The Effect Of Ginkgo Biloba Extract On T Lymphocytes And Its Relationship With PKC In Asthma

IntroductionAmong the masses factors of mechanism of asthma, it was widely acknowledged that T-lymphocytes were one of the most important factors to chronic airway inflammation of asthma. The exacerbation and development of asthma was closely associated with T-lymphocytes (it is mainly Th2 cells) while it was activating, prolifering and producing amount of cytokines such as interleukin 4 (IL-4), IL-5 and so on. PKC is one of the major regulatory enzymes involved in the control of wide variety of physiological processes including T lymphocytes differentiation, proliferation, and activation. The role of PKC in T lymphocytes differentiation and activation has been extensively investigated. The evidence indicated that the differentiation and activation of T lymphocytes, when triggered through the T cell receptor, is dependent on PKC activity. PKC alpha is one of twelve diverse isotypes of PKC gene family. More and more investigation indicated that PKC alpha involves and play a main role in the T lymphocyte differentiation and activation. In severe asthma, glucocorticosteroids was the most effectively drugs to inhibit the airway inflammation. However, it has much adverse effects when it is used systemic. To minimize the side effects or avoid the use of steroids, other less toxic immunotherapeutic agents including Ginkgo biloba extracts based on an understanding of the pathogenesis of asthma have been proposed. Ginkgo biloba, an ancient plant, its standardized extract of leaves has been extensively used in disease of cardiovascular system and cerebrovascular system. Ginkgo biloba extract has been mentioned in the traditional Chinese pharmacopoeia, and Chinese has used the ginkgo leaf for the treatment of bronchial asthma and bronchitis for many centuries. However, so far, the effects of Ginkgo biloba extract on the activation and function of T-lymphocytes, an important population among immune effector cells ininflammatory responses of airway inflammation of asthma, and PKC singnals conducts in that, has been very limited studies. Therefore, to acquire more information of effect of EGb on treatment asthma and the mechanism, it is necensary to systemicly investigating the effect of EGb on T-lymphocytes function and the relationship between T-lymphocytes and PKCa.The results will be benefit for the treatment and prevention of asthma.Part 1The Effect of Ginkgo biloba Extract on Production of cytokines of Thl/Th2 and Protein Kinase Ca Expression in Peripheral Blood T Lymphocytes of AsthmaChapter 1Effects of Ginkgo Biloba Extract on Proliferation and Apoptosis of T Lymphocytes Cultured in vitro of the Rat Model for AsthmaObjective: to study the partial therapeutic mechanism of Ginkgo Biloba extract in asthmatic patient treatment, we detected the effects of Ginkgo Biloba extract on proliferation of T lymphocytes cultured in vitro of the rat model for asthma.Methods: 14 Wistar rats were randomly divided into two groups, 7 rats were sensitized as asthmatic model and the others as healthy controls. T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) of the rats, and were cultured in vitro with Ginkgolide B (BN-52021 group) or Ginkgo Biloba extract 761 (EGb761 group) in different concentration or without any of them (control group). After different period of time culture, the effect of Ginkgo Biloba extract on T lymphocytes viability was measured using the MTT proliferation assay, and the effect of BN-52021 on T lymphocytes apoptosis was analysis by Flow Cytometry.Results: Compared with the control group, BN-52021 could significantly suppress the proliferation of T lymphocytes in vitro of healthy and sensitized rat (P<0.05). The effects of suppression were enhanced by the concentration ofBN-52021 increasing and the time prolonging. To the effects of the BN-52021's suppression, there is a significant difference between the healthy and the asthmatic model rat (P<0.05). However, EGb761 had a contrary pharmic effect on the in vitro culture T lymphocytes' proliferation as the concentration varied from low to high level, it promote T lymphocytes growth at low level and suppressed it at high level. Compared with the control group there were a significant difference (P<0.05). With the concentration of BN-52021 increasing, the rate of T lymphocytes apoptosis is increasing. There is a significant changes of rate among the different concentration levels (P<0.01).Conclusions: Ginkgolide Biloba Extract had a different effect on the T lymphocytes cultured in vitro because it had different ingredients. They had a different effect on T lymphocytes proliferation between healthy and asthmatic rat. Ginkgolide B was the mainly for suppressing the T lymphocyte proliferation among the ingredients, and it probably could increasing the rate of apoptosis of T lymphocytes while they were cultured with them in vitro.Chapter 2 The Effect of Ginkgo biloba Extract on Production of Interleukin-2,4,5 andProtein Kinase Co Expression in Peripheral Blood T Lymphocytes Culture inVitro of Asthmatic RatsObjective To investigate therapeutic effect of Ginkgo biloba extract on bronchial asthma.Methods Fourteen SD rats were randomly divided into two groups, 7 rats were sensitized as asthmatic model and the others as healthy controls. T lymphocytes were isolated from peripheral blood mononuclear cells of rats, and were cultured in different conditions, blank group, group treated with Phorbol Myristate Acetate (PMA), group treated with Ginkgolide B (BN-52021), group treated with PMA + BN-52021, group treated with PMA+ Ro31-8220 (an inhibitor of PKC ), group treated with PMA+ Ro31-8220 + BN-52021. After 72 h, we detected the levels of IL-2, 4, 5 mRNA in each groups with reverse transcriptase polymerase chainreaction (RT-PCR), and detected the activity of PKCα by checking the ratio of PKCα in membrane to cytoplasm in T lymphocytes with Western blotting.Results The quantity of IL-2, 4 and 5 mRNA in blank group of asthmatic models (respectively 0.58 ± 0.086, 1.03 ± 0.12 and 0.48 ± 0.08) were significantly increased as compared to those in blank group of normal rats (respectively 0.45 ± 0.03, 0.35 ± 0.08 and 0.15 ± 0.05) (PO.05). The quantity of IL-2, 4 and 5 mRNA decreased significantly when the T lymphocytes of asthmatic model rats were cultured with BN-52021 (respectively 0.49± 0.05, 0.55± 0.09 and 0.27±0.05) (P<0.05). The ratio of IL-2/IL-4 mRNA in normal rats was 1.27±0.20, significantly higher than that in asthmatic model rats (0.60±0.18) (PO.01), and BN-52021 could significantly increase the ratio when the T lymphocytes of asthmatic model rats were cultured with it (PO.01). BN-52021 could significantly counteract the levels of IL-2, 4 and 5 mRNA that increased by PMA (P<0.05), but the decreasing effect was less than the Ro31-8220, a specific inhibitor of PKCα (P<0.05). And BN-52021 couldn't further counteract significantly the levels of IL-2, 4 and 5 mRNA that increased by PMA which had significantly decreased by the Ro31-8220. The expression ratio of PKCα in membrane and cytoplasm of T lymphocytes of asthmatic model rats was significantly higher than that of normal rats (P<0.01), and BN-52021 could significantly decrease the ratio when the T lymphocytes were cultured with it (PO.05). Furthermore, it also can significantly counteract the expression ratio of PKCα in membrane and cytoplasm which increased by PMA (P<0.05). The expression ratio of PKCα in membrane and cytoplasm was significantly positively correlated with the level of IL-4 mRNA in T lymphocytes cultured in vitro in asthmatic model rats group (n=42, r=0.845, P<0.01).Conclusion Ginkgo biloba extract could depress the expression of IL2, 4 and 5 mRNA of asthmatic model rat T lymphocytes in vitro. However, the inhibition effects were distinct in degree, so the balance of IL-2/IL-4 was changed according to the relative variability. Ginkgo biloba extract also could decrease the expression ratio of PKCα in membrane and cytoplasm. It is possible that Ginkgo biloba extractinfluences the production of interleukin and the balance of Thl/Th2 in the asthmatic model rats through its influence to the PKC signal pathway.Chapter 3 The Effects of the Ginkgo Biloba Extract on the Expression of Interleukin-2,Interleukin-4, and Interleukin-5 Regulated by Protein Kinase Cot in Peripheral Blood T Lymphocytes Cultured in Vitro from Asthmatic PatientsObjective To investigate whether the secretion of T helper (Th) interleukin(IL), such as IL-2, IL-4 and IL-5, are regulated by protein kinase Cα (PKCα) in asthmatic T lymphocytes cultured in vitro, and the probably mechanism of the effects of Ginkgo biloba Extract (EGb) to asthma.Methods Peripheral blood T lymphocytes were isolated from 12 asthmatic patients and 10 health volunteers, and were cultured in vitro in different groups: blank group, group cultured with Phorbol Myristate Acetate (PMA), group cultured with Ginkgolide B (BN 52021), group cultured with PMA + BN 52021, group cultured with PMA+ Ro31-8220(a specific inhibitor of PKCα), and group cultured with PMA+ Ro31-8220 + BN 52021, respectively. After 72 h, we detected the levels of IL-2, 4, 5 mRNA in each groups with reverse transcriptase polymerase chain reaction (RT-PCR), and detected the activity of PKCα by checking the ratio of PKCα in membrane to cytoplasm in T lymphocytes with Western Blot.Results The levels of IL-2, 4 and 5 mRNA in blank group of asthmatic patients (0.68 ± 0.103, 1.14 ± 0.12 and 0.56 ± 0.06, respectively) were significantly increased than those in blank group of healthy control (0.55 ± 0.05, 0.41 ± 0.07 and 0.24 ± 0.04, respectively, P<0.05). The levels of IL-2, 4 and 5 mRNA decreased significantly when the T lymphocytes from asthmatic patients were cultured with BN-52021(0.59±0.06, 0.67±0.09 and 0.36±0.05, respectively, P<0.05). The ratio of IL-2/IL-4 mRNA in T lymphocytes from normal controls was significantly higher than that from asthmatic patients (1.36±0.21 and 0.71±0.18, respectively, P<0.01), and BN52021 could significantly increased the ratio when the T lymphocytes from asthmatic patients were cultured with it (P<0.01). BN52021 could significantlydecreased the levels of IL-2, 4 and 5 mRNA that increased by the PMA (P<0.05), but its effects were less than Ro31-8220(P<0.05). The BN52021 couldn't further significantly decreased the levels of IL-2, 4 and 5 mRNA which had significantly decreased by the Ro31-8220 in the group cultured with PMA+ Ro31-8220 + BN52021. The activity of PKCα in T lymphocytes from asthmatic patients were significantly higher than that of healthy controls (P<0.01), and BN52021 could significantly decreased the ratio when T lymphocytes were cultured with it (P<0.05). Furthermore, it also can significantly decrease the activity of PKCα which increased by the PMA (P<0.05). The activity of PKCα were significantly correlated with the level of mRNA of IL-4 and IL-5 in T lymphocytes cultured in vitro (r=0.885 and r=0.938, respectively, P<0.01).Conclusions EGb could depress the level of IL2, IL-4 and IL-5 mRNA in T lymphocytes from asthmatic patients cultured in vitro. However its ability for inhibition was difference in degree among different interleukins, and therefore it could recover the imbalance of IL-2/IL-4 in asthmatic T lymphocytes. EGb also could decrease the activity of PKCα in asthmatic T lymphocytes. We concluded that EGb influenced on the production of interleukin and the balance of Thl/Th2 in the asthmatic patients probable partly through its influence on the PKC signal pathway.Part 2The Investigation of the Effect of Extract of Ginkgo Biloba on the Expression of PKC in airway Inflammation Cells and the level of Th2-cytokines in AirwaySecretion of AsthmaChapter 1The Effect of Extract of Ginkgo Biloba on the Expression of PKCα in the Inflammation Cells and the level of IL-4 in Alveolar lavage of Asthmatic RatObjectives To investigate the effect and possible mechanism of the Extract of Ginkgo biloba (EGb) in the asthma therapy.Methods Forty-eight rats were classified randomly 8 groups; these were normal control group, asthmatic control group, asthmatic rats treated with glucocorticosteroid 1, 2 and 4 weeks groups, and asthmatic rats treated with EGb 1, 2 and 4 weeks groups. The expression of PKCα in the alveolar lavage cells was detected using immunocytochemistry and positive staining cells in different cells were counted. Interleukin-4 (IL-4) in alveolar lavage supernatants was detected using enzyme-linked immunosorbent assay.Results The percentage of eosinophils (EOS), lymphocytes in alveolar lavage cells, the ratio of PKCα positive expressing in lymphocytes and total cells of alveolar lavage and the concentration of IL-4 in alveolar lavage supernatants, all that in rat asthmatic models were higher than that of the controls (P<0.05). And glucocorticosteroid could decrease significantly the amount of eosinophils and lymphocytes in airway of the asthmatic rats (P<0.05), and it could suppress significantly the expressing of PKCα in lymphocytes and total cells of alveolar lavage (P<0.05), and it also could depress significantly the secretion of IL-4 in airway inflammation cells of asthmatic rats (P<0.05). There were significantly higher in the groups of EGb treatment 1, 2 weeks than the groups treatment with glucocorticosteroid in the same items were detected (respectively P<0.05). There was no significantly different between group treated with EGb 4 weeks and group treated with glucocorticosteroid in the same items were detected (P>0.05). There was no significantly different between group treated with EGb 1 week and asthmatic control group in the same items were detected (P>0.05). The same items were detected in group treated with EGb 2 weeks were significantly lower than that in group treated with EGb 1 week (P<0.05), but were significantly higher than that in group treated with EGb 4 weeks (P<0.05). The ratio of PKCα positive expressing in total cells of alveolar lavage was significantly correlated to the percentage of EOS , lymphocytes in alveolar lavage cells, and the concentration of IL-4 in alveolar lavage supernatants (n=18, respectively r=0.641, r=0.69, r=0.625, respectively P<0.01).Conclusions EGb could decrease the amount of eosinophils and lymphocytes in airway of the asthmatic rats, and it could suppress the expressing of PKCα in lymphocytes and total cells of alveolar lavage, and it also could depress significantly the secretion of IL-4 in airway inflammation cells of asthmatic rats. However, the effect of that was less than that of glucocorticosteroid, but the effect would improve with the time prolonged. The probably mechanism of the EGb in the asthma therapy is that it suppress the expression of PKCα in airway inflammation cells and thus then depress the secretion of IL-4, decrease the amount of eosinophils and lymphocytes in airway of asthmatic rats.Chapter 2The Effect of Inhaled Glucocorticosteroid on Expression of PKCα in the Inflammation Cells and Producing of IL-5 in Induced Sputum of AsthmaPatientsObjectives To investigate the Expression of PKCα in the Inflammation Cells and the producing of interleukin-5(IL-5) in induced sputum in asthma patients and the effect of inhaled glucocorticosteroid on them.Methods 29 asthma patients were classified 2 groups, 14 patients were treated with fluticasone propionate 2 weeks and the others were treated with fluticasone propionate 4 weeks. All individuals needed followed up and a method to induce sputum with inhaled hypertonic saline(4%～5%) was respectively used in them. 14 health volunters were as control. The expression of PKCα in the cells was detected using immunocytochemistry and positive percentage in different cells were counted. Interleukin-5(IL-5) in sputum supernatants was detected using enzyme-linked immunosorbent assay.Results The percentage of eosinophils(11.1%±4.3%) , lymphocytes(4.3%±2.6%) and the concentration of IL-5(64.9 ng/L±46.0ng/L) in asthmas were higher than the eosinophils(0.9%± 0.9%) , lymphocytes(1.2% ± 0.8%) and the concentration of IL-5(64.9 ng/L±46.0ng/L) in the controls(P<0.05). The concentration of IL-5 is correlated to the percentage positive of PKCα in inflammation cells (P<0.01). Thepositives percentage of PKCα and the concentration of IL-5 in all groups were lower than treated before(P<0.05), and they are correlated to the forced expiratory volume in first second (FEV1).Conclusions PKCα in the inflammation cells and IL-5 may play an important part in the airway inflammation., and the signal transduction of the PKCα may be one of the mechanisms of the airway inflammation in asthma patients. It is powerful to the glucocorticosteroid in asthma therapy. The probably mechanism is that glucocorticosteroid suppress the expression of PKCα in airway inflammation cells and then decrease the producing of IL-5 in the airway.Chapter 3The Effect of Ginkgo Biloba Extract on the Expression of PKCα in theInflammation Cells and the Level of IL-5 in Induced Sputum of AsthmaPatientsObjectives To investigate the effect of the EGb on the therapy of asthma and the probable mechnisms.Methods 75 asthma patients were classified into 4 groups, treated with fluticasone propionate 2 weeks or 4 weeks, or treated with fluticasone propionate and EGb 2 weeks or 4 weeks. 15 health volunteers were as normal control. All individuals needed followed up. Induced sputum after inhaling hypertonic saline (4%～5%) was performed respectively to them. Lung ventilatory function and forced expiratory volume in one second (FEV1) were measured to all individuals. The numbers of different cells in induced sputum were calculated after cells stained with Wright's staining. The expression of PKCα in the cells was detected with immunocytochemistry and positive percentage in different cells were counted and calculated. Interleukin-5 (IL-5) in sputum supernatants was detected with enzyme-linked immunosorbent assay.Results The percentage of eosinophils (14.3±2.7) %, lymphocytes (7.9±2.6) %, PKCα positive inflammation cells (80.7±9.1) % and the concentration of IL-5 (56.8±10.8) ng/L in asthmatic patients are higher than that percentage of theeosinophils (1.1±0.4) % , lymphocytes (2.4±0.7)%, PKCα positive inflammation cells (13.6±3.4) % and the concentration of IL-5 (9.7±4.3) ng/L in the controls (P<0.05), and it was significantly lower than that after they were treated with fluticasone propionate or fluticasone propionate and EGb. However, it was still significantly higher than that of the controls. Compared to the group treatment with glucocorticosteroid 2 weeks, there was no significantly decreasing in the percentage of eosinophils, lymphocytes, PKCα positive inflammation cells and the induced sputum supernatant levels of IL-5. Compared to the group treatment with glucocorticosteroid 2 or 4 weeks, There was a significantly decrease in the same items in the group treated with fluticasone propionate and EGb 4 weeks. The levels of IL-5 in supernatant of induced sputum was significantly correlated positive to the percentage of PKCα positive inflammation cells and the percentage of eosinophils in the induced sputum in groups of asthma patients (respectively: n=150, r= 0.83, P<0.01; n=150, r=0.76, P<0.01). The forced expiratory volume in first second are significantly correlated negative to the percentage of PKCα positive inflammation cells and the levels of IL-5 in supernatant of induced sputum in groups of asthma patients (respectively: n=150, i= -0.77, P<0.01; n=150, r= -0.64, P<0.01).Conclusions EGb could significantly decrease the infiltration of inflammation cells such as eosinophils and lymphocytes in airway of bronchial asthma. It could relieve the airway inflammation in some degree. Part mechanism of that probably is that EGb could decrease the activation of the PKCα in the inflammation cells and then decrease the induced sputum supernatant levels of IL-5. EGb was probably as glucocorticosteroid complement in therapy of asthma, but it took much longer time to display the effect of treatment of asthma than glucocorticosteroid.