ObjectiveTo investigate whether intestinal deficiency of lanosterol synthase （LSS）， a key enzyme in cholesterol synthesis， influences the progression of metabolic dysfunction-associated steatotic liver disease （MASLD） by regulating intestinal cholesterol absorption and immune response. MethodsLSS heterozygous knockout （LSS^+/-） mice and wild-type （WT） controls were generated using CRISPR/Cas9 technology and fed either a high-fat diet （HFD） or regular chow （CHOW）. The model was validated by genotyping. Hepatic steatosis was assessed by HE and oil red O staining. Immunohistochemistry was used to detect the localization and expression of NPC1L1 and CD36 proteins in the intestine. Western blot analysis was performed to measure JNK phosphorylation and TLR4 protein levels in intestinal tissues. Real-time quantitative polymerase chain reaction （qPCR） was employed to examine the mRNA expression of TLR4 and IL-6. ResultsLSS^+/- mice were successfully validated by genotyping and reduced intestinal LSS protein expression. HE and oil red O staining of liver sections showed that， compared with WT mice fed a CHOW diet， WT mice fed a HFD exhibited a marked increase in hepatic lipid vacuoles. In contrast， compared with HFD-fed WT mice， HFD-fed LSS^+/- mice displayed significantly attenuated hepatic lipid deposition and reduced serum ALT levels （P<0.05）. Immunohistochemical analysis revealed that， compared with WT mice， the expression of the cholesterol absorption protein NPC1L1 in the intestinal villi of LSS^+/- mice was downregulated under both CHOW and HFD conditions （P_HFD<0.001）. Conversely， the expression of the fatty acid transporter CD36 was upregulated in the intestines of LSS^+/- mice （P_CHOW<0.05， P_HFD<0.01）. Western blot analysis demonstrated that， compared with WT mice， TLR4 protein expression in the intestines of LSS^+/- mice significantly increased under both CHOW and HFD conditions （both P<0.05）. JNK phosphorylation level was significantly elevated in LSS^+/- mice under CHOW condition （both P<0.05）. Under HFD condition， total JNK protein expression increased， but its phosphorylation level showed no significant change. qPCR analysis showed that， compared with WT mice， the mRNA levels of TLR4 （P_CHOW<0.01， P_HFD<0.000 1） and IL-6 （P_CHOW<0.001， P_HFD<0.01） were significantly upregulated in the intestines of LSS^+/-mice. ConclusionLSS deficiency counteracts hepatic lipid deposition by orchestrating a synergistic reprogramming involving restricted intestinal cholesterol absorption， enhanced fatty acid utilization， and activation of immune pathways， suggesting intestinal LSS as a potential therapeutic target of MASLD.

ObjectiveTo investigate the effect of hypoxia on pemetrexed （PE）-mediated apoptosis in non-small cell lung cancer cells. MethodsA549 cells were cultured under 21% O₂ （normoxia） or 1% O₂ （hypoxia）， and treated with 8 μmol/L PE for 48 h. The cells were divided into normoxic control group， normoxic vehicle group， normoxic drug-treated group， hypoxic control group， hypoxic vehicle group and hypoxic drug-treated group. Trypan blue staining was used to observe cell death after PE intervention under normoxia and hypoxia. Flow cytometry was adopted to detect cell apoptosis rate. Wound healing assay was used to assess cell migration. Western blot was performed to detect the protein levels of MRP-1， Cleaved Caspase-3， E-Cadherin and N-Cadherin. ResultsTrypan blue staining results showed that the cell death rate was reduced in PE-treated cells under hypoxia compared with the normoxic PE group （P<0.001）. Flow cytometry results indicated that PE induced cell apoptosis under both normoxic and hypoxic conditions.However， the apoptosis rate of the normoxic PE group was significantly higher than that of the hypoxic PE group （P<0.000 1）. Wound healing assay showed that the migration distance of A549 cells decreased after PE treatment for 24 h and 48 h under normoxia and hypoxia when compared with the control group （P<0.05）， while no significant difference was found between the normoxic PE group and the hypoxic PE group. Western blot results revealed that MRP-1 expression was markedly higher in hypoxic PE-treated cells than in normoxic PE-treated cells （P<0.000 1）. Compared with the control group， Cleaved Caspase-3 expression was upregulated in A549 cells following PE intervention under normoxia （P<0.05）， whereas no obvious alteration in Cleaved Caspase-3 expression was detected in hypoxic PE-treated cells. Under both normoxic and hypoxic conditions， PE treatment downregulated the expression of E-Cadherin （P<0.000 1，P<0.001） and N-Cadherin （P<0.05，P<0.000 1）. In addition， the expression levels of E-Cadherin and N-Cadherin in the hypoxic PE group were significantly lower than those in the normoxic PE group （all P<0.000 1）. ConclusionHypoxia significantly attenuates the pro-apoptotic effect of PE on A549 cells and blocks Cleaved Caspase-3 activation. This mechanism may be related to hypoxia-induced MRP-1 overexpression and reduced drug accumulation. Interfering with MRP-1 or restoring the activity of the apoptosis pathway may serve as a novel strategy to overcome hypoxia-induced drug resistance.

Objective: To investigate the effect of loss of function of lanosterol synthase( LSS) gene on intestinal function in a mouse model of non-alcoholic fatty liver disease( NAFLD) induced by a high-fat diet． Methods: LSS gene heterozygous knockout C57 mice ( LSS + / －) were established using the CRISRP / Cas9 system．After being fed a high-fat diet with 60% fat content for 6 months，the fat deposition in liver tissues was detected by HE and Oil red O staining，the morphological changes of small intestine tissue were detected by HE staining．The changes in total cholesterol content in intestinal tissue were detected by kits．The gastrointestinal motility function of mice was detected by phenol red paste．The intestinal permeability was detected by Evans blue staining，and the expression of LSS，tight junction protein ( Claudin) -1，Claudin-5，cluster of differentiation 36 ( CD36) ，and Niemann-Pick type C1-like 1 protein ( NPC1L1) proteins in small intestinal tissues were detected by Western blot． Results : The results of HE and Oil red O staining of liver tissues showed that liver fat deposition in LSS gene heterozygous knockout mice was lower than that in wild-type mice in the high-fat diet group．The total cholesterol content in intestinal tis- sue of LSS gene heterozygous knockout mice decreased ( P ＜0. 01) ，but no morphological differences were ob- served between the two groups of mice by HE staining of intestinal tissues．The gastrointestinal motility function of LSS gene heterozygous knockout mice did not show significant changes．The intestinal permeability of LSS gene het- erozygous knockout mice in the high-fat diet group decreased as detected by Evans blue ( P＜0. 05) ．The expres- sion levels of Claudin-5 protein in the intestinal tissue of LSS gene heterozygous knockout mice in the high-fat diet group increased ( P ＜0. 05 ) ，while the expression of LSS protein in the intestinal tissues of LSS heterozygous knockout mice decreased ( P ＜0. 05) ． Conclusion In the NAFLD model induced by a high-fat diet，LSS gene heterozygous knockout reduces liver fat deposition induced by a high-fat diet and improves intestinal barrier function by regulating cholesterol metabolism in intestinal tissues and up-regulating the expression of Claudin-5．

Objective : To explore the changes in behavior and spatial memory of C57BL/6J female mice of different ages (youth , middle-aged , and elderly) . Methods: C57BL/6J female mice were divided into female youth group (YG group) , female middle-aged group ( MG group) and female elderly group ( OG group) according to age. The Morris water maze test measured spatial memory ability , and the open field and elevated cross maze test observed activity level and anxiety level. Western blot was used to determine the protein expressions of CREB , CaMKⅡ(pan) and CaMKⅡ(p) in the hippocampus of the brain tissues of female mice in each group. Results: Compared with the YG group , the weight of the MG group and the OG group significantly increased (P < 0. 01 , P < 0. 001) . Compared with the OG group , the third quadrant escape latency and the number of crossings in the YG group and MG group were shortened , and the difference was not statistically significant. Compared with the OG group , there was a statistically significant difference in the exercise speed in the open field of the YG group (P < 0. 01) , there was no significant difference in the movement speed in the open field of the MG group , the number of entries into the central zone significantly increased in the MG group ( P < 0. 05 ) , and there was no significant difference in the number of entries in the YG group (P > 0. 05) . Compared with the OG group , the YG group had a statistically significant difference in the elevated cross maze (P < 0. 05) , the MG group had no statistically signif- icant difference in the elevated cross maze , and the number of closed arm entries in the YG group and MG group significantly increased (P < 0. 001 , P < 0. 01) . Compared with the YG group , the relative expression level of CaMKⅡ(pan) in the OG group was statistically significant ( P < 0. 05 ) , while the relative expression level of CaMKⅡ(pan) in the MG group was not statistically significant ( P > 0. 05) . Conclusion With the increase of age , the weight of C57BL/6J female mice gradually increased , the activity level and desire to explore gradually de- creased , the spatial memory ability also declined , and the anxiety level and anxiety-like behavior increased. This study helps to reveal the effect of age on the activity level and cognitive function of females , and provides a refer- ence for studying cognitive and memory decline in older females.

Objective : To investigate the effect of laminarin(LAM) on nonproliferative diabetes retinopathy by high throughput sequencing(RNA-seq). Methods : The diabetes model was established by intraperitoneal injection of streptozotocin(STZ), and the effect of LAM on diabetic mice was observed.C57BL/6 mice were randomly divided into three groups: Control group, Model group, and LAM group, with 8 mice in each group. After 8 weeks of modeling, the LAM group received a 4-week intraperitoneal injection of LAM treatment. Changes in blood glucose and body weight of the three groups of mice were recorded, HE staining was performed to examine retinal lesions, and RNA-seq was used to identify differentially expressed genes(DEGs) in diabetic retinopathy(DR) under the action of STZ and LAM. Results : STZ successfully established the model of DR, and LAM reduced the blood sugar in diabetic mice to a certain extent and improved the pathological morphology of retinal structural looseness in diabetic mice. After RNA-seq analysis of DEGs, it was found that there were a total of 214 DEGs in the retina of the Model group mice compared to the Control group. Enrichment analysis revealed that DR could exacerbate the lesions through the PI3K Akt signaling pathway. There were a total of 42 DEGs in the retina of the Model group and LAM group mice, and enrichment showed that LAM improved the lesions through the neutrophil extracellular trap pathway. Early growth response factor 1(Egr1), FBJ osteosarcoma oncogene(Fos), nuclear receptor subfamily 4A member 1(Nr4a1), and salt-induced kinase 1(Sik1) were regulated by STZ, and LAM significantly regulated their expression, which might be closely related to LAM′s treatment of diabetic retinopathy. Conclusion DEGs can exacerbate the severity of diabetic retinopathyviathe PI3K-Akt signaling pathway. LAM can mitigate diabetic retinopathyviathe neutrophil extracellular trap pathway. Egr1, Fos, Nr4a1, and Sik1 are key genes involved in LAM treatment of STZ-induced DR.

Objective : To investigate the role of endoplasmic reticulum stress in the occurrence and development of fatty liver induced by high fat. Methods : In the high-fat Drosophila model, the high-fat group was fed with high-fat medium, while the control group was fed with normal medium; in the mouse fatty liver model, the high-fat group was fed with high-fat diet, and the control group was fed with normal diet; in the HepG2 cell steatosis model, the high-fat group was induced by palmitic acid(PA), and the control group was cultured with DMEM. The fat body size of the third instar larvae of Drosophila melanogaster was photographed. Steatosis in mice liver and HepG2 cells was observed by H&E and Oil Red staining. The expression levels of ATF6, Bip and CHOP in the third instar larvae, liver tissues of mice and HepG2 cells were analyzed by quantitative real-time polymerase chain reaction(qPCR) and Western blot. Results : In Drosophila model, fat body and fat storage were obviously increased in high fat fed flies when compared with control group. The formation of liver fat droplets and cells vacuolation were confirmed by H&E and Oil Red staining in mice livers fed with high fat and HepG2 cells with palmitic acid treatment. The expression levels of ATF6, Bip and CHOP were significantly increased in third instar larvae and mice livers fed with high fat and palmitic acid treated HepG2 cells with palmitic acid treatment. Conclusion High fat may induce the occurrence and development of hepatic steatosis by activating endoplasmic reticulum stress.

Objective:To investigate the clinical characteristics and management status of children with Turner syndrome (TS) in China.Methods:As a cross-sectional study, 1 089 TS patients were included in the database of the National Collaborative Alliance for the Diagnosis and Treatment of Turner Syndrome from August 2019 to November 2023. Clinical characteristics (growth development, sexual development, organ anomalies, etc.), karyotypes, auxiliary examinations, and treatments were collected and analyzed.Results:Among the 1 089 TS cases, 809 were recorded karyotypes. The karyotype distribution was as follows: 45, X in 317 cases (39.2%), X chromosome structural variants (including partial deletions of p or q arm, ring chromosome, and marker chromosome) in 89 cases (11.0%), 45, X/46, XX mosaicism in 158 cases (19.5%), mosaicism with X chromosome structural variants in 209 cases (25.8%), and presence of Y chromosome material in 36 cases (4.4%). Among the 824 TS cases, the age of diagnosis was 9.7(6.4, 12.2) years, with a height standard deviation score (HtSDS) of -3.1±1.2. Five hundred and fifty three cases underwent growth hormone (GH) stimulation test, and 352 cases (63.7%) had GH peak values <10 μg/L and 75.9% (577/760) had low IGF1 levels, with IGF1 SDS ≤-2 accounting for 38.2% (290 cases). Among 471 cases aged ≥8 years, 132 cases (28.0%) showed spontaneous sexual development (mean bone age (11.0±1.7) years), 10 cases had spontaneous menarche (mean bone age (12.0±2.2) years), and 2 cases had regular menstrual cycles. Common physical features included cubitus valgus (311 cases (28.5%)), neck webbing (188 cases (17.2%)), low posterior hairline (185 cases (17.0%)), shield chest (153 cases (14.0%)), high arched palate (127 cases (11.6%)), short fourth metacarpal (43 cases (3.9%)), and spinal abnormalities (38 cases (3.5%)). Congenital cardiovascular and urogenital anomalies occurred in 91 cases (19.4%) and 66 cases (12.0%)respectively. Abdominal ultrasound in 33 cases (7.2%) indicated fatty liver, hepatomegaly, intrahepatic bile duct stones, and splenomegaly. Among 23 cases undergoing oral glucose tolerance test (OGTT) test, 2 were diagnosed with diabetes mellitus and 4 with impaired glucose tolerance. Following diagnosis, 669 cases (80.7%) received rhGH treatment at a chronological age of (9±4) years and bone age of (8.3±3.2) years. Additionally, 112 cases (19.4%) received sex hormone replacement therapy starting at the age of (14±4) years and bone age of (12.6±1.2) years.Conclusions:The karyotypes of 45, X and mosaicism were most common in Chinese children with TS. The clinical manifestations were mainly short stature and gonadal dysplasia. However, a few TS children could be in the normal range of height, and some cases among those aged of ≥8 years old had spontaneous sexual development. Some exhibited physical features, congenital cardiovascular and urogenital anomalies, and dysfunction of the hypothalamic-pituitary-IGF1 axis. Moreover, a few of them developed impaired glucose tolerance and diabetes mellitus. Following diagnosis, most of the patients received rhGH treatment, and a few of them received sex hormone replacement therapy.

Objective To investigate the affecting factors of postoperative restenosis in patients with Trans Atlantic Inter-Society Consensus-Ⅱ(TASC-Ⅱ)type C or D atherosclerosis obliterans(ASO)treated with endovascular intervention.Methods Eighty-one patients who underwent endovascular interventional treatment for ASO were included and were followed up continuously for two years after the procedure.Also,the pre-and post-treatment data of the restenosis group and the nonstenosis group were compared and statistically analyzed to clarify the affecting factors of restenosis as well as the value of the related factors in predicting the postoperative restenosis in the preoperative period.Results The incidence of restenosis in 81 patients was 40.74％within two years after the interventional treatment.The unifactorial results showed that the comparison of ankle brachial index(ABI),age,gender,smoking history,body mass index(BMI),procedure,number of stent placement,lesion length,hyperlipidemia,coronary heart disease,diabetes mellitus,and hypertension between the two groups was not statistically significant(P＞0.05);the comparison of blood biochemical indexes in the levels of hypersensitive C-reactive protein(hs-CRP),fibrinogen,neutrophil-lymphocyte ratio(NLR),homocysteine(Hcy),and cystatin C(Cys C)showed a difference of statistically significant(P＜0.05);binary logistic regression results for fibrinogen level,hs-CRP,NLR,Hey,and Cys C were all independent risk factors for postoperative restenosis[odds ratio(OR)=40.501,4.507,4.381,1.509,and 23.094,P＜0.05].The results of receiver operating characteristic(ROC)curves showed that hs-CRP,NLR,and Cys C could effectively predict ASO postoperative restenosis,with area under the curve(AUC)of 0.683,0.637,and 0.632,and cutoff values of 4.225,3.465,and 1.000,respectively(P＜0.05).Conclusion Post-interventional vascular restenosis in ASO patients is correlated with the levels of fibrinogen,hs-CRP,NLR,Hcy,and Cys C.Also,hs-CRP,NLR,and Cys C may be used as blood biochemical indexes to predict post-interventional vascular restenosis in ASO patients.

Objective To explore the correlation of serum Chemerin level with disease activity and the ratio of T helper 17/regulatory T cells(Th17/Treg)in patients with rheumatoid arthritis(RA).Methods A total of 180 patients with RA who were admitted to our hospital were regarded as the observation group.According to the DAS28 score,the observation group was divided into the high activity group(60 cases),the moderate activity group(60 cases)and the low activity group(60 cases).Another 180 healthy people who underwent physical examination in our hospital during the same period were regarded as the control group.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum levels of Chemerin,interleukin-9(IL-9),interleukin-10(IL-10)and interleukin-17(IL-17).Flow cytometry was used to detect the Th17/Treg ratio.Spearman correlation analysis was applied to analyze the correlation between serum Chemerin level and DAS28 score.Pearson correlation analysis was used to analyze the correlation between serum Chemerin level and Th17,Treg cell percentage and Th17/Treg ratio.Results The results of this study showed that the serum level of Chemerin was higher in the observation group than that in the control group(P＜0.05).The serum Chemerin level was positively correlated with DAS28 score(P＜0.05).Serum Chemerin levels and DAS28 scores decreased in turn in the high,moderate and low activity groups(P＜0.05).The percentage of Th17 cells and the ratio of Th17/Treg were higher in the observation group than those in the control group,and the percentage of Treg cells was lower in the observation group than that in the control group(P＜0.05).The level of IL-10 was lower in the observation group than that in the control group,while levels of IL-17 and IL-9 were higher in the observation group than those in the control group(P＜0.05).The results of Pearson correlation analysis showed that serum Chemerin level was positively correlated with the percentage of Th17 cells and the ratio of Th17/Treg,and negatively correlated with the percentage of Treg cells(P＜0.05).Conclusion Serum Chemerin level is elevated in patients with RA,which is closely related to disease activity and Th17/Treg ratio.

Objective To investigate the clinical utility of iFlow color flow coding imaging technology in the diagnosis of lower extremity arteriosclerosis obliterans(LEASO).Methods A total of 106 patients diagnosed with LEASO between March 2022 and October 2023 were included as the LEASO group,while 80 volunteers without arterial disease but matched with LEASO were selected as the control group.Both groups underwent digital subtrac-tion angiography(DSA),and iFlow color flow coding imaging technology was employed to assess time to peak(TTP)in the femoral head and ankle regions.The difference value of TTP between these two regions was calculated,along with measurement of ankle-brachial index(ABI).Results There were no significant differences in age,sex,body mass index,smoking history,hypertension history,diabetes history,coronary heart disease history and TTP in the femoral head between the two groups(P＞0.05).However,the TTP in the ankle area and the difference values of TTP in the LEASO group were significantly higher than those in the control group(P＜0.05).The com-parison of TTP in the femoral head region among patients with different Rutherford classifications and between patients with left and right lesions in the LEASO group showed no statistical significance(P＞0.05).Furthermore,a negative correlation was observed between Rutherford classification and both TTP values in ankle joint region as well as TTP difference value(P＜0.05),indicating that higher Rutherford classification is associated with lower TTP values.Pearson test results revealed a significant negative correlation between TTP values and ankle joint region/TTP difference value of LEASO patients with ABI(P＜0.05).Receiver operating characteristic curve analysis demonstrated that both TTP values in ankle joint region and TTP difference value are effective diagnostic indicators for LEASO;moreover,Delong test indicated that area under ROC curve for TTP difference value was significantly higher than that for TTP value alone(P＜0.05)Conclusion iFlow color flow coding imaging technology enables quantitative assessment of both TPP values within ankle joint region as well as their differences which can be utilized for diagnosis of LEASO.