Experimental Study on Immunoloregulation Function of Icariin in Vitro and in Vivo
|School||Hebei Medical University|
|Course||Clinical Laboratory Diagnostics|
|Keywords||ICA cord blood dendritic cells cytotoxicity immunosuppression immunoregulatory|
Objective: In order to study the effect of ICA on immune system and immunocyte in vitro and in vivo. Meanwhile, we approached immunoregulation mechanism of ICA initially. For the final purpose, this study provides some experimental bases to clinical application of ICA to cure certain diseases, such as tumor.Methods:1 The cord blood monocytes, which were collected from health and uterogestation parturient were isolated by density gradient centrifugation using lymphocyte isolating solution under axenic condition, and dendritic cells (DCs) were induced by GM-CSF and IL-4.2 The morphological characteristics of DCs from untreated group, TNF-αgroup and ICA group were observed under inverted microscope and transmission election microscope.3 Expressions of CD1a, CD83, CD80 and CD86 on DCs of untreated group, TNF-αgroup and ICA group, and the cell cycles and expressions of CD54, CD18 on B-MD-C1 (ADR+/+) cells treated by ICA (0, 12.5, 25, 50, 100μg/ml) for 12hours were detected by flow cytometry.4 The level of IL-12 and IFN-γin the culture supernatant of DCs from untreated group, TNF-αgroup and ICA group were detected by ELISA.5 The proliferation of T cells stimulated by DCs, and the inhibition effect of ICA on cell growth of B-MD-C1 (ADR+/+) in treated or untreated groups (12.5, 25, 50, 100μg/ml ICA and control) for different treatment times (24h, 48h and 72h) were determined by MTT method.6 The cytotoxicity of CIK cells against B-MD-C1 (ADR+/+) cell, which were treated by ICA (0, 12.5, 25, 50, 100μg/ml) for 12hours were investigated by LDH release method. 7 Expression of CD54, CD18 mRNA in B-MD-C1 (ADR+/+) cells after treated with ICA (0, 1.25, 25, 50μg/ml) for 12h were detected by semi-quantitative RT-PCR.8 The sixty C57BL/6j mice of 7-8-week-old were divided into six groups randomly, and constructed immunosuppression models on them besides control group, the others were divided into experimental group, positive group and model group, and then the three groups were injected with cyclophosphamide (Cy) in abdominal cavity one time (300mg/kg). In the second day, the mice of experimental group were given ICA (150, 80, 40 mg/kg·day) by intragastric administration, the positive group were given Shenqi Fuzheng Injection (1ml/day); the model group were given normal saline (NS). All the mice were treated for 10 days. The mice of control group were raised normally. To observe the survival condition of the mice everyday, after the lasted given drugs for 12 hours, all the mice were killed, and the thymus, spleen, peritoneal macrophage, bone marrow and peripheral blood were removed for the succedent experiments.9 Morphology of thymus of the mice were observed under optical microscope after HE staining.10 The proliferation of lymphocyte of the mice were determined by MTT method.11 The level of TNF-αand IL-12 that were produced by peritoneal macrophage of mice were detected by ELISA.12 The cytotoxicity of the peritoneal macrophage of the mice were investigated by LDH release method.13 Morphological images of bone marrow and peripheral blood smears of the mice were detected by Wright-Giemsa’s staining.14 Population of WBC, RBC and PLT in the peripheral blood were detected with automated blood cell counter (ABCC).15 The number of bone marrow cells in the single femur was counted under microscope.Results: 1 After 5 days cultured with GM-CSF and IL-4, the human CBMC developed into immature DCs with typical morphological characteristics, the cells were small and a little dendrites on the cellular surface, and they were grown in clustering.2 After 10 days cultured with cytokines and ICA, the CBMC that were stimulated by ICA for 5 days developed into mature DCs with typical morphological characteristics, the cells enlarged and were irregular in shape, many thin and long dendrites on the cellular surface, abundance of cytoplasm with more mitochondrion and rough endoplasmic reticulum, the caryon heterochromatin were under the karyolemma, and fewer lysosome and vacuolus.3 The result of phenotype analysis showed that the expressions of CD1a, CD83, CD80 and CD86 on mature DCs surface were up-regulated significantly by ICA, compared to the control group untreated with stimulating factor(P<0.05), in which most significant change occurred at CD80, reached (90.44±0.81)%. The expression of CD54 and CD18 on B-MD-C1 (ADR+/+) cells surface were also up-regulate by ICA in concentration-dependent manner. The expression of CD54 and CD18 on B-MD-C1 (ADR+/+) cells surface between every treated group, the difference was significant(P<0.01), the cells after treatment with different doses of ICA, compared to control group, the difference was significant(P<0.01). The results of FCM showed that ICA could arrest cell cycle of B-MD-C1 (ADR+/+) cells at S phase.4 ICA enhanced obviously the IL-12 and IFN-γproduction by DCs(P<0.01), but there was no significance difference between ICA group and TNF-αgroup(P>0.05).5 ICA-treated DCs stimulated markedly the proliferation of T cell (P<0.05), and the ability displayed rising tendency with the DC/T ratio. ICA (12.5~100μg/ml) could inhibited the proliferation of B-MD-C1 (ADR+/+) cells in vitro (P<0.05), the growth inhibition was in a time-and dose-dependent manner. The greatest inhibition rate was 78.74% observed in the treatment group of B-MD-C1 (ADR+/+) cells with the concentration of 100μg/ml ICA for 72 hours.6 ICA could enhance the susceptibility of B-MD-C1 (ADR+/+) cells to CIK in concentration-dependent manner and E/T-dependent manner, compared to control group, the difference was significant (P<0.05).7 The results analized by semi-quantitative RT-PCR showed that CD54, CD18 mRNA were up regulated after B-MD-C1 (ADR+/+) cells were treated with different concentrations of ICA for 12h, there was statistically significant differences compared to the control group (P<0.05).8 The immunosuppression models of the mice that were induced by cyclophosphamide were successfully established. The locomotor activity of the mice of the model group were feebly, the foodintake and hydroposia were not so good, one of them was died in the 6th day, however, the control group, positive group and experimental group were normal, none of them was died.9 Great changes have taken place in thymus of the mice of model group, and there were apomorphosis and necrosis of adipocyte around the thymus; there were partial fill of adipose tissue in the thymus of ICA low dose group, and no necrosis obviously, there were no changes in thymus of high dose, middle dose and Shenqi Fuzheng Injection groups.10 ConA enhanced obviously the proliferation of spleen T cell of high dose of ICA, middle dose and Shenqi Fuzheng Injection groups, compared to model group, the difference was significant (P<0.05); and LPS could enhanced the proliferation of spleen T cell of high dose of ICA, compared to model group, the difference was significant (P<0.05), however, it has no effects on the proliferation of spleen B cell of middle dose of ICA and low dose(P>0.05).11 Compared with control group, the level of TNF-αand IL-12 that were produced by peritoneal macrophage of the mice of model group was decreased markedly, after treated with ICA for 10 days, there was no significance difference in the experimental group, positive group and control group(P>0.05).12 The cytotoxicity of peritoneal macrophage of the mice of model group was decreased markedly(P<0.01), ICA of different concentration could all enhanced the cytotoxicity of peritoneal macrophage, compared to model group, the difference was significant(P<0.05); high dose and positive group, compared with control group, there was no significance difference(P>0.05).13 In the bone marrow and peripheral blood smear, we can see the karyocyte hyperplasia in the control group, but hematopoietic depression in the model group; after treated with ICA, the hematopoietic depression was improved.14 Result of automated blood cell counter (ABCC) revealed that the WBC, RBC and PLT of model group were all decreased markedly (P<0.01), after treated with ICA for 10 days, all of them were improved.15 After treated with Cy for 10 days, the number of bone marrow cells in single femur of NS group was 7.03×106/femur, compared with control group (10.36×106/femur) was decreased markedly(P<0.01), after treated with middle dose, high dose of ICA and Shenqi Fuzheng Injection, the number of bone marrow cells in the single femur, compared with control group, there was no significance difference(P>0.05).Conclusion:1 ICA induced the differentiation and maturation of DCs derived from CBMC in vitro, up-regulated the surface markers, cytokines production and increased the ability to stimulate the proliferation of T cell.2 ICA could up-regulated the expression of LFA-1/ICAM-1 on the B-MD-C1 (ADR+/+) cells, enhance the immunogenicity of them, and make them easy to recognized by immunologically competent cell.3 ICA protected immune organs of immunosuppression mice.4 ICA could inhibit the proliferation of B-MD-C1 (ADR+/+) cells in vitro and in a dose-and time-dependent manner, and arrest cell cycle of B-MD-C1 (ADR+/+) cells at S phase.5. ICA enhanced T and B cell proliferation of mice spleen significantly, and the killing ability of peritoneal macrophage of the mice as well, stimulated the production of TNF-αand IL-12, enhanced the function of cellular immunity and humoral immunity of immunosuppression mice.6 ICA improved the hematopoiesis of bone marrow of the mice, raised the number of bone marrow and peripheral blood cells, and indicated that it was good at improving the hematopoiesis of bone marrow.7 ICA possessed immunologic enhancement significantly, and great potent in clinical application.