| Objective:To investigate the effect of forsythoside A(FA) on the percentage of CD4+CD25+regulatory T cell(Treg) in peripheral blood, the expression of Foxp3, IL-10andTGF-β1of endotoxemia mouse induced by LPS. By observe the effect of FA on thecell viability, the percentage of Treg, the expression of transcription factor Foxp3,cytokines IL-10and TGF-β1in lymphocyte induced by LPS after48hours. In orderto explore the possible mechanism of immunomodulatory effect of FA.Methods:1Effect of FA on Treg in endotoxemia mouse1.1Model design: intraperitoneal injection of LPS to prepare endotoxemia micemodels.1.2Experimental groups: a total of48BABL/C mice were randomly dividedinto six groups: Control control group(Control), endotoxemia model group(LPS),anti-CD25group, and the high, middle, small dose FA-treated group. The Control,LPS, and anti-CD25group were injected with Control saline0.5ml/d for7days. Among,anti-CD25group was injected with anti-CD25as a positive control in the next day.Three drug groups were injected with FA80mg/kg,20mg/kg,5mg/kg for7days.At theeighth day, every group except control was injected by LPS. Blood and spleen tissuespecimens were taken4hours later to test.1.3Indicators of detection:①Observe the performence of mice.②Tregpercentage in peripheral blood was measured by flow cytometry.③The expression ofFoxp3mRNA in spleen tissue was tested by real-time PCR.④The expression ofFoxp3protein in spleen tissue was tested by Western blot.⑤The leaves of IL-10andTGF-β1in blood serum were tested by ELISA.2Effects of FA on mouse lymphocyte induced by LPS2.1The extraction of primary lymphocytes: The spleen was carefully polishedwith a200mesh filter cloth, and then filtered with a400mesh filter cloth. Finally we prepared primary single cell suspension by the methord of density gradientcentrifugation.2.2Experimental groups: the experiment was divided into six groups: Controlcontrol group(Control), endotoxemia model group(LPS), FA high, medium and lowdose group, PMB group. Control group: cells were cultured Controlly.LPS group:adding LPS at final concentration of100ng/ml for48h. FA high, medium and lowdose group and PMB group: adding respectively FA320ug/ml,80ug/ml,20ug/ml andPMB10ug/ml pretreated cells2h, then stimulated cultur48h by100ng/ml LPS.2.3Indicators of detection:①MTT detected the cell viability.②Flow cytometrymeasured the percentage of Treg in toal lymphocytes.③Real-Time PCR assay theexpression of Foxp3mRNA.④Western blot was used to detect the expression ofFoxp3protein.⑤ELISA assay IL-10and TGF-β1levels of cell culture medium.Results:1Effect of FA on Treg in endotoxemia mouse1.1General performance of mouse: compared with the Control group, mice inLPS group were lack of energy and unresponsive while crawling. Anti-CD25anddrug group had no significant anomalies.1.2Change of Treg percentage in mouse peripheral blood:Treg ratio in LPSgroup increased significantly (P<0.01).Compared with LPS group, we found thathigh-dose FA can significantly lower Treg proportion in peripheral blood(P<0.01).1.3The expression of Foxp3mRNA and protein in mouse spleen tissue:compared to Control group, the expression of Foxp3in LPS group raised verysignificantly(P<0.01).The intervention of anti-CD25and FA can markedly decreasedtits expression by dose-dependent(P<0.01,P<0.05).1.4Leavels of IL-l0and TGF-β1in mouse blood serum:the leavel of IL-10inLPS group was significantly higher than Control(P<0.01). Depletion of Treg byanti-CD25and treatment by FA can effectively reduce IL-10content in mouseserum(P<0.01, P<0.05). Changes of TGF-β1was significantly inferior to IL-10.2Effects of FA on mouse lymphocyte induced by LPS2.1Changes in cell viability: compared with Control group, the cell viability ofLPS group significantly decreased(P<0.01).Pretreated with FA can significantly improve it with dose-dependent manner(P<0.01,P<0.05). Moreover, Control group andhigh-dose FA pretreatment showed no statistically significant (P>0.05).2.2Changes of Treg percentage in total lymphocytes:Treg percentage of Controlgroup is5%~15%. Treg of LPS group risen very significantly(P<0.01).After theintervention with PMB and FA, the ratio decreased in a dose-dependent manner(P<0.05).2.3Expression of Foxp3mRNA and protein: It was found that Foxp3had a basicexpression in lymphocytes under Control condition.Stimulated by LPS for48h, it wasincreased significantly than Control(P<0.01). Adding varying degrees FA couldmitigate this change(P<0.05),and the effect of high-dose group was the most obvious.2.4The levels of IL-10and TGF-β1: after the stimulation of LPS, the contents ofIL-10and TGF-β1in culture were significantly increased(P<0.01),FA pretreatmentcan reduce its leavel(P<0.01,P<0.05)with a dose-dependent manner.Conclusion:Giving FA by intraperitoneal injection can lower the leavel of Treg in peripheralblood of endotoxemia mouse, and the expression of Foxp3. FA preconditioning canenhance the cell viability of lymphocyte induced by LPS. Therefore, It was verifiedthat FA had significant immunomodulatory effect from both cells and animals. Itstherapeutic mechanism may be related to the down-regulation of Treg，interference ofFoxp3expression and secretion of cytokine IL-10and TGF-β1．... |
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