LCD Affects DLCs Cell Through The TLR9-MyD88-NF-κB Signaling Pathway

Objective:Study on LCD affects protein expressions of MyD88,P-IRAK1,NF-κB in TLR9-MyD88-NF-κB signaling pathway.To explore mechanism of increasing the curative effect,reducing side effects,with LCD treating on SLE,besides,it will provide a new method for the treatment on SLE.Method:Collect and separate clinical strong positive ds-DNA of 8 SLE patients’ serum.And preparate immune complex(IC)for the next experiment.Cultivate KG-1,then use PMA and TNF-α inducing KG-1 into DLCs(Dendritic like cells).After inducting,we use flow cytometry to detect type of the inducted cells.Next,we divide into 4 groups,which are blank group,control group,DXM group,LCD group.Blank group is without any drug treatment,only using RPMI-1640 to culture 18h;Control group adds IC to stimulate and culture 18h;DXM group firstly adds IC to stimulate and culture 6h,and then add DXM to culture 12h;LCD group firstly adds IC to stimulate and culture 6h,and then add LCD to culture 12h.Extract Total RNA from cells,using Realtime-PCR method to detect TLR9,MyD88 mRNA transcription level;In addition,collect protein from cells,using Western blot method to detect TLR9,MyD88,p-IRAK1,NF-κB protein expression.Result:Using flow cytometry to detect the type of cells which from KG-1 induced.The result is this type of induced cells is the same to DCs,which express CD80,CD54.In the inducted cells from KG-1,the protein expressions of MyD88,p-IRAK1,NF-κB all increase.And the transcriptional levels of TLR9、MyD88 mRNA also increase,compared with blank group,these differences with statistical significance(P<0.05);Compared with control group,DXM group TLR9,MyD88 mRNA,MyD88 protein expression level raised,these differences are not statistically significant(P>0.05),but TLR9,p-IRAK1,NF-κB protein expression level all decrease,and the differences have statistical significance(p<0.05);Compared with control group,the protein expressions of MyD88,p-IRAK1,NF-κB all decrease,and the transcriptional levels of TLR9、MyD88 mRNA also decrease in LCD group,these differences with statistical significance(P<0.05).Conclusion:Using IC to stimulate the induced cells which is one type of DLCs fromKG-1,TLR9-MyD88-NF-κB signaling pathway and its downstream important signaling molecules are obviously activated.LCD through by cutting down on TLR9,MyD88 mRNA transcription expression,decreases the expression of many important signaling proteins in the TLR9-MyD88-NF-κB pathway,such as TLR9,MyD88,P-IRAK1.Cutting down activation levels of TLR9-MyD88-NF-κB signaling pathway,reducing the expression of proinflammatory factor,these may be the important mechanism that LCD has the effects on treating SLE,which can increase the curative effect,reduce side effects.