Seung-Hoon Baek

Modulation of Mitochondrial Function and Autophagy Mediates Carnosine Neuroprotection Against Ischemic Brain Damage

Background and Purpose— Despite the rapidly increasing global burden of ischemic stroke, no therapeutic options for neuroprotection against stroke currently exist. Recent studies have shown that autophagy plays a key role in ischemic neuronal death, and treatments that target autophagy may represent a novel strategy in neuroprotection. We investigated whether autophagy is regulated by carnosine, an endogenous pleiotropic dipeptide that has robust neuroprotective activity against ischemic brain damage. Methods— We examined the effect of carnosine on mitochondrial dysfunction and autophagic processes in rat focal ischemia and in neuronal cultures. Results— Autophagic pathways such as reduction of phosphorylated mammalian target of rapamycin (mTOR)/p70S6K and the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II were enhanced in the ischemic brain. However, treatment with carnosine significantly attenuated autophagic signaling in the ischemic brain, with improvement of brain mitochondrial function and mitophagy signaling. The protective effect of carnosine against autophagy was also confirmed in primary cortical neurons. Conclusions— Taken together, our data suggest that the neuroprotective effect of carnosine is at least partially mediated by mitochondrial protection and attenuation of deleterious autophagic processes. Our findings shed new light on the mechanistic pathways that this exciting neuroprotective agent influences.

Recent Methodology in Ginseng Analysis

As much as the popularity of ginseng in herbal prescriptions or remedies, ginseng has become the focus of research in many scientific fields. Analytical methodologies for ginseng, referred to as ginseng analysis hereafter, have been developed for bioactive component discovery, phytochemical profiling, quality control, and pharmacokinetic studies. This review summarizes the most recent advances in ginseng analysis in the past half-decade including emerging techniques and analytical trends. Ginseng analysis includes all of the leading analytical tools and serves as a representative model for the analytical research of herbal medicines.

Pectic polysaccharides from Panax ginseng as the antirotavirus principals in ginseng.

To evaluate the antidiarrheal effect of ginseng, the active principals of ginseng were studied in vitro model of rotavirus infection, the leading cause of severe diarrhea. Two pectic polysaccharides, named as GP50-dHR (56.0 kDa) and GP50-eHR (77.0 kDa), were purified from hot water extract of ginseng by bioassay-linked fractionation. Both polysaccharides rescued cell viability from rotavirus infection dose-dependently (IC50 are 15 and 10 microg/mL, respectively). Both polysaccharides had common structural features of homogalacturonan backbone with hairy regions of rhamnogalacturonan type I. Arabinose-rich side chains with abundant branch points were unique in GP50-eHR and may contribute to a greater antirotavirus effect of GP50-eHR than GP50-dHR. Because homogalacturonan itself did not show an antirotavirus effect, hairy regions might be functional sites. Of note, the antirotavirus effect of both polysaccharides resulted from inhibiting rotavirus attachment to cells. Together with a wide range of noncytotoxicity, these findings suggest that ginseng polysaccharides are viable therapeutic options for rotavirus diarrhea.

Autophagy sustains the survival of human pancreatic cancer PANC-1 cells under extreme nutrient deprivation conditions

Pancreatic ductal adenocarcinomas are an extremely aggressive and devastating type of cancer with high mortality. Given the dense stroma and poor vascularization, accessibility to nutrients is limited in the tumor microenvironment. Here, we aimed to elucidate the role of autophagy in promoting the survival of human pancreatic cancer PANC-1 cells exposed to nutrient-deprived media (NDM) lacking glucose, amino acids, and serum. NDM inhibited Akt activity and phosphorylation of p70 S6K, and induced AMPK activation and mitochondrial depolarization. NDM also time-dependently increased LC3-II accumulation, number of GFP-LC3 puncta, and colocalization between GFP-LC3 and lysosomes. These results suggested that autophagy was progressively activated through Akt- and AMPK-mTOR pathway in nutrient-deficient PANC-1 cells. Autophagy inhibitors (chloroquine and wortmannin) or silencing of Atg5 augmented PANC-1 cell death in NDM. In cells exposed to NDM, chloroquine and wortmannin induced apoptosis and Z-VAD-fmk inhibited cytotoxicity of these inhibitors. These data demonstrate that autophagy is anti-apoptotic and sustains the survival of PANC-1 cells following extreme nutrient deprivation. Autophagy modulation may be a viable therapeutic option for cancer cells located in the core of solid tumors with a nutrient-deficient microenvironment.

Modulation of lipid kinase PI4KIIα activity and lipid raft association of presenilin 1 underlies γ-secretase inhibition by ginsenoside (20S)-Rg3.

Background: Cerebral elevation and accumulation of amyloid β-peptide is an invariant feature of Alzheimer disease. Results: Natural compound (20S)-Rg3, a PI4KIIα activator, modulates γ-secretase activity in lipid rafts by increasing levels of phosphoinositides. Conclusion: Activation of a key phospholipid synthetic pathway by a natural product regulates γ-secretase activity. Significance: We identify a novel molecular mechanism for the regulation of γ-secretase activity by (20S)-Rg3. Amyloid β-peptide (Aβ) pathology is an invariant feature of Alzheimer disease, preceding any detectable clinical symptoms by more than a decade. To this end, we seek to identify agents that can reduce Aβ levels in the brain via novel mechanisms. We found that (20S)-Rg3, a triterpene natural compound known as ginsenoside, reduced Aβ levels in cultured primary neurons and in the brains of a mouse model of Alzheimer disease. The (20S)-Rg3 treatment induced a decrease in the association of presenilin 1 (PS1) fragments with lipid rafts where catalytic components of the γ-secretase complex are enriched. The Aβ-lowering activity of (20S)-Rg3 directly correlated with increased activity of phosphatidylinositol 4-kinase IIα (PI4KIIα), a lipid kinase that mediates the rate-limiting step in phosphatidylinositol 4,5-bisphosphate synthesis. PI4KIIα overexpression recapitulated the effects of (20S)-Rg3, whereas reduced expression of PI4KIIα abolished the Aβ-reducing activity of (20S)-Rg3 in neurons. Our results substantiate an important role for PI4KIIα and phosphoinositide modulation in γ-secretase activity and Aβ biogenesis.

A newly synthesized macakurzin C-derivative attenuates acute and chronic skin inflammation: The Nrf2/heme oxygenase signaling as a potential target.

Impaired immune responses in skin play a pivotal role in the development and progression of chemical-associated inflammatory skin disorders. In this study, we synthesized new flavonoid derivatives from macakurzin C, and identified in vitro and in vivo efficacy of a potent anti-inflammatory flavonoid, Compound 14 (CPD 14), with its underlying mechanisms. In lipopolysaccharide (LPS)-stimulated murine macrophages and IFN-γ/TNF-α-stimulated human keratinocytes, CPD 14 significantly inhibited the release of inflammatory mediators including nitric oxide (NO), prostaglandins, and cytokines (IC50 for NO inhibition in macrophages: 4.61μM). Attenuated NF-κB signaling and activated Nrf2/HO-1 pathway were responsible for the anti-inflammatory effects of CPD 14. The in vivo relevance was examined in phorbol 12-myristate 13-acetate (TPA)-induced acute skin inflammation and oxazolone-induced atopic dermatitis models. Topically applied CPD 14 significantly protected both irritation- and sensitization-associated skin inflammation by suppressing the expression of inflammatory mediators. In summary, we demonstrated that a newly synthesized flavonoid, CPD 14, has potent inhibitory effects on skin inflammation, suggesting it is a potential therapeutic candidate to treat skin disorders associated with excessive inflammation.

Protective effect of ginsenosides Rk3 and Rh4 on cisplatin-induced acute kidney injury in?vitro and in?vivo

Background Nephrotoxicity is the major side effect in cisplatin chemotherapy. Previously, we reported that the ginsenosides Rk3 and Rh4 reduced cisplatin toxicity on porcine renal proximal epithelial tubular cells (LLC-PK1). Here, we aimed to evaluate the protective effect of ginsenosides Rk3 and Rh4 on kidney function and elucidate their antioxidant effect using in vitro and in vivo models of cisplatin-induced acute renal failure. Methods An enriched mixture of ginsenosides Rk3 and Rh4 (KG-KH; 49.3% and 43.1%, respectively) was purified from sun ginseng (heat processed Panax ginseng). Cytotoxicity was induced by treatment of 20μM cisplatin to LLC-PK1 cells and rat model of acute renal failure was generated by single intraperitoneal injection of 5 mg/kg cisplatin. Protective effects were assessed by determining cell viability, reactive oxygen species generation, blood urea nitrogen, serum creatinine, antioxidant enzyme activity, and histopathological examination. Results The in vitro assay demonstrated that KG-KH (50 μg/mL) significantly increased cell viability (4.6-fold), superoxide dismutase activity (2.8-fold), and glutathione reductase activity (1.5-fold), but reduced reactive oxygen species generation (56%) compared to cisplatin control cells. KG-KH (6 mg/kg, per os) also significantly inhibited renal edema (87% kidney index) and dysfunction (71.4% blood urea nitrogen, 67.4% creatinine) compared to cisplatin control rats. Of note, KG-KH significantly recovered the kidney levels of catalase (1.2-fold) and superoxide dismutase (1.5-fold). Conclusion Considering the oxidative injury as an early trigger of cisplatin nephrotoxicity, our findings suggest that ginsenosides Rk3 and Rh4 protect the kidney from cisplatin-induced oxidative injury and help to recover renal function by restoring intrinsic antioxidant defenses.

Protective effect of Korean Red Ginseng against FK506-induced damage in LLC-PK1 cells

Background Compound FK506 is an immunosuppressant agent that is frequently used to prevent rejection of solid organs upon transplant. However, nephrotoxicity due to apoptosis and inflammatory response mediated by FK506 limit its usefulness. In this study, the protective effect of Korean Red Ginseng (KRG) against FK506-induced damage in LLC-PK1 pig kidney epithelial cells was investigated. Methods LLC-PK1 cells were exposed to FK506 with KRG and cell viability was measured. Western blotting and RT-PCR analyses evaluated protein expression of MAPKs, caspase-3, and KIM-1. TLR-4 gene expression was assessed. Caspase-3 activities were also determined. The number of apoptotic cells was measured using an image-based cytometric assay. Results The reduction in LLC-PK1 cell viability by 60μM FK506 was recovered by KRG cotreatment in a dose-dependent manner. The phosphorylation of p38, p44/42 MAPKs (ERK), KIM-1, cleaved caspase-3, and TLR-4 mRNA expression was increased markedly in LLC-PK1 cells treated with 60μM FK506. However, with the exception of p-ERK, elevated levels of p-p38, KIM-1, cleaved caspase-3, and TLR-4 mRNA expression were significantly decreased after cotreatment with KRG. Activity level of caspase-3 was also attenuated by KRG cotreatment. Moreover, image-based cytometric assay showed that apoptotic cell death was increased by 60μM FK506 treatment, whereas it was decreased after cotreatment with KRG. Conclusion Taken together, these results suggest that the molecular mechanism of KRG in the FK506-induced nephrotoxicity may lead to the development of an adjuvant for the inhibition of adverse effect FK506 in the kidney.

Role of Autophagy in Endothelial Damage and Blood–Brain Barrier Disruption in Ischemic Stroke

The global burden of neurological diseases including stroke has significantly increased,1 and an urgent need exists to develop new treatment strategies. Impairment of autophagic regulation has been observed in diseases including neurodegenerative diseases and ischemic stroke, suggesting that modulation of autophagy could be a potential therapeutic target.2 Endothelial cells (ECs) maintain homeostasis by regulating the vascular tone and permeability and endothelial dysfunction is associated with diverse cardiovascular diseases (CVDs). The integrity of the blood–brain barrier (BBB), which shows selective permeability for substances into the brain, is significantly impaired under ischemic stroke. This review focuses on autophagy in endothelial dysfunction in the context of ischemic stroke and potential targets for therapeutic manipulation. Autophagy or autophagocytosis is an evolutionarily conserved mechanism for the degradation and recycling of cellular organelles and protein.2 It occurs continually at basal levels in cells and contributes to the maintenance of cellular homeostasis. When external nutrient supplies are limited, cells attempt to generate their energy by degrading and recycling macromolecules and cellular organelles by autophagy.3 Autophagy is also an important defense mechanism against stress including oxidative stress and infection, enabling cellular repair, or clearance of pathogens.1,2 Defects in autophagy flux may lead to the accumulation of damaged or senescent proteins and abnormal protein aggregates, and this is closely associated with human diseases including neurodegenerative, cardiovascular, and metabolic diseases, as well as cancer.2,4 In contrast to the protective role of autophagy in maintaining basal cellular homeostasis, excessive autophagy may also cause dysregulation of catabolic activity and maladaptation to cellular stress, leading to autophagic cell death.5 Accumulating evidence shows that modulating the level of autophagy by targeting specific regulatory molecules in the autophagy machinery may impact disease onset or disease …

New metabolites of hongdenafil, homosildenafil and hydroxyhomosildenafil

HighlightsThe new metabolites of hongdenafil, homosildenafil, and hydroxyhomosildenafil were determined using LC‐Q TOF‐MS/MS.Biological samples of rats for in vivo study and human liver microsome for in vitro study were determined and compared.Major metabolites were identified at m/z 461.1966 or 439.2455 by piperazine N‐dehydroxyethylation or N‐deethylation.From five to seven metabolites were identified in hongdenafil, homosildenafil and hydroxy homosildenafil treated samples.These new metabolites could be fundamental data for the toxicity study of sildenafil analogues and forensic science fields. ABSTRACT Recently, illegal sildenafil analogues have emerged, causing serious social issues. In spite of the importance of sildenafil analogues, their metabolic profiles or clinical effects have not been reported yet. In this study, new metabolites of illegal sildenafil analogues such as hongdenafil, homosildenafil, and hydroxyhomosildenafil were determined using liquid chromatography quadrupole‐time of flight mass spectrometry (LC‐Q‐TOF‐MS) and tandem mass spectrometry (LC‐Q‐TOF‐MS/MS). To prepare metabolic samples, in vitro and in vivo studies were performed. For in vivo metabolites analysis, urine and feces samples of rats treated with sildenafil analogues were analyzed. For in vitro metabolites analysis, human liver microsomes incubated with sildenafil analogues were extracted and analyzed. All metabolites were characterized by LC‐Q‐TOF‐MS and LC‐Q‐TOF‐MS/MS. As a result, five, six, and seven metabolites were determined in hongdenafil, homosildenafil, and hydroxyhomosildenafil treated samples, respectively. These results could be applied to forensic science and other analytical fields. Moreover, these newly identified metabolites could be used as fundamental data to determine the side effect and toxicity of illegal sildenafil analogues.