OBJECTIVE: To observe the protective effect of Fisetin on sepsis-associated brain injury and explore its possible mechanism from the perspective of ferroptosis. METHODS: Sprague-Dawley (SD) rats (6-8-week-old male) were randomly divided into three groups: sham operation group (Sham group), colonic ligation and puncture (CLP) induced sepsis model group (CLP group) and Fisetin preprocessing group (CLP+Fisetin group), with 18 rats in each group (12 for observing survival rate and 6 for indicator testing). The CLP+Fisetin group was given Fisetin solution 50 mg×kg^-1×d^-1 by gavage continuously for 5 days before CLP, with dimethyl sulfoxide (DMSO) as the solute, while Sham group and CLP group were given the same dose of DMSO. The model was established at 2 hours after the last gavage. The general condition of each group of rats were observed, and the 10-day mortality were record. The behavioral testing (new object recognition experiment, elevated cross maze experiment) were performed after 7 days of modeling. After 24 hours of modeling, nerve reflex scoring was performed, and then the rats were euthanized and brain tissue was collected. The pathological changes of brain tissue were observed under a microscope by hematoxylin-eosin (HE) staining, the deposition of iron ion in brain tissue was observed by Prussian blue staining. The content of iron in brain tissue was determined by tissue iron kit, and the content of malondialdehyde (MDA) in brain tissue was determined by colorimetry. The expressions of tumor necrosis factor-α (TNF-α), neuron damage marker S100β, nuclear factor E2-related factor 2 (Nrf2), heme oxygenases-1 (HO-1) and glutathione peroxidase 4 (GPX4) were detected by Western blotting. RESULTS: On day 10 post-operation, 12, 3, and 7 animals survived in the Sham group, CLP group, and CLP+Fisetin group, respectively. Compared with the Sham group, rats in the CLP group showed significantly decreased nerve reflex score, new object discrimination index and open arm dwell time. HE staining showed arranged disorderly of neuronal cells, cytoplasm deep staining, nuclear condensation, unclear structures, neuron loss, and significant inflammation in the hippocampus in the hippocampus. Prussian blue staining showed iron ion deposition in the brain tissue. The contents of iron and MDA in brain tissue were elevated, and the expressions of TNF-α and S100β were up-regulated, while the expressions of Nrf2, HO-1, and GPX4 were down-regulated. Compared with the CLP group, the CLP+Fisetin group showed significantly increased neurological reflex score (7.33±1.15 vs. 4.67±1.53), improved new object discrimination index (0.44±0.02 vs. 0.32±0.04), and longer open arm dwell time (minutes: 78.33±9.29 vs. 41.15±9.64). Neuronal cells in the hippocampus were more organized, with less cytoplasmic staining, nuclear condensation, reduced neuronal loss, and fewer inflammatory cells. Iron ion deposition was reduced, and the contents of iron ions and MDA in brain tissue were decreased [iron ion (μg/g): 151.27±14.90 vs. 224.69±17.64, MDA (μmol/g): 470.0±44.3 vs. 709.3±65.4]. The expressions of TNF-α and S100β were significantly decreased (TNF-α/GAPDH: 0.651±0.060 vs. 0.896±0.022, S100β/GAPDH: 0.685±0.032 vs. 0.902±0.014), while the expressions of Nrf2, HO-1, and GPX4 were significantly increased (Nrf2/GAPDH: 0.708±0.108 vs. 0.316±0.112, HO-1/GAPDH: 0.694±0.022 vs. 0.538±0.024, GPX4/GAPDH: 0.620±0.170 vs. 0.317±0.039). All differences were statistically significant (all P < 0.05). CONCLUSION Fisetin pretreatment can inhibit ferroptosis and reduce sepsis-associated brain injury by Nrf2/HO-1/GPX4 pathway.
Animals ; Ferroptosis/drug effects* ; Rats, Sprague-Dawley ; NF-E2-Related Factor 2/metabolism* ; Sepsis/complications* ; Male ; Rats ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Neurons/drug effects* ; Signal Transduction ; Brain Injuries/metabolism* ; Flavonols ; Flavonoids/pharmacology* ; Heme Oxygenase-1/metabolism* ; Heme Oxygenase (Decyclizing)

"Toxicity Testing in the 21st Century—A Vision and Strategy"proposed by the National Research Council of US has brought innovative directives and objectives for toxicity evaluation and risk assessment,pushing forward the next generation of toxicity testing and risk assessment.In this initiative,the concept of adverse outcome pathways(AOPs)has emerged as a prominent methodology,capturing the attention of toxicologists and researchers due to its promising applications in recent years.The quantitative AOP(qAOP)is an extension of the adverse outcome pathway,which is built upon the foundational qualitative adverse outcome pathway model and leverages mathematical frame-works to depict dose-response and/or response-response relationships.This article reviews the princi-ples and advancement surrounding qAOP,introduceds two prevalent methodologies for constructing qAOP,Bayesian network models and regression models,and demonstrates diverse applications of qAOP.Actual cases are used to underscore the transformative role of qAOP in contemporary toxicology and risk assessment practices.

Objective:To investigate the immunoregulatory effects of Glycyrrhiza uralensis ethanol extract(GUEE) on the maturation of dendritic cells (DCs) and the adjuvant effect of GUEE on OVA in na?ve BALB/c mice and an ovalbumin (OVA)-induced asthma mouse model. Methods:GUEE was prepared, and the effects of different concentrations of GUEE on the maturation of DCs and the secreted cytokines as well as the effects of GUEE on bacterial lipopolysaccharide (LPS)-induced DC maturation were examined in vitro. The effect of GUEE on the morphology of mouse bone marrow derived DCs was observed using microscopy. Molecular expression levels on the surface of DCs were detected using flow cytometry. The levels of interleukin-1β (IL-1β), IL-6, IL-12, and tumor necrosis factor-α (TNF-α) in the supernatant of DCs cultures were measured by enzyme-linked immunosorbent assay (ELISA). The maturation status of DCs was detected by flow cytometry by injecting different concentrations of GUEE into the paws of mice and isolating the draining lymph nodes 24 h later. The naive BALB/c mice were co-immunized with OVA, and the changes in regulatory T cells (Treg) were detected by flow cytometry. An OVA-protein-induced mouse asthma model was established to investigate whether GUEE as a tolerogenic adjuvant has an antigen-specific therapeutic effect on asthmatic mice. Pulmonary pathological changes were analyzed by hematoxylin-eosin staining (HE) and PAS staining. OVA-specific antibodies in serum and the frequencies of Tregs, CD4 + IFN-γ + and CD4 + IL-4 + T cells in the spleen were detected by ELISA and flow cytometry, respectively. Results:GUEE suppressed DCs maturation induced by LPS both in vitro and in vivo (all P<0.05), and reduced proinflammatory cytokine production, including IL-1β, IL-6, IL-12 and TNF-α in the absence or presence of LPS (all P<0.05). Moreover, co-immunization with OVA and GUEE increased the amount of Tregs in na?ve BALB/c mice ( P<0.05). In OVA-induced asthmatic mice, OVA and GUEE co-immunization and GUEE alone treatment substantially ameliorated the inflammation of lung tissues, decreased the levels of IgG 1 and the amount of CD4 + IL-4 + T cells, and increased the amount of Tregs (all P<0.05). Conclusions:GUEE alone or as the tolerogenic adjuvant can ameliorate allergic diseases through inhibition of DC maturation and type 2 helper T cell responses and induction of Tregs.

Objective To investigate the effects of water extracts of Lepidium meyenii walp (LMWE) collected from two different places in Xinjiang on the maturation and function of dendritic cells (DCs) and to evaluate their roles in immunoregulation. Methods Water extracts of LMWE growing in Tashikuergan County (Ta xian) and A La gou of Xinjiang were prepared and named as LMWE-T and LMWE-A,respectively. Bone marrow-derived DCs and splenocytes isolated from C57BL/6 mice were treated with different concentrations of polysaccharide extracts from LMWE-T/A. Effects of LMWE-T/A on the per-centage and apoptosis of DC,expression of co-stimulatory molecules and secretion of cytokines were detected by flow cytometry and ELISA. MTT assay was used to analyze the proliferation of splenocytes. Changes in the functions of DC were evaluated by mixed lymphocyte reaction(MLR). Results LMWE-T/A had no in-fluence on the percentage and viability of DC. Expression of CD40 and CD86,and secretion of TNF-α,IL-12p40 and IFN-γ were significantly increased by LMWE-T/A treatment in a dose-dependent manner. LMWE-T/A treatment enhanced the functions of DCs and also dose-dependently promoted the proliferation of splenocytes. LMWE-A was more effective than LMWE-T in promoting CD86 expression,IFN-γ secretion and splenocyte proliferation. Pretreatment with TAK-242,an inhibitor of Toll-like receptor 4(TLR4),could sig-nificantly inhibit the regulatory effects of LMWE-T/A on CD40 expression and the secretion of TNF-α and IL-12p40. Conclusion This study shows that LMWE could promote the maturation of DC through TLR4 signaling pathway,enhance the functions of DC without side effects on DC survival,and increase the prolif-eration of splenocytes,indicating that LMWE has a potential immunopotentiating effect. LMWE-A has better effects than LMWE-T on immune enhancement.

OBJECTIVE: To evaluate the safety of the circular round window and discus anatomic landmarks of posterior wall of internal auditory canal by investigating the microscopic anatomy of internal auditory canal area of the retrosigmold approach, which can provide the anatomical basis for acoustic neutrinomas surgery. METHOD: Fifteen adult cadaver heads (30 sides) fixed with formalin were used in the study. The retrosigmold approach operations were imitated to dissect the blood vessels and nerves in internal auditory canal area by opening round bony window and removing posterior wall of internal auditory canal. RESULT: Fifteen specimens of 30 sides circular bone window were opened without injury with transverse sinus and sigmoid sinus. The vertical distance between the highest point of bone window margo superior and the lowest point of transverse sinus margo inferior was (4.02 ± 0.32) mm. The vertical distance from the most anterior point of bone window leading edge to the most posterior point of sigmoid sinus trailing edge was (6.31 ± 0.43) mm. The internal auditory canal tubercle located in the anterior superior position of internal auditory canal. The vertical distance from the highest point of internal auditory canal tubercle to the upper margin of internal auditory canal was (2.31 ± 0.32) mm. To expose the whole internal auditory canal, the length and width of the internal auditory canal posterior wall removal was (7.29 ± 0.32) mm, (4.12 ± 0.29) mm. Within this removal range, no case of cochlea, semicircular canal or venous was injured in 30 specimens. CONCLUSION The method of opening round window through retrosigmold approach is simple, practial and convenient. With little variation and easiness of location, the sinternal auditory canal tubercle can be used in the identification of the internal auditory canal. When exposing the whole internal auditory canal, the removal scope of the posterior wall should be paid more attention to, in order to avoid the damage of cochlea, semicircular canal and jugular bulb.
Adult ; Cranial Sinuses ; Ear Canal ; Ear, Inner ; Humans ; Round Window, Ear ; anatomy & histology ; Semicircular Canals ; anatomy & histology ; Temporal Bone

Objective In order to study the difference of depression-like behaviours among three widelyused stress models in rats.Methods The new-born Spragne-Dawley rats were randomly divided into maternal deprivation(MD) group(n=27),chronic mild stress(CMS) group (n=29),dual stress(DS) group ( n=31 ) and control (C) group ( n =30) on postnatal day 1.MD rats only received maternal deprivation.CMS rats only received chronic mild stress when 10 weeks old.DS rats received both maternal deprivation and chronic mild stress.Control rats received no experimental handling.Behavior tests including forced swimming test and sucrose consumption which were carried out to evaluate rats' depression-like behaviors in the thirteenth weeks.The extend time of floating and sucrose preference ratio should be recorded in the forced swimming test and sucrose consumption test respectively to reflect the behavior of helplessness and anhedonia of rats.Results In the forced swimming test,the extend floating time of MD group( 119.30 ± 65.56) s,CMS group ( 145.00 ± 80.24) s and DS group ( 170.03 ±61.75 )s were longer than the control group(81.14 ± 52.40)s (F =11.53,P ＜ 0.01 ).In the sucrose consumption test,the MD group(0.32 ± 0.22) had a low sucrose consumption.The comparison of the MD group and CMS group (0.43 ± 0.28 ) to the control group (0.54 ± 0.28 ) had significant differences (F =4.33,P ＜ 0.01 ).In these four groups,no sexual difference was found in the forced swimming test and sucrose consumption test ( all P ＞ 0.05 ).Conclusion The results suggest that MD,CMS and DS may induce some kind of depression-like behaviors in rodents such as anhedonia or the behavior of helplessness and the depression-like behaviors induced by different stresses are similar in male and female individuals.

In this paper is recommended a highly sensitive and reagent-safe method to determine plasma heamoglobin (FHb) in viscacha hemolytic test. The 2,4-dichlorophenol method (2,4-DCP) of Trinder reaction has been improved. The performance of 2,4-DCP is verified. The sensitivity of 2,4-DCP is 2.39 times that of phenol method. It is well used with run precision and day-to-day precision. The reaction color is stable. The reference value FHb is 1-36.7 mg/L. Sodium citric is an excellent anticoagulant liquid to keep erythrocyte. The 2,4-DCP method is neither carcinogenic nor poisonous;it is suitable for viscacha hemolytic test in clinical and biomedical engineering.
Chlorophenols ; Coombs Test ; methods ; Hemoglobins ; analysis ; Hemolysis ; Humans ; Sensitivity and Specificity

Objective To conduct a screening survey of glycophorin (GP) variants and observe the content changes of sialic acid (SA) and total sulfhydryl (SH) groups on the erythrocyte membranes among residents in a tertian malaria hyperendemic area of Guizhou Province.Methods GP variants were detected in the erythrocyte hemolysates of 173 local residents at two villages of Libo County by SDS-PAGE on 10% to 15% gradients gel and Western immunoblotting. Their SA and total SH group contents were estimated in erythrocyte membranes by spectrophotometric methods. 114 healthy subjects in Changsha and 49 individuals at a neighbouring village of the above area showing low morbidity of malaria served as normal and endemic controls respectively.Results Three distinct types of GP variants were found among 19 propositi in this hyperendemic area. The incidence of GP variants was 7.9% (8/101) at Yaolu Village whose population was mainly composed of Yao ethnic group;while that of Buyi ethnic group at Maolan Village was higher (15.3%; 11/72).The erythrocyte membrane contents of SA in residents at both villages exhibited a very significant tendency of decline (P<0.01), whereas those of total SH groups increased prominently in residents of Yaolu Village only (P<0.05).Conclusions The frequency of GP variants in this hyperendemic area does not depend upon the severity of malarial prevalence. The evident reduction of SA contents in the residents may be related to the breaking down of the SA residues on membrane GPs by the invasion of Plasmodium vivax.