Chlorogenic acid 및 인동등 ethyl acetate 분획의 비장 및 흉선 세포에서의 유전자 발현 분석을 통한 면역조절효과 Immunomodulatory effects of chlorogenic acid and ethyl acetate fraction from Lonicera japonica on cytokine gene expression profiles in spleen and thymus원문보기
Objective : Lonicera japonica contains anti complementary polysaccharides and polyphenolic compound. Among these polyphenolic substances, chlorogenic acid is the major active component of this plant. However, the immunological mechanisms for these activities, have not been elucidated, nor the active...
Objective : Lonicera japonica contains anti complementary polysaccharides and polyphenolic compound. Among these polyphenolic substances, chlorogenic acid is the major active component of this plant. However, the immunological mechanisms for these activities, have not been elucidated, nor the active components. To clarify immunomodulatory effects of those we examined the relationship between the activity of CD8+ T cell-mediated lysis and the frequency of cytokine profiles in spleen, thymus (especially IFN-${\gamma}$, IL-4, GM-CSF etc.) expressing CD8+ T cells activated by IL-2. Methods : To study immunomodulatory effects ethyl acetate fraction from Lonicera japonica, chlorogenic acid on cytokine gene expression from spleen, thymus cells, RT-PCR was performed after quantitative normalization for each gene by a densitometry using ${\beta}$-actin gene expression. A modified standard $^{51}Cr$-release assay was used to measure cytotoxic activities of cytotoxic T cells. Spleen, thymus cells from NOD mice were stained with CD3, CD4, CD44, CD69 in staining buffer and analyzed by two color flow cytometry. Results : We showed that ethyl acetate fraction from Lonicera japonica in combination with IL-2 resulted in a significant enhancement of PCR products for IFN-${\gamma}$, IL-4, IL-10, GM-CSF, IL-6 and cytotoxtic CD8+ T cell proportion in spleen and thymus T cells in NOD mice. This suggests that IFN-${\gamma}$, IL-6 like IL-4 may be acting as a regulatory rather than proinflammatory cytokine. Conclusions : In conclusion, based on the results of the present study which showed that ethyl acetate fraction from Lonicera japonica and chlorogenic acid upregulating cytokine gene expression in spleen and thymus, we are tempted to speculate that some of the therapeutic efficacies such as anti-diabetic activity of Lonicera japonica are due to the immunomodulatory its ethylacetate fraction and chlorogenic acid.
Objective : Lonicera japonica contains anti complementary polysaccharides and polyphenolic compound. Among these polyphenolic substances, chlorogenic acid is the major active component of this plant. However, the immunological mechanisms for these activities, have not been elucidated, nor the active components. To clarify immunomodulatory effects of those we examined the relationship between the activity of CD8+ T cell-mediated lysis and the frequency of cytokine profiles in spleen, thymus (especially IFN-${\gamma}$, IL-4, GM-CSF etc.) expressing CD8+ T cells activated by IL-2. Methods : To study immunomodulatory effects ethyl acetate fraction from Lonicera japonica, chlorogenic acid on cytokine gene expression from spleen, thymus cells, RT-PCR was performed after quantitative normalization for each gene by a densitometry using ${\beta}$-actin gene expression. A modified standard $^{51}Cr$-release assay was used to measure cytotoxic activities of cytotoxic T cells. Spleen, thymus cells from NOD mice were stained with CD3, CD4, CD44, CD69 in staining buffer and analyzed by two color flow cytometry. Results : We showed that ethyl acetate fraction from Lonicera japonica in combination with IL-2 resulted in a significant enhancement of PCR products for IFN-${\gamma}$, IL-4, IL-10, GM-CSF, IL-6 and cytotoxtic CD8+ T cell proportion in spleen and thymus T cells in NOD mice. This suggests that IFN-${\gamma}$, IL-6 like IL-4 may be acting as a regulatory rather than proinflammatory cytokine. Conclusions : In conclusion, based on the results of the present study which showed that ethyl acetate fraction from Lonicera japonica and chlorogenic acid upregulating cytokine gene expression in spleen and thymus, we are tempted to speculate that some of the therapeutic efficacies such as anti-diabetic activity of Lonicera japonica are due to the immunomodulatory its ethylacetate fraction and chlorogenic acid.
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가설 설정
In this study, we analyzed the effect of ethyl acetate fraction and chlorogenic acid on the expression of genes expression profiles associated with immunomodulatory activity.
제안 방법
Blood glucose analysis was performed every week using a Lifescan One Touch Profile Meter (Issy les Mouline aux, France) with one drop of blood sampled from the tail. A 1.
0g/lcalibration standard solution was assayed at least every samples. In this study, attention was focused on any sign of glycemic disturbance. The threshold for positivity was set at the mean of the pretest values plus two standard errors.
대상 데이터
All antibodies(CD3, CD4, CD44, CD69) for flow cytometric analysis were purchased from Becton Dickinson (BD) PharMingen (SanDiego,CA). Spleen, thymus cells from NOD mice were stained with the indicated antibodies in staining buffer(PBS containing 1% FBS and 0.
The voucher specimens are deposited in our laboratory (Department of Herbology, College of Oriental Medicine, Sanji University Wonju 220-702, Republic of Korea). Chlorogenic acid were purchased from Sigma-Aldrich (Sigma-Aldrich,Korea). Ethylacetate (EtOAc), 1-butanol (BuOH), chloroform (CHCL3) were purchased from Merck (Darmstate, Germany).
Chlorogenic acid were purchased from Sigma-Aldrich (Sigma-Aldrich,Korea). Ethylacetate (EtOAc), 1-butanol (BuOH), chloroform (CHCL3) were purchased from Merck (Darmstate, Germany). Water was prepared in a Milli-Q water purification system (Millipore, Bedford, MA,USA).
The sample of Lonicera japonica were purchased from Buyeo-gun agriculture technology center (Buyeo, Korea). The voucher specimens are deposited in our laboratory (Department of Herbology, College of Oriental Medicine, Sanji University Wonju 220-702, Republic of Korea). Chlorogenic acid were purchased from Sigma-Aldrich (Sigma-Aldrich,Korea).
데이터처리
All data represent the standard deviation of at least three different determinants and were compared using Student's unpaired t-test.
For statistical analysis of data, P-values were analyzed using a unpaired Student's t-test software program (Startview 5.1; Abacus Concepts, Berkeley,CA).
성능/효과
In our results, IL-2, IFN-γ, IL-4, GM-CSF, IL-6 gene expression were positively regulated by all three major families of transcription factors, these findings suggest that there may be significant overlap in T-cells between the signal transduction circuits that activate transcription of above genes.
This results suggest that IFN-γ, IL-6 like IL-4 may be acting as a regulatory rather than proinflammatory cytokine and immunomodulation of ethyl acetate fraction from Lonicera japonica may be due to several transcription factors including NFAT as well as through indirect effects on the transcriptional activity of NF-κB and AP-1.
Thus, based on the results of the present study which showed that ethyl acetate fraction from Lonicera japonica and chlorogenic acid upregulating cytokine gene expression in spleen and thymus, we are tempted to speculate that some of the therapeutic efficacies such as anti-diabetic activity of Lonicera japonica are due to the immunomodulatory its ethylacetate fraction and chlorogenic acid. Moreover, its mechanisms are involved by activating NF-kB, NFAT and AP-1.
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