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2022 Vol. 52, No. 12 Published: 2022-12-25   947 The role of Tim-3 in regulating macrophage polarization in the occurrence and development of Crohn’s disease LIN Daopo, SHAO Xiaoxiao, HU Dingyuan, WU Hao, JIANG Yi. DOI: 10.3969/j.issn.2095-9400.2022.12.001 Objective: To explore the clinical effect and possible mechanism of T cells immunoglobulin domain and mucin domain protein 3, mucin 3 (Tim-3) in Crohn’s disease (CD) based on bioinformatics techniques. Methods: The Microarray data of expression profile (GSE112366, GSE75214, GSE16879 and GSE186582) were obtained from the common Gene Expression Omnibus (GEO). GEO2R online tool was used to analyze the expression level of Tim-3 in CD and healthy control. The mRNA expression of Tim-3 in normal tissues, intestinal tissues with active CD and inactive CD were observed. Then the relationship between Tim-3 mRNA expression and simplified endoscopy of Crohns’s disease score (SES-CD) was analyzed by Spearman rank correlation analysis. Subsequently, the expression level of Tim-3 in intestinal tissue of CD patients was analyzed before and after Infliximab (IFX) or Ustekinumab (UST) treatment. ROC curve analysis was performed to evaluate the mRNA expression of Tim-3 at week 0 in intestinal tissue of active CD in predicting the effect of biological agents. Cibersort online tool was used to estimate the proportion of M0, M1 and M2 macrophages in intestinal tissue of CD patients in each dataset, and analyze the correlation between Tim-3 expression and macrophage proportion in intestinal tissue. Next, the intestinal tissues of 12 patients with active CD and 15 age and gender-matched healthy controls were collected from the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, and the expression levels of Tim-3, CD68 (macrophage marker) as well as the number of CD68+ Tim-3+ macrophages in intestinal tissues were measured by immunofluorescence method.Finally, the expression of Tim-3 and M1, M2-related cytokines at mRNA level in colon tissues of 6 patients with active CD and 8 healthy controls were determined by real time fluorescence quantitative PCR. Results: In all 4 datasets, it was found that compared with normal intestinal tissues, the mRNA expression level of Tim-3 in intestinal tissues of active CD patients was significantly higher. The expression level of Tim-3 in intestinal tissue of active CD patients was also significantly higher than of inactive CD patients. The expression level of Tim-3 in intestinal tissue of active CD patients was positively correlated with SES-CD score (r=0.39, P<0.001). After IFX/UST treatment, the expression of Tim-3 in intestinal tissue of CD patients was lower than before IFX/UST treatment. In addition, the expression level of Tim-3 in intestinal tissue of IFX-responsive group was lower than that of non-responsive group. At week 4 or 6 of Infliximab treatment, ROC curve analysis showed that the expression level of Tim-3 in intestinal tissue of patients with active CD at week 0 has predictive value for the efficacy of IFX (AUC=0.70, P=0.04). Cibersort analysis showed that the mRNA expression level of Tim-3 in intestinal tissue was positively correlated with the infiltration level of M0 and M1 macrophages in intestinal tissue of active CD patients. Immunofluorescence results showed that the expressions of CD68 and Tim-3 in inflamed intestinal mucosa of CD patients were higher than healthy controls (P<0.05). In addition, the proportion of CD68+ Tim-3+ double-positive macrophages was also significantly increased in the inflammatory sites of CD patients. The mRNA expression level of Tim-3 in intestinal tissue of active CD patients was significantly higher than healthy controls, and was positively correlated with the expression level of M1-related cytokines TNF-α and IL-1β. Conclusion: Tim-3 may play an important role in the development of CD by affecting the polarization of M1 macrophages. Meanwhile, it may predict the efficacy of early IFX. 2022 Vol. 52 (12): 947-955 [Abstract] ( 410 ) HTML (1 KB)  PDF (1969 KB)  ( 577 ) 956 Effect of methylene blue analogues on glutamate-induced HT22 neuron cell injury and its mechanism CHEN Ziwei, CHEN Qiuhe, CAI Yu, HOU Shanshan, LIU Fuhe DOI: 10.3969/j.issn.2095-9400.2022.12.002 Objective: To investigate the effect of methylene blue (MB) analogues (2-Chlorophenothiazine,Neutral red, Azure A and Azure B) on glutamate-induced HT22 neuronal cell injury and its mechanism. Methods:The toxicity effect of 2-Chlorophenothiazine (2-C), Azure A (A-A), Azure B (A-B) and Neutral red (NR) on HT22 neuronal cells were detected by MTT. The protect effect of methylene blue analogues against glutamateinduced cell death was detected by MTT and LDH release assay; ROS production was measured by H2DCF-DA staining; siRNA was used to download the expression of MEF2D. Western blot was used to detect the protein levels of MEF2D, ND6, Akt and GSK-3β. Results: LD50 of compound was calculated according to the results of MTT experiment,: MB (50.66 μmol/L), 2-C (175.90 μmol/L), A-A (152.40 μmol/L), A-B (140.40 μmol/L), NR (73.19 μmol/L). Except for the NR, the other compounds had significant protective effect on glutamate-induced oxidative damage of HT22 cells. The EC50 value of each compound was calculated as follows: MB (42.13 nmol/L),2-C (34.32 nmol/L), A-A (48.96 nmol/L), A-B (44.42 nmol/L). MB, 2-C, A-B and A-A significantly reduced glutamate-induced ROS generation in HT22 cells detected by H2DCF-DA staining at 100 nmol/L. However, NR had no obvious effect on glutamate-induced ROS generation in HT22 cells. MB, 2-C, A-B and A-A elevated the protein level of MEF2D (P<0.05). To investigate the role of MEF2D in the neuroprotective effect of MB analogues against glutamate-induced cell death, we knocked down MEF2D by siRNA in HT22 cells (P<0.05). MTT results showed that the knockdown of MEF2D abolished part of neuroprotective effect of MB, 2-C, A-B and A-A against glutamate in HT22 cells. Further studies on the mechanism showed that 2-C, A-B, A-A and MB could activate Akt, increase GSK-3β phosphorylation (P<0.05) and inactivate GSK-3β, which may be related to the activation of MEF2D and ND6. Conclusion: Compared with MB, 2-C has lower cytotoxicity, and stronger damage activity to the anti-glutamate-induced cells.Compounds with phenothiazine structure such as 2-C, A-B,A-A and MB have excellent antioxidant activity. The Akt/GSK-3β/MEF2D/ND6 pathway may be related to its antioxidant activity. 2022 Vol. 52 (12): 956-964,971 [Abstract] ( 549 ) HTML (1 KB)  PDF (2764 KB)  ( 679 ) 965 Analysis of children’s thyroid hormone and sex hormone levels in Wenzhou area LI Shanshan, NI Li, XU Xiaojie, ZHU Lidan, DING Hongxiang. DOI: 10.3969/j.issn.2095-9400.2022.12.003 Objective: To explore the biological reference interval of children in Wenzhou area by analyzing their gender and age distribution of thyrotropin (TSH), thyroxine (T4), 3, 5 ,3’ triiodothyronine (T3), free thyroxine (FT4), free triiodothyroxine (FT3), luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (PROG), prolactin (PRL), estradiol (E2) and testosterone (T). Methods: Healthy children aged 1-18 years were selected from those who underwent physical examination between August 2018 and December 2021 in the Children’s Health Care Department of the Second Affiliated Hospital of Wenzhou Medical University. They were divided into 6 groups as 1-3 years old (male and female group), 4-8 years old (female group), 4-10 years old (male group), 9-18 years old (female group) and 11-18 years old (male group). With excluding outliers, 783 qualified cases were obtained. The serum hormone level was measured by SIEMENS ADVIA Centaur XP System chemiluminescence analyzer. The comparison between groups was analyzed by t test, approximate t test or Kruskal-Wallis test, and the correlation s analyzed by Spearman. Results: The levels of FT3, LH, FSH, PRL,E2 and T were significantly different between boy and girl in different age groups (P<0.05). In boys, age was negatively correlated with T3, T4 (r=-0.419, -0.347, P<0.05), while it showed strong positive correlation with LH, FSH, PROG, T (r=0.768, 0.678, 0.535, 0.703, P<0.05). Girls’ age was negatively correlated with T3,T4, FT3 (r=-0.486, -0.318, -0.424, P<0.05), while it was positively correlated with LH, FSH, PROG, E2, T(r=0.811, 0.335, 0.574, 0.584, 0.744, P<0.05). The correlation coefficients of Tanner stage and LH, FSH, PRL,PROG, E2, T in adolescent male and female groups were 0.641, 0.204, 0.308, 0.479, 0.604, 0.686 and 0.664,0.399, 0.436, 0.485, 0.621, 0.649 respectively. It’s positive correlation (P<0.01). Based on the thyroid hormone and sex hormone levels of children of different age and gender groups, the reference intervals of 11 hormones for children aged 1-18 in Wenzhou area were preliminarily formulated. Conclusion: There’re differences in the level of thyroid and sex hormones in children between the findings of this study and the European reference intervals currently used in our department, which is of clinical significance in terms of regional reference intervals. 2022 Vol. 52 (12): 965-971 [Abstract] ( 332 ) HTML (1 KB)  PDF (1578 KB)  ( 560 ) 972 The role of granzyme B-perforin in selectively inducing apoptosis of macrophages in pancreatitis based on KEGG pathway enrichment analysis KONG Hongru, YU Haohui, CHEN Geer, CHEN Zimiao, DAI Shengjie, SUN Xuecheng, JIN Yuepeng, YANG Wenjun, SUN Hongwei DOI: 10.3969/j.issn.2095-9400.2022.12.004 Objective: To investigate the effect of granzyme B (GRB)-perforin (PFP) on macrophage apoptosis during selective induction of pancreatitis. Methods: Human mononuclear macrophage cell line (TPH-1) was stimulated by tumor necrosis factor-α (TNF-α) to simulate the macrophage microenvironment during acute pancreatitis. The optimum concentrations of TNF-α, PFP and GRB were screened by CCK8 test. Apoptosis was detected by flow cytometry in control group, TNF-α group, PFP+GRB group and TNF-α+PFP+GRB group. The cell samples were divided into three groups: normal cell group (NC group), TNF-α stimulation group (Treat1 group) and TNF-α+PFP+GRB stimulation group (Treat2 group). Three groups of cell RNA were extracted for transcriptomic analysis. Gene expression was detected by principal component analysis, differential gene analysis and enrichment analysis. Seven differentially expressed genes (RAB37, GPA33, USH1C, SYTL1, MSRB3, GPER1, CD1B) were selected in the “cytokine - cytokine receptor interaction pathway” to verify the accuracy of RNA-seq by fluorescence quantitative PCR. Results: Compared to NC group and Treat1 group, the apoptosis rate of Treat2 group was significantly higher. The difference between NC group and Treat1 group and Treat2 group was obvious, with more significant genes, while the difference between Treat1 group and Treat2 group was unconspicuous, but the change of Treat2 group was greater than that of Treat1 group in NC group. Enrichment analysis showed that the differential genes were mainly concentrated in the “cytokine - cytokine receptor interaction pathway”. The results of fluorescence quantitative PCR and RNA-seq were highly consistent. Conclusion:GRB-PFP selectively induces apoptosis of macrophages in pancreatitis and acts as a “cytokine - cytokine receptor interaction pathway” to down-regulate the expression of cytokines and reduces the inflammatory response. 2022 Vol. 52 (12): 972-980 [Abstract] ( 367 ) HTML (1 KB)  PDF (2738 KB)  ( 763 ) 981 Distribution characteristics of Pseudomonas aeruginosa T3SS and T6SS and their correlation with drug resistance ZHOU Beibei, WANG Lingbo, ZHANG Xiucai, CHEN Lijiang, WANG Zhongyong,ZHOU Tieli DOI: 10.3969/j.issn.2095-9400.2022.12.005 Objective: To understand the Type III secretion system (T3SS) and Type VI secretion system(T6SS) gene distribution characteristics of Pseudomonas aeruginosa (P. aeruginosa) from sputamentum and blood and their correlation with drug resistance. Methods: A total of 92 strains of P. aeruginosa (61 strains isolated from sputamentum, and 31 strains isolated from blood) in the First Affiliated Hospital of Wenzhou Medical University were collected from 2016 to 2018. The PCR method was used to detect the T3SS effector protein encoding genes exoS, exoT, exoU, exoY and pscC and the T6SS effector protein encoding genes tssB1, tse1, tse2,tse3, tssB2, pldA, tssB3, and pldB. The agar dilution method was used to detect the sensitivity of the strains to commonly used clinical antibacterial drugs. The correlation between different infections and the carrying status of genes encoded by the secretory system was investigated, and further analysis of their relationship with drug resistance was made. Results: The detection rate of T3SS protein encoding gene exoT and T6SS protein genes tssB1, tse2, tssB2, and pldB in sputamentum and blood was as high as > 90%. In addition, the carrying rate of T3SS protein gene exoU in strain from sputum was significantly higher than from blood (P<0.05), and the drug resistance rate of strains to imipenem derived from sputum was significantly higher than those from blood (P<0.05). Spearman correlation analysis showed exoU was positively correlated to pldA (r=0.474, P<0.01).There was no significant difference among strains with different T3SS and T6SS gene profiles against commonly antibiotics (P>0.05). Conclusion: The pldA gene often coexists with the virulence gene exoU in P. aeruginosa. The carrying rate of T3SS effector protein encoding gene exoU and the resistance against imipenem in P. aeruginosa from sputum is significantly higher than that of strains from blood. 2022 Vol. 52 (12): 981-986 [Abstract] ( 425 ) HTML (1 KB)  PDF (1357 KB)  ( 543 ) 987 The composition of intestinal flora in infants with functional constipation: an analysis based on 16S rDNA sequencing LI Hui, WANG Juan, YUAN Xinhua DOI: 10.3969/j.issn.2095-9400.2022.12.006 Objective: To explore the structure and composition of intestinal flora in infants with functional constipation by 16S rDNA sequencing and provide evidence for clinical treatment. Methods: From January to December 2020, 17 infants with functional constipation treated in Huzhou Maternity and Child Health Care Hospital were selected as the observation group, and 15 normal infants were assigned as the control group. With feces collected from the two groups, the 16S rDNA amplicon sequencing method was used to detect the intestinal bacteria to analyze the composition and compare the results. Results: There was no significant difference in the Alpha diversity of intestinal flora between the two groups (P>0.05), but there was significant difference in the Beta diversity (P<0.01). At the phylum level, Actinobacteria, Firmicutes, and Proteobacteria were the most abundant flora in the intestinal microbial communities of the two groups of children. Among them, the abundance of Verrucomicrobia in the constipation group was higher than that in the control group (P<0.05). At the genus level,Bifidobacterium was the dominant species in the two groups, and the abundance of Lactobacillus and Akkermansia in the intestinal tract of children with constipation was higher than that in the control group (P<0.05). The abundance of Bifidobacterium species, Microflora verrucosa, Achmania and Lactobacillus paracasei in the constipation group was statistically different, while that of Bifidobacterium dentatum, Actinomyces, Actinomycetidae,Actinomycetes and Lactobacillus rhamnosus in the control group was statistically different (P<0.05). Conclusion:There are changes in the structure and diversity of intestinal flora in children with functional constipation, There may be a correlation between constipation and intestinal flora. 2022 Vol. 52 (12): 987-992 [Abstract] ( 309 ) HTML (1 KB)  PDF (1707 KB)  ( 514 ) 993 Phenotype and gene mutation analysis of a family with hereditary antithrombin deficiency ZHOU Xingxing, XIE Yaosheng, XIE Haixiao, WANG Mingshan DOI: 10.3969/j.issn.2095-9400.2022.12.007 Objective: To analyze the coagulation index and genotype of a patient with hereditary antithrombin (AT) deficiency and his family members, and to explore its molecular pathogenesis. Methods:The blood coagulation indexes such as plasma AT activity (AT:A) and AT antigen (AT:Ag) in the peripheral blood of each family member were detected on Stago instrument; the peripheral blood DNA was extracted and sequenced, and the gene mutation sites were located. The effect of mutations on protein function was analyzed by bioinformatics software. Results: The AT:A of the proband and his maternal grandmother, father, mother and younger brother all decreased to varying degrees, and AT:Ag decreased synchronously. There were no obvious abnormalities in PC:A and PS:A in all family members, showing type I AT Defects. Genetic analysis showed that the proband had a heterozygous missense mutation of c.1A>G (p.Tyr2stop) in exon 1 of SERPINC1 gene and a heterozygous synonymous mutation of c.1005G>A in exon 5; his father carried c.1A>G heterozygous missense mutation, his grandmother, mother and younger brother carry c.1005G>A heterozygous synonymous mutation.Conservation analysis showed that Tyr2 was highly conserved among homologous species. Analysis made by three online bioinformatics software, i.e., MutationTaster, PolyPhen-2 and LRT, showed that p.Tyr2stop mutation was “pathogenic and harmful”; protein model analysis showed that the p.Tyr2stop mutation could cause premature termination of AT gene translation, resulting in a truncated protein. Conclusion: The AT:A and AT:Ag reduction to different degrees in the proband and family members was related to the c.1A>G heterozygous missense mutation and the c.1005G>A heterosynonymous mutation in the SERPINC1 gene. 2022 Vol. 52 (12): 993-998 [Abstract] ( 375 ) HTML (1 KB)  PDF (1751 KB)  ( 578 ) 999 Children with MPPH I syndrome due to missense variant of PIK3R2 gene: A case analysis RUAN Miaohua,HU Sisi, ZHOU Xiaojun, SHI Jiamin, CHEN Yuan, ZHU Mianmian, WANG Qiu, WANG Dan. DOI: 10.3969/j.issn.2095-9400.2022.12.008 Objective: To summarize the clinical features and make a genetic analysis of a patient with megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome I (MPPH I). Methods: We retrospectively collected the clinical manifestations, imaging examinations and genetic analysis of a neonate with MPPH type I which was diagnosed at the Department of Neonatology of the First Affiliated Hospital of Wenzhou Medical University in January 2021. Literature was retrieved to summarize the clinical and genetic characteristic of MPPH type I casued by PIK3R2 variant. Whole exome sequencing was carried out for the proband. Candidate variant was verified by Sanger sequencing. Results: The proband was found to harbor a heterozygous missense c.1117G>A (p.Gly373Arg) variant of the PIK3R2 gene, which was unreported previously in China. The variant was not detected in either parent. The clinical characteristics of the 16 cases reported in the relevant literature review mainly presented a constellation of neurodevelopmental disorder, including megalencephaly, polymicrogyria,and hydrocephalus, and multiple neurological symptoms, including intellectual disability and epilepsy. PIK3R2 gene variants were identified, and most of them were p.Gly373Arg variant in exon 10 (fourteen cases), followed by p.Leu401Pro (one case), p.Asp557His (one case). Conclusion: The PIK3R2 gene c.1117G>A (p.Gly373Arg) variant may underlay the MPPH I syndrome in this patient. Hydrocephalus may be related to MPPH syndrome.Prenatal diagnosis is recommended for subsequent pregnancies. 2022 Vol. 52 (12): 999-1004 [Abstract] ( 464 ) HTML (1 KB)  PDF (2008 KB)  ( 518 ) 1005 Analysis of thyroid endoscopic surgery by two-hole trans-subclavian approach JIN Chun DOI: 10.3969/j.issn.2095-9400.2022.12.009 Objective: To investigate the safety and feasibility of the new two-hole trans-subclavian approach in thyroid endoscopic surgery. Methods: The clinical data of 32 patients who underwent two-hole trans-subclavian thyroid endoscopic surgery in the Department of Thyroid Surgery, the First Affiliated Hospital of Wenzhou Medical University from May 26, 2022 to October 28, 2022 were retrospectively analyzed. The operation time, the length of postoperative hospital stay and the postoperative complications were recorded.Results: All operations were successfully completed under endoscopy approach except the second case who was transferred to open surgery due to bleeding when the flap was separated. The operation time was 103 min (range:45 to 195 min), and the postoperative hospital stay was 4 days (range: 3 to 5 days). One Postoperative wound infection was observed, which was cured with conservative treatment. No recurrent laryngeal nerve injury or postoperative bleeding was observed. Conclusion: The two-hole trans-subclavian thyroid endoscopic surgery is a safe and feasible approach, which is valuable for further clinical application. 2022 Vol. 52 (12): 1005-1008 [Abstract] ( 336 ) HTML (1 KB)  PDF (1858 KB)  ( 772 ) 1020 The functions and drug development of ecto-nucleotide pyrophosphatase/phosphodiesterase-1 LIU Yitang,DING Zihan, CHENG Wenqing, CAI Zheyang, SHEN Linjie, GUO Wei DOI: 10.3969/j.issn.2095-9400.2022.12.014 The extracellular adenosine metabolism system, which is involved in numerous and varied physiological and pathological processes, has drawn wide interest as targets for new drug development.However, the research progress of drug development targeting ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) is rare. Recently, the roles of ENPP1 have received progressive attention after the breakthrough finding as the dominant 2’3’-cyclic GMP-AMP dinucleotides-hydrolase. Here, having briefly introduced the structure of ENPP1, we focus on its physiological and pathological functions in biomineralization and tumor, as well as the research and development of enzyme replacement therapy and small molecule drugs targeting ENPP1. 2022 Vol. 52 (12): 1020-1025 [Abstract] ( 789 ) HTML (1 KB)  PDF (1505 KB)  ( 1320 ) 1026 Exploration and practice of reforming the professional title system in colleges and universities against the background of rectifying the “Five Overemphasized Indicators for Evaluation”——a case study of Wenzhou Medical University KE Xintong, LIN Xinbiao, ZHANG Yi, WANG Fang DOI: 10.3969/j.issn.2095-9400.2022.12.015 Against the background of rectifying the “Five Overemphasized Indicators for Evaluation (namely papers, honorary titles, professional titles, degrees and awards)”, it becomes an important task for Chinese colleges and universities to explore the reform of the professional title system for professional and technical personnel.In accordance with the policy documents and taking into consideration of the actual situation of the university,Wenzhou Medical University endeavors to deepen the reform of the professional title system by initiating the following six points. First, the university follows the principle of the Party exercising leadership over personnel and in the work of professional title reform. Second, a diversified evaluation system is established in line with many and varied disciplines by adopting category-oriented evaluation. Third, a quantitative indicator system for professional title evaluation is constructed by adding more evaluation dimensions. Fourth, the peer review system is perfected. Fifth, a “priority channel” is set up for special talents. Last, an information platform for professional title evaluation is built. The case illustrated by Wenzhou Medical University provides reference for the practice of reforming professional title system in the context of Chinese colleges and universities fostering virtue through education in the new era. 2022 Vol. 52 (12): 1026-1029 [Abstract] ( 276 ) HTML (1 KB)  PDF (1319 KB)  ( 588 ) 1030 Exploration on the mixed mode of “Three Links and Three Blends” in the teaching of Pharmacology course ZHAO Yeli, CHEN Jun, QIAN Jianchang, LI Junwei, CHEN Bingbing DOI: 10.3969/j.issn.2095-9400.2022.12.016 This teaching reform has constructed and explored a new mixed teaching mode of “three links and three blends” in Pharmacology course. “Three Links” means the effective linkage between online and offline,proper integration of large and small courses and deep connection between schools and hospitals; “Three Blends” refers to the integration of moral education and curriculum development, the integration of basic sciences and clinical subjects, and the integration of theory and practice. This teaching mode can improve students’ autonomous learning ability, apply theory to practice, and internalize the professionalism of “sincere doctors with boundless love” into the course teaching. The questionnaire results show that the students are satisfied with this innovative teaching mode. This teaching reform improves the students’ learning interest, teamwork ability and job competency. In this way, the teaching quality has been further improved. 2022 Vol. 52 (12): 1030-1032,封三 [Abstract] ( 225 ) HTML (1 KB)  PDF (1416 KB)  ( 450 )

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