). The secondary endpoints included the 3-month CR rate (3m-CR rate), adverse events rate, recurrence rate, and pregnancy rate in one year after CR. Results: (1) A total of 292 patients with EH were enrolled in the study with the median age of 39 years (31-45 years). A total of 135 SH patients were randomly assigned to MPA group (n=67) and DG group (n=68), and 157 CH patients were randomly assigned to MPA group (n=79) and DG group (n=78). (2) Among 292 patients, 205 patients enrolled into the primary endpoint analysis, including 92 SH patients and 113 CH patients, with 100 patients in MPA group and 105 in DG group, respectively. The 6m-CR rate of MPA group and DG group were 90.0% (90/100) and 88.6% (93/105) respectively, and there were no statistical significance (χ²=0.11, P=0.741), with the rate difference (RD) was -1.4% (95%CI:-9.9%-7.0%). Stratified by the pathology types, the 6m-CR rate of SH patients was 93.5% (86/92), and MPA group and DG group were respectively 91.1% (41/45) and 95.7% (45/47); and the 6m-CR rate of CH patients was 85.8% (97/113), and MPA group and DG group were 89.1% (49/55) and 82.8% (48/58) respectively. The 6m-CR rates of the two treatments had no statistical significance either (all P>0.05). A total of 194 EH patients enrolled into the secondary endpoint analysis, including 88 SH patients and 106 CH patients, and 96 patients in MPA group and 98 in DG group, respectively. The 3m-CR rate of SH patients were 87.5% (77/88), while the 3m-CR rates of MPA group and DG group were 90.7% (39/43) and 84.4% (38/45), respectively; the 3m-CR rate of CH patients was 66.0% (70/106), and MPA group and DG group had the same 3m-CR rate of 66.0% (35/53). No statistical significance was found between the two treatments both in SH and CH patients (all P>0.05). (3) The incidence of adverse events between MPA group and DG group had no statistical significance (P>0.05). (4) A total of 93 SH patients achieved CR, and the cumulative recurrence rate in one year after CR were 5.9% and 0 in MPA group and DG group, respectively. While 112 CH patients achieved CR, and the cumulative recurrence rate in one year after CR were 8.8% and 6.5% in MPA group and DG group, respectively. There were no statistical significance between two treatment groups (all P>0.05). Among the 93 SH patients, 10 patients had family planning but no pregnancy happened during the follow-up period. Among the 112 CH patients, 21 were actively preparing for pregnancy, and the pregnancy rate and live-birth rate in one year after CR in MPA group were 7/9 and 2/7, while in DG group were respectively 4/12 and 2/4, and there were no statistical significance in pregnancy rate and live-birth rate between the two treatment groups (all P>0.05). Conclusions: Compared with MPA, DG is of good efficacy and safety in treating EH. DG is a favorable alternative treatment for EH patients.
Female ; Humans ; Adult ; Medroxyprogesterone Acetate/adverse effects* ; Endometrial Hyperplasia/pathology* ; Dydrogesterone/adverse effects* ; Hyperplasia ; Prospective Studies