Keyhole limpet hemocyanin induced Th1/Th2 imbalance in splenocytes of Balb/C mice / 中国组织工程研究

BACKGROUND:Establishing a characteristic.stable and repeatable model of Th1/Th2 imbalance in animals,is the key of studying the mechanism of Th1/Th2 imbalance.OBJECTIVE:To observe the characteristics of Th1/Th2 imbalance in splenocytes derived from Balb/C mice immnnized by keyhole limpet hemocyanin(KLH).DESIGN:A randomized control exploratory experiment.SETTING:Hebei Provincial Forensic Laboratory.Institute of Basic Medicine,Hebei Medical University.MATERLALS:The experiment was carried out in the Hebei Provincial Forensic Laboratory,Institute of Basic Medicine,Hebei Medical University from September 2005 to January 2007.Balb/C mice were adopted in this study.and all the disposals were in accordance with the guidance of animal ethics.METHODS:Balb/C mice were immunized with KLH emulsified in complete Freund's adjuvant(CFA),splenocytes were acquired,and the peak of cytokine secretion was determined in 3 groups:KLH groups of 6.25 mg,kg.12.5 mg,kg and 25 mg/kg.According to the immunizing dose and immunizing frequency.mice were divided into 7 groups:KLH groups of 6.25 mg/kg,12.5 mg/kg and 25 mg/kg,secondary immunity groups of 6.25 mg/kg,12.5 mg/kg and 25 mg/kg,as well as control group.According to the determined levels of IgG1 and IgG2a in blood serum.mice were divided into KLH group of 6.25 mg/kg and control group.MAIN OUTCOME MEASURES:Mice splenocytes supematant was detected with enzyme linked immunosorbent assay (ELISA)for the production of Th1 cytokines interferon (IFN)-γ,interleukin(IL)-2.IL-12 p40 and Th2 cytokines IL-4 and IL-5.The levels of Th1 antibody IgG2a and Th2 antibody IgG1 in blood serum were also detected by ELISA.RESULTS:The spleen derived from KLH-immunized mice appeared hypertrophy,and the number of splenocytes was manifold.Splenocytes restimulated with KLH in vitro produced much more IFN-γ,IL-2,IL-4,IL-5,but not IL-12p40.IL-2 secretion was obviously elevated after incubated for 24 hours and achieved pinnacle at 48 hours;productions of IL-4,IL-5 and IFN-γ were elevated after 24 hours,and increased gradually to 96 hours;IL-12p40 production was very low at every time point.Using different doses of KLH inlmunity once or twice,could all lead to the elevated productions of IL-2,IL-4,IL-5,IFN-γ,and the elevation of IL-4/IFN-γ ratio,but the secondary immunity group of 6.25 mg/kg KLH showed obviously higher levels than other groups(P＜0.01).The level of KLH specific antibody IgG2a and IgG1,especially IgG1 was elevated in blood serum of KLH-immunized mice.CONCLUSION:Balb/C mice immunizad with KLH emulsified in CFA can indce a Th2 predominant imbalance in splenocytes.

Effects of cholecystokinin octapeptide on TNF- α- induced IL- 6 expression and its possible molecular mechanismin rat synovial cell strain RSC-364 / 中国病理生理杂志

AIM: To investigate the effect of sulfated cholecystokinin octapeptide (CCK -8 ) on TNF -α induced IL - 6 mRNA expression, NF - κB activation in the rat fibroblast - like synovial cell strain RSC - 364 and its possible receptor mechanisms. METHODS: RSC -364 cells were stimulated with TNF - α( 10 μg/L) in the presence or absence of sCCK- 8( 10-8 - 10-6 mol/L) or/and CCK receptor antagonist proglumide(2 mg/L). IL -6 and CCK receptor A/B (CCK- AR/CCK/BR) mRNA expression were assayed by reverse transcription polymerase chain reaction (RT- PCR) at 3 h after stimulation, and nuclear factor - κB (NF - κB) binding activity was analyzed by electrophoretic mobility shift assay (EMSA) at lh after stimulation. At 30 min of stimulation the IκB protein level in cytoplasma was measured by Western blotting. RESULTS: Both CCK - AR and CCK - BR were constitutively expressed on RSC - 364. sCCK - 8, at concentrations from 10-8 mol/L to 10 -6 mol/L, significantly increased IL - 6 mRNA expression, CCK - AR and CCK - BR mRNA expression, NF - κB binding activity and IκB protein degradation. The effects of sCCK - 8 on NF - κB activity and IκB degradation level were attenuated by CCK receptor antagonist proglumide. CONCLUSION: sCCK - 8 upregulats TNF - α- induced IL - 6 mRNA expression by NF - κB pathway through its receptor on rat synoviocytes, suggesting its possible regulatory role in the pathogenesis of rheumatoid arthritis.

Cholecystokinin octapeptide inhibits the in vitro expression of CD14 in rat pulmonary interstitial macrophages induced by lipopolysaccharide / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study the effect of cholecystokinin octapeptide (CCK-8) on lipopolysaccharide (LPS)-stimulated pulmonary interstitial macrophages (PIM) in vitro.</p><p><b>METHODS</b>PIM were isolated and cultured in the presence or absence of LPS, CCK-8, proglumide (the antagonist of CCK receptors) and vehicle. The expression of membrane CD14 (mCD14) protein was assayed by flow cytometry and soluble CD14 (sCD14) in the supernatant was analyzed semi-quantitatively by Western blot. TNF-alpha in the supernatant was detected with ELISA.</p><p><b>RESULTS</b>CCK-8, at concentrations of 10(-7) mol/L and 10(-6) mol/L, significantly inhibited the expression of mCD14. Release of sCD14 and TNF-alpha in the supernatant was up-regulated by LPS (1 microg/ml) but reduced by CCK-8. The effect of CCK-8 was inhibited by proglumide.</p><p><b>CONCLUSION</b>CCK-8 negatively modulated several functions of LPS-stimulated PIM through CCK receptors. This may be one of the mechanisms for CCK-8 to alleviate inflammation in lung tissue during endotoxemia.</p>

Brain injury induced by ischemia-reperfusion of rat hindlimbs and its mechanisms / 中国病理生理杂志

AIM:To investigate the pathologic changes in the brain and its underlying mechansims during ischemia-reperfusion of rat hindlimbs.METHODS:SD rats were divided into the normal(N), sham(S), 4 h ischemia without reperfusion(I), and 4 h ischemia-2, 6,12,18 or 24 h reperfusion (I-R) groups at random. Ischemia and ischemia-reperfusion were established with the occlusion or/and re-opening of the terminal of abdominal aorta, respectively. The pathologic changes in the brain tissue were morphologically observed. The expression of inducible nitric oxide synthase (iNOS) mRNA, and iNOS protein and the nitrotyrosine, a marker of peroxynitrite (ONOO-)，in the brain tissue were detected with RT-PCR and immunohistochemical technique, respectively. The brain superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents were spectraphotometrically measured.RESULTS:Hydropic degeneration and severe injury to neurons were only showed in I-R group. Expressions of iNOS mRNA and protein were demonstrated in I-R, I and S groups, which were maximal in I-R 6 h group. iNOS positive neurons and microglias were more spread in I-R 6 h group than those in S and I groups. NT positive neurons were localized in the cerebral cortex and hippcampus of I-R 6 h group. The contents of MDA markedly increased, while the activity of SOD significantly decreased in I-R 6 h group compared to the N, S and I groups. There were no significant changes in MDA and SOD in N, S and I groups.CONCLUSION:Severe ischemia-reperfusion of rat hindlimbs could induce brain injury, and its mechanisms might be related to enhanced expression of iNOS-NO-ONOO- in the brain.

Effects of cholecystokinin octapeptide on TNF-?-induced IL-6 expression and its possible molecular mechanism in rat synovial cell strain RSC-364 / 中国病理生理杂志

AIM:To investigate the effect of sulfated cholecystokinin octapeptide(CCK-8) on TNF-? induced IL-6 mRNA expression,NF-?B activation in the rat fibroblast-like synovial cell strain RSC-364 and its possible receptor mechanisms.METHODS:RSC-364 cells were stimulated with TNF-?(10 ?g/L) in the presence or absence of sCCK-8(10-8-10-6 mol/L) or/and CCK receptor antagonist proglumide(2 mg/L).IL-6 and CCK receptor A/B(CCK-AR/CCK/BR) mRNA expression were assayed by reverse transcription polymerase chain reaction(RT-PCR) at 3 h after stimulation,and nuclear factor-?B(NF-?B) binding activity was analyzed by electrophoretic mobility shift assay(EMSA) at 1h after stimulation.At 30 min of stimulation the I?B protein level in cytoplasma was measured by Western blotting.RESULTS:Both CCK-AR and CCK-BR were constitutively expressed on RSC-364.sCCK-8,at concentrations from 10-8 mol/L to 10-6 mol/L,significantly increased IL-6 mRNA expression,CCK-AR and CCK-BR mRNA expression,NF-?B binding activity and I?B protein degradation.The effects of sCCK-8 on NF-?B activity and I?B degradation level were attenuated by CCK receptor antagonist proglumide.CONCLUSION:sCCK-8 upregulats TNF-?-induced IL-6 mRNA expression by NF-?B pathway through its receptor on rat synoviocytes,suggesting its possible regulatory role in the pathogenesis of rheumatoid arthritis.

Inhibitory effects of CCK-8 on NF-?B activities stimulated by LPS in rat PIMs / 中国病理生理杂志

AIM: To investigate the inhibitory effects of cholecystokinin octapeptide(CCK-8) on nuclear factor-?B(NF-?B) activities stimulated by lipopolysaccharide(LPS) by using forskolin,the activator of adenylate cyclase,and PKA inhibitor H-89 in rat pulmonary interstitial macrophages(PIMs).METHODS: PIMs were isolated and purified.EMDA was applied to detect NF-?B activities and Western blotting was used to analyze the I?B-? protein level in rat PIMs.RESULTS: The NF-?B activity was not detected in normal control rat PIMs.The NF-?B activity in LPS-treated rat PIMs was obviously higher than that in control group(P0.05).The NF-?B activity in CCK+LPS group and LPS+Fsk group were obviously lower than that in LPS group(P

Cholecystokinin octapeptide inhibits tumor necrosis factor-? transcription and nuclear factor-?B activity induced by lipopolysaccharide in rat pulmonary interstitial macrophages / 中国病理生理杂志

AIM: To elucidate the anti-inflammatory mechanism of cholecystokinin octapeptide (CCK-8). METHODS: The pulmonary interstitial macrophages (PIMs) from rats were stimulated with LPS (1 mg?L~(-1)) in the presence or absence of CCK-8 (10~(-8)-10~(-6) mol?L~(-1)) or/and CCK receptor antagonist proglumide (2 mg?L~(-1)). The expression of TNF-? mRNA was assayed by reverse transcription polymerase chain reaction (RT-PCR) at 3 h of the stimulation, and nuclear factor-?B (NF-?B) binding activity was analyzed by electrophoretic mobility shift assay (EMSA) at 1 h of stimulation. The I?B? protein level in the cytoplasma at 30 min of the stimulation was detected by Western blot. RESULTS: CCK-8, at concentrations from 10~(-8) mol?L~(-1) to 10~(-6) mol?L~(-1) obviously inhibited LPS-induced TNF-? mRNA expression and NF-?B binding activity in a dose-dependent manner. Stimulation with LPS resulted in a reduction of I?B? protein level in PIMs, which was elevated by CCK-8. The effects of CCK-8 on NF-?B activity and I?B protein level were attenuated by CCK receptor antagonist proglumide. CONCLUSION: CCK-8 inhibits LPS-induced TNF-? mRNA expression by regulating NF-?B activity in rat PIMs, which is mediated through CCK receptors and inhibition of I?B? degradation. This represents one of the anti-inflammatory mechanisms of CCK-8.

Inhibitory effect of cholecystokinin octapeptide on expression of CD14 on rat pulmonary interstitial macrophages induced by lipopolysaccharide in vitro / 中国病理生理杂志

AIM: To study the effect of cholecystokinin octapeptide(CCK-8) on lipopolysaccharide (LPS)-stimulated pulmonary interstitial macrophage(IM) in vitro. METHODS: Pulmonary IM were isolated and cultured in the presence of LPS, CCK-8, proglumide(the antagonist of CCK receptors) and vehicle alone or together. The expression of mCD14 protein was assayed by flow cytometry, and sCD14 in the supernatant was analyzed semi-quantitatively by Western blot, and TNF-? in the supernatant was detected with ELISA. RESULTS: CCK-8, at concentrations from 10 -7 mol/L to 10 -6 mol/L inhibited significantly the expression of mCD14, the release of sCD14 and TNF-? to the supernatant up-regulated by LPS(1 mg/L). The effect of CCK-8 was inhibited by proglumide. CONCLUSION: CCK-8 modulated negatively several functions of LPS-stimulated pulmonary IM through CCK receptors, which may be one of the mechanisms for CCK-8 to alleviate the inflammation in lung tissues during endotoxemia.

Brain injury induced by ischemia-reperfusion of rat hindlimbs and its mechanisms / 中国病理生理杂志

AIM: To investigate the pathologic changes in the brain and its underlying mechansims during ischemia-reperfusion of rat hindlimbs.METHODS: SD rats were divided into the normal(N), sham(S), 4 h ischemia without reperfusion(I), and 4 h ischemia-2, 6,12,18 or 24 h reperfusion (I-R) groups at random. Ischemia and ischemia-reperfusion were established with the occlusion or/and re-opening of the terminal of abdominal aorta, respectively. The pathologic changes in the brain tissue were morphologically observed. The expression of inducible nitric oxide synthase ( iNOS ) mRNA, and iNOS protein and the nitrotyrosine, a marker of peroxynitrite (ONOO -),in the brain tissue were detected with RT-PCR and immunohistochemical technique, respectively. The brain superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents were spectraphotometrically measured.RESULTS: Hydropic degeneration and severe injury to neurons were only showed in I-R group. Expressions of iNOS mRNA and protein were demonstrated in I-R, I and S groups, which were maximal in I-R 6 h group. iNOS positive neurons and microglias were more spread in I-R 6 h group than those in S and I groups. NT positive neurons were localized in the cerebral cortex and hippcampus of I-R 6 h group. The contents of MDA markedly increased, while the activity of SOD significantly decreased in I-R 6 h group compared to the N, S and I groups. There were no significant changes in MDA and SOD in N, S and I groups.CONCLUSION: Severe ischemia-reperfusion of rat hindlimbs could induce brain injury, and its mechanisms might be related to enhanced expression of iNOS -NO-ONOO - in the brain.

Effect of CCK-8 on IL-12 secreted in murine bone marrow-derived dendritic cell induced by LPS / 中国药理学通报

Aim To investigate the effects of cholecystokinin-octopeptide on IL-12 secreted in murine bone marrow-derived dendritic cells induced by lipopolysaccharide.Methods The CCK receptor subtypes were investigated by immunofluorescence in murine bone marrow-derived dendritic cells.Enzyme linked immunosorbent assay and Western blot were used to estimate the contents of IL-12 and p38MAPK activity.Results CCK-1R and CCK-2R were detected in BM-DC;CCK-8(at concentrations of 10-10,10-8,10-6 mol?L-1)could significantly increase the secretion of IL-12 in the LPS-induced DC, and LPS-activated p38MAPK activity in a dose-dependent manner.The effect of CCK-8 was reduced partially by CR1409(a CCK-1R antagonist) and CR2945(a CCK-2R antagonist).Conclusion CCK-8 could dose-dependently increase the expressions of IL-12 in LPS-induced DC,probably by promoting p38MAPK activity and the effect was mediated by CCK1 and CCK2 receptors.

Cholecystokinin octapeptide modulates T-lymphocyte subsets in KLH-immunized mice / 中国病理生理杂志

AIM: To study the effects of cholecystokinin octapeptide (CCK-8) on T-lymphocyte subsets in keyhole limpet haemocyanin (KLH)-immunized mice.METHODS: Female BALB/c mice were immunized with KLH and injected with different dosages of CCK-8 or saline simultaneously. Positive CD4+and CD8+ T cells in peripheral blood or splenocytes were measured by flow cytometry. The production and mRNA level of Th1 cytokine, IFN-? and Th2 cytokine, IL-4 in the splenocytes were evaluated by ELISA and RT-PCR, respectively. In addition, lung tissue sections were made with HE staining.RESULTS: CCK-8 downregulated the positive CD4+and CD8+ T lymphocytes both in peripheral blood and in splenocytes in KLH-immunized mice, resulting in the reduction of CD4+/CD8+ ratio.CCK-8 improved the production of IFN-?, elevated IFN-? gene transcription, whereas cut down the production of IL-4, decreased IL-4 gene transcription. CCK-8 lightened the pulmonary inflammation induced by KLH.CONCLUSION: CCK-8 inhibits CD4+T lymphocytes activation, Th2 cytokine mRNA expression and protein production in KLH-immunized mice, indicating that CCK-8 modulates adaptive immune response.

Inhibitory effect of CRE-decoy ODN on the upregulation of CCK a nd fosB mRNA induced by chronic morphine administration in SK-N-SH cells / 中国病理生理杂志

AIM: To investigate the inhibitory effect s of a synthetic CRE-transcription factor decoy oligodeoxynucleotide (CRE-decoy ODN) on the upregulation of the expression of cholecystokinin (CCK) and fosB mRN A induced by chronic morphine administration in SK-N-SH cells. METHODS: The CRE cis-element, TGACGTCA, was palindromic, a sy nthetic single-stranded phosphorothioate oligodeoxynucleotide composed of the CR E sequence self-hybridizes to form a duplex/hairpin. The CRE-palindromic decoy a nd control oligodeoxynucleotides were added to the medium (1 h before exposure t o morphine) at 150 nmol/L in the presence of cationic lipid DOTAP. After the cel ls were treated with 100 ?mol/L morphine for 48 h, 10 ?mol/L naloxone was use d for 15 min. The effects of CRE-decoy ODN on the DNA-binding activity of CREB, the expression of CCK and fosB mRNA were detected by electrophoresis mobi lity shift assay (EMSA) and RT-PCR, respectively. The stability of cell-incorpo rated [ 32P]-labeled CRE-decoy ODN was extracted with phenol:chloroform a nd then subjected to 20% nondenaturing polyacrylamide gel electrophoresis and au toradiography. RESULTS: Chronic morphine administration and acute naloxone-prec ipitated withdrawal significantly activated the DNA-binding activity of CREB and the expression of CCK and fosB mRNA in SK-N-SH cells. The CRE-decoy ODN pen etrated into the cells, specifically downregulated these indexes. CONCLUSIONS: CRE-decoy ODN can significantly downregulates the e xpre ssion of CCK and fosB mRNA through specifically suppressing the DNA-binding activity of CREB activated by chronic morphine administration in SK-N-SH cells.

Binding characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages / 中国病理生理杂志

AIM: To investigate the binding characteristics of cholecystokinin receptors in rat pulmonary interstitial macrophages (PIMs). METHODS: The PIMs were isolated from rat lung tissues, purified using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. The PIM membrane was obtained by supercentrifuge. Receptors for CCK in PIMs were examined using [~3H] labeled CCK-8S as ligand. The specificity of [~3H]-CCK-8S binding to PIMs membrane and the subtypes of CCK receptors were determined by competitive inhibition experiments with CCK-8S, CCK-A and CCK-B receptors selective antagonists (CR1409 and CR2945). The effects of time and incubation temperature on the specific binding were also observed. RESULTS: The specific binding of [~3H]-CCK-8S was not detected in normal rat PIMs, but was detected in the rat administrated with LPS for 48 h. The capacity of ligand-receptor binding was dependent on the incubation temperature and time. Scatchard analysis of the saturation curves suggested that the presence of CCK receptors with high affinity [Kd=(0.68?0.28)mmol/L] and low binding capacity [Bmax=(32.50?2.70) pmol?g~(-1) protein] in PIMs. By means of competitive inhibition studies, the specific binding of [~3H]-CCK-8S to rat PIMs was inhibited by unlabelled CCK-8S [IC_(50)=(3.20?1.13) nmol/L], CCK-AR specific antagonist CR 1 409 [IC_(50)=(0.19?0.06)?mol/L] and CCK-BR specific antagonist CR 2945[IC_(50)=(2.30?0.80)nmol/L]. CONCLUSION: These results suggest the presence of two subtypes of CCK-AR and CCK-BR and provide a structural basis for CCK to play a pivotal role in PIMs.

Changes of HO - 1 expression in kidney following ischemia and reperfusion of limbs and their significance in rats / 中国病理生理杂志

AIM: To detect the changes of heme oxygenase-1 (HO-1) expression in kidney following ischemia-reperfusion of hindlimbs and to elucidate their significance. METHODS: Health SD rats were randomly divided into normal (N), sham (S), 4h ischemia without reperfusion (I), 4 h ischemia-2, 6, 12, 18 or 24 h reperfusion (I-R) groups and I-R18h+zinc protoporphyrin (ZnPP) group. I-R was established using the occlusion for 4 h and re-opening for 2-24 h of the femoral arteries. In I-R 18 h+ZnPP group, ZnPP (5 ?mol?kg~(-1) body weight) was intravenously injected 6 h and 12 h after reperfusion, respectively. The expression of HO-1 mRNA in kidney was detected with reverse transcription-polymerase chain reaction (RT-PCR). The expression and location of HO-1 protein were detected with immunohistochemical technique. The observation of pathologic changes of kidney was made following the inhibition of HO-1 by ZnPP. RESULTS: The relative expression level of HO-1 mRNA significantly increased in I-R group, compared to those in the control groups, It was maximal in I-R 18 h group, and thereafter expression level of HO-1 mRNA decreased, however significant expression was still detected in I-R 24 h group (P

Changes of iNOS gene expression in kidney following ischemia and reperfusion of limbs and their significance in rats / 中国病理生理杂志

AIM: To detect the changes of inducible nitric oxide synthase (iNOS) expression in kidney following ischemia-reperfusion(I-R)of hindlimbs and to elucidate their significance. METHODS: I-R was established using the occlusion of the femoral arteries for 4 h and re-opening for 2-24 h in rats. The expression of iNOS mRNA,and iNOS protein and the nitrotyrosine (NT),a marker of peroxynitrite (ONOO -),in renal tissue were detected with reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical technique,respectively. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents in renal tissue were spectraphotometrically measured. The observation of pathologic changes of renal tissue was made following the inhibition of iNOS by aminoguanidine (AG). RESULTS: Compared with control group,the relative expression level of iNOS mRNA significantly increased in I-R group. There were more iNOS and more NT positive product in the epithelial cells of renal proximal convoluted tubules and thick segments of Henle′ loops in I-R group than control group. The contents of MDA markedly increased,while the activity of SOD significantly decreased in I-R group,compared to those in the control groups. The pathologic changes of kidney became milder in I-R group following the inhibition of iNOS by AG. CONCLUSION: The expression of iNOS mRNA and protein in renal tissue were significantly upregulated,excess induction of NO contributed to the kidney injury during the I-R of hindlimbs.

Changes of HO-1 gene expression in brain following ischemia and reperfusion of limbs / 中国病理生理杂志

AIM: To detect the changes of heme oxygenase-1(HO-1) expression in brain following ischemia-reperfusion of hindlimbs in rats and to elucidate their significance. METHODS: Ischemia-reperfusion was established using the occlusion for 4 h and re-opening for 2-24 h of the femoral arteries. The expression of HO-1 mRNA in brain was detected with reverse transcription-polymerase chain reaction (RT-PCR). The expression and location of HO-1 protein were detected with immunohistochemical technique. The observation of pathologic changes of brain was made following the inhibition of HO-1 by zinc protoporphyrin (ZnPP). RESULTS: The relative expression level of HO-1 mRNA significantly increased in I-R group, compared to those in control group. It was maximal in I-R 12 h group, and thereafter expression level of HO-1 mRNA decreased, however significant expression was still detected in I-R 24 h group (P