ObjectiveTo investigate the regulatory mechanism of thioredoxin binding protein (TXNIP)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway in the occurrence and development of breast cancer.MethodsThe resected 15 cases of breast cancer tissues and their adjacent tissues in our hospital from September 2019 to June 2020 were selected, and the immunohistochemistry was used to detect the expression levels of TXNIP and NLRP3 in breast cancer and its adjacent tissues. Three kinds of breast cancer cell lines (MDA-MB231, MCF-7 and SKBR3) and normal breast epithelial cell line (HMEC) were collected. Western blot was used to detect the relative expression levels of TXNIP and NLRP3 in three kinds of breast cancer cell lines and HMEC cell line. MDA-MB231 cancer cells were divided into blank control group (normal culture without any treatment), TXNIP overexpression group (Ad-TXNIP group, transfected with adenovirus vector carrying TXNIP overexpression sequence), Ad-TXNIP negative control group (Ad-eGFP1 group, transfected of empty adenovirus vector without TXNIP overexpression sequence), NLRP3 overexpression group (Ad-NLRP3 group, transfected with adenovirus vector containing NLRP3 overexpression sequence), TXNIP and NLRP3 overexpression co-transfection group (Ad-TXNIP+Ad-NLRP3 group, co-transfection of adenovirus vector carrying TXNIP and NLRP3 overexpression sequence), TXNIP overexpression and Ad-NLRP3 negative control (Ad-eGFP2) co-transfection group (Ad-TXNIP+Ad-eGFP2 group,co-transfection of adenovirus vector carrying TXNIP overexpression sequence and empty adenovirus without NLRP3 overexpression sequence). After 24 hours of transfection and culture, CCK-8 method was used to detect the MDA-MB231 cells proliferation. Transwell chamber method was used to detect MDA-MB231 cells migration and invasion. Nude mice tumorigenicity test was used to detect the tumorigenicity of the MDA-MB231 cells in vivo. Western blot was used to detect the expressions of TXNIP, NLRP3, proliferation marker protein (Ki-67), caspase-1, vascular endothelial growth factor (VEGF), interleukin (IL)-1β, IL-18 and caspase-1 precursor protein (pro-caspase-1) in the MDA-MB231 cells.ResultsCompared with the adjacent tissues, the relative expression level of TXNIP decreased (P<0.05) and the relative expression level of NLRP3 increased (P<0.05) in breast cancer tissues. Compared with normal breast epithelial cell line (HMEC cell line), the relative expression levels of TXNIP in MDA-MB231, MCF-7 and SKBR3 breast cancer cell lines were decreased (P<0.05), and the relative expression levels of NLRP3 were increased (P<0.05). Compared with the blank control group, the relative expression levels of TXNIP, NLRP3, IL-1β, IL-18, pro-caspase-1 and caspase-1 were increased (P<0.05), the relative expression levels of Ki-67 and VEGF, the proliferation activity, invasion and migration ability of MDA-MB231 cells and tumor weight were decreased (P<0.05) in the Ad-TXNIP group and the Ad-NLRP3 group. Compared with the Ad-TXNIP group and the Ad-NLRP3 group, the relative expression levels of TXNIP, NLRP3, IL-1β, IL-18, pro-caspase-1 and caspase-1 were further increased (P<0.05), the relative expression levels of Ki-67 and VEGF, the proliferation activity, invasion and migration ability of MDA-MB231 cells and tumor weight were further decreased (P<0.05) in the Ad-TXNIP+Ad-NLRP3 group.ConclusionsIn breast cancer tissues and breast cancer cell lines, TXNIP is low expression and NLRP3 is high expression. They can interact with each other to promote pyroptosis and inhibit the proliferation, invasion and migration of breast cancer cells.
Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.
ObjectiveTo investigate the influence of heat shock protein A2 (HSPA2) on the biological behavior of pancreatic adenocarcinoma cells and its mechanism. MethodsThe expressions of HSPA2 were determined in the human pancreatic adenocarcinoma cell lines (PANC-1, BxPC-3, and AsPC-1) using the Western blot. Subsequently, the cells with the lowest and highest HSPA2 expressions among these three lines were selected for conducting overexpression and knockdown experiments targeting HSPA2, respectively. The cellular proliferation, cell clonogenesis, migration, and invasion capabilities were assessed using MTT, clonogenic assay, and Transwell assay, respectively. Additionally, the impact of HSPA2 on the expression of key markers of epithelial-mesenchymal transition (EMT) was examined using the Western blot. The potential target molecules of HSPA2 were identified through immunoprecipitation assay and mass spectrometry. The rescue experiments further explored the regulatory relation between the HSPA2 and its target molecules. The influence of HSPA2 on pancreatic adenocarcinoma growth was investigated through establishment of xenograft tumor model in nude mice. ResultsThe HSPA2 exhibited the lowest expression in the PANC-1 cells and the highest expression in the AsPC-1 cells among the three cell lines. Subsequent functional studies demonstrated that the overexpression of HSPA2 in the PANC-1 cells markedly promoted proliferation, cell clonogenesis, migration, and invasion, while the knockdown of HSPA2 expression in the AsPC-1 cells markedly inhibited these processes. The Western blot analysis further showed that the HSPA2 overexpression downregulated E-cadherin expression and upregulated N-cadherin and Vimentin expressiones, whereas the HSPA2 knockdown produced opposite effects. The rescue experiments indicated that the HSPA2 promoted the EMT in pancreatic adenocarcinoma cells by upregulating Yes associated protein (YAP). The subcutaneous xenograft tumor experiments in the nude mice showed that the HSPA2 knockdown inhibited tumor growth. ConclusionThe results of this study suggest that HSPA2 promotes EMT via upregulating YAP, which facilitates proliferation, migration, and invasion of pancreatic adenocarcinoma cells.
Objective To investigate the effects of NGF on the prol iferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts (HDFs), and to explore the function of NGF on the wound heal ing. Methods The 3rd generation of HDFs were incubated with various concentrations of NGF (0, 25, 50, 100, 200 and 400 ng/mL), the cell prol iferation was measured with MTT assay. After treated with NGF at 0, 100 ng/mL, the cell cycle of HDFs was determined by flow cytometry (FCM). Hydroxyprol ine and real-time fluorescence quantitative PCR (FQ-PCR) were used to measure collagen synthesis at protein level and mRNA level respectively. The in vitro cell scratch wound model was set up to observe the effect of NGF (0, 50, 100 and 200 ng/mL) on the migration of HDFs after 24 hours of culture. Results Absorbance value of HDFs for different concentrations of NGF (0, 25, 50, 100, 200, and 400 ng/ mL) showed that NGF did not influence the prol iferation of HDFs (P gt; 0.05). When HDFs were treated with NGF at 0 and 100 ng/mL, the result of FCM analysis showed that percentage of HDFs in G0/G1, S, G2/M phases were not changed (P gt; 0.05). Compared with control group, the expression of Col I and Col III were not significantly different, measured by both hydroxyprol ine and FQ-PCR (P gt; 0.05). The rates of HDFs’ migration at various concentrations of NGF (0, 50, 100, 200 ng/ mL) were 52.12% ± 6.50%, 80.67% ± 8.51%, 66.33% ± 3.58%, and 61.19% ± 0.97%, respectively, indicating that NGF could significantly enhanced the migration of HDFs at 50 and 100 ng/mL (P lt; 0.05). Conclusion NGF does not influence prol iferation, mitotic cycle and collagen synthesis of HDFs, but significantly enhanced migration in an in vitro model of wounded fibroblasts.
ObjectiveTo observe the effects of aquaporin 1 (AQP1) on the proliferation and migration of endothelial progenitor-endothelial progenitor cells (EPC).MethodsBone marrow cells of AQP1 wild-type (WT) (n=6) and knockout-type (KO) mice (n=6) were isolated and differentiated into EPC in vitro. Immunofluorescence was used to detect cell surface antigens to identify EPC. Live cell kinetic imaging and quantification technology, transwell migration assays, as well as scratch test were used to compare the function of EPC between AQP1 WT and KO mice.ResultsEPC culture showed that cells were initially suspended and gradually adhered to typical mesenchymal stem cells within 7 days. After cultured on special medium for endothelial cells they were adhered and differentiated, and fusiform or polygonal, paving stone-like EPC were observed around 14 days. When cultured by special medium of EPC, CD133 and CD31 were positively detected after 7 days, and CD34 and Flk-1 were positively detected after 14 days. Positive expression of AQP1 was only detected in EPC of AQP1 WT mice. Functional studies of EPC revealed there was no significant difference in the proliferation of EPC between AQP1 WT and KO group mice. Transwell assay showed that EPC migration ability of AQP1 KO mice was significantly weaker than that of WT mice. The scratch healing ability of EPC in AQP1 KO mice was significantly lower than that of WT mice.ConclusionsEPC initially shows the characteristics of stem cells and with the prolongation of culture time, EPC gradually shows the characteristics of endothelial cells. AQP1 affects the EPC migration rather than proliferation.
Objective To explore the effects of DNA cross-linking repair 1B (DCLRE1B) gene on the migration and invasion ability of hepatocellular carcinoma cell. Methods Bioinformatics analysis was used to analyze the expression of DCLRE1B mRNA in hepatocellular carcinoma, and its relationship with the prognosis and related influencing factors of patients. Immunohistochemical staining was used to detect the expression of DCLRE1B protein in resected hepatocellular carcinoma tissues and their corresponding normal liver tissues. The DCLRE1B gene silenced Huh7 and HepG2 hepatocellular carcinoma cell lines were constructed by lentivirus, and the transfected effect was detected by Western blot. The migration and invasion of DCLRE1B silenced hepatocellular carcinoma cells were detected by scratch test and Transwell method. The changes of genes related to epithelial mesenchymal transformation (EMT) after DCLRE1B silencing were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results ① The biological information analysis results showed that: The mRNA expression of DCLRE1B was highly expressed in a variety of tumors including hepatocellular carcinoma (P<0.05). The mRNA expression of DCLRE1B was associated with the TNM staging of tumor (P<0.05). The relative expression level of DCLRE1B mRNA in hepatocellular carcinoma patients was related to their prognosis. The overall survival situation (P=0.038) and progression free survival situation (P=0.005) of hepatocellular carcinoma patients in the high expression group were worse than those in the low expression group. Univariate and multivariate Cox analysis showed that the expression of DCLRE1B gene was an independent factor affecting the prognosis of hepatocellular carcinoma (P<0.05). ② The positive rate of DCLRE1B protein expression in resected hepatocellular carcinoma tissues was higher than that in normal liver tissues (P<0.05). ③ Cell experiment results showed that: After stable silencing DCLRE1B gene of hepatocellular carcinoma cell (Huh7 and HepG2) constructed by lentivirus, the expression of DCLRE1B protein was significantly down regulated (P<0.05). After silencing DCLRE1B gene, the migration and invasion ability of hepatocellular carcinoma cells were significantly decreased (P<0.05). After silencing DCLRE1B, the mRNA expressions of E-cadherin, matrix metalloproteinase 9, and β-catenin were up regulated (P<0.05), and the mRNA expressions of N-cadherin and Vimentin were down regulated (P<0.05), but the mRNA expression of zinc finger transcription factor had no significant change, and the difference was not statistically significant (P＞0.05). Conclusion Silencing DCLRE1B gene can inhibit the migration and invasion ability of hepatocellular carcinoma cells, and its mechanism may be related to the process of EMT.
ObjectiveTo investigate the prognostic value of high mobility group protein 1 (HMGB1) in patients with ventilator-associated pneumonia (VAP). MethodsA total 118 VAP patients admitted between March 2013 and March 2015 were recruited in the study. The patients were divided into a death group and a survival group according to 28-day death. Baseline data, HMGB1, C-reactive protein (CRP), clinical pulmonary infection score (CPIS), acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) and sepsis-related organ failure assessment (SOFA) scores were collected on 1st day (d1), 4th day (d4), and 7th day (d7) after VAP diagnosis. The possible prognostic factors were analyzed by univariate and logistic multivariate analysis. ResultsThere were 87 cases in the survival group and 31 cases in the death group. Age, female proportion, body mass index, HMGB1 (d1, d4, d7), APACHEⅡ (d1, d4, d7) and SOFA (d1, d4, d7) scores were all higher in the death group than those in the survival group (all P < 0.05). HMGB1 (d4, P=0.031), APACHEⅡ (d4, P=0.018), SOFA (d4, P=0.048), HMGB1(d7, P=0.087), APACHEⅡ(d7, P=0.073) and SOFA (d7, P=0.049) were closely correlated with 28-day mortality caused by VAP. Multivariate analysis revealed that HMGB1 (d4, HR=1.43, 95%CI 1.07 to 1.78, P=0.021), SOFA (d4, HR=1.15, 95%CI 1.06 to 1.21, P=0.019) and HMGB1 (d7, HR=1.27, 95%CI 1.18 to 1.40, P=0.003) were independent predictors of death in the VAP patients. ROC curve revealed HMGB1 (d4, d7) and SOFA (d4) with area under ROC curve of 0.951, 0.867 and 0.699. ConclusionIndividual HMGB1 level can be used as a good predictor of the short-outcomes of VAP.
Objective To observe the protective effects of unfractionated heparin (UFH) on high-mobility group box-1 protein (HMGB1) induced increased permeability of endothelial cells, and investigate the protective mechanism of UFH on HMGB1 induced defective expression of zonula occludens-1 (ZO-1). Methods Human umbilical vascular endothelial cells (HUVECs) were culturedin vitro and divided into 4 groups (n=5), namely a control group, a HMGB1 group (100 ng/ml), a heparin group (UFH 10 U/ml), a HMGB1/heparin group (100 ng/ml HMGB1 + UFH 10 U/ml). Endothelial cell viability was measured by methyl thiazolyl tetrazolium (MTT) colorimetric method. Endothelial permeability was determination by Transwell chamber method. Immunofluorescence and laser confocal microscopy were used to assess the distribution of ZO-1. The protein expressions of tight junction protein ZO-1 and nuclear factor kappa B (NF-κB) were detected by Western blot. Results HMGB1 (100 ng/ml) had no inhibitory effect on endothelial cell viability (P＞0.05). UFH pretreatment could reduce the permeability increment of endothelial cells induced by HMGB1. UFH pretreatment could reduce the close loop reduction and damage of ZO-1 induced by HMGB1, enhance the fluorescence intensity and expression of ZO-1, and decrease the NF-κB translocation. Conclusions UFH can protect HMGB1-mediated defect of ZO-1 expression and increased permeability of the endothelial cells. The mechanism may be related to the decreased nuclear translocation of NF-κB.
Objective To study the migration of Schwann cells from the nerve autograft in the acellular nerve allograft of the rats in vivo. Mehtods The sciatic nerves (20 mm long) of the SD rats were harvested and prepared for the acellular nerve grafts by the chemical extraction. Then, they were observed by the gross view, HE staining, and Antilamininstaining, respectively. Another 32 female SD rats weighing 250-300 g were obtained for the study. A 2-mm-long nerve autograft was interposed between the two 10-mm-long nerve allografts to form a 22-mm-long composite. Then, the composite was placed in the muscle space, together with a sole 22-mm-long nerve allograftas a control. They were harvested at 5，10，15 and 20 days, respectively, and were then given the HE staining and the S-100 staining. Results The acellular nerve graft was semitransparent under the gross view. HE staining showed that no cell was observed within the nerve graft. Anti-laminin staining showed that the basal membrane was partially interrupted, with a positive result (dark brown). All the nerve grafts in both the groups exhibited the existenceof the cells. The S-100 positive cells were observed from the 15th day at the far ends of the two allografts of the composite; however, there were no suchcells observed within the sole nerve allograft. Conclusion Schwann cells from the sciatic nerves (2 mm- long) of the rats can migrate in the acellular nerve allograft to the far ends of the neighboring 10-mm-long nerve allografts at 15 days after operation, which offers the theoretical basis forthe repair of the longrange nerve defect by the composite of the acellular nerve allografts with the interposed nerve autograft.
Objective To investigate the effect of resveratrol (RES) on inflammation-induced cartilage endplate (CEP) degeneration, and its regulatory mechanism on high mobility group box-1 protein (HMGB1) signaling pathway. Methods The intervertebral CEP cells of Sprague Dawley (SD) rats aged 3 weeks were extracted and identified by toluidine blue staining and immunofluorescence staining of rabbit anti-rat collagen type Ⅱ. The cell counting kit 8 (CCK-8) method was used to screen the optimal concentration of RES on intervertebral CEP cells. Gene chip analysis was used to determine the target of RES on intervertebral CEP cells. Interleukin 1β (IL-1β) was used to construct the intervertebral CEP cell degeneration model caused by inflammation and the 7-8-week-old SD rat intervertebral disc degeneration model, and pcDNA3.1-HMGB1 (pcDNA3.1) was used as the control of RES effect. Flow cytometry and TUNEL staining were used to detect the apoptotic rate of intervertebral CEP cells and rat intervertebral disc tissue cells, respectively. ELISA kit was used to detect the content of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in the cell supernatant and rat serum. Western blot was used to detect the expressions of HMGB1, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (p-ERK), B cell lymphoma/leukemia 2 gene (Bcl-2), and Bcl-2-associated X protein (Bax). ResultsThe extracted cells were identified as rat intervertebral CEP cells. CCK-8 method screened out the highest activity of intervertebral CEP cells treated with 30 μmol/L RES. The gene chip analysis confirmed that the HMGB1-ERK signal was the target of RES. Both cell experiments and animal experiments showed that RES treatment can significantly down-regulate the apoptosis rate of intervertebral CEP cells, inhibit the release of TNF-α, and increase the content of IL-10; and down-regulate the expressions of HMGB1, p-ERK, and Bax, and increase Bcl-2; and pcDNA3.1 could partially reverse these effects of RES, and the differences were all significant (P<0.05). ConclusionRES can significantly inhibit the apoptosis of intervertebral CEP cells induced by inflammation, which is related to inhibiting the expression of HMGB1.