Effect Mechanism Of E-selectin And The Intervention Of Drugs In Vinorelbine Induced Infusion Phlebitis

Infusion phlebitis is a common clinical drug-induced disease with high morbidity and perniciousness, bringing more unnecessary physiological or psychological pain and additional medical cost to patient, interfering their normal compliance and curative effect of pharmacotherapy severely. This is a kind of severe and frequent adverse drug reactions. However, there is little efficient and special way to prevent or cure the side effect. To develop such a standard prevent and treatment program, more basic theory should be established by research.Without comprehensive basic study, many scattered clinical observation resulted discrepant outcomes and could not be applied to practice. Many infusion phlebitis caused by the infusion drugs such as antibiotics and chemotherapeutic agents. Vinorelbine (VIN) is a semisynthetic vinca alkakoid derived from vinblastin that has been shown to be active in a variety of malignancies. Although VIN has a better toxicity profile than other vinca alkaloids;it remains a vesicant and venous irritant. Frequent and intolerant phlebitis limited the use of this effective agent and decreased the life-quality of patients. Effective prophylaxis should be based on an understanding of the possible pathogenesis.Recently, some literature has reported laboratory data about antibiotics associated with infusion phlebitis. These data showed that the incidence of infusion phlebitis may be related to the up-regulation of the expression of some adhesion molecules such as ICAM-1 on endothelial cells. One of a published clinical study indicated that cimetidine (CIM), a classic antagonist to H₂ histamine receptor, showed a significant preventive effect from VIN inducing infusion phlebitis. Similarly, other studies on tumor metastasis approved that CIM can inhibit cancer cell adhesion to endothelial cells and prevent metastasis by blocking E-selectin expression. Do these two effects of CIM have a same mechanism? Does E-selectin play an important role in the pathogenesis of infusion phlebitis induced by vinca alkakoid? And by what kinds of pathway does it work?To discuss the problems above, we did the studies below. Firstly, we established an animal test model of vinorelbine-induced infusion phlebitis, and took a pathological observation during the phlebitis formation. We graded and scored the histopathologicalchanges according the degrees of loss of venous endothelial cells, inflammatory cell infiltration, edema and thrombus, respectively. Secondly, we investigated the expression and regulation by drugs of E-selectin on vessel endothelial cells of animals and cultured ECV304 endothelial cells by irnmunohistochemistry, ELISA, flow cytometry. Furthermore, we undertook an adhesion experiment between the endothelial cells and neutrophils using a Rose Bengal staining assay, which indicated that the adhesion function of endothelial cells was enhanced with the stimulation of VIN and weakened after pretreatment of CIM and DMF (dimethylfumarate). These changes on functions were consistent with that of the expression of E-selectin. Using a real-time PCR method, we measured the mRNA of E-selectin on the fourth step, which showed consistent results compared to the expression of protein expression of E-selectin and indicated that the effect site of VIN and other test drugs were in the transcription phase. At last, we measured the level of total NF-KBp65, activated NF-icBp65 and Ser536 phosphorylated NF-icBp65 by an ELISA method. The result indicated that the up-regulation by VIN and the down-regulation by CIM and DMF to E-selectin were in a NF-kB dependent manner.Part I: Histopathological study of infusion phlebitis induced by vinorelbine and its influence factors1. Rabbits and mice were administered by VIN in intravenous infusion or injection approach, respectively. Experiment conditions including drug intervention time, sampling site, drug concentration and infusion rate were optimized in the rabbit model, according the pathological findings including loss of venous endothelial cells, inflammatory cell infiltration and thrombus. The optimized conditions were that 5 mg/kg of VIN were infused in a rate of 12 mL/h, concentration of 10 mg/mL to rabbit auricular vein, then sampled after 24 h at the number 2 sampling site (0-1 cm region in front of the catheter tip).2. Grading and scoring the histopathological changes of rabbits and mice treated by different drugs was done according the degrees of loss of venous endothelial cells, inflammatory cell infiltration, edema and thrombus, respectively. Statistical results indicated that CIM could not block the emergence of pathological changes above (P > 0.05), but can weaken the degree of them (P < 0.05). It could be used to lighten the symptom of infusion phlebitis in clinic.3. Several serum indexes were measured by ELISA including adhesion molecules such as sICAM-1, sVCAM-1, sE-selectin and sP-selectin, and indexes of the balance of fibrinolysissuch as t-PA and PAI-1 before and after the drug administration. The results showed that sE-selectin level can be up-regulated from (0.920±0.522) ng/mL to (1.534±0.449) ng/mL (P < 0.05) after VIN treatment. CIM can down-regulated it from (1.715±0.354) ng/mL to (1.012±0.659) ng/mL (P < 0.05). It indicated that the expression of E-selectin is associated with the infusion phlebitis induced by VIN. The level of t-PA was decreased by 35.41 % after the stimulation of VIN, which indicated a disorder of the balance of fibrinolysis was induced by VIN.Part II The expression drug regulation of E-selectin during the infusion phlebitis induced by vinorelbine1. The expression of E-selectin in rabbits and mice models and in ECV304 endothelial cells were measured by immunohistochemistry. It showed that VIN can enhance the expression of E-selectin while CIM can weaken its expression.2. Soluble E-selectin in culture medium of ECV304 cells were measured before and after the stimulation of VIN and other test drugs. It increased from (930.939±103.040) pg/mL to (1278.467±62.197) pg/mL (P < 0.01) by the stimulation of VIN;and decreased to (1070.267±103.040) pg/mLCP < 0.05) by CIM. However, the changes of soluble ICAM-1 didn't show a correlation to the drug interventions.3. Measurement of the E-selectin on ECV304 plasmalemma was undertaken by flow cytometry technology. It also showed a consistent result to the ELISA results above. It confirms the conclusion that VIN can up-regulate and CIM and DMF can down-regulate the expression of E-selectin.4. ELISA determination also was used to detect the sE-selectin in 9 patients' serum who were diagnosed as lung cancer and were receiving chemotherapy including VIN. 4 hours after the adminstration, the level was moved from (31.50±28.65) ng/mL to (43.08±44.49) ng/mL {P <0.05).All of the results above indicated that E-selectin plays an important role in the pathogenesis of infusion phlebitis induced by vinorelbine. Thus, regulation of the expression of E-selectin like CIM may intervention the formation of infusion phlebitis.Part III The effect of vinorelbine on adhesion function of endothelial cells toneutrophil and its relativity to the expression of E-selectinChanges of adhesion function of endothelial cells to neutrophil were detected using a Rose Bengal staining and colorimetric method on ECV304 cells. Neutrophil was separated from human venous blood. The optical density of the system increased markedly after VIN stimulation (P < 0.05), which means the adhesion rate of the two kinds of cells enhanced by the drug. The value of adhesion rate raised as 4.3 folds much as the baseline. Observation by phase-contrast microscope could find the changes of adhesion rate. Both of two observations also showed the inhibition of CIM and DMF, which decreased the adhesion rate by 74.34 % and 84.65 % {P < 0.01), respectively.Findings above indicated that VIN enhances the adhesion function of endothelial cells to neutrophil through up-regulating the expression of E-selectin. Injuries of endothelium and inflammatory cell infiltration and penetration may occur through this mechanism and infusion phlebitis episodes then. CIM may weaken the syndrome of phlebitis by inhibiting the expression of E-selectin. So it may be used to prevent the infusion phlebitis in clinic.Part IV Gene regulation to the expression of E-selectin by vinorelbineThe mRNA of E-selectin of ECV304 cells were measured using a real-time PCR method. Marked increase of mRNA of E-selectin was observed after the stimulation of VIN, which was lightly lower than the level stimulated by TNF-a. CIM showed an effective inhibition of mRNA of E-selectin, which decreased the VIN and TNF-a increased level of mRNA by 52.94% and 52.47%, respectively. DMF showed a more marked inhibition of mRNA, which down-regulated the levels by 68.10% and 85.52%, respectively.The results by real-time PCR indicated that the stimulation effect of VIN and the inhibition effect of CIM and DMF presents earlier than protein expression and in the transcription phase. The stimulation signal of drugs makes the expression of mRNA of E-selectin, and then induces the expression of E-selectin, which mediates the following cascade reactions of infusion phlebitis.Part V Primary investigation of the relationship between the expression of E-selectin and the NF- * B pathway1. The expression of the transcription factor NF-kB in mice and in ECV304 cells after the stimulation of VIN was examined by imrnunohistochemistry. Weaker positive expression of NF-kB p65 was observed before the drug stimulation and it was strengthened after VIN or TNF-a treatment. CIM and DMF could partly weaken the positive expression of it. This indicated that the expression of E-selectin induced by drugs above is associated with NF-kB signal transduction pathway.2. The level of total NF-KBp65, activated NF-KBp65 and Ser536 phosphorylated NF-KBp65 in the cytoplasm and nucleus of ECV304 cells were measured by an ELISA method, respectively. Results showed that VIN didn't interfere with the activation and Ser536 phosphorylation of NF-icBp65, but it may improve the entry of NF-KBp65 to nuclear of endothelial cells. CIM and DMF also showed a interference with the entry of NF-KBp65 to nuclear, because the level in nucleus were decreased after its pretreatment That indicated that regulation of VIM, CIM and DMF to E-selectin are in a NF-kB dependent manner.In conclusion, a system study on relationship of the expression of E-selectin in endothelial cells and the pathogenesis of infusion phlebitis induced by vinorelbine, a representative chemotherapeutic drug with high potency of infusion phlebitis was undertaken. Both the in vivo and in vitro studies were done in animal models with rabbits and mice, in clinical patients and in ECV304 endothelial cells, respectively. Many qualitative and quantitative technology including histopathology, immunohistochemistry, ELISA, adhesion experiment, flow cytometry and real-time PCR were applied in order to investigate the expression of the protein and mRNA of E-selectin, the adhesion function of the cell, and the signal transduction manner of it. The most results of the study indicated that vinorelbine induces infusion phlebitis in a NF-kB dependent manner: The drug stimulation enhance the entry of NF-kB to nucleus, which binding to the promotor region of E-seletcin and trigger the expression of E-seletcin mRNA. The E-seletcin is expressed then and mediates the adhesion between endothelial cells and neutrophils. After that, injuries of endothelium andinflammatory cell infiltration and penetration occur and cause infusion phlebitis at last. Cimetidine and dimethyfumarate can inhibit the entry of NF-kB to nucleus, and then inhibit of the downstream reactions. They may be developed to use to prevent infusion phlebitis in clinic.