Female ICR Mice Research Articles

Oral Toxicity and Hypotensive Influence of Sericin-Derived Oligopeptides (SDOs) from Yellow Silk Cocoons of Bombyx mori in Rodent Studies.

Sericin-derived oligopeptides (SDOs) from yellow silk cocoons exhibit antihypertensive and hypoglycemic properties in both in vitro and in vivo studies. This study investigated the acute toxicity of SDOs as a novel food for human consumption using female ICR mice and Wistar rats, as well as the chronic toxicity test on both sexes of Wistar rats. Clinical chemistry, hematology, and histopathological studies revealed that SDOs were safe for a single dose of 2000 mg kg-1 body weight (BW) and daily oral administration of 50, 100, and 200 mg kg-1 BW for six months. The chronic toxicity study additionally measured the rats' systolic blood pressure (SBP) and blood sugar monthly as they slowly aged. In the 2nd month for male rats and the 4th month for both sexes, SDOs had a significant hypotensive effect on Wistar rats' blood pressure, lowering it from 130 mmHg to a plateau at 110-115 mmHg. In contrast, the blood pressure of the control rats exceeded 140 mmHg after five months. Nonetheless, the hypoglycemic effect was not observed. Measurements of SBP and blood glucose in aged rats during chronic toxicity tests yielded insights beyond ordinary toxicity, including the health and fitness of the lab rats, perhaps resulting in novel discoveries or areas of study that justify the sacrifice of the animals' lives.

Tap Water Shorts the Lifespan of Mice with Prolonged Exposure to Fractionated γ-Radiation

Assessing the role of social factors in the formation of radioresistance in chronic exposure and finding ways to increase it are important for understanding the mechanisms of the damaging effects of radiation and developing practical methods for reducing radiation risk in professionals exposed to ionizing radiation and patients undergoing radiation therapy. In this work, we investigated the ability to influence the lifespan of animals exposed to γ-radiation fractionally for a long time by replacing drinking water from tap water to distilled water. Female ICR mice (CD-1) were exposed to total 60Co γ radiation weekly in fractions for 33 weeks starting at 9 weeks of age. The dose of a single irradiation was 50 mGy, the average dose rate was 2 mGy/sec. The total radiation dose was 1.65 Gy. Control non-irradiated mice and irradiated animals were divided into 2 groups. The first received tap water, and the second received distilled water throughout the experiment. Non-irradiated animals kept on tap water showed a statistically insignificant (log-rank test, p = 0.483) reduction in average life expectancy compared to mice kept on distilled water. In animals after exposure to 60Co γ-radiation, a statistically significant (p = 0.0013) decrease in life expectancy was noted when kept on tap water and statistically insignificant (p = 0.1511) when kept on distilled water. Tap water and irradiation showed a clear synergy with a combined effect on the body of mice, expressed in a more than threefold decrease in the period of post-radiation shortening of life expectancy. Distilled water reduced the rate of death of irradiated animals and modified the rate of death of non-irradiated animals. Our data demonstrate that the reduction in life expectancy of mice kept on tap water caused by long-term fractionated irradiation can be reduced when animals are kept on distilled water.

Evaluating the Effects of Perinatal Exposures to BPSIP on Hepatic Cholesterol Metabolism in Female and Male Offspring ICR Mice

Evaluating the Effects of Perinatal Exposures to BPSIP on Hepatic Cholesterol Metabolism in Female and Male Offspring ICR Mice

Photobiomodulation ameliorates ovarian aging by alleviating oxidative stress and inflammation damage and improving mitochondrial function

Ovarian aging is a serious clinical concern. Few safe and effective methods are currently available to improve ovarian functions. Photobiomodulation (PBM) is a safe and noninvasive physical therapy that can modulate a series of biological processes. Recently, several studies have noted its potential to improve the function of ovary and reproductive cells. However, the effects of PBM treatment on natural ovarian aging remain unclear. In this study, we used a naturally reproductive aging mouse model to observe the effect of PBM on ovarian function. Young and aged female ICR mice were treated with or without PBM for 2 months. PBM was performed using a semiconductor InGaAlP laser emitting at 650 nm (80 mW, 6.7 mW/cm2 for 5 or 10 min, resulting in a dose of 2 or 4 J/cm2, respectively). After treatment, the effects of PBM and its role in oxidative stress, inflammation, and mitochondrial function were investigated. We found that PBM (4 J/cm2) effectively recovered the levels of sex hormones, increased the number of primordial and growing follicles, improved angiogenesis, and decreased cell apoptosis in naturally aged mice. Moreover, PBM reduced oxidative stress, inhibited chronic ovarian inflammation, and improved mitochondrial function in aged ovaries. Similar protective effects of PBM were observed in a hydrogen peroxide-induced oxidative stress model of human granulosa cell line (KGN) in vitro. Increased cell viability, cell proliferation, hormone secretion, mitochondrial membrane potential, and adenosine triphosphate levels and decreased apoptosis and oxidative stress were detected in KGN cells after PBM treatment. Collectively, this study suggest that PBM treatment is beneficial for restoring ovarian function in naturally reproductive aging mice and has a significant protective effect against oxidative stress damage in KGN cells. The mechanisms underlying the benefits of PBM in ovarian aging include antioxidant stress, reduction of inflammation, and preservation of mitochondrial function. Therefore, this study emphasizes the potential of PBM as a therapeutic intervention to ameliorate ovarian aging.

Ninjinyoeito ameliorates anorexia and changes in peptide YY and ghrelin levels of cisplatin-treated mice

We explored the effect of Ninjinyoeito (NYT) on cisplatin-induced anorexia, which reduces cancer patient survival. Both gastrointestinal motility and plasma concentrations of gastrointestinal peptides were assessed. Nine-week-old ICR female mice received intraperitoneal cisplatin injections (10 mg/kg) and daily oral NYT doses of 300 mg/kg (NYT300) or 1000 mg/kg (NYT1000). Plasma levels of gastrointestinal peptides were measured at 3 and 6 days after cisplatin injection. Gastrointestinal motility was assessed by analyzing the concentration of phenol red marker within sections of the gastrointestinal tract. Cisplatin-injected mice showed a decrease in daily food intake, but this effect was attenuated on day 5 with NYT1000 administration. Although plasma ghrelin levels were reduced on day 3 in cisplatin-treated mice, NYT1000 administration ameliorated this decrease. However, there were no differences in ghrelin levels among all groups on day 6. Levels of peptide YY (PYY) were elevated in the plasma of cisplatin-injected mice on days 3 and 6. Administration of NYT300 and NYT1000 suppressed the increase in PYY levels on day 6 but not on day 3. Gastrointestinal motility was impaired on day 6 in cisplatin-treated mice, but NYT1000 administration attenuated this effect. Our results suggest that NYT improves cisplatin-induced anorexia by suppressing alterations in ghrelin and PYY levels and by increasing gastrointestinal motility. Therefore, NYT may be a promising candidate for alleviating cisplatin-induced anorexia.

Effect of 28 days treatment of baricitinib on mechanical allodynia, osteopenia, and loss of nerve fibers in an experimental model of type-1 diabetes mellitus.

Type-1 diabetes mellitus (T1DM) is associated with numerous health problems, including peripheral neuropathy, osteoporosis, and bone denervation, all of which diminish quality of life. However, there are relatively few therapies to treat these T1DM-related complications. Recent studies have shown that Janus kinase (JAK) inhibitors reverse aging- and rheumatoid arthritis-induced bone loss and reduce pain associated with peripheral nerve injuries, and rheumatoid arthritis. Thus, we assessed whether a JAK1/JAK2 inhibitor, baricitinib, ameliorates mechanical pain sensitivity (a measure of peripheral neuropathy), osteoporosis, and bone denervation in the femur of mice with T1DM. Female ICR mice (13 weeks old) received five daily administrations of streptozotocin (ip, 50mg/kg) to induce T1DM. At thirty-one weeks of age, mice were treated with baricitinib (po; 40mg/kg/bid; for 28 days) or vehicle. Mechanical sensitivity was evaluated at 30, 33, and 35 weeks of age on the plantar surface of the right hind paw. At the end of the treatment, mice were sacrificed, and lower extremities were harvested for microcomputed tomography and immunohistochemistry analyses. Mice with T1DM exhibited greater blood glucose levels, hind paw mechanical hypersensitivity, trabecular bone loss, and decreased density of calcitonin gene-related peptide-positive and tyrosine hydroxylase-positive axons within the marrow of the femoral neck compared to control mice. Baricitinib treatment significantly reduced mechanical hypersensitivity and ameliorated sensory and sympathetic denervation at the femoral neck, but it did not reverse trabecular bone loss. Our findings suggest that baricitinib may represent a new therapeutic alternative to treat T1DM-induced peripheral neuropathy and bone denervation.

Non-nucleoside O6-methylguanine-DNA methyltransferase inhibitors in murine spontaneous tumor experimental chemotherapy in vivo

Alkylating chemotherapy agents are well-established for inducing DNA lesions that result in apoptosis in cancer cells. However, the efficacy of these agents is often diminished due to the activity of the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), which confers resistance to chemotherapy by catalyzing dealkylation reactions. Recent studies have identified novel non-nucleoside MGMT inhibitors with promising properties. In this study, we evaluated the effectiveness of these novel non-nucleoside MGMT inhibitors in combination with alkylating chemotherapy in vivo. Our experimental model involved ICR female mice that spontaneously developed malignant tumors. These mice were treated with a combination of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and new MGMT inhibitors. We analyzed tumor growth dynamics and observed the levels of MGMT and other proteins using western blot analysis. Our findings demonstrated that the addition of MGMT inhibitors significantly improved the tumor growth-inhibiting effects of the alkylating chemotherapy. Tumor growth was more effectively suppressed in the mice receiving the combination therapy compared to those receiving the alkylating agents alone. Additionally, MGMT levels were significantly reduced following the combined treatment. Furthermore, the active form of caspase 3 was detected in treated tumors, suggesting that the reduction in tumor growth may be mediated through an apoptotic pathway. These results underscore the potential for these novel MGMT inhibitors to enhance the efficacy of alkylating agents in cancer therapy, holding substantial promise for improving therapeutic outcomes against tumors that exhibit high MGMT activity.

Effects of Selective and Mixed-Action Kappa and Delta Opioid Receptor Agonists on Pain-Related Behavioral Depression in Mice.

We recently developed a series of nalfurafine analogs (TK10, TK33, and TK35) that may serve as non-addictive candidate analgesics. These compounds are mixed-action agonists at the kappa and delta opioid receptors (KOR and DOR, respectively) and produce antinociception in a mouse warm-water tail-immersion test while failing to produce typical mu opioid receptor (MOR)-mediated side effects. The warm-water tail-immersion test is an assay of pain-stimulated behavior vulnerable to false-positive analgesic-like effects by drugs that produce motor impairment. Accordingly, this study evaluated TK10, TK33, and TK35 in a recently validated assay of pain-related behavioral depression in mice that are less vulnerable to false-positive effects. For comparison, we also evaluated the effects of the MOR agonist/analgesic hydrocodone (positive control), the neurokinin 1 receptor (NK1R) antagonist aprepitant (negative control), nalfurafine as a selective KOR agonist, SNC80 as a selective DOR agonist, and a nalfurafine/SNC80 mixture. Intraperitoneal injection of dilute lactic acid (IP lactic acid) served as a noxious stimulus to depress vertical and horizontal locomotor activity in male and female ICR mice. IP lactic acid-induced locomotor depression was alleviated by hydrocodone but not by aprepitant, nalfurafine, SNC80, the nalfurafine/SNC80 mixture, or the KOR/DOR agonists. These results suggest that caution is warranted in advancing mixed-action KOR/DOR agonists as candidate analgesics.

P-580 Benzophenone interrupted maturation of oocytes in vitro by promoting mitochondrial activity and inflammatory response in mice

Abstract Study question As an endocrine disrupting chemical, does benzophenone affect the competency of oocytes? Summary answer Benzophenone interfered with the in vitro maturation of oocytes and increased the number of mitochondria in oocytes by inducing of inflammatory response. What is known already Benzophenone (BP) is the organic compound used in ultraviolet (UV) filters due to its ability to absorb UV radiations and the applications of BP have a wide range in commercial/industrial products such as sunscreens, shampoos, lipsticks, food packaging, and plastics. However, BP is suspected to be an endocrine disrupting chemical (EDC) that can cause adverse effects on the endocrine system by mimicking or blocking the response of hormones in the body. Study design, size, duration Intraperitoneal injection of pregnant mare serum gonadotropin was performed at 7-week-old female ICR mice for acquire the number of available cumulus-oocyte-complexes (COCs). After 46 hours post injection, COCs were harvested from antrum follicles in the ovary and cultured in in-vitro maturation medium for 16 hours. After that, oocytes were retrieved. Participants/materials, setting, methods During in vitro maturation, BP was treated at COCs and Metaphase II (MII) oocytes were separated from retrieved oocytes. Intracellular tubulin of MII oocytes was stained for spindle alignment and their mitochondria were quantified by Mito-tracker and quantitative PCR (qPCR). Also, the expression levels of inflammation-related genes were confirmed by reverse transcription-qPCR. Main results and the role of chance In the present study, MII rates (MII/total oocyte) of each group were 77.50% of control, 79.17% of 1 μM BP, 68.10% of 10 μM BP, and 59.35% of 100 μM BP. Compared with the control group, the MII rate were significantly reduced at 100 μM of BP-treated group. We evaluated the morphology of meiotic spindle in MII oocytes with well-organized tubulin fibers and tightly aligned chromosomes. Abnormal morphology of spindle and chromosomal misalignment were found in the oocytes from BP-treated group. Because it has been known that having the optimal number of functional mitochondria is important for the maturation of oocytes, mitochondrial DNA (mtDNA) copy number and the distribution of mitochondria were confirmed in retrieved MII oocytes. There was a significant 4.2-fold increase in the number of mtDNA copies in 100 μM BP treatment group compared to the control group. Also, the number of activated mitochondria was raised when BP was treated, which was assessed using Mitotracker. Additionally, levels of inflammation-related genes (IL-1β, IL-6) were elevated in BP treatment groups. Limitations, reasons for caution Oocytes used in this study were collected from mice, not human. And since in vitro fertilization did not perform by using the MII oocytes, we couldn’t confirm whether BP-treated MII oocytes were capable of fertilization. Wider implications of the findings As industries evolve, novel chemicals, including EDCs, are being developed, used, and exposed to humans. Therefore, we need to understand the effects of these chemicals and assess their reproductive toxicity. Collectively, we suggest that BP causes an inflammatory response which activate mitochondria and has detrimental effects on the oocyte’s maturation. Trial registration number not applicable

P-548 An investigation into the mechanisms of active enhancers regulating the initial lineage differentiation in mammals

Abstract Study question What role do active enhancers play in determining the fate of early embryonic cells? Are there differences in active enhancers between mouse and human blastocysts? Summary answer Tissue-specific active enhancers in inner cell mass (ICM) and trophoblast (TE) are essential for blastocyst formation, regulating gene expression through interactive loops with promoters. What is known already At the two-cell stage of nuclear transfer embryos, the absence of interaction between Zscan4d promoter and distal active enhancer leads to reduced Zscan4d expression, resulting in lower developmental rates. Overexpressing the demethylase Kdm4d alleviates reprogramming-resistant H3K9me3 modification, restoring the structural loop between the Zscan4d promoter and active enhancer and enhancing developmental efficiency. These findings strongly suggest a close association between the formation of regulatory chromatin interactions in early embryonic development and histone modifications. Indicating active enhancers marked with H3K27ac may regulate differentiation gene expression through distal interactions in blastocyst. Study design, size, duration Analyze variations in histone H3K27ac modification, chromatin accessibility, higher-order structures, and transcriptomes during mouse embryo differentiation from 8-cell stage to ICM and TE formation. Identify tissue-specific active enhancers in ICM and TE, validate their gene expression regulation, explore their role in early differentiation-associated genes in mice. Compare and clarify similarities and differences in enhancer regulation mechanisms during early embryo development in humans and mice. Participants/materials, setting, methods The 8-cell, morula and blastocyst stage embryos were collected from the oviducts and uterus of super-ovulated female ICR mice mated with male mice. The clinical human blastocysts were collected and separated the ICM and TE. Low-input multi-omics sequencing, including Cut-Tag, ATAC-seq, Hi-C, and RNA-seq, was performed on the embryonic samples. After analyzing and identifying active enhancers, tissue-specific ones were selected and validated for their impact on gene expression via CRISPR-Cas9 knockout in embryos. Main results and the role of chance Inhibiting ep300 expression in mouse embryos through siRNA interference reduces blastocyst formation from 90% to approximately 40%. Introducing the small molecule inhibitor A485 at the 8-cell stage, blocking the active center of CBP/p300 enzymes, completely arrests embryos at the morula stage, preventing their development into blastocysts. Emphasizing the crucial role of correctly establishing active enhancers marked by H3K27ac modification in embryonic differentiation. Multi-omics data demonstrates high repeatability and quality. PCA clustering analysis reveals a clear differentiation path in the distribution of H3K27ac-modified histones, chromatin accessibility sites, and the transcriptome from the 8-cell stage to the blastocyst. Differences in H3K27ac histone modification distribution between the ICM and TE indicate the presence of tissue-specific active enhancers in blastocyst. The disparities in H3K27ac modification distribution between blastocyst ICM and TE primarily occur in distal gene regions rather than proximal ones. Moreover, these distal enhancers exhibit a positive correlation with the expression of lineage-specific genes, suggesting a probable regulatory role of distal enhancers in gene modulation during embryonic differentiation. Tissue-specific enhancer-promoter (EP) interaction loops emerge during embryonic differentiation, allowing direct interaction between tissue-specific active enhancers and promoters, thereby activating the expression of tissue-specific genes. Limitations, reasons for caution While DNA sequences with H3K27ac modification are currently considered markers for active enhancers, H3K4me modification is another histone marker for enhancers. However, enhancers associated with H3K4me modification may not necessarily be in an activated state. Future research can simultaneously incorporate appropriate detection methods for H3K4me modification. Wider implications of the findings After elucidating the mechanisms through which active enhancers regulate mouse differentiation, comparing the differences in active enhancers during the first lineage differentiation process between mice and humans can help identify factors that enhance human blastocyst quality. This can potentially improve clinical outcomes in assisted reproduction. Trial registration number not applicable

Streptococcus thermophilus iHA318 Improves Dry Eye Symptoms by Mitigating Ocular Surface Damage in a Mouse Model.

Dry eye is a complicated ocular surface disease that causes discomfort, visual disturbance, and frequently observed ocular surface damage. Emerging hypotheses suggest probiotics may help relieve dry eye symptoms by modulating inflammation and oxidative stress. This study aimed to investigate the therapeutic effects of Streptococcus thermophilus iHA318 probiotics on dry eye using in vitro assays and an in vivo murine model of ultraviolet B (UVB) radiation-induced dry eye. In vitro analyses revealed that S. thermophilus iHA318® exhibited antioxidant activity and anti-inflammatory effects by inhibiting reactive oxygen species production and suppressing inflammatory cytokines. For the in vivo study, female ICR mice were assigned to normal control, UVB-induced dry eye, and UVB+iHA318 treatment groups. UVB exposure significantly decreased tear volume and tear film breakup time (TBUT) compared to normal controls. Supplementation with S. thermophilus iHA318® via oral gavage markedly improved tear production and TBUT on day 7 post-UVB exposure. Ocular surface photography demonstrated improved gradings of corneal opacity, smoothness, and lissamine green staining in the iHA318 group versus the UVB group. Topographical analysis further revealed improvement in the UVB-induced corneal irregularities by iHA318 treatment. Collectively, these results indicate that S. thermophilus iHA318 exerts a protective effect against dry eye symptoms by mitigating oxidative stress and inflammation, thereby preserving tear film stability and ocular surface integrity. This probiotic strain represents a promising therapeutic approach for managing dry eye syndrome.

Exposure to Benzo(a)pyrene Enhances Acetaminophen-Induced Liver Injury in Mice at Non-Hepatotoxic Doses

Acetaminophen (APAP) is a widely used analgesic, especially for children. Its primary mechanism involves inhibiting cyclooxygenase enzymes and activating the endocannabinoid and TRPV1 systems. Though its toxicity is low, it can harm the liver in a dose-dependent manner. Low APAP doses can also increase pollutant-induced liver damage. Little is known about interactions between APAP and benzo[a]pyrene (B[a]P). This study aimed to assess if co-exposure to non-hepatotoxic doses of B[a]P and APAP causes liver injury in mice, exploring the underlying mechanisms. Female ICR mice received 50 mg/kg B[a]P or a vehicle for three days, followed by 200 mg/kg APAP or a vehicle. Liver injury was assessed through histopathological examination, serum transaminase activity, and gene expression analysis. In the B[a]P/APAP group, several histology changes were observed, including ballooning injury, steatosis, necrosis, inflammation, and apoptosis. Transaminase levels correlated with histopathological scores, and there was an increase in hepatic cytochrome P450 family 1 subfamily a member 1 (Cyp1a1) mRNA levels and a decrease in aryl hydrocarbon receptor (Ahr), cytochrome P450 family 2 subfamily e polypeptide 1 (Cyp2e1), superoxide dismutase 1 (Sod1), peroxisome proliferator activated receptor gamma (Ppar-γ), and caspase 3 (Casp3). This suggests that prior exposure to B[a]P makes mice more susceptible to APAP-induced liver injury, involving changes in gene expression related to metabolism, redox balance, and cell proliferation. Therefore, using therapeutic APAP doses after exposure to B[a]P could lead to liver injury.

Impact of high-fat diet on ovarian epigenetics: Insights from altered intestinal butyric acid levels

ObjectiveTo investigate the effects of a high-fat diet (HFD) on the gut bacterium Roseburia intestinalis and butyric acid levels, and to assess their impact on ovarian function and epigenetic markers in mice. MethodsA total of 20 female ICR mice aged 4 weeks were randomly assigned to two groups and fed either a control diet (CD) or an HFD for 36 weeks. Post-intervention, ileal contents were analyzed for the quantification of butyric acid using ELISA, while feces were obtained for Roseburia intestinalis expression assessment via qPCR. Histological evaluations of intestinal and ovarian tissues included H&E and Alcian Blue-Periodic Acid Schiff (AB-PAS) staining, alongside immunohistochemical analysis for F4/80, and immunofluorescent detection of Occludin, ZO-1, 5 mC, and H3K36me3. Ovarian health was assessed through follicle counts and morphological evaluations. Statistical analyses were performed using GraphPad Prism 8.0, with P < 0.05 considered significant. ResultsAfter 36 weeks, the HFD group showed significantly higher body weight compared to the CD group (P < 0.01). The HFD led to a decrease in Roseburia intestinalis and butyric acid levels, a reduction in intestinal goblet cells, and an increase in intestinal inflammation. Histological analyses revealed impaired ovarian follicular development and enhanced inflammation in the HFD mice, with immunofluorescent staining showing downregulation of the ovarian epigenetic markers 5 mC and H3K36me3. ConclusionOur study demonstrates that long-term HFD negatively impacts ovarian function and epigenetic regulation. We found decreased levels of the gut bacterium Roseburia intestinalis and its metabolite, butyric acid, which contribute to these adverse effects. Additionally, the associated intestinal inflammation and compromised mucosal barrier may contribute to these adverse outcomes on female reproductive health.

Characterization of Japanese Encephalitis Virus Isolated from Persistently Infected Mouse Embryo Cells.

Japanese encephalitis virus (JEV) has a positive-sense single-stranded RNA genome and belongs to the genus Flavivirus of the family Flaviviridae. Persistent JEV infection was previously shown in pig blood cells, which act as a natural reservoir of this virus. We aimed to determine the pathogenicity factors involved in persistent JEV infection by analyzing the pathogenicity and genome sequences of a virus isolated from a persistent infection model. We established persistent JEV infections in cells by inoculating mouse fetus primary cell cultures with the Beijing-1 strain of JEV and then performing repeated infected cell passages, harvesting viruses after each passage while monitoring the plaque size over 100 generations. The virus growth rate was compared among Vero, C6/36, and Neuro-2a cells. The pathogenicity was examined in female ICR mice at several ages. Additionally, we determined the whole-genome sequences. The 134th Beijing-1-derived persistent virus (ME134) grew in Vero cells at a similar rate to the parent strain but did not grow well in C6/36 or Neuro-2a cells. No differences were observed in pathogenicity after intracerebral inoculation in mice of different ages, but the survival time was extended in older mice. Mutations in the persistent virus genomes were found across all regions but were mainly focused in the NS3, NS4b, and 3'NCR regions, with a 34-base-pair deletion found in the variable region. The short deletion in the 3'NCR region appeared to be responsible for the reduced pathogenicity and growth efficiency.

Bergenin attenuates triptolide-caused premature ovarian failure in mice based on the antioxidant activity

Tripterygium wilfordii (TW) preparations have been utilized in China for treating rheumatoid arthritis and autoimmune diseases. However, their clinical use is limited due to reproductive toxicity, notably premature ovarian failure (POF). Our study aimed to investigate the effect and mechanism of bergenin in attenuating POF induced by triptolide in mice. POF was induced in female ICR mice via oral triptolide administration (50 μg/kg) for 60 days. Mice received bergenin (25, 50, 100 mg/kg, i.g.) or estradiol valerate (EV) (0.1 mg/kg, i.g.) daily, 1 h before triptolide treatment. In vitro, ovarian granulosa cells (OGCs) were exposed to triptolide (100 nM) and bergenin (1, 3, 10 μM). Antioxidant enzyme activity, protein expression, apoptosis rate, and reactive oxygen species (ROS) levels were assessed. The results showed that triptolide-treated mice exhibited evident atrophy, along with an increase in atretic follicles. Bergenin (50, 100 mg/kg) and EV (0.1 mg/kg), orally administered, exerted significant anti-POF effect. Bergenin and EV also decreased apoptosis in mouse ovaries. In vitro, bergenin (1, 3, 10 μM) attenuated triptolide-induced OGCs apoptosis by reducing levels of apoptosis-related proteins. Additionally, bergenin reduced oxidative stress through downregulation of antioxidant enzymes activity and overall ROS levels. Moreover, the combined use with Sh-Nrf2 resulted in a reduced protection of bergenin against triptolide-induced apoptosis of OGCs. Together, bergenin counteracts triptolide-caused POF in mice by inhibiting Nrf2-mediated oxidative stress and preventing OGC apoptosis. Combining bergenin with TW preparations may effectively reduce the risk of POF.

Hypoxia Leads to Diminished Ovarian Reserve in an Age-Dependent Manner

Objectives: Perinatal hypoxia causes premature activation and initiation of growth in dormant follicles, leading to diminished ovarian reserve. An indirect mechanism such as the release of stress-related hormones may influence ovarian follicle recruitment under hypoxic conditions. We wanted to determine whether hypoxic ovarian damage results from increased follicle growth and “burnout” or from increased apoptosis and whether this damage is age-dependent. Design: Animal study was conducted. Participants/Materials, Setting, Methods: Using adult 6-week-old (n = 8) and one-day-old newborn (n = 20) ICR (CD-1) female mice, ovarian follicular counts were conducted on H&E-stained sections. Methods: Immunohistochemistry was performed on sections stained with Ki-67, anti-Caspase 3, and anti-FOXO3A. Results: Exposure to hypoxia resulted in significantly reduced proportion of primordial follicles versus normoxia in both adult dams and newborn pups (3.17 ± 2.75 vs. 17.89 ± 4.4%; p = 0.004; 40.59 ± 14.88 vs. 81.92 ± 31.56%, p = 0.001, respectively), concomitant with increased growing-primary and secondary follicles, and more pronounced in adult dams versus newborn pups (6-fold vs. 2-fold, respectively). Ki67 staining revealed higher scores of cell proliferation in follicular granulosa cells after exposure to hypoxia than normoxia. However, Caspase 3 and Foxo3A staining did not show any differences in these markers of apoptosis in oocytes, granulosa cells, theca cells, or stromal cells when exposed to hypoxia versus normoxia. Limitations: The current study has several limitations; first, the sample size for each group is relatively small, which could limit the generalizability of the findings. Second, the study used an ex vivo culture system, which may not fully capture the complex interactions that occur in the whole animal. Third, the exposure to hypoxia only lasted for 3 h, which may not be long enough to observe all the potential effects. In addition, the study only analyzed specific markers of apoptosis in a few cell types, and other cell types or apoptotic pathways might be involved. Lastly, the study provides evidence for accelerated follicular activation and decreased ovarian reserve, but the underlying mechanisms are not fully explored. Conclusions: Direct tissue hypoxia led to premature activation and initiation of growth in dormant follicles leading to diminished ovarian reserve. Hypoxic damage is age-dependent, with adult ovaries more susceptible than newborn ovaries. These findings support the possibility of follicular “burn out” as a potential mechanism responsible for hypoxia-induced loss of ovarian reserve.

Effect of Shoutai Pills on expression of key glycolytic proteins and apoptosis related factors at maternal fetal interface in mouse model of threatened abortion with syndrome of kidney deficiency

This study aims to explore the mechanism of Shoutai Pills in treating threatened abortion. According to the random number table method, ICR female mice were randomized into a normal group, a model group, a dydrogesterone group, and a Shoutai Pills group, with 15 mice in each group. Mice were administrated with normal saline(normal and model groups) or the suspension of Shoutai Pills or dydrogesterone by gavage at 9:00 am every day. At 16:00 every day, mice in the normal group were administrated with an equal volume of distilled water, while those in the model, Shoutai Pills, and dydrogesterone groups were administrated with hydrocortisone solution by gavage for 4 consecutive days. ICR female and male mice were caged in a ratio of 2∶1 during the pre-estrous or estrous period. From the first day of pregnancy, drug administration was continued for 5 consecutive days. On day 6, mice were administrated with mifepristone by gavage to establish the model of kidney deficiency-induced abortion. On day 6 of pregnancy, 10 female ICR mice were randomly selected from each group, and the uterus was collected for observation of the pathological changes of trophoblasts at the maternal-fetal interface by hematoxylin-eosin(HE) staining. The protein levels of key enzymes of glycolysis, hexokinase 2(HK2), enolase 1(ENO1), pyruvate kinase M2(PKM2), and lactate dehydrogenase A(LDHA), were determined by Western blot and immunofluorescence. The expression of apoptosis-related proteins including B cell lymphoma-2(Bcl-2), Bcl-2-associated protein X(Bax), and cysteinyl aspartate-specific proteinase-3(caspase-3) was determined by Western blot and real-time PCR. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling was employed to examine apoptosis. The embryo loss rate of the remaining five female mice was calculated by trypan blue staining method on day 14 of pregnancy. On day 14 of pregnancy, the embryo loss rate of the normal group was 5.00%, which was lower than that(27.78%) in the model group(P&lt;0.05). Dydrogesterone and Shoutai Pills groups showed reduced embryo loss rates(10.26% and 7.50%, respectively) compared with the model group. On day 6 of pregnancy, compared with the normal group, the model group showed down-regulated expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and up-regulated expression of Bax and caspase-3(P&lt;0.05). Compared with the model group, dydrogesterone and Shoutai Pills up-regulated the expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and down-regulated the expression of Bax and caspase-3(P&lt;0.05). Compared with that in the normal group, the apoptosis rate in the model group increased(P&lt;0.05). Compared with the model group, dydrogesterone and Shoutai Pills reduced the apoptosis rate(P&lt;0.05). In conclusion, Shoutai Pills can reduce the embryo loss rate and protect embryos by promoting aerobic glycolysis at the maternal-fetal interface and inhibiting the apoptosis of trophoblasts in mice.

Effects of Repeated Treatment with the Monoacylglycerol Lipase Inhibitor MJN110 on Pain-Related Depression of Nesting and Cannabinoid 1 Receptor Function in Male and Female Mice.

MJN110 inhibits the enzyme monoacylglycerol lipase (MAGL) to increase levels of the endocannabinoid 2-arachidonoylglycerol , an endogenous high-efficacy agonist of cannabinoid 1 and 2 receptors (CB1/2R). MAGL inhibitors are under consideration as candidate analgesics, and we reported previously that acute MJN110 produced partial antinociception in an assay of pain-related behavioral depression in mice. Given the need for repeated analgesic administration in many pain patients and the potential for analgesic tolerance during repeated treatment, this study examined antinociceptive effects of repeated MJN110 on pain-related behavioral depression and CB1R-mediated G-protein function. Male and female ICR mice were treated daily for 7 days in a 2 × 2 design with (a) 1.0 mg/kg/d MJN110 or its vehicle followed by (b) intraperitoneal injection of dilute lactic acid (IP acid) or its vehicle as a visceral noxious stimulus to depress nesting behavior. After behavioral testing, G-protein activity was assessed in lumbar spinal cord (LSC) and five brain regions using an assay of CP55,940-stimulated [35S]GTPɣS activation. As reported previously, acute MJN110 produced partial but significant relief of IP acid-induced nesting depression on day 1. After 7 days, MJN110 continued to produce significant but partial antinociception in males, while antinociceptive tolerance developed in females. Repeated MJN110 also produced modest decreases in maximum levels of CP55,940-induced [35S]GTPɣS binding in spinal cord and most brain regions. These results indicate that repeated treatment with a relatively low antinociceptive MJN110 dose produces only partial and sex-dependent transient antinociception associated with the emergence of CB1R desensitization in this model of IP acid-induced nesting depression. SIGNIFICANCE STATEMENT: The drug MJN110 inhibits monoacylglycerol lipase (MAGL) to increase levels of the endogenous cannabinoid 2-arachidonoylglycerol and produce potentially useful therapeutic effects including analgesia. This study used an assay of pain-related behavioral depression in mice to show that repeated MJN110 treatment produced (1) weak but sustained antinociception in male mice, (2) antinociceptive tolerance in females, and (3) modest cannabinoid-receptor desensitization that varied by region and sex. Antinociceptive tolerance may limit the utility of MJN110 for treatment of pain.

Pediococcus pentosaceus MIANGUAN Enhances the Immune Response to Vaccination in Mice.

Increasing evidence shows that some probiotics can improve vaccine responses as adjuvants. This study aimed to evaluate the effect of Pediococcus pentosaceus MIANGUAN (PPM) on SARS-CoV-2 vaccine-elicited immune response in mice. Six-week-old female ICR mice were primed and boosted with SARS-CoV-2 vaccine intramuscularly at weeks 0 and 4, respectively. Mice were gavaged with PPM (5 × 109CFU/mouse) or PBS (control) for 3days immediately after boosting vaccination. Compared to the control, oral PPM administration resulted in significantly higher levels of RBD-specific IgG binding antibodies (> 2.3-fold) and RBD-specific IgG1 binding antibodies (> 4-fold) in the serum. Additionally, PPM-treated mice had higher titers of RBD-specific IgG binding antibodies (> 2.29-fold) and neutralization antibodies (> 1.6-fold) in the lung compared to the control mice. The transcriptional analyses showed that the B cell receptor (BCR) signaling pathway was upregulated in both splenocytes and BAL cells in the PPM group vs. the control group. In addition, the number of IFN-γ-producing splenocytes (mainly in CD4 + T cells as determined by flow cytometry) in response to restimulation of RBD peptides was significantly increased in the PPM group. RNA sequencing showed that the genes associated with T cell activation and maturation and MHC class II pathway (CD4, H2-DMa, H2-DMb1, H2-Oa, Ctss) were upregulated, suggesting that oral administration of PPM may enhance CD4 + T cell responses through MHC class II pathway. Furthermore, PPM administration could downregulate the expression level of proinflammatory genes. To conclude, oral administration of PPM could boost SARS-CoV-2 vaccine efficacy through enhancing the specific humoral and cellular immunity response and decrease the expression of inflammation pathways.

Toxicological Evaluation of a Polyherbal Formulation (18KHT01) and Validation of UPLC-DAD Method for Quality Control.

Background: 18KHT01 is a novel synergistic composition of Quercus acutissima, Camellia sinensis, Geranium thunbergii, and a small portion of Citrus limon. Our previous report demonstrated that the 18KHT01 exhibits potent antiobesity effects, with synergistic antioxidant, antiadipogenic, and antiobesity activities in diet-induced obese mice. This study explores the toxicity profile and quality control parameters of the 18KHT01 formulation. Methods: Broad-spectrum acute and subacute oral toxicity studies were performed using male and female ICR mice. In order to simultaneous analysis of the 18KHT01 formulation, an ultraperformance liquid chromatography coupled with a diode array detector (UPLC-DAD) method was developed and validated using six marker compounds. Results: Acute oral toxicity evaluation of 18KHT01, administered at single high doses of 2, 2.5, 3, and 5 g/kg, identified 2 g/kg as the no-observed adverse effect level (NOAEL). The LD50 (50% lethal dose) and LD100 (100% lethal dose) of 18KHT01 for male ICR mice were 3.99 and 7.77 g/kg, and those for the female mice were 2.94 and 4.70 g/kg, respectively. In addition, a 30-day repeated dose oral subacute toxicity evaluation indicated that 18KHT01 is safe below 500 mg/kg/day for long-term administration in ICR mice of either sex. UPLC-DAD method validation revealed that each calibration curve for the marker compounds showed good linearity, as well as the validation parameters such as precision, specificity, and accuracy met the acceptance criteria. Conclusion: The present study evidenced the toxicological profile of 18KHT01 polyherbal formulation in mice as well as developed a simple, rapid, and accurate chromatographic method for quality control.