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Objective To investigate the effect of high fat diet feeding on mitochondrial structure and function. Methods Male C57BL/6J mice at the age of 4 weeks were used in this study. After 6 weeks of regular diet(RD)or high-fat diet(HF)feeding, the high fat-induced obesity phenotype was confirmed by body weight measurement and liver histopathology. RNA was isolated from the liver tissue of RD and HF mice and the expression profiles were detected using RNA-seq. Differentially expressed genes between RD and HF mice were analyzed using BRB-ArrayTools. DAVID online tools were applied to analyze the GO and KEGG pathways. Transmission electron microscopy and western blotting were performed to observe the mitochondrial ultrastructure and quantified the expression of function-related proteins. Results Compared with the RD mice,the body weight gain was faster in the HF mice. The size of the lipid droplets was bigger in the HF-fed mouse liver tissue. Multiple pathway analysis all identified that these major gene changes were related to mitochondria. The mitochondrial deformation,enlarged or even destruction was observed in the high fat diet group observed by transmission electron microscopy. This observation was further confirmed by detecting of the expression of genes in the HF liver mitochondria. The levels of MFN1 and PHB1 were significantly increased, while the level of FKBP51 was significantly decreased. Conclusions FKBP51 is involved in the high-fat-induced mitochondrial damage via morphological and structural damages of mitochondria.
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Objective To study the function of Fkbp51 in the heart and liver by analyzing the differential RNA expression profiles in the wild-type mice (WT) and Fkbp51 knockout (KO) mice, and to elucidate the role of Fkbp51 gene in metabolic pathways in the heart and liver.Methods Using the second generation of high-throughput gene sequencing technology, the mRNA expression profiles of heart and liver were sequenced in WT and Fkbp51 KO mice.The data of sequencing of heart tissues were analyzed by DEGseq, and the results of sequencing of liver tissues were analyzed by BRB-Array Tools.The differential genes of the heart and liver in the mice were screened respectively.Gene ontology (GO) analysis and KEGG pathway analysis were performed to analyze the differentially expressed genes using the online tool DAVID.In addition, the differential genes of the two organ tissues were analyzed by Venn diagram.The interaction network of proteins was analyzed using the STRING database.Results (1) The absence of Fkbp51 led to changes in mRNA expressions of heart-related signal pathways such as vascular smooth muscle contraction, chemokine, retinol, and MAPK signaling pathways.(2) The lack of Fkbp51 mostly induced changes in cholesterol synthesis and metabolism, lipid metabolism, redox and other related genes and pathways in the liver.(3) In the heart and liver, Fkbp51 deletionresult ed in four co-differential genes, among them, down-regulation of Rnaset2b, Hmga1 and Fkbp51, while Cyp2b10 was down-regulated in the heart but up-regulated in the liver.All these proteins may interact with HSP90 protein and participat in the metabolism of heart and liver tissues.Conclusions Fkbp51 is involved in different metabolic and gene expression regulation pathways of heart and liver, and the roles are both independent and interrelated.
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Objective To determine if fetal stem cells can enter the maternal circulation during pregnancy and re-pair the injuries of maternal heart.Methods C57 female mice at the age of 6-8 weeks were randomly assigned to three groups:sham control, surgery without pregnancy, and surgery with pregnancy ( n=8,eath group) .The control sham group was developed by opening and closing of the chest.The other two groups underwent heart surgery.The myocardial infarc-tion ( MI) model was induced by ligation of the left anterior descending coronary artery.Half of the surgical mice mated with e-GFP transgenic male mice, and another half group was not.Electrocardiogram ( ECG) and echocardiographic images were recorded at pre-operation, post-operation and postpartum.The collected data were used to evaluate the heart function. The GFP expression was detected by immunohistochemistry and q-PCR.Results When compared with the sham group, both the ischemia surgery groups with and without pregnancy, the ECG ST segment was significantly increased.This meas-urement indicated that the myocardial ischemia surgery was successful, and no significant difference in the ST segments be-tween two ischemia surgery groups was found.However, when ECG was measured in the surgical mice after postpartum, their myocardial ischemia was dramatically improved when compared with that of the ischemia surgery only mice.Echocar-diographic images also indicated that both the surgery groups had myocardial ischemia, however, no significant difference was observed in the pregnant mice before and after postpartum.The order of the cardiac function indexes from high to low was the sham group, surgery with pregnancy group, and surgery with no pregnancy group;in particular, the cardiac func-tion of pregnancy group was significantly enhanced compared with that of the surgery with no pregnancy group (P<0.05). More importantly, both immunofluorescence and q-PCR results showed that the embryonic stem cell translocation through circulation system with GFP expression in the heart of pregnancy group, while negative in other two groups.Conclusions Embryonic stem cells can be transferred into the maternal circulation of pregnant mice, and play a role in the repairing of their cardiac injuries.
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Objective To investigate the effect of protein phosphatase 5 (PP5) on lipid metabolism in the PP5 knockout (KO) mice.Methods Male PP5 KO and wild type (WT) mice at the age of 6 weeks were used in this study. In order to study the effect of high fat diet ( HFD) feeding, the body weight was measured.The liver histology was examined by HE and oil red O staining.To further verify PP5 functions in the adipogenesis, in vitro experiment was carried out using mouse embryonic fibroblasts (MEF).Western blotting and real-time PCR were performed to quantified the expression of lipid metabolism-related genes in the liver tissues.Results Compared with the WT mice, the body weight gain was slower in the KO mice.The size of the lipid droplets was smaller and the quantity was less in the KO mouse liver tissue.In vitro study revealed that the KO mouse MEF cells showed less differentiated adipocytes with smaller lipid droplets than the WT MEF cells.This observation was further confirmed by detecting the expression of adipogenesis-related genes in the HFD liver.The markers of adipocyte differentiation, such as CD36, AP2, PPARγ2, and Glut4, were significantly decreased, while energy expenditure-related markers, such as phosphorylation of GR and expression of UCP1, were significantly increased.Conclusions Protein phosphatase 5 may play a regulatory role in the mouse lipid metabolism through regulating the de-phosphorylation of p-GR and enhancing the expression of UCP1.
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Objective The purpose of this study was to establish a rapid method for synthesis and detection of guild RNA (gRNA) which is an essential component in CRISPR/Cas9 knockout technology .Methods First, the Nkp46 gRNA core fragment was synthesized as amplification template .Second , the forward and reversed primers of the matched gRNA were designed using the Nkp46 gene as reference sequence .Third, the DNA fragment of Nkp46 gRNA was amplified by PCR technology using the synthesized gRNA core fragment as template .The gRNA was reversely transcribed in vitro u-sing amplified DNA fragment as template .The efficiency and specificity of gRNA and its interaction with Cas 9 were detec-ted in vitro.Results The specificity and activity of Nkp46 gRNA were high .The obtained gRNA interacted with Cas 9 en-zyme and successfully cut the target double-stranded DNA at the designed site .Conclusions The method for synthesis and detection of gRNA established in this study is faster than the original method , and the created gRNA is fully functional .
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Objective The goal of this study is to understand the function of FKBP51 in resistant to high fat diet-induced obesity using FKBP51 knockout ( KO) mice and in vitro adipocyte differentiation.Methods Four-week old male FKBP51 KO and wild type ( WT) mice were fed separately with regular or high fat diet for 6 weeks.The body weight and food consumption were recorded weekly, the energy expenditure differences ( O2 consumption, CO2 production, respiratory exchange ratio, and heat production) of each group were monitored using the MM-100 metabolism cages system for 24 hours, then the liver from the above animals were stained with the Oil red-O to detect the lipid accumulation and the expression of metabolic genes.In addition, induction of adipocyte differentiation of immortalized MEF cells from WT and FKBP51 KO mice were used to observe the effect of FKBP51 gene on lipogenesis.Results Compared to WT mice, FKBP51 KO mice has less weight increment, and less lipid accumulation in the liver, but with no difference on food consumption during high-fat diet fed.Moreover, FKBP51 KO mice exhibited more O2 consumption, CO2 production and heated production under both RD and HF diet conditions.The PEPCK, G6Pase and UCP-1 genes up-regulation.In addition, lipid content was reduced in FKBP51 gene deficient MEF cells after adipocyte differentiation.Conclusions The FKBP51 gene plays an important role in high fat diet-induced obesity through the energy metabolism enhancement and lipogenesis inhibition.