Regulatory Effect Of Baicalin, Sinomenine And Gemmaromone On TLR8 In THP-1 Cells

Objective : TLR8 is an important receptor molecule involved in innate immune response and specific immune response,but the studies on the mechanism of Chinese herbal medicine of TLR8 are few.Therefore,we selected those anti-inflammatory antiviral Chinese medicine which has been reported : baicalin,sinomenine,germacrone,plumbagin and gallic acid.The main objective of this study is to investigate whether baicalin,sinomenine etc.can play a immune role by regulating the TLR8.The results of this study will lay the foundation for the development of anti-inflammatory and antiviral drugs in the future.Methods:(1)We isolated normal human peripheral blood mononuclear cells(PBMCs)by the density gradient centrifugation method and used baicalin,sinomenine,germacrone,plumbagin,gallic acid and Resiquimod(R848)stimulate PBMCs 12 h and 24 h.The total RNA were extracted from the cells and detected by semi-quantitative RT-PCR method using the specific primer for the expression of TLR8 after baicalin,sinomenine and other monomers of Chinese traditional herbs stimulated PBMCs 12 h or 24 h.We screened out drugs that regulate the activity of TLR8.(2)CCK-8 method was used to determine the effect of the monomers which are screened included baicalin,sinomenine and gemmaromone on the viability of THP-1 cells after 12 h and 24 h.The corresponding drug concentration range was established.(3)Real-time q PCR was used to determine the transcription level changes of TLR8 gene of THP-1.ELISA was used to detect the density of IL-6,IL-12 in the THP-1 cells' up-layer culture liquid.The transcription level changes of TLR8 gene of normal human PBMCs stimulated by baicalin,sinomenine,germacrone were determined by q PCR.The expression of TLR8 in mononuclear cells with THP-1 cells was compared.Results:(1)The relative expression of TLR8 m RNA were increased by baicalin of the drugs group after 12h(P<0.05).Compared with the R848 control group,the TLR8 m RNA relative expression of THP-1 cells which was intervened 24 h with sinomenine after R848 had stimulatied was significantly decreased(P<0.01).The group treated with gemmaromone after 12 h had synergistic effect but no significant difference(P>0.05).Compared with the blank group,plumbagin and allic acid had no significant effect(P>0.05).(2)When the activation of baicalin of the drugs group was 400?mol/L,THP-1 cells were less affected.Low-dose baicalin(100?mol/L)will be beneficial to the growth and reproduction of THP-1 cells.When the germacrone concentration maintained at 200?mol/L(P<0.01),THP-1 cells were less affected.After the R848 stimulation,the concentration of sinomenine in the drug intervention group was 400?mol/L,which had little effect on the activation of THP-1 cells.(3)Compared with blank group,the expression of TLR8 m RNA increased by baicalin and germacrone in the drugs group.The secretion of IL-6 and IL-12 in THP-1 cells was inhibited by 400?mol/L baicalin(P<0.05).Germacrone group's content of IL-12 was significantly increased(P<0.01).The IL-6 level was decreased with high dose of germacrone group(P<0.05).After R848 stimulatied,Sinomenine down-regulated TLR8 m RNA of THP-1 cells(P<0.01).Sinomenine inhibited THP-1 cells secreting IL-6 and IL-12(P<0.01),and its inhibitory effect also increaseed with time.One of the founds in validation experiments was that 400?mol/L baicalin and 200?mol/L germacrone up-regulated the expression of TLR8 m RNA in peripheral blood mononuclear cells(P<0.05).Another was that the expression of TLR8 m RNA of Human PBMCs was down-regulated by sinomenine(after R848 stimulatied)(P<0.01).The results of this study are consistent with the results of baicalin,sinomenine and gemcitabine on THP-1 cells.Conclusion:Sinomenine,baicalin and germacrone have a moderating effect on THP-1 cells and human peripheral blood mononuclear cells.Baicalin and germacrone could improve he expression of TLR8 of THP-1 cells.The expressions of the common downstream factors(IL-6,IL-12)of TLR4 and TLR8 were decreased when baicalin stimulatied.This result confirmed that baicalin may not influence the human natural immunity by TLR8.Germacrone by raising the expression of TLR8 in THP-1 cells promoted the secretion of IL-12 and inhibited IL-6,which could illustrate less anti-inflammatory effects of germacrone may be related to the expression of TLR8.Sinomenine could significantly reduce the expression of TLR8 of THP-1 cells and human PBMCs.