2형 당뇨병 동물 모델에서 SGLT2 억제제의 신장 세포 노화 억제 효과

학위논문 (석사) -- 서울대학교 대학원 : 의과대학 의학과, 2021. 2. 조영민.Introduction Sodium-glucose cotransporter 2 (SGLT2) inhibitors prevent the progression of diabetic kidney disease. However, the mechanisms underlying this renoprotective benefit are not completely understood. I evaluated whether SGLT2 inhibition reduces cellular senescence in the kidney and investigated the molecular pathways involved in the renoprotective effect. Methods Dapagliflozin (1 mg/kg), glimepiride (2.5 mg/kg), or vehicle was administered daily via oral gavage for 8 weeks in db/db mice. Expression levels of aging marker genes (p21, p16, p53, γH2AX) were measured in the kidney using real-time RT-PCR, immunohistochemistry, and western blot analysis. For in vitro analysis, HK-2 cells, a human renal tubular epithelial cell line, were pretreated with H2O2 to induce cellular senescence, and the levels of aging markers were measured after treatment with β-hydroxybutyrate (β-HB) or NRF2-specific siRNA. Results Mesangial expansion was ameliorated in the dapagliflozin-treated db/db (db/db+SGLT2i) group. Besides, in the db/db+SGLT2i group, the size of the glomerulus was smaller, and albuminuria was markedly reduced compared with the vehicle-treated db/db (db/db+vehicle) group. Expression levels of aging marker genes (p21, p16, p53, γH2AX) in the kidney were increased in the db/db+vehicle group compared with the db/+ group, and this increase was markedly reversed in the db/db+SGLT2i group, but not in the glimepiride-treated db/db (db/db+SU) group. In the kidneys of mice in the db/db+SGLT2i group, oxidative stress was also reduced compared with those in the db/db+vehicle and db/db+SU groups. Dapagliflozin increased plasma β-HB, which reduced H2O2-induced DNA damage and senescence in HK-2 cells. β-HB-induced NRF2 nuclear translocation mediated anti-senescent effects by inducing antioxidant pathways. Conclusion Dapagliflozin, an SGLT2 inhibitor, prevented the progression of diabetic kidney disease by inhibiting cellular senescence and oxidative stress in the kidneys via ketone-induced NRF2 activation.서론 Sodium-glucose cotransporter 2 (SGLT2) 억제제는 당뇨병성 신증의 진행을 억제한다고 알려져 있다. 그러나 그 기전에 대해서는 아직 완전히 밝혀져 있지 않다. 본 연구에서는 SGLT2 억제제가 신장 세포의 노화를 억제하는 지 알아보고, 그 분자적 기전을 밝히고자 한다. 방법 Dapagliflozin (1 mg/kg/day), glimepiride (2.5 mg/kg/day), 또는 vehicle을 db/db 생쥐에 8 주간 경구로 투약했다. db/db 생쥐의 신장 세포 노화는 p21, p16, p53, γH2AX 유전자를 real-time RT-PCR, 면역염색, 웨스턴 블롯 등의 방법으로 측정하였다. 또한, in vitro에서 HK-2 신장 세포를 H2O2로 전처리하여 세포 노화를 유도하고, β-hydroxybutyrate (β-HB) 또는 siNRF2를 처리하여 노화 관련 표현형을 조사하였다. 결과 Dapagliflozin은 db/db 생쥐의 신장에서 메산지움 확장을 억제하였고, 사구체의 크기를 줄였으며, 알부민 배설을 감소시켰다. 노화 마커인 p21, p16, p53, γH2AX 유전자의 신장 내 발현 수준은 db/+ 그룹과 비교하여, db/db+vehicle 그룹에서 증가하였고, 이 증가는 db/db+SGLT2i 그룹에서 감소하였지만, db/db+SU 그룹에서는 감소하지 않았다. 또한, db/db+SGLT2i 그룹의 산화 스트레스 수준은 db/db+vehicle, db/db+SU 그룹과 비교하여 유의하게 감소하였다. Dapagliflozin은 혈장 β-HB 농도를 증가시켰고, β-HB는 HK-2 세포에서 H2O2로 유도된 DNA 손상 및 노화 현상을 감소시켰다. 또한, β-HB 처리는 NRF2를 핵 내로 이동시켰고, 이는 항산화 경로를 활성화하여 항노화 효과를 매개하였다. 결론 SGLT2 억제제인 dapagliflozin은 혈중 케톤 농도 증가를 통해 신장 세포의 NRF2 활성화를 매개하여 산화 스트레스를 억제함으로써 신장 세포의 노화를 방지하고, 이에 따라 당뇨병성 신증의 진행을 억제하였다.Introduction 1 Material and Methods 4 Results 11 Discussion 38 Acknowledgements 42 References 43 국문초록 48Maste

Chitinase 3-Like 1 Contributes to Food Allergy via M2 Macrophage Polarization

Purpose: Food allergy is a hypersensitive immune response to specific food proteins. Chitinase 3-like 1 (CHI3L1, also known as YKL-40 in humans or BRP-39 in mice) is associated with various chronic diseases, such as cancer, rheumatoid arthritis, and allergic disease. CHI3L1 is involved in allergen sensitization and type 2 helper T (Th2) inflammation, but the role of CHI3L1 in food allergy remains unclear. In this study, we sought to investigate the role of CHI3L1 in the development of food allergy. Methods: We measured serum levels of CHI3L1 in food allergic patients. Food allergy was induced in wild-type (WT) and CHI3L1 null mutant (CHI3L1-/-) BALB/c mice with ovalbumin (OVA). We investigated Th2 immune responses, M2 macrophage polarization, and mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) signaling pathways, and also performed transcriptome analysis. Results: Serum levels of CHI3L1 were significantly higher in children with food allergy compared with those in healthy controls. Furthermore, CHI3L1 expression levels were elevated in WT mice after OVA treatment. Food allergy symptoms, immunoglobulin E levels, Th2 cytokine production, and histological injury were attenuated in food allergy-induced CHI3L1-/- mice compared with those in food allergy-induced WT mice. CHI3L1 expression was increased in OVA-treated WT intestinal macrophages and caused M2 macrophage polarization. Furthermore, CHI3L1 was involved in the extracellular signal-regulated kinases (ERK) and AKT signaling pathways and was associated with immune response and lipid metabolism as determined through transcriptome analysis. Conclusions: CHI3L1 plays a pivotal role in Th2 inflammation and M2 macrophage polarization through MAPK/ERK and PI3K/AKT phosphorylation in food allergy.ope

NLRX1 knockdown attenuates pro-apoptotic signaling and cell death in pulmonary hyperoxic acute injury

Hyperoxia is frequently used for treating acute respiratory failure, but it can cause acute lung injury. Nucleotide-binding domain and leucine-rich-repeat-containing family member X1 (NLRX1) is localized in mitochondria and involved in production of reactive oxygen species, inflammation, and apoptosis, which are the features of hyperoxic acute lung injury (HALI). The contribution of NLRX1 to HALI has not previously been addressed. Thus, to investigate the role of NLRX1 in hyperoxia, we generated a murine model of HALI in wild-type (WT) and NLRX1−/− mice by exposure to > 95% oxygen for 72 h. As a result, NLRX1 expression was elevated in mice exposed to hyperoxia. In acute lung injury, levels of inflammatory cells, protein leakage, cell cytotoxicity, and pro-inflammatory cytokines were diminished in NLRX1−/− mice compared to WT mice. In a survival test, NLRX1−/− mice showed reduced mortality under hyperoxic conditions, and apoptotic cell death and caspase expression and activity were also lower in NLRX1−/− mice. Furthermore, levels of the MAPK signaling proteins ERK 1/2, JNK, and p38 were decreased in NLRX1-deficient mice than in WT mice exposed to hyperoxia. The study shows that a genetic deficit in NLRX1 can suppress hyperoxia-induced apoptosis, suggesting that NLRX1 acts as a pivotal regulator of HALI. © 2023, The Author(s).ope

Predicting allergic diseases in children using genome-wide association study (GWAS) data and family history

The recent rise in the prevalence of chronic allergic diseases among children has increased disease burden and reduced quality of life, especially for children with comorbid allergic diseases. Predicting the occurrence of allergic diseases can help prevent its onset for those in high risk groups. Herein, we aimed to construct prediction models for asthma, atopic dermatitis (AD), and asthma-AD comorbidity (also known as atopic march) using a genome-wide association study (GWAS) and family history data from patients of Korean heritage. Among 973 patients and 481 healthy controls, we evaluated single nucleotide polymorphism (SNP) heritability for each disease using genome-based restricted maximum likelihood (GREML) analysis. We then compared the performance of prediction models constructed using Least Absolute Shrinkage and Selection Operator (LASSO) and penalized ridge regression methods. Our results indicate that the addition of family history risk scores to the prediction model greatly increase the predictability of asthma and asthma-AD comorbidity. However, prediction of AD was mostly attributable to GWAS SNPs.ope

German Cockroach Extract Induces Matrix Metalloproteinase-1 Expression, Leading to Tight Junction Disruption in Human Airway Epithelial Cells

PURPOSE: Cockroach exposure is a pivotal cause of asthma. Tight junctions are intercellular structures required for maintenance of the barrier function of the airway epithelium, which is impaired in this disease. Matrix metalloproteinases (MMPs) digest extracellular matrix components and are involved in asthma pathogenesis: MMP1 is a collagenase with a direct influence on airway obstruction in asthmatics. This study aimed to investigate the mechanism by which German cockroach extract (GCE) induces MMP1 expression and whether MMP1 release alters cellular tight junctions in human airway epithelial cells (NCI-H292). MATERIALS AND METHODS: mRNA and protein levels were determined using real-time PCR and ELISA. Tight junction proteins were detected using immunofluorescence staining. Epithelial barrier function was measured by transepithelial electrical resistance (TEER). The binding of a transcription factor to DNA molecules was determined by electrophoretic mobility shift assay, while the levels of tight junction proteins and phosphorylation were determined using Western blotting. RESULTS: GCE was shown to increase MMP1 expression, TEER, and tight junction degradation. Both an inhibitor and small interfering RNA (siRNA) of MMP1 significantly decreased GCE-induced tight junction disruption. Furthermore, transient transfection with ETS1 and SP1 siRNA, and anti-TLR2 antibody pretreatment prevented MMP1 expression and tight junction degradation. An extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor also blocked MMP1 release, ETS1/SP1 DNA binding, and tight junction alteration. CONCLUSION: GCE treatment increases MMP1 expression, leading to tight junction disruption, which is transcriptionally regulated and influenced by the ERK/MAPK pathway in airway epithelial cells. These findings may contribute to developing novel therapeutic strategies for airway diseases.ope

Effect of Central Line Bundle Compliance on Central Line-Associated Bloodstream Infections

PURPOSE: The present study aimed to evaluate the effect of central line (CL) bundle compliance on central line-associated bloodstream infections (CLABSIs) in different departments of the same hospital, including the intensive care unit (ICU) and other departments. MATERIALS AND METHODS: The four components of the CL bundle were hand hygiene, use of maximal sterile barrier precautions, chlorhexidine use, and selection of an appropriate site for venous access. Compliance of the CL bundle and CLABSIs were measured for every department [emergency room (ER), ICU, general ward (GW), and operating room (OR)]. A total of 1672 patients were included over 3 years (August 2013 through July 2016). RESULTS: A total of 29 CLABSI episodes (1.73%) were identified, and only 53.7% of the patients completed CL bundles. The performance rates of all components of the CL bundle were 22.3%, 28.5%, 36.5%, and 84.6% for the ER, ICU, GW, and OR, respectively. The highest CLABSI rate was observed in patients of the ICU, for whom all components were not performed perfectly. Conversely, the lowest CLABSI rate was observed for patients of GWs, for whom all components were performed. Among individual components, femoral insertion site [relative risk (RR), 2.26; 95% confidence interval (CI), 1.09-4.68], not using a full body drape (RR, 3.55; 95% CI, 1.44-8.71), and not performing all CL bundle components (RR, 2.79; 95% CI, 1.19-6.54) were significant variables associated with CLABSIs. CONCLUSION: This study provides direct evidence that completing all CL bundle components perfectly is essential for preventing CLABSIs. Customized education should be provided, according to specific weaknesses of bundle performance.ope

Acute Esophageal Necrosis Associated with Esophageal Foreign Body Injury and the Development of Pneumomediastinum

A 74-year-old woman complained of dysphagia and hemoptysis after ingesting a fragment of crab shell while eating crab salted-fermented fish products, and presented dyspnea that had lasted for three days. Computed tomography indicated pneumomediastinum. Laboratory results revealed acute renal failure. The patient experienced respiratory distress and shock over the days following her initial presentation. Upper gastrointestinal endoscopy revealed black pigmentation of the esophageal mucosa from the middle to lower esophagus. Despite intensive care, the patient’s condition deteriorated and she died. This is the first case of acute esophageal necrosis associated with esophageal foreign body injury and the development of pneumomediastinum reported in Korea.ope

IgE reactivity to carbohydrate moieties of glycoproteins in wheat allergy

Carbohydrate moieties of different glycoproteins, such as cross-reactive carbohydrate determinants (CCDs) and galactose α-1,3-galactose, can induce IgE reactivity with varied clinical significance. In this study, the possible participation of glycan from wheat gliadin, with respect to its IgE-binding capacity, was investigated in children with food allergies to wheat. Total IgE and wheat-specific IgE quantification, documentation of history, and/or oral food challenge (OFC) were performed for 52 children. Subjects with positive wheat-specific IgE were characterized as the symptomatic group, never-exposed group, or asymptomatic group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and glycan detection in gliadin were performed. IgE binding to gliadin and deglycosylated gliadin was measured by immunoblotting and ELISA. Gliadin-specific IgE was detected and correlated with wheat-specific IgE in the symptomatic, never-exposed, and asymptomatic groups. The glycan range overlapped significantly with the gliadin range. Deglycosylation of gliadin reduced the allergenicity of gliadin. In gliadin, the allergenicity of the glycan portion was greater in the symptomatic group than in the never-exposed and asymptomatic groups. We conclude that N-glycan in gliadin might exhibit allergenicity as a possible carbohydrate epitope in wheat allergy in children.ope

Clusterin Deficiency Exacerbates Hyperoxia-Induced Acute Lung Injury

Exposure to high oxygen concentrations leads to generation of excessive reactive oxygen species, causing cellular injury and multiple organ dysfunctions and is associated with a high mortality rate. Clusterin (CLU) is a heterodimeric glycoprotein that mediates several intracellular signaling pathways, including cell death and inflammation. However, the role of CLU in the pathogenesis of hyperoxic acute lung injury (HALI) is unknown. Wild-type (WT) and CLU-deficient mice and cultured human airway epithelial cells were used. Changes in cell death- and inflammation-related molecules with or without hyperoxia exposure in cells and animals were determined. Hyperoxia induced an increase in CLU expression in mouse lungs and human airway epithelial cells. Mice lacking CLU had increased HALI and mortality rate compared with WT mice. In vitro, CLU-disrupted cells showed enhanced release of cytochrome c, Bax translocation, cell death and inflammatory cytokine expression. However, treatment with recombinant CLU attenuated hyperoxia-induced apoptosis. Moreover, the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses revealed metabolic pathways, hematopoietic cell lineage, response to stress and localization and regulation of immune system that were differentially regulated between WT and CLU-/- mice. These results demonstrate that prolonged hyperoxia-induced lung injury is associated with CLU expression and that CLU replenishment may alleviate hyperoxia-induced cell death.ope

A comparison of 24- vs. 48-week peginterferon plus ribavirin in patients with genotype 1 chronic hepatitis

Background/Aims: The standard therapy for patients with genotype 1 chronic hepatitis C (CHC) is a combination of peginterferon and ribavirin for 48 weeks. However, the most appropriate duration of treatment remains to be established because of treatment-related side effects and cost. The aim of this study was to compare the efficacies of 24- and 48-week treatments, and to assess the efficacy of split 24-week therapy (a further 24 weeks of treatment in patients with relapse after the initial 24 weeks of treatment). Methods: A total of 130 patients with genotype 1 CHC was treated between June 2004 and December 2006. Patients with undetectable HCV RNA at 24 weeks of treatment (as assessed by qualitative PCR assay; n=101 patients) were allowed to choose either 24 or 48 weeks as the duration of their treatment; 51 patients chose the 24-week treatment regimen and the remainder chose the 48-week regimen. Patients who relapsed after 24 weeks of treatment were treated for further 24 weeks. The sustained virologic response (SVR) of each treatment group was analyzed. Results: The SVR rate was higher in patients treated for 48 weeks than in those treated for 24 weeks (74.0% vs. 52.9%, P=0.028). In the multivariate analysis, age < 50 years, platelets ≥ 150,000/mm3, and treatment duration for 48 weeks remained significant independent predictors of SVR. Fourteen of the 24 patients who relapsed in the 24-week treatment group received split 24-week therapy, and 6 patients (42.9%) achieved SVR. The overall SVR rate did not differ significantly between the 24-week treatment group, including those who underwent 24-week split therapy (64.7%), and the 48-week treatment group (64.7% vs. 74%, P=0.311). Conclusions: The 24-week plus additional split 24-week therapy following failure is a useful treatment strategy for patients with genotype 1 CHCope