Objective:To investigate the effects and possible mechanisms of caffeic acid phenethyl ester (CAPE) on the replication, amplification, and fibre formation of prions (PrP Sc). Methods:The CCK8 assay was used to detect the cell viability of the prion-infected cell model SMB-S15 after CAPE treatment for 3 days and 7 days and the maximum safe concentration of CAPE for SMB-S15 was obtained. The cells were treated with a concentration within a safe range, and the content of PrP Sc in the cells before and after CAPE treatment was analyzed by western blot. Protein misfolding cycle amplification (PMCA) and western blot were used to assess changes in PrP Sc level in amplification products following CAPE treatment. Real-time-quaking induced conversion assay (RT-QuIC) technology was employed to explore the changes in fibril formation before and after CAPE treatment. The binding affinity between CAPE and murine recombinant full-length prion protein was determined using a molecular interaction assay. Results:CCK8 cell viability assay results demonstrated that treatment with 1 μmol/L CAPE for 3 and 7 days did not exhibit statistically significant differences in cell viability compared to the control group (all P<0.05). However, when the concentration of CAPE exceeded 1 μmol/L, a significant reduction in cell viability was observed in cells treated with CAPE for 3 and 7 days, compared to the control group (all P<0.05). Thus, 1 μmol/L was determined as the maximum safe concentration of CAPE treatment for SMB-S15 cells. The western blot results revealed that treatment with CAPE for both 3 and 7 days led to a detectable reduction in the levels of PrP Sc in SMB-S15 cells (all P<0.05). The products of PMCA experiments were assessed using western blot. The findings revealed a significant decrease in the levels of PrP Sc (relative grey value) in the PMCA amplification products of adapted-strains SMB-S15, 139A, and ME7 following treatment with CAPE, as compared to the control group (all P<0.05). The RT-QuIC experimental results demonstrated a reduction in fibril formation (as indicated by ThT peak values) in CAPE-treated mouse-adapted strains 139A, ME7, and SMB-S15, as well as in SMB-S15 cells infected with prions. Furthermore, CAPE exhibited varying degrees of inhibition towards different seed fibrils formation, with statistically significant differences observed (all P<0.05). Notably, CAPE exhibited a more pronounced inhibitory effect on ME7 seed fibrils. Molecular interaction analyses demonstrated significant binding between CAPE and murine recombinant prion protein, and the association constant was (2.92±0.41)×10 -6 mol/L. Conclusions:CAPE inhibits PrP Sc replication, amplification, and fibril formation in vitro possibly due to specific interactions with the prion protein at the molecular level.

Objective:To investigate the effects and possible mechanisms of caffeic acid phenethyl ester (CAPE) on the replication, amplification, and fibre formation of prions (PrP Sc). Methods:The CCK8 assay was used to detect the cell viability of the prion-infected cell model SMB-S15 after CAPE treatment for 3 days and 7 days and the maximum safe concentration of CAPE for SMB-S15 was obtained. The cells were treated with a concentration within a safe range, and the content of PrP Sc in the cells before and after CAPE treatment was analyzed by western blot. Protein misfolding cycle amplification (PMCA) and western blot were used to assess changes in PrP Sc level in amplification products following CAPE treatment. Real-time-quaking induced conversion assay (RT-QuIC) technology was employed to explore the changes in fibril formation before and after CAPE treatment. The binding affinity between CAPE and murine recombinant full-length prion protein was determined using a molecular interaction assay. Results:CCK8 cell viability assay results demonstrated that treatment with 1 μmol/L CAPE for 3 and 7 days did not exhibit statistically significant differences in cell viability compared to the control group (all P<0.05). However, when the concentration of CAPE exceeded 1 μmol/L, a significant reduction in cell viability was observed in cells treated with CAPE for 3 and 7 days, compared to the control group (all P<0.05). Thus, 1 μmol/L was determined as the maximum safe concentration of CAPE treatment for SMB-S15 cells. The western blot results revealed that treatment with CAPE for both 3 and 7 days led to a detectable reduction in the levels of PrP Sc in SMB-S15 cells (all P<0.05). The products of PMCA experiments were assessed using western blot. The findings revealed a significant decrease in the levels of PrP Sc (relative grey value) in the PMCA amplification products of adapted-strains SMB-S15, 139A, and ME7 following treatment with CAPE, as compared to the control group (all P<0.05). The RT-QuIC experimental results demonstrated a reduction in fibril formation (as indicated by ThT peak values) in CAPE-treated mouse-adapted strains 139A, ME7, and SMB-S15, as well as in SMB-S15 cells infected with prions. Furthermore, CAPE exhibited varying degrees of inhibition towards different seed fibrils formation, with statistically significant differences observed (all P<0.05). Notably, CAPE exhibited a more pronounced inhibitory effect on ME7 seed fibrils. Molecular interaction analyses demonstrated significant binding between CAPE and murine recombinant prion protein, and the association constant was (2.92±0.41)×10 -6 mol/L. Conclusions:CAPE inhibits PrP Sc replication, amplification, and fibril formation in vitro possibly due to specific interactions with the prion protein at the molecular level.

OBJECTIVE: To analyze the relationship between Chemokine IP10 and its receptor CXCR3 during prion infection. METHODS: We investigated the increases in IP10 signals, primarily localized in neurons within the brains of scrapie-infected mice, using western blotting, ELISA, co-immunoprecipitation, immunohistochemistry, immunofluorescence assays, and RT-PCR. RESULTS: Both CXCR3 levels and activation were significantly higher in the brains of scrapie-infected mice and prion-infected SMB-S15 cells. Enhanced CXCR3 expression was predominantly observed in neurons and activated microglia. Morphological colocalization of PrP ^C/PrP ^Sc with IP10/CXCR3 was observed in scrapie-infected mouse brains using immunohistochemistry and immunofluorescence. immunohistochemistry (IHC) analysis of whole brain sections further revealed increased accumulation of IP10/CXCR3 specifically in brain regions with higher levels of PrP ^Sc deposits. Co-immunoprecipitation and biomolecular interaction assays revealed the molecular interactions between PrP and IP10/CXCR3. Notably, a significantly larger amount of IP10 accumulated within prion-infected SMB-S15 cells than in the normal partner cell line, SMB-PS. Importantly, resveratrol treatment effectively suppressed prion replication in SMB-S15 cells, thereby restoring the accumulation and secretion pattern of cellular IP10 similar to that observed in SMB-PS cells. CONCLUSION Our data demonstrate that the activation of IP10/CXCR3 signaling in prion-infected brain tissues coincides with PrP ^Sc deposition. Modulation of IP10/CXCR3 signaling in the brain represents a potential therapeutic target for mitigating the progression of prion diseases.
Animals ; Receptors, CXCR3/metabolism* ; Mice ; Chemokine CXCL10/metabolism* ; Brain/metabolism* ; Scrapie/metabolism* ; Signal Transduction ; PrPSc Proteins/metabolism*

Objective:To analyze the expression of prion protein in papillary thyroid carcinoma and its relationship with related clinical factors.Methods:Specimens from 60 patients with papillary thyroid cancer (PTC) diagnosed by pathology and 60 patients with benign thyroid lesions (BTL) were collected, and the prion proteins in the samples were detected and analyzed by immunohistochemistry, Western blotting and immunofluorescence. Statistical analysis was performed on the relationship between protein expression and patient-related clinical factors.Results:Immunohistochemistry showed a higher rate of prion positivity in papillary thyroid cancer tissue than in benign thyroid lesions ( χ2=36.310, P<0.001). Immunoblotting and immunofluorescence method show that prion protein expression is significantly higher in papillary thyroid cancer tissue than in patients with benign thyroid lesions[Immunoblot: (1.288±0.153)U/L vs. (0.667±0.036)U/L, t=6.855, P<0.01; Immunofluorescence: [Green fluorescence, 20X: (12.911±1.947)U/L vs. (36.322±3.797)U/L, t=9.502, P<0.001; Red fluorescence, 20X: (6.259±2.062)U/L vs. (28.229±2.611)U/L, t=11.437, P<0.001]. The expression of prion protein was significantly higher in clinical grade III papillary thyroid cancer tissues than in stage I and II patients, and all were significantly different (Stage I over III: χ2=6.301, P<0.05; Stage II over III: χ2=5.683, P<0.05). There was no correlation with the presence or absence of lymphatic metastases and no statistically significant correlation with the age or sex of the patients. Conclusions:Prion protein expression was increased in papillary thyroid carcinoma and correlated with clinical stage of the tumor. This study provides experimental evidence for prion protein as a molecular marker for targeted diagnosis and treatment of papillary thyroid cancer.

Objective:To observe the expression of α-enolase in laryngeal carcinoma patients and its relationship with the infection with HPV16/18.Methods:The expression of α-enolase protein in 98 cases was detected by immunohistochemical method and the relationship between α-enolase and clinical stage was analyzed. The expression of HPV16/18 was detected by PCR, sequencing and immunohistochemistry, the relationship between HPV16/18 expression and α-enolase expression was analyzed.Results:Immunohistochemical result showed that the total positive rate of α-enolase expression was 89.8%, and the total positive rate of HPV gene and protein detection was 40.8%. The localization of α-enolase protein tended to shift from cytoplasm and nucleus expression to more cytoplasmic expression only.Conclusions:With the progression of clinical course and HPV16/18 infection, the expression of α-enolase protein showed a tendency of localization to the cytoplasm. Detection of α-enolase protein can provide laboratory data reference for early diagnosis and treatment of laryngeal carcinoma.

Objective:To observe the expression and clinical significance of galectin-3 and HPV16/18-E6 proteins in laryngeal carcinoma patients.Methods:Immunohistochemistry and Western blot method were used to observe the expressions of galectin-3 protein and HPV16/18-E6 protein in laryngeal carcinoma tissues of 60 patients. SAS 9.4 software was used to analyze the relationship between the galectin-3 protein and the clinical stage severity, pathological grades and lymph node metastasis in all the cases. Results:The result of immunohistochemical examinations showed that the positive rate of galectin-3 protein expression was 78.3% (47/60), and the Western blot result showed that the galectin-3 protein expression in tumor tissues was significantly higher than that in adjacent normal tissues. Statistical analysis showed that galectin-3 expression was correlated with the severity of clinical staging, differentiation of the tumor and lymph node metastasis of laryngeal cancer. There was no correlation between the expression of galectin-3 protein and the 16/18-E6 protein of human papillomavirus.Conclusions:The expression of galectin-3 protein showed a gradually increasing trend with the aggravation of the clinical severity of laryngeal carcinoma, providing certain experimental data for the early diagnosis and treatment of laryngeal cancer.

Objective: To observe the expression and clinical significance of galectin-1 in Chinese laryngeal squamous cell carcinoma patients. Methods: Immunohistochemical technique was used to test the galecin-1 protein expression in the 50 laryngeal squamous cell carcinoma patients and Western blot method was used to test the galecin-1 protein expression in the 3 of the patients. SAS 9.4 software was used to analyze the relationship between the galectin-1 protein and the clinical degrees, pathological grades and lymph node metastasis in all the cases. Results: In the 50 laryngeal squamous cell carcinoma patients, the positive rate of galectin-1 protein was 74% (37/50). The expression of galectin-1 protein was correlated with the severity of clinical staging, pathological differentiation and lymph node metastasis. Conclusions High expression of galectin protein related with the occurrence and development of head and neck squamous cell carcinomas.