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Aim To study the effect of proliferation and activation of vascular smooth muscle cells (VSMCs) induced by activated the complement alternative pathway and intervention. Methods Normal human plasma was specifically activated the complement alternative pathway by incubated with cobra venom factor (CVF). Exposed VSMCs to the activated complement products, the change of cell morphology and the cell viability were assayed by inverted phase contrast microscope and MTT method, respectively. The supernatant was assayed for expression of E-selectin, ICAM-1 and VCAM-1 by using ELISA reagent kits. The protein expression levels of p-NF-kB p65, NF-kB p65 and IKK were assayed by Western blot. The nucleus transcriptional activity of NF-ΚB p65 was tested by the dual luciferase reporter assay system. Pyrrolidine dithiocarbamate (PDTC) was used to intervene the proliferation and activation of VSMCs. Results VSMCs were activated and induced to proliferation after exposed to the products of activated complement alternative pathway. The expressions of E-selectin, VCAM-1 and ICAM-1 were up-regulated. The contents of ICAM-1 and VCAM-1 reached the peak at 6 h and the E-selectin increased significantly at 12 h. Meanwhile, the phosphorylation level of NF-ΚB p65, nucleus transcriptional activity of NF-ΚB p65 and the protein expression of IKK and N F - Κ B p65 increased. The above mentioned changes were clearly inhibited by PDTC. Conclusions The activated complement alternative pathway can initiate proliferation and activation of VSMCs, and its mechanism goes hand in hand with activation of N F - Κ B signaling pathway. PDTC, a specific inhibitor of N F - K B, can effectively inhibit the proliferation and activation of VSMCs.
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Aim To explore the protective effect and mechanism of icariin ( ICA ) on acute lung injury ( ALI) in mice induced by activation of the comple-ment alternative pathway. Methods 32 healthy KM mice were randomly divided into four groups: the nor-mal group, the model group, the PDTC group and the Icariin group, which received 7-day intragastric admin-istration respectively. Then cobra venom factor ( CVF) was used to activate specifically complement alternative pathway to induce acute lung injury in mice by intrave-nous injection. Myeloperoxidase ( MPO ) activity of lung homogenate, the cell count and the protein con- tent of bronchoalveolar lavage fluid ( BALF ) were measured. The concentration of IL-6, TNF-α, P-selec-tin and ICAM-1 in BALF and serum were determined by ELISA. The pathological change of lung tissue was observed by HE staining. The phosphorylation of NF-κB p65 in lung tissues was checked by immunohisto-chemistry. The effect of the transcriptional activity of NF-κB signal pathway in microvascular endothelial cells was measured by employing dual-luciferase re-porter assay system. Results ICA reduced MPO ac-tivity of lung homogenate, the cell count and the con-tent of IL-6, TNF-α, P-selectin in BALF obviously. The level of TNF-α, P-selectin and ICAM-1 in serum was decreased, the pulmonary inflammatory cell infil-tration was reduced, the phosphorylation of NF-κB p65 in lung was inhibited significantly and the transcrip-tional activity of NF-κB was also down-regulated. Con-clusion ICA can alleviate acute inflammatory re-sponse of ALI mice induced by activation of the com-plement alternative pathway. The mechanism may be highly related to the inhibition of inflammatory cell in-filtration in lung tissue, the down-regulation of phos-phorylation of NF-κB p65 and nuclear transcriptional activity.
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Aim To investigate the change of coagulation and fibrinolysis functions of human microvascular endothelial cells (HMEC) induced by activated complement alternative pathway and effect of pyrrolidine-dithiocarbamate (PDTC) and resveratrol (Res) on intervention.Methods Normal human serum was activated by cobra venom factor (CVF).After exposure of HMEC to activated complement for various time points, supernatant was removed and assayed for activities of hydrolysing chromogenic substrate and affecting activated partial thromboplastin time (APTT) and prothrombin time (PT).The cells exposed to activated complement were collected and washed, and then the cell suspension was assayed for activity of affecting coagulation function of normal plasma.Lastly, the coagulation and fibrinolysis functions of HMEC pretreated with PDTC and Res were also investigated after HMEC was exposed to activated complement alternative pathway.Results The hydrolysis activity of chromogenic substrate of supernatant was up-regulated significantly after HMEC exposed to activated complement alternative pathway.The supernatant induced APTT decreased significantly, and also shortened PT.The cell suspension of various time points induced APTT decreased significantly, and also shortened PT by suspension of 6 h time point.PDTC and Res failed to inhibit the up-regulation of the chromogenic hydrolysis activity, but Res showed significant intervention on decrease of APTT, and PDTC had better effect on inhibiting the decrease of PT than that of Res.Conclusion Activated complement alternative pathway can induce abnormality of coagulation and fibrinolysis functions of HMEC, and PDTC and Res can affect this change.
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Aim To study the development of acute lung inflammation in mice induced by activation of the complement alternative pathway and the changes of the related indicators, and to provide an ideal pathological model of acute lung inflammation in mice for drug screening and intervention. Methods Cobra venom factor( CVF) was used to activate complement alterna-tive pathway of SPF Kunming mice by intravenous injection. According to different sampling time, the mice were divided into 15 min, 30 min, 1 h, 2 h, 6 h group, and the parallel PBS control groups were set at the same time. Lung coefficient, lung water content, myeloperoxidase ( MPO ) activity, BALF cell number and protein content were tested. The pathological changes of lung tissue were observed by HE staining. The concentration of IL-6 , TNF-α, P-selectin and ICAM-1 in bronchoalveolar lavage fluid ( BALF ) and serum were determined by ELISA. Results CVF caused pulmonary inflammatory cell infiltration in mice obviously. Compared with PBS groups, MPO activity of lung tissue, BALF cell and the protein concentration were significantly increased. The contents of IL-6, TNF-α, P-selectin in BALF and serum were in-creased, and the content of ICAM-1 in serum was also increased. The content of P-selectin in BALF reached the first peak at 30 min point, the content of IL-6 and TNF-α in BALF reached the first peak at 1 h point, but the indicators had no further changes at 2 h point, and all the indicators rose again at 6 h point. The lev-els of IL-6 and TNF-α in serum reached peak at 1 h point,then the content showed lower levels at the sub-sequent time points. The levels of P-selectin and ICAM-1 in serum increased along the time. Lung coef-ficient, lung water content and ICAM-1 of the BALF showed no significant alteration. Conclusion The ac-tivation of the complement alternative pathway can lead to acute lung inflammation in mice and the inflammato-ry response is the most obvious at 30 min to 1 h. The study could provide an ideal pathological model of a-cute lung inflammation in mice for drug screening and intervention.
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Aim To investigate the effect of resveratrol ( Res) , PDTC and AG490 on adhesion molecules ex-pression induced by product of activated complement alternative pathway on human microvascular endothelial cells ( HMECs) and the possible mechanisms. Meth-ods HMECs were exposed to the product of comple-ment alternative pathway activation, then the superna-tant was removed to detect the concentration of malond-ialdehyde ( MDA ) with TBA method. ELISA method was used to detect the expression of soluble ICAM-1 , VCAM-1 ( and E-selectin) in the culture supernatant. Res, PDTC and AG490 with different concentrations were used to determine their effect on cell oxidation level and adhesion molecules expression. The phospho-rylation of NF-κB p65 was detected by Western blot, and the intervention of Res, PDTC and AG490 was as-sayed by the same way. Results The activation of complements alternative pathway resulted in the phos-phorylation of NF-κB p65 , and increased the concen-tration of MDA and up-regulated the expression of ICAM-1, VCAM-1 and E-selectin. Res reduced the concentration of MDA. Res, PDTC and AG490 inhibi-ted the phosphorylation of NF-κB p65 . Res and PDTC showed similar inhibition on expression of ICAM-1 and VCAM-1 , while exhibiting little effect on expression of E-selectin, and AG490 significantly inhibited the ex-pression of the above adhesion molecules. Conclusions Res, PDTC and AG490 could inhibit the expression of adhesion molecules induced by activated complement alternative pathway, the inhibition of NF-κB pathway activation was involved in their mechanism, and JAK2 may be a more important intervention target in regula-ting adhesion molecule expression.
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ObjectiveTo investigate synergistic effect of donor livers blocked by recipient blood serum (RS) and cobra venom factor (CVF) treatment to inhibit hyperacute rejection (HAR) happened in liver xenotransplantation.MethodsThe SD rat blood serum was prepared for pre-perfusing the donor livers before experiment.24 pairs of guinea-pig (GP) and Sprague-Dawley (S.D.) rats were choiced respectively and pair-matched between GP donor and rat recipient randomly.Before transplantation,donor livers of GPs were pre-perfused by 0.5％ SD rat serum.Paired animals were divided into 4 groups randomly such as donor liver perfused by RS,recipient treated by CVF,RS+ CVF performed and Ringer solution as a control.The orthotopic liver xenotransplantations was performed with two-cuff technique.The survival time and liver function of recipients,morphological and pathological changes of rat livers were observed.ResultsThere was no piebaldism change on the recipient liver from experimental group.The survival time of recipients from RS+CVF group [(161.5±30.9) min]was longer than that of control[(45.2 ± 13.9) min] and CVF[(125.2 ± 25.5) min] or RS groups [(88.1±19.7) min] (P＜0.05).The ALT in serum of recipients from RS+CVF [(63.2±13.9)U/L]was lower than that from congtrol group [(126.1±23.3)U/L](P＜0.01) and CVF group [(79.9±18.1)U/L](P＜0.05) or RS group [(106.1±19.3)U/L](P＜0.01) The histological damages including thrombosis,interstitial bleeding and edema of recipient liver from RS+CVF group were alleviated markebly than that of other groups (P＜0.05).ConclusionThere was a synergistic effect to inhibit HAR happened in liver xenotransplantation by blocking the donor liver with recipient blood serum and CVF treatment significantly.
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PURPOSE: We designed a pig to canine liver xenotransplantation model to study the diverse immunologic and hemodynamic consequences that follow xenotransplantation and hyperacute rejection. METHODS: The animals were divided into two groups: the cobra venom factor and Gadolinium chloride treatment group (CVF+Gd group) (3 cases) and the control group (3 cases). The donor pig's whole liver was harvested, and then the harvested pig's whole liver was transplanted into a dog after the dog underwent left hepatectomy. After reperfusion of the graft, blood samples were taken 20, 40 and 60 minutes after reperfusion, and the liver, lung and kidney tissues were taken 1 hour after reperfusion. RESULTS: In the control group, the grafts showed a patchy hypoperfused liver surface and it felt rubbery solid compared to the CVF+Gd group. The serum total protein, albumin, fibrinogen and platelets decreased abruptly and there were no significant differences between the two groups. The serum PT, PTT and FDP were increased in both groups and the CVF+Gd group showed a more obtuse slope than the control group. We could not find any intravascular pathologic changes on the microscopic findings of the graft. Only scant intravascular fibrin deposition was found. Hepatocellular vacuolization and sinusoidal dilatation were also found. There were patches of necrosis without any zonal distribution, intrasinusoidal neutrophil sequestration and interstitial hemorrhage. These findings were milder in the CVF+Gd group. CONCLUSION: The pig to canine partial auxiliary liver xenotransplantation model is feasible and it is a good model before starting to perform pig to primate liver xenotransplantation. In the CVF+Gd group, pathologic findings like patch hepatocyte necrosis etc. were less severe. As there were no corresponding vascular pathologic findings, these findings are not the direct effect of CVF and gadolinium treatment, and so other factors like Ischemia- reperfusion injury should be considered.
Subject(s)
Animals , Dogs , Humans , Blood Platelets , Elapid Venoms , Complement System Proteins , Dilatation , Fibrin , Fibrinogen , Fluconazole , Formycins , Gadolinium , Hemodynamics , Hemorrhage , Hepatectomy , Hepatocytes , Kidney , Kupffer Cells , Liver , Lung , Necrosis , Neutrophils , Primates , Rejection, Psychology , Reperfusion , Reperfusion Injury , Ribonucleotides , Tissue Donors , Transplantation, Heterologous , TransplantsABSTRACT
Aim To investigate the inhibitory effect of atrase B on human platelet aggregation induced by activated complement.Methods By employing CVF to activate complement,the effect of atrase B on gel filtered platelet aggregation induced by activated complement was measured by turbidimetry and the expression of P-selectin and GPⅡb/Ⅲa on platelet membrane were detected by flow cytometry.Results Atrase B inhibited platelet aggregation and the expression of P-selectin and GPⅡb/Ⅲa on platelet membrane induced by activated complement.Conclusion Anticomplementary protein atrase B from Naja atra venom can significantly inhibit platelet activation and aggregation induced by activated complement.
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Recently xenotransplantation has been thought as a final solution for the controi of donor organ shortage in allograft. In order to be a ciinicai entity, xenotransplantation has many obstacles such as hyperacute rejection and delayed xenogratt rejection as a potent immunologic reaction, zoonosis and ethical problems. We already reported the eariy immunoiogic events occuring soon after xenograft in animal model, in which natural antibody and complement have a crucial roie in rejection response. As a further step for the prolongation of graft survival, we used anticomplement agent (cobra venom factor, CVF) in the same model. Graft survival in discordant (guinea pig-to-rat) xenogratt was extended from 30.6 minutes to 2 days following singie injection of CVF, which showed similar pattern of rejection with the concordant xenogratt in terms of time of rejection response after grafting. In this setting antibody response in the blood did not show any difference between that of pre CVF and post CVF, even though IgM response was more pronounced than IgG. The complement activity in the blood showed marked suppression following CVF injection. Intragraft complement gene (C3 mRNA) expression in CVF injected discordant showed delayed response in a similar pattern like that of concordant xenograft. Interestingly enough intragraft anticomplement gene expression showed the simiiar pattern of response with the complement. From these results we can conclude that anticomplement agent (CVF) extended the graft survival in discordant xenograft upto the level of concordant xenograft by shifting the complement activation response from that of discordant to concordant xenograft.
Subject(s)
Rats , AnimalsABSTRACT
AIM To establish the model of exudative pleurisy induced by alternatively activating complement and to study protection of anti inflammatory drugs on it. METHODS Purified cobra venom factor (CVF) was injected into the pleura, and then exudate volumn, total leukocyte count, protein content and histamine concentration were determined. RESULTS CVF, injected into rat pleura at dose of 0 08～2 mg?kg -1 , induced exudative inflammation in dose dependent and time dependent manner. Both diphehydramine and isoprenaline reduced significantly exudate volume, total leukocyte count, protein content and histamine concentration, while indomethacin, dexamethasone and cyclophosphamide also reduced exudate volume, total leukocyte count and protein content, although they did change the release of histamine. DLF, one of the toxic components in cobra venom, potentiated CVF induced inflammation. CONCLUSION CVF can induce significantly exudative pleurisy sensitive to anti inflammatory agents. DLF has augmentation on CVF induced inflammation.