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Objective To explore the changes of renal cortical energy metabolism and its related molecular mechanisms in rats with progressive kidney disease.Methods A rat model of 5/6 nephrectomy was established as the model of progressive nephropathy.Rats were divided into surgical group (5/6Nx group) and sham-operated group (Sham group).Respectively,the rats were sacrificed at 1 week and 12 weeks after completing the model,and their blood,urine sample and kidney specimens were collected.Blood urea nitrogen,serum creatinine and 24 h urine protein were used to evaluate the renal function.Pathological changes in renal tissue were detected by PAS staining and Sirius red staining.The renal cortical energy metabolites were made quantitative analysis by liquid chromatography-mass spectrometry-based targeted metabolomics.The mRNA expressions of inflammatory cytokines (IL-6,IL-1β),fibrosis factors (fibronectin,collagen-1),glycolytic and tricarboxylic acid (TCA) cycle related enzymes were confirmed by real-time PCR.The protein expressions of fibrotic proteins (fibronectin,collagen-1),silent information regulator 1 (SIRT-1) and liver kinase B1 (LKB1) were tested by Western blotting.Results Compared with those in Sham group,the renal function indexes increased,the renal tissue pathological damage was obvious,the mRNA expressions of renal cortical inflammatory and fibrosis factors increased,and fibrotic proteins also increased in 5/6Nx group rats at 1 week and 12 weeks (all P ＜ 0.05),meanwhile,kidney damage worsened over time.Compared with those in Sham group,in the renal cortex of 5/6Nx group glycolytic metabolite lactate,the TCA cycle metabolites (citrate,isocitrate,oxaloacetate) and the oxidized phosphorylation metabolite reduced coenzyme Ⅰ were up-regulated (all P ＜ 0.05),but adenosine triphosphate (ATP) was no change at 1 week,then the abnormal metabolites increased further at 12 weeks,such as the down-regulation of pyruvate,oxidized coenzyme Ⅰ and ATP (all P ＜ 0.05).The pentose phosphate pathway metabolites (reduction and oxidized coenzyme Ⅱ) shows no statistical significant difference in the two group (all P ＞ 0.05).Compared with those in Sham group,in the 5/6Nx group the mRNA expressions of glycolytic enzyme hexokinase 2 and lactate dehydrogenase a were upregulated in the renal cortex at 1 week,whereas the mRNA expressions of pyruvate dehydrogenase α,pyruvate dehydrogenase β and succinate dehydrogenase of the TCA cycle related enzymes were downregulated (all P ＜ 0.05).Meanwhile,renal abnormal metabolic enzyme mRNA expressions were further increased in the 5/6Nx group at 12 weeks.The protein levels of SIRT-1 and LKB1 were not significantly different in the renal cortex of two group rats at 1 week,while SIRT-1 and LKB1 levels decreased in 5/6Nx group than those in Sham group at 12 weeks (all P＜ 0.05).Conclusions During the progression of nephropathy,rats accompanied with renal fibrosis and inflammatory have energy metabolism changes in the renal cortex which accompanies.The features of metabolic changes are manifested as enhanced glycolysis and decreased oxidative phosphorylation,which is aggravated gradually.Its mechanism is related to the inhibition of energy-regulating proteins LKB1 and SIRT-1.
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Objective To study the molecular mechanism and biological significance of GPR30 activating HER2 in MCF-7 breast cancer cells with low expresses HER2.Methods Western blot was adopted to examine the phosphorylation of HER2 and the downstream signaling molecular ERK1/2 after 17-β-estradiol(E2),4-OHT(the active metabolite of tamoxifen) or G-1 (the GPR30 agonist) treatment in MCF-7 cells.After different inhibitors such as G-15 (the GPR30 antagonist),AG1478(EGFR tyrosine inhibitor),AG825 (HER2 tyrosine inhibitor),PP2 (Src family kinase inhibitor)or GM6001 (MMP inhibitor) pretreated for 2 h,the phosphorylation of HER2 and ERK1/2 were further analyzed.Finally,the altered migration and invasive capability of MCF-7 cells were detected by Transwell method.Results HER2 and ERK1/2 were activated in MCF-7 cells after E2,4-OHT or G-1 treatment and these changes could be inhibited by G-15,AG1478,AG825,PP2 or GM6001 pretreatment.The enhancement of G-1-induced migration and invasion ability in MCF-7 cells could also be inhibited by those inhibitors too.Conclusion GPR30 promotes the migration and invasion of MCF-7 cells through activating HER2-ERK1/2 signal transduction pathway.
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Objective To study the prevalence and associated factors of loneliness in individuals with speech disability.Methods Using multi-stage stratified random cluster sampling method,170 community-residing ver-bally disabled persons were selected and administered with a general information questionnaire,one single -item loneliness self-rating question and social support scale.A total of 204 study questionnaires were distributed to the subjects;170 subjects(mean age:43.1±13.7 years)completed the survey.Results As high as 46.47% (79/170)of these verbally disabled individuals reported to feel lonely often.Females (OR=2.45),unemployment (OR=2.95), first and second degrees of disability (OR=4.35),co-existence of chronic illnesses (OR=6.50)and low utiliza-tion of social support (OR=2.58)were significantly associated with the increased risk of loneliness in persons with speech disability (P =0.002~0.046).Conclusion Loneliness is highly prevalent in individuals with speech disabili-ty.Verbally disabled persons,who are female,unemployed,severely disabled,and chronically ill and have a low use of social support,are the target population of mental health services.
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Acute coronary syndrome (ACS)is a group of clinical syndromes whose pathological basis is complete or incomplete occlusive thrombosis secondary to coronary atherosclerotic plaque rupture or erosion.Inflammatory me-diators,such as P-selectin and high sensitive C reactive protein (hsCRP),possess very important clinical significance for diseased degree,progression and prognosis of ACS.This article made an overview on researches about the rela-tionship among P-selectin,hsCRP and occurrence and development of ACS.
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Objective To construct the lentivirus vector containing the hsa‐miR‐424 gene ,and identify the expression level of miR‐424 in cells .Research the influence of hsa‐miR‐424 on proliferation of cervical cancer Hela cell line .Methods Using the human genomic DNA as template to design the upper and lower primers for synthesis of miR‐424 ,and amplifying the target fragment by polymerase chain reaction (PCR) .Recover the products and conduct sequencing after connecting it into the pMD18T vector .Ampli‐fy the product by PCR template as pMD18T‐miR424 ,and insert the fragment expressing pMD18T‐miR424 into the vector of pLen‐tis‐CMV‐GFP‐MCS‐PGK‐PURO after enzyme cutting to construct the pLentis‐CMV‐GFP‐miR424‐PGK‐PURO .Package the com‐pound with pMD2 .G and pSPAX2 in 293T cell to produce the lentivirus ,and using the supernatant containing lentivirus to infect the Hela cell line .Results The sequencing result proved the sequence of miR‐424 in plasmid vector was correct ,which proved the construction of lentivirus was successful and the target lentivirus was obtained .The expression of miR‐424 almost rise 60 times af‐ter infected the cervical cancer Hela cell by the carrier .The result of M TT method suggested :the cervical cancer Hela cell lines have slowed proliferation with infection miR‐424 lentivirus .Conclusion The miR‐424 lentivirus vector was constructed successfully and the high efficacy expression miR‐424 cell line was established and stable .The cervical cancer Hela cell were infected with the super‐natant containing lentivirus ,inhibited the proliferation of Hela cell successfully ,and laid a good foundation for subsequent research .
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AIM: To study the effects of apolipoprotein (apo) A-Ⅰon ATP binding cassette transporter A1 (ABCA1) degradation and cholesterol efflux in THP-1 macrophage-derived foam cells. METHODS: After exposure of the cultured THP-1 macrophage-derived foam cells to apolipoprotein A-Ⅰ for different time, cholesterol efflux, ABCA1 mRNA and protein level were determined by liquid scintillation counting, reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The mean ABCA1 fluorescence intensity on THP-1 macrophage-derived foam cells was detected by flow cytometry. RESULTS: ApoA-Ⅰ markedly increased ABCA1-mediated cholesterol efflux from THP-1 macrophage-derived foam cells. This was accompanied by an increase in the content of ABCA1. ApoA-Ⅰ did not alter ABCA1 mRNA abundance. Thiol protease inhibitors increased the level of ABCA1 protein and slowed its decay in THP-1 macrophage-derived foam cells, whereas none of the proteosome-specific inhibitor lactacystin, other protease inhibitors, or the lysosomal inhibitor NH_4Cl showed such effects. The apoA-Ⅰ mediated cellular cholesterol efflux was enhanced by thiol protease inhibitors. CONCLUSION: Thiol protease inhibitors might provide an alternative way to upregulate ABCA1 protein. This strategy is especially appealing since it may mimic the stabilizing effect of the natural ligands apoA-Ⅰ.
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AIM: To explore the effect of ovariectomization on Alzheimer-like phosphorylation of Tau in hippocampus of Sorague-Dawlery rats. METHODS: An animal model was developed using ovariectimized (OVX) rats, and the phosphorylation of Tau protein was measured by Western blot. RESULTS: The levels of phosphorylated Tau at PHF-1epitope were elevated in ovariectimized rat brain hippocampus 4 weeks and 8 weeks after ovariectomization, when compared with sham-OVX rats (P0.05). CONCLUSION: Ovariectomization may induce Alzheimer-like hyperphosphorylation of Tau protein in brain hippocampus of rats. [