1.Quality Evaluation and Analysis of Key Problems in Rukuaixiao Preparations
Rong CHEN ; Chao ZHANG ; Shuisheng ZHONG ; Wei CHEN ; Enyun TU ; Yaqiong WANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):146-155
ObjectiveBased on multi-index and multi-method detection, the quality of Rukuaixiao preparations was systematically evaluated from the perspective of characteristic components, and the existing problems were analyzed. MethodsLiquid chromatography-mass spectrometry(LC-MS) for the determination of 16 characteristic components was established to evaluate the quality of 129 batches of Rukuaixiao preparations. High performance liquid chromatography(HPLC) was established to determine the contents of salvianolic acids and tanshinones, investigate the rationality of quality control index of Salviae Miltiorrhizae Radix et Rhizoma in the standard for Rukuaixiao preparations in the 2020 edition of Pharmacopoeia of the People's Republic of China(hereinafter referred to as Chinese Pharmacopoeia) (volume Ⅰ), and trace the causes of significant difference among different batches. The processing and different extraction methods of Vaccariae Semen were tested, analyzing the impact of formulation changes across different editions of Chinese Pharmacopoeia. The LC-MS was established for determining the changes in the ratio of toosendanin and isotoosendanin after water extraction of Toosendan Fructus. The contents of active components in different parts of Gleditsiae Spina were determined to identify the reason of the low contents of characteristic components in some enterprises. ResultsBased on the comprehensive analysis of the samples from different dosage forms, the contents of characteristic components of Vaccariae Semen and Gleditsiae Spina in tablets from manufacturer B and granules from manufacturer D were significantly higher than those in tablets from manufacturer A, and tablets and capsules from manufacturer C. The contents of tanshinones in some batches of products from manufacturer A were abnormally high, potentially linked to the use of 70% ethanol reflux during extraction of Salviae Miltiorrhizae Radix et Rhizoma. All samples from manufacturer C exhibited abnormally high proportions of salvianic acid A and 3,4-dihydroxybenzaldehyde(salvianolic acid degradation products) to the total seven phenolic acids, indicating a risk of blindly pursuing tanshinol content and neglecting more effective components. The extraction rate of the characteristic components from Vaccariae Semen by decocting with water was obviously higher than that by reflux extraction with 70% ethanol. However, using the stir-fried Vaccariae Semen could reduce the loss of ingredients. The content ratio of toosendanin and isotoosendanin decreased from the crude herb to the prepared medicine when Toosendan Fructus was prepared by water decoction. The reason for the low component content of Gleditsiae Spina may be attributed to manufacturers using excessive non-medicinal parts in their formulations. ConclusionIt is suggested that enterprises should ensure the quality of raw material inputs, especially those without quality-control items in the standard, reduce the use of non-medicinal parts, and prohibit arbitrary alterations to manufacturing methods or processes. It is also recommended that Vaccariae Semen in Rukuaixiao capsules and granules should be changed to the stir-fried processed products. Isotoosendanin should be taken into consideration in the drug supervision when Toosendan Fructus is prepared into medicine by water decoction. Salvianolic acid B should be set as a quality control index for Salviae Miltiorrhizae Radix et Rhizoma when revising the pharmacopoeia standard of Rukuaixiao preparations.
2.Quality Evaluation and Analysis of Key Problems in Rukuaixiao Preparations
Rong CHEN ; Chao ZHANG ; Shuisheng ZHONG ; Wei CHEN ; Enyun TU ; Yaqiong WANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):146-155
ObjectiveBased on multi-index and multi-method detection, the quality of Rukuaixiao preparations was systematically evaluated from the perspective of characteristic components, and the existing problems were analyzed. MethodsLiquid chromatography-mass spectrometry(LC-MS) for the determination of 16 characteristic components was established to evaluate the quality of 129 batches of Rukuaixiao preparations. High performance liquid chromatography(HPLC) was established to determine the contents of salvianolic acids and tanshinones, investigate the rationality of quality control index of Salviae Miltiorrhizae Radix et Rhizoma in the standard for Rukuaixiao preparations in the 2020 edition of Pharmacopoeia of the People's Republic of China(hereinafter referred to as Chinese Pharmacopoeia) (volume Ⅰ), and trace the causes of significant difference among different batches. The processing and different extraction methods of Vaccariae Semen were tested, analyzing the impact of formulation changes across different editions of Chinese Pharmacopoeia. The LC-MS was established for determining the changes in the ratio of toosendanin and isotoosendanin after water extraction of Toosendan Fructus. The contents of active components in different parts of Gleditsiae Spina were determined to identify the reason of the low contents of characteristic components in some enterprises. ResultsBased on the comprehensive analysis of the samples from different dosage forms, the contents of characteristic components of Vaccariae Semen and Gleditsiae Spina in tablets from manufacturer B and granules from manufacturer D were significantly higher than those in tablets from manufacturer A, and tablets and capsules from manufacturer C. The contents of tanshinones in some batches of products from manufacturer A were abnormally high, potentially linked to the use of 70% ethanol reflux during extraction of Salviae Miltiorrhizae Radix et Rhizoma. All samples from manufacturer C exhibited abnormally high proportions of salvianic acid A and 3,4-dihydroxybenzaldehyde(salvianolic acid degradation products) to the total seven phenolic acids, indicating a risk of blindly pursuing tanshinol content and neglecting more effective components. The extraction rate of the characteristic components from Vaccariae Semen by decocting with water was obviously higher than that by reflux extraction with 70% ethanol. However, using the stir-fried Vaccariae Semen could reduce the loss of ingredients. The content ratio of toosendanin and isotoosendanin decreased from the crude herb to the prepared medicine when Toosendan Fructus was prepared by water decoction. The reason for the low component content of Gleditsiae Spina may be attributed to manufacturers using excessive non-medicinal parts in their formulations. ConclusionIt is suggested that enterprises should ensure the quality of raw material inputs, especially those without quality-control items in the standard, reduce the use of non-medicinal parts, and prohibit arbitrary alterations to manufacturing methods or processes. It is also recommended that Vaccariae Semen in Rukuaixiao capsules and granules should be changed to the stir-fried processed products. Isotoosendanin should be taken into consideration in the drug supervision when Toosendan Fructus is prepared into medicine by water decoction. Salvianolic acid B should be set as a quality control index for Salviae Miltiorrhizae Radix et Rhizoma when revising the pharmacopoeia standard of Rukuaixiao preparations.
3.Development status among infants at ages of 0 to 36 months in Xiaoshan District
LI Qing ; ZHONG Bihua ; SUN Jiarui ; DAI Fengpo ; DING Yina ; MIAO Xiangqing ; FU Yaxian ; TU Yuyan ; TAN Wenjuan ; YU Yinfei
Journal of Preventive Medicine 2024;36(3):255-259
Objective:
To learn the status and influencing factors of development among infants at ages of 0 to 36 months in Xiaoshan District, Hangzhou City, so as to provide the reference for promoting healthy development of infants.
Methods:
Infants at ages of 0-36 months who underwent physical examination in Child Health Clinic of Xiaoshan District Community Health Service Center were selected in 2022. General data of infants and their mothers were collected through questionnaires, and the development status of infants was screened by Age and Stages Questionnaire (third edition). Factors affecting the development status were identified using a multivariable logistic regression model.
Results:
A total of 2 519 infants were investigated, including 1 339 males (53.16%) and 1 180 females (46.84%). There were 608 infants with abnormal development of at least one functional area of communication (CM), gross motor (GM), fine motor (FM), problems solving (CG) and personal-social (PS). The abnormal rate was 24.14%, and the abnormal rates of the above functional areas were 9.77%, 6.59%, 7.98%, 6.39% and 9.33%, respectively. Multivariable logistic regression analysis showed that gender (male, OR=1.563, 95%CI: 1.191-2.052), mother's childbearing age (≥35 years, OR=1.411, 95%CI: 1.001-1.988), mother's educational level (lower than junior college, OR=1.460, 95%CI: 1.116-1.912) were factors affecting abnormal development of CM; preterm birth (OR=2.323, 95%CI: 1.315-4.103) was factors affecting abnormal development of GM; gender (male, OR=1.654, 95%CI: 1.225-2.232) was factors affecting abnormal development of FM; gender (male, OR=1.511, 95%CI: 1.086-2.102) and mode of delivery (cesarean section, OR=1.460, 95%CI: 1.060-2.010) were factors affecting abnormal development of CG; gender (male, OR=1.340, 95%CI: 1.019-1.763) and birth weight (low birth weight, OR=1.985, 95%CI: 1.149-3.432) were factors affecting abnormal development of PS.
Conclusions
The rate of abnormal development among infants at ages of 0 to 36 months in Xiaoshan District is 24.14%. Gender, preterm birth, mode of delivery, birth weight, mother's childbearing age and mother's educational level could affect the development status of infants.
4.Machine-learning-based models assist the prediction of pulmonary embolism in autoimmune diseases: A retrospective, multicenter study
Ziwei HU ; Yangyang HU ; Shuoqi ZHANG ; Li DONG ; Xiaoqi CHEN ; Huiqin YANG ; Linchong SU ; Xiaoqiang HOU ; Xia HUANG ; Xiaolan SHEN ; Cong YE ; Wei TU ; Yu CHEN ; Yuxue CHEN ; Shaozhe CAI ; Jixin ZHONG ; Lingli DONG
Chinese Medical Journal 2024;137(15):1811-1822
Background::Pulmonary embolism (PE) is a severe and acute cardiovascular syndrome with high mortality among patients with autoimmune inflammatory rheumatic diseases (AIIRDs). Accurate prediction and timely intervention play a pivotal role in enhancing survival rates. However, there is a notable scarcity of practical early prediction and risk assessment systems of PE in patients with AIIRD.Methods::In the training cohort, 60 AIIRD with PE cases and 180 age-, gender-, and disease-matched AIIRD non-PE cases were identified from 7254 AIIRD cases in Tongji Hospital from 2014 to 2022. Univariable logistic regression (LR) and least absolute shrinkage and selection operator (LASSO) were used to select the clinical features for further training with machine learning (ML) methods, including random forest (RF), support vector machines (SVM), neural network (NN), logistic regression (LR), gradient boosted decision tree (GBDT), classification and regression trees (CART), and C5.0 models. The performances of these models were subsequently validated using a multicenter validation cohort.Results::In the training cohort, 24 and 13 clinical features were selected by univariable LR and LASSO strategies, respectively. The five ML models (RF, SVM, NN, LR, and GBDT) showed promising performances, with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.962-1.000 in the training cohort and 0.969-0.999 in the validation cohort. CART and C5.0 models achieved AUCs of 0.850 and 0.932, respectively, in the training cohort. Using D-dimer as a pre-screening index, the refined C5.0 model achieved an AUC exceeding 0.948 in the training cohort and an AUC above 0.925 in the validation cohort. These results markedly outperformed the use of D-dimer levels alone.Conclusion::ML-based models are proven to be precise for predicting the onset of PE in patients with AIIRD exhibiting clinical suspicion of PE.Trial Registration::Chictr.org.cn: ChiCTR2200059599.
5.Application of nasal endoscopic Draf Ⅱ-Ⅲ frontal sinus surgery in recurrent frontal sinus infection and sinus formation after craniocerebral trauma
Cao LYU ; Xiaobin HUANG ; Jie CHEN ; Zhong LAN ; Yi TU ; Xiaohong YANG ; Zhong BAI
Chinese Archives of Otolaryngology-Head and Neck Surgery 2024;31(4):248-250
OBJECTIVE To investigate the application of endoscopic Draf Ⅱ-Ⅲ frontal sinus surgery in the treatment of recurrent frontal sinus infection and fistula formation after craniocerebral trauma.METHODS There were 8 cases of recurrent frontal sinus infection after craniocerebral trauma,the main manifestations were headache,recurrent frontal infection,discharge of pus,fistula formation.The average onset time was 43.25 months.The patients underwent DRAF Ⅱ-Ⅲ frontal sinus surgery under nasal endoscopy,including Draf Ⅱa 2,Draf Ⅱb 5,and Draf Ⅲ1,respectively.During the operation,the frontal sinus ostium was expanded.It was found that bone wax blocked the frontal sinus ostium in the frontal sinus.The bone wax was removed,and the frontal sinus drainage was smooth.No facial incision was made in all patients.RESULTS There were 8 patients with frontal infection who were cured after surgery.No cerebrospinal fluid rhinorrhea or intracranial infection occurred during or after operation.After discharge,the outpatient follow-up review was conducted in 1,3,6,and 12 months.It was found that the frontal sinus remained unobstructed.The frontal sinus did not become infected again,and the fistula gradually healed.CONCLUSION Draf Ⅱ-Ⅲ frontal sinus surgery under nasal endoscopy is an effective way to treat recurrent frontal sinus infection and fistula formation after craniocerebral trauma.
6.A multicenter study of neonatal stroke in Shenzhen,China
Li-Xiu SHI ; Jin-Xing FENG ; Yan-Fang WEI ; Xin-Ru LU ; Yu-Xi ZHANG ; Lin-Ying YANG ; Sheng-Nan HE ; Pei-Juan CHEN ; Jing HAN ; Cheng CHEN ; Hui-Ying TU ; Zhang-Bin YU ; Jin-Jie HUANG ; Shu-Juan ZENG ; Wan-Ling CHEN ; Ying LIU ; Yan-Ping GUO ; Jiao-Yu MAO ; Xiao-Dong LI ; Qian-Shen ZHANG ; Zhi-Li XIE ; Mei-Ying HUANG ; Kun-Shan YAN ; Er-Ya YING ; Jun CHEN ; Yan-Rong WANG ; Ya-Ping LIU ; Bo SONG ; Hua-Yan LIU ; Xiao-Dong XIAO ; Hong TANG ; Yu-Na WANG ; Yin-Sha CAI ; Qi LONG ; Han-Qiang XU ; Hui-Zhan WANG ; Qian SUN ; Fang HAN ; Rui-Biao ZHANG ; Chuan-Zhong YANG ; Lei DOU ; Hui-Ju SHI ; Rui WANG ; Ping JIANG ; Shenzhen Neonatal Data Network
Chinese Journal of Contemporary Pediatrics 2024;26(5):450-455
Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75%(27/36);boys accounted for 64%(23/36).Among the 36 neonates,31(86%)had disease onset within 3 days after birth,and 19(53%)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61%)had left cerebral infarction and 13(36%)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75%)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72%)and seizures in 10 neonates(34%).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33%,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44%(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.
7.Establishment of UPLC-DAD fingerprint of raw and vinegar Bupleurum bupleurum and study on spectral effect relationship of anti-hepatic fibrosis
Ni-Ping CHEN ; Yan WANG ; Yan DONG ; Yang-Xin XIAO ; Ji-Yuan TU ; Yan-Ju LIU ; Zhong-Shi ZHOU
Chinese Pharmacological Bulletin 2024;40(6):1145-1152
Aim To establish the fingerprint of raw bupleurum and vinegar bupleurum,investigate the difference in their anti-liver fibrosis effects,and ex-plore the relationship between the chemical composition of raw bupleurum and vinegar bupleurum and their an-ti-liver fibrosis efficacy.Methods The fingerprints of 10 batches of raw bupleuri and 10 batches of bupleuri were established by UPLC method.The liver fibrosis cell model in vitro was established by TGF-β induced LX-2 hepatic stellate cells.The liver fibrosis cell mod-el was analyzed with collagen type Ⅰ(col1a1)and α-smoothmuscleactin.The expression of α-SMA protein was used as the pharmacodynamic index.MetaboAna-lyst5.0 was used to screen the difference markers af-fecting the quality of raw bupledges and vinegar bu-pledges with VIP value>1 as the criterion.Orthogo-nal partial least squares discriminant analysis(OPLS-DA)was used to screen the main components of raw bupleurum and vinegar bupleurum against liver fibro-sis.Results There were 18 peaks in the UPLC fin-gerprints of raw bupleurum and vinegar bupleurum,and the analysis results showed that there were nine main differences between raw bupleurum and vinegar bupleurum,among which peaks 9,7 and 6 could be considered as bupleurin d,bupleurin a and bupleurin f.The results of spectral effect relationship showed that the main components of bupleurum anti-liver fibrosis were peaks 11,12,14,15 and 18.Conclusions The established fingerprint method of raw bupleurum and vinegar bupleurum is simple and feasible,and the important components of anti-liver fibrosis activity are screened through the spectrum effect relationship,which provides a basis for clarifying the material basis of anti-liver fibrosis effect of raw bupleurum and vine-gar bupleurum.
8.Clinical application of four-hook needle combined with holographic image in robot-assisted partial nephrectomy for completely intrarenal tumors
Qiubo XIE ; Yu ZHOU ; Lei GAO ; Zhong TU ; Jian SONG ; Renhao WANG ; Xiang LI ; Guan ZHANG ; Le ZHANG ; Tiejun PAN
Chinese Journal of Urology 2024;45(5):343-347
Objective:To investigate the safety and efficacy of four-hook needle combined with holographic image in robot-assisted partial nephrectomy for completely intrarenal tumors.Methods:The clinical data of 8 patients with completely intrarenal tumors treated by robot-assisted partial nephrectomy with four-hook needle combined with holographic image admitted to General Hospital of Central Theater Command from October 2023 to December 2023 were retrospectively analyzed. There were 6 males and 2 females, with average age of (44.5±12.0) years old. Tumors of 6 cases were in the left side and 2 cases in the right side. The maximum diameter of the tumor was (23.2±8.1) mm. The R. E.N.A.L. score was (9.0±1.4). The preoperative serum creatinine (Scr) was (73.1±14.7) μmol/L. CT-guided four-hook needle was used to locate the edge of completely intrarenal tumor before surgery. During the operation, the tumor was precisely resected under the guidance of hologram and four-hook needle. Perioperative data of patients were collected and analyzed.Results:All the tumor were successfully resected under the guidance of four-hook needle and hologram without conversion to radical nephrectomy or open surgery. The mean operative time was (117.0±14.5) min, the mean hot ischemia time was (20.2±5.1) min, the mean intraoperative blood loss was (75.0±17.3) ml, and the average hospitalization time was (9.5±1.3) days.The one week postoperative Scr was (73.2±14.8) μmol/L, which had no significant difference with that of before operation ( P=0.952). None of them received blood transfusion. The pathology results of 8 patients were clear cell renal cell carcinoma, and the surgical margins were negative. Conclusions:For completely intrarenal tumors, the four-hook needle combined with the hologram can guide the surgeon to quickly locate the tumor, accurately resect the tumor, reduce perioperative complications, and is safe and effective.
9.Clinical treatment guideline for pulmonary blast injury (version 2023)
Zhiming SONG ; Junhua GUO ; Jianming CHEN ; Jing ZHONG ; Yan DOU ; Jiarong MENG ; Guomin ZHANG ; Guodong LIU ; Huaping LIANG ; Hezhong CHEN ; Shuogui XU ; Yufeng ZHANG ; Zhinong WANG ; Daixing ZHONG ; Tao JIANG ; Zhiqiang XUE ; Feihu ZHOU ; Zhixin LIANG ; Yang LIU ; Xu WU ; Kaican CAI ; Yi SHEN ; Yong SONG ; Xiaoli YUAN ; Enwu XU ; Yifeng ZHENG ; Shumin WANG ; Erping XI ; Shengsheng YANG ; Wenke CAI ; Yu CHEN ; Qingxin LI ; Zhiqiang ZOU ; Chang SU ; Hongwei SHANG ; Jiangxing XU ; Yongjing LIU ; Qianjin WANG ; Xiaodong WEI ; Guoan XU ; Gaofeng LIU ; Junhui LUO ; Qinghua LI ; Bin SONG ; Ming GUO ; Chen HUANG ; Xunyu XU ; Yuanrong TU ; Liling ZHENG ; Mingke DUAN ; Renping WAN ; Tengbo YU ; Hai YU ; Yanmei ZHAO ; Yuping WEI ; Jin ZHANG ; Hua GUO ; Jianxin JIANG ; Lianyang ZHANG ; Yunfeng YI
Chinese Journal of Trauma 2023;39(12):1057-1069
Pulmonary blast injury has become the main type of trauma in modern warfare, characterized by externally mild injuries but internally severe injuries, rapid disease progression, and a high rate of early death. The injury is complicated in clinical practice, often with multiple and compound injuries. Currently, there is a lack of effective protective materials, accurate injury detection instrument and portable monitoring and transportation equipment, standardized clinical treatment guidelines in various medical centers, and evidence-based guidelines at home and abroad, resulting in a high mortality in clinlcal practice. Therefore, the Trauma Branch of Chinese Medical Association and the Editorial Committee of Chinese Journal of Trauma organized military and civilian experts in related fields such as thoracic surgery and traumatic surgery to jointly develop the Clinical treatment guideline for pulmonary blast injury ( version 2023) by combining evidence for effectiveness and clinical first-line treatment experience. This guideline provided 16 recommended opinions surrounding definition, characteristics, pre-hospital diagnosis and treatment, and in-hospital treatment of pulmonary blast injury, hoping to provide a basis for the clinical treatment in hospitals at different levels.
10.The mechanism of action and prognostic value of Dynamin 3 in gastric cancer
Ruhong TU ; Gildas Eric Sita Emmanuel ; Qing ZHONG ; Chaohui ZHENG ; Ping LI ; Jianwei XIE ; Jiabin WANG ; Jianxian LIN ; Jun LU ; Qiyue CHEN ; Longlong CAO ; Mi LIN ; Changming HUANG
Chinese Journal of Digestive Surgery 2023;22(9):1100-1112
Objective:To investigate the mechanism of action and prognostic value of Dynamin 3 (DNM3) in gastric cancer.Methods:The bioinformatic analysis, experimental study and retrospective cohort study was conducted. The clinicopathological data, fresh gastric cancer tissues, paired normal tissues and the corresponding paraffin sections of 153 gastric cancer patients who underwent radical gastrectomy in Fujian Medical University Union Hospital from January 2013 to July 2018 were collected. Tissues and the corresponding paraffin sections were subjected to quanti-tative real-time polymerase chain reaction, immunoblotting assay, flow cytometric cell cycle assay and immunohistochemical staining, respectively, and clinicopathological data were used for prognostic analysis. The stomach adenocarcinoma (STAD) dataset from the Cancer Genome Atlas (TCGA) database was collected for bioinformatic analysis. Observation indicators: (1) DNM3 gene expression in TCGA-STAD in gastric cancer; (2) mutations and copy number alterations of DNM3 in gastric cancer; (3) methylation level of promoter of DNM3 in gastric cancer; (4) relative protein expression of DNM3 and p53 in gastric cancer; (5) DNM3 correlation and enrichment analysis; (6) ratio of G0/G1 phase, S phase and G2/M phase of cell cycle progression; (7) correlation between immune cell infiltration and DNM3 in gastric cancer; (8) correlation between results of immunohistochemical (IHC) staining and clinical features; (9) analysis of independent factors influencing 5-year overall survival rate of gastric cancer patients. Measurement data with normal distribution were represented as Mean±SD, and comparison among multiple groups was conducted using the ANOVA and further comparison between two groups was conducted using the LSD. Comparison between two groups was conducted using the t test. Measurement data with skewed distribution were represented as M( Q1, Q3), and comparison between groups was conducted using the Mann-Whitney U test. Count data were described as absolute numbers or percentages, and compari-son between groups was conducted using the chi-square test or Fisher exact probability. Comparison of ordinal data was conducted using the rank sum test. The Pearson correlation coefficient or Spearman correlation coefficient was used to test the correlation between groups. Univariate and multivariate analyses were conducted using the COX proportional risk regression model. The Kaplan-Meier method was used to draw survival curves and calculate survival rates, and the Log-Rank test was used for survival analysis. The Benjamini-Hochberg false discovery rate correction was used for adjusting of the P-value. Results:(1) DNM3 gene expression in TCGA-STAD. The expression levels of DNM3 gene in the 27 tumor tissues and paired normal tissues of the TCGA-STAD database were 0.775(0.605,1.161) and 1.216(0.772,1.681), showing a significant difference between them ( Z=?2.64, P<0.05). The messenger RNA (mRNA) expression levels of DNM3 gene in 48 pairs of gastric cancer tissues and paired normal tissues of the author′s center were 4.370(2.870,6.040) and 2.520(0.850,4.170), showing a significant difference between them ( Z=?4.39, P<0.05). (2) Mutations and copy number alterations of DNM3 in gastric cancer. There were 16 gastric cancer patients in the TCGA-STAD database with DNM3 mutation or somatic copy number alterations, including 6 cases with missense mutations, 1 case with truncated mutation, 8 cases with copy number gain and 1 case with copy number loss. The mRNA expression levels of DNM3 gene before and after mutation in the 370 gastric cancer patients of the TCGA-STAD database were 6.13(5.40,7.08) and 5.02(3.98,5.46), showing a significant difference between them (Log 2FC=?1.11, Z=?2.59, P<0.05). (3) Methylation level of promoter of DNM3 in gastric cancer. There were 372 gastric cancer patients in the TCGA-STAD database undergoing DNM3 methylation and mRNA examinations, and the results showed that levels of methylation and mRNA expression of DNM3 was 0.198 (-0.458, 0.301) and 6.014 (5.141, 6.628), respectively. The levels of methylation in DNM3 was negatively correlated with its mRNA expression ( r=?0.38, P<0.05). Results of follow-up in 32 patients showed that the 3-year overall survival rate of 16 cases with high levels of methylation in DNM3 and 16 cases with low levels of methylation in DNM3 was 18.8% and 41.3%, respectively, showing a significant difference between them ( hazard ratio=1.40, P<0.05). Results of immunoblot-ting assay showed that the relative expression level of DNM3 protein in the AGS cells treated with 0, 0.5, and 1.0 μmol/L of 5-azacytidin was 0.270±0.020, 0.357±0.051 and 0.599±0.039, respectively, showing a significant difference among the three groups ( F=57.84, P<0.05). The relative expression level of DNM3 protein in the HGC-27 cells treated with 0, 0.5, and 1.0 μmol/L of 5-azacytidin was 0.316±0.038, 0.770±0.031 and 0.877±0.052, respectively, showing a significant difference among the three groups ( F=156.30, P<0.05). (4) Relative protein expression of DNM3 and p53 in gastric cancer. Results of immunoblotting assay showed that the relative expression of DNM3 and p53 protein was 0.688±0.047 and 0.872±0.041 in the AGS cells transfected with pCMV-DNM3 plasmid, versus 0.249±0.029 and 0.352±0.020 in the AGS cells transfected with control plasmid, showing significant differences in the above indicators between the two types of cells ( t=13.77,19.74, P<0.05). The relative expression of DNM3 and p53 protein was 0.969±0.069 and 1.464±0.081 in the HGC-27 cells transfected with pCMV-DNM3 plasmid, versus 0.456±0.048 and 0.794±0.052 in the HGC-27 cells transfected with control plasmid, showing significant differences in the above indicators between the two types of cells ( t=10.57, 12.06, P<0.05). (5) DNM3 correlation and enrichment analysis. Results of correlation analysis showed that DNM3 was positively correlated with genes such as RBMS3, CNTN4 and PDE1A ( r=0.52, 0.52, 0.50, P<0.05) and negatively correlated with genes such as SLC25A39, PAICS and GAPDH ( r=?0.41, ?0.40, ?0.40, P<0.05) in gastric cancer. Results of gene set enrichment analysis showed that the set of genes related to ribosome and oxidative phosphorylation were upregulated in gastric cancer patients with DNM3 low expression [normalized enrichment score (NES)=?3.30, ?2.16, P<0.05], while the set of genes related to immunomodulatory interactions between lymphocytes and non-lymphoid cells were upregulated in gastric cancer patients with DNM3 high expression (NES=1.67, P<0.05). Results of gene ontology analysis showed that the low expression of DNM3 was associated with the separation of mitotic sister chromatid (No.0000070), nonsense-mediation of nuclear transcriptional mRNA catabolic process, sister chromatid separation (No.0000819), nuclear transcriptional mRNA catabolic process and regulation of oxidative phos-phorylation (NES=?2.29, ?3.10, ?2.33, ?2.56, ?2.68, P<0.05). Results of Kyoto encycl opedia of genes and genomes analysis showed that metabolic pathway related to ribosome and oxidative phosphory-lation were upregulated and crosstalked in gastric cancer with low expression of DNM3 (NES=?3.34, ?2.21, P<0.05). (6) Ratio of G0/G1 phase, S phase and G2/M phase of cell cycle progression. Results of flow cytometric cell cycle experiments showed that the proportions of G0/G1 phase, S phase and G2/M phase in the cell cycle was 65.1%±3.0%, 17.3%±3.0% and 17.6%±1.0% in the AGS cells transfected with pCMV-DNM3 plasmid, versus 53.4%±4.0%, 26.3%±2.0% and 20.3%±3.0% in the AGS cells transfected with control plasmid, showing significant differences in the proportions of G0/G1 phase and S phase in the two types of cells ( t=4.05, 4.32, P<0.05). (7) Correlation between immune cell infiltration and DNM3 in gastric cancer. Results of immune cell infiltration examination showed that the expression level of DNM3 was positively associated with mast cells, NK cells, pDCs, B cells, follicular helper T cells, effector memory T cells, T cells, central memory T cells, CD8 T cells, DC cells, macrophages, γ-δ T cells (Tgd), iDCs and eosinophils infiltration (Spearman correlation coefficients as 0.41, 0.29, 0.26, 0.20, 0.22, 0.22, 0.13, 0.16, 0.15, 0.14, 0.14, 0.17, 0.18, 0.22, P<0.05) and negatively associated with Th17 cell, Th2 cells and NK CD56 dim cells infiltration ( r=?0.18, ?0.23, ?0.10, P<0.05). (8) Correlation between results of IHC staining and clinical features. Results of IHC staining analysis showed that the IHC score of DNM3 was 3(2,4) in the 105 gastric cancer tissues, versus 6(4,9) in the 105 paired normal tissues, showing a significant difference between them ( Z=-7.35, P<0.05). There were significant differences in gender, tumor location and N stating between the 70 patients with low expression of DNM3 and the 35 patients with high expression of DNM3 ( χ2=4.29, 7.67, 6.86, P<0.05). (9) Analysis of independent factors influencing 5-year overall survival rate of gastric cancer patients. Results of multivariate analysis showed that stage pT3?4 and low IHC score of DNM3 were independent risk factors for 5-year overall survival rate of gastric cancer patients ( hazard ratio=1.91, 0.51, 95% confidence interval as 1.06?3.43, 0.26?0.98, P<0.05). The 5-year overall survival rate was 44.3% in patients with low expression of DNM3, versus 65.7% in gastric cancer patients with high expression of DNM3, showing a significant difference between them ( χ2=5.02, P<0.05). Conclusion:DNM3 is a tumor suppressor and an independent predictor of poor prognosis for gastric cancer, which may regulate gastric cancer cell cycle and immunosuppression in the tumor microenvironment through methylation.


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