Effects Of Rabdosia Trifoliata On TLR4/NF-κB/NLRP3 Signaling Pathway In Kupffer Cells

Objective: To study the effect of Rabdosia trifoliata on TLR4/NF-κB/NLRP3 signaling pathway in Kupffer cells.Methods: 1.The samples of Rabdosia trifoliata were extracted with water and precipitated with alcohol,and then concentrated into extractum,which was the experimental sample.2.Cell grouping and treatment:(1)Effect of Rabdosia trifoliata extract on TLR4/NF-κ B/NLRP 3 signaling pathway of Kupffer cells(KC).KC cells covered about 90% of the well bottom,with three parallel wells at each concentration.Set blank control group: add serum-free DMEM medium;Model control group(LPS): RPMI-1640 complete medium containing LPS;Colchicine group(positive group): after colchicine pretreatment for 1 h,the liquid medicine was discarded and RPMI-1640 complete medium containing LPS was added;Rabdosia trifoliata extract: after pretreatment of Rabdosia trifoliata extract for 1 hour,discard the liquid medicine and add RPMI-1640 complete medium containing LPS;TLR4 blocker: after pretreatment with TLR4 blocker for1 h,the blocker was discarded and RPMI-1640 complete medium containing LPS was added.TLR4 blocker+Rabdosia trifoliata water extract: after pretreatment with TLR4 blocker for 1h,the blocker was discarded,after treatment with Rabdosia trifoliata extract for 1h,the liquid medicine was discarded,and then RPMI-1640 complete medium containing LPS was added.(2)the effect of herbal serum from rabdosia trifoliata on TLR4/NF-κB/NLRP3 signaling pathway in kupffer cells KC cells covered about 90% of the well bottom,with three parallel wells at each concentration.Set blank control group: add serum-free RPMI-1640medium;Model control group(LPS): DMEM medium containing LPS;Blank control serum +LPS group: after pretreatment of blank control serum for 1 hour,the liquid medicine was discarded and RPMI-1640 complete medium containing LPS was added;Colchicine-containing serum group: after pretreatment of colchicine-containing serum for 1 hour,the liquid medicine was discarded and RPMI-1640 complete medium containing LPS was added;Drug-containing serum group of Rabdosia trifoliata: after pretreatment of drug-containing serum of Rabdosia trifoliata for 1 hour,the liquid medicine was discarded and RPMI-1640 complete medium containing LPS was added;Group TLR4 blocker: after pretreatment with TLR4 blocker for 1 hour,the blocker was discarded and RPMI-1640 complete medium containing LPS was added.Group TLR4blocker+rabdosia trifoliata drug-containing serum: after pretreatment with TLR4 blocker for 1h,the blocker was discarded,and after treatment with rabdosia trifoliata drug-containing serum for 1h,the liquid medicine was discarded,and then RPMI-1640 complete medium containing LPS was added to continue the culture.After treatment,cells and cell supernatants were collected in each group,and the corresponding indexes were detected.3.Index detection: Collecting cells,detecting the m RNA expression of TLR4,IκBα,Caspase-1 and NLRP3 by RT-PCR,and detecting the protein expression of TLR4,IκBα,p-IκBα,Caspase-1 and NLRP 3 by Western Blot.The expression regions of TLR4,IκBα,p-IκBα,Caspase-1,NLRP3 and NF-κBp65 were detected by immunofluorescence,and the cell supernatants were collected.the contents of IL-1β,IL-18,TNF-α and IL-6were detected by ELISA Results:(1)The effect of Rabdosia trifoliata extract on KC TLR4/NF-κB/NLRP3 signal pathway1.compared with the blank control group,the expressions of TLR4 m RNA,IκBαm RNA,Caspase-1m RNA and NLRP3 m RNA in the model control group were significantly increased(P<0.01).Compared with the model control group,the expressions of TLR4 mrna,IκBαm RNA,Caspase-1m RNA and NLRP3 m RNA in colchicine group,Rabdosia trifoliata water extract group,TLR4 blocker and tlr4 blocker+Rabdosia trifoliata water extract group were significantly decreased(p < 0.01).Compared with TLR4 blocker group,the expressions of TLR4 mrna,IκBαm RNA,Caspase-1m RNA and NLRP3 m RNA in tlr4blocker+Rabdosia trifoliata water extract group were significantly decreased(P<0.05)2.Compared with the blank control group,the expressions of TLR4,IκBα,p-IκBα,Caspase-1 and NLRP3 in the model control group were significantly increased(P<0.01).Compared with the model control group,the expressions of TLR4,p-IκBα,Caspase-1 and NLRP3 in colchicine group were significantly decreased(p < 0.05).The expression of IκBα and p-IκBα protein decreased in the water extract group(P < 0.05).The expression of TLR4 and p-IκBα protein decreased in TLR4 blocker group(p < 0.05).The expression of TLR4,p-IκBα,Caspase-1 and NLRP3 protein in TLR4 blocker+water extract of rabdosia trifoliata decreased.Compared with TLR4 blocker group,the expression of p-IκBα and Caspase-1 protein in TLR4 blocker+water extract of Rabdosia trifoliata group decreased,and the difference was statistically significant(P<0.05).3.Compared with the blank control group,the expression of TLR4,IκBα,p-IκBα,Caspase-1,NLRP3 and NF-κBp65 protein in the model control group increased significantly(P<0.01).Compared with the model control group,the expression of TLR4,IκBα,p-IκBα,Caspase-1,NLRP3,NF-κBp65 and histone in the water extract of Rabdosia trifoliata were significantly decreased in colchicine group,TLR4 blocker group and TLR4 blocker+Rabdosia trifoliata water extract group(P<0.05).Compared with TLR4 blocker group,TLR4,IκBα,p-IκBα,Caspase-1,NLRP3,NF-κBp65 protein expression in TLR4 blocker+Rabdosia trifoliata water extract group decreased,with no significant difference(P>0.05).4.Compared with the blank control group,the expression of IL-6,IL-1β,IL-18 and TNF-α in the model control group increased(P<0.01).Compared with the model control group,the expressions of IL-6,IL-1β,IL-18 and TNF-α in colchicine group,rabdosia trifoliata extract group,TLR4 blocker group and TLR4 blocker+rabdosia trifoliata water extract group were significantly decreased(P<0.05).Compared with TLR4 blocker group,the expressions of IL-1β,IL-18 and TNF-α in TLR4blocker+rabdosia trifoliata water extract group were significantly lower(P<0.05).(2)the effect of herbal serum from rabdosia trifoliata on KC TLR4/NF-κB/NLRP3 signaling pathway1.compared with the blank control group,TLR4 m RNA,IκBαm RNA,Caspase-1m RNA and NLRP3 m RNA in the model control group were significantly increased(P<0.01).Compared with the model control group,the expressions of TLR4 mrna,IκBαm RNA,Caspase-1m RNA and NLRP3 m RNA in colchicine-containing serum group,rabdosia trifoliatacontaining serum group,TLR4 blocker group and tlr4 blocker+rabdosia trifoliata-containing serum group were significantly decreased(P<0.01).Compared with TLR4 blocker group,the expressions of TLR4 mrna,IκBαm RNA and Caspase-1m RNA in tlr4 blocker+rabdosia trifoliata serum group were decreased(P<0.05).2.Compared with the blank control group,the expression of TLR4,IκBα,p-IκBα,Caspase-1 and NLRP3 protein in the model control group increased significantly(P<0.01).Compared with the model control group,the protein expressions of TLR4,IκBα,p-IκBα,Caspase-1 and NLRP3 in colchicine serum group decreased(p < 0.05).The expression of TLR4 and p-IκBα protein decreased in the serum group containing rabdosia trifoliata(p < 0.05).The expression of TLR4,IκBα and p-IκBα protein decreased in TLR4 blocker group(p < 0.05).The expression of TLR4,IκBα,p-IκBα,Caspase-1 and NLRP3 protein in TLR4 blocker+rabdosia trifoliata serum group decreased significantly(P<0.01).Compared with TLR4 blocker group,the expression of p-IκBα and NLRP3 protein in TLR4blocker+rabdosia trifoliata serum group decreased significantly(P<0.01).3.Compared with the blank control group,the expression of TLR4,IκBα,p-IκBα,Caspase-1,NLRP3 and NF-κBp65 protein in the model control group increased significantly(P<0.01).Compared with the model control group,the expression of IκBα protein in LPS+blank serum group decreased(P < 0.05).The expressions of TLR4,IκBα,p-IκBα,Caspase-1,NLRP3 and NF-κBp65 protein in colchicine-containing serum group,rabdosia trifoliata-containing serum group,TLR4 blocker group and TLR4blocker+rabdosia trifoliata-containing serum group were significantly decreased(P<0.05).Compared with TLR4 blocker group,TLR4,NLRP3,NF-κBp65,IκBα,p-IκBα and Caspase-1 protein expression in TLR4blocker+rabdosia trifoliata serum group had no significant difference(P>0.05).4.Compared with the blank control group,the expressions of IL-1β,IL-18,TNF-α and IL-6 in the model control group were significantly increased(P<0.01).Compared with the model control group,the expressions of IL-1β,IL-18,TNF-α and IL-6 in colchicine-containing serum group,rabdosia trifoliata-containing serum group and TLR4blocker+rabdosia trifoliata-containing serum group were significantly decreased(P<0.05).Compared with TLR4 blocker group,the expression of IL-1β and IL-18 decreased significantly in TLR4 blocker+rabdosia trifoliata serum group(P<0.05).Conclusion: the results of this study show that rabdosia trifoliata can inhibit the gene expression of TLR4,IκBα,NF-κBp65,NLRP3 and Caspase-1,inhibit the phosphorylation of IκBα protein and reduce the expression of IL-1β,IL-6,IL-18 and TNF-α.The results indicated that rabdosia trifoliata inhibited the activation of Kupffer cells and downregulated the expression and release of inflammatory factors by downregulating the activation of TLR4/NF-κB/NLRP3 signaling pathway.Therefore,we can speculate that the anti-hepatic fibrosis mechanism of Rabdosia trifoliata is to inhibit the activation of TLR4/NF-κB/NLRP3 signal pathway,thereby down-regulating the expression and release of inflammatory factors,weakening the inflammatory damage of liver cells and achieving the anti-hepatic fibrosis effect.