Objective To investigate the effect of FUNDC1 tyrosine phosphorylation site mutations on mitophagy in H9c2 myocardial cells by constructing tyrosine site mutant plasmids (Y11 and Y18) of the FUN14 domain-containing protein 1 (FUNDC1). Methods The mutant plasmids constructed by whole-gene synthesis were transfected into rat myocardial H9c2 cells and divided into five groups: empty plasmid group, FUNDC1 overexpression group, Y11 mutant group, Y18 mutant group, and Y11 combined with Y18 mutant group. The viability of H9c2 cells was assessed using the CCK-8 assay. Additionally, tetramethylrhodamine ethyl ester (TMRE) staining was utilized to detect mitochondrial membrane potential. The protein expression levels of FUNDC1, translocase of the outer mitochondrial membrane 20 (TOM20), and cytochrome c oxidase IV (COX IV) were detected by Western blot analysis. Confocal microscopy was used to evaluate transfection efficiency as well as the co-localization of mitochondria and lysosomes. Results The FUNDC1 overexpression, Y11, Y18, and Y11 combined with Y18 mutant plasmids were successfully constructed. After plasmid transfection, widespread GFP fluorescence expression was observed under confocal microscopy. Compared with the empty plasmid group, FUNDC1 protein expression levels were significantly increased in the FUNDC1 overexpression group, Y11 mutation group, Y18 mutation group, and Y11 combined with Y18 mutation group, while cell viability and mitochondrial membrane potential showed no significant changes. Compared to the empty plasmid group, cells transfected with Y18 and Y11 combined with Y18 mutant plasmids showed increased TOM20 and COX IV expression levels and decreased mitochondrial-lysosomal co-localization. Conclusion Transfection with FUNDC1 Y18 or Y11 combined with Y18 mutant plasmids inhibited mitophagy in H9c2 myocardial cells.
Animals ; Rats ; Mitophagy/genetics* ; Myocytes, Cardiac/cytology* ; Mitochondrial Proteins/metabolism* ; Mutation ; Phosphorylation ; Tyrosine/genetics* ; Cell Line ; Membrane Proteins/metabolism* ; Membrane Potential, Mitochondrial

Objective: To study the role of rs 12145833 polymorphism of SDCCAG 8 gene in the intervention of childhood obesity, so as to provide scientific basis for formulating personalized intervention measures based on genetic background in children with obesity. Methods: From September 2018 to June 2019, a total of 393 children aged 8-10 years in Beijing were enrolled in a cluster randomized controlled trial. Eight schools were randomly allocated into intervention group and control group at a ratio of 1∶1. Saliva DNA samples were collected to detect rs 12145833 polymorphism of SDCCAG 8 gene. The intervention group received a comprehensive intervention, while the control group received usual practice. Intervention measures included diet improvement, sports, school amd family sport. The obesity related indicators were measured at baseline and after the end of intervention 1 academic year. Multiple linear regression and Logistic regression were used to analyze the interaction between genes and intervention on obesity indicators. Results: In the intervention group, children with TT genotype of rs 12145833 of the SDCCAG 8 gene had less increase in systolic( β=4.56, 95%CI=1.84-7.28, P <0.01) and diastolic blood pressure( β=2.59, 95%CI=0.45-4.73, P <0.05) than those with GT and GG genotypes. In the control group, the systolic blood pressure of children with TT genotype increased more than those with GT and GG genotype( β=-2.86, 95%CI=-5.63--0.83, P <0.05). There was an interaction between rs 12145833 polymorphism of SDCCAG 8 gene and intervention on systolic blood pressure, diastolic blood pressure and body fat percentage in children( P <0.05). Conclusion Children with TT genotype of rs 12145833 in the SDCCAG 8 gene are more sensitive to obesity intervention than those with GG and GT genotypes, especially in the improvement of systolic blood pressure, diastolic blood pressure and body fat percentage. Further trials to study the role of rs 12145833 polymorphism of SDCCAG 8 gene in the intervention of childhood obesity among different ethnic populations are needed.

Objective:To investigate the clinical efficacy of gastroscopic 'sandwich’ injection and sequential laparoscopic splenectomy combined with pericardial devascularization in the treatment of gastric variceal bleeding.Methods:The retrospective cohort study was conducted. The clinical data of 52 patients with cirrhotic portal hypertension combined with gastric variceal bleeding who were admitted to Affiliated Hospital of Jiaxing University between March 2011 and October 2019 were collected. There were 33 males and 19 females, aged (63±9)years, with a range of 41-83 years. Of the 52 patients, 31 undergoing gastroscopic 'sandwich’ injection and sequential laparoscopic splenectomy combined with pericardial devascularization and 21 undergoing laparoscopic splenectomy combined with pericardial devascularization were allocated into sequential group and laparoscopic group, respectively. Observation indicators: (1) surgical situations; (2) complications; (3) changes in gastric varices after treatment; (4) follow-up. Follow-up was performed using telephone interview combined with outpatient examination to detect survival of patients up to December 2019. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was analyzed by the t test. Measurement data with skewed distribution were represented as M (range), and comparison between groups was analyzed by the Mann-Whitney U test. Count data were expressed as absolute numbers, and comparison between groups was analyzed using the chi-square test or Fisher exact probability. Ordinal data was analyzed by nonparametric rank sum test. Results:(1) Surgical situations: patients of sequential group and laparoscopic group underwent surgery successfully. The operation time and volume of intraoperative blood loss of the sequential group were (112±16)minutes and (57±11)mL, respectively, versus (103±14)minutes and (55±9)mL of the laparoscopic group, showing no significant difference in the above indicators between the two groups ( t=0.963, 1.052, P>0.05). (2) Complications: 11 patients in the sequential group had postoperative complications including 1 case with perioperative bleeding, 2 cases with postoperative gastrointestinal rebleeding, 4 cases with ascites, 4 cases with portal vein thrombosis. There was no death in the sequential group. Sixteen patients in the laparoscopic group had postoperative complications including 2 cases with perioperative bleeding, 6 cases with postoperative gastrointestinal rebleeding, 4 cases with ascites, 4 cases with portal vein thrombosis. Three patients died in the laparoscopic group. There was no significant difference in the cases with perioperative bleeding or cases with ascites between the two groups ( P>0.05) and no significant difference in the cases with portal vein thrombosis or death between the two groups ( χ2=0.082, 0.082, P>0.05). There was a significant difference in the cases with postoperative gastrointestinal rebleeding between the two groups ( P<0.05). Cases with postoperative gastrointestinal rebleeding, cases with ascites, cases with portal vein thrombosis in the sequential group were cured after the treatment of gastroscopy, low salt diet combined with diuretic or low dose warfarin, respectively. Of the 6 patients with postoperative gastrointestinal rebleeding in the laparoscopic group, 3 were cured after the treatment of gastroscopy and 3 died due to failure to rescue in time. Cases with ascites and cases with portal vein thrombosis in the laparoscopic group were cured after the treatment of low salt diet plus diuretic or low dose warfarin, respectively. (3) Changes in gastric varices after treatment: at postoperative 6 months, 31 patients in the sequential group were diagnosed with negative gastric varices; 15 of 21 patients in the laparoscopic group were diagnosed with negative gastric varices, 3 cases were diagnosed with obvious gastric varices and 3 cases were diagnosed with severe gastric varices. There was a significant difference in the cases with gastric varices between the two groups ( Z=-3.128, P<0.05). At postoperative 12 months, 29 patients in the sequential group and 13 patients in the laparoscopic group were diagnosed with negative gastric variceal. There were 2 patients in the sequential group diagnosed with obvious gastric varices, and 8 patients in the laparoscopic group diagnosed with gastric varices including 3 cases with obvious gastric varices and 5 cases with severe gastric varices. There was a significant difference in the cases with gastric varices between the two groups ( Z=-2.933, P<0.05). Cases with obvious gastric varices in the sequential group were cured after the treatment of gastroscopy. Cases with obvious or severe gastric varices in the laparoscopic group were cured after the treatment of gastroscopy except 1 died due to massive gastrointestinal hemorrhage. (4) Follow-up: 52 patients were followed up for 1-8 years, with a median time of 4 years. All the 31 patients in the sequential group and 18 ptients in the laparoscopic group survived. Conclusion:Gastroscopic 'sandwich’ injection and sequential laparoscopic splenectomy combined with pericardial devascularization in the treatment of gastric variceal bleeding has low recurrence rate of varicosity and low incidence of rebleeding.

Objective: To investigate the feasibility of echocardiography-guided closed-chest repeated intraventricular blood sampling in mice, and to clarify the maximum blood volume that can be collected by this method, and whether the method can be used for long-term repeated blood collection in mice. Methods: Twenty-four male C57BL/6J mice (10-14 weeks old) were divided into the terminal experiment group (n=4, for investigating the maximum blood amount that could be sampled at one time), the repeated 0.5 ml blood collection group (n=10, sampling 0.5 ml whole blood each time, once every two days for consecutive 4 weeks), and the repeated 0.75 ml blood collection group (n=10, sampling 0.75 ml whole blood each time, once every two days for consecutive 4 weeks). High-frequency echocardiography was used to display the largest section of the left ventricle, guiding the insulin syringe needle through the thorax into the left ventricle for blood collection. In the repeated 0.5 ml blood collection group, echocardiography was used to detect the cardiac structure and function before blood collection, three minutes after blood collection, and one week after the last (the 14th) blood collection. Results: We successfully performed echocardiography-guided closed-chest intraventricular blood sampling, with an average operating time (88±19)s per mouse, and a maximum blood volume (1.43±0.11)ml per mouse. In the repeated 0.5 ml blood collection group, heart rate, left ventricular ejection fraction, left ventricular fractional shortening, left ventricular end-diastolic dimension and left ventricular posterior wall end-diastolic thickness remained uncganged before the first blood collection and after 4 weeks of repeated blood collection (all P>0.05). No death in the repeated 0.5 ml blood collection group. However, in the 0.75 ml blood collection group, two mice died before the end point. Conclusions The echocardiography-guided closed-chest intraventricular blood sampling is a safe, minimally invasive, convenient and efficient method, and can be used repeatedly for long-term blood collection in mice.

AIM:We investigated how AT 1-R stimulated by mechanical stresses induces cardiac fibrosis .METHODS:We produced in vivo cardiac pressure overload model in angiotensinogen knockout ( ATG-/-) mice and in vitro mechanically-stretched cell model in cultured neonatal cardiac cells of ATG-/-mice both lack the participation of Ang II .RESULTS: Pressure overload for 4 weeks in ATG-/-mice induced myocardial hypertrophy accompanied by the significant interstitial fibrosis , however , the TGF-β, a key regulatory factor of fibrosis, was not significantly increased in these ATG-/-mice.Meanwhile, the inhibitor for AT1-R significantly inhibited mechani-cal stress-induced cardiac fibrosis in these ATG-/-models whereas inhibition of TGF-βdid not.CONCLUSION:The results showed that mechanical stress-induced fibrotic responses through AT 1-R required the phosphorylation of Smad 2 but not the involvement of TGF-β.