| Objective:To establish a rat model of chronic alcoholic liver injury complicated with gouty arthritis,and to study the effects of the total extract of Lagotis brachystachya Maxim on the Toll-like receptor（TLR）/myeloid differentiation factor 88（MyD88）/nuclear transcription factorκB（NF-κB）,NOD-like receptor protein 3（NLRP3）,and Janus kinase（JAK）-signal transduction and transcriptional activator（STAT）signaling pathways,and to explore the mechanism of the total extract of L.brachystachya Maxim against chronic alcoholic liver injury complicated with gouty arthritis.Thirty-two monomeric compounds contained in the L.brachystachya Maxim were selected to act on the THP-1 cell inflammation model established by Monosodium Urate（MSU）combined with Lipopolysaccharide（LPS）and the alcohol-injured HepG2 cells,respectively,to screen out the monomeric compounds that were effective for the two cell models,and study the possible mechanism based on TLR/MyD88/NF-κB,NLRP3,JAK/STAT,and MAPK signaling pathways,to finally obtain the monomeric compounds that can simultaneously produce therapeutic effects on the two cell models,and to explore the pharmacodynamic material basis and molecular mechanism of L.brachystachya Maxim.Method:1.Seventy rats were randomly divided into a normal group,a model group,a positive drug colchicine group(0.3 mg·kg-1),a positive drug bifendate group(100mg·kg-1),and a L.brachystachya Maxim(2.0,1.0,and 0.5 g·kg-1)dose group.The drug was given at 10 ml·kg-1 at 9:00 a.m.every day and 56-degree Hongxing Erguotou was given by gavage at 2:00 p.m.With 4 ml·kg-1 as the initial concentration,the drug was increased by 2 ml·kg-1 per week to 10 ml·kg-1 for maintenance.The drug was given by gavage for consecutive 8 weeks.The gouty arthritis model was established by articular injection of sodium urate（MSU）crystal while the alcohol was given in the eighth week.The toe volumes of each period were measured.At the end of the eighth week,blood was taken out after the last dose,and serum levels of uric acid（UA）,xanthine oxidase（XOD）and total bilirubin（TBIL）were detected.The levels of serum interleukin-1β（IL-1β）were detected by enzyme-linked immunosorbent assay（ELISA）.The levels of superoxide dismutase（SOD）and malondialdehyde（MDA）were detected with 10%liver tissue homogenate.The expressions of MyD88,Caspase-1,NF-κB,STAT3,TLR4,NLRP3,and JAK2 proteins in the liver and synovial membrane were detected by Western Blot.Hematoxylin and eosin（HE）staining was used to observe the pathological morphology of the liver and synovial membrane.2.THP-1 cells were induced to extend out of the pseudopodia for 48h and adhere to the wall using a final concentration of 100ng/ml PMA.The CCK-8 method was used to determine the effect of colchicine（0.002μM）and drugs（1.25 and 0.625μM）on the activity of THP-1 cells.After 48h of PMA induction,all the groups except the blank group were stimulated by adding a culture medium containing LPS for 3h,and then MSU crystals were added to establish an acute gout cell model.After the administration group was dosed based on modeling,it was placed in the incubator for continuous culture for 24 h.IL-1βcytokine was detected by Elisa for active drug screening.The screened effective monomer compounds were further verified by detecting TNF-αand IL-6 by Elisa.Western blotting was used to detect the protein expression levels of MyD88,Caspase-1,pNF-κB,NF-κB,TLR2,TLR4,STAT3,NLRP3,and JAK2 in THP-1 cells of the normal group,the model group,the colchicine group,and the effective compound group.3.The CCK-8 method was used to detect the effects of different concentrations（1%,2%,3%,4%,4.5%,5%,5.5%,and 6%）of absolute ethanol on the survival rate of HepG2 cells.The CCK-8 method was used to detect the effects of different concentrations（25,12.5,6.25,3.125,1.5625,0.78125,0.390625,and 0μM）of compounds on the survival rate of HepG2 cells.The protective effects of compounds（1.5625,0.78125μM）on the alcohol-induced injury of HepG2 cells were measured by the CCK-8 method.After the monolayer culture was placed in a 96-well plate for 24h in HepG2 cells,the medium was discarded.A blank medium was added to the blank group and model group,and a medium containing compounds with different concentrations was added to the administration group.The cells were incubated at 37C for 1 h with 5%CO2 and added with a certain concentration of ethanol.After the cells continuously interacted for 48h,the cells and the supernatant were treated accordingly for subsequent experiments.The levels of ALT and AST in the cell supernatant were detected by the enzyme labeling method,and effective monomer compounds were screened based on the results.The levels of IL-1β,IL-6,and TNF-αin the cell supernatant were detected by Elisa.The expression levels of MyD88,NF-κB,TLR4,Caspase-1,NLRP3,JAK2,STAT3,P38MAPK,p-P38MAPK,and JNKproteins in HepG2 cells were detected by Western blot.Result:1.（1）Compared with the normal group,the swelling of rats’feet in the model group was significantly increased at each time point（P<0.01）;Serum TNF-α,IL-1β,IL-6,ALT,AST,TC,TG,AKP,TBIL,XOD,and UA levels were significantly increased（P<0.01）.The MDA level in 10%liver tissue homogenate was significantly increased（P<0.01）,and the SOD level was significantly decreased（P<0.01）.The protein expression levels of MyD88,Caspase-1,NF-κB,STAT3,TLR4,NLRP3,and JAK2 in liver tissue and synovial tissue were up-regulated to varying degrees（P<0.05,P<0.01）.（2）Compared with the model group,the swelling of rats’feet decreased significantly after 12h（P<0.01）;Serum TNF-α,IL-1β,and IL-6 were all decreased to different degrees（P<0.05,P<0.01）.Serum UA levels of rats in colchicine group and medium and low dose groups were significantly reduced（P<0.05,P<0.01）.The levels of XOD,ALT,AST,TC,TG,ALP,AKP,and TBIL were decreased to different degrees in the bifendate group and the high,middle,and low dose groups（P<0.05,P<0.01）.The protein expression levels of MyD88,Caspase-1,NF-κB,STAT3,TLR4,NLRP3,and JAK2 in the liver and synovial tissues of rats in each group were down-regulated（P<0.05,P<0.01）.The pathological section states of liver and synovial tissues in rats of each group were significantly improved.2.（1）The effects of different groups on THP-1 cell viability were detected by CCK-8 cytotoxicity kit.Compared with the control group,colchicine at the concentration of 0.002μM had no significant effect on THP-1 cell viability.Each monomer compound had no significant effect on THP-1 cell viability at concentrations of 1.25 and 0.625μM;Therefore,this concentration was chosen to interfere with the THP-1-induced injury of cells by LPS in combination with MSU.（2）Possible effective monomer compounds were screened out according to the detection results of IL-1β,and the detection results of TNF-αand IL-6 were continued.Compared with the normal group,the model group was significantly increased（P<0.01）.Compared with the model group,the colchicine group and the three compounds including floxuridine B,lucerne-7-O-glucoside,and geniposide with an administration concentration of 1.25μm were compared.The levels of IL-1β,TNF-α,and IL-6 of 8compounds（echinacoside,quercetin,hydrocyanic,apigenin,luteolin-7-O-β-D-glucopyranoside,quercetin-7-O-glucoside,apigenin-7-O-β-D-glucoside and 8-epi brucine）at the administration concentration of 0.625μM were significantly decreased（P<0.01,P<0.05）.（3）Western blotting showed that compared with the normal group,the protein contents of MyD88,TLR2,TLR4,pNF-κB/NF-κB,NLRP3,Caspase-1,JAK2,and STAT3 were significantly increased in the model group（P<0.05,P<0.01）.Compared with the model group,the protein expression levels of TLR4 and PNF-κB/NF-κB were significantly decreased in the colchicine group（P<0.01）.The eleven compounds including floxuridine B,echinacoside,quercetin,hydrocyanic,lucerne-7-O-glucoside,apigenin,luteolin-7-O-β-D-glucopyranoside,quercetin-7-O-glucoside,apigenin-7-O-β-D-glucoside,8-epi strychnine monophosphate,and genocide could callback MyD88,TLR2,TLR4,pNF-κB/NF-κB to different degrees.Some of the protein expression levels of NLRP3,Caspase-1,JAK2,and STAT3 showed significant differences（P<0.05,P<0.01）.3.（1）Through the CCK-8 test,the alcohol concentration with the cell viability of50%–70%was selected and 4.5%was determined to be the optimal alcohol concentration for modeling.The CCK-8 method detected no significant effect of each group on HepG2 cell viability at concentrations of 1.5625 and 0.78125μM.The CCK-8 method was used to detect the effect of monomer compounds on alcohol-injured HepG2 cells,which showed a certain protective effect.Compared with the normal group,the cell survival rate in the model group was significantly reduced（P<0.01）.Compared with the model group,the cell viability of the biphenyl diester group was significantly increased（P<0.05）.A concentration of 1.5625μM of plantamajoside,floxuridine B,plantain D,kaempferol-7-O-glucoside,kaempferol glucuronide,quercetin,luteolin-7-glucuronide,luteolin,lucerne,procyanidin,lucerne-7-O-glucoside,kaempferol,apigenin,luteolin-7-O-β-D-glucopyranose,quercetin-7-O-glucoside,Among the 18 compounds including apigenin-7-O-D-glucoside,8-epistrychnine monophosphate,andβ-sitosterol,the four compounds including verbascoside,astragaloside,vanilloid,and 4-hydroxyphenylethanol at the concentration of 0.78125 significantly increased the cell viability（P<0.01,P<0.05）.（2）ALT and AST enzyme activities were detected by ELISA kit and compared with the normal group,ALT and AST enzyme activities in the model group were significantly increased（P<0.01）;Compared with the model group,the AST enzyme activity of the bifendate group was significantly decreased（P<0.05）.The enzyme activities of ALT and AST in the 1.5625μm plantamajoside,quercetin,luteolin-7-glucuronide,luteolin,kaempferol,and 0.78125μm lagotiside C,echinacoside,lucerne-7-O-glucoside,apigenin,and luteolin-7-O-β-D-glucopyranoside groups were significantly decreased（P<0.01,P<0.05）in the administration group.（3）Detection of expression levels of IL-1β,TNF-αand IL-6 inflammatory factors in cell supernatant by（3）Elisa:Compared with the normal group,the levels of IL-1β,TNF-αand IL-6 in the model group were significantly increased（P<0.01）;Compared with the model group,the levels of IL-1β,TNF-αand IL-6 in the bifendate group were significantly reduced（P<0.05）.In the administration group,the levels of nine IL-1βcompounds such as plantamajoside,lagotiside C,echinacoside,quercetin,luteolin-7-glucuronic acid,luteolin,procyanidin,kaempferol,and luteolin-7-O-β-D-glucopyranose were significantly decreased（P<0.01）.The level of eight compounds TNF-αsuch as lagotiside C,echinacoside,quercetin,luteolin-7-glucuronic acid,lucerne-7-O-glucoside,kaempferol,apigenin and luteolin-7-O-β-D-glucopyranose are remarkably reduce（P<0.01）,The levels of IL-6 of 11 compounds,including plantamajoside,lagotiside C,echinacoside,quercetin,luteolin-7-glucuronic acid,luteolin,procyanidin,lucerne-7-O-glucoside,kaempferol,apigenin,and luteolin-7-O-β-D-glucopyranose,were significantly decreased（P<0.01）.（4）Western blot analysis showed that compared with the normal group,the protein contents of MyD88,TLR4,NF-κB,NLRP3,Caspase-1,JAK2,STAT3,p-P38/P38,and JNK in the model group were significantly increased（P<0.01,P<0.05）.Compared with the model group,the protein expression levels of TLR4 and pNF-κB/NF-κB in the colchicine group were significantly decreased（P<0.01）.The eleven compounds including plantamajoside,lagotiside C,echinacoside,quercetin,luteolin-7-glucuronic acid,luteolin,hydrocyanic,lucerne-7-O-glucoside,kaempferol,apigenin,and luteolin-7-O-β-D-glucopyranose could callback the protein expression levels of MyD88,TLR4,NF-κB,NLRP3,Caspase-1,JAK2,STAT3,p-P38/P38 and JNK in different degrees（P<0.05,P<0.0.05）.Conclusion:The total extract of L.brachystachya Maxim showed a certain therapeutic effect on the rat model of chronic alcoholic liver injury with gouty arthritis,which might be based on the effects of TLR/MyD88/NF-κB,NLRP3 and JAK-STAT signaling pathways.Through studying the effect of 32 monomer compounds contained in the L.brachystachya Maxim on MSU combined with LPS-injured THP-1 cells and alcohol-injured HepG2 cells,11 compounds such as floxuridine b are screened to be effective on THP-1 cells of a gout inflammation model,and 11 compounds such as plantamajoside are screened to be effective on alcohol-injured HepG2 cells,Among them,six compounds such as echinacoside,quercetin,hyprocyanin,lucerne-7-O-glucoside,apigenin and luteolin-7-O-β-D-glucopyranoside were effective on two cell models at the same time,and they might be the main active components in the treatment of alcoholic liver injury combined with gouty arthritis in L.brachystachya Maxim,which might exert effects through TLR/MyD88/NF-κB,NLRP3,JAK-STAT,and MAPK signaling pathways. |
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