Characterization of changes in global gene expression in the hearts and kidneys of transgenic mice overexpressing human angiotensin-converting enzyme 2 / 한국실험동물학회지

Human angiotensin-converting enzyme 2 (hACE2) has recently received a great attention due to it play a critical role as SARS-CoV receptor in the infection of human body. However, no further analysis for gene regulation has been performed in target tissues of model mice during hACE2 overproduction. To characterize changes in global gene expression in the hearts and kidneys of rtTA/hACE2 double transgenic (dTg) mice in response to hACE2 overexpression, total RNA extracted from these tissues from dTg mice after doxycycline (Dox) treatment was hybridized to oligonucleotide microarrays. Briefly, dTg mice were generated by cross-mating pα-MHC/rtTA Tg mice with pTRE/hACE2 Tg mice. The expression level of hACE2 protein was determined to be high in hearts, kidneys, and brains of dTg mice, whereas lung, liver, and testis tissues expressed low levels. The level of hACE2 was significantly enhanced in hearts and kidneys of the Dox+dTg group compared to that in Vehicle+dTg mice although consistent levels of mouse ACE2 (mACE2) remained in the same tissues. Based on the microarray analysis of heart tissue, 385 genes were differentially expressed, including 168 upregulated and 217 downregulated, when comparing non-Tg and Vehicle+dTg mice, whereas 216 genes were differentially expressed, including 136 upregulated and 80 downregulated, between Vehicle+dTg and Dox+dTg mice. In the kidneys, 402 genes were differentially expressed, including 159 upregulated and 243 downregulated, between non-Tg and Vehicle+dTg mice. Dox-treated dTg mice exhibited the differential expression of 4735 genes including 1636 upregulated and 3109 downregulated. Taken together, these findings suggested that several functional groups and individual genes can be considered biomarkers that respond to hACE2 overexpression in dTg mice. Moreover, our results provided a lot of useful information to predict physiological responses when these dTg mice are applied as a susceptible model for novel coronavirus (SARS-CoV, COVID-19) in both vaccine and drug development.

Chemopreventive effects of garlic and mugwort mixture extract on Helicobacter pylori-associated mouse gastric carcinogenesis / 대한수의학회지

Garlic and mugwort have long been used in traditional medicine to prevent various diseases. Several in vitro studies have reported protective efficacies of garlic and mugwort in cases of gastric cancer. In the present study, we investigated the cancer preventive effects of garlic and mugwort mixture extract (GME) in a Helicobacter (H.) pylori-associated gastric carcinogenesis mouse model. To induce gastric cancer, C57BL/6 mice were treated with N-methyl-N-nitrosourea and H. pylori. Various concentrations of GME (0, 100, 500, and 1,000 ppm) were then fed to the mice for 38 weeks, after which the tumor tissues were examined for histopathology, mucin histochemistry and beta-catenin. The incidence of gastric tumors was significantly lower in the highest dose GME-treated mice (46.7%) than control mice (85.7%) (p < 0.05). The multiplicity and size of tumors were also significantly reduced by GME feeding in a dose-dependent manner (p < 0.01). Furthermore, GME suppressed the H. pylori-associated chronic inflammation measured by histologic grading of H. pylori density, chronic gastritis, glandular atrophy and intestinal metaplasia in non-tumorous gastric mucosae. Our data suggest that GME suppresses gastric tumorigenesis via suppression of H. pylori-associated chronic inflammation.

Chemopreventive effects of garlic and mugwort mixture extract on Helicobacter pylori-associated mouse gastric carcinogenesis / 대한수의학회지

Garlic and mugwort have long been used in traditional medicine to prevent various diseases. Several in vitro studies have reported protective efficacies of garlic and mugwort in cases of gastric cancer. In the present study, we investigated the cancer preventive effects of garlic and mugwort mixture extract (GME) in a Helicobacter (H.) pylori-associated gastric carcinogenesis mouse model. To induce gastric cancer, C57BL/6 mice were treated with N-methyl-N-nitrosourea and H. pylori. Various concentrations of GME (0, 100, 500, and 1,000 ppm) were then fed to the mice for 38 weeks, after which the tumor tissues were examined for histopathology, mucin histochemistry and beta-catenin. The incidence of gastric tumors was significantly lower in the highest dose GME-treated mice (46.7%) than control mice (85.7%) (p < 0.05). The multiplicity and size of tumors were also significantly reduced by GME feeding in a dose-dependent manner (p < 0.01). Furthermore, GME suppressed the H. pylori-associated chronic inflammation measured by histologic grading of H. pylori density, chronic gastritis, glandular atrophy and intestinal metaplasia in non-tumorous gastric mucosae. Our data suggest that GME suppresses gastric tumorigenesis via suppression of H. pylori-associated chronic inflammation.

Angiogenic effects of recombinant thymosin beta4 in a mouse hindlimb ischemia model / 동물의과학연구지

Recombinant thymosin beta4 (rTbeta4) has been reported to migrate and promote vascularization, wound-healing, and hair growth in a mouse hindlimb ischemia model of peripheral vascular disease. C57BL/6 mice (11-weeks-old) were anesthetized and an ischemic model was made by cutting the right aorta femoralis. The ischemic group was intraperitoneally administered with saline (300 microL/mouse) and the muscular administration group received rTbeta4 (150 microg in 300 microL of saline) or rTbeta4 (150 microg in 300 microL saline) to the abdominal cavity at 3-day intervals for 21 days. Myoatrophy of the ischemic group was observed compared to the normal control group. Generation of adjacent vessels was carried out in the rTbeta4 administration group compared to the ischemic group. The biopsy results showed significant fibrosis around the muscular undersurface and perimysium in the musculus quadriceps femoris of the ischemic group, whereas partial fibrosis was observed in the perimysium and endomysium in the rTbeta4 administration group. Immunostaining indicated that expression levels of hypoxiainducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor-1 (VEGF-1), and endothelial nitric oxide synthase (eNOS) in the rTbeta4 group were higher than those of the ischemic group. Western blotting showed that expression levels of HIF-1alpha, VEGF-1, and eNOS in the rTbeta4 group were higher than those of the ischemic group. In conclusion, rTbeta4 increases expression levels of HIF-1alpha, VEGF-1, and eNOS, resulting in angiogenesis.

Effects of administration of IH901, a ginsenoside intestinal metabolite, on muscular and pulmonary antioxidant functions after eccentric exercise

This study was conducted to investigate whether administration of IH901, a ginseng intestinal metabolite, ameliorates exercise-induced oxidative stress while preserving antioxidant defense capability in rat skeletal muscles and lung. Eight adult male Sprague-Dawley rats per group were randomly assigned to the resting control, exercise control, resting with IH901 (25, 50, and 100 mg/kg) consumption (R/IH901), or exercise with IH901 (25, 50, and 100 mg/kg) consumption (E/IH901) group. The trained groups ran 35 min 2 days/week for 8 weeks. To analyze the IH901-training interaction, serum biochemical analysis, lipid peroxidation, citrate synthase, protein oxidation, antioxidant and superoxide dismutase in skeletal muscles and lung tissue were measured. Compared to the exercise control group, animals that consumed IH901 had significantly increased exercise endurance times (p < 0.05) and decreased plasma creatine kinase and lactate dehydrogenase levels (p < 0.05), while those in the E/IH901 groups had increased citrate synthase and anti-oxidant enzymes and decreased lipid peroxidation and protein oxidation (p < 0.05). In conclusion, IH901 consumption in aging rats after eccentric exercise has beneficial effects on anti-inflammatory and anti-oxidant activities through down-regulation of pro-inflammatory mediators, lipid peroxidation, and protein oxidation and up-regulation of anti-oxidant enzymes.

Historical control data from 13-week repeated toxicity studies in Crj:CD (SD) rats / 한국실험동물학회지

Reference ranges of standard experimental parameters are useful for comparisons in toxicology. The aim of this study was to collect data from 13-week repeated toxicity studies in Crl:CD (SD) rats, a strain widely used for toxicity and efficacy research, for establishing domestic reference values. Data on body weight, food consumption; urinalysis, hematological, and blood biochemical parameters; and organ weights were collected from 11 toxicity studies in 220 Crl:CD (SD) rats (110 males and 110 females). The studies had been performed at a single testing facility over the last 5 years and involved animals sourced from a single breeder. The findings were collated as means, standard deviations, percentages, and ranges. Urine volume, uterus weight, eosinophil, and basophil counts, and triglyceride, total bilirubin, and gamma-glutamyl transpeptidase levels showed standard deviations of 30% or more. These historical control data would help to interpret the effects of test substances in routine toxicity and efficacy studies.

In vitro antioxidant properties of a ginseng intestinal metabolite IH-901 / 한국실험동물학회지

IH-901 (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol or compound K) is a final intestinal bacterial metabolite of ginseng in humans. It has various pharmacologic effects such as antiaging, immunopotentiation, antistress, and antimetastatic activities. We analyzed the antioxidant activities of IH-901 using several assays including: total antioxidant activity, reductive potential, 1,1-diphenyl-2-picryl-hydrazyl, hydroxyl, superoxide and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays, a nitric oxide scavenging assay and a lipid peroxidation assay. At concentrations of 25 and 100 microg/mL, IH-901 inhibited lipid peroxidation of a linoleic acid emulsion with a potency comparable to ascorbic acid and butylated hydroxyanisole. The reductive potential of IH-901 increased in a concentration-dependent manner. IH-901 exhibited strong DPPH, hydroxyl, superoxide and ABTS radical scavenging activities. IH-901 was also an effective inhibitor of lipid peroxidation, although IH-901 had only a mild scavenging activity against nitric oxide. These results suggest that IH-901 may be a useful antioxidant agent against reactive oxygen species.

Exposure to genistein does not adversely affect the reproductive system in adult male mice adapted to a soy-based commercial diet

Genistein, a soybean-originated isoflavone, is widely consumed by humans for putative beneficial health effects but its estrogenic activity may affect adversely the development of male reproductive system. Five-week-old ICR mice were purchased and fed with a soybean-based Purina Chow diet until 6 months of age. The animals were exposed by gavage to genistein (2.5 mg/kg/day) or 17beta-estradiol (7.5 microgram/kg/day) for five weeks. Corn oil was used for the negative control. The animals were fed the caseinbased AIN-76A diet throughout the experimental periods. There were no significant differences in body and organ weights of mice among experimental groups. No significant differences in sperm counts and sperm motile characteristics were found between the control and the genistein groups. Treatment of 17beta-estradiol caused a significant decrease in epididymal sperm counts compared to the control (p<0.05). The level of phospholipid hydroxide glutathione peroxidase in the epididymis of mice exposed to genistein was significantly higher than that of the control mice (p<0.05). 17beta-estradiol treatment caused a reduction of germ cells in the testis and hyperplasia of mucosal fold region in the prostate of mice. Genistein treatment did not cause any lesion in the testis, epididymis, and prostate. These results suggest that dietary uptake of genistein at adult stage of life may not affect male reproductive system and functions.

Autoradiographic Study on the Effect of AG60 to the DNA Synthesis of Gastric Epithelium of Mouse Inoculated with Ehrlich Carcinoma / 대한해부학회지

To study the tumor-suppression effect of a newly developed anti-tumor agent AG60 [acriflavine (1) : guanosine (1) composition, Taerim Pharm. Co., Seoul, Korea], each Ehrlich carcinoma (10(7) cells)-inoculated mouse received the subcutaneous injection of 0.2 ml of saline, 5 mg/kg of AG60, and 30 mg/kg of AG60 per day for a week. The day following the last injection, each mouse was injected with a single dose of 0.7 microcurie/g of methyl-3H-thymidine (25Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed, and gastric tissues were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 micrometer-thick sections. Deparaffinized sections were coated with autoradiographic emulsion EM 1 (Amersham Lab. England) in a dark room and dried and were placed in a light-tight box. The sections were exposured for 5 weeks in the dark room, and were then developed in D-19 developer. Labeled indices (mean number of labeled cells per 100 epithelial cells in the isthmus) were observed and calculated. The results are as follows; 1. On histological study, gastric mucosa had no morphological changes following the injection of AG60. 2. On autoradiographic study, labeled grains of 3H-thymidine were restricted on the isthmus portion of the gatric gland. 3. On autoradiographic study, labeling indicies of gastric epithelial cells of normal control, experimental control, AG60 (5 mg/kg)-treated and AG60 (30 mg/kg)-treated groups were 21.9+/-0.28%, 18.8+/-0.03%, 21.6+/-1.61% and 6.3+/-0.93%, respectively. These result suggest that AG60 is expected as one of most effective anticancer drugs, and the dosage under 5 mg/kg of AG60 does not result any defect on the DNA synthesis in gastric epithelial cells.

Fluorescent Study on the Tissue-Distribution of Acriflavine-Guanosine Compound (AG60) / 대한해부학회지

The study was carried out to evaluate the tissue-distribution of acriflavine or AG60 (acriflavine-guanosine compound, 1 : 1), the newly developed anticancer remedy. Successful access or distribution of a drug to specific tissue is important to attack the cancer cells in the same area. But it also means that the drug may disturb the activities of labelled tissues or cells. On the other hand, unlabelled elements may survive from massive treatment with the drug. In this study, distribution of acriflavine or AG60 in Yac-1 leukemic cells (0.25~25 microgram/ml) and in the tissues of rats or mice (5~50 mg/kg) were evaluated. Yac-1 cells showed prominent fluorescence on the heterochromatin and more or less prominent fluorescence on the nucleoplasm, cytoplasm and plasma membrane. Cytotoxicity of AG60 led to morphologic changes such as bleb- or baloon-formation on the surface, general swelling of the cell, and lysis of the cell. Following the subcutaneous administration of acriflavine or AG60 (5~50 mg/kg) to the Ehrlich carcinoma-inoculat-ed rats or mice, most tissues including cancer cells showed acriflavine-fluorescence with some exception. The nuclei of cells of tissues were labelled more strongly than those of cytoplasm. Fluorescence was especially strong over biliary tree, renal corpuscle, gastrointestinal mucous coat, and bronchial mucous coat. But parenchymal components of central nervous system did not show any fluorescence. As shown in Yac-1 cells treated with AG60, the drug strongly attached to nucleic acids, and it induced swelling and disintegration of cancer cells. Fast turn-over of AG60 was seen in the secretory passages of bile juice, urine, gastrointestinal mucin, and bronchial mucin. The results show that AG60 could reach most tissues except parenchymes of central nervous system.

Anticancer Effect of AG60 (Acriflavine-Guanosine Compound) on the Ehrlich Cancer Cells Light Microscopic, Autoradiographic and Electron Microscopic Study / 대한해부학회지

To evaluate the effect and working mechanism of a newly developed anti-cancer drug, AG60 (acriflavine-guanosine compound, Taerim Pharm. Co. Seoul, Korea), histotologic, autoradiographic and electron microscopic studies were carried out. For the histologic study, each Ehrlich carcinoma cells (10(7) cells)-inoculated mouse was subcutaneously injected with saline (0.2 ml), 10 mg/kg of AG60, or 30 mg/kg of AG60, every other day, respectively. Animals were sacrified on the 14th day from the first injection, and tumor masses were fixed in 10% formalin solution. Tissue sections of the tumor were stained with hematoxylin and eosin. For the electron microscopic study, Ehrlich carcinoma (10(7) cells)-inoculated mice were subcutaneously injected every other day with saline (0.2 ml) or 30 mg/kg of AG60, respectively. The day after 7th injection (14th day), animals were sacrified, small piece of tumor masses were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution followed by fixation in 2% osmium tetroxide solution. Ultrathin sections were counter stained with uranyl acetate-lead citrate solutions, and observed with JEM 100CX electron microscope. For the autoradiographic study, each Ehrlich carcinoma (10(7) cells)-inoculated mouse was injected every day with 0.2 ml of saline, 5 mg/kg of AG60, or 30 mg/kg of AG60, respectively. The day following the last injection, each animal was given a single dose of 0.7 micricurie/g of methyl-3H-thymidine (Amersham Lab., England) through the tail vein. Seventy minutes after the thymidine injection, animals were sacrified, tumor masses were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 micrometer-thick sections. Deparaffinzied sections were dipped in the autradiographic emulsion E1 (Amersham Lab., England) and dried and stocked in the dark room. Filmed sections were exposured five weeks in the dark room, and were developed in the developer. Labeled indices (mean number of labeled cells per 100 cancer cells) and labeled grain indices (mean number of labeled silver grains per one cancer cell, and total granule numbers per every 100 cancer cell) were observed and calculated. The results were as follows : 1. On histological study, massive apoptosis were occured following the injection of AG60. Only small number of live cancer cells were observed. 2. On electron microscopic study, massive apoptotic figures including fragmentation of nuclei and cytoplasms, multiple nucleoli, condensation of nucleus and cytoplasm, deep invaginations and microcleft formations of nuclei, margination of heterochromatin along the inner nuclear membrane and microcleft , etc. were noticed. Giant cells represent the "tumor cell-tumor cell emperipolesis", and many of them seem to be in process of "cytolytic emperipolesis". 3. On autoradiographic study, labeled grains of 3H-thymidine were suppressed to only 11%~5% of control cancer cells following AG60 administrations. Discussed on the above experiments, it is suggested that severe suppression of DNA, RNA and protein syntheses by AG60 induce massive apoptosis of cancer cells. AG60 is expected as one of most effective anticancer drugs for the cytostatic therapy, the disease stabilization, the improved quality of life, the prolongation of life, and possibly the chemoprevention.