Drug Development from Natural Products Based on the Pathogenic Mechanism of Asthma.

Asthma is a chronic inflammatory disease of the pulmonary system associated with many wheeze-to-sleep apnea complications that may lead to death. In 2019, approximately 262 million patients suffered from asthma, and 455 thousand died from the disease worldwide. It is a more severe health problem in children and older adults, and as the aging of society intensifies, the problem will continue to worsen. Asthma inducers can be classified as indoor and outdoor allergens and can cause asthma due to their repeated invasion. There are several theories about asthma occurrence, such as the imbalance between Th1 and Th2, inflammation in the pulmonary system, and the abnormal apoptosis/cell proliferation of cells related to asthma. Although there are many medications for asthma, as it is an incurable disease, the purpose of the drugs is only to suppress the symptoms. The current drugs can be divided into relievers and controllers; however, as they have many adverse effects, such as immune suppression, growth retardation, promotion of cataracts, hyperactivity, and convulsions, developing new asthma drugs is necessary. Although natural products can have adverse effects, the development of asthma drugs from natural products may be beneficial, as some have anti-asthmatic effects such as immune modulation, anti-inflammation, and/or apoptosis modulation.

Therapeutic treatments for diabetes mellitus-induced liver injury by regulating oxidative stress and inflammation.

Diabetes mellitus (DM) is a metabolic disease that affects all systems in the body, including the liver. Numerous studies have reported that chronic DM etiology and pathogenesis complications implicate oxidative stress, generating reactive oxygen species, such as superoxide anions and free radicals. In addition, pro-inflammatory reactions are also underlying functions closely related to oxidative stress that further exacerbate pathological DM states. The liver is especially susceptible to hyperglycemia-induced oxidative stress and the related inflammation. Thus, anti-oxidation and anti-inflammation therapies are promising strategies for treating liver damage. This review summarizes therapeutic treatments attenuating the generation of oxidative stress and pro-inflammation, which also cause DM-induced liver injury. Although the treatments have several impediments to be solved, these remedies may have clinically important implications under the absence of effective drugs for the damaged liver in DM patients.

Chlorogenic acid attenuates pro-inflammatory response in the blood of streptozotocin-induced diabetic rats.

BACKGROUND: Chlorogenic acid (CGA) has been shown to reduce pro-inflammation by scavenging reactive oxygen species (ROS) and reactive nitrogen species. In this study, the anti-inflammatory effect of CGA was expanded to streptozotocin (STZ)-induced diabetic rats. The inter-relationships among oxidative stress, pro-inflammation, and cytochrome P450 (CYP) 1A enzymes were also investigated in peripheral blood mononuclear cells (PBMC) of STZ-diabetic rats. RESULTS: The levels of pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor-alpha, increased by approximately 3.4- and 2.9-fold, respectively, and the albumin concentration decreased in the serum of STZ-induced diabetic rats compared to normal rats. The C-reactive protein (CRP) values also increased by about 3.8-fold higher, indicating that STZ induced an inflammation in the blood of STZ-diabetic rats. The expression levels and catalytic activities of CYP1A enzymes were elevated by approximately 2.2-2.5- and 4.3-6.7-fold, respectively, in the PBMC of STZ-treated rats. A decrease in the amount of PBMC-bound albumin was also observed. In contrast, the levels of cytokines and CRP in serum and the activities of CYP1A enzymes in PBMC were significantly reduced in CGA-treated diabetic rats in a CGA concentration-dependent manner. In addition, STZ-mediated elevation of ROS in serum and PBMC was decreased by the CGA administration. However, the CGA treatment did not change the enhanced blood glucose level and expression of CYP1A enzymes by STZ. STZ-mediated decrease in the levels of serum and PBMC-bound albumin was not also restored by the CGA administration. CONCLUSIONS: These results suggest that CGA could be used to treat type 1 diabetes-induced inflammation.

Multiple Porous Synthetic Bone Graft Comprising EngineeredMicro-Channel for Drug Carrier and Bone Regeneration.

Due to high demand but limited supply, there has been an increase in the need to replace autologous bone grafts with alternatives that fulfill osteogenic requirements. In this study, two different types of bone grafts were tested for their drug carrying abilities along with their osteogenic properties. Two different types of alendronate-loaded bone grafts, Bio-Oss (bovine bone graft) and InRoad (biphasic synthetic bone graft) were observed to see how different concentrations of alendronate would affect the sustained release to enhance osteogenesis. In this study, defected ovariectomize-induced osteoporotic rat calvarias were observed for 28 days with three different concentrations of alendronate (0 mg, 1 mg, 5 mg) for both Bio-Oss and InRoad. A higher concentration (5 mg) allowed for a more controlled and sustained release throughout the 28-day comparison to those of lower concentrations (0 mg, 1 mg). When comparing Bio-Oss and InRoad through histology and Micro-CT, InRoad showed higher enhancement in osteogenesis. Through this study, it was observed that alendronate not only brings out robust osteogenesis with InRoad bone grafts, but also enhances bone regeneration in an alendronate-concentration-dependent manner. The combination of higher concentration of alendronate and multiple porous bone graft containing internal micro-channel structure of InRoad resulted in higher osteogenesis with a sustained release of alendronate.

Korean Red Ginseng affects ovalbumin-induced asthma by modulating IL-12, IL-4, and IL-6 levels and the NF-κB/COX-2 and PGE2 pathways.

BACKGROUND: Asthma is an incurable hyper-responsive disease of the pulmonary system that is caused by various allergens, including indoor and outdoor stimulators. According to the Global Asthma Network, 339 million people suffered from asthma in 2018, with particularly severe forms in children. Numerous treatments for asthma are available; however, they are frequently associated with adverse effects such as growth retardation, neurological disorders (e.g., catatonia, poor concentration, and insomnia), and physiological disorders (e.g., immunosuppression, hypertension, hyperglycemia, and osteoporosis). METHODS: Korean Red Ginseng has long been used to treat numerous diseases in many countries, and we investigated the anti-asthmatic effects and mechanisms of action of Korean Red Ginseng. Eighty-four BALB/c mice were assigned to 6 treatment groups: control, ovalbumin-induced asthma group, dexamethasone treatment group, and 3 groups treated with Korean Red Ginseng water extract (KRGWE) at 5, 25, or 50 mg/kg/day for 5 days. Anti-asthmatic effects of KRGWE were assessed based on biological changes, such as white blood cell counts and differential counts in the bronchoalveolar lavage fluid, serum IgE levels, and histopathological changes in the lungs, and by examining anti-asthmatic mechanisms, such as the cytokines associated with Th1, Th2, and Treg cells and inflammation pathways. RESULTS: KRGWE affected ovalbumin-induced changes, such as increased white blood cell counts, increased IgE levels, and morphological changes (mucous hypersecretion, epithelial cell hyperplasia, inflammatory cell infiltration) by downregulating cytokines such as IL-12, IL-4, and IL-6 via GATA-3 inactivation and suppression of inflammation via NF-κB/COX-2 and PGE2 pathways. CONCLUSION: KRGWE is a promising drug for asthma treatment.

Respiratory and Systemic Toxicity of Inhaled Artificial Asian Sand Dust in Pigs.

Air pollution, particularly caused by Asian sand dust (ASD) and particulate matter (PM), has become one of the leading threats to public health. However, the majority of studies have primarily focused on epidemiological assessment, and in vivo toxicities of certain air pollutants have been poorly elucidated in medium/large-size laboratory animals. To investigate the impact of ASD in domestic animals, 16 Landrace pigs were exposed to an artificial ASD sandstorm for 6 h. All animals were divided in four cages, and a commercial yellow soil was used for generating artificial mineralogical particles. Blood samples were collected, and necropsies were performed before exposure and 6, 12, 24, and 72 h after exposure. Complete blood cell count and the levels of serum biochemical enzymes, blood gas, electrolytes, and a variety of inflammatory cytokines were evaluated. In addition, histopathological examination was conducted. Various test results proved acute lower airway disorders with systemic inflammation in pigs. To our knowledge, this study is the first to describe experimental research in domestic animals concerning the damage caused by artificial ASD exposure. The results of this study suggest that ASD has importance in terms of not only public health but also of ultimate economic losses in the pork industry.

Protective effects of Erythronium japonicum and Corylopsis coreana Uyeki extracts against 1,3-dichloro-2-propanol-induced hepatotoxicity in rats.

Erythronium japonicum (E. japonicum) and Corylopsis coreana Uyeki (C. coreana Uyeki, Korean winter hazel) have been shown to significantly decrease 1,3-dichloro-2-propanol (1,3-DCP)-induced generation of reactive oxygen species and CYP2E1 activity in HuH7, human hepatocytes. In this study, we expanded upon the previous study and investigated the effects of E. japonicum and C. coreana Uyeki extracts on 1,3-DCP-induced liver damage in rats. The pre-treatment of rats with these extracts alleviated a decrease in body weight and reduced 1,3-DCP-induced increase in catalytic activities of hepatic enzymes, such as aspartate aminotransferase and alanine aminotransferase, in the serum. Moreover, treatment with the extracts restored the 1,3-DCP-induced decreases in anti-oxidant enzyme activities, such as the activities of superoxide dismutase and catalase, in the rat liver. Histopathological studies also strongly supported the results of enzyme activities. These results suggest a possibility that the extracts of E. japonicum and C. coreana Uyeki can be a remedy for alleviating 1,3-DCP-induced liver damage in animals.

Extracts from Erythronium japonicum and Corylopsis coreana Uyeki reduce 1,3-dichloro-2-propanol-mediated oxidative stress in human hepatic cells.

In this study, it was demonstrated that 1,3-dichloro-2-propanol (1,3-DCP) induced oxidative stress and cell death in HuH7, human hepatocytes. The protective effects of Erythronium japonicum (E. japonicum) and Corylopsis coreana Uyeki (C. coreana Uyeki) extracts against 1,3-DCP-treated cells were also investigated. First, the activities of superoxide dismutase (SOD) and catalase (CAT) were diminished by the treatment of 1,3-DCP. Moreover, 1,3-DCP stimulated the expression and catalytic activity of cytochrome P450 2E1 (CYP2E1), an enzyme that generates reactive oxygen species in the liver. In contrast, co-treatment of 1,3-DCP with the extracts significantly decreased ROS generation and inhibited CYP2E1 activity without affecting its expression. The co-administration of extracts also restored the activities of SOD and CAT reduced by 1,3-DCP and protected against 1,3-DCP-mediated cell death. In conclusion, these results suggest that 1,3-DCP induces oxidative stress through the elevated CYP2E1 level, which is inhibited by the extracts, protecting cells against the effects of 1,3-DCP.

Wicking Property of Graft Material Enhanced Bone Regeneration in the Ovariectomized Rat Model.

BACKGROUND: Recruitment and homing cells into graft materials from host tissue is crucial for bone regeneration. METHODS: Highly porous, multi-level structural, hydroxyapatite bone void filler (HA-BVF) have been investigated to restore critical size bone defects. The aim was to investigate a feasibility of bone regeneration of synthetic HA-BVF compared to commercial xenograft (Bio-Oss). HA-BVF of 0.7 mm in average diameter was prepared via template coating method. Groups of animals (n = 6) were divided into two with normal (Sham) or induced osteoporotic conditions (Ovx). Subsequently, subdivided into three treated with HA-BVF as an experiment or Bio-Oss as a positive control or no treatment as a negative control (defect). The new bone formation was analyzed by micro-CT and histology. RESULTS: At 4 weeks post-surgery, new bone formation was initiated from all groups. At 8 weeks post-surgery, new bone formation in the HA-BVF groups was greater than Bio-Oss groups. Extraordinarily greater bone regeneration within the Ovx-HA group than Sham-Bio-Oss or Ovx-Bio-Oss group (p < 0.05). CONCLUSION: This study suggests that the immediate wicking property of HA-BVF from host tissue activates a natural healing cascade without the addition of exogeneous factors or progenitor cells. HA-BVF may be an effective alternative for repairing bone defects under both normal and osteoporotic bone conditions.

Diacylglycerol in Cationic Nanoparticles Stimulates Oxidative Stress-Mediated Death of Cancer Cells.

Previous reports have suggested that cargo-free cationic nanoparticles (cNP) consisting of cationic monovalent lipids, such as 1,2-oleoyl-3-trimethylammonium-propane (DOTAP), induce reactive oxygen species (ROS) generation and toxicity in cells. In addition, cNP containing six lysine residues (6K) and cargo (6K-cNP) exerted synergistic effects on ROS production and cell death in cancer cells. In this study, we investigated the effect of diacylglycerol (DAG) derived from egg phosphatidylcholine in nanoparticles (NP) on ROS-mediated cellular toxicity. When DAG was incorporated into cNP (D-cNP) or 6K-cNP (6K-D-cNP) up to 7.8 mol% at the expense of DOTAP, and treated with cells, ROS generation in cancer cells increased further in a DAG concentration-dependent manner compared with those of both cNP without DAG. Concomitantly, cancer cell viability was more decreased upon the treatment with DAG-containing cNP. Moreover, D-cNP or 6K-D-cNP exhibited enhanced uptake into cells under endocytosis-inhibited conditions. Taken together, these results suggested that the presence of DAG in NP stimulated the interaction of NP with cancer cells and the resulting ROS-mediated cytotoxicity.

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from Mycobacterium smegmatis.

OBJECTIVES: To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs. RESULTS: CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H2O2 and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates. CONCLUSIONS: CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3.

Enzymatic conversion of natural glycosides to their corresponding hydroxylated products using cytochromes P450 has significant advantages over synthetic chemistry and even enzyme-catalyzed glycosylation of chemicals. At present, the basic strategy for making glycosides of stilbenoid compounds is to use the glycosylation activity of enzymes, such as glycosyltransferases. Here, an efficient synthesis of a valuable (E)-astringin, a piceatannol glucoside, was developed using CYP102A1 via the highly regioselective C-3' hydroxylation of polydatin, a resveratrol glucoside. (E)-astringin is a high added value compound found in plants and wine. Benzylic hydroxylation of polydatin provides an attractive route to (E)-astringin, a catechol product. Thus far, chemical and enzymatic methods of producing (E)-astringin have not been developed. In the present study, a set of CYP102A1 mutants from Bacillus megaterium was found to catalyze regioselective hydroxylation of polydatin at the C-3' position to generate an (E)-astringin, a piceatannol glucoside.

Regioselective C-H hydroxylation of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human metabolite.

OBJECTIVES: To find a simple enzymatic strategy for the efficient synthesis of the expensive 5'-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate. RESULTS: The practical synthetic strategy for the 5'-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5'-OH omeprazole sulfide with a high conversion yield (85-90 %). CONCLUSIONS: A highly efficient synthesis of 5'-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

Cargo-Free Nanoparticles Containing Cationic Lipids Induce Reactive Oxygen Species and Cell Death in HepG2 Cells.

Nanoparticles (NPs) containing cationic monovalent lipids such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA), have been widely used for the delivery of nucleic acid such as small-interfering RNA and polypeptide to cells as cancer therapies and vaccine development. Several previous reports have suggested that cationic liposomes induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. Here, we systematically investigated the effects of DOTAP- or DOTMA-containing NPs without any cargo on the human carcinoma cells, HepG2. Treatment with NPs containing DOTAP or DOTMA increased the production of cellular ROS, such as H2O2 and lipid peroxidation, in HepG2 cells and concomitantly decreased cell viability. These effects were dependent on the lipid concentration, surface density of cationic lipids, and particle size of NPs. However, neutral NPs consisting of 1,2-dioleoyl-3-phosphocholine did not elicit the effective ROS generation or cell death regardless of the lipid concentration and particle size. The present study suggests that DOTAP- and DOTMA-NPs are able to induce cancer cell death through production of ROS in the absence of any therapeutic cancer reagents. These results also provide a rational background for the design of delivery systems using cationic lipid-based NP formulations.

Transformation of Escherichia coli and protein expression using lipoplex mimicry.

We investigated a "one-step" method for transformation of and protein expression in Escherichia coli (E. coli) using a complex of n-stearylamine, a cationic lipid, and plasmid DNA, which mimics lipoplex-based approaches. When E. coli cells were treated with the cationic lipid-plasmid complex, the transformation efficiencies were in the range of approximately 2-3 × 10(6) colony-forming units. Further increase in the efficiency was obtained by co-treatment with calcium chloride (or rubidium chloride) and the complexes. Moreover, after DNA transfer, E. coli cells successfully expressed plasmid-encoded proteins such as cytochrome P450s and glutathione-S-transferase without overnight incubation of the cells to form colonies, an indispensable step in other bacterial transformation methods. In this study, we provide a simple method for E. coli transformation, which does not require the preparation of competent cells. The present method also shortens the overall procedures for transformation and gene expression in E. coli by omitting the colony-forming step.

New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity.

Plant phytochromes are photoreceptors that mediate a variety of photomorphogenic responses. There are two spectral photoisomers, the red light-absorbing Pr and far-red light-absorbing Pfr forms, and the photoreversible transformation between the two forms is important for the functioning of phytochromes. In this study, we isolated a Tyr-268-to-Val mutant of Avena sativa phytochrome A (AsYVA) that displayed little photoconversion. Interestingly, transgenic plants of AsYVA showed light-independent phytochrome signaling with a constitutive photomorphogenic (cop) phenotype that is characterized by shortened hypocotyls and open cotyledons in the dark. In addition, the corresponding Tyr-303-to-Val mutant of Arabidopsis (Arabidopsis thaliana) phytochrome B (AtYVB) exhibited nuclear localization and interaction with phytochrome-interacting factor 3 (PIF3) independently of light, conferring a constitutive photomorphogenic development to its transgenic plants, which is comparable to the first constitutively active version of phytochrome B (YHB; Tyr-276-to-His mutant). We also found that chromophore ligation was required for the light-independent interaction of AtYVB with PIF3. Moreover, we demonstrated that AtYVB did not exhibit phytochrome B activity when it was localized in the cytosol by fusion with the nuclear export signal and that AsYVA exhibited the full activity of phytochrome A when localized in the nucleus by fusion with the nuclear localization signal. Furthermore, the corresponding Tyr-269-to-Val mutant of Arabidopsis phytochrome A (AtYVA) exhibited similar cop phenotypes in transgenic plants to AsYVA. Collectively, these results suggest that the conserved Tyr residues in the chromophore-binding pocket play an important role during the Pr-to-Pfr photoconversion of phytochromes, providing new constitutively active alleles of phytochromes by the Tyr-to-Val mutation.

Evidence that phytochrome functions as a protein kinase in plant light signalling.

It has been suggested that plant phytochromes are autophosphorylating serine/threonine kinases. However, the biochemical properties and functional roles of putative phytochrome kinase activity in plant light signalling are largely unknown. Here, we describe the biochemical and functional characterization of Avena sativa phytochrome A (AsphyA) as a potential protein kinase. We provide evidence that phytochrome-interacting factors (PIFs) are phosphorylated by phytochromes in vitro. Domain mapping of AsphyA shows that the photosensory core region consisting of PAS-GAF-PHY domains in the N-terminal is required for the observed kinase activity. Moreover, we demonstrate that transgenic plants expressing mutant versions of AsphyA, which display reduced activity in in vitro kinase assays, show hyposensitive responses to far-red light. Further analysis reveals that far-red light-induced phosphorylation and degradation of PIF3 are significantly reduced in these transgenic plants. Collectively, these results suggest a positive relationship between phytochrome kinase activity and photoresponses in plants.

Selenium supplementation restores the decreased albumin level of peripheral blood mononuclear cells in streptozotocin-induced diabetic mice.

Previously, it has been suggested that the phenotypic level of albumin in peripheral blood mononuclear cells (PBMC) decreased in streptozotocin (STZ)-induced diabetic rats. Concomitantly, the production of oxidative stresses was also elevated in the diabetic PBMC compared to that of normal control. These results suggest the close relationship between PBMC-albumin and its antioxidant roles. Here, we expanded the previous studies and investigated the effect of selenium supplementation as inorganic (sodium selenate) forms on the levels of albumin expression and oxidative stress in PBMC of STZ-induced diabetic mice. Selenium intake recovered the decreased albumin levels to those of normal mice and reduced the production of reactive oxygen species (ROS). These results support that selenium intake may alleviate the etiology and pathology of PBMC in type 1 diabetic mice by restoring the decrease in albumin contents and the production of ROS.

New Surface-Treatment Technique of Concrete Structures Using Crack Repair Stick with Healing Ingredients.

This study focused on the development of a crack repair stick as a new repair method along with self-healing materials that can be used to easily repair the cracks in a concrete structure at the construction site. In developing this new repair technique, the self-healing efficiency of various cementitious materials was considered. Likewise, a crack repair stick was developed to apply to concrete structures with 0.3 mm or lower crack widths. The crack repair stick was made with different materials, such as cement, an expansive material (C12A7), a swelling material, and calcium carbonate, to endow it with a self-healing property. To verify the performance of the crack repair stick for concrete structures, two types of procedures (field experiment and field absorption test) were carried out. As a result of such procedures, it was concluded that the developed crack repair stick could be used on concrete structures to reduce repair expenses and for the improved workability, usability, and serviceability of such structures. On the other hand, to evaluate the self-healing performance of the crack repair stick, various tests were conducted, such as the relative dynamic modulus of elasticity test, the water tightness test, the water permeability test, observation via a microscope, and scanning electron microscope (SEM) analysis. From the results, it is found that water leakage can be prevented and that the durability of a concrete structure can be improved through self-healing. Also, it was verified that the cracks were perfectly closed after 28 days due to application of the crack repair stick. These results indicate the usability of the crack repair stick for concrete structures, and its self-healing efficiency.

Functional characterization of steroid hydroxylase CYP106A1 derived from Bacillus megaterium.

In this study, we examined the catalytic activity of CYP106A1 from the Bacillus megaterium American Type Culture Collection 14581 strain. The CYP106A1 gene was cloned from B. megaterium, heterologously expressed in Escherichia coli, and purified. Potential electron partners and possible bacterial CYP106A1 substrates were identified by examining the oxidative activity toward a set of steroids in the presence of several reductase systems. The activities of CYP106A1 in a reconstituted system could not be achieved using rat NADPH-P450 reductase or a putidaredoxin reductase-putidaredoxin pair. However, the spinach redox proteins, a ferredoxin reductase-ferredoxin pair, were found to be efficient redox partners for CYP106A1. CYP106A1 catalyzes the hydroxylation of a set of steroids including testosterone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and 11-deoxycortisol to produce monohydroxylated products as the major metabolites. These results suggest that CYP106A1 would be useful for the bioconversion of steroid hormones to hydroxylated products that can be used for industrial applications.