1.METTL3-mediated m6A methylation regulates lipopolysaccharide-induced endothelial cell permeability changes
Jianfeng WANG ; Huilin YU ; Youxin YU ; Junhui SONG ; Chengying MENG ; Wei JIANG ; Delin HU
Acta Universitatis Medicinalis Anhui 2024;59(6):1023-1028
Objective To explore the molecular mechanism of N6-methyladenosine(m6A)methylation mediated by methyltransferase 3(METTL3)in regulating lipopolysaccharide(LPS)-induced endothelial cell permeability changes.Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro.HUVECs were treated with LPS 50,125,250,500,1 000,2 000 ng/ml for 24 h.METTL3 mRNA expression was detected by Real-time PCR.After HUVECs were intervened with 500 ng/ml for 24 h,the methylation level of m6A was detec-ted,and cell permeability was measured by cell permeability test.Real-time PCR and Western blot were used to detect mRNA and protein expression of intercellular junction proteins(Claudin-5,Occludin and VE-caherin).METTL3 overexpressed stable cell lines were constructed to measure the changes of m6A methylation level and per-meability of endothelial cells during METTL3 overexpression.Results Compared to the control group,LPS inhibi-ted the expression of HUVECs METTL3 mRNA,decreased the methylation of m6A,increased the cell permeabili-ty,and decreased the mRNA and protein expression of intercellular junction proteins(Claudin-5,Occludin and VE-Caherin).When METTL3 was overexpressed,the m6A methylation levels of endothelial cells were enhanced,and the increase of endothelial cell permeability induced by LPS was reversed.Conclusion METTL3-mediated m6A methylation can improve the permeability of endothelial cells induced by sepsis.
2.Effects of Tongfengning(痛风宁) on Intestinal Flora and Intestinal Uric Acid Metabolism in Model Mice of Hyperuri⁃cemia of Spleen Deficiency with Exuberance of Dampness Syndrome
Yingjie ZHANG ; Xiao MAO ; Yan XIAO ; Tangyan CAI ; Jiemei GUO ; Peng CHEN ; Peng ZHANG ; Jun LIU ; Yu CHEN ; Mengting QIU ; Youxin SU
Journal of Traditional Chinese Medicine 2023;64(21):2232-2240
ObjectiveTo explore the possible mechanisms of Tongfengning (痛风宁, TFN) in treating hyperuricemia (HUA) of spleen deficiency with exuberance of dampness syndrome. MethodsTen of 60 mice were randomly selected, and were fed with regular diet as the control group, while the remaining 50 mice were fed with high-fat and high-sugar diet combined with excessive exercise and potassium oxonate-allopurinol suspension to establish an HUA animal model of syndrome of spleen deficiency with exuberance of dampness. After the successful modeling, in order to better observe the effects of TFN on the intestinal microbiota of the model mice, a mixed antibiotic suspension was administered by gavage to induce further dysbiosis of the intestinal microbiota in the model mice. Fifty sucessfully modeled mice were randomly divided into model group, TFN group, allopurinol group, probiotics group, and an allopurinol + probiotics group, 10 in each group. The TFN group was administered TFN liquid at a dosage of 19.11 g/(kg·d) by gavage. The allopurinol group was administered allopurinol suspension at a dosage of 78 mg/(kg·d) by gavage. The probiotics group was administered live combined Bifidobacterium and Lactobacillus tablets suspension at a dosage of 3 g/(kg·d) by gavage. The allopurinol + probiotics group was administered allopurinol at a dosage of 78 mg/(kg·d) and live combined Bifidobacterium and Lactobacillus tablets suspension at a dosage of 3 g/(kg·d) by gavage. The control group and model group were administered normal saline at a dosage of 19.11 ml/(kg·d) by gavage. The interventions were continued for 21 days. In order to maintain a stable high blood uric acid state, all groups but the control group continued modeling while receiving drug intervention. The changes in spleen deficiency syndrome scores, blood uric acid levels, microbial community structure, acetic acid and butyric acid content in intestinal lavage fluid, adenosine deaminase (ADA) and xanthine oxidase (XOD) content in small intestine tissue, as well as ATP-binding cassette transporter G2 (ABCG2), glucose transporter 9 (GLUT9) protein and mRNA expression in the small intestine tissue were compared among the groups of mice. ResultsCompared with the control group, the model group showed increased spleen deficiency syndrome scores, blood uric acid levels, relative abundance of phylum Firmicutes, Firmicutes/Bacteroidetes ratio, abundance of Bacteroides genus, Klebsiella genus, and Enterococcus genus, acetic acid content in intestinal lavage fluid, ADA and XOD content in small intestine tissue, as well as GLUT9 protein and mRNA expression (P<0.05). The number of operational taxonomic units (OTUs) of intestinal microbiota, relative abundance of Bacteroidetes phylum, abundance of Lactobacillus genus and uncultured Bacteroides genus, butyric acid content in intestinal lavage fluid, and ABCG2 protein and mRNA expression in small intestine tissue were significantly decreased (P<0.05). Compared with the model group, in the group treated with TFN, probiotics, and allopurinol + probiotics, the spleen deficiency syndrome score, blood uric acid level, relative abundance of Firmicutes, acetic acid content in intestinal lavage fluid, ADA and XOD content in small intestine tissue, GLUT9 protein and mRNA expression significantly decreased. The number of gut microbiota OTUs, relative abundance of proteobacteria, butyric acid content in intestinal lavage fluid, ABCG2 protein and mRNA expression in small intestine tissue significantly increased (P<0.05). In the probiotics group, the ratio of Firmicutes to Bacteroidetes decreased. In the TFN group, the abundance of Lactobacillus and uncultured Bacteroidetes significantly increased, while the abundance of Parabacteroides, Klebsiella, and Enterococcus significantly decreased (P<0.05). Compared with the TFN group, allopurinol group and the probiotics group showed elevated blood uric acid levels, abundance of Bacteroidetes, ADA and XOD levels in intestinal tissue, and GLUT9 mRNA expression. The relative abundance of Firmicutes, abundance of lactobacilli, and ABCG2 mRNA expression significantly decreased. The probiotics group showed elevated GLUT9 protein expression in intestinal tissue. The probiotics group and the allopurinol plus probiotics group showed significantly higher scores for spleen deficiency syndrome in mice, and lower levels of butyric acid in mouse intestinal lavage fluid. The allopurinol group showed decreased numbers of OTUs in mouse intestinal flora, decreased abundance of proteobacteria, and butyric acid levels in intestinal lavage fluid. The allopurinol group also showed decreased ABCG2 protein expression in intestinal tissue, increased acetic acid levels in intestinal lavage fluid, increased abundance of Klebsiella, and significantly elevated GLUT9 protein expression (P<0.05). ConclusionsThe treatment of HUA with TFN may be associated with the regulation of intestinal probiotics (such as lactobacilli) and pathogenic bacteria (such as Klebsiella), as well as the production of bacterial metabolites such as acetic acid and butyric acid. It may also involve reducing the expression of ADA and XOD in the intestines, decreasing intestinal uric acid production, upregulating the expression of intestinal epithelial urate transporter ABCG2, downregulating GLUT9 expression, and promoting intestinal uric acid excretion. These factors are related to the syndrome of spleen deficiency with exuberance of dampness.
3.Microvascular changes of macular edema secondary to branch retinal vein occlusion treated with intravitreal conbercept injection
Zhikun YANG ; Weihong YU ; Youxin CHEN
Chinese Journal of Ocular Fundus Diseases 2021;37(9):675-680
Objective:To observe the changes of macular microvascular structure in eyes with macular edema secondary to branch retinal vein occlusion (BRVO-ME) after intravitreal injection of conbercept and analyze its relationship with visual function and central retinal thickness (CRT).Methods:A prospective clinical study. From July 2018 to June 2019, 21 eyes of 21 patients with unilateral temporal BRVO-ME diagnosed in the Department of Ophthalmology of Peking Union Medical College Hospital were included in the study. Among them, there were 14 eyes of 14 males and 7 eyes of 7 females; the average age was 58.0±8.3 years. There were 13 eyes and 8 eyes with occlusion of the superior temporal and inferior temporal branches of the retinal vein, respectively. The affected area was defined as the side of the venous obstruction. All the affected eyes underwent best-corrected visual acuity (BCVA) and optical coherence tomography angiography (OCTA) examination. The BCVA was tested using the international standard logarithmic visual acuity chart, which was converted into the logarithmic minimum angle of resolution (logMAR) visual acuity during statistical analysis. All the eyes were treated with intravitreal injection of conbercept once a month for 3 months, and then treated as needed. A 3 mm × 3 mm scan centered on fovea was obtained and the vascular density of superficial capillary plexus (SCP) and deep capillary plexus (DCP), fovea avascular zone (FAZ) area, perimeter of FAZ (PERIM), acircularity index (AI), foveal vascular density in a 300 μm wide region around FAZ (FD-300) and central retinal thickness (CRT) were measured. The follow-up time after treatment was 6 months. The vascular density and FAZ parameters were compared before and after treatment by paired t test. The correlations of BCVA, CRT and vascular density, FAZ area and the other parameters at 6 months after treatment were analyzed by linear regression analysis. Results:Before treatment, the logMAR BCVA of the eyes was 0.506±0.159, and the CRT was 375.4±81.3 μm; 6 months after treatment, the logMAR BCVA of the eyes was 0.294±0.097, and the CRT was 266.3±46.7 μm. There was a statistically significant difference of logMAR BCVA and CRT between the eyes before and after treatment ( t=8.503, 9.843; P<0.05). There was no statistically significant difference in the overall vascular density of SCP and DCP before and 6 months after treatment ( t=-0.091, -0.320; P>0.05). The foveal vascular density decreased, and the difference was statistically significant ( t=8.801, 3.936; P<0.05). The vascular density of DCP of the affected area increased, and the difference was statistically significant ( t=-2.198, P<0.05). Compared with those before treatment, the FAZ area and PERIM of the affected eyes had an increasing trend, while AI and FD-300 had a decreasing trend, and the differences were statistically significant ( t=-18.071, -12.835, 2.555, 8.610; P<0.05). The linear regression analysis showed that BCVA and FAZ area 6 months after treatment have significant correlation ( t=2.532, P=0.024). Conclusion:CRT decreased and BCVA increased after intravitreal injection of conbercept in BRVO-ME eyes. After treatment, the foveal vascular density of SCP and DCP decreased while the vascular density of DCP of the affected area increased. The FAZ increased and the PERIM and AI decreased during follow-up. The BCVA was significantly correlated with the FAZ area 6 months after treatment.
4.Research progress in retinal vascular changes for cardiovascular disease assessment and prediction
Hongzhe ZHANG ; Weihong YU ; Youxin CHEN
Journal of Chinese Physician 2021;23(5):641-644
In recent years, the prevalence of cardiovascular disease (CVD) in China has been increasing year by year, and it has become a major public health concern. The prevention and control of chronic CVD is inseparable from effective early screening. However, due to the imbalance in the level of economic and social development in various parts of China, the universality and accessibility of screening in some areas still need to be improved. The fundus retinal blood vessel is the only vessel in the whole body that can be directly observed. Studies have found that its morphological and functional changes are related to the occurrence and development of many CVD, and it has the potential to become a tool for assessment and prediction. This review intends to introduce the research progress of retinal blood vessels in the evaluation and prediction of cardiovascular disease in recent years.
5.Application of artificial intelligence reading label system in diabetic retinopathy grading training of junior ophthalmologists and medical students
Ruoan HAN ; Weihong YU ; Huan CHEN ; Mingyue LUO ; Youxin CHEN
Journal of Chinese Physician 2021;23(5):650-653
Objective:To evaluate the efficiency of using artificial intelligence reading label system in diabetic retinopathy (DR) grading training among junior ophthalmologists and medical students.Methods:520 diabetic fundus images were randomly divided into 8 groups with 65 images in each group. 13 junior ophthalmologists and medical students were selected as the research objects. Each of them read 8 groups of pictures and evaluated the DR grading of each fundus image. The sensitivity, specificity and diagnostic test consistency (Q-kappa value) of grading results were analyzed with the DR grading given by 3 senior ophthalmologists as the gold standard. The average Q-kappa values of 13 subjects were compared between the first four times and the last four times.Results:Through 8 round reading, the average Q-kappa was elevated from 0.67 to 0.81. Average Q-kappa of round 1 to 4 was 0.77, and average Q-kappa of round 5 to 8 was 0.81. The participants were divided into two groups. Participants in group 1 were junior ophthalmologists and participants in group 2 were medical students. Average Q-kappa of group 1 was elevated from 0.71 to 0.76. Average Q-kappa of group 2 was elevated from 0.63 to 0.84.Conclusions:The artificial intelligence reading label system was a useful tool in training junior ophthalmologists and medical students in doing diabetic retinopathy grading.
6.Comparative study of lateral position and half lithotomy position in Asian proximal femur intramedullary nail antirotation system internal fixation for treatment of elderly patients with femoral inter-trochanteric fractures
Xiaohua ZHANG ; Shuqing ZHOU ; Bowen YANG ; Luxin YU ; Ming ZHU ; Youxin LIAO ; Jian YE ; Qiuwen ZHU
Chinese Journal of Trauma 2020;36(6):503-508
Objective:To compare the therapeutic effect of lateral position and half lithotomy position in Asian proximal femur intramedullary nail antirotation system (PFNA-II) for treating the elderly patients with femoral intertrochanteric fractures.Methods:A retrospective case control study was made on 141 patients with femoral intertrochanteric fractures admitted to Jiangjin Central Hospital from January 2016 to September 2017, including 54 males and 87 females, aged 65-99 years (mean, 80.4 years). According to AO classification, there were 42 patients with type A1 fractures, 88 with type A2 and 11 with type A3. Of all, 74 patients were stabilized by PFNA-II internal fixation in lateral position (lateral position group) and 67 patients by PFNA-II internal fixation in half lithotomy position (half lithotomy position group). The postural placement time, total incision length, operative time, intraoperative blood loss, fluoroscopy frequency, tip-apex distance, reduction quality, fracture healing time, postoperative complications and Harris hip function at 12 months after surgery were compared between the two groups.Results:All patients were followed up for 12-18 months (mean, 12.5 months), except that 13 patients were lost after 9 months, an average of 12.5 months. There were no statistically significant differences in postural placement time, operative time, fracture healing time, and Harris hip score between the two groups ( P>0.05). While significant differences were seen between lateral position group and half lithotomy position group regarding the incision length [(6.5±1.3)cm vs. (7.5±1.5)cm], intraoperative blood loss [(84.3±3.1)ml vs. (90.4±3.9)ml], fluoroscopy frequency [(13.1±1.9)times vs. (11.2±1.2)times], tip-apex distance [(20. 6±2.2)mm vs. (24.4±1.8)mm], good rate of reduction quality (80% vs. 85%) and implant related complications (5% vs. 2%) ( P<0.05 or 0.01). Conclusion:For treatment of elderly patients with intertrochanteric fractures, compared to the lateral position, the half lithotomy position in PFNA-II internal fixation can reduce frequency of fluoroscopy, improve quality of fracture reduction and reduce implant-related complications.
7.Validation and application of an artificial intelligence robot assisted diagnosis system for diabetic retinopathy
Shaohui GAO ; Xuemin JIN ; Zhaoxia ZHAO ; Weihong YU ; Youxin CHEN ; Yuhui SUN ; Dayong DING
Chinese Journal of Experimental Ophthalmology 2019;37(8):669-673
Objective To evaluate the performance of an artificial intelligence ( AI ) assisted diagnosis system for diabetic retinopathy ( DR) based on deep learning theory. Methods Diagnostic performance of a robot assisted diagnosis system called SongYue for DR was trained by using 25297 retinal images tagged by fundus doctors from multiple hospitals in China. Four types of DR detection model consisting of abnormal DR,referable DR,severe non-proliferative and proliferative DR as well as proliferative DR according to fundus leisions identification were established. The ability of the system to distinguish DR was determined by using receiver operator characteristic (ROC) analysis,sensitivity and specificity of the system. Results SongYue system achieved an area under the ROC curve ( AUC) of 0. 920 for successfully distinguishing normal images from those DR with a sensitivity of 96. 0%at a specificity of 87. 9%. The AUC of SongYue for referable DR was 0. 925,sensitivity was 90. 4%,and specificity was 95. 2%. For severe non-proliferative and proliferative DR,AUC was 0. 845,sensitivity was 72. 7%,and specificity was 96. 2%. For proliferative DR, AUC was 0. 855, sensitivity was 73. 5%, and specificity was 97. 3%. Conclusions SongYue robot assisted diagnosis system has high AUC,sensitivity and specificity for identifying DR, showing good clinical applicable benefits.
8. Regulation of hypoxia inducible factor-1α on permeability of vascular endothelial cells and the mechanism
Delin HU ; Youxin YU ; Rong LIANG ; Shunying ZHOU ; Shengliang DUAN ; Zhiyong JIANG ; Chengying MENG ; Wei JIANG ; Huan WANG ; Yexiang SUN ; Linsen FANG
Chinese Journal of Burns 2019;35(3):209-217
Objective:
To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α) on permeability of rat vascular endothelial cells and the mechanism.
Methods:
Twelve male Sprague-Dawley rats aged 35 to 38 days were collected and vascular endothelial cells were separated and cultured. The morphology of cells was observed after 4 days of culture, and the following experiments were performed on the 2nd or 3rd passage of cells. (1) Rat vascular endothelial cells were collected and divided into blank control group, negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were transfected with GV248 empty plasmid, recombinant plasmid respectively containing HIF-1α interference sequence 1, interference sequence 2, and interference sequence 3 with liposome 2000. Cells in blank control group were only transfected with liposome 2000. After transfection of 24 h, expression levels of HIF-1α mRNA and protein of cells in each group were respectively detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction and Western blotting (the same detecting methods below) . The sequence with the highest interference efficiency was selected. (2) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α low expression group, with 3 wells in each group. Cells in blank control group were only transfected with liposome 2000, and cells in negative control group and HIF-1α low expression group were respectively transfected with GV248 empty plasmid and low expression HIF-1α recombinant plasmid selected in experiment (1) with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α in each group were detected. (3) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α high expression group, with 3 wells in each group. Cells in blank control group were transfected with liposome 2000, and cells in negative control group and HIF-1α high expression group were respectively transfected with GV230 empty plasmid and HIF-1α high expression recombinant plasmid with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α of cells in each group were detected. (4) After transfection of 24 h, cells of three groups in experiment (1) and three groups in experiment (2) were collected, and mRNA and protein expressions of myosin light chain kinase (MLCK), phosphorylated myosin light chain (p-MLC), and zonula occludens 1 (ZO-1) of cells were detected. Data were processed with one-way analysis of variance and
9.Intravitreal aflibercept versus photodynamic therapy in Chinese patients with neovascular age-related macular degeneration: outcomes of the SIGHT study
Xiaoxin LI ; Youxin CHEN ; Junjun ZHANG ; Xun XU ; Feng ZHANG ; Chui Ming Gemmy Cheung ; Yu RUI ; Kazmi HUSAIN ; Sowade OLAF ; Oliver Zeitz on behalf of the SIGHT study group
Chinese Journal of Ocular Fundus Diseases 2019;35(2):156-165
Objective To assess the efficacy and safety ofintravitreal aflibercept injection (IAI) compared with photodynamic therapy (PDT) in the treatment of Chinese patients with predominantly classic subfoveal choroidal neovascularization (CNV) lesions secondary to neovascular age-related macular degeneration (nAMD).Methods A randomized,double-blind,multi-center phase-3 clinical trial lasting for 52weeks (from December 2011 to August 2014).Subjects were randomized in a 3:1 ratio to either IAI group or PDT-to-IAI group.Subjects in the IAI group received 2 mg IAI at baseline and at week 4,8,16,24,32,40,48,with sham injection at week 28,36.Subjects in the PDT-to-IAI group were forced to receive PDT once at baseline and more time at week 12,24 if PDT retreatment conditions were met.Sham injections were given in PDT-to-IAI group at baseline and at week 4,8,16 and 24,followed by 2 mg IAI at week 28,32,36,40,48.The primary outcome of efficacy were the change in mean Best Corrected Visual Acuity (BCVA) from baseline to week 28,and that of week 52.Safety evaluation included the percentage of subjects who suffered treatment emergent adverse events (TEAEs).Results Among the 304 subjects enrolled,there were 228 and 76 cases in IAI group and PDT-to-IAI group respectively.At week 28,the changes of mean BCVA in IAI group,PDT-to-IAI group compared to baseline were +14.0,+3.9 letters,respectively.At week 52,the changes of mean BCVA in two groups were + 15.2,+8.9 letters respectively with the difference of +6.2 letters (95%CI 2.6-9.9,P=0.000 9).At week 52,the mean foveal retinal thickness in the two groups decreased by-189.6,-170.0 μm,respectively.Subjects with the most BCVA increase in IAI group were those aged <65,and those with active CNV lesion area <50% of total lesion area.The most common TEAEs in IAI group and PDT-to-IAI group are macular fibrosis [11.8% (27/228),6.6% (5/76)] and BCVA decline [6.6% (15/228),21.1% (16/76)].There were 3 cases of arterial thromboembolic events defined in the antiplatelet experimental collaboration group,but all were considered unrelated to interventions.Conclusions The efficacy of aflibercept is superior to that of PDT in nAMD patients in China.The therapeutic effect of aflibercept persisted to week 52 in all subjects.The rate of adverse events was consistent with the safety data of aflibercept known before.
10. Influence of three-level collaboration network of pediatric burns treatment in Anhui province on treatment effects of burn children
Zhengguo XIA ; Xianliang ZHOU ; Weichang KONG ; Xingzhao LI ; Junhui SONG ; Linsen FANG ; Delin HU ; Chen CAI ; Yizhong TANG ; Youxin YU ; Chunhua WANG ; Qinglian XU
Chinese Journal of Burns 2018;34(3):143-148
Objective:
To explore the influence of three-level collaboration network of pediatric burns in Anhui province on treatment effects of burn children.
Methods:
The data of medical records of pediatric burn children transferred from Lu′an People′s Hospital and Fuyang People′s Hospital to the First Affiliated Hospital of Anhui Medical University from January 2014 to December 2015 and January 2016 to September 2017 (before and after establishing three-level collaboration network of pediatric burns treatment) were analyzed: percentage of transferred burn children to hospitalized burn children in corresponding period, gender, age, burn degree, treatment method, treatment result, occurrence and treatment result of shock, and operative and non-operative treatment time and cost. Rehabilitation result of burn children transferred back to local hospitals in 2016 and 2017. Data were processed with


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