AIM:To investigate the immunosuppressive effects of M2-like tumor-associated macrophages(M2-TAMs)on CD8+T cells within the tumor microenvironment of esophageal cancer.METHODS:Multiplex fluores-cence immunohistochemistry was used to analyze the distribution of immune cells in esophageal cancer tissues.An in vitro co-culture system was established,and flow cytometry along with Calcein-AM/PI staining was employed to assess the im-pact of M2-TAMs on CD8+T cell function.The GEPIA database was utilized to evaluate the prognostic significance of PDCD1 expression in esophageal cancer patients and to analyze the correlations between gene expressions.Immunohisto-chemistry(IHC)was performed to detect the expression of TGF-β1 in esophageal cancer tissues.Flow cytometry and en-zyme-linked immunosorbent assay(ELISA)were used to measure PD-1,IFN-γ and TNF-α expression in CD8+T cells fol-lowing treatment with a TGF-β1 inhibitor.RESULTS:Compared with early-stage(stage I)esophageal cancer patients,the patients with advanced disease(stages Ⅱ to Ⅳ)exhibited dynamic changes in the infiltration of CD4+T cells,CD8+T cells,Tregs,and M2-TAMs within tumor tissues,with significant correlations observed among these cell populations(P<0.05).The distribution of M2-TAMs and Tregs was positively correlated with poor prognosis(P<0.05),while that of CD8+T cells was negatively correlated(P<0.05).In contrast,CD4+T cell infiltration showed no significant association with clinical outcomes(P>0.05).Co-culture of CD8+T cells with M2-TAMs resulted in significant downregulation of CD107a,granzyme B,IFN-γ and TNF-α expression(P<0.01).Additionally,M2-TAM-treated CD8+T cells co-cultured with esophageal cancer cells led to reduced apoptosis of cancer cells.High expression of PDCD1 was significantly associated with poor prognosis(P<0.05),and significant correlations were observed between CD8A and PDCD1 expression,as well as between TGF-β1 and CD274 gene expression(P<0.01).TGF-β1 was also significantly associated with CD163+macro-phage infiltration and the progression of esophageal cancer(P<0.05).Treatment with a TGF-β1 inhibitor in the M2-TAM and CD8+T cell co-culture system significantly down-regulated PD-1 expression and increased the secretion of IFN-γ and TNF-α(P<0.01).CONCLUSION:The TGF-β1 derived from M2-TAMs inhibits the antitumor activity of CD8+T cells in the esophageal cancer microenvironment,suggesting potential therapeutic targets for overcoming immunosuppression in esophageal cancer.

Objective:To investigate the mechanisms of baicalin in treating septic acute liver injury through a combination of network pharmacology and animal experiments.Methods:Thirty male C57BL/6 mice (6 weeks old) were divided into five groups ( n=6): control group (normal saline), model group [lipopolysaccharide (LPS) 10 mg/kg, intraperitoneal injection], low-dose baicalin group (10 mg/kg), high-dose baicalin group (20 mg/kg), and baicalin-only group (20 mg/kg, without LPS). Baicalin was administered orally for 14 consecutive days prior to modeling. Mice were sacrificed 24 h after LPS injection. Alanine transaminase, aspartate transaminase liver tissue histopathology were measured; neutrophil infiltration was visualized using immunofluorescence; mRNA expression levels of interleukin (IL)-1β, IL-17, IL-6, and tumor necrosis factor (TNF)-α were detected by RT-qPCR; and the expression of Toll-like receptor 4 (TLR4) and phosphorylated nuclear factor (NF)-κB proteins were analyzed by Western blotting. Results:In the LPS model group, the ALT, AST, and histopathological injury score were (148.60±22.02) U/L, (81.58±11.59) U/L, and 8.50(7.75, 9.25), respectively. These indicators were significantly reduced in the high-dose baicalin group with (77.90±16.79) U/L, (49.92±14.89) U/L, and 1.00(1.00, 2.25) (all P<0.05). Compared with the LPS group, neutrophil infiltration in the liver of high-dose baicalin group was also significantly reduced [1.18%(0.98%, 1.22%) vs. 6.13%(5.41%, 8.69%), P<0.05]. RT-qPCR results showed that the relative mRNA expression levels of inflammatory cytokines IL-1β [(1.03±0.06) vs. (2.60±0.34)], IL-17 [(1.21±0.12) vs. (2.94 ± 0.39)], IL-6 [(1.37±0.26) vs. (2.73±0.18)], and TNF-α [(1.18±0.10) vs. (3.30±0.92)] were significantly decreased in the high-dose baicalin group compared with the LPS group (all P<0.05). Western blot analysis revealed that the relative protein expression levels of TLR4 [(1.25±0.13) vs. (1.73±0.06)] and phosphorylated NF-κB [(1.25±0.25) vs. (1.79±0.12)] were also significantly lower in the high-dose baicalin group (both P<0.05). Conclusion:Baicalin reduces liver injury in septic mice by downregula-ting the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-17, potentially through the inhibition of the TLR4/NF-κB signaling pathway.

Objective:To establish and evaluated a logistic regression model for predicting the acute biliary pancreatitis (ABP) based on liver-injury related indexes.Methods:Clinical data of 210 patients diagnosed with acute pancreatitis (AP) at the Affiliated Hospital of Jiangnan University from October 2020 to December 2022 were retrospectively analyzed, including 113 males and 97 females, with a median age of 52 years (range, 43 to 58). Among these, 88 were diagnosed with ABP and 122 with acute non-biliary pancreatitis (ANBP). Additionally, a test cohort was created using data from 101 AP patients diagnosed between January and December 2023, including 60 males and 41 females, with a median age of 53 years (range, 43 to 63). Based on the original dataset, univariate and multivariate logistic regression analyses were conducted to identify the factors influencing ABP. A prediction probability formula (Pre) was then established based on the multivariate results. The effectiveness of each indicator in predicting ABP was evaluated using the receiver operating characteristic (ROC) curve. The ROC curve analysis determined the optimal cutoff value of Pre, which was subsequently used to diagnose ABP and ANBP in the test cohort.Results:Multivariate logistic regression analysis showed the factors influencing ABP include direct bilirubin (DBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholinesterase (CHE), and fibrinogen (FIB). Based on the multivariate analysis results, the prediction probability formula (Pre) for ABP was established as follows: P=1/{1+ exp[-(4.807+ 0.134×DBIL-1.859×AST/ALT-0.0003×CHE-0.387×FIB)]}. ROC curve analysis revealed that the area under the curve (AUC) for Pre in predicting ABP was 0.858, with an optimal cutoff value of 0.56, at which the sensitivity was 69.3% and the specificity was 91.0%. Using the cutoff value of 0.56 for Pre, ABP was diagnosed when Pre≥0.56 and ANBP was diagnosed when Pre<0.56. This criterion was applied to diagnose patients in the test cohort, where the sensitivity and specificity of Pre for diagnosing ABP were 86.1% and 92.3%, respectively.Conclusion:The logistic regression model based on liver injury-related indicators is a valuable tool for clinically assessing the incidence of ABP.

Objective:To analyze the changes of serum metabolites in patients with acute pancreatitis (AP) by non-targeted metabolomics method.Methods:Serum samples and clinical data of 15 AP patients hospitalized in the Affiliated Hospital of Jiangnan University from August to September 2024 were collected and included in the AP group, including 9 males and 6 females, aged (55.4±15.3) years. The serum and clinical data of 25 patients with colon polyps in the same hospital during the same period of time were collected, including 15 males and 10 females, aged (61.2±11.5) years, and were included in the control group. Serum metabolomic detection was performed using the ultra-high performance liquid chromatography tandem Fourier transform mass spectrometer. The modeling method was orthogonal partial least square discriminant analysis, and principal component analysis was performed on the data matrix to screen the differential metabolites in serum of AP patients. The Kyoto Encyclopedia database of Genes and Genomes was used to annotate differential metabolites, and the pathway of differential metabolite enrichment was analyzed by software.Results:The principal component analysis showed that the contribution ratio of the first principal component was 15.1%, the proportion of the second principal component was 10.8%, and the total proportion of the two was 25.9%. In principal component analysis, two groups of samples can be clearly distinguished and show obvious clustering characteristics. According to the analysis of OPLS-DA model, there were significant differences in serum metabolic profiles between AP group and control group. There were 683 differentially expressed metabolites between the two groups, with 367 differentially expressed metabolites up-regulated compared with the control group and 316 differentially expressed metabolites down-regulated compared with the control group. It is mainly Phosphatidic Acid (Lte4/8: 0) (+ 218%), Omeprazole Sulphone (-38%), and 2-(Propylthio) Nicotinic Acid (2-propyl thionicotinic acid) (-58%), Gein (salicyricetin) (-47%) and so on. Pathway enrichment analysis showed that the differential metabolites in AP patients were mainly concentrated in citric acid cycle, arginine biosynthesis and glycerophospholipid metabolism pathways.Conclusion:Serum metabolites in AP patients change significantly, including citric acid cycle, arginine biosynthesis, glycerophospholipid metabolism.

Objective:To explore the effects of Levothyroxine Sodium (L-T4) treatment on coagulation function and adverse pregnancy outcomes in patients with hypothyroidism during pregnancy in early and mid-to-late pregnancy.Methods:A total of 120 patients with hypothyroidism during pregnancy who were treated by L-T4 in Linyi Central Hospital from Mar. 2021 to Mar. 2024 were included, and their clinical data were retrospectively analyzed. According to the gestational age of the patients at the time of treatment, 64 cases with a gestational age of <12 weeks were included in the early pregnancy group, and 56 cases with a gestational age of ≥12 weeks were included in the mid-to-late pregnancy group. The serum thyroid hormone indexes, coagulation function indexes, adverse pregnancy outcomes, and neuropsychological development of the offspring were compared between the two groups. Statistical analyses were conducted using independent/paired t-tests and chi-square tests.Results:After treatment, there were no significant differences in the thyroid hormone indexes and coagulation function indexes between the early pregnancy group and the middle and late pregnancy group ( P>0.05). The incidence of fetal growth restriction and gestational hypertension in the early pregnancy group was 10.94% (7/64) and 17.19% (11/64), and that in the middle and late pregnancy group was 26.79% (15/56) and 33.93% (19/56), respectively. The incidence in the early pregnancy group was significantly lower than that in the middle and late pregnancy group ( χ2=5.010, 4.464, P<0.05). The developmental quotient (DQ) of the offspring in the early pregnancy group at 6 months of age in various areas of neuropsychological development (social behavior, language, adaptability, fine motor skills and gross motor skills) was significantly higher than that in the middle and late pregnancy group. The DQ of the first trimester group was (97.82±7.25) points, (94.59±6.85) points, (95.87±8.02) points, (96.08±7.25) points, and (98.34±8.07) points. The DQ of the second trimester group was (92.54±7.06) points, (90.45±6.14) points, (91.01±7.51) points, (91.24±7.08) points, and (92.74±7.38) points. The DQ of the first trimester group was significantly higher than that of the second trimester group ( t=4.029, 3.466, 3.411, 3.688, 3.946, P<0.05) . Conclusion:L-T4 treatment in early or mid- to late-pregnancy patients with gestational hypothyroidism can effectively improve their thyroid hormone levels and coagulation function, but treatment in early pregnancy is beneficial to reduce the incidence of adverse pregnancy outcomes such as fetal growth restriction and alleviate the impact of the disease on the neuropsychological development of offspring.

AIM:To investigate the immunosuppressive effects of M2-like tumor-associated macrophages(M2-TAMs)on CD8+T cells within the tumor microenvironment of esophageal cancer.METHODS:Multiplex fluores-cence immunohistochemistry was used to analyze the distribution of immune cells in esophageal cancer tissues.An in vitro co-culture system was established,and flow cytometry along with Calcein-AM/PI staining was employed to assess the im-pact of M2-TAMs on CD8+T cell function.The GEPIA database was utilized to evaluate the prognostic significance of PDCD1 expression in esophageal cancer patients and to analyze the correlations between gene expressions.Immunohisto-chemistry(IHC)was performed to detect the expression of TGF-β1 in esophageal cancer tissues.Flow cytometry and en-zyme-linked immunosorbent assay(ELISA)were used to measure PD-1,IFN-γ and TNF-α expression in CD8+T cells fol-lowing treatment with a TGF-β1 inhibitor.RESULTS:Compared with early-stage(stage I)esophageal cancer patients,the patients with advanced disease(stages Ⅱ to Ⅳ)exhibited dynamic changes in the infiltration of CD4+T cells,CD8+T cells,Tregs,and M2-TAMs within tumor tissues,with significant correlations observed among these cell populations(P<0.05).The distribution of M2-TAMs and Tregs was positively correlated with poor prognosis(P<0.05),while that of CD8+T cells was negatively correlated(P<0.05).In contrast,CD4+T cell infiltration showed no significant association with clinical outcomes(P>0.05).Co-culture of CD8+T cells with M2-TAMs resulted in significant downregulation of CD107a,granzyme B,IFN-γ and TNF-α expression(P<0.01).Additionally,M2-TAM-treated CD8+T cells co-cultured with esophageal cancer cells led to reduced apoptosis of cancer cells.High expression of PDCD1 was significantly associated with poor prognosis(P<0.05),and significant correlations were observed between CD8A and PDCD1 expression,as well as between TGF-β1 and CD274 gene expression(P<0.01).TGF-β1 was also significantly associated with CD163+macro-phage infiltration and the progression of esophageal cancer(P<0.05).Treatment with a TGF-β1 inhibitor in the M2-TAM and CD8+T cell co-culture system significantly down-regulated PD-1 expression and increased the secretion of IFN-γ and TNF-α(P<0.01).CONCLUSION:The TGF-β1 derived from M2-TAMs inhibits the antitumor activity of CD8+T cells in the esophageal cancer microenvironment,suggesting potential therapeutic targets for overcoming immunosuppression in esophageal cancer.

Objective:To explore the effects of Levothyroxine Sodium (L-T4) treatment on coagulation function and adverse pregnancy outcomes in patients with hypothyroidism during pregnancy in early and mid-to-late pregnancy.Methods:A total of 120 patients with hypothyroidism during pregnancy who were treated by L-T4 in Linyi Central Hospital from Mar. 2021 to Mar. 2024 were included, and their clinical data were retrospectively analyzed. According to the gestational age of the patients at the time of treatment, 64 cases with a gestational age of <12 weeks were included in the early pregnancy group, and 56 cases with a gestational age of ≥12 weeks were included in the mid-to-late pregnancy group. The serum thyroid hormone indexes, coagulation function indexes, adverse pregnancy outcomes, and neuropsychological development of the offspring were compared between the two groups. Statistical analyses were conducted using independent/paired t-tests and chi-square tests.Results:After treatment, there were no significant differences in the thyroid hormone indexes and coagulation function indexes between the early pregnancy group and the middle and late pregnancy group ( P>0.05). The incidence of fetal growth restriction and gestational hypertension in the early pregnancy group was 10.94% (7/64) and 17.19% (11/64), and that in the middle and late pregnancy group was 26.79% (15/56) and 33.93% (19/56), respectively. The incidence in the early pregnancy group was significantly lower than that in the middle and late pregnancy group ( χ2=5.010, 4.464, P<0.05). The developmental quotient (DQ) of the offspring in the early pregnancy group at 6 months of age in various areas of neuropsychological development (social behavior, language, adaptability, fine motor skills and gross motor skills) was significantly higher than that in the middle and late pregnancy group. The DQ of the first trimester group was (97.82±7.25) points, (94.59±6.85) points, (95.87±8.02) points, (96.08±7.25) points, and (98.34±8.07) points. The DQ of the second trimester group was (92.54±7.06) points, (90.45±6.14) points, (91.01±7.51) points, (91.24±7.08) points, and (92.74±7.38) points. The DQ of the first trimester group was significantly higher than that of the second trimester group ( t=4.029, 3.466, 3.411, 3.688, 3.946, P<0.05) . Conclusion:L-T4 treatment in early or mid- to late-pregnancy patients with gestational hypothyroidism can effectively improve their thyroid hormone levels and coagulation function, but treatment in early pregnancy is beneficial to reduce the incidence of adverse pregnancy outcomes such as fetal growth restriction and alleviate the impact of the disease on the neuropsychological development of offspring.

Objective:To investigate the mechanisms of baicalin in treating septic acute liver injury through a combination of network pharmacology and animal experiments.Methods:Thirty male C57BL/6 mice (6 weeks old) were divided into five groups ( n=6): control group (normal saline), model group [lipopolysaccharide (LPS) 10 mg/kg, intraperitoneal injection], low-dose baicalin group (10 mg/kg), high-dose baicalin group (20 mg/kg), and baicalin-only group (20 mg/kg, without LPS). Baicalin was administered orally for 14 consecutive days prior to modeling. Mice were sacrificed 24 h after LPS injection. Alanine transaminase, aspartate transaminase liver tissue histopathology were measured; neutrophil infiltration was visualized using immunofluorescence; mRNA expression levels of interleukin (IL)-1β, IL-17, IL-6, and tumor necrosis factor (TNF)-α were detected by RT-qPCR; and the expression of Toll-like receptor 4 (TLR4) and phosphorylated nuclear factor (NF)-κB proteins were analyzed by Western blotting. Results:In the LPS model group, the ALT, AST, and histopathological injury score were (148.60±22.02) U/L, (81.58±11.59) U/L, and 8.50(7.75, 9.25), respectively. These indicators were significantly reduced in the high-dose baicalin group with (77.90±16.79) U/L, (49.92±14.89) U/L, and 1.00(1.00, 2.25) (all P<0.05). Compared with the LPS group, neutrophil infiltration in the liver of high-dose baicalin group was also significantly reduced [1.18%(0.98%, 1.22%) vs. 6.13%(5.41%, 8.69%), P<0.05]. RT-qPCR results showed that the relative mRNA expression levels of inflammatory cytokines IL-1β [(1.03±0.06) vs. (2.60±0.34)], IL-17 [(1.21±0.12) vs. (2.94 ± 0.39)], IL-6 [(1.37±0.26) vs. (2.73±0.18)], and TNF-α [(1.18±0.10) vs. (3.30±0.92)] were significantly decreased in the high-dose baicalin group compared with the LPS group (all P<0.05). Western blot analysis revealed that the relative protein expression levels of TLR4 [(1.25±0.13) vs. (1.73±0.06)] and phosphorylated NF-κB [(1.25±0.25) vs. (1.79±0.12)] were also significantly lower in the high-dose baicalin group (both P<0.05). Conclusion:Baicalin reduces liver injury in septic mice by downregula-ting the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-17, potentially through the inhibition of the TLR4/NF-κB signaling pathway.

Objective:To establish and evaluated a logistic regression model for predicting the acute biliary pancreatitis (ABP) based on liver-injury related indexes.Methods:Clinical data of 210 patients diagnosed with acute pancreatitis (AP) at the Affiliated Hospital of Jiangnan University from October 2020 to December 2022 were retrospectively analyzed, including 113 males and 97 females, with a median age of 52 years (range, 43 to 58). Among these, 88 were diagnosed with ABP and 122 with acute non-biliary pancreatitis (ANBP). Additionally, a test cohort was created using data from 101 AP patients diagnosed between January and December 2023, including 60 males and 41 females, with a median age of 53 years (range, 43 to 63). Based on the original dataset, univariate and multivariate logistic regression analyses were conducted to identify the factors influencing ABP. A prediction probability formula (Pre) was then established based on the multivariate results. The effectiveness of each indicator in predicting ABP was evaluated using the receiver operating characteristic (ROC) curve. The ROC curve analysis determined the optimal cutoff value of Pre, which was subsequently used to diagnose ABP and ANBP in the test cohort.Results:Multivariate logistic regression analysis showed the factors influencing ABP include direct bilirubin (DBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholinesterase (CHE), and fibrinogen (FIB). Based on the multivariate analysis results, the prediction probability formula (Pre) for ABP was established as follows: P=1/{1+ exp[-(4.807+ 0.134×DBIL-1.859×AST/ALT-0.0003×CHE-0.387×FIB)]}. ROC curve analysis revealed that the area under the curve (AUC) for Pre in predicting ABP was 0.858, with an optimal cutoff value of 0.56, at which the sensitivity was 69.3% and the specificity was 91.0%. Using the cutoff value of 0.56 for Pre, ABP was diagnosed when Pre≥0.56 and ANBP was diagnosed when Pre<0.56. This criterion was applied to diagnose patients in the test cohort, where the sensitivity and specificity of Pre for diagnosing ABP were 86.1% and 92.3%, respectively.Conclusion:The logistic regression model based on liver injury-related indicators is a valuable tool for clinically assessing the incidence of ABP.

Objective:To analyze the changes of serum metabolites in patients with acute pancreatitis (AP) by non-targeted metabolomics method.Methods:Serum samples and clinical data of 15 AP patients hospitalized in the Affiliated Hospital of Jiangnan University from August to September 2024 were collected and included in the AP group, including 9 males and 6 females, aged (55.4±15.3) years. The serum and clinical data of 25 patients with colon polyps in the same hospital during the same period of time were collected, including 15 males and 10 females, aged (61.2±11.5) years, and were included in the control group. Serum metabolomic detection was performed using the ultra-high performance liquid chromatography tandem Fourier transform mass spectrometer. The modeling method was orthogonal partial least square discriminant analysis, and principal component analysis was performed on the data matrix to screen the differential metabolites in serum of AP patients. The Kyoto Encyclopedia database of Genes and Genomes was used to annotate differential metabolites, and the pathway of differential metabolite enrichment was analyzed by software.Results:The principal component analysis showed that the contribution ratio of the first principal component was 15.1%, the proportion of the second principal component was 10.8%, and the total proportion of the two was 25.9%. In principal component analysis, two groups of samples can be clearly distinguished and show obvious clustering characteristics. According to the analysis of OPLS-DA model, there were significant differences in serum metabolic profiles between AP group and control group. There were 683 differentially expressed metabolites between the two groups, with 367 differentially expressed metabolites up-regulated compared with the control group and 316 differentially expressed metabolites down-regulated compared with the control group. It is mainly Phosphatidic Acid (Lte4/8: 0) (+ 218%), Omeprazole Sulphone (-38%), and 2-(Propylthio) Nicotinic Acid (2-propyl thionicotinic acid) (-58%), Gein (salicyricetin) (-47%) and so on. Pathway enrichment analysis showed that the differential metabolites in AP patients were mainly concentrated in citric acid cycle, arginine biosynthesis and glycerophospholipid metabolism pathways.Conclusion:Serum metabolites in AP patients change significantly, including citric acid cycle, arginine biosynthesis, glycerophospholipid metabolism.