Analysis of mRNA-miRNA interaction network reveals the role of CAFs-derived exosomes in the immune regulation of oral squamous cell carcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) have significant tumor regulatory functions, and CAFs-derived exosomes (CAFs-Exo) released from CAFs play an important role in the progression of oral squamous cell carcinoma (OSCC). However, a lack of comprehensive molecular biological analysis leaves the regulatory mechanisms of CAFs-Exo in OSCC unclear. METHODS: We used platelet derived growth factor-BB (PDGF-BB) to induce the transformation of human oral mucosa fibroblast (hOMF) into CAFs, and extracted exosomes from the supernatant of CAFs and hOMF. We validated the effect of CAFs-Exo on tumor progression by exosomes co-culture with Cal-27 and tumor-forming in nude mice. The cellular and exosomal transcriptomes were sequenced, and immune regulatory genes were screened and validated using mRNA-miRNA interaction network analysis in combination with publicly available databases. RESULTS: The results showed that CAFs-Exo had a stronger ability to promote OSCC proliferation and was associated with immunosuppression. We discovered that the presence of immune-related genes in CAFs-Exo may regulate the expression of PIGR, CD81, UACA, and PTTG1IP in Cal-27 by analyzing CAFs-Exo sequencing data and publicly available TCGA data. This may account for the ability of CAFs-Exo to exert immunomodulation and promote OSCC proliferation. CONCLUSIONS: CAFs-Exo was found to be involved in tumor immune regulation through hsa-miR-139-5p, ACTR2 and EIF6, while PIGR, CD81, UACA and PTTG1IP may be potentially effective targets for the treatment of OSCC in the future.

[Pilose antler polypeptide CNT14 promoted proliferation and migration of human oral mucosa fibroblast cells].

PURPOSE: To explore the effect of pilose antler polypeptides CNT14 on proliferation and migration of human oral mucosa fibroblast (hOMF) cells and the related molecular mechanism. METHODS: The biosafety of pilose antler polypeptides CNT14 on hOMF cells was verified by live-dead cell staining kit.CCK-8 assay was used to detect the effect of pilose antler polypeptides CNT14 on hOMF cell proliferation. The effect of pilose antler polypeptides CNT14 on hOMF cell migration was detected by scratch test. Western blot was used to detect the expression of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in hOMF cells stimulated by pilose antler polypeptides CNT14. The effect of Smad2 inhibitors on fibroblast activation induced by pilose antler polypeptides CNT14 was evaluated.The model of keratinized gingival defect was established in New Zealand white rabbits, and the regenerated gingival tissue was stained with H-E. The expression levels of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in the gingival tissues of regenerated New Zealand white rabbits were detected by immunohistochemistry, and the ability of pilose antler polypeptides CNT14 to promote regeneration of oral gingival tissues was verified. Statistical analysis was performed with SPSS 20.0 software package. RESULTS: The survival rate of hOMF cells was above 95% after treated with pilose antler polypeptides CNT14. After stimulation of hOMF cells with pilose antler polypeptides CNT14, the proliferation and migration rates of hOMF cells were increased compared with the control group (P＜0.05). The expression of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in hOMF cells stimulated by pilose antler peptide CNT14 was increased, and the difference was statistically significant(P＜0.05). The expression of α-SMA in fibroblasts induced by Smad2 inhibitor was decreased. In animal experiments, H-E staining showed that the inflammatory response of oral mucosal wounds of New Zealand white rabbits treated with CNT14 was less than that of the control group. Immunohistochemical staining results showed that the expressions of α-SMA, TGF-ß1, Smad2 and p-Smad2 in the regenerated gingival tissues of New Zealand white rabbits treated with CNT14 were significantly increased compared with those in the control group on the 9th and 11th days within the gingival wounds(P＜0.05). CONCLUSIONS: Pilose antler polypeptides CNT14 has good biosafety and can promote the proliferation and migration of human oral mucosa fibroblast cells, and the expression levels of α-SMA, TGF-ß1, Smad2 and p-Smad2 were increased, promoting the regeneration of gingival tissues.

Comprehensive immunogenomic landscape analysis of prognosis-related genes in head and neck cancer.

Head and neck cancer is the sixth most common malignancy around the world, and 90% of cases are squamous cell carcinomas. In this study, we performed a systematic investigation of the immunogenomic landscape to identify prognostic biomarkers for head and neck squamous cell carcinoma (HNSCC). We analyzed the expression profiles of immune-related genes (IRGs) and clinical characteristics by interrogating RNA-seq data from 527 HNSCC patients in the cancer genome atlas (TCGA) dataset, including 41 HPV+ and 486 HPV- samples. We found that differentially expressed immune genes were closely associated with patient prognosis in HNSCC by comparing the differences in gene expression between cancer and normal samples and performing survival analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to annotate the biological functions of the differentially expressed immunogenomic prognosis-related genes. Two additional cohorts from the Oncomine database were used for validation. 65, 56 differentially expressed IRGs was associated with clinical prognosis in total and HPV- samples, respectively. Furthermore, we extracted 10, 11 prognosis-related IRGs from 65, 56 differentially expressed IRGs, respectively. They were significantly correlated with clinical prognosis and used to construct the prognosis prediction models. The multivariable ROC curves (specifically, the AUC) were used to measure the accuracy of the prognostic models. These genes were mainly enriched in several gene ontology (GO) terms related to immunocyte migration and receptor and ligand activity. KEGG pathway analysis revealed enrichment of pathways related to cytokine-cytokine receptor interactions, which are primarily involved in biological processes. In addition, we identified 63 differentially expressed transcription factors (TFs) from 4784 differentially expressed genes, and 16 edges involving 18 nodes were formed in the regulatory network between differentially expressed TFs and the high-risk survival-associated IRGs. B cell and CD4 T cell infiltration levels were significantly negatively correlated with the expression of prognosis-related immune genes regardless of HPV status. In conclusion, this comprehensive analysis identified the prognostic IRGs as potential biomarkers, and the model generated in this study may enable an accurate prediction of survival.

Identification of cancer hallmark-associated gene and lncRNA cooperative regulation pairs and dictate lncRNA roles in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumour in the oral and maxillofacial region. Numerous cancers share ten common traits ("hallmarks") that govern the transformation of normal cells into cancer cells. Long non-coding RNAs (lncRNAs) are important factors that contribute to tumorigenesis. However, very little is known about the cooperative relationships between lncRNAs and cancer hallmark-associated genes in OSCC. Through integrative analysis of cancer hallmarks, somatic mutations, copy number variants (CNVs) and expression, some OSCC-specific cancer hallmark-associated genes and lncRNAs are identified. A computational framework to identify gene and lncRNA cooperative regulation pairs (GLCRPs) associated with different cancer hallmarks is developed based on the co-expression and co-occurrence of mutations. The distinct and common features of ten cancer hallmarks based on GLCRPs are characterized in OSCC. Cancer hallmark insensitivity to antigrowth signals and self-sufficiency in growth signals are shared by most GLCRPs in OSCC. Some key GLCRPs participate in many cancer hallmarks in OSCC. Cancer hallmark-associated GLCRP networks have complex patterns and specific functions in OSCC. Specially, some key GLCRPs are associated with the prognosis of OSCC patients. In summary, we generate a comprehensive landscape of cancer hallmark-associated GLCRPs that can act as a starting point for future functional explorations, the identification of biomarkers and lncRNA-based targeted therapy in OSCC.

Hyperthermia inhibited the migration of tongue squamous cell carcinoma through TWIST2.

BACKGROUND: Hyperthermia has been shown promising in the treatment of head and neck squamous cell carcinoma (HNSCC); however, the mechanism underlying hyperthermia reducing tumor metastasis is poorly elucidated. TWIST2, an important transcription factor of epithelial-mesenchymal transition (EMT), plays a critical role in the tumor progression and metastasis. The role of TWIST2 in tongue squamous cell carcinoma (TSCC) and its association with hyperthermia still have not been reported. METHOD: The correlations between TWIST2 expression and the clinical-pathologic characteristics of 89 patients with TSCC were evaluated by immunohistochemical staining. TSCC cell lines transfected with siRNA against TWIST2 were heated for 40 min at 42.5°C, and the migration capability of cells was examined by migration assay. Xenograft tumors in nude mice were treated by hyperthermia, and TWIST2 expression was measured. RESULTS: Our data showed that TWIST2 expression was associated with the metastasis of human TSCC. In Tca8113 and Cal-27 cells, TWIST2-siRNA treatment can reduce cell migration ability and has no effect on the cell proliferation and apoptosis. Hyperthermia can decrease the level of TWIST2 in TSCC and inhibit the migration of cells. CONCLUSIONS: This demonstrated that hyperthermia might decrease the migration of Tca8113 and Cal-27 cells by reducing TWIST2 expression. Altogether, these findings suggest an as yet undescribed link between TWIST2 and hyperthermia in TSCC.

[Protective effect of recombinant TM-N and recombinant soluble RAGE in a mouse model of acute hepatic failure].

OBJECTIVE: To evaluate the roles of N-terminal lectin-like domain of thrombomodulin (TM-N) and receptor for advanced glycation end products (RAGE) in acute hepatic failure using a mouse model system. METHODS: Acute hepatic failure was induced in Kunming mice by intraperitoneal injection of D-galactosamine (D-Galn at 600 mg/kg) and lipopolysaccharide (LPS at 5 mug/kg) and mice were divided into groups for injection with saline, recombinant (r)TM-N protein, or recombinant soluble (rs)RAGE protein. Unmanipulated model mice served as the negative controls. Effects on liver expression of high mobility group box-1 (HMGB1) were detected by immunohistochemistry and real time RT-PCR. Effects on serum levels of tumor necrosis factor-alpha (TNFa) and interleukin-1 beta (IL)-1b were quantified by ELISA. RESULTS: Treatment with rTM-N and rsRAGE both alleviated the acute liver damage induced by D-Galn/LPS exposure, and decreased the hepatic expression of HMGB1 as well as the serum levels of TNFa and IL-1b. CONCLUSION: Intraperitoneal delivery of rTM-N and rsRAGE can alleviate acute liver damage by modulating the expression of necrosis- and inflammation-related factors.

Induction of lung epithelial cell transformation and fibroblast activation by Yunnan tin mine dust and their interaction.

Tumor-stroma interactions play a significant role in tumor development and progression. Our study employed an in vitro co-culture model of epithelial cells and fibroblasts to investigate the mechanism of and interaction between lung epithelial cell transformation and fibroblast activation induced by Yunnan tin mine dust. Epithelial cell transformation was evaluated using concanavalin A agglutination and anchorage-independent growth assays, and fibroblast activation was assessed via immunohistochemistry. The TGF-ß1/Smad pathway was monitored by Western blot analysis and ELISA. We found concanavalin A agglutination and anchorage-independent growth assays of dust-exposed epithelial cells were positive, dust-exposed fibroblasts expressed α-SMA, and during the mine dust-induced tumorigenesis, TGF-ß1/Smad signaling pathway changed. In conclusion, Yunnan tin mine dust is able to induce the malignant transformation of bronchial epithelial cells and fibroblast activation. Epithelial cells are the main target of mine dust. Bronchial epithelial cell transformation and fibroblast activation are correlated and synergistic. Their interdependence is related to the TGF-ß1/Smad signaling pathway.

[Role of protein kinase C-delta in hyperthermia-induced apoptosis in tongue squamous cell carcinoma Tca8113 cells].

OBJECTIVE: To study the role of protein kinase C-delta (PKC-delta) in hyperthermia-induced apoptosis in human tongue squamous cell carcinoma Tca8113 cells. METHODS: Tca8113 cells were treated at 43 degrees C in a heating water bath for 0, 40, 80, 120 min after pretreatment with Rottlerin, a specific inhibitor of PKC-delta, and equal volume dimethyl sulfoxide (DMSO) for 30 min, respectively. The cells were stained by propidium iodide (PI) and Rhodamine 123 to analysis apoptotic rate and the changes of mitochondrial transmembrane potential by flow cytometry (FCM). The total proteins were extracted for Western blotting analysis of activation and proteolysis of PKC-delta, and for colorimetric assay of relative activity of Caspase-3. RESULTS: Hyperthermia could induce proteolysis and activation of PKC-delta, and this was attenuated by Rottlerin. Apoptotic rate, decreasing of mitochondrial transmembrane potential and activity of Caspase-3 which being induced by hyperthermia in Tca8113 cells were inhibited by PKC-delta specific inhibitor Rottlerin. There were significantly statistical differences in apoptosis rates, mitochondrial transmembrane potential and activity of Caspase-3 between Rottlerin- and non-Rottlerin-pretreated cells after hyperthermia for 40, 80, 120 min (P < 0.01). CONCLUSION: Activated PKC-delta may facilitate hyperthermia-induced apoptosis in Tca8113 cells, and may be one of the mechanisms of apoptosis induced by hyperthermia.

Hyperthermia-induced apoptosis in Tca8113 cells is inhibited by heat shock protein 27 through blocking phospholipid scramblase 3 phosphorylation.

PURPOSE: Hyperthermia induces tumour cell apoptosis through the mitochondrial apoptotic pathway; however, the signal transduction mechanism underlying this process still needs to be fully elucidated. Phospholipid scramblase 3 (PLS3), a target of protein kinase C-delta (PKC-delta), resides in mitochondria and plays pivotal roles in regulating apoptotic response. Activated PLS3 facilitates cardiolipin (CL) translocation from the mitochondrial inner membrane to the outer leaflet of the mitochondrial outer membrane and triggers apoptosis. MATERIALS AND METHODS: The tongue squamous cell carcinoma Tca8113 cells were transfected or co-transfected using Lipofectamine 2000 with plasmids pCMV-6xHis-PLS3, pCMV-6xHis-PLS3 (T21A), pHA-PKC-delta, pHA-PKC-delta-KD (K376R), pHA-Hsp27, and empty control plasmid pcDNA3.1. The transfected cells were heated in water bath at 43 degrees C for 20 min, 40 min and 60 min. Assessments of apoptosis and redistribution of mitochondrial cardiolipin were performed by flow cytometry. PLS3, PKC-delta, Hsp27, phosphorylation of PLS3 and PLS3/PKC-delta interaction were detected by western blotting. RESULTS: In our study the results show that elevated levels of the wild-type PLS3, but not the PLS3 (T21A) mutant, is able to increase hyperthermia-induced CL translocation and apoptosis. Wild-type PKC-delta facilitates PLS3 phosphorylation, PKC-delta/PLS3 interaction, and CL translocation, which consequently promote apoptosis. In contrast, heat shock protein 27 (Hsp27) blocks PKC-delta-induced PLS3 phosphorylation, suppresses PKC-delta/PLS3 interaction and CL translocation, and inhibits apoptosis. CONCLUSIONS: Our findings suggest that phosphorylation of PLS3 by PKC-delta is involved in the hyperthermia-induced apoptotic signal transduction pathway in Tca8113 cells, and that Hsp27 blocks this pathway to suppress hyperthermia-induced apoptosis.

[Protective effect of recombinant HMGB1 A box protein in mouse with acute hepatic failure].

OBJECTIVE: To evaluate the effect of recombinant HMGB1 A box protein in mouse with acute hepatic failure. METHOD: Acute hepatic failure was induced by D-galactosamine and lipopolysaccharide in mice. After injection with saline (control) or recombinant HMGB1 A box proteins, the expression of HMGB1 in liver tissues was detected by immunohistochemistry and RT-PCR, and the serum TNFalpha and IL-1beta were quantified. RESULTS: rHMGB1-Abox protein alleviated the acute liver damage. rHMGB1-Abox protein treatment decreased the expression of HMGB1 in liver tissues and reduced the serum levels of TNFalpha and IL-1beta. CONCLUSION: rHMGB1-Abox protein can alleviate the acute liver damage and inhibit the expression of HMGB1.

[The effects of different PAP domains on hepatitis B virus replication].

OBJECTIVE: To investigate the effects of different PAP domains on hepatitis B virus replication. METHODS: The full length and two truncated PAP mutants were cloned into a eukaryotic expression plasmid, and were transfected into HepG2.2.15 cells using lipofectamine 2000. 3 days after transfection, the medium and cells were collected. HBsAg and HBeAg were measured using ELISA. The titers of HBV DNA were quantified using fluorogenic quantitative PCR (FQ-PCR). HepG2 cells were used to determine the cytotoxicity of the plasmids transfection by MTT assays. RESULTS: The inhibitory effect on HBV replication of the C-terminal 25 amino acids deleted PAP mutant (pXF3H-PAP14) was not significantly different from that of the full length PAP (pXF3H-PAP12) (Chi-square test = 0.5, 2.0, 0.02, probability value more than 0.05), however, the cytotoxicity of pXF3H-PAP14 was lower than that of pXF3H-PAP12 (Chi-square test = 7.7, probability value less than 0.01). Both N-terminal 69 amino acids deleted mutant and C-terminal 25 amino acids deleted mutant had no cytotoxicity and no antiviral activity. CONCLUSION: C-terminal 25 amino acid of PAP is related to cytotoxicity but not related to antiviral activity of PAP. N-terminal 69 amino acid of PAP is related to the anti-HBV effect of PAP.

Thermochemotherapy of lower lip squamous cell carcinoma without metastases: an experience of 31 cases.

INTRODUCTION: The aim of this study was to evaluate the efficacy, functional and aesthetic results, and safety of a novel treatment, thermochemotherapy, for lower lip squamous cell carcinoma (LLSCC) without metastases. PATIENTS AND METHODS: A combination of local hyperthermia delivered by a 915MHz microwave heating system and the chemotherapy of pingyangmycin (bleomycin A(5) hydrochloride) (PYM) and methotrexate (MTX), was administered to 31 patients of LLSCC twice per week for a period of 4.5-7.5 weeks. Patients with complete response (CR) have been followed up for a full five-year period, whereas partial response (PR) patients were excluded for further analysis. The local control of tumour, functional and cosmetic outcomes, recurrence, regional lymph node and distant metastases, and complications were assessed by clinical and imaging examination. RESULTS: Clinical CR was observed in twenty-nine (93.55%) patients and PR in two (6.45%), the total response rate was 100%, while the adverse effects were extremely minimal and tolerable in all 31 patients including 6 elderly patients with a compromised general condition. All 29 CR, including 8 extensive lesions, achieved excellent cosmetic and functional preservation. During the follow-up period, local relapse was seen in 1 case, 1 patient died, and the remainder obtained a complete remission. CONCLUSION: This clinical study suggests that thermochemotherapy may be a feasible treatment for primary LLSCC without cervical metastases, especially for patients with extensive lesions and a compromised general condition.

[Expression of fragile histidine triad (FHIT) protein and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust].

OBJECTIVE: To study the expression and significance of fragile histidine triad (FHIT) and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust. METHODS: Every second generation of immortalized human bronchial epithelial cells (BEAS-2B) and human embryo lung fibroblasts (WI-38) were exposed to 100 µg/ml Yunnan tin mine dust for 72 h, until the ninth generation. The cells were subsequently co-cultured from the 11th generation. Experimental setup: B group, B (W) group, B (W 100) group, B100 group, B100 (W) group, B100 (W100) group. The expressions of FHIT and Ki-67 in epithelial cells were determined by the method of immunocytochemistry at the 16th, 26th and 36th generation. The percentage of Ki-67 positive cells was calculated as proliferation index. RESULTS: The expression of FHIT was observed in BEAS-2B cells. The expression levels of FHIT among B group, B (W) group and B (W 100) group had not instinctive difference. At the 16th generation, the expression of FHIT in the B100 group was decreased compared with that in the B group and the expression of FHIT between B100 (W) group and B100 (W100) group was lower than that in the B100 group. At the 26th generation, the expression of FHIT was decreased compared with that at the 16th generation in the B100, B100 (W) and B100 (W100) groups. However, At the 36th generation, positive expression were observed again in the B100, B100 (W) and B100 (W100) groups and the expression levels were in incremental order. At the 16th, 26th and 36th generation, the proliferation indexes of B group, B (W) group and B (W 100) group were all < 3%. The proliferation indexes of B100, B100 (W) and B100 (W100) were increased step by step with the generation elongation. CONCLUSIONS: FHIT could be a target at which Yunnan tin mine dust induces transformation of BEAS-2B cells. The proliferation activation of BEAS-2B cells can be improved by Yunnan tin mine dust.

[Interaction between malignant transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust].

OBJECTIVE: To study the interaction between transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust. METHODS: (1) The immortalized human bronchial epithelial cell line BEAS-2B and human embryo lung fibroblast cell line WI-38 were grown in MEM medium containing 5% and 10% FBS, respectively, at 37 degrees C and 5% CO2 with saturated humidity. The cells were subcultured every 6 days. BEAS-2B cells and WI-38 cells were induced with Yunnan tin mine dust on every other generation at the concentration of 100 microg/ml. From the 11th generation, the cells were co-cultured. Epithelial cell transformation was tested using concanavalin A (ConA) agglutination and anchorage-independent growth assays. The cell cycles were analyzed through flow cytometry. The expressions of alpha-SMA in fibroblasts were determined with immunocytochemistry. RESULTS: (1) Cell morphology of mine dust-exposed epithelial cells began to transform at the 28th generation. Similar transformations were observed with mine dust-induced epithelial cells co-cultured with fibroblasts from the 20th generation and mine dust-induce epithelial cells co-cultured with mine dust-induced fibroblasts from the 16th generation. ConA agglutination assay and anchorage-independent growth assays were negative in normal BEAS-2B cells. At the 26 th generation, the agglutination test result of the mine dust-exposed epithelial cells was positive. Co-cultured with fibroblasts and mine dust-exposed fibroblasts, the agglutination time of the mine dust-exposed epithelial cells became short. Epithelial cell anchorage-independent growth assay was positive for mine dust-exposed epithelial cells co-cultured with fibroblasts at the 36th generations and for mine dust-exposed epithelial cells co-cultured with mine dust-exposed fibroblasts at the 26th generations. The clone formation rate of the 26th generation was 6.00 per thousand +/- 1.00 per thousand and 15.33 per thousand +/- 2.52 per thousand respectively, with the significant differences (P < 0.05). With generation adding, the portion of S phase increased for mine dust-exposed epithelial cells. (2) At the 26th generations, fibroblasts expressed alpha-SMA. Co-cultured with epithelial cell, the alpha-SMA expression of fibroblasts increased. Especially, positive cell numbers and intensity of staining dramatically increased with generation adding. CONCLUSIONS: (1) The tin mine dust can induce malignant transformation of human pulmonary epithelial cells BEAS-2B and activation of fibroblasts WI-38. (2) The epithelial cells are major target in carcinogenesis induced by Yunnan tin mine dust. (3) Transformation of epithelia and activation of fibroblasts co-evolve in the developing process of induced lung cancer by Yunnan tin mine dust.

[Estrogen protects the dopaminergic neurons in substantia nigra against damage induced by 6-hydroxydopamine].

Substantial evidence strongly implies that sensory gating P50 (also called P50 auditory evoked potential, P50) and dopaminergic neurotransmitters are related. In animal experiment, P50 can be recorded in an awake and quiet state with freedom of movement. Until now there is lack of animal experimental data on the supportive effect of estrogen on function of dopaminergic neurons in substantia nigra (SN) in physiological state. In the present study, female Sprague-Dawley (SD) rats were used as subjects. The animals were divided randomly into four groups: (1) control group (normal animals); (2) Parkinson's disease (PD) model group: the right SN was lesioned with 6-hydroxydopamine (6-OHDA); (3) PD model with bilateral ovariectomized group (OVX-PD): bilateral ovariectomy was performed before administration with 6-OHDA; (4) estrogen + PD model with bilateral ovariectomized group (OVX-E(2)-PD): physiological dose of estrogen was given to the bilateral ovariectomy animals before administration with 6-OHDA. P50 induced by two brief acoustic stimuli were recorded in the right SN and the number of TH(+) dopaminergic neurons in the SN stained by immunohistochemistry was calculated after the determination of P50. The results showed that in the PD model group, the testing/conditioning (T/C) ratio of P50 decreased by 40.60% and the number of TH(+) cells in the right SN decreased by 64.74% as compared with that in the control group (P<0.01); In the OVX-PD group, the T/C ratio of P50 decreased by 45.88% and the number of TH(+) cells was reduced by 57.26% as compared with that in the PD group (P<0.01). Administration with 6-OHDA into the SN pars compacta of ovariectomized rats caused more decrease in the number of TH(+) cells as well as more damage to the function of sensory gating in SN. While in OVX-E(2)-PD group, intramuscular injection with estrogen at physiological dose 3 d before 6-OHDA administration decreased the degree of damage to the SN functionally and morphologically, and its degree of injury corresponded to PD group. These results indicate that the mechanism of protection of dopaminergic neurons in the SN provided by physiological level of estrogen is by promoting the resistibility of the neurons to harmful stimulation. If the gonads are resected resulting in a lack of estrogen, the degree of injury to the function and morphology of dopaminergic neurons in SN induced by 6-OHDA increases. Replacement of estrogen at physiological level on time is necessary. Sensory gating P50 in SN may reflect dynamically the protection of estrogen against dopaminergic neurons depletion in vivo.

Rosiglitazone prevents murine hepatic fibrosis induced by Schistosoma japonicum.

AIM: To evaluate the effect of rosiglitazone in a murine model of liver fibrosis induced by Schistosoma japonicum infection. METHODS: A total of 50 mice were randomly and averagely divided into groups A, B, C, D and E. The mice in group A served as normal controls, while those in the other four groups were infected with Schistosoma japonicum to induce the model of liver fibrosis. Besides, the mice in groups C, D and E were treated with praziquantel, rosiglitazone and praziquantel plus rosiglitazone, respectively. NF-kappaB binding activity and expression of PPAR gamma-mRNA were determined by Western blot assay and real-time quantitative PCR. Radioimmunonassay technique was used to detect the serum content changes of TNF-alpha and IL-6. Histological specimens were stained with HE. Expression of TGF-beta1, a-smooth muscle actin and type I and type III collagen was detected by immunohistochemistry and multimedia color pathographic analysis system. RESULTS: Inflammation and fibrosis in the rosiglitazone plus praziquantel treatment group (group E) were lightest among the mice infected with Schistosoma (P < 0.05). To further explore the mechanism of rosiglitazone action, we found that rosiglitazone can significantly increase the expression of PPAR gamma [E: -18.212 +/- (-3.909) vs B: -27.315 +/- (-6.348) and C: -25.647 +/- (-5.694), P < 0.05], reduce the NF-kappaB binding activity (E: 88.89 +/- 19.34 vs B: 141.11 +/- 15.37, C: 112.89 +/- 20.17 and D: 108.89 +/- 20.47, P < 0.05), and lower the serum level of TNF-alpha (E: 1.613 +/- 0.420 ng/mL vs B: 2.892 +/- 0.587 ng/mL, C: 2.346 +/- 0.371 ng/mL and D: 2.160 +/- 0.395 ng/mL, P < 0.05) and IL-6 (E: 0.106 +/- 0.021 ng/mL vs B: 0.140 +/- 0.031 ng/mL and C: 0.137 +/- 0.027 ng/mL, P < 0.05) in mice with liver fibrosis. Rosiglitazone can also substantially reduce the hepatic expression of TGF-beta1, alpha-SMA type I and type III collagen in mice with liver fibrosis. CONCLUSION: The activation of PPAR gamma by its ligand can retard liver fibrosis and suggest the use of rosiglitazone for the treatment of liver fibrosis due to Schistosoma japonicum infection.

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro.

AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over-length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 mug/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 microg and 2.0 microg plasmid pXF3H-PAP, the levels of HBV nucleocapside-associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 mug plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%. CONCLUSION: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.

[In vitro study on Hep G2 cell infected by hepatitis C virus].

OBJECTIVE: To establish hepatitis C virus (HCV) infected cell model which is similar to the infection in vivo and can support HCV to replicate for a long time. METHODS: After infected with HCV-positive serum, Hep G2 cells were cultured for 60 days. Nested RT-PCR was used to detect plus and minus HCV RNA in cultured cells and supernatants. RESULTS: Plus HCV RNA was detected intermittently in Hep G2 cells during 2-30 days, minus HCV RNA was detected during 3-30 days after infection, the detection rate was similar to plus HCV RNA. Plus and minus HCV RNA can be still intermittently detected during 31-60 days after infection. However, the detection rate gradually declined. Plus HCV RNA was also found intermittently positive in the supernatant, and the detection rate was consistent to that in cells. Minus HCV RNA was not detected in the supernatant. CONCLUSION: Hep G2 cells were susceptible to HCV, and could support HCV to replicate for a relatively long time. Hep G2 is an ideal HCV infection cell model.

[Effect of phosphorothioate antisense Bcl-xL oligodeoxynucleotides on apoptosis and thermosensitivity of BcaCD885 cells].

OBJECTIVE: To study the effect of phosphorothioate antisense Bcl-xL oligodeoxynucleotides on apoptosis and thermosensitivity of BcaCD885 cells. METHODS: After phosphorothioate antisense Bcl-xL oligodeoxynucleotides were transfected into BcaCD885 cells. The characteristics of apoptotic cells were evaluated by morphological observation and TUNEL staining. Apoptotic rate and Bcl-xL protein expression were analyzed with flow cytometry. The influence of phosphorothioate antisense Bcl-xL oligodeoxynucleotides on apoptotic rate of BcaCD885 cells induced by hyperthermia with 43 degrees C 40 min was also examined through flow cytometry. RESULTS: The BcaCD885 cells transfected with phosphorothioate antisense Bcl-xL oligodeoxynucleotides displayed apoptotic morphological features. The Bcl-xL protein expression level of these cells was down-regulated significantly compared with the controlled group (P < 0.05). The apoptotic rate of these cells induced by hyperthermia was increased significantly compared with the controlled group (P < 0.05). CONCLUSION: Phosphorothioate antisense Bcl-xL oligodeoxynucleotides can induce apoptosis and improve thermosensitivity of BcaCD885 cells.

[Expression of IL-2 and TNF-alpha in the liver and the effect of injection of these cytokines on liver fibrosis in mice infected with Schistosoma japonicum].

OBJECTIVE: To detect the expression of IL-2 and TNF-alpha in the liver at different period postinfection of Schistosoma japonicum and their effect on liver fibrosis after supplementary injection of these cytokines. METHODS: Mice were infected with schistosome cercariae and divided into 3 groups. Two groups were injected (i.p.) every other day with IL-2 and TNF-alpha respectively for consecutive 4 wk. The third group and an uninfected group of normal mice were regarded as control. The ABC immunohistochemistry and pathologic image multimedia quantification system were applied to detect activity of IL-2 and TNF-alpha. RESULTS: The level of IL-2 and TNF-alpha in the liver in infected but untreated group slowly decreased (from 8, 11, 14 to 18 wk). The supplementary injection of the cytokines at 6 wk postinfection in the two groups increased the cytokines significantly, the level of IL-2 or TNF-alpha was higher at 1-8 wk after the last injection than that of both infected and uninfected control groups (P < 0.01). The granulomatous inflammation and fibrosis in the livers of the two groups were slighter than that of the control. CONCLUSION: At the 6th wk postinfection with egg deposition, exogenous supplementation with TNF-alpha or IL-2 induces enhanced expression of the two kinds of cytokines, corresponding to a diminished degree of the liver granulomatous inflammation and fibrosis.