Objective To explore serum levels of serine/threonine protein kinase 4(MST4)and heat shock protein 70(HSP70)in children with idiopathic immune thrombocytopenia(ITP)and their clinical signifi-cance.Methods Totally 98 children with ITP admitted to Northwest Women and Children's Hospital from April 2019 to April 2023 were retrospectively selected as the ITP group,and 50 healthy children who under-went physical examination during the same period were selected as the control group.Enzyme linked immu-nosorbent assay was used to detect serum levels of MST4 and HSP70,and the serum MST4 and HSP70 levels in children with different ITP levels were compared.The correlation between the indicators were analyzed by Pearson correlation.Logistic regression model was used to screen the prognostic factors of ITP,and the as-sessment value of serum MST4 and HSP70 on ITP prognosis was analyzed by subject working characteristic curve.Results Serum MST4,HSP70,and CD8 in the ITP group were higher than those in the control group,while PLT,CD3+,CD4+,CD4+/CD8+were lower than those in the control group,with statistical significance(P＜0.05).Serum MST4 and HSP70 levels in mild group,moderate group and severe group were increased successively,with statistical significance(P＜0.05).Correlation analysis showed that serum MST4 and HSP70 were positively correlated with CD8+(P＜0.05),and negatively correlated with PLT,CD3+,CD4+,CD4+/CD8+(P＜0.05).The disease course,serum MST4 and HSP70 of ITP children in the poor prognosis group were higher than those in the good prognosis group,and the differences were statistically significant(P＜0.05).Logistic regression analysis showed that the course of disease(OR=1.579,P＜0.001),serum MST4(OR=1.451,P＜0.001)and serum HSP70(OR=1.442,P＜0.001)were independent risk factors af-fecting the prognosis of children with ITP.The area under the curve of serum MST4 and HSP70 combined in the assessment of poor prognosis of ITP children was larger than that of serum MST4 and HSP70,and the difference was statistically significant(Z=4.568,4.672,both P＜0.001).Conclusion The elevated serum MST4 and HSP70 levels in children with ITP are related to the severity of the disease and cellular immune function.The combination of the two has a high evaluation value for the prognosis of children with ITP.

Inner ear diseases are common in the field of otolaryngology,including hearing loss,tinnitus and peripheral vestibular dysfunction.Their pathogenesis is relatively complex,which is one of the hot spots in current research.A large number of studies have demonstrated that sleep disorder is an important inducement of inner ear diseases.This paper reviews the impact of sleep deprivation on inner ear diseases in order to pro-vide a theoretical basis for the mechanisms of sleep deprivation on inner ear diseases.

Objective To investigate the role of TLR4/NF-κB signaling pathway in mediating sleep depriva-tion induced endolymphatic hydrops.Methods A total of 30 healthy sprague-dawley(SD)rats were randomly di-vided into the control group、big platform control group,and 2 w,3 w,4 w sleep deprivation group,with 6 rats in each group.Modified multiple platform method was adopted to establish the rat sleep deprivation model.Before and after the experiment,the open field and acoustic brain-stem response(ABR)was conducted to evaluate the behavior and hearing level.After ABR test,blood samples were collected from abdominal aorta,and serum levels of TNF-αand MCP-1 were detected by ELISA.The cochlea was dissected,the severity of endolymphatic hydrops was as-sessed by calculating the ratio of the cross sectional area of the membranous cochlear duct(SM)to that of the mem-branous cochlear duct+scale vestibuli(SM+SV).Positive expression of IL-1β,TNF-α,MCP-1,TLR4,NF-κB P65 in rat cochlear tissues was detected via immunohistochemical staining.After the experiment,the changes of hearing level,the severity of endolymphatic hydrops and TLR4/NF-κB signaling pathway related proteins and down-stream inflammatory factors expression level were observed.The correlation between TLR4/NF-κB signaling path-way and hearing level and endolymphatic hydropsin rats was analyzed.Results ABR results showed an increased threshold of wave Ⅱ in the sleep deprivation group compared to those of the control group and big platform control group(P＜0.05).The rate of hydrops was 0％in control and big platform control groups,16.67％in 2w sleep deprivation group and 25％in 3 w and 4 w sleep deprivation group.The concentrations of TNF-αand MCP-1 in ser-um of rats in sleep deprivation groups were higher than those in control and big platform control groups,and the 4w sleep deprivation group were statistically significant compared with control and big platform control groups.The ex-pressions of IL-1β,TNF-α,MCP-1,TLR4 and NF-κB P65 in the cochlear spiral ganglion,spiral canal,stria vascu-laris and spiral ligament of rats in sleep deprivation groups were higher than those in control and big platform control groups.Conclusion Sleep deprivation may induce endolymphatic hydrops by the TLR4/NF-κB signaling pathway.

Ménière disease(MD)is a complex inner ear disorder with an uncertain cause and multiple contrib-uting factors.Affective disorders,such as anxiety,depression,insomnia,and other mood disorders,are common mental illnesses characterized by mood instability.There is a reciprocal relationship between affective disorders and MD,leading to a vicious cycle that worsens the progression of MD.This paper provides a comprehensive summary of the mechanisms linking affective disorders to MD,including changes in hormone levels and tissue damage caused by inflammation.It also explores how affective disorders influence MD through their effects on hormones and in-flammation.Additionally,the feasibility and effectiveness of psychotherapy in treating MD are discussed,with the aim of offering potential avenues for future MD treatment.

Objective:By detecting the levels of proteins in the Toll-like receptor-4/nuclear factor-κB （TLR4/NF-κB） signaling pathway and downstream proinflammatory cytokines in peripheral blood of patients with Meniere's disease （MD）, Pittsburgh Sleep Quality Index （PSQI） scores were collected to investigate the correlation between sleep disorders and MD and the role of TLR4/NF-κB signaling pathway in mediating sleep disorders inducing MD. Methods:Thirty-two MD patients and 20 family members of patients without middle ear and inner ear related diseases were selected. Basic data, PSQI and fasting peripheral blood of all subjects were collected. Enzyme linked immunosorbent assay.The levels of interleukin-1β（IL-1β）, tumor necrosis factor-α（TNF-α）, monocyte chemokine-1（MCP-1）, Toll-like receptor 4（TLR4） and nuclear factor-κB（NF-κB） in peripheral blood were detected by ELISA, and the data were statistically analyzed. Results:①PSQI score of MD group was higher than that of normal control group, and the difference was statistically significant（P<0.01）; The scores of every factors of PSQI in MD group were higher than those in normal control group, and the scores of factors 2, 4 and 6 were significantly different from those in normal control group. ②In the MD group, there were 18 patients with sleep disorders, with a prevalence rate of 56.25%, including 6 males with a prevalence rate of 50.00% and 12 females with a prevalence rate of 60.00%. ③The levels of five test indexes in MD group, sleep disorder group and non-sleep disorder group were higher than those in control group, and the levels of TLR4 and NF-κB in MD group were significantly different from those in control group（P<0.05）. The levels of IL-1β, TNF-α, TLR4 and NF-κB in sleep disorder group were significantly different from those in control group（P<0.05）. The levels of five test indexes in non-sleep disorder group were not statistically significant compared with those in control group. The levels of five test indexes in the MD sleep disorder group were higher than those in the MD group and the non-sleep disorder group, with no statistical significance. The levels of five test indexes in MD group were higher than those in non-sleep disorder group, with no statistical significance（P>0.05）. Conclusion:①Sleep disorders may be one of the important predisposing factors of some MD, and the effects of sleep disorders on MD are different between the sexes. ②Sleep disorders may activate TLR4/NF-κB signaling pathway to induce MD. The selection of TLR4/NF-κB signaling pathway related proteins and downstream pro-inflammatory factor inhibitors to intervene MD may provide a new idea for protecting the hearing balance function of MD.
Female ; Humans ; Male ; Meniere Disease ; NF-kappa B/metabolism* ; Signal Transduction ; Sleep Deprivation ; Toll-Like Receptor 4 ; Tumor Necrosis Factor-alpha/metabolism*

Objective: To investigate genotypes and drug susceptibility of the 100 strains of Candida glabrata isolated from 100 women (including 50 pregnant women) in order to study the drug-resistance and gene polymorphism, and to investigate the correlation of drug-resistance, gestation and gene polymorphism. Methods: Multilocus sequence typing (MLST) technique was introduced to identify sequences of 6 housekeeping genes from 100 isolates of Candida glabrata. The results were compared with sequence information in MLST databases by Clustalx software to determine a strain allelic profile and sequence type (ST). Drawing the phylogenetic tree by weighted paired group average method and the minimal spanning tree method of MEGA6.0 software, the microevolution and relationship between different strains were analyzed. ATB FUNGUS semi-automatic system was used to test the drug susceptibility. Fisher analysis method was used to analyze the correlation between the genotypes and pregnancy.Ridit analysis method was used to analyze the correlation between the genotypes and drug susceptibility. Results: The 100 isolates belonged to 34 clone sequences. There were 53 isolates belonged to ST-7 ( 26 isolates of pregnancy), 7 isolates belonged to ST-3 (3 isolates of pregnancy), 6 isolates belonged to ST-19 (5 isolates of pregnancy ), 3 isolates belonged to ST-15 (1 isolate of pregnancy), 2 isolates belonged to ST-10 (2 isolates of pregnancy), 1 isolate belonged to the other types of ST. Total of 100 isolates of Candida glabrata were 100% sensitive to fluorocytosine and amphotericin. The effect of itraconazole was poor with the sensitive rate of 20%. The resistance rates of fluconazole and voriconazole were 4% and 1% respectively. All genotypes were sensitive to voriconazole except ST-X1. In the correlation between genotype and itraconazole resistance, ST-7 as the standard group, the Ridit values in the group of ST-15, ST-19 and other types of ST were not included the mean Ridit value of the standard group in itraconazole (0.5). The system evolution tree was built using the neighbor-joining method (NJ) . All genotypes could be divided into 3 groups, as Ⅰ, Ⅱ, Ⅲ. Group Ⅰ had 44 cases, group Ⅱ had 53 cases , group Ⅲ had 3 cases . All the collected clinical strains had small genetic distances during molecular evolution. Conclusions ST-7 was the dominant genotype in Guiyang. No correlation between different STs and patients′ pregnancy was found. The different drug susceptibility in itraconazole between ST-7, ST-15, ST-19 and other STs were found. The Candida glabrata associated with VVC showed highly discrimative diversity. However, the phylogenic analysis exhibited genetic similarity among the strains studied.(Chin J Lab Med, 2018, 41: 596-600)

Objective To explore the relationship between the immune function of cellin peripheral blood with the virureplication and hepatitiviru(HCV)-cAg expression in the patientwith chronihepatiti(CHC) .MethodPeripheral blood lymphocytesubpopulation ,HCV-Rnand HCV core antigen (HCV-cAg) in 63 healthy people undergoing the physical exami-nation (control group) and 85 caseof CHC(Chgroup) were analyzed by the flow cytometry ,real-time Pcand ELIS,respec-tively .ResultThe percentageof total cell,T4 cell,T8 cell,double negative cell(DN) and double positive cell(DP) in the Chgroup were (67 .37 ± 10 .43)% ,(37 .11 ± 10 .28)% ,(21 .63 ± 8 .87)% ,(7 .80 ± 4 .57)% and (0 .20 ± 0 .29)% , respectively ,the absolute contentwere in turn (0 .70 ± 0 .44) × 109/L ,(0 .37 ± 0 .22) × 109/L ,(0 .22 ± 0 .17) × 109/L ,(0 .08 ± 0.06)×109/Land(0.19±0.68)×107/L,respectively.TheratioofT4/T8was(2.18±1.26)% .Theresultsindicatedthatthe percentage of T8 cellin the Chpatientwadecreased obviously (P＜0 .01) ,which resulted in the ratio of T4/T8 raising(P＜0 .05);meanwhile ,the absolute contentof the total cell,T4 cell,T8 celland Dnwere all decreased obviously (P＜0 .05);moreove,the percentage of T4 celland Dnin the patientwith HCV-Rnpositive and HCV-cAg positive wasignificantly in-creased (P＜0 .05) .Conclusion When HCV replicating in the patientwith CHC,the T lymphocyte subpopulation haobviouab-normity .The low immune function or immune tolerance ofT cells may be the important cause of recurrence and uncurability of CHC.

Background Recent researches show that oxidative stress is involved in the progress of keratoconus.Nuclear factor-E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a critical role in the defense against oxidative stress,but its function in keratoconus is unclear.Objective To investigate the differences of Nrf2-ARE signaling activation and matrix degenerating enzymes between keratoconus and normal corneal stromal cells.Methods Corneal stromal cells were isolated from keratoconus and normal cornea by using dispase and collagenase digestion.The cells were treated with hydrogen peroxide (H2O2) to mimic in vivo oxidative stress condition.Reactive oxygen species (ROS) production was measured by fluorescence substrate DCHF-DA incubation.Nrf2 level and the expression of Nrf2-ARE downstream antioxidant genes were analyzed by Western blot and real-time quantitative-PCR(RT-qPCR).The activity of matrix degenerating enzymes,including urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR) system and matrix metalloproteinase-2 (MMP-2) were assessed by Western blot and gelatin zymography respectively.Results In normal culture,keratoconus corneal stromal cells assumed increased basal ROS and Nrf2 level when compared with normal cells(t =18.155,P＜0.01).However,after H2O2 treatment,the keratoconus corneal stromal cells showed increased ROS production,while decreased Nrf2 translocation and no significant difference in expression levels of Nrf2-ARE downstream antioxidant genes (Nrf2:t =62.123,P＜ 0.01 ; (nicotinamide adenine dinucleotide phosphate quinine oxidoreductase-1 [NQO-1]:t =2.209,P =0.092 ; hemo oxygenase-1 [HO-1]:t =0.293,P =0.784 ; superoxide dismutase [SOD2]:t =0.749,P =0.495).The contents of uPA-uPAR and the activity of MMP-2 also showed a higher level in keratoconus corneal stromal cells than normal cells,with significant differences between them (t =19.164,15.458,4.818,all at P＜0.01).Conclusions The defect of Nrf2-ARE signaling activation exists in the keratoconus corneal stromal cells,and correlats with the abnormal expression level of stromal degeneration enzymes,which suggests that the defect of Nrf2-ARE signaling activation may be involved in the progression of keratoconus.

Background Diclofenac sodium eye drops,pranoprofen eye drops and bromfenac sodium hydrate eye drops are three clinical commonly used nonsteroidal anti-inflammatory drugs(NSAIDs).The variation of cytoxicity among these drugs and whether the cytoxicity is related to the supplements are also unknown.Objective This study was to compare the cytotoxicity of three non-steroidal anti-inflammatory eye drops and their active components with cultured human corneal epithelial cells (HCECs) in vitro,and to discuss toxic origins of these drugs.Methods HCECs were cultured in different drugs with the final concentration of 0.10％,0.05％,0.02％ and 0.01％.Cell proliferation was evaluated by MTT assay.Then,0.002％ eye drops (1∶50) was added,and the migration and damage of the cells were deceted by transwell migration assay and lactate dehydrogenase (LDH) assay.Results The cytotoxicity of three nonsteroidal anti-inflammatory eye drops on HCECs was concentration-dependent (all at P=0.00).Diclofenac sodium eye drops showed the most dominant effects on the proliferation,migration and damage of HCECs among the three eye drops,while bromfenac sodium eye drops showed the least effect on the cell damage (proliferation:Fdrug =20.25,P=0.00;migration:F =103.43,P =0.00;damage:Fdrug =164.16,P =0.00).Compared with the eye drops,their active components showed less cytoxicity.Pranoprofen appeared the least effects on the proliferation,migration and damage of HCECs (proliferation:Fdrug =332.27,P =0.00;migration:F =332.27,P =0.00;damage:Fdrug=154.83,P=0.00).Conclusions The cytotoxicity ofdiclofenac sodium eye drops is more obvious than that of pranoprofen eye drops or bromfenac sodium hydrate eye drops.The cytotoxicity of the three eye drops originates from their supplements or the interaction between the supplements and active components.

Background Limbal stem cells (LSCs) play an important role on the stability of corneal epithelium and corneal transparency.Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor can promote cell proliferation and reduce apoptosis,such as human embryonic stem cells and karatin epithelial cells.Objective This study was to investigate the improving effect of Y-27632,a ROCK inhibitor,on the activity of rabbit LSCs in corneal preservation medium.Methods Corneal preservation solution was prepared by adding 12.5％ chondroitin sulfate,10.0％ low molecular dextran,20.0 mg/L dexamethasone,100 mg/L tobramycin sulfate,9.5 g/L Hepes and 0.375 mg/L L-glutamine in MEM.The corneas of New Zealand white rabbits were collected and preserved in the corneal preservation solution with or without Y-27632 for 4,7,14 days,and the density and morphology of corneal endothelial cells were examined by using 0.25％ trypan blue staining and 0.2％ alizarin red staining.Isolated corneal epithelial cells were seeded on 3T3 feeder layer and cultured for 7-10 days until colonies formation.Colony shape of LSCs was observed under the light microscope,and colony-formation efficiency was analyzed after Giemsa staining by Image J software.Results The morphology and density of corneal endothelial cells were normal in the corneal preservation solution with and without Y-27632 for 4 days.In the seventh day after preservation,the cells remained the regular hexagon in shape in the preservation solution with Y-27632,however,the cellular membrane was slightly shrinking with the positive staining for alizarin red in the preservation solution without Y-27632.The density of corneal endothelial cells in the corneal preservation solution without Y-27632 was (2 262-± 75) cells /mm2,while in the preservation solution with Y-27632 was (2 425 ±95) cells/mm2(P＜0.001).The cloning spheres of LSCs were similar in preservation solution both with and without Y-27632 in the freshly isolated cornea or preserved corneas and exhibited more cells inside.But in 7 days and 14 days after preservation,the cloning spheres were much smaller in the preservation solution without Y-27632 group than those in the preservation with Y-27632 group.No significant differences were found in the cloning-formation rate and survival rate of corneal epithelial cells in corneas freshly isolated or preserved for 4 days in both groups (all at P＞0.05).In 7 days and 14 days after preservation,the active rates of corneal epitheli.al cells were (73.00±2.12)％ and (56.00±0.71)％ in the preservation solution with Y27632,which were significantly higher than (66.00 ± 4.00) ％ and (49.00 ± 0.71) ％ in the preservation solution without Y-27632,showing statistically significant differences between them (t =3.098,P =0.018;t =9.798,P =0.000).In addition,the cloning-formation rates of LSCs were (11.05±0.21)％ and (3.10±1.97)％ in the preservation solution with Y-27632 in 7 days and 14 days after preservation,revealing significantly elevation in comparison with (2.05 ± 1.20) ％ and (0.40 ±0.14) ％ in the preservation solution without Y-27632 (t =18.107,P =0.000;t=3.184,P=0.017).Conclusions Y-27632 promotes the vitality and cloning-formation ability of LSCs in corneal preservation medium,suggesting its potential use during storage of cornea.