1.Succinate/GPR91 promotes mitochondrial damage in vascular endothelial cells through DHODH/CoQ10
Wenhua QIN ; Chuchu YUAN ; Yuhui SUN ; Bo YU ; Dangheng WEI
Chinese Journal of Arteriosclerosis 2024;32(6):466-472
Aim To explore the effect of succinate/G protein coupled receptor 91(GPR91)on mitochondria in vascular endothelial cells and its regulatory mechanisms.Methods Transmission electron microscopy,Western blot and fluorescence microscopy were used to observe the effects of succinate analogues diethyl succinate(DS),GPR91 agonist and inhibitor on the mitochondrial morphology,cristae,cristate homeostasis related proteins reactive oxygen species(ROS)content,Ca2+concentration,mitochondrial membrane potential,the expression of dihydroorotate dehydrogenase(DHODH)and oxidized coenzyme Q10(CoQlO).Fluorescence probes were used to observe the effect of DHODH inhib-itor and CoQ10 on ROS level and Ca2+concentration of endothelial cells.Results After DS treatment,the mitochon-dria showed pyknosis and mitochondrial volume significantly decreased,electron density of the mitochondrial membrane in-creased,and the number of cristae decreased in endothelial cells;the expression of cristae homeostasis related proteins MIC60 decreased by 23%,while cellular ROS level and Ca2+concentration increased;mitochondrial membrane potential decreased(P<0.05 or P<0.01).After GPR91 agonist treatment,the expression of cristae homeostasis related proteins MIC60 decreased by 31%,meanwhile,cellular ROS level increased by 27%and Ca2+concentration increased by 36%;mitochondrial membrane potential decreased(P<0.05 or P<0.01).After GPR91 inhibitor treatment,the expression of cristae homeostasis related proteins MIC60 increased by 22%and ATP5I increased by 40%;the levels of ROS decreased by 41%and Ca2+concentration decreased by 67%;and the mitochondrial membrane potential was restored to normal(P<0.05 or P<0.01).After DS treatment,the expression of DHODH decreased by 43%and the level of oxidized CoQ10 in-creased by 120%(P<0.05 or P<0.01).After GPR91 agonist treatment,the expression of DHODH decreased by 22%and the level of oxidized CoQ10 increased by 36%(P<0.05 or P<0.01).After GPR91 inhibitor treatment,the expres-sion of DHODH increased by 40%and the level of oxidized CoQ10 decreased by 39%(P<0.01).After DHODH inhibi-tor treatment,the ROS level increased by 20%and Ca2+concentration increased by 28%,and mitochondrial membrane po-tential reduced at same time(P<0.05 or P<0.01).Exogenous oxidized CoQ10 inhibited ROS production by 30%and decreased Ca2+concentration by 20%(P<0.05 or P<0.01).Conclusion Succinate/GPR91 promotes mitochondrial damage in endothelial cells,and its mechanism may relate to down-regulating the expression of DHODH and inhibiting the reduction of CoQ10 by affecting the mitochondrial cristae homeostasis.
2.Biological characteristics of Escherichia coli phage and Staphylococcus aureus phage isolated from sewage
Xiangni WANG ; Caiqin MA ; Jinren LIU ; Na LIU ; Xiying WANG ; Jiajia LU ; Chuchu KANG ; Xingmin SHI ; Wang YUAN
Journal of Xi'an Jiaotong University(Medical Sciences) 2023;44(5):823-829
【Objective】 Escherichia coli phage (ECP) and Staphylococcus aureus phage (SAP) isolated from sewage were used as research objects, and their biological characteristics were analyzed to provide new experimental materials for the application of phages. 【Methods】 ECP and SAP were purified and cultured by double-layer agar method. Then a series of biological characteristics of these two phages were preliminarily analyzed by electron microscope observation, optimal multiplicity of infection (MOI) test, one-step growth curve test, temperature, pH, chloroform and ultraviolet sensitivity tests, respectively. 【Results】 The results of biological characteristics showed that ECP and SAP were both virulent phages, belonging to myoviridac family. Their optimal MOI was 10-1, and they had strong resistance to ultraviolet light. The cleavage volume of ECP was 76.3 PFU/cell, while that of SAP was 8.3 PFU/cell. ECP had a wide range of temperature tolerance and could stably survive at 30-50 ℃, while SAP was more sensitive to temperature and could be completely inactivated at 50 ℃ for 1 h. ECP could maintain a good lysis activity in the range of pH 5-11, while SAP in the range of pH 6-9. ECP had strong resistance to chloroform and was non-membranous phage, while SAP was more sensitive to chloroform and was a membranous phage. 【Conclusion】 ECP and SAP are both virulent phages and have strong resistance to ultraviolet light. The lysability, temperature, pH, and chloroform tolerance of ECP are stronger than those of SAP.