Mangiferin Promotes IL-10 Production By Activating JAK2-STAT3 And ERK Pathways In Murine Splenic Mononuclear Cells

Objectives:Our previous experiments have proved that mangiferin?MA?can up-regulate the levels of regulatory B cells?Bregs?and interleukin 10?IL-10?in murine splenic mononuclear cells?MNCs?.Based on the existing results,the aim of this study is to further explore the molecular mechanism by which MA up-regulates Bregs and IL-10 levels in murine splenic MNCs,and to provide molecular basis for the application of mangiferin in the prevention and treatment of chronic graft-versus-host disease?cGVHD?.Methods:Male BALB/c mice were sacrificed by cervical dislocation,and then their splenic MNCs were isolated by red blood cell lysis buffer.First,we examined whether MA could active JAK2-STAT3,ERK,TLR4-MyD88 signal pathways.In this part,the cells were randomly divided into control group?0.1%DMSO?and experimental groups?50uM,100uM,150uM,200uM MA?.The protein expression of JAK2,p-JAK2,STAT3,p-STAT3,ERK,p-ERK,TLR4 and MyD88 were detected by western blot,and the mRNA level of TLR4 was detected by RT-PCR.Then we explored whether MA increased Bregs and IL-10 levels by activating these signal pathways above,at the presence of inhibitors.And in this part,the cells were divided into three groups:the control group?0.1%DMSO?,the 200uM MA group,the 200uM MA+inhibitor?AG490 or HO3867 or U0126?group.The IL-10 concentration in cell supernatant was detected by ELISA,and the Bregs level in MNCs was measured by flow cytometry.Results:1.MA promoted IL-10 expression by activating JAK2-STAT3 signaling pathway,but the Bregs level was not affected by this signaling:?1?MA could active JAK2-STAT3 pathway.Compared with the control group,MA significantly increased the protein levels of p-JAK2 and p-STAT3 in a dose-dependent manner,particularly in 200uM MA group?P=0.000,P=0.008?,but the levels of JAK2and STAT3 between the two groups had no significant difference?P>0.05?.?2?AG490 and HO3867 could inhibit the activation of JAK2-STAT3 signal pathway by MA.The western blot results showed that the protein levels of p-JAK2 and p-STAT3 in MA+inhibitor group?AG490 or HO3867?were much lower than those in MA group(AG490:P_p-JAK2=0.043,P_p-STAT3=0.016;HO3867:P_p-STAT3=0.001).?3?AG490 and HO3867 could inhibit the promotion of IL-10 secretion by MA in MNCs.ELISA results showed that the IL-10 level in MA+inhibitor group was decreased when compared with the MA group?AG490:P=0.0057;HO3867:P=0.0333?,but Bregs levels between the two groups had no significant difference?P>0.05?.2.MA promoted IL-10 expression by activating ERK signaling,but the Bregs level was not affected by this signal pathway:?1?MA up-regulated ERK signaling.Compared with the control group,MA significantly increased the protein level of p-ERK?P=0.045,0.016,0.103,0.047?,but it did not affect the expression of ERK?P>0.05?.?2?U0126 inhibited the phosphorylation of ERK by MA.The western blot results showed that the protein level of p-ERK in MA+U0126 group was much lower than that in MA group?P=0.0079?.?3?U0126 could inhibit the promotion of IL-10 level by MA in MNCs.ELISA results showed that the IL-10 level in MA+U0126 group was decreased when compared with the MA group?P=0.0046?,but Bregs levels between the two groups had no significant difference?P>0.05?.3.MA may promote Bregs level and IL-10 production by activating TLR4-MyD88signaling pathway in MNCs:?1?RT-PCR results showed that,compared with the control group,TLR4 mRNA levels were increased in MA groups?P=0.042,0.114,0.02,0.031?.TLR4?P=0.049,0.09,0.003,0.001?and MyD88(P_100uM=0.036)protein levels measured by Western blot in MA groups also significantly increased than those in the control group.Conclusion:This study complements and extends the results of our previous experiments.The results suggest that MA promote IL-10 production in murine splenic MNCs by activating JAK2-STAT3 and ERK signaling pathways.MA may enhance the immunoregulatory activity of Bregs,but not increase the level of Bregs through activating these two signal pathways.It may provide a molecular mechanism for the anti-cGVHD effect of MA.However,the molecular mechanism by which MA up-regulated the Bregs level still need further studies,as well as the activity of MA in vivo.