Correlation between clinical stages of HBV chronic infection and co-expression of CD244 and PD-1 in HBV-specific CTLs

AIM: To investigate the co-expression of cluster of differentiation 244 (CD244) and programmed death 1 protein (PD-1) on hepatitis B virus (HBV) antigen-specific cytotoxic T-lymphocytes (CTLs) in chronic hepatitis B (CHB) patients and its correlation with the clinical stages of HBV chronic infection. METHODS: Eighty-one CHB subjects with human leukocyte antigen-A2 (HLA-A2) positive, including 20 cases of acute-on-chronic liver failure (ACLF), 20 cases of severe chronic hepatitis B (s-CHB), 34 cases of mild and moderate chronic hepatitis B (m-CHB) and 7 cases of immune tolerant stage (IT) of chronic hepatitis B as well as 14 healthy controls (HC), were enrolled in this study. The co-expression of CD244 and PD-1 was analyzed in virus-specific CD8⁺ T-cells derived from peripheral blood using major histocompatibility complex class I (MHC-I) pentamers targeting immunodominant epitopes of HBV core antigen (18-27). RESULTS: In the patients with chronic HBV infection, virus-specific CD8+T-cells with co-expression of CD244 and PD-1 were at increased levels than those in total CD8+ T-cells (67.48%±17.16% vs 14.01%±7.97%, P<0.01) in the peripheral blood. Among different clinical stages, increased level of CD244 expression coincided with increased expression of PD-1 in m-CHB compared with IT of CHB (73.08%±8.63% vs 53.11%±18.05%, P<0.05), which was followed by decreased co-expression level in ACLF (63.11%±13.87% vs 72.05%±16.86%, P<0.05) and restoration of the ability to secrete IFN-γ (30.95%±20.29% vs 13.63%±10.46%, P<0.05). CONCLUSION: CD244 and PD-1 are highly co-expressed in HBV-specific CTLs in the patients with s-CHB and m-CHB, and are decreased in ACLF following the restoration of IFN-γ secretion. The severity of CHB may be correlated with CD244 and PD-1 co-expression in HBV-specific CTLs.     

Effect of mineralocorticoid receptor gene silencing by RNA interference on proliferation and apoptosis of murine macrophage cell line RAW 264.7

AIM: To establish stable knockdown of mineralocorticoid receptor (MR) expression through short hairpin RNA (shRNA)-mediated silencing in murine RAW 264.7 macrophages. METHODS: Stable MR silencing in RAW 264.7 cells was achieved by recombinant shRNA plasmid targeting murine MR gene via liposome-mediated transfection, followed by G418 selection. The efficacies of plasmid transfection and MR silencing in G418-resistant cells were verified by immunofluorescent microcopy and real-time PCR, respectively. Proliferative activity of MR-silencing cell line was analyzed by CCK-8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. RESULTS: MR gene expression was down-regulated by 70% compared with the negative control (NC) plasmid transfection. In addition, MR-silencing cells exhibited lower proliferative activity compared with NC and wide type RAW 264.7 cells (P<0.05), along with reduced proliferation index of 31.0%±1.3% (P<0.05), compared with the wide type cells (37.2%±0.5%) and the NC cells (37.5%±1.6%). In resting state, the apoptotic rate in wide type, NC and MR-silencing cells were 2.18%±0.36%, 6.65%±0.81% and 7.70%±1.34%, respectively, and no statistical difference was observed between NC and MR-silencing cells (P>0.05). CONCLUSION: MR gene silencing inhibits the proliferation of RAW 264.7 macrophages, but has no obvious effect on the apoptosis of the resting state cells.     

Balance of Treg/Th17 in synovium of collagen-induced arthritis rats and impact of TNF-α blockage therapy

AIM: To investigate the balance of Treg/Th17 in synovium of collagen-induced arthritis (CIA) and the impact of tumor necrosis factor α(TNF-α) blockage therapy. METHODS: Rat CIA model was established by bovine II collagen injection. The pathological score was evaluated by HE staining and toluidine blue staining. The TNF-α level in plasma was measured by ELISA. The expression of Treg/Th17 in synovium was detected by double staining immunofluorescence. RESULTS: The plasma level of TNF-α in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (P<0.01), whereas no significant difference was found between TNFR-Fc treatment group and control group (P>0.05). No significant difference between CIA group and control group in the ratio of CD4⁺Foxp3⁺Treg cells/CD4⁺ cells in synovium (23.12%±4.93% vs 24.66%±5.82%, P>0.05) was observed, whereas the ratio in TNFR-Fc treatment group was significantly increased(33.07%±5.14%). The ratio of CD4⁺RORγt⁺Th17 cells/CD4⁺ cells in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (9.74%±2.23% vs 1.00%±0.59%, 5.63%±1.76%, P<0.01). CONCLUSION: Differentiation disturbance of Treg/Th17 exists in the synovium of CIA rats. TNFR-Fc may restore the balance of Treg/Th17 by inhibiting Th17 cell differentiation and inducing the production or accumulation of Treg.     

The ex vivo expansion characteristic of endothelial progenitor cells

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34⁺ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34⁺ and CD34^- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34⁺ and CD34^-,total MNC led to a significant increase in the expansion of CD34⁺ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34⁺, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34⁺ and AC133⁺cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34⁺ and CD34^- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.     

Urinary concentration of aquaporin-2 water channel protein at different stages of chronic heart failure rats

AIM: To study urinary concentration of aquaporin-2 water channel protein(AQP2) at different stages of chronic heart failure(CHF) rats and its relation to hyponatremia. METHODS: Male Sprague-Dawley rats(200~250 g) underwent either a left coronary artery ligation (a model of CHF) or a sham-operation. At different stage after surgery, urinary AQP2 concentration was measured by Western blot. 24-hours urine volume, serum sodium and urine osmolality were measured at the same time. RESULTS: There were two peaks of urinary excretion of AQP2 in severe heart failure rats model: one was the third day after operation, the other was the 9th week. Serum sodium and urine osmolality were significantly different in CHF rats as compared with sham-operated rats. Seven weeks after surgery, urinary AQP2 concentration was increased significantly insevere CHF rats compared with the mild ones and the control ones(365%±103% vs 179%±81% and 99%±48%, P＜0.01).CONCLUSION: The variation of urinary AQP2 excretion at different stages after ligation showed that the expression of AQP2 gene was increased obviously in CHF rats, and its expression level was higher as the heart failure became more severe.This is the important reason of heart failure with hyponatremia.     

Effects of NBP on expression of VEGF and HIF-1α in HUVECs under the condition of oxygen-glucose deprivation

AIM: To investigate the mechanism that dl-3-n-butylphthalide (NBP) protects cells from the induced by oxygen-glucose deprivation (OGD).METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Endothelial injury was assessed by measuring the changes of chromatin morphology and MTT method. The protein levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were determined by immunofluorescence and quantitatively analyzed with the software IPP. Western blotting was applied to verify the results.RESULTS: NBP at the concentrations of 0.01 to 100 μmol/L dose-dependently protected against OGD-induced cell damage. Compared with OGD group, NBP enhanced OGD-induced the expression of VEGF and HIF-1α, and the difference was statistically significant. The expression of VEGF and HIF-1α reached to the peak at the time points of 6 h and 8 h after OGD, respectively.CONCLUSION: Under the condition of OGD, NBP enhances the expression of HIF-1α in HUVECs, subsequently promotes the expression of downstream VEGF, and eventually elevates the survival of the cells.     

Effect of activation of stimulator cells on expression of CD69 by responder T cells in mixed lymphocyte reaction

AIM: To study the influence of status of stimulator cells on activation of responder T cells in mixed lymphocyte reaction (MLR), so as to provide some basis for clinical transplantation. METHODS: Stimulator cells were pretreated differently before mixed lymphocyte culture (MLC) to change their functional status, fluorescence conjugated antibodies and flow cytometry were used to detect expression of CD69 by responder T cells at several different time points.RESULTS:The expression percentages of CD69 by responder T cells in MLCa group(stimulator cells were pre-activated)were significantly higher than those in MLC group(stimulator cells were not pre-activated)at 24,48and 72 hours of culture,respectively(5.21%±0.24%vs 1.98%±0.33%,29.81%±0.85%vs 20.65%±1.00%and 39.61%±1.62%vs 13.49%±0.60%,P<0.01). CONCLUSION: Our results showed that with pre-activated stimulator cells, expression of CD69 by responder T cells could be significantly elevated in MLR.     

Analysis of cytokines derived from murine yolk sac endothelial cells cultured in vitro

AIM:To purify murine yolk sac endothelial cells (mYS-EC) and investigate the cytokines mRNA expression in mYS-EC. METHODS:The murine yolk sacs were digested with 0.1% collagenase, resuspended in DMEM and counted after digestion and centrifugation. The yolk sac adherent cells were cultured in DMEM containing 15% FBS with 10% mBMEC-CM or 5μg/L VEGF, ECGF and bFGF. The phagocytose function and expression of vWF were evaluated via particle phagocytosis and immunohistochemistry method. Atlas cDNA expression array was used for analysis of cytokine expression in mYS-EC. RESULTS:Colonies consisting of pure yolk sac endothelial cells were obtained in liquid culture system containing 15% FBS and 10% mBMEC-CM or 5μg/L VEGF, ECGF and bFGF. For complete purification of the endothelial cells, subsequent passage was also necessary. Cellular cord formed during passage culture. The endothelial cells were round or oval sharp in morphology, positive in phagocytosis and factor VIII related antigen (von Willebrand's Factor, vWF). The mRNA expressions of cytokines, such as TGF-β2, TNF-α, IFN-γ, FL, BMP-4, MIP-1β, BMP-2A, FLT2, endothelin 2, thymosin β10, IL-6, IL-13, IL-9, SCYA5 and ACBP were detected in mYS-ECs. CONCLUSION:mYS-EC was purified and expanded in vitro. The mRNA expression of 15 kinds of cytokines was detected in mYS-ECs by Atlas arrays.     

Effect of antisense VEGF gene on VEGF expression in human renal cell carcinoma

AIM: To construct eukaryotic expression vector carrying human antisense VEGF gene and to study its effect on VEGF expression and growth of renal cell carcinoma. METHODS: VEGF gene was cloned. Antisense VEGF gene was inserted into eukaryotic expression vector pcDNA3.1(-), then using restrict enzyme to confirm the result. The vector was transfected into renal cell carcinoma and positive clone was selected by using G418. The VEGF expression was detected by using RT-PCR and immunohistochemical method. The growth of cells was measured by MTT and cell cycle by FCM. RESULTS: Antisense VEGF gene eukaryotic expression vector was successfully constructed. Compared with empty vector group and control group, the amount of VEGF mRNA expression and VEGF protein were decreased in the antisense VEGF group, the difference was significant. There was no significant difference between empty vector group and control group. The cell growth in the antisense VEGF group became slower, and the percent of G1 phase increased and the percent of S phase decreased. CONCLUSION: Antisense VEGF gene decreases the VEGF mRNA and protein expression in renal cell carcinoma and inhibits the growth of renal carcinoma. Antisense VEGF gene may be used in gene therapy of renal carcinoma.     

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro via cardiotrophin 1

AIM: To investigate the effects of cardiotrophin 1 (CT-1) on differentiation of swine bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte-like cells in vitro.METHODS: MSCs were isolated and proliferated from Tibet miniswine. Adipogenic and osteogenic potentials were identified. MSCs were divided into 4 groups for induction: untreated group, 5-azacytidine (5-Aza) group,CT-1 group and 5-Aza combined with CT-1 group. After induction for 4 weeks, the expression of cardiac cell markers including α-actin and cardiac troponin-T (cTnT) was estimated by immunofluorescence staining. Finally, the rates of red fluorescence positive-staining cells were calculated. RESULTS: The expression of α-actin in the 4 groups by red fluorescence staining was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 29.90%±4.76%, significantly higher than that in 5-Aza group (17.73%±2.34%, P<0.01), CT-1 group (6.63%±0.55%, P<0.01) and untreated group (1.62%±0.09%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.05). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01). The expression of cTnT in the 4 groups was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 36.50%±4.09%, significantly higher than that in 5-Aza group (14.37%±1.65%, P<0.01), CT-1 group (7.50%±0.61%, P<0.01) and untreated group (1.12%±0.23%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.01). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01).CONCLUSION: Appropriate concentrations of 5-Aza (10 μmol/L) and CT-1 (0.1 μg/L) induce swine bone marrow MSCs to differentiate into cardiomyocyte-like cells in vitro. CT-1 combined with 5-Aza significantly increases the differentiation rate.     

Study on polarized Th1/Th2 cell in vitro from adult human peripheral blood

AIM: To investigate the details of CD4⁺ T cell polarized to Th1/Th2 in vitro. METHODS: After isolated the PBMCs and blood-plasma from adult human peripheral blood by Ficoll-Hypaque centrifugation, the PBMC culture procedure with or without the self-blood -plasma was applied to polarize T cells in vitro, these cells were polarized by PHA(20 mg/L),non-PHA respectively. The polarized rates of Th cell after 24 h,48 h,72 h were estimated respectively by flow cytometry following two-color immunofluorescent staining. RESULTS: CD4⁺T cell would polarize to Th1/Th2 two subsets after self-cytokines and PHA activation in vitro. The polarized rates of T cell after cultured for 24 h,48 h and 72 h were (13.28%±1.59%)/(12.70%±1.65%),(17.19%±1.03%)/(17.50%±1.30%),(19.49%±2.87%)/(18.58%±1.49%) respectively, but the polarized rates of T cell were very low if without self-blood-plasma. The difference between them was significant. The ratios of Th1/Th2 cells were about 1. CONCLUSION: CD4⁺T cell from adult human peripheral blood would polarize to Th1/Th2 two subsets in the presence of self-blood-plasma and PHA(20 mg/L) in vitro, and the cell number of Th1 and Th2 would be in balance.     

Dysfunction of proteasome inhibits the proliferation of hBMSCs via activation of P53

AIM: To investigate the role of P53 in decreased cell proliferation and proteasomal activity during culture expansion of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: The proteasomal activity and expression level of P53 in early-passage (passage 3~4) and late-passage (passage≥14) hBMSCs were observed. Early-passage hBMSCs were divided into DMSO control group, MG132 group (treated with 10 μmol/L MG132 for 4 h) and pifithrin-α(PET-α) + MG132 group (pretreatment with 20 μmol/L PFT-α for 1 h then exposure to MG132 for 4 h). The proliferation and cell cycle distribution of the cells were measured by BrdU incorporation assay and PI staining. To further confirm the effect of P53 inhibitor on late-passage hBMSCs, the cells were incubated with 20 μmol/L PFT-α and the BrdU-positive cells were counted. RESULTS: Accompanied by reduced proteasomal activity, the expression level of P53 in late-passage hBMSCs was up-regulated by 16.89%±4.44% compared with early-passage cells. Application of the proteasome inhibitor MG132 reduced the proliferation of early-passage hBMSCs, and the percentage of BrdU-positive cells dropped to 33.36%±2.24%. However, MG132-induced decrease in cell proliferation was partially reversed by pretreatment with PFT-α. BrdU-positive cells in PFT-α + MG132 group were increased to 49.23%±2.67%. The cell cycle distribution had no significant difference between PFT-α + MG132 group and DMSO group, and the higher proliferation index was found in PFT-α + MG132 group but not in MG132 group. Inhibition of P53 activity in late-passage hBMSCs by PFT-α promoted the cell proliferation, and the percentage of BrdU-positive cells was higher than that in control group. CONCLUSION: Long-term expansion of hBMSCs in vitro reduces proteasomal activity, which in turn activates P53 to suppress cell proliferation through blocking cell cycle progression.     

Effects of eicosapentaenoic acid on expression of nuclear factor κB and release of cytokines in human umbilical vein endothelial cells stimulated by lipopolysaccharide

AIM: To investigate the effects of eicosapentaenoic acid(EPA) on the expression of nuclear factor kappa B(NF-κB) and release of vascular endothelial growth factor(VEGF), IL-1α and IL-6 in cultured human umbilical vein endothelial cells(HUVECs) stimulated by lipopolysaccharide(LPS).METHODS: HUVECs were obtained from cell strain and cultured in vitro. HUVECs were divided into 4 groups: control group, LPS group, 0.030 g/L EPA treatment group and 0.050 g/L EPA treatment group. The cells were cultured with LPS alone in LPS group and incubated with EPA for 1 h in the EPA pretreatment groups at the concentrations of 0.030 g/L and 0.050 g/L before LPS stimulation. Twenty-four hours after stimulated by LPS, the protein expression of NF-κB p65 in HUVECs were assessed by Western blotting analysis at different time points. The production of VEGF, IL-1α and IL-6 in cultured HUVECs was evaluated by ELISA. The effects of EPA on the protein expression of NF-κB p65 and the production of VEGF, IL-1α and IL-6 in HUVECs challenged by LPS were also determined.RESULTS: Compared with control group, the protein expression of NF-κB p65 was significantly increased in HUVECs induced by LPS and was inhibited by EPA. Compared with control group, the protein expression of VEGF, IL-1α and IL-6 was dramatically increased in HUVECs induced by LPS and most of the increase was inhibited by EPA.CONCLUSION: LPS enhances the protein expression of NF-κB and the release of VEGF, IL-1α and IL-6. EPA inhibits the protein expression of NF-κB, and the production of VEGF and the inflammatory cytokines in cultured HUVECs stimulated by LPS, indicating that EPA may be useful for preventing and treating neovascular and inflammatory diseases.     

Regulatory role of CCN1 in lipopolysaccharide-induced mouse acute lung injury

AIM: To observe the cellular location and expression change of cysteine-rich protein 61 (CYR61/CCN1) in lung tissues of the mice with lipopolysaccharide (LPS) intratracheal instillation, and to clarify the regulatory role of CCN1 expression in mediating inflammatory response. METHODS: The expression change of CCN1 in the lung tissues in vivo was observed by the method of immunohistochemistry, and immunofluorescence was employed to certify the cellular location of CCN1 in bronchial epithelial cells. Bronchial epithelial 16HBE cells were cultured in vitro, and the expression of CCN1 under the condition of LPS stimulation was quantified by RT-qPCR and Western blot with or without specific inhi-bitors of ERK1/2, JNK, P38 and PI3K signaling pathways. The mRNA expression levels of interleukin-6 (IL-6), IL-8, transformrg growth factor-β (TGF-β) and vascular endothlial growth factor (VEGF) were measured by RT-qPCR under the condition of recombinant CCN1 exposure or transfection with CCN1-siRNA. RESULTS: The results of immunohistochemistry indicated that CCN1 was primarily located in bronchial epithelium. The results of immunofluorescence revealed that CCN1 was localized in the cytoplasm. The specific inhibitors of ERK1/2, JNK, P38 and PI3K signaling pathways reversed the up-regulation of CCN1 upon LPS stimulation. Exposure to recombinant CCN1 resulted in the up-regulation of IL-6, IL-8, TGF-β and VEGF, while LPS-related up-regulation of IL-6, IL-8, TGF-β and VEGF was blocked by silencing of CCN1. CONCLUSION: Airway epithelium-derived CCN1 is up-regulated under the condition of lung injury and the regulatory mechanism involves ERK1/2, JNK, P38 and PI3K signal transduction pathways. CCN1 acts as an inflammatory mediator in amplification of inflammatory response, laying theoretical basis for the potential molecular therapeutic target of acute lung injury.     

Effects of flavonoids isolated from Scutellaria stem and leaf on expression of NMDAR and VEGF in chronic cerebral ischemia rats

AIM: To study the effects of flavonoids isolated from Scutellaria stem and leaf (SSF) on the expression of N-methyl-D-aspartate receptor (NMDAR) and vascular endothelial growth factor (VEGF) in chronic cerebral ischemia rats. METHODS: The model of chronic cerebral ischemia was established by bilateral carotid artery occlusion for 2 months in female SD rats. The effects of SSF on mRNA expression of NMDAR in hippocampus and VEGF in cerebral cortex were evaluated by the method of RT-PCR. RESULTS: Compared with the sham group, the expression of NMDAR1, NMDAR2A and NMDAR2B in hippocampus and VEGF in cerebral cortex were significantly increased (P<0.01). However, the cerebral ischemia rats daily and orally administered with SSF at doses of 17.5 mg·kg^-1·d^-1, 35 mg·kg^-1·d^-1 and 70 mg·kg^-1·d^-1 for 38 days appeared that the mRNA expression of NMDAR1, NMDAR2A and NMDAR2B in hippocampus was obviously reduced (P<0.05), and the mRNA content of VEGF in the cortex (P<0.05) was increased. CONCLUSION: SSF decreases the expression of NMDAR in hippocampus, increases the expression of VEGF in cerebral cortex of cerebral ischemia rats, suggesting that the neuroprotective effect of SSF may be exerted by influencing the production of NMDAR and VEGF in the brain.     

Effect of scutellarin on VEGF expression in human retinal pigment epithelial cells and retinas of diabetic rats

AIM: To evaluate the influence of scutellarin on the expression of vascular endothelial growth factor (VEGF) in high glucose-treated human retinal pigment epithelial cell line ARPE-19 and to observe the effects of scutellarin on the protein expression of VEGF, p-ERK and VEGFR2 in the retinas of type II diabetic rats. METHODS: Cultured ARPE-19 cells were divided into normal control group, scutellarin group, high glucose group and high glucose+scutellarin group. The protein levels of VEGF, p-ERK and VEGFR2 were measured by Western blot. The VEGF release in ARPE-19 cells was detected by ELISA. Normal rats were randomly divided into normal control group and scutellarin group. Diabetic rat model was established by feeding with high-fat diet and injecting with streptozocin, and randomly divided into diabetes group and diabetes treated with scutellarin group. After 16 weeks, the eyes were removed. The morphological changes of the retinas were observed under light microscope with HE staining, and histopathological score was recorded. The expression of VEGF in the retinas was observed by the method of immunohistochemistry. RESULTS: Compared with normal control group, the protein levels of VEGF, p-ERK and VEGFR2 in the ARPE-19 cells decreased in scutellarin group, but increased in high glucose group. The histopathological score of the retinas showed significant difference among diabetes group, diabetes treated with scutellarin group and normal control group, and no significant difference between normal control group and scutellarin group was observed. The expression of VEGF increased in diabetic group and was significantly higher than that in scutellarin treatment group (P<0.05). CONCLUSION: Scutellarin inhibits the increased protein le-vels of VEGF, p-ERK and VEGFR2 in ARPE-19 cells, and decreases the expression of VEGF in the retinas of diabetic rats. The suppression of the diabetic retinopathy development by scutellarin may be partly involved in the ERK/MAPK pathway.     

Effects of IL-6 and AG490 on growth of diffuse large B-cell lymphoma and Burkitt lymphoma transplanted into nude mice

AIM: To observe the effects of interleukin-6 (IL-6) and AG490 on diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) transplanted into nude mice, and to explore the effects of STAT3 activation on growth of these kinds of lymphoma in nude mice and its related mechanisms. METHODS: The nude mouse models with DLBCL and BL were established by transplantation with OCI-LY8 cells and Raji cells, respectively, and were divided into 3 groups:control group, IL-6 group and AG490 group. The body weight of mice and tumor size were measured. Western blot and immunohistochemical staining were used to detect the protein levels of p-STAT3, survivin and vascular endothelial growth factor (VEGF), and real-time PCR was used to detect the mRNA expression of survivin and VEGF. RESULTS: The tumorigenic rate of 2 kinds of tumor cell lines in nude mice was 83.3% (25/30) totally. The tumorigenicity of OCI-LY8 cells (66.7%, 10/15) was significantly lower than that of Raji cells (100%, 15/15) (P<0.05). The tumor size and body weight on days 9 and 10 in IL-6 group increased as compared with the control group, and the total difference value of tumor size between day 1 and day 10 in IL-6 group was obviously larger than that in control group (P<0.05). The positive protein of p-STAT3 was found in the nucleus, while the positive expression of survivin and VEGF was found in the cytoplasm. As compared with control group, the expression of survivin and VEGF was significantly increased (P<0.05), while the protein level of p-STAT3 was not significantly increased in IL-6 group of DLBCL. The protein levels of p-STAT3 and VEGF were significantly decreased (P<0.05), while the expression of survivin did not significantly decreased in AG490 group of DLBCL. The p-STAT3 and VEGF levels significantly increased (P<0.05) in IL-6 group of BL, while the levels of 3 kinds of proteins significantly deceased (P<0.05) in AG490 group of BL, as compared with control group. No statistical difference of mRNA expression of survivin and VEGF among IL-6, AG490 and control groups was observed. CONCLUSION: IL-6 and AG490 affect the growth of DLBCL and BL through activation of STAT3 pathway. The activated STAT3 participates in pathogenesis and progress of DLBCL and BL by up-regulating the expression of survivin and VEGF.     

Phenytoin inhibited changes in nitric oxide of rat hippocampus induced by stress

AIM: To investigate the changes in nNOS and iNOS expression of hippocampal CA3 pyramidal neurons and NO₂^-/NO₃^- level of hippocampal homogenate of rats induced by stress, and to explore the effect of phenytoin on them. METHODS: Rats were subjected to forced-swimming stress, phenytoin was administered(ip) at 30 min before stress. Using the immunohistochemistry and the computerized image technique, the expression levels of nNOS and iNOS of rat hippocampal CA3 pyramidal neurons were assayed quantitatively, and the NO₂^-/NO₃^- level of hippocampal homogenate was also measured using nitric acid deoxidize enzyme method. RESULTS: The nNOS average grey degree of hippocampal CA3 pyramidal neurons was significantly lower in stress group (155.42±3.77)than that in control group(164.54±4.62)and in stress plus phenytoin group(164.27±2.55)(P<0.01); The iNOS relative sectional area proportion of hippocampal CA3 pyramidal neuron was significantly larger in stress group(5.87%±2.90%) than that in control group (0.90％±0.89%) and in strers plus phenytoin groups (0.90%±0.88%)(P<0.01); The NO₂^-/NO₃^- level of hippocampal homogenate was significantly higher in stress group(42.75 umol/L±14.49 umol/L)than that in control group(21.23 umol/L±6.99 umol/L)and in stress plus phenytoin group(18.40 umol/L±8.11 umol/L)(P<0.01). CONCLUSION: It is suggested that the stress could induce nNOS and iNOS expression in CA3 pyramidal neurons and excessive production of NO in hippocampus of rats, which could be inhibited by phenytoin.     

The clinical application of fluorescently-labeled monoclonal antibody against P-selectin

AIM: To investigate the clinical significance in determination of the P-selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P-selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently-labeled SZ-51 by direct FCM comparing with indirect FCM and enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of P-selectin on platelet membrane is higher in DM (23.92%±15.83%), in hyperlipidemia (18.34%±9.46%) and in cerebral infarction (19.32%±10.38%) than normal subjects (3.38%±1.11%) (P<0.01). In addition, similar results on P-selectin were obtained by indirect FCM and ELISA in patients with DM and cerebral infarction. CONCLUSION: FITC-labeled SZ-51-IgG can be used in FCM, and it would be a new and sensitive method in detecting platelet activation.