Artesunate (ART) is a semi-synthetic derivative of artemisinin with well-established antimalarial activity. In this study, we demonstrate that ART treatment markedly attenuates disease severity in dextran sulfate sodium (DSS)–induced colitis. ART significantly reduces the accumulation of inflammatory neutrophils, monocytes, Th17 cells, and IL-17-producing ILCs and γδT cells in the colon, accompanied by suppression of pro-inflammatory gene expression, including Tnf, Il6, and Il1b, and upregulation of anti-inflammatory mediators such as Tgfb and Il10. In parallel, ART restores the expression of intestinal tight junction proteins ZO-1 and claudin-1, indicating improved epithelial barrier integrity. ART treatment inhibits the IL-17 production from both Th17 cells and other lymphocytes. ART also reshapes the gut microbiota, which contributes to protection against DSS-induced colitis. Notably, mesalazine co-treatment with ART augmented its protective efficacy against DSS-induced colitis. Collectively, these findings identify ART as a potent modulator of intestinal inflammation and barrier function, highlighting its therapeutic potential for mucosal inflammatory diseases.

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Tensin2 (TNS2) is a focal adhesion-associated protein that can negatively regulate insulin/insulin-like growth factor 1 (IGF-1) signaling, but its role in skeletal muscle regeneration remains unclear. To address this, we analyzed a TNS2-related immunoreactive signal detected with an anti-phospho-TNS2 (Tyr483) antibody during C2C12 myogenic differentiation and in a cardiotoxin (CTX)-induced tibialis anterior (TA) injury model, and assessed regeneration in Tns2^nph mutant mice. Under basal conditions, Tns2^nph mice showed no obvious abnormalities in muscle histology, myofiber size, or phosphorylation of Akt and p70 ribosomal protein S6 kinase (p70S6K), a canonical downstream effector of mTORC1 signaling, compared with wild-type (WT) mice, although the soleus muscle/body weight ratio was slightly increased. In C2C12 cells, the anti-phospho-TNS2 (Tyr483)-reactive signal was stronger during proliferation than during differentiation. In vivo, this signal increased transiently during the early phase of CTX-mediated regeneration. After injury, Tns2^nph mice showed a greater number of centrally nucleated fibers at day 4, larger myofibers at day 7, and a higher TA muscle/body weight ratio at day 15. In addition, phosphorylation of p70S6K was increased in regenerating muscle in Tns2^nph mice. These findings suggest that TNS2-related signaling may act as a context-dependent modulator of regenerative myofiber growth, possibly through augmented anabolic signaling during early regeneration.

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Pre-mRNA splicing is a conserved post-transcriptional regulatory process in eukaryotes, and mis-splicing is associated with numerous diseases. Therefore, experimentally induced exon skipping provides an important strategy for exon-level analysis of gene function. Exons can be broadly classified by their usage patterns across transcripts: some are shared by all transcripts, whereas others are selectively used across transcripts. Skipping the former affects all transcripts and often results in gene-level effects, while skipping the latter reflects functional diversity across transcripts. Therefore, considering characteristics of target exons is important to enhance interpretability of exon-level functional analysis. Recent advances in base editing enable exon skipping by introducing point mutations at splice sites, providing an approach for exon-level analysis. Accordingly, several guide RNA (gRNA) design tools have been developed for the purpose of inducing exon skipping. However, existing tools do not provide exon-level properties, including exon usage patterns, reading frame, and coding status. As a result, selecting target exons appropriate for specific research objectives remains difficult. Here we developed AltEx-BE, an automated gRNA design tool for base editing–mediated splice site disruption. AltEx-BE uses transcript annotations and reference genome sequences as inputs to classify each exon by usage pattern and to generate gRNA candidates compatible with several base editing tools. The tool also provides annotations, including coding status, to facilitate interpretation of outcomes of exon skipping. The tool promotes base editing–mediated exon skipping experiments across model organisms and enhances the interpretability of experimental outcomes.

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Endometriosis, a chronic disease that affects about 10% of women of reproductive age, is characterized by severe pain and growth of endometrium-like tissues outside the uteri. Although several theories have been advanced for the cause of endometriosis, no theory fully explains why endometriosis occurs. For this study, we strove to elucidate a new mechanism of endometriosis leading to establishment of a new therapeutic strategy for its treatment. In our murine endometriosis model, a marked hypoxic state was induced in the peritoneal cavity, as indicated by increased expression of HIF-1α in peritoneal exudate cells. Moreover, innate immune response, such as TLR4 and IL-1β expression, was upregulated. To restore the hypoxic milieu in endometriosis, we chose resveratrol (RSV), a natural polyphenolic compound, as a hypoxia-reducing agent. As expected, RSV administration decreased the number of HIF-1α–positive cells. Furthermore, RSV administration reduced the size and weight of endometriotic lesions significantly. However, RSV administration did not change TLR4 and IL-1β expression levels in peritoneal exudate cells. In the process of seeking mechanisms of endometriosis other than TLR4-dependent and IL-1β-dependent pathways, we detected autoantibody deposition in endometriotic lesions. In endometriotic lesions from RSV-administered mice, accumulation of autoantibodies was inhibited compared to a control group. As described herein, we proposed a new concept by which autoantibody deposition can cause endometriosis, raising the possibility of RSV as a promising therapeutic option for endometriosis.

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Intrauterine adhesion (IUA) is a significant contributor to uterine infertility, primarily characterized by endometrial fibrosis. Although diacerein exhibits anti-inflammatory and anti-fibrotic properties, its therapeutic potential in IUA remains unclear. This study investigated the protective effects of diacerein in an IUA rat model induced by mechanical injury. Histological analysis revealed that diacerein treatment alleviated endometrial damage, and immunohistochemical staining confirmed the restoration of CK19 and CK18 expression, indicating improved epithelial integrity and regeneration. Diacerein mitigated endometrial fibrosis by inhibiting epithelial-mesenchymal transition (EMT), as evidenced by increased E-cadherin and decreased N-cadherin expression, likely via suppression of TGFβ/SMAD2 signaling. Diacerein exerted anti-inflammatory effects in IUA rats. Notably, diacerein inhibited ferroptosis by reducing lipid peroxidation, limiting Fe²⁺ accumulation, and modulating ferroptosis-related proteins, including ACSL4, SLC7A11, GPX4, and FTH1. The protective effects of diacerein were mirrored by ferroptosis inhibitor Ferrostatin-1 treatment but reversed by ferroptosis inducer Erastin, confirming that diacerein alleviates endometrial fibrosis in IUA rats through ferroptosis suppression. Mechanistically, diacerein modulated the NRF2/HMGB1 signaling pathway, restoring NRF2 and HO1 levels while downregulating HMGB1 expression. Collectively, these findings suggest that diacerein effectively attenuates ferroptosis-mediated fibrosis in IUA, highlighting ferroptosis inhibition as a promising therapeutic strategy for IUA management.

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Signal transducer and activator of transcription 3 (STAT3) is critical in psoriasis, but keratinocyte-derived STAT3 remains unclear. This study investigated the effect of keratinocyte-specific STAT3 knockout on imiquimod (IMQ)-induced psoriasis-like dermatitis. Keratinocyte-specific STAT3 knockout (cKO) mice and littermate controls (LC) received daily topical 5% IMQ to induce psoriasis-like lesions. Lesion severity was assessed by PASI score, histopathology by H&E staining, and systemic inflammation by spleen index and serum CXCL1, CCL20, and IL-22 levels (ELISA). Skin expression of inflammation, chemokine, and STAT3 pathway related factors was examined by IHC, qRT-PCR, and Western blot. In vitro, IL-17A‑stimulated HaCaT cells were used with siRNA-mediated STAT3 knockdown; changes in STAT3 signaling and downstream factors were assessed by qRT-PCR and Western blot. In vivo, keratinocyte-specific STAT3 knockout significantly alleviated IMQ-induced skin lesions, reduced PASI score, improved barrier function, decreased spleen index, and lowered serum CXCL1, CCL20, and IL-22. It also suppressed IL-22, CXCL1, and CCL20 expression in skin and inhibited STAT3 activation. In vitro, IL-17A increased STAT3 phosphorylation, which was blocked by STAT3 knockdown, along with suppression of IL-22 and CXCL1 upregulation.

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Medetomidine-midazolam-butorphanol (MMB) anesthesia is widely used in laboratory rodents but is frequently accompanied by peri- and post-anesthetic hypothermia. This study examined whether co-administration of nicardipine could attenuate hypothermia associated with MMB anesthesia in rats, thereby testing the concept that modulation of peripheral vascular tone can complement thermal management. Rats received MMB alone (0Ni) or MMB combined with nicardipine at 0.25 or 0.5 mg/kg. Body temperature was assessed under warmed and non-warmed conditions, and systolic blood pressure (SBP) and heart rate (HR) were monitored to evaluate cardiovascular effects. Under warmed conditions, body temperature profiles did not differ among groups. Under non-warmed conditions, however, nicardipine-treated rats maintained higher body temperatures during recovery, and both nicardipine groups showed significantly higher recovery-phase AUC values than the 0Ni group. Nicardipine did not significantly alter induction or recovery timing. In parallel, nicardipine produced a dose-dependent reduction in SBP and an increase in HR, particularly at 0.5 mg/kg. These findings suggest that nicardipine partially attenuates post-anesthetic hypothermia during recovery from MMB anesthesia under non-warmed conditions and may serve as a complementary approach to thermal management. However, the marked SBP reduction observed at 0.5 mg/kg warrants caution in practical application.

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Vulvovaginal candidiasis, a common infection of the female reproductive tract, poses heightened risks when occurring during pregnancy (pVVC). Although Honghe Fujie lotion (HFL), a traditional Chinese medicine, is clinically used for mycotic vaginitis, its therapeutic efficacy and mechanisms in pVVC remain unclear. In this study, HPLC-MS was used to analyze and identify the compounds in HFL. This study investigated HFL’s effects through in vitro and in vivo approaches. A murine model of vaginal C. albicans infection was established to evaluate the therapeutic effect of HFL on pregnancy-associated vulvovaginal candidiasis (pVVC). Vaginal fungal load was quantified by analyzing vaginal lavage fluid, while the impact of HFL on adverse pregnancy outcomes was assessed through placental developmental analysis. Furthermore, the effects of HFL on pVVC-induced vaginitis and NLRP3 inflammasome activation were examined using immunofluorescence, enzyme-linked immunosorbent assay, and western blot. The results included: (1) High performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analysis identified 545 chemical constituents in HFL. (2) HFL significantly inhibited Candida albicans (C. albicans) growth and biofilm metabolic activity in vitro; (3) in a murine pVVC model, HFL reduced vaginal fungal load and mitigated adverse pregnancy outcomes, while also restoring trophoblast cell distribution in placental tissues; (4) mechanistically, HFL suppressed NLRP3 inflammasome activation, leading to Caspase-1 activity downregulation and subsequent impairment of pro-inflammatory cytokine secretion. These findings demonstrate that HFL alleviates pVVC by targeting the NLRP3 signaling pathway, providing a preclinical foundation for its therapeutic application in pregnancy-associated VVC.

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Autoimmune thyroiditis (AIT) is a systemic autoimmune disorder that extends beyond thyroid dysfunction and is associated with disturbances in pancreatic endocrine secretion and glucose metabolism. The present study aimed to evaluate the effects of selected plant flavonoids—dihydroquercetin, chrysoeriol, luteolin, resveratrol, kaempferol, and thamiflaside—administered alone or in combination with selenium on pancreatic hormone secretion, glycemic control, and hepatic glycogen content in an experimental rat model of AIT. AIT was induced in male Wistar rats using thyroglobulin and Freund’s adjuvant. After confirmation of autoimmune thyroiditis, animals received flavonoid treatment (20 mg/kg body weight), with or without selenium, for 14 days. Serum concentrations of insulin, glucagon, amylin, and somatostatin were determined by immunoassay. Glycated hemoglobin (HbA1c) was measured as an indicator of long-term glycemic control, and hepatic glycogen content was assessed in liver homogenates. AIT induction resulted in marked metabolic dysregulation characterized by reduced insulin and amylin secretion, elevated glucagon levels, impaired somatostatin regulation, increased HbA1c, and depletion of hepatic glycogen stores. Flavonoid treatment produced compound-specific corrective effects. Luteolin and kaempferol, particularly in combination with selenium, showed the strongest efficacy by restoring pancreatic hormone balance, reducing hyperglucagonemia, improving HbA1c levels, and replenishing hepatic glycogen toward physiological ranges. Dihydroquercetin and chrysoeriol exerted moderate protective effects, whereas resveratrol and thamiflaside showed limited efficacy. In conclusion, autoimmune thyroiditis induces significant disturbances in pancreatic endocrine function and hepatic glucose metabolism, while flavonoid–selenium combinations may represent promising strategies for correcting AIT-associated metabolic dysfunctions.

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Safflower yellow (SY), a bioactive compound from Carthamus tinctorius L., possesses potent antioxidant and anti-inflammatory properties and may exert protective effects on vulnerable brain regions during acute global ischemia. Therefore, this study aimed to evaluate the neuroprotective effects of SY against cerebral ischemia-reperfusion (IR) injury in mice, focusing on oxidative status, hippocampal neuronal survival, and white matter integrity. Forty male ICR mice were assigned to Sham-veh, IR-veh, IR-SY50, and IR-SY100 groups. Pretreatment with SY or vehicle was performed for 2 weeks before IR induction by 30-minute bilateral common carotid artery occlusion and 24-hour reperfusion. Brain infarct volume, oxidative status, and histological changes in the hippocampus and corpus callosum were evaluated. Significantly increased lipid peroxidation and cerebral infarction, depletion of CAT activity, selective neuronal death in the CA1 region, and loss of corpus callosum white matter integrity were clearly depicted after IR induction. Pretreatment with SY doses of 50 and 100 mg/kg significantly attenuated brain infarct volume, together with upregulated superoxide dismutase and catalase activities. Histological analysis revealed a dose-dependent therapeutic threshold. Only SY at 100 mg/kg significantly prevented neuronal death in the CA1 region and preserved corpus callosum white matter integrity. Pretreatment with SY exhibits neuroprotective effects against IR primarily by enhancing endogenous antioxidant defenses. Crucially, while biochemical improvements are achievable at lower doses, a higher therapeutic dose is required to preserve neuronal and white matter structural integrity in selectively vulnerable brain regions.

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Depression affects females more than males, with responses to antidepressants also exhibiting a distinct difference between sexes. Stress models in rodents have predominantly been developed for males, and their application in females remains challenging. This study investigated how post stress sensory contact with male mice affects the behavioral outcomes of female C57BL/6JJcl (B6) mice subjected to vicarious social defeat stress (VDS). We compared three types of VDS paradigms in female mice: the conventional paradigm and two adapted protocols that eliminated sensory contact with male ICR mice during the post-stress period under either single-housed (SH) or pair-housed (PH) conditions. In the conventional paradigm, female B6 mice did not exhibit a significant increase in social avoidance. In contrast, in the adapted paradigm, social avoidance behavior was observed in the PH group, indicating that post stress sensory contact with male ICR affects the stress susceptibility of female VDS model. Furthermore, the estrous cycle stage did not affect social avoidance behavior in each group. Next, we compared two different housing conditions to examine the appropriate method for eliminating sensory contact in female VDS model. SH mice showed reduced feed intake, increased water consumption, and attenuated body weight gain, regardless of VDS exposure. These findings suggest that post-stress sensory contact with a male mouse may influence social behavior in female mice exposed to VDS. The present study highlights the potential impacts of these experimental variables when interpreting behavioral outcomes in female VDS models.

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Mammalian hibernation is an adaptive strategy for surviving harsh cold seasons and food scarcity by suppressing the metabolic rate and thermogenesis. The Syrian hamster (Mesocricetus auratus) (hereafter termed as hamsters), a small mammalian hibernator, hibernates upon exposure to winter-like, short-photoperiod and cold (SP-Cold) conditions throughout a year in a laboratory, and therefore is a useful animal model obtained from a commercial breeder to study mechanisms of hibernation. However, the rates of hibernation occurrence often vary among experiments and facilities, the reason for which is largely unclear. Here we examined the effects of diet given to hamsters during post-weaning stages on hibernation occurrence. Hamsters fed either a high-nutrient (H) diet suitable for reproduction or a standard (STD) diet during the post-weaning period (from 3 or 4 weeks of age to sexual maturation) were exposed to winter-like conditions, and the occurrence of hibernation was assessed. Females showed a higher hibernation induction rate (over 80%) regardless of the post-weaning diet type than males. Approximately half (52%) of the males fed the STD diet from 3 weeks hibernated, whereas males fed the STD diet from 4 weeks or the H diet showed markedly lower hibernation induction rates (≤33%). These results suggest that female Syrian hamsters are more prone to hibernation than males and that the hibernation induction rate in male hamsters is influenced by diet during a critical developmental window between 3 and 4 weeks of age.

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Insulin resistance (IR) and hepatic steatosis are key pathological features of type 2 diabetes mellitus (T2DM). Fraxin, a natural flavonoid with potential metabolic regulatory properties, has been proposed to modulate metabolic disorders. This study aimed to investigate whether fraxin protects against IR and steatosis via Sirt1/AMPK/mTOR-mediated autophagy activation. A T2DM rat model was established using high-fat diet (HFD) feeding combined with streptozotocin (STZ) injection. Rats were treated with fraxin (25 or 50 mg/kg) for 4 weeks, followed by assessment of fasting blood glucose (FBG), insulin (FINS), HOMA-IR index, serum lipid profiles and liver function. Histopathological examination (H&E and Oil Red O staining) evaluated hepatic steatosis. BRL 3A cells exposed to palmitic acid (PA) were treated with fraxin (40 or 80 μM), and glucose uptake, lipid accumulation, and protein expression (IRS-1 phosphorylation, GLUT-2 translocation, autophagy markers and Sirt1/AMPK/mTOR pathway components) were analyzed. The specific autophagy inhibitor chloroquine (CQ) was used to verify whether the regulatory effects of fraxin were dependent on autophagy activation. Fraxin significantly reduced FBG, FINS, HOMA-IR, and dyslipidemia in diabetic rats. In PA-treated BRL 3A cells, fraxin enhanced glucose uptake, reduced lipid droplets, and restored IRS-1 phosphorylation and GLUT-2 membrane localization. Mechanistically, fraxin activated autophagy and modulated the Sirt1/AMPK/mTOR pathway, which were abolished by the Sirt1 inhibitor EX527. In conclusion, fraxin alleviates IR and hepatic steatosis in HFD/STZ-induced diabetic rats by activating autophagy through Sirt1/AMPK/mTOR pathway, suggesting its potential as a therapeutic agent for diabetes-associated metabolic disorders.

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The Lepus yarkandensis (L. yarkandensis) is an endemic species inhabiting the arid ecosystem of the Tarim Basin, characterized by extreme dryness and scarce water resources. In contrast, Oryctolagus cuniculus (O. cuniculus) is adapted to temperate environments rich in water. Over the course of long-term evolution, the L. yarkandensis has likely developed unique physiological and histological adaptations to cope with the survival challenges posed by such arid conditions. This study aims to investigate the differences between the L. yarkandensis and O. cuniculus in terms of hematological and biochemical blood parameters, renal histology, water-salt regulation capacity, and the expression of glomerular filtration barrier proteins, in order to investigate the adaptive strategies of the L. yarkandensis to drought environments. The results showed that the L. yarkandensis exhibited higher red blood cell counts, hematocrit levels, urine osmolality, and total urinary protein levels compared to O. cuniculus, while the urine pH was lower. Histological analysis revealed increased collagen content in the outer medulla and more complex tubular structures in the kidneys of the L. yarkandensis. Molecular analysis further demonstrated upregulated expression of glomerular filtration barrier proteins nephrin, podocin, and CD2AP, alongside downregulated expression of laminin and WT1. In summary, these findings provide insights into the potential physiological and molecular adaptations of L. yarkandensis, laying the groundwork for future research into the evolution of desert fauna.

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Noda epileptic rat (NER) is a mutant model for epilepsy that exhibits spontaneous generalized tonic-clonic seizure. Epileptogenesis of NER remains to be elucidated; but it is detected an insertion of an endogenous retrovirus sequence in intron 2 of the PHD finger protein 24 (Phf24) gene. PHF24 is widely expressed in the soma of neurons and neuropil in the wild-type rat brain and spinal cord but significantly less expressed in NER. In addition, the characteristic PHF24 expressions were noted in the soma of specific populations of the inhibitory interneurons. Here, we first examined the morphology of the neurons and synapses in the brain of NER to evaluate the epileptogenesis of NER. In NER, the total number of neurons was decreased, but the inhibitory neurons were increased. Inhibitory synapses increased while excitatory synapses tended to decrease in NER. Secondary, we examined Phf24-null rat without symptom of seizures in the same manner and evaluate a contribution of PHF24 to the epileptogeneis of NER. Interestingly, the opposite trend was observed in Phf24-null rats, with a decrease in the inhibitory neurons and synapses, an increase trend in the excitatory synapses. PHF24 is considered to play an important role in maintaining of the inhibitory neurons in the rat brain. We conclude that the reduction of PHF24 might lead to impaired inhibitory signals and increase susceptibility to epilepsy in NER. The histopathological changes of NER in the present study may represent a secondary change to repeated seizures.

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Repeated surgical oocyte collection in mice enables the harvest of multiple cohorts of eggs from a single female, thereby reducing the need for animal use. Traditionally, oocyte collection for the mouse requires euthanasia, and only two sequential collections have been reported. The most efficient superovulation protocol involves the use of inhibin antiserum; however, repeated administration may pose a risk of anaphylactic reactions. In this study, we used severely immunodeficient NOG mice to evaluate the feasibility of performing three serial oocyte collections with inhibin antiserum treatments. We found that three consecutive cycles of antiserum administration followed by surgical retrieval cycles were achieved without mortality. Although the number of mice from which oocytes could be collected decreased over successive cycles, oocyte retrieval remained feasible. The application of the anti-adhesion film improved the number of mice from which oocytes could be recovered. Furthermore, embryos derived from the third retrieval developed to term after transfer. However, compared with the control group, we were unable to achieve a proportional increase in the number of retrieved oocytes corresponding to the number of collection cycles. Further improvements to this repeated collection method may include increasing the number of consecutive retrievals, optimizing the protocol to enhance both the oocytes yield and the fertilization rates. Such advances could increase oocyte yield per female, enable the mass production of embryos from fewer animals.

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Sex differences have been observed in the onset of type 2 diabetes, with men being more likely to develop the disease than women. There is a clear sex difference in the development of diabetes in obese type 2 diabetes model ZFDM rats, with all males developing diabetes, but none of the females. In the present study, we examined the effects of sex hormones on the development of diabetes and gene expression in the pancreatic islets of ZFDM rats. All experiments were performed using 6-week-old ZFDM rats homozygous for the leptin receptor mutation (fatty, fa). Orchiectomy did not affect the development of diabetes but slightly suppressed non-fasting blood glucose levels in male ZFDM rats. Except for one diabetic rat, ovariectomy did not affect non-fasting blood glucose levels but impaired glucose tolerance in female ZFDM rats, which was accompanied by accelerated islet fibrosis and pancreatic β-cell loss. Administration of the female hormone (17β-estradiol, E2) did suppress the development of diabetes in male ZFDM rats. Pair feeding matched with the E2-administered group also suppressed the development of diabetes in male ZFDM rats. Direct E2 treatment of the pancreatic islets of diabetic male ZFDM rats changed the expression of genes related to type 2 diabetes and insulin secretion closer to those of obese non-diabetic male ZF rats. These results suggest that female hormones have the suppressive effects on the development of diabetes, whereas male hormones, if any, have the opposite effects in ZFDM rats.

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This article released online on March 23, 2020 as advance publication was withdrawn from consideration for publication in Experimental Animals at author’s request.

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