• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.25-32
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• 2021

Inflammatory reactions activated by lipopolysaccharide (LPS) of gram-negative bacteria can lead to severe septic shock. With the recent emergence of multidrug-resistant gram-negative bacteria and a lack of efficient ways to treat resulting infections, there is a need to develop novel anti-endotoxin agents. Antimicrobial peptides have been noticed as potential therapeutic molecules for bacterial infection and as candidates for new antibiotic drugs. We previously designed the 9-meric antimicrobial peptide Pro9-3 and it showed high antimicrobial activity against gram-negative bacteria. Here, to further examine its potency as an anti-endotoxin agent, we examined the anti-endotoxin activities of Pro9-3 and elucidated its mechanism of action. We performed a dye-leakage experiment and BODIPY-TR cadaverine and limulus amebocyte lysate assays for Pro9-3 as well as its lysine-substituted analogue and their enantiomers. The results confirmed that Pro9-3 targets the bacterial membrane and the arginine residues play key roles in its antimicrobial activity. Pro9-3 showed excellent LPS-neutralizing activity and LPS-binding properties, which were superior to those of other peptides. Saturation transfer difference-nuclear magnetic resonance experiments to explore the interaction between LPS and Pro9-3 revealed that Trp3 and Tlr7 in Pro9-3 are critical for attracting Pro9-3 to the LPS in the gram-negative bacterial membrane. Moreover, the anti-septic effect of Pro9-3 in vivo was investigated using an LPS-induced endotoxemia mouse model, demonstrating its dual activities: antibacterial activity against gram-negative bacteria and immunosuppressive effect preventing LPS-induced endotoxemia. Collectively, these results confirmed the therapeutic potential of Pro9-3 against infection of gram-negative bacteria.

• Biomolecules & Therapeutics
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• 제31권2호
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• pp.200-209
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• 2023

Patients with non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) amplification or sensitive mutations initially respond to the tyrosine kinase inhibitor gefitinib, however, the treatment becomes less effective over time by resistance mechanism including mesenchymal-epithelial transition (MET) overexpression. A therapeutic strategy targeting MET and EGFR may be a means to overcoming resistance to gefitinib. In the present study, we found that picropodophyllotoxin (PPT), derived from the roots of Podophyllum hexandrum, inhibited both EGFR and MET in NSCLC cells. The antitumor efficacy of PPT in gefitinib-resistant NSCLC cells (HCC827GR), was confirmed by suppression of cell proliferation and anchorage-independent colony growth. In the targeting of EGFR and MET, PPT bound with EGFR and MET, ex vivo, and blocked both kinases activity. The binding sites between PPT and EGFR or MET in the computational docking model were predicted at Gly772/Met769 and Arg1086/Tyr1230 of each ATP-binding pocket, respectively. PPT treatment of HCC827GR cells increased the number of annexin V-positive and subG1 cells. PPT also caused G2/M cell-cycle arrest together with related protein regulation. The inhibition of EGFR and MET by PPT treatment led to decreases in the phosphorylation of the downstream-proteins, AKT and ERK. In addition, PPT induced reactive oxygen species (ROS) production and GRP78, CHOP, DR5, and DR4 expression, mitochondrial dysfunction, and regulated involving signal-proteins. Taken together, PPT alleviated gefitinib-resistant NSCLC cell growth and induced apoptosis by reducing EGFR and MET activity. Therefore, our results suggest that PPT can be a promising therapeutic agent for gefitinib-resistant NSCLC.

• Korean Circulation Journal
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• 제54권5호
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• pp.233-252
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• 2024

Background and Objectives: Myocardial ischemia-reperfusion injury (MIRI) refers to the damage of cardiac function caused by restoration of blood flow perfusion in ischemic myocardium. However, long non-coding RNA prostate androgen regulated transcript 1 (PART1)'s role in MIRI remain unclear. Methods: Immunofluorescence detected LC3 expression. Intermolecular relationships were verified by dual luciferase reporter assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and transferase-mediated dUTP nick-end labeling (TUNEL) assays analyzed cell viability and apoptosis. The release of lactate dehydrogenase was tested via enzyme-linked immunosorbent assay (ELISA). Left anterior descending coronary artery surgery induced a MIRI mouse model. Infarct area was detected by 2,3,5-triphenyltetrazolium chloride staining. Hematoxylin and eosin staining examined myocardial injury. ELISA evaluated myocardial marker (creatine kinase MB) level. Results: PART1 was decreased in hypoxia/reoxygenation (H/R) induced AC16 cells and MIRI mice. PART1 upregulation attenuated the increased levels of Bax, beclin-1 and the ratio of LC3II/I, and enhanced the decrease of Bcl-2 and p62 expression in H/R-treated cells. PART1 upregulation alleviated H/R-triggered autophagy and apoptosis via miR-302a-3p. Mechanically, PART1 targeted miR-302a-3p to upregulate transcription factor activating enhancer-binding protein 2C (TFAP2C). TFAP2C silencing reversed the protected effects of miR-302a-3p inhibitor on H/R treated AC16 cells. We further established TFAP2C combined to dual-specificity phosphatase 5 (DUSP5) promoter and activated DUSP5. TFAP2C upregulation suppressed H/R-stimulated autophagy and apoptosis through upregulating DUSP5. Overexpressed PART1 reduced myocardial infarction area and attenuated MIRI in mice. Conclusion: PART1 improved the autophagy and apoptosis in H/R-exposed AC16 cells through miR-302a-3p/TFAP2C/DUSP5 axis, which might provide novel targets for MIRI treatment.

• Korean Circulation Journal
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• 제53권3호
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• pp.151-167
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• 2023

Background and Objectives: Acute myocardial infarction (AMI) often occurs suddenly and leads to fatal consequences. Ferroptosis is closely related to the progression of AMI. However, the specific mechanism of ferroptosis in AMI remains unclear. Methods: We constructed a cell model of AMI using AC16 cells under oxygen and glucose deprivation (OGD) conditions and a mice model of AMI using the left anterior descending (LAD) ligation. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide was employed to determine cell viability. The levels of lactate dehydrogenase, creatine kinase, reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and iron were measured using corresponding kits. Dual luciferase reporter gene assay, RNA-binding protein immunoprecipitation, and RNA pull-down were performed to validate the correlations among AC005332.7, miR-331-3p, and cyclin D2 (CCND2). Hematoxylin and eosin staining was employed to evaluate myocardial damage. Results: AC005332.7 and CCND2 were lowly expressed, while miR-331-3p was highly expressed in vivo and in vitro models of AMI. AC005332.7 sufficiency reduced ROS, MDA, iron, and ACSL4 while boosting the GSH and GPX4, indicating that AC005332.7 sufficiency impeded ferroptosis to improve cardiomyocyte injury in AMI. Mechanistically, AC005332.7 interacted with miR-331-3p, and miR-331-3p targeted CCND2. Additionally, miR-331-3p overexpression or CCND2 depletion abolished the suppressive impact of AC005332.7 on ferroptosis in OGD-induced AC16 cells. Moreover, AC005332.7 overexpression suppressed ferroptosis in mice models of AMI. Conclusions: AC005332.7 suppressed ferroptosis in OGD-induced AC16 cells and LAD ligation-operated mice through modulating miR-331-3p/CCND2 axis, thereby mitigating the cardiomyocyte injury in AMI, which proposed novel targets for AMI treatment.

• Molecules and Cells
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• 제41권3호
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• pp.198-206
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• 2018

Aortic dissection (AD) is a catastrophic disease with high mortality and morbidity, characterized with fragmentation of elastin and loss of smooth muscle cells. Although AD has been largely attributable to polymorphisms defect in the elastin-coding gene, tropoelastin (TE), other undermined factors also appear to play roles in AD onset. Here, we investigated the effects of post-transcriptional control of TE by microRNAs (miRNAs) on elastin levels in aortic smooth muscle cells (ASMC). We found that miR-144-3p is a miRNA that targets TE mRNA in both human and mouse. Bioinformatics analyses and dual luciferase reporter assay showed that miR-144-3p inhibited protein translation of TE, through binding to the 3'-UTR of the TE mRNA. Interestingly, higher miR-144-3p levels and lower TE were detected in the ASMC obtained from AD patients, compared to those from non-AD controls. In a mouse model for human AD, infusion of adeno-associated viruses (serotype 6) carrying antisense for miR-144-3p (asmiR-144-3p) under CAG promoter significantly reduced the incidence and severity of AD, seemingly through enhancement of TE levels in ASMC. Thus, our data suggest an essential role of miR-144-3p on the pathogenesis of AD.

• Korean Circulation Journal
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• 제53권6호
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• pp.387-403
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• 2023

Background and Objectives: Myocardial ischemia and reperfusion injury (MIRI) has high morbidity and mortality worldwide. We aimed to explore the role of long noncoding RNA lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1) in cardiomyocyte pyroptosis. Methods: Hypoxia/reoxygenation (H/R) injury was constructed in human cardiomyocyte (HCM). The level of LOXL1-AS1, miR-761, phosphatase and tensin homolog (PTEN) and pyroptosis-related proteins was monitored by quantitative real-time polymerase chain reaction or western blot. Flow cytometry examined the pyroptosis level. Lactate dehydrogenase (LDH), creatine kinase-MB and cardiac troponin I levels were detected by test kits. Enzyme-linked immunosorbent assay measured the release of inflammatory cytokines. Dual-luciferase assay validated the binding relationship among LOXL1-AS1, miR-761, and PTEN. Finally, ischemia/reperfusion (I/R) animal model was constructed. Hematoxylin and eosin staining assessed morphological changes of myocardial tissue. NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and casepase-1 expression was determined by immunohistochemistry. Results: After H/R treatment, LOXL1-AS1 and PTEN were highly expressed but miR-761 level was suppressed. LOXL1-AS1 inhibition or miR-761 overexpression increased cell viability, blocked the release of LDH and inflammatory cytokines (interleukin [IL]-1β, IL-18), inhibited pyroptosis level, and downregulated pyroptosis-related proteins (ASC, cleaved caspase-1, gasdermin D-N, NLRP3, IL-1β, and IL-18) levels in HCMs. LOXL1-AS1 sponged miR-761 to up-regulate PTEN. Knockdown of miR-761 reversed the effect of LOXL1-AS1 down regulation on H/R induced HCM pyroptosis. LOXL1-AS1 aggravated the MIRI by regulating miR-761/PTEN axis in vivo. Conclusions: LOXL1-AS1 targeted miR-761 to regulate PTEN expression, then enhance cardiomyocyte pyroptosis, providing a new alternative target for the treatment of MIRI.

• 대한환경공학회지
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• 제35권12호
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• pp.897-905
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• 2013

본 연구에서는 불용성 토양 휴믹물질(HS)인 토양 휴민(Hu)을 대상으로 페난트린(PHE)과의 흡착상수($K_{OC}$, n)를 조사하였고, 각 휴민 분자의 물질특성과 흡착상수와의 상관관계를 조사하였다. 토양 휴민은 한라산 토양을 포함한 국내 5개 지역의 토양과 국제휴믹학회(IHSS) 표준토양 및 이탄토(Peat)에서 분리한 7종을 사용하였으며, 원소성분비 및 고체 $^{13}C$ NMR을 이용한 탄소형태별 분포(%) 등을 조사하였다. 토양 휴민은 알킬탄소를 주요 성분으로 하는 높은 지방족 탄소함량(57.1~72.3%)을 가진 분자구조 특성을 보였으며, 추출원별 알킬탄소의 함량($C_{Al-H,C}$, %)은 화강암 기원의 토양 Hu (26~42) > 화산재토양 기원의 HL Hu (23.9) > Peat Hu (14.0)의 순으로 나타났다. 토양 휴민의 물질특성과 PHE 흡착상수의 상관성 해석결과, 유기탄소 표준화분배계수($K_{OC}$, mL/g) 값은 알킬탄소 함량(%)과 높은 상관성($r^2$ = 0.77, p < 0.05)을 보인 반면, Freundlich plot을 통해 얻은 비선형 흡착상수(n)는 H,C-치환 방향족탄소 함량($C_{Ar-H,C}$, %)과 높은 상관성($r^2$ = (-)0.74, p < 0.05)을 보였다. $K_{OC}$ 값은 분자 극성도(PI, N+O)/C)와도 높은 상관성($r^2$ = (-)0.74, p < 0.1)을 보여, 분자극성도 값도 소수성유기물의 흡착능 예측에 유용한 물질특성 인자임을 확인하였다. 이상의 결과로 부터 토양휴민 분자 내 알킬탄소의 함량이 높거나, 분자극성도가 낮을수록 PHE의 흡착능이 증가하며, 방향족탄소 함량이 높을수록 흡착의 비선형성(nonlinear sorption)이 증가하는 경향이 있음을 알 수 있었으며, 이러한 토양 휴민의 PHE 흡착특성은 dual reactive mode 흡착모델에 적용하여 해석하였다.

• International Journal of Stem Cells
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• 제15권4호
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• pp.359-371
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• 2022

Background and Objectives: The goal of this study was to investigate the mechanism of mesenchymal stem cell (MSC)-derived microRNA (miR)-150-5p-expressing exosomes in promoting skin wound healing through activating PI3K/AKT pathway by PTEN. Methods and Results: Human umbilical cord (HUC)-MSCs were infected with miR-150-5p overexpression and its control lentivirus, and HUC-MSCs-derived exosomes (MSCs-Exos) with stable expression of miR-150-5p were obtained. HaCaT cells were induced by H₂O₂ to establish a cellular model of skin injury, in which the expression of miR-150-5p and PTEN and the phosphorylation of PI3K and AKT were evaluated. HaCaT cells were transfected with pcDNA3.1-PTEN or pcDNA3.1 and then cultured with normal exosomes or exosomes stably expressing miR-150-5p. Cell proliferation was inspected by CCK-8. Cell migration was detected by scratch test and cell apoptosis by flow cytometry. The starBase tool was used to predict the binding site of miR-150-5p to PTEN. Dual-luciferase reporter assay and RIP assay were applied to assess the interaction between miR-150-5p and PTEN. In H₂O₂-induced HaCaT cells, the miR-150-5p expression decreased, and PTEN expression increased in a concentration-dependent manner. MSCs-Exos promoted the growth and migration of H₂O₂-induced HaCaT cells and inhibited their apoptosis. In addition, overexpression of exosomal miR-150-5p enhanced the protective effect of MSCs-Exos on H₂O₂-induced HaCaT cells; PTEN overexpression in HaCaT cells partially restrained miR-150-5p-mediated inhibition on H₂O₂-induced injury in HaCaT cells. PTEN was a target gene of miR-150-5p. MiR-150-5p regulated PI3K/AKT pathway through PTEN. Conclusions: MSCs-derived miR-150-5p-expressing exosomes promote skin wound healing by activating PI3K/AKT pathway through PTEN.