| Schistosomiasis is one of the major public health problems in China.Nearly 40 kinds of mammal hosts can be infected by Schistosoma Japonicum.Different kinds of host presented different suitability for S.Japonicum infection.Different kinds of host have unique micro-environments,which will effect the growth,development,maturation and the paring of schistosome,and eventually have different histopathological changes.MicroRNAs(miRNAs)are a class of small noncoding RNAs that regulate gene expression at post transcription level which discovered in recent years.MicroRNA 223(miR-223)is a highly expressed miRNA in myeloid cells.Previous research showed that miR-223 was highly-expressed in the tissues of BALB/c mouse,Wistar rat and M.fortis infected with S.Japonicum.Indicating miR-223 played an imprtant role in hosts’ innate immune system against Schistosomiasis.But limited reports about the regulation mechanism of miR-223 was found.In this research,we compare and analyze the expression of miR-223 from different kinds of hosts infected with S.Japonicum,we investigate the difference between RAW264.7 macrophages stimulated by SEA and SSA,and we learn the regulation function of miR-223 after transfected into macrophages and SEA,SSA stimulation.This research may be helpful for the understanding of regulation function of miR-223 in hosts innate immunity against Schistosomiasis.1.Expression of miR-223 among different suitable rodent hosts and different period after infected with S.Japonicum.We infect suitable host BALB/c mouse,non-suitable host Wistar rats and non-permissive host M.Fortis with cercaria,and collect the serum in 3d,7d and 10 d after infecion,then we detect the expression of miR-223 from different suitable rodent hosts by qPCR.Results showed significent difference of miR-223 expression among different suitable rodent hosts.An increase in miR-223 expression level was observed as time prolonging in mouse.While a first rise then down trend was seen in rats.And a decrease trend was found in M.Fortis.In addition,the quantity of miR-223 in mouse is higher than that of rats,and the quantity of miR-223 in rats is higher than that of M.Fortis.Indicating that miR-223 showed a special regulation function in different suitable rodent hosts against Schistosomiasis infection,and it may influence the living,growing and developing process of S.Japonicum in hosts’ micro-environments.2.Expression of miR-223 from macrophages stimulated with different antigen.We detect the expression of miR-223 from macrophages RAW264.7 after stimulated by TLR ligand,SEA and SSA.Result showed a decrease in miR-223 expression level was observed in macrophages treated with POLY(I:C)(TLR3 ligand)and LPS(TLR4 ligand).An increase in miR-223 expression level was seen in macrophages stimulated by PAM3CSK4(TLR2 ligand).The expression of miR-223 decrease after macrophages was stimulated by SSA,an opposite result occured while macrophages was stimulated by SEA.Indicating that miR-223 can regulate macrophages stimulated by SEA and SSA with TLR signaling pathways.3.Regulation of miR-223 on macrophages stimulated with different antigen.To clearify the regulation function of miR-223 on macrophages stimulated with SEA and SSA,we over-expressed miR-223 in macrophages,we stimulate macrophages with SEA and SSA,and observed the changes of TLR signaling molecules,inflammatory factors,macrophages surface molecule and iNOs.When macrophages was stimulated by SEA,expression of TLR1、2、3、4 increased in various degree.Among which,the expression of TLR1 and TLR2 was significent(P<0.05).Expression of MyD88 falled,TRIF rised,and inflammatory factors like IL-1β,IL-10,IL-6,TNF-αboth increased.When macrophages was stimulated by SSA,expression of TLR1,4 increased significently(P<0.05),while TLR2 decreased.The expression of MyD88 rised,TRIF falled,and IL-6 was not obvious.Indicating that TLR signaling molecules on macrophages may identify and activate SEA and SSA,which may induce production of immune effector molecules and eventually play an important role in hosts’ innate immunity against schistosomiasis.we transfected macrophages with miR-223 mimics before stimulated by SEA,and the data showed that despite TLR2,the expression of TLR1,TLR3 and TLR4 were all lower than that of the negative control group,among which TLR4 was the most sinificent.TLR signaling molecules MyD88 rised,TRIF falled.Inflammatory factors IL-6 、 IL-1 β,IL-10 increased,while TNF-α decreased.But compared with macrophages stimulated by SEA without miR-223 transfection,the expression of inflammatory factors reduced,among which IL-6 was the most significent,followed by IL-1β.Indicating that miR-223 may down regulate the expression of TLR1,2,3,4and TRIF,up regulate the expression of MyD88,and eventually induce the down regulation of inflammatory factors like IL-6、IL-1β、IL-10 and TNF-α.We stimulate macrophages by SSA after miR-223 transfection,and the data showed that despite TLR4,the expression of TLR1,TLR2 and TLR3 were all lower than that of the negative control group.compared with macrophages stimulated by SSA without miR-223 transfection,the expression of TLR3 was not obvious,TLR1,4decreased,TLR2 increased.Expression of TLR signaling molecules MyD88 and TRIF were all reduced.Expression of inflammatory factors IL-1 β and IL-10 growed,TNF-αand IL-6 reduced.Single SSA stimulated TNF-α expression level was higher than that of miR-223 transfection group,other inflammatory factors expression level decreased,among which IL-1βwas the most significent.Indicating that miR-223 can regulate macrophages stimulated by SSA with down regulation of TLR1,TLR4 and MyD88,upregulation of TLR2 and TRIF.And eventually induce the down regulation of IL-1β、IL-10 and IL-6.In this study,we compared and analyzed the expression of miR-223 from different suitable rodent hosts infected with S.Japonicum.We discussed the regulation function of miR-223 on macrophages stimulated by SEA and SSA.This research may provide useful insights to the understanding of regulation mechanism of miR-223 on macrophages innate immunity against schistosomiasis. |
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