OBJECTIVES: To explore the value of ferroptose-related genes in the diagnosis and prediction of ulcerative colitis (UC). METHODS: We used UC dataset from the GEO database to screen for differentially expressed genes (DEGs) in UC. The DEGs related to ferroptositis were screened from the FerrDb database and their functions were analyzed. The hub genes were identified by constructing the protein-protein interaction network (PPI), the differences in immune infiltration levels between UC and the control group were evaluated using CIBERSORT, and the diagnostic values of the hub genes for UC were verified by using the training set. In a mouse model of UC, we examined the expression levels of the hub genes in the colon tissues of the mice using real-time fluorescence quantitative PCR (qPCR). RESULTS: We identified a total of 76 DEGs related to ferroptosis. Functional enrichment analysis showed that these genes were significantly enriched in ferroptosis and hypoxia pathways. The PPI network identified 10 hub genes, and 9 of them were highly expressed in UC. Analysis of immune cell infiltration showed that 27 cell types were significantly increased in UC (P<0.05), and the immune checkpoints-related genes had the strongest correlation with the hub gene PPARG (P<0.05). Verification analysis using the training set showed that P4HB, PPARG and STAT3 had the best predictive value for UC (P<0.05). In the UC mouse model, the expression of PPARG was significantly decreased and the expressions of P4HB and STAT3 were significantly increased in the colon tissues of the mice as compared with the normal mice. CONCLUSIONS Ferroptose-related genes have significant value for diagnosis and prediction of UC.
Colitis, Ulcerative/genetics* ; Animals ; Mice ; Ferroptosis/genetics* ; Humans ; Protein Interaction Maps ; Disease Models, Animal ; Gene Expression Profiling ; STAT3 Transcription Factor/genetics*

Objective To study the role and influence of basic Alkaline ceramidase 1 on mucosal barrier and immune regulation in ulcerative colitis(UC).Methods Acer1 knockout mice(Acer1 KO)were constructed,and the UC model was induced by continuous drinking water of sodium dextran sulfate(DSS)for 7 days,and the severity of symptoms of UC disease in C57 and Acer1 KO mice was compared.The expression levels of intestinal mucosal barriers(ZO-1,Occludin,Claudin,JAMA)were detected by RT-PCR and immunohistochemistry.Systemic immune response levels(IL-1b、IL-6、IL-23、IL-17、IL-10,and TNF-a)were assessed by ELISA and intestinal inflammatory infiltration levels(TNF-a、IL-1b、IL-6、IL-17、IL-21,etc.)were assessed by RT-PCR.Results There was no significant difference between C57 and Acer1 KO mice in free drinking water.In the DSS induced UC model,compared with C57 mice,the survival rate of Acer1 KO mice decreased,the weight decreased significantly,the mechanical barrier and mucus barrier protein expression levels decreased significantly,the intes-tinal epithelial barrier was seriously damaged,the inflammatory response was strong,and the cytokine infiltration was obvious,with statistical differences(P<0.05).Conclusion Acer1 can inhibit inflammatory infiltration by maintaining the integrity of intestinal mucosal barrier,preventing endotoxin,and delaying the progression of UC.

Objective To study the role and influence of basic Alkaline ceramidase 1 on mucosal barrier and immune regulation in ulcerative colitis(UC).Methods Acer1 knockout mice(Acer1 KO)were constructed,and the UC model was induced by continuous drinking water of sodium dextran sulfate(DSS)for 7 days,and the severity of symptoms of UC disease in C57 and Acer1 KO mice was compared.The expression levels of intestinal mucosal barriers(ZO-1,Occludin,Claudin,JAMA)were detected by RT-PCR and immunohistochemistry.Systemic immune response levels(IL-1b、IL-6、IL-23、IL-17、IL-10,and TNF-a)were assessed by ELISA and intestinal inflammatory infiltration levels(TNF-a、IL-1b、IL-6、IL-17、IL-21,etc.)were assessed by RT-PCR.Results There was no significant difference between C57 and Acer1 KO mice in free drinking water.In the DSS induced UC model,compared with C57 mice,the survival rate of Acer1 KO mice decreased,the weight decreased significantly,the mechanical barrier and mucus barrier protein expression levels decreased significantly,the intes-tinal epithelial barrier was seriously damaged,the inflammatory response was strong,and the cytokine infiltration was obvious,with statistical differences(P<0.05).Conclusion Acer1 can inhibit inflammatory infiltration by maintaining the integrity of intestinal mucosal barrier,preventing endotoxin,and delaying the progression of UC.

Objective To assess the effect of platycodin D(PD)for alleviating pulmonary fibrosis in mice and explore the underlying mechanism.Methods C57BL/6J mouse models of pulmonary fibrosis induced by bleomycin injection into the airway were treated with daily intragastric administration of 10 mg/kg PD for 28 days.The changes of pulmonary fibrosis and the expression and distribution of transient receptor potential cation channel subfamily C member 6(TRPC6)were evaluated with immunohistochemistry,HE staining and Sirius Red staining.Western blotting was used to detect α-SMA expression in the lung tissues of the mice.Primary cultures of mouse lung fibroblasts were pretreated with PD(2.5,5.0,and 10 μmol/L)or larixyl acetate(LA;10 μmol/L)before exposure to 10 ng/mL transforming growth factor-β1(TGF-β1),and the changes in cell survival rate,expressions of collagen I,α-SMA and TRPC6,reactive oxygen species(ROS)production,mitochondrial membrane potential,and cell proliferation capacity were assessed.Network pharmacology analysis was performed to explore the mechanism by which PD alleviated pulmonary fibrosis.Results PD treatment significantly alleviated pulmonary fibrosis and reduced α-SMA expression in BLM-induced mouse models(P<0.05).In TGF-β1-induced primary mouse lung fibroblasts,PD effectively inhibited the cell proliferation,reduced ROS production(P<0.0001),rescued the reduction of mitochondrial membrane potential(P<0.001),and inhibited the expressions of α-SMA and collagenⅠ(P<0.05).Network pharmacology analysis suggested that TRPC6 mediated the effect of PD for alleviating pulmonary fibrosis.Immunohistochemistry showed that PD significantly reduced TRPC6 expression in the lung tissues of BLM-induced mice.In primary mouse lung fibroblasts,PD significantly inhibited TGF-β1-induced TRPC6 expression(P<0.05),and LA treatment obviously lowered the expression levels of TRPC6,α-SMA and collagenⅠ(P<0.05).Conclusion PD alleviated pulmonary fibrosis in mice possibly by down-regulating TRPC6 and reducing ROS production.

Objective:To explore the relationship between the expression of tumor necrosis factor receptor-associated factor 5 (TRAF5) and interferon regulatory factor 5 (IRF5) in intestinal mucosa of patients with ulcerative colitis and gut microbiota and intestinal mucosal barrier function.Methods:A total of 126 patients with ulcerative colitis in Qingdao Municipal Hospital from April 2021 to April 2023 were collected, according to the condition, there were 76 patients in the active phase and 50 patients in the remission phase, another 50 patients with intestinal polyps were taken as controls, intestinal mucosal tissues were collected from patients in three groups. Immunohistochemical staining was applied to detect the expression of TRAF5 and IRF5, the relationship between the expression of TRAF5 and IRF5 in intestinal mucosa of patients with ulcerative colitis and clinical symptoms, gut microbiota, and intestinal barrier function indicators was analyzed. Spearman method was applied for correlation analysis.Results:The positive expression rates of TRAF5 and IRF5 in intestinal mucosa of patients with active phase and remission phase ulcerative colitis were higher than those of patients with intestinal polyps: 85.53% (65/76) and 80.00% (40/50) vs. 40.00% (20/50), 81.58% (62/76) and 76.00% (38/50) vs. 50.00% (25/50), and the difference was statistically significant ( P<0.05); the expression of TRAF5 and IRF5 in intestinal mucosa of patients with ulcerative colitis was related to diarrhea, abdominal pain, purulent stool, tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), C-reactive protein (CRP), erythrocyte sedimentation rate, Baron endoscopic score, inflammatory bowel disease questionnaire score, Mayo index score and Geboes index ( P<0.05). Compared with patients with negative expression of TRAF5 and IRF5, patients with positive expression of TRAF5 and IRF5 had fewer bifidobacteria and lactobacilli, and more enterobacteria and enterococcus: (8.72 ± 0.43) cfu/g vs. (6.85 ± 0.47) cfu/g, (9.74 ± 0.31) cfu/g vs. (8.26 ± 0.27) cfu/g, (9.73 ± 0.46) cfu/g vs. (11.06 ± 0.48) cfu/g, (7.64 ± 0.31) cfu/g vs. (8.47 ± 0.34) cfu/g; (8.82 ± 0.44) cfu/g vs. (6.73 ± 0.47) cfu/g, (9.13 ± 0.30) cfu/g vs. (8.22 ± 0.27) cfu/g, (11.09 ± 0.48) cfu/g vs. (9.87 ± 0.46) cfu/g, (7.76 ± 0.32) cfu/g vs. (8.48 ± 0.34) cfu/g, and the difference was statistically significant ( P<0.05). The levels of diamine oxidase, lipopolysaccharide and D-lactic acid in patients with positive expression of TRAF5 and IRF5 were obviously higher than those in patients with negative expression of TRAF5 and IRF5: (12.18 ± 2.75) mg/L vs. (7.56 ± 2.49) mg/L, (76.14 ± 13.86) ng/L vs. (37.57 ± 12.51) ng/L, (18.15 ± 4.83) U/L vs. (9.87 ± 3.25) U/L; (12.39 ± 2.72) mg/L vs. (7.65 ± 2.66) mg/L, (77.21 ± 13.79) ng/L vs. (40.87 ± 13.04) ng/L, (18.36 ± 4.75) U/L vs. (10.67 ± 3.86)U/L, and the difference was statistically significant ( P<0.05). The expression of TRAF5 in the intestinal mucosa of patients with ulcerative colitis was negatively correlated with the numbers of bifidobacteria and lactobacilli ( r = - 0.645 and - 0.646; P<0.05), and positively correlated with the number of enterobacteria, number of enterococcus, diamine oxidase, lipopolysaccharide and D-lactic acid ( r = 0.629, 0.589, 0.509, 0.606 and 0.596; P<0.05). The expression of IRF5 was negatively correlated with the numbers of bifidobacteria and lactobacilli ( r = - 0.701 and - 0.690; P<0.05), and positively correlated with the number of enterobacteria, number of enterococcus, diamine oxidase, lipopolysaccharide and D-lactic acid ( r = 0.690, 0.624, 0.605, 0.595 and 0.568; P<0.05). Conclusions:The positive rates of TRAF5 and IRF5 in intestinal mucosa of patients with ulcerative colitis is high, which is closely related to the imbalance of gut microbiota and the damage of intestinal mucosal barrier function.

Objective To assess the effect of platycodin D(PD)for alleviating pulmonary fibrosis in mice and explore the underlying mechanism.Methods C57BL/6J mouse models of pulmonary fibrosis induced by bleomycin injection into the airway were treated with daily intragastric administration of 10 mg/kg PD for 28 days.The changes of pulmonary fibrosis and the expression and distribution of transient receptor potential cation channel subfamily C member 6(TRPC6)were evaluated with immunohistochemistry,HE staining and Sirius Red staining.Western blotting was used to detect α-SMA expression in the lung tissues of the mice.Primary cultures of mouse lung fibroblasts were pretreated with PD(2.5,5.0,and 10 μmol/L)or larixyl acetate(LA;10 μmol/L)before exposure to 10 ng/mL transforming growth factor-β1(TGF-β1),and the changes in cell survival rate,expressions of collagen I,α-SMA and TRPC6,reactive oxygen species(ROS)production,mitochondrial membrane potential,and cell proliferation capacity were assessed.Network pharmacology analysis was performed to explore the mechanism by which PD alleviated pulmonary fibrosis.Results PD treatment significantly alleviated pulmonary fibrosis and reduced α-SMA expression in BLM-induced mouse models(P<0.05).In TGF-β1-induced primary mouse lung fibroblasts,PD effectively inhibited the cell proliferation,reduced ROS production(P<0.0001),rescued the reduction of mitochondrial membrane potential(P<0.001),and inhibited the expressions of α-SMA and collagenⅠ(P<0.05).Network pharmacology analysis suggested that TRPC6 mediated the effect of PD for alleviating pulmonary fibrosis.Immunohistochemistry showed that PD significantly reduced TRPC6 expression in the lung tissues of BLM-induced mice.In primary mouse lung fibroblasts,PD significantly inhibited TGF-β1-induced TRPC6 expression(P<0.05),and LA treatment obviously lowered the expression levels of TRPC6,α-SMA and collagenⅠ(P<0.05).Conclusion PD alleviated pulmonary fibrosis in mice possibly by down-regulating TRPC6 and reducing ROS production.

Animals ; Humans ; Thymic Stromal Lymphopoietin ; Pyroglyphidae/metabolism* ; Cytokines/metabolism* ; Asthma/metabolism* ; Respiratory System ; Epithelial Cells/metabolism*

Objective:To investigate the effects of oleanolic acid on the growth and migration of keloid fibroblasts.Methods:Keloid tissue samples from 9 patients in the Department of Plastic Surgery of General Hospital of Northern Theater were collected and fibroblasts were cultured in vitro. Fibroblasts were treated with different concentrations of oleanolic acid and divided into three groups: control group added 0.9% NaCl; 5 μmol/L oleanolic acid group added 5 μmol/L oleanolic acid; 10 μmol/L oleanolic acid group added 10 μmol/L oleanolic acid. MTT assay was used to detect cell proliferation; flow cytometry was used to detect cell cycle. Annexin V propidium iodide (AV-PI) staining was used to detect cell apoptosis. Transwell assay was used to detect the migration of oleanolic acid. Western blotting and real-time PCR were used to detect the expression of related proteins and mRNA activity. Each group was made in triplicate. Analysis of variance was used to compare the data among the three groups. LSD- t test was used for pairwise comparison, and P<0.05 was considered to be statistically significant. Results:MTT result showed that oleanolic acid could inhibit the proliferation of cells. After 24 hours, the proliferation of cells in 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were 0.660±0.020 and 0.460±0.020, respectively, compared with 0.780±0.001 in the control group, F=114.4, P<0.001. Compared with the control group, the difference was statistically significant ( t=5.94, P<0.001, t=15.60, P<0.001); flow cytometry showed that the cell cycle G1/S phase transduction was blocked, 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were significantly inhibited. The percentage of G1 phase cells in the 5 μmol/L oleanolic acid group was significantly higher than that in the control group ( t=3.14, P=0.030, t=6.38, P< 0.001). AⅤ-PI staining showed that the number of apoptotic cells in the 5 μmol/L oleanolic acid group (0.9%) and 10 μmol/L oleanolic acid group (3.4%) was significantly higher than that in the control group (0.4%), and the difference among the three groups was F=119.6, P<0.001. Transwell assay showed that the migration number of cells in 5 μmol/L oleanolic acid group (57.13 ± 2.65) and 10 μmol/L oleanolic acid group (42.15 ± 2.55) was significantly lower than that in control group (72.27± 3.32), F=101.3, P<0.001. Compared with the control group, the difference was statistically significant ( t=6.50, P<0.001, t=14.41, P<0.001). Western blotting showed that oleanolic acid could inhibit the expression of Cyclin D1, Bcl-2, Bax and MMP2. Compared with the control group, 5 μmol/L oleanolic acid t=8.70, P<0.001, t=5.00, P=0.040, t=12.41, P<0.001, t=10.46, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=31.61, P<0.001, t=23.17, P<0.001, t=12.11, P<0.001, t=44.52, P<0.001. Real-time PCR reaction showed that the mRNA activity levels of Cyclin D1, Bcl-2, Bax, MMP2 were also inhibited. Compared with the control group, 5 μmol/L oleanolic acid t=5.42, P< 0.001, t=3.11, P=0.040, t=16.11, P<0.001, t=11.71, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=51.78, P<0.001, t=30.89, P<0.001, t=10.64, P<0.001, t=17.10, P< 0.001. Conclusions:Oleanolic acid (5 μmol/L and 10 μmol/L) can inhibit the proliferation and migration of keloid fibroblasts and induce apoptosis of keloid fibroblasts after treating keloid fibroblasts for 24 hours, which can inhibit the growth of keloid and be used for the prevention and treatment of keloid.

Objective:To investigate the effects of oleanolic acid on the growth and migration of keloid fibroblasts.Methods:Keloid tissue samples from 9 patients in the Department of Plastic Surgery of General Hospital of Northern Theater were collected and fibroblasts were cultured in vitro. Fibroblasts were treated with different concentrations of oleanolic acid and divided into three groups: control group added 0.9% NaCl; 5 μmol/L oleanolic acid group added 5 μmol/L oleanolic acid; 10 μmol/L oleanolic acid group added 10 μmol/L oleanolic acid. MTT assay was used to detect cell proliferation; flow cytometry was used to detect cell cycle. Annexin V propidium iodide (AV-PI) staining was used to detect cell apoptosis. Transwell assay was used to detect the migration of oleanolic acid. Western blotting and real-time PCR were used to detect the expression of related proteins and mRNA activity. Each group was made in triplicate. Analysis of variance was used to compare the data among the three groups. LSD- t test was used for pairwise comparison, and P<0.05 was considered to be statistically significant. Results:MTT result showed that oleanolic acid could inhibit the proliferation of cells. After 24 hours, the proliferation of cells in 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were 0.660±0.020 and 0.460±0.020, respectively, compared with 0.780±0.001 in the control group, F=114.4, P<0.001. Compared with the control group, the difference was statistically significant ( t=5.94, P<0.001, t=15.60, P<0.001); flow cytometry showed that the cell cycle G1/S phase transduction was blocked, 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were significantly inhibited. The percentage of G1 phase cells in the 5 μmol/L oleanolic acid group was significantly higher than that in the control group ( t=3.14, P=0.030, t=6.38, P< 0.001). AⅤ-PI staining showed that the number of apoptotic cells in the 5 μmol/L oleanolic acid group (0.9%) and 10 μmol/L oleanolic acid group (3.4%) was significantly higher than that in the control group (0.4%), and the difference among the three groups was F=119.6, P<0.001. Transwell assay showed that the migration number of cells in 5 μmol/L oleanolic acid group (57.13 ± 2.65) and 10 μmol/L oleanolic acid group (42.15 ± 2.55) was significantly lower than that in control group (72.27± 3.32), F=101.3, P<0.001. Compared with the control group, the difference was statistically significant ( t=6.50, P<0.001, t=14.41, P<0.001). Western blotting showed that oleanolic acid could inhibit the expression of Cyclin D1, Bcl-2, Bax and MMP2. Compared with the control group, 5 μmol/L oleanolic acid t=8.70, P<0.001, t=5.00, P=0.040, t=12.41, P<0.001, t=10.46, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=31.61, P<0.001, t=23.17, P<0.001, t=12.11, P<0.001, t=44.52, P<0.001. Real-time PCR reaction showed that the mRNA activity levels of Cyclin D1, Bcl-2, Bax, MMP2 were also inhibited. Compared with the control group, 5 μmol/L oleanolic acid t=5.42, P< 0.001, t=3.11, P=0.040, t=16.11, P<0.001, t=11.71, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=51.78, P<0.001, t=30.89, P<0.001, t=10.64, P<0.001, t=17.10, P< 0.001. Conclusions:Oleanolic acid (5 μmol/L and 10 μmol/L) can inhibit the proliferation and migration of keloid fibroblasts and induce apoptosis of keloid fibroblasts after treating keloid fibroblasts for 24 hours, which can inhibit the growth of keloid and be used for the prevention and treatment of keloid.