• Molecules and Cells
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• v.45 no.8
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• pp.575-587
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• 2022

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metal-free porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible "bulge" loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

• Biomolecules & Therapeutics
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• v.30 no.5
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• pp.447-454
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• 2022

Few studies have evaluated the role of autophagy in the development of oxaliplatin (OXT) resistance in colon cancer cells. In this study, we compared the role of autophagy between SNU-C5 colon cancer cells and OXT-resistant SNU-C5 (SNU-C5/OXTR) cells. At the same concentration of OXT, the cytotoxicity of OXT or apoptosis was significantly reduced in SNU-C5/OXTR cells compared with that in SNU-C5 cells. Compared with SNU-C5 cells, SNU-C5/OXTR cells exhibited low levels of autophagy. The expression level of important autophagy proteins, such as autophagy-related protein 5 (Atg5), beclin-1, Atg7, microtubule-associated proteins 1A/1B light chain 3B I (LC3-I), and LC3-II, was significantly lower in SNU-C5/OXTR cells than that in SNU-C5 cells. The expression level of the autophagy-essential protein p62 was also lower in SNU-C5/OXTR cells than in SNU-C5 cells. In SNU-C5/OXTR cells, the production of intracellular reactive oxygen species (ROS) was significantly higher than that in SNU-C5 cells, and treatment with the ROS scavenger N-acetylcysteine restored the reduced autophagy levels. Furthermore, the expression of antioxidant-related nuclear factor erythroid 2-related factor 2 transcription factor, heme oxygenase-1, and Cu/Zn superoxide dismutase were also significantly increased in SNU-C5/OXTR cells. These findings suggest that autophagy is significantly reduced in SNU-C5/OXTR cells compared with SNU-C5 cells, which may be related to the production of ROS in OXT-resistant cells.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.311-318
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• 2023

Background: The beneficial effects of compound K (CK) on different chronic diseases have been shown to be at least related to antioxidant action. Nevertheless, since its antioxidant activity in human retinal pigment epithelial (RPE) cells is still unknown, here we investigated whether CK alleviates oxidative stress-stimulated damage in RPE ARPE-19 cells. Methods: The cytoprotective consequence of CK in hydrogen peroxide (H₂O₂)-treated cells was evaluated by cell viability, DNA damage, and apoptosis assays. Fluorescence analysis and immunoblotting were performed to investigate the inhibitory action of CK on reactive oxygen species (ROS) production and mitochondrial dysfunction. Results: H₂O₂-promoted cytotoxicity, oxidative stress, DNA damage, mitochondrial impairment, and apoptosis were significantly attenuated by CK in ARPE-19 cells. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation level and its shuttling to the nucleus were increased, which was correlated with upregulated activation of heme oxygenase-1 (HO-1). However, zinc protoporphyrin, a blocker of HO-1, significantly abrogated the preventive action of CK in H₂O₂-treated ARPE-19 cells. Conclusion: This study indicates that activation of Nrf2/HO-1 signaling by CK plays an important role in rescuing ARPE-19 cells from oxidative cellular damage.

• Journal of The Korean Society of Integrative Medicine
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• v.11 no.3
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• pp.159-169
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• 2023

Purpose : Though other Citrus spp. have reported their anti-inflammatory and antioxidative activities in previous studies, the biological activity of Kiyomi (Citrus unshiu × C. sinensis) has not been reported yet. Therefore, this study attempted to analyze the anti-inflammatory mechanisms of Kiyomi leaf ethanol extract (KLEE) in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Methods : The cytotoxic effect of KLEE in RAW 264.7 cells was determined by WST-1 assay. Bacterial endotoxin, the concentration of nitric oxide (NO) was analyzed by the Griess reaction. In addition, Western blot analysis was applied to measure the protein expression level of inducible NO synthase (iNOS). The phosphorylated status of the critical inflammatory transcription factor, nuclear factor (NF)-𝜅B, and its upstream signaling molecules, phosphoinositide 3-kinase (PI3K)/Akt as well as mitogen-activated protein kinases (MAPKs), were also measured by Western blot analysis. Results : KLEE was not cytotoxic up to a concentration of 200 ㎍/㎖, and protein expression levels of iNOS and cyclooxygenase (COX)-2, enzymes that counteract NO and prostaglandin (PG) E2 production, were inhibited by KLEE treatment. The phosphorylated status of PI3K/Akt as well as MAPKs including extracellular regulated kinase (ERK), c-jun NH2kinase (JNK), and p38, were significantly attenuated by KLEE treatment in LPS stimulated RAW 264.7 cells. Moreover, one of phase II enzymes, heme oxygenase (HO)-1 which has known for its anti-inflammatory capacity, was strongly induced by KLEE treatment. Conclusion : Consequently, KLEE treatment significantly attenuated the production of NO as well as the expression levels of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. The inflammatory transcription factor, NF-𝜅B, as well as its upstream signaling molecules, PI3K/Akt and MAPKs, were also diminished by KLEE treatment with statistical significance in LPS-stimulated RAW 264.7 cells. These results suggest that KLEE might be a promising candidate for the attenuation of inflammatory disorders.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.387-397
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• 2023

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

• Journal of Microbiology and Biotechnology
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• v.33 no.4
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• pp.463-470
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• 2023

This study confirmed the change in functional composition and alcohol-induced acute liver injury in Aloe arborescens after fermentation. An acute liver injury was induced by administration of ethanol (3 g/kg/day) to C57BL/6J mice for 5 days. A fermented A. arborescens Miller leaf (FAAL) extract was orally administered 30 minutes before ethanol treatment. After fermentation, the emodin content was approximately 13 times higher than that of the raw material. FAAL extract significantly attenuated ethanol-induced aspartate aminotransferase, alanine aminotransferase, and triglyceride increases in serum and liver tissue. Histological analysis revealed that FAAL extract inhibits inflammatory cell infiltration and fat accumulation in liver tissues. The cytochrome P450 2E1, superoxide dismutase, and glutathione (GSH), which involved in alcohol-induced oxidative stress, were effectively regulated by FAAL extract in serum and liver tissues, except for GSH. FAAL also maintained the antioxidant defense system by upregulating heme oxygenase 1 and nuclear factor erythroid 2-related factor 2 protein expression. In addition, FAAL extract inhibited the decrease in alcohol dehydrogenase and aldehyde dehydrogenase activity, which promoted alcohol metabolism and prevented the activation of inflammatory response. Our results suggest that FAAL could be used as a potential therapeutic agent for ethanol-induced acute liver injury.

• Journal of Nutrition and Health
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• v.56 no.6
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• pp.589-601
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• 2023

Purpose: Baicalein, a natural flavone found in herbs, exhibits diverse biological activities. Nonalcoholic steatohepatitis (NASH) is an irreversible condition often associated with a poor prognosis. This study aimed to evaluate the effects of baicalein on the development of NASH in mice. Methods: Male C57BL/6J mice were randomly divided into four groups. Three groups were fed a methionine-choline-deficient (MCD) diet to induce NASH and were simultaneously treated with baicalein (at doses of 50 and 100 mg/kg) or vehicle only (sodium carboxymethylcellulose) through oral gavage for 4 weeks. The control group was fed a methionine-choline-sufficient (MCS) diet without the administration of baicalein. Results: The baicalein treatment significantly reduced serum levels of alanine aminotransferase and aspartate aminotransferase, suggestive of reduced liver damage. Histological analysis revealed a marked decrease in nonalcoholic fatty liver activity scores induced by the MCD diet in the mice. Similarly, baicalein treatment at both doses significantly attenuated the degree of hepatic fibrosis, as examined by Sirius red staining, and hepatocellular death, as examined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Baicalein treatment attenuated MCD-diet-induced lipid peroxidation, as evidenced by lower levels of hepatic malondialdehyde and 4-hydroxynonenal, demonstrating a reduction in oxidative stress resulting from lipid peroxidation. Moreover, baicalein treatment suppressed hepatic protein levels of 12-lipoxygenase (12-Lox) induced by the MCD diet. In contrast, baicalein enhanced the activities of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Additionally, baicalein treatment significantly reduced hepatic non-heme iron concentrations and hepatic ferritin protein levels in mice fed an MCD diet. Conclusion: To summarize, baicalein treatment suppresses hepatic lipid peroxidation, 12-Lox expression, and iron accumulation, all of which are associated with the attenuation of NASH progression.

• The Korea Journal of Herbology
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• v.39 no.1
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• pp.23-29
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• 2024

Objectives : Polyporus umbellatus is a medicinal mushroom that has been used for over thousands years in Chinese medicine as a powerful diuretic to relieve fluid retention and edema. Dermal papilla is located at the bottom of the hair follicle and connected to the blood vessels where it gets the nutrients and oxygen to nurture hair follicle. This study examined the mechanism through which the ethanol extract of Polyporus umbellatus (EPU) promoted the proliferation of human dermal papilla cells (HHDPCs). Methods : To estimate the proliferative effects of EPU on HHDPCs, cell viability was estimated by thiazolyl blue tetrazolium bromide (MTT) assay. Western blotting was used to investgate the activation of ERK, phosphoinositide 3-kinase (PI3K)/Akt, β-catenin, GSK-3β and heme oxygenase-1 (HO-1). Cells were treated with inhibitors of ERK and Akt prior to EPU treatment. Results : EPU promoted the proliferation of HHDPCs and the phosphorylation of ERK and Akt in dose dependent manner. However, the proliferative effect of EPU on HHDPCs was inhibited by pre-treatment of ERK inhibitor (PD98059) and Akt inhibitor (LY294002). Furthermore, EPU respectively stimulated the protein expression of β-catenin and phosphorylated GSK-3β. EPU significantly increased the protein expression levels of proliferation and cytoprotection related genes such as Bcl-2, SIRT-1, and HO-1 in cells. Conclusion : This results suggest that EPU promoted the proliferation of HHDPCs via activating PI3K/Akt and Wnt/β-catenin signaling pathway in HHDPCs.

• Biomolecules & Therapeutics
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• v.32 no.4
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• pp.467-473
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• 2024

In this study, we investigated the potential protective effects of (+)-afzelechin (AZC), a natural compound that is derived from Bergenia ligulata, on lipopolysaccharide (LPS)-induced inflammatory responses. AZC is known to have antioxidant, anticancer, antimicrobial, and cardiovascular protective properties. However, knowledge regarding the therapeutic potential of AZC against LPS-induced inflammatory responses is limited. Thus, we investigated the protective attributes of AZC against inflammatory damage caused by LPS exposure. We examined the effects of AZC on heme oxygenase (HO)-1, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) in LPS-activated human umbilical vein endothelial cells (HUVECs). In addition, the effects of AZC on the expression of iNOS, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β were analyzed in the lung tissues of LPS-injected mice. Data revealed that AZC promoted the production of HO-1, inhibited the interaction between luciferase and nuclear factor (NF)-κB, and reduced the levels of COX-2/PGE2 and iNOS/NO, thereby leading to a decrease in the signal transducer and activator of transcription (STAT)-1 phosphorylation. Moreover, AZC facilitated the nuclear translocation of Nrf2, increased the binding activity between Nrf2 and the antioxidant response elements (AREs), and lowered the expression of IL-1β in the LPS-treated HUVECs. In the animal model, AZC significantly reduced the expression of iNOS in the lung tissue structure and the TNF-α level in the bronchoalveolar lavage fluid. These findings demonstrate that AZC possesses anti-inflammatory properties that regulate iNOS through the inhibition of both NF-κB expression and p-STAT-1. Consequently, AZC has potential as a future candidate for the development of new clinical substances for the treatment of pathological inflammation.

• Korean Journal of Radiology
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• v.25 no.8
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• pp.726-741
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• 2024

Recent advancements in Alzheimer's disease treatment have focused on the elimination of amyloid-beta (Aβ) plaque, a hallmark of the disease. Monoclonal antibodies such as lecanemab and donanemab can alter disease progression by binding to different forms of Aβ aggregates. However, these treatments raise concerns about adverse effects, particularly amyloid-related imaging abnormalities (ARIA). Careful assessment of safety, especially regarding ARIA, is crucial. ARIA results from treatment-related disruption of vascular integrity and increased vascular permeability, leading to the leakage of proteinaceous fluid (ARIA-E) and heme products (ARIA-H). ARIA-E indicates treatment-induced edema or sulcal effusion, while ARIA-H indicates treatment-induced microhemorrhage or superficial siderosis. The minimum recommended magnetic resonance imaging sequences for ARIA assessment are T2-FLAIR, T2^* gradient echo (GRE), and diffusion-weighted imaging (DWI). T2-FLAIR and T2^* GRE are necessary to detect ARIA-E and ARIA-H, respectively. DWI plays a role in differentiating ARIA-E from acute to subacute infarcts. Physicians, including radiologists, must be familiar with the imaging features of ARIA, the appropriate imaging protocol for the ARIA workup, and the reporting of findings in clinical practice. This review aims to describe the clinical and imaging features of ARIA and suggest points for the timely detection and monitoring of ARIA in clinical practice.