1.Effect of total secondary ginsenosides on apoptosis and energy metabolism of H9c2 cells under hypoxia based on mitochondrial biogenesis.
Zhong-Jie YUAN ; Yue XIAO ; Zhen LIU ; Ai-Qun ZHANG ; Bin LI ; Shang-Xian GAO
China Journal of Chinese Materia Medica 2025;50(5):1255-1266
This study explores the effect of total secondary ginsenosides(TSG) on apoptosis and energy metabolism in H9c2 cells under hypoxia and its potential mechanisms. H9c2 cell viability was observed and the apoptosis rate was calculated to determine suitable intervention concentrations of TSG, antimycin A complex(AMA), and coenzyme Q10(CoQ10), along with the duration of hypoxia. H9c2 cells at the logarithmic phase were divided into a normal group, a model group, a TSG group, an AMA group, a TSG+AMA group, and a CoQ10 group. All groups, except the normal group, were treated with their respective intervention drugs and cultured under hypoxic conditions. Adenosine triphosphate(ATP) content and creatine kinase(CK) activity were measured using an ATP chemiluminescence assay kit and a CK colorimetric assay kit. Flow cytometry was used to assess apoptosis rates, and Western blot evaluated the expression levels of apoptosis-related proteins, including B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), cysteinyl aspartate-specific protease(caspase)-3, caspase-8, and caspase-9, as well as mitochondrial biogenesis-related proteins peroxisome proliferator-activated receptor-γ coactivator 1α(PGC-1α), estrogen-related receptor-α(ERRα), nuclear respiratory factor(NRF)-1, NRF-2, peroxisome proliferator activated receptor-α(PPARα), and Na~+-K~+-ATPase. RT-PCR was employed to analyze the mRNA expression of mitochondrial biogenesis factors, including PGC-1α, ERRα, NRF-1, NRF-2, PPARα, mitochondrial transcription factor A(TFAM), mitochondrial cytochrome C oxidase 1(COX1), and mitochondrial NADH dehydrogenase subunit 1(ND1), ND2. The selected intervention concentrations were 7.5 μg·mL~(-1) for TSG, 10 μmol·L~(-1) for AMA, and 1×10~(-4) mol·L~(-1) for CoQ10, with a hypoxia duration of 6 h. Compared with the normal group, the model group showed decreased ATP content and CK activity, increased apoptosis rates, decreased Bcl-2 expression, and increased Bax, caspase-3, caspase-8, and caspase-9 expression in H9c2 cells. Additionally, the protein and mRNA expression levels of mitochondrial biogenesis-related factors(PGC-1α, ERRα, NRF-1, NRF-2, PPARα), mRNA expression of TFAM, COX1, and ND1, ND2, and protein expression of Na~+-K~+-ATPase in mitochondrial DNA, were also reduced. In the TSG and CoQ10 groups, ATP content and CK activity increased, and apoptosis rates decreased compared with those in the model group. The TSG group showed decreased protein expression of apoptosis-related proteins Bax, caspase-3, caspase-8, and caspase-9, increased protein and mRNA expression of mitochondrial biogenesis factors PGC-1α, ERRα, NRF-1, and PPARα, and increased NRF-2 protein expression and TFAM mRNA expression in mitochondrial DNA. Conversely, in the AMA group, ATP content and CK activity decreased, the apoptosis rate increased, Bcl-2 expression decreased, and Bax, caspase-3, caspase-8, and caspase-9 expression increased, alongside reductions in PGC-1α, ERRα, NRF-1, NRF-2, PPARα protein and mRNA expression, as well as TFAM, COX1, ND1, ND2 mRNA expression and Na~+-K~+-ATPase protein expression. Compared with the TSG group, the TSG+AMA group exhibited decreased ATP content and CK activity, increased apoptosis rates, decreased Bcl-2 expression, and increased Bax, caspase-3, caspase-8, and caspase-9 expression, along with decreased PGC-1α, ERRα, NRF-1, NRF-2, and PPARα protein and mRNA expression and TFAM, COX1, and ND1, ND2 mRNA expression. Compared with the AMA group, the TSG+AMA group showed increased CK activity, decreased apoptosis rate, increased Bcl-2 expression, and decreased Bax, caspase-8, and caspase-9 expression. Additionally, the protein and mRNA expression of PGC-1α, ERRα, NRF-1, PPARα, mRNA expression of TFAM, COX1, ND1, ND2, and Na~+-K~+-ATPase protein expression increased. In conclusion, TSG enhance ATP content and CK activity and inhibit apoptosis in H9c2 cells under hypoxia, and the mechanisms may be related to the regulation of PGC-1α, ERRα, NRF-1, NRF-2, PPARα, and TFAM expression, thus promoting mitochondrial biogenesis.
Apoptosis/drug effects*
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Ginsenosides/pharmacology*
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Energy Metabolism/drug effects*
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Mitochondria/metabolism*
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Animals
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Rats
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Cell Line
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Cell Hypoxia/drug effects*
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Organelle Biogenesis
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Adenosine Triphosphate/metabolism*
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Humans
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Cell Survival/drug effects*
2.Chinese Medicine for Treatment of COVID-19: A Review of Potential Pharmacological Components and Mechanisms.
Qian-Qian XU ; Dong-Dong YU ; Xiao-Dan FAN ; He-Rong CUI ; Qian-Qian DAI ; Xiao-Ying ZHONG ; Xin-Yi ZHANG ; Chen ZHAO ; Liang-Zhen YOU ; Hong-Cai SHANG
Chinese journal of integrative medicine 2025;31(1):83-95
Coronavirus disease 2019 (COVID-19) is an acute infectious respiratory disease that has been prevalent since December 2019. Chinese medicine (CM) has demonstrated its unique advantages in the fight against COVID-19 in the areas of disease prevention, improvement of clinical symptoms, and control of disease progression. This review summarized the relevant material components of CM in the treatment of COVID-19 by searching the relevant literature and reports on CM in the treatment of COVID-19 and combining with the physiological and pathological characteristics of the novel coronavirus. On the basis of sorting out experimental methods in vivo and in vitro, the mechanism of herb action was further clarified in terms of inhibiting virus invasion and replication and improving related complications. The aim of the article is to explore the strengths and characteristics of CM in the treatment of COVID-19, and to provide a basis for the research and scientific, standardized treatment of COVID-19 with CM.
Humans
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Drugs, Chinese Herbal/pharmacology*
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COVID-19 Drug Treatment
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SARS-CoV-2/drug effects*
;
COVID-19/therapy*
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Medicine, Chinese Traditional/methods*
;
Antiviral Agents/pharmacology*
;
Animals
3.Effect of Kuanxiong Aerosol on Perioperative Coronary Microcirculation in Patients with Unstable Angina Undergoing Elective PCI: A Pilot Randomized Controlled Trial.
Zi-Hao LIU ; Wen-Long XING ; Hong-Xu LIU ; Ju-Ju SHANG ; Ai-Yong LI ; Qi ZHOU ; Zhen-Min ZHANG ; Zhi-Bao LI ; Ke-Ji CHEN
Chinese journal of integrative medicine 2025;31(3):206-214
OBJECTIVE:
To evaluate the immediate effect of Kuanxiong Aerosol (KXA) on perioperative coronary microcirculation in patients with unstable angina (UA) suffering from elective percutaneous coronary intervention (PCI).
METHODS:
From February 2021 to July 2023, UA inpatients who underwent PCI alone in the left anterior descending (LAD) branch were included. Random numbers were generated to divide patients into the trial group and the control group at a ratio of 1:1. The index of coronary microcirculation resistance (IMR) was measured before PCI, and the trial group was given two sprays of KXA, while the control group was not given. IMR was measured again after PCI, cardiac troponin I (cTnI) and creatine kinase isoenzyme-MB (CK-MB) were detected before and 24 h after surgery, and major cardiovascular adverse events (MACEs) were recorded for 30 days. The data statistics and analysis personnel were blinded.
RESULTS:
Totally 859 patients were screened, and 62 of them were involved into this study. Finally, 1 patient in the trial group failed to complete the post-PCI IMR and was excluded, 30 patients were included for data analysis, while 31 patients in the control group were enrolled in data analysis. There was no significant difference in baseline data (age, gender, risk factors, previous history, biochemical index, and drug therapy, etc.) between the two groups. In addition, differences in IMR, cTnI and CK-MB were not statistically significant between the two groups before surgery. After PCI, the IMR level of the trial group was significantly lower than that of the control group (19.56 ± 14.37 vs. 27.15 ± 15.03, P=0.048). Besides, the incidence of perioperative myocardial injury (PMI) was lower in the trial group, but the difference was not statistically significant (6.67% vs. 16.13%, P=0.425). No MACEs were reported in either group.
CONCLUSIONS
KXA has the potential of improving coronary microvascular dysfunction. This study provides reference for the application of KXA in UA patients undergoing elective PCI. (Registration No. ChiCTR2300069831).
Humans
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Percutaneous Coronary Intervention
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Male
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Microcirculation/drug effects*
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Female
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Angina, Unstable/physiopathology*
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Pilot Projects
;
Middle Aged
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Aged
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Drugs, Chinese Herbal/pharmacology*
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Aerosols
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Troponin I/blood*
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Coronary Circulation/drug effects*
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Elective Surgical Procedures
4.Analysis of influencing factors of perioperative ischemic stroke in non-cardiac and non-neurosurgical surgeries
Ya-Zhen BAI ; Tong-Tong ZHENG ; Meng-Nan FAN ; Yi-Ru SHANG ; Gan-Qin DU ; Qi-Zhi FU
Medical Journal of Chinese People's Liberation Army 2024;49(10):1117-1122
Objective To explore the incidence and risk factors of perioperative ischemic stroke in non-cardiac and non-neurosurgical surgeries and its correlation with preoperative risk assessment of cerebrovascular events,so as to guide perioperative risk management.Methods A retrospective study was conducted on 40 patients aged≥18 years who underwent non-cardiac and non-neurosurgical surgeries and experienced perioperative ischemic stroke in the First Affiliated Hospital of Henan University of Science and Technology from January 2015 to January 2022,forming the stroke group.A control group of 160 patients without perioperative ischemic stroke was selected in a 1:4 case-control ratio,matched for gender,age,date of operation,and the surgeon.Clinical data and preoperative risk assessment of cerebrovascular events(including the single or combined application of head CT/MRI,transcranial Doppler ultrasound,carotid ultrasound,and neurological consultation)of the two groups of patients were collected and statistically analyzed.Multiple logistic regression analysis was used to identify risk factors associated with perioperative ischemic stroke.Results The incidence of perioperative ischemic stroke was 0.042%.Multiple logistic analysis results showed that hypertension(OR=7.858,95%CI 2.175-28.388,P=0.002),hyperlipidemia(OR=4.457,95%CI 1.320-15.049,P=0.016),renal insufficiency(OR=8.277,95%CI 1.480-46.282,P=0.016),and intraoperative hypotension(OR=3.862,95%CI 1.211-12.317,P=0.022)were independent risk factors for perioperative ischemic stroke in non-cardiac and non-neurological surgeries;preoperative cerebrovascular risk assessment(OR=0.130,95%CI 0.031-0.542,P=0.005)was a protective factor against it.Conclusions The incidence of perioperative ischemic stroke in non-cardiac and non-neurosurgical surgery is low but has a poor prognosis.Hypertension,hyperlipidemia,renal insufficiency,and postoperative hypotension are risk factors for perioperative ischemic stroke,while preoperative cerebrovascular event risk assessment is beneficial to reducing its incidence.
5.Development and validation of predictive model for 28-day mortality in very older patients with sepsis
Qiujing LI ; Na SHANG ; Zhen WANG ; Tiecheng YANG ; Shubin GUO
Chinese Journal of Emergency Medicine 2024;33(4):542-548
Objective:To develop and validate a predictive nomogram for 28-day mortality among very older patients with sepsis, to identify high-risk patients early and improve prognosis.Methods:This study was conducted from January 1, 2022, to November 30, 2022. Very older patients aged≥80 years with sepsis admitted to the emergency department of Beijing Chao-Yang Hospital, Capital Medical University were consecutively recruited. Their clinical data within 24 h of admission and 28-day mortality was recorded. The participants were divided into training (70%) and validation cohort (30%) (random number). In the training cohort, the risk factors of 28-day mortality were selected via least absolute shrinkage and selection operator (LASSO) regression analysis and multivariable Cox proportional hazard model, and a nomogram was developed. The prediction model was verified in validation cohort.Results:In total, 507 very older patients with sepsis were included, among which the mortality rate was 31.2%. In training cohort, the independent risk factors for 28-day mortality were identified: increased age [hazard ratio ( HR)=1.059, 95% confidence interval (95% CI)=1.017-1.103, P=0.005], cognitive impairment ( HR=2.100, 95% CI=1.322-3.336, P=0.002), frailty ( HR=2.561, 95% CI=1.183-5.545, P=0.017), decreased mean arterial pressure ( HR=0.987, 95% CI=0.976-0.998, P=0.017), decreased prealbumin ( HR=0.997, 95% CI=0.994-1.000, P=0.040), increased blood urea nitrogen ( HR=1.028, 95% CI=1.010-1.045, P=0.001), increased procalcitonin ( HR=1.008, 95% CI=1.001-1.016, P=0.019) via LASSO regression analysis and multivariable Cox regression analysis. The nomogram was developed using these seven predictors. In the training and validation cohorts, the calibration curves, time-dependent AUC curves, and decision curve analysis showed that the nomogram had good calibration degree, discrimination and clinical net benefits. Conclusions:Increased age, cognitive impairment, frailty, decreased mean arterial pressure, decreased prealbumin, increased blood urea nitrogen, and increased procalcitonin are independent risk factors for 28-day mortality in very older patients with sepsis. The nomogram, which included the seven predictors, have good predictive performance, and might be helpful for prognosis assessment.
6.Design of GIS-based 3D playback system for flight human-plane data
La-Mei SHANG ; Yu-Fei QIN ; Wen WANG ; Wan-Qi LI ; Da-Long GUO ; Xiao-Chao GUO ; Juan LIU ; Zhen TIAN ; Ting-Ting CUI ; Yu-Bin ZHOU
Chinese Medical Equipment Journal 2024;45(10):14-19
Objective To develop a GIS-based 3D playback system for the flight human-plane data to realize the fusion of pilots'airborne flight data and physiological data.Methods The 3D playback system was developed with the Browser/Server(B/S)architecture,micro-server model,Java language and Spring Cloud technology framework,which was composed of three functional modules for flight process reproduction,physiological situational awareness and critical event calibration analysis.Results The system developed achieved time synchronization and data fusion of airborne flight data and physiological data with a time synchronization frequency of 1 Hz and a refresh rate of not less than 120 frames/s.Conclusion The system developed with high safety,stability,reliability and accuracy facilitates pilot in-flight physiological monitoring and fusion and simultaneous display of airborne flight data and physiological data,which can be used as an important platform for decision-making support in flight training.[Chinese Medical Equipment Journal,2024,45(10):14-19]
7.Comparison of the effects of CR and PS prostheses in the treatment of knee osteoarthritis
Shangz-Eng WANG ; Bei ZHANG ; Zhen WANG ; Shang MA ; Deyang RUANGZHANG ; Zhiying YIN ; Yunqi ZHU ; Kunpeng HU ; Shao CHENG
The Journal of Practical Medicine 2024;40(9):1251-1256
Objective To compare the effect of posterior cruciate ligament retaining(CR)and posterior cruciate substituting(PS)prosthesis in knee arthroplasty.Methods 64 patients with knee osteoarthritis were admitted to our department from June 2021 to June 2022 and whom met the selection criteria were randomly selected and divided into observation group and control group(n = 32).CR prosthesis was used in the observation group,and PS prosthesis was used in the control group.Operation time,postoperative drainage volume,difference of hemoglobin(HGB)before and after operation,postoperative hospital stay,postoperative range of motion at 7 d,14 d,45 d,3 months and 1 year were recorded.ROM,visual analog pain Scale(VAS),Hospital for special surgery(HSS)score,and maximum forward and backward displacement of knee joint and the regular radiographs were also recorded,too.Results The two groups were similar in terms of operation time,postoperative hospital stay,postoperative VAS score and maximum anterior and posterior displacement of knee joint,with P values greater than 0.05,which had no statistical significance.The postoperative drainage volume in the observation group was significantly less than that in the control group,with statistical significance(P<0.05).The HGB difference of observation group was significantly lower than that of control group,with statistical significance(P<0.05).There was no statistically significant differ-ence in ROM and HSS scores between the two groups before surgery;the ROM and HSS scores of the control group at 7,14 and 45 days after surgery was better than that of the observation group,with statistical significance(P<0.05);the ROM and HSS scores of the two groups at 3 months after surgery was not statistically significant.The ROM and HSS scores in the observation group was better than that in the control group at 6 months and 1 year after operation,and the difference was statistically significant(P<0.05).Conclusions CR prosthesis is superior to PS prosthesis in terms of intraoperative blood loss and post-operative drainage volume,which can reduce surgical risk.Both kinds of prosthesis can achieve good knee stability after surgery,while PS prosthesis has better early clinical effect,while CR prosthesis has better long-term clinical effect.
8.Effects of Aβ receptor PirB on mouse astrocyte proliferation and reactive astrogliosis
Yuan-Jie ZHAO ; Zhen-Jie TUO ; Pei-Jun SHANG ; Jin-Wen YANG ; Xiao-Hua ZHANG
Medical Journal of Chinese People's Liberation Army 2024;49(1):82-90
Objective To observe the effects of amyloid-β(Aβ)receptor PirB on mouse astrocyte proliferation and reactive astrogliosis in vitro.Methods Mouse primary astrocytes were cultured,and divided into control group,Aβ group,Aβ+0.2 μmol/L PEP group,Aβ+0.4 μmol/L PEP group,Aβ+Fluspirilene group,Aβ+GFP-LV group,and Aβ+mPirB-LV group.The mouse astrocytes were treated with soluble PirB extracellular peptide PEP or PirB inhibitor Fluspirilene,respectively,to inhibit endogenous PirB receptor,or overexpressed PirB gene via lentivirus transfection and then treated with Aβ1-42 oligomers.The proliferation of astrocytes was observed by RTCA and EdU methods,and the mRNA expression levels of S-100 calcium-binding protein B(S-100β),Vimentin,Nestin and amyloid precursor protein(APP)associated with reactive astrogliosis of astrocytes were observed by real-time PCR,and the expression level of glial fibrillary acid protein(GFAP)was detected by Western-blotting.Results The results of RTCA monitoring showed that normalized cell index(NCI)values of each group decreased sharply after treatment,and then increased gradually and tended to be stable.The results of EdU staining showed that the proliferative activity of astrocytes was significantly enhanced in the Aβ group(P<0.05)compared with control group;Compared with Aβ group,cell proliferation activity in Aβ + 0.2 μmol/L PEP group,Aβ+0.4 μmol/L PEP group and Aβ+Fluspirilene group were significantly decreased(P<0.01 or P<0.001).The results of real-time PCR showed that compared with control group,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβ group were significantly increased(P<0.05);Compared with Aβ group,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβ+0.4 μmol/L PEP group were significantly decreased(P<0.01);Compared with Aβ+GFP-LV group,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβ +mPirB-LV group were significantly increased(P<0.05).The results of Western blotting showed that compared with control group,the expression of GFAP in Aβ group was significantly increased(P<0.05);Compared with Aβ group,the expression of GFAP in Aβ+0.4 μmol/L PEP group was significantly decreased(P<0.05).Conclusions PirB is an upstream molecule which could regulate astrocyte proliferation and reactive astrogliosis,and inhibiting PirB receptor in astrocytes may be a potential treatment for Alzheimer's disease.
9.Establishment and application for evaluation system of pathogen detection rate indicators before antimicrobial treatment
Lei-Lei GUO ; Hong-Ying QIN ; Sai-Nan XU ; Zhen-Zhen WU ; Yi ZHANG ; Shang-Shu ZHANG ; Hong-Kai LIAN
Chinese Journal of Infection Control 2024;23(6):712-718
Objective To establish an evaluation system of pathogen detection rate of hospitalized patients before antimicrobial treatment based on process and outcome indicators,and study its application effect.Methods Hospi-talized patients with therapeutic antimicrobial treatment in a tertiary first-class hospital from July 2022 to June 2023 were selected as the study objects.Difference in process and outcome indicators were compared between before(Ju-ly-December 2022,control group)and after(January-June 2023,intervention group)the implementation of inter-vention in the evaluation system for pathogen detection rate of hospitalized patients before antimicrobial treatment.Results 41 577 and 27 052 hospitalized patients received therapeutic antimicrobial agents were included in the inter-vention group and control group,respectively.Pathogen detection rate and the accurate pathogen detection rate in the intervention group were both higher than those in the control group(74.2%vs 62.8%and 90.3%vs 82.5%,respectively),both with statistically significant differences(both P<0.001).The goal achievement rate and im-provement rate for pathogen detection rate were 237.5%and 18.2%respectively,the goal achievement rate and im-provement rate for accurate pathogen detection rate were 104.0%and 9.5%respectively.For the process indica-tors,the conformity rate of detection and diagnosis,accurate collection rate,timely transfer rate,acceptance rate of pathogen specimens in the intervention group were all higher than those in the control group,differences were all statistically significant(all P<0.001).For the outcome indicators,detection rates of targeted indicators and blood culture specimens,pathogen detection rate before treatment of restricted and special grade antimicrobial agent,as well as detection rate of sterile body fluid specimens in the intervention group were all higher than those in the con-trol group,with statistical significance(all P<0.05).In addition,the positive rate of blood culture specimens in-creased from 18.5%before intervention to 21.7%after intervention,with statistically significant difference(P<0.05).Conclusion The establishment of indicator evaluation system can improve the detection rate and accurate detection rate of pathogens before antimicrobial treatment,as well as the management quality and connotation of indicators.
10.Analysis of prenatal ultrasound signs and prevention strategies for missed diagnosis and misdiagnosis in Apert syndrome
Huijuan LIN ; Limin WANG ; Li ZHENG ; Chaoxiang YANG ; Ning SHANG ; Zhen XIAO ; Fangfang XU
Chinese Journal of Ultrasonography 2024;33(9):784-790
Objective:To summarize the key prenatal ultrasound diagnosis features of Apert syndrome, analyze the causes of missed diagnosis and misdiagnosis, and propose corresponding preventive strategies.Methods:A retrospective analysis was made on the medical records and prenatal ultrasound images of 15 fetuses (including 14 cases referred from other hospitals) who underwent prenatal ultrasound examination in Guangdong Women and Children Hospital from August 2014 to May 2022 and were eventually clinically confirmed as Apert syndrome by induction or after birth. By conducting a comparative analysis, particularly focusing on the initial and final diagnoses of referral cases, the key ultrasound diagnostic points of Apert syndrome and the causes for missed and misdiagnosis were summarized.Results:①Diagnostic accuracy: Among the 15 fetuses, 11 cases (73.3%) were correctly diagnosed by prenatal ultrasound and 4 cases (26.7%) were missed diagnosis and misdiagnosis. For the 14 referral cases, only 2 cases (14.3%) were correctly identified in the initial diagnosis at the referring hospital (14.3%), and 12 cases (85.7%) were missed missed diagnosis and misdiagnosis. ②Detection rate of ultrasound signs: In the 15 fetuses with Apert syndrome, the detection rate of " cloverleaf" skull was 13.3% (2/15), premature coronal suture was 66.7% (10/15), the " brain shadowing sign" and flat occiput were both 93.3% (14/15), prominent forehead, hypertelorism and bilateral syndactyly of hands were all 100% (15/15), and bilateral syndactyly of feet was 73.3% (11/15). ③Analysis of missed diagnosis and misdiagnosis: Among the 4 cases of missed diagnosis and misdiagnosis in our hospital, premature closure of coronal suture, " brain shadowing sign", flat occiput and hypertelorism were all not recognized. Among these, 3 cases also missed the prominent forehead, bilateral syndactyly of hands and feet. Additionally, 1 case of bilateral syndactyly of hands was misdiagnosed as partial absence of metacarpals and phalangess.Conclusions:In the prenatal ultrasound diagnosis of fetal Apert syndrome, the symmetric syndactyly of both hands serves as an extremely important diagnostic clue. The " cloverleaf" skull is not common. The premature closure of coronal suture as a direct diagnostic sign with a high detection rate, highlighting its significance in the diagnostic of Apert syndrome. Furthermore, the high detection rates of characteristic ultrasound features such as prominent forehead, flat occiput, " brain shadowing sign" and hypertelorism could help to improve the accuracy of prenatal ultrasound diagnosis for Apert syndrome and effectively reduce missed and misdiagnosis.

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