ObjectiveQi deficiency and phlegm dampness syndrome is a common type of clinical traditional Chinese medicine（TCM） syndrome. However， there is no standard， scientific， and accurate report on the construction of animal models of Qi deficiency and phlegm dampness syndrome. This study aims to construct a mouse model of Qi deficiency and phlegm dampness syndrome by using a multi-factor composite modeling method and to evaluate the model. MethodsTwenty-one C57BL/6 mice were randomly divided into three groups with seven mice in each group， which were the normal group， model group， and Shenling Baizhusan （SLBZ） group. The control group was fed with ordinary diet and kept in a normal environment. The model group and SLBZ group were fed with a high-fat diet in a high-humidity environment. Swimming with heavy weights until exhaustion and gavage with cold water or lard were used to establish the mouse model of Qi deficiency and phlegm dampness syndrome. In order to test the syndrome by prescription， mice in the SLBZ group were treated with SLBZ for 14 days after model construction. The exhaustive swimming time， body weight， serum lipid levels， tongue changes， "Qi deficiency and phlegm dampness" assessment scale score， and cecal index of mice in each group were measured. The feces of each group of mice were sent for metagenomics and metabolome sequencing， and the changes in intestinal flora and metabolites were analyzed. ResultsAfter the modeling of Qi deficiency and phlegm dampness syndrome， the exhaustive swimming time of mice was obviously shortened （P<0.01）. The serum total cholesterol， low density lipoprotein cholesterol， and non-high density lipoprotein cholesterol of mice were significantly increased （all P<0.01）. The tongue of mice was significantly different from that of the normal group， and the score of the assessment scale was significantly higher than that of the control group （P<0.01）. Cecal index decreased significantly （P<0.01）. The serum lipid level， tongue image， assessment scale score， and cecal index were reversed in the SLBZ group. Metagenomic and metabolome sequencing results showed that intestinal flora and fecal metabolites were significantly changed in mice with Qi deficiency and phlegm dampness syndrome. Akkermansia_muciniphila， Faecalibaculum_rodentium， Eubacterium_plexicaudatum， Eubacterium sp 14_2， Candida glabrata， Romboutsia_ilealis， Turicibacter sp TS3， and other bacteria had significant changes， and the expressions of intestinal metabolites such as chenodeoxycholic acid， choline， L-phenylalanine betaine， and 2-phenylbutyric acid were significantly changed. Related metabolic pathways such as linoleic acid metabolism， primary bile acid biosynthesis， lysine degradation， arginine biosynthesis， and alpha-linolenic acid metabolism were affected. ConclusionThe Qi deficiency and phlegm dampness model of mice can be constructed by the multi-factor composite modeling method of high-fat diet feeding， high-humidity environment feeding， exhaustive swimming with heavy weight， and intragastric administration with cold water or lard. The blood lipid level， tongue change， score of "Qi deficiency and phlegm dampness assessment scale"， cecal index， and changes in related intestinal flora and metabolites of mice can be used as key indicators for model evaluation.

Objective: To investigate the current status of emotional and behavioral problems among preschool children in Hefei, and to explore their association with the family rearing environment, providing scientific evidence for improving the family rearing environment and promoting children s mental health. Methods: From March to May 2023, a stratified cluster sampling method was applied to select 1 893 preschool children from five kindergartens in Hefei. Data on children s emotional and behavioral problems and family rearing environment were collected through electronic questionnaires. The Strengths and Difficulties Questionnaire (Parent Version) was employed to assess children s emotional and behavioral problems. Binary Logistic regression was conducted to analyze their association with family rearing environment. Results: Among preschool children, the prevalence rates of emotional symptoms, conducts, hyperactivity/inattention, peer relationship difficulties, and prosocial behavior were 9.7% (184 cases), 37.7% (713 cases), 16.7% (316 cases), 48.5% (918 cases), and 17.5% (332 cases), respectively. The abnormal detection rate of total difficulties score was 18.3% (346 cases). Binary Logistic regression analysis showed that higher maternal education level (postgraduate degree), parental daily interaction time ≥1 h, daily outdoor activity time for children ≥2 h, consistent family rearing attitudes, and nightly sleep duration for children ≥8 h were associated with lower abnormal risks of total difficulties score in preschool children [ OR(95%CI )=0.48(0.24-0.95), 0.66(0.49-0.90), 0.65(0.48-0.87), 0.39(0.29-0.52), 0.71(0.51-0.99)，all P < 0.05 ]. Conclusion The abnormal detection rates of emotional and behavioral problems among preschool children in Hefei are at moderate levels, and family rearing environment is related to children s emotional and behavioral problems.

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 construct and evaluate a mouse model that simulates the pathological progression of immune checkpoint inhibitor-associated pneumonitis (CIP), in order to address the critical issue of the lack of standardized animal models in this research field. MethodsThirty-five SPF-grade 6-week-old male C57BL/6J mice were randomly assigned to a control group (n=5), a bleomycin (BLM) group (n=15), and a BLM+programmed death-1 (PD-1) monoclonal antibody (BLM+PD-1) group (n=15). Corresponding drugs were administered via intraperitoneal injection for intervention. The general condition, body weight changes, survival rate, and pulmonary histopathological alterations were systematically monitored. ResultsCompared with the BLM group, the BLM+PD-1 group exhibited significantly more severe pulmonary fibrosis, characterized by progressive body weight loss and more severe deterioration of general conditions. Histopathological examination showed that pulmonary inflammation was alleviated 4 weeks post-modeling in the BLM group. In contrast, pulmonary lesions continued to worsen in the BLM+PD-1 group, with no evidence of resolution of inflammation 28 days post-modeling. Quantitative immunohistochemical analysis revealed that the positive expression rate of interleukin-17A (IL-17A) in lung tissues reached 34.28% in the BLM+PD-1 group 14 days post-modeling, which was markedly higher than that in the BLM group (24.02%, P < 0.000 1). The positive expression rate of α-smooth muscle actin (α-SMA) was 12.40% in the BLM+PD-1 group 14 days post-modeling, nearly twice the level in the BLM group (5.87%). 28 days post-modeling, the positive expression rate of α-SMA remained at 7.72% in the BLM+PD-1 group, while it declined to 3.38% in the BLM group. ConclusionThe addition of a PD-1 inhibitor to bleomycin treatment accelerates the progression of pulmonary interstitial fibrosis and effectively recapitulates the pathological process of lung injury and fibrosis driven by aberrant immune activation during clinical immunotherapy. Accordingly, this study successfully establishes a dynamic CIP animal model recapitulating the progression from early inflammatory response to pulmonary fibrosis. This model addresses the critical shortage of animal models for this condition and offers an experimental platform for further mechanistic studies and the development of therapeutic strategies.

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 analyze the factors influencing the development of refeeding syndrome (RFS) in patients with sepsis and its impact on clinical prognosis. METHODS: A retrospective case-control study method was used to collect the clinical data of patients with sepsis admitted to the intensive care unit (ICU) of Qingdao Municipal Hospital from December 2018 to December 2023. The patients were divided into RFS and non-RFS groups according to whether RFS occurred, and the basic data, nutritional status and assessment scale, laboratory indicators, nutritional intake, medical history and prognosis were compared between the two groups. Binary multifactorial Logistic regression analysis was used to screen the influencing factors of the occurrence of RFS in patients with sepsis. RESULTS: A total of 544 patients with sepsis were finally enrolled, of whom 250 did not develop RFS and 294 developed RFS, with an incidence of 54.0%. Compared with the non-RFS group, the patients in the RFS group had lower body mass index (BMI), albumin, prealbumin, baseline electrolytes (serum phosphorus, serum potassium, and serum magnesium), creatinine-height index, and protein intake, and had higher nutritional risk screening 2002 (NRS2002) score, sequential organ failure assessment (SOFA) score, calorie intake, and the proportions of feedings during the 48 hours of ICU admission, history of diabetes and septic shock. Binary multifactorial Logistic regression analysis showed that BMI [odds ratio (OR) = 0.910, 95% confidence interval (95%CI) was 0.857-0.947, P < 0.001], SOFA score (OR = 1.166, 95%CI was 1.085-1.254, P < 0.001), albumin (OR = 0.946, 95%CI was 0.902-0.991, P = 0.019), baseline serum phosphorus (OR = 0.343, 95%CI was 0.171-0.689, P = 0.003), baseline serum potassium (OR = 0.531, 95%CI was 0.377-0.746, P < 0.001), creatinine-height index (OR = 0.891, 95%CI was 0.819-0.970, P = 0.008), caloric intake (OR = 1.108, 95%CI was 1.043-1.178, P = 0.001), protein intake (OR = 0.107, 95%CI was 0.044-0.260, P < 0.001), and feedings during the 48 hours of ICU admission (OR = 0.592, 95%CI was 0.359-0.977, P = 0.040) and septic shock (OR = 0.538, 95%CI was 0.300-0.963, P = 0.037) were independent influence factors on the occurrence of RFS in septic patients. Of the 544 patients, 267 died at 28 days, with a mortality of 49.1%. The 28-day mortality of patients in the RFS group was significantly higher than that in the non-RFS group [54.4% (160/294) vs. 42.8% (107/250); χ² = 7.302, P = 0.007]. 544 patients had a length of ICU stay of 20 (17, 24) days. The patients in the RFS group had a significantly longer length of ICU stay than that in the non-RFS group [days: 20 (17, 25) vs. 19 (17, 23); Z = -2.312, P = 0.021]. CONCLUSIONS The incidence of RFS in septic patients is high. Factors influencing the occurrence of RFS in septic patients include BMI, SOFA score, albumin, baseline serum phosphorus, baseline serum potassium, caloric intake, protein intake, feeding within 48 hours of ICU admission, and septic shock. RFS prolongs the length of ICU stay and increases the 28-day mortality in patients with sepsis.
Humans ; Retrospective Studies ; Sepsis/complications* ; Prognosis ; Refeeding Syndrome/etiology* ; Case-Control Studies ; Intensive Care Units ; Male ; Nutritional Status ; Female ; Risk Factors ; Middle Aged ; Logistic Models ; Body Mass Index ; Aged

OBJECTIVE: To explore the effects and mechanisms of the C-X-C motif chemokine ligand 12/C-X-C motif chemokine receptor 4 (CXCL12/CXCR4) signaling axis on apoptosis and autophagy in SH-SY5Y neuronal cells subjected to oxygen-glucose deprivation/reperfusion (OGD/R) model in vitro. METHODS: SH-SY5Y cells were divided into the following groups: OGD/R group and non-OGD/R group, with the OGD/R group subjected to OGD/R modeling and the non-OGD/R group receiving no treatment. Cells were also divided into CXCL12⁺ and CXCL12^- groups; the CXCL12⁺ group received 0.1 mg/L exogenous recombinant CXCL12 (rhCXCL12) at reoxygenation, while the CXCL12^- group did not. Another set of cells was divided into CXCL12+AMD3100 and CXCL12 groups; the CXCL12+AMD3100 group was pretreated with 2.5 mg/L AMD3100, a CXCR4 inhibitor, for 2 hours before OGD/R and received both 2.5 mg/L AMD3100 and 0.1 mg/L rhCXCL12 at reoxygenation, whereas the CXCL12 group received rhCXCL12 only. Additionally, cells were divided into small interfering RNA CXCR4 (siCXCR4) and small interfering RNA negative control (siNC) groups; the siCXCR4 group underwent CXCR4 knockdown before OGD/R modeling and received 0.1 mg/L rhCXCL12 at reoxygenation, while the siNC group, transfected with a negative control, received the same treatment. Protein expression of autophagy-related 16 (ATG16), microtubule-associated protein 1 light chain 3 (LC3), aquaporin-3 (AQP3), and CXCR4 was detected by Western blotting. Apoptosis rate and CXCR4 expression were measured by flow cytometry. RESULTS: Compared with the non-OGD/R group, the OGD/R group showed a significantly increased apoptosis rate and markedly decreased protein expression levels of ATG16, LC3, AQP3, and CXCR4 (all P < 0.05). CXCR4 fluorescent expression was also significantly reduced, suggesting that OGD/R simultaneously affects neuronal apoptosis and autophagy while inhibiting CXCR4 and AQP3 expression in SH-SY5Y cells. Compared with the CXCL12^- group, the CXCL12⁺ group exhibited no significant change in apoptosis rate but demonstrated significantly increased protein expression of ATG16, LC3, and AQP3 (ATG16/GAPDH: 1.21±0.10 vs. 1.00±0.00; LC3/β-actin: 1.22±0.10 vs. 1.00±0.00; AQP3/β-actin: 1.26±0.04 vs. 1.00±0.00; all P < 0.05). CXCR4 expression was also significantly enhanced (fluorescence intensity: 1.19±0.05 vs. 1.00±0.00, P < 0.05), indicating that CXCL12 may promote autophagy in OGD/R-injured SH-SY5Y cells via the CXCR4/AQP3 pathway. Compared with the CXCL12 group, the CXCL12+AMD3100 group showed no significant difference in apoptosis rate but significantly lower protein levels of ATG16 and LC3 (ATG16/GAPDH: 0.75±0.08 vs. 1.00±0.00; LC3/GAPDH: 0.86±0.07 vs. 1.00±0.00; both P < 0.05), suggesting that CXCL12 induces autophagy in OGD/R SH-SY5Y cells through CXCR4. Compared with the siNC group, the siCXCR4 group showed no significant change in apoptosis rate but significantly reduced protein expression of ATG16, LC3, AQP3, and CXCR4 (ATG16/GAPDH: 0.76±0.06 vs. 1.00±0.00; LC3/GAPDH: 0.79±0.11 vs. 1.00±0.00; AQP3/GAPDH: 0.81±0.05 vs. 1.00±0.00; CXCR4/GAPDH: 0.86±0.04 vs. 1.00±0.00; all P < 0.05), indicating that CXCR4 knockdown suppresses OGD/R-induced autophagy in SH-SY5Y cells likely via AQP3. CONCLUSIONS The CXCL12/CXCR4 signaling axis can regulate OGD/R-induced autophagy in SH-SY5Y cells through AQP3 without affecting apoptosis, indicating a role for this pathway in neuronal autophagy during cerebral ischemia/reperfusion injury.
Humans ; Receptors, CXCR4/metabolism* ; Chemokine CXCL12/metabolism* ; Autophagy ; Glucose/metabolism* ; Apoptosis ; Neurons/cytology* ; Oxygen/metabolism* ; Signal Transduction ; Cell Line, Tumor ; Cell Hypoxia ; Benzylamines ; Cyclams

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 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．