1.Screening of ARVC desmosomal protein gene mutation in people from Yunnan unexplained sudden death area in Xiangyun County, Dali Prefecture, Yunnan Province
Lin MA ; Yuebing WANG ; Xue TANG ; Wenjuan LI ; Chunli DUAN ; Puping LEI ; Yanmei XI ; Mengyao SUN ; Yi DONG
Chinese Journal of Endemiology 2021;40(8):605-609
Objective:To investigate the mutation of desmosomal protein gene of arrhythmogenic right ventricular cardiomyopathy (ARVC) in people from Yunnan unexplained sudden death (YUSD) area in Xiangyun County, Dali Prefecture, Yunnan Province, and to explore the etiological relationship between the mutation of ARVC desmosomal protein gene and YUSD.Methods:The autopsy cardiac blood sample of YUSD case ( n = 1) and the peripheral venous blood samples of the same time case ( n = 1) and relatives of YUSD case ( n = 16) were collected in Xiangyun County. Blood DNA was extracted for PCR amplification and sequencing of a total of 97 exons of the ARVC desmosomal protein genes [plakophilin 2 (PKP2), junction plakoglobin (JUP), desmoplakin (DSP), desmoglein 2 (DSG2) and desmocollin 2 (DSC2)] were conducted by Sanger method. At the same time, basic information and genetic family of YUSD case, the same time case and relatives of YUSD case were investigated, and gene mutations were comprehensively analyzed. Results:The YUSD case and the same time case carried JUP, DSP and DSG2 gene mutations. Among the relatives of YUSD case, 2, 14, 16, 15 and 4 cases had mutations in PKP2, JUP, DSP, DSG2 and DSC2 genes, respectively. The YUSD case, the same time case and the relatives of YUSD case carried 6 identical mutation sites: JUP gene exon 3 c.213 T>C synonymous mutation, exon 14 c.2089 A>T missense mutation; DSP gene exon 19 c.2631 G>A synonymous mutation, exon 24 c.8472 G>C synonymous mutation; DSG2 gene exon 8 c.861 C>T synonymous mutation, and exon 15 c.3321 T>C synonymous mutation.Conclusion:In Xiangyun County, six identical mutation sites (JUP gene c.213 T>C and c.2089 A>T, DSP gene c.2631 G>A and c.8472 G>C, DSG2 gene c.861 C>T and c.3321 T>C) carried by YUSD case, the same time case and the relatives of YUSD case may be related to the incidence of some YUSD cases.
2.Effects of stress-induced protein Sestrin2 on necroptosis of dendritic cells induced by lipopolysaccharide
Mengyao WU ; Renqi YAO ; Yu DUAN ; Lu WANG ; Liyu ZHENG ; Pengyi HE ; Ning DONG ; Yao WU ; Yongming YAO
Chinese Critical Care Medicine 2024;36(3):237-243
Objective:To investigate the effect of stress-induced protein Sestrin2 (SESN2) on necroptosis of mouse dendritic cell (DC) induced by lipopolysaccharide (LPS) combined with zVAD, a panaspartate-specific cysteine protease (caspase) inhibitor.Methods:The DC2.4 cell line derived from the bone marrow of mouse in the 3rd to 10th generations was cultured. The cells were stimulated with LPS for 0 hour, 6 hours, 12 hours, and 24 hours, and grouped according to the stimulation time points. Western blotting was performed to determine the protein expression of SESN2 in each group. Overexpression empty lentivirus (NC), SESN2 gene overexpression RNA sequence lentivirus (SESN2 LV-RNA), small interfering empty lentivirus (NS), and SESN2 gene small interfering RNA sequence lentivirus (SESN2 siRNA) were transfected into DC2.4 cells. After 72 hours of transfection, cell fluorescence expression was observed under the inverted fluorescence microscope. Cells in each transfection group were stimulated with LPS for 24 hours. The blank control groups were set up and cultured with phosphate buffered saline (PBS) for 24 hours. Western blotting was performed to measure SESN2 protein expression. In the same groups as above, cells were stimulated with LPS+zVAD for 24 hours. The blank control groups were set up and cultured with PBS for 24 hours. Western blotting was used to determine the expression of mixed lineage kinase domain-like protein (MLKL) and phosphorylated-MLKL (p-MLKL). The p-MLKL levels and the number of positive cells were observed using laser scanning confocal microscopy. The necroptotic cell ratios were assessed by both flow cytometry and Hoechst staining.Results:Compared to the LPS 0 hour group, the expression of SESN2 in the LPS 24 hours group showed a significant increase. Therefore, 24 hours was chosen as the subsequent stimulation time point. After successful lentivirus transduction and 24 hours of cultivation, the MLKL phosphorylation level in the SESN2 siRNA+LPS+zVAD group was significantly higher than that in the NS+LPS+zVAD group. The MLKL phosphorylation in the SESN2 LV-RNA+LPS+zVAD group was significantly lower than that in the NC+LPS+zVAD group. The MLKL phosphorylation levels in both the NS+LPS+zVAD group and the NC+LPS+zVAD group were obviously higher than those in the NS+PBS group and the NC+PBS group, respectively. Laser scanning confocal microscopy showed that the trends in quantity and fluorescence intensity of p-MLKL protein expressions were consistent with the above results. The results from flow cytometry analysis and Hoechst staining showed that the rates of cell necrotic apoptosis in SESN2 siRNA+LPS+zVAD group were significantly higher than those in NS+LPS+zVAD group [flow cytometry analysis: (30.800±1.153)% vs. (20.800±1.114)%, Hoechst staining: (75.267±0.451)% vs. (46.267±3.371)%, both P < 0.05], indicating that knocking down SESN2 further exacerbated the occurrence of necroptosis. The necrotic apoptosis rates in SESN2 LV-RNA+LPS+zVAD group were significantly lower than those in NC+LPS+zVAD group [flow cytometry analysis: (7.160±0.669)% vs. (19.240±2.322)%, Hoechst staining: (32.433±3.113)% vs. (48.567±4.128)%, both P < 0.05], indicating that overexpressing SESN2 reversed such response and markedly reduced the proportion of necroptotic cells compared to the corresponding empty vector group. Conclusion:SESN2 exhibits an inhibitory effect on necroptosis of DC in sepsis. Targeted SESN2 expression may regulate the process of DC-mediated immune response in sepsis.
3.Research on Objective Characteristics of Tongue Manifestation in 315 Patients with Coronary Heart Disease
Mengyao DUAN ; Chuhao WANG ; Yuquan TAN ; Kun LIAN ; Xintian SHOU ; Yang JIANG ; Boyan MAO ; Zhixi HU
Journal of Traditional Chinese Medicine 2024;65(9):921-927
ObjectiveTo investigate the objective characteristics of tongue manifestations in patients with coronary heart disease (CHD). MethodsA total of 315 participants with CHD were recruited in the CHD group, and 211 healthy participants who underwent physical examination were recruited as the healthy control group. In addition, according to the common comorbidities (primary hypertension, carotid atherosclerosis, type 2 diabetes mellitus, fatty liver, hyperlipidaemia, heart failure, and cerebral infarction) in 315 participants with CHD, each comorbidity was classified into a group of comorbidities with that disease and a group of non-comorbidities. Tongue images were captured using a TFDA-1 tongue diagnostic instrument to characterise the tongue body (TB) and tongue coating (TC), comparing the RGB, HIS, and Lab colour spaces in the chromaticity index (R, red; G, green; B, blue; H, hue; I, intensity; S, saturation; L, lightness; a, red-green axis; b, yellow-blue axis), the tongue coating thickness index (per-All), contrast (CON), angular second moment (ASM), entropy (ENT), and mean (MEAN) in texture metrics. ResultsCompared with the healthy control group, the characteristic indexes of tongue body in CHD group showed lower TB-R, TB-G, TB-B, TB-I, TB-L and higher TB-H, TB-b; and the characteristic indexes of tongue coating in CHD group showed lower TC-R, TC-B and higher TC-CON, TC-MEAN, TC-H, TC-b (P<0.05 or P<0.01). Compared with non-combined primary hypertension group, CHD combined primary hypertension group showed higher per-All, TB-G, TB-L, and lower TB-a, TC-a (P<0.05); compared with the non-combined carotid atherosclerosis group, CHD combined carotid atherosclerosis group showed higher TB-CON, TB-ENT, TB-MEAN, and lower TB-ASM (P<0.05 or P<0.01); compared with the non-combined type 2 diabetes mellitus group, CHD combined type 2 diabetes mellitus group showed lower per-All and higher TB-H (P<0.05 or P<0.01); compared with the non-combined fatty liver group, CHD combined fatty liver group showed higher TB-CON, TB-MEAN, TB-ENT, and lower TB-ASM and TC-S (P<0.05 or P<0.01); compared with the non-combined hyperlipidaemia group, CHD combined hyperlipidaemia group showed lower TB-S and TB-a (P<0.05); compared with non-combined heart failure group, CHD combined heart failure group showed lower TB-R, TB-G, TB-I, TB-L, and higher TB-a (P<0.05 or P<0.01); compared with non-combined cerebral infarction group, CHD combined cerebral infarction group showed higher TC-CON, TC-ENT, TC-MEAN, and lower TC-ASM (P<0.05 or P<0.01). ConclusionCompared to healthy individuals, patients with CHD tend to have darker tongue colours and rougher TC textures. Compared with non-comorbidity participants, those with primary hypertension tended to be lighter tongue colour and thicker tongue coating, those with carotid atherosclerosis had paler tongue body, those with type 2 diabetes mellitus had thinner tongue coating, those with fatty liver disease had paler tongue body and whiter tongue colour, those with hyperlipidaemia and heart failure had paler tongue colour, and those with cerebral infarction had rougher tongue texture.