| Background and objectivesSepsis is uncontrolled proinflammatory response triggered with recognition of pathogen associated molecular patterns(PAMPs)by pattern recognition receptor(PRRs)under conditions of pathogen infection.Lipopolysaccharide(LPS),the major component of the out membrane on Gram-negative Bacteria,is the most deeply investigated among the defined PAMPs.LPS is more important in triggering sepsis.Therefore,it is of vital importance to the prevention and treatment of sepsis through investigation of the blocking and clearing mechanisms of LPS.Kukoamine B(KB)is an alkaloid compound isolated from a traditional Chinese herb Cotex Lycii in our previous study.It has been successfully obtained through chemical synthesis.KB effectively neutralizes LPS,inhibits its proinflammatory bioactivity and significantly improves the survival of sepsis model animals.KB has been testified in a phase?clinical trial.The mechanism of KB is due to its high binding affinity with LPS,which results in the formation of KB/LPS complex.However,it is less clear about the distribution,metabolism and possible accumulative toxicity of the KB/LPS complex,which is meaningful for better elucidating the mechanisms and safety of KB.To determine the above mentioned issues,we labeled LPS,KB,cells and receptors with fluorescein and utilized laser scanning confocal microscopy(LSCM),flow cytometry(FCM)and siRNA interference to explore the possible mechanisms which may explain the distribution and metabolism of KB/LPS complex.Methods1.The effect of KB/LPS complex to the distribution of LPS in vivoLPS labeled by FITC(FITC-LPS)for tracing of KB/LPS complex,then FITC-LPS(1mg/kg)mixed with KB(3.6mg/kg)and vibrated at 37? for 30 min in vitro for preparation of KB/FITC-LPS complex.Then the LPS or KB/LPS complex was administrated in C57 BL/6 mice via tail vein injection.Then peripheral blood samples were collected 1,4,8 and 24 hours respectively and the fluorescence intensity in plasma was detected to determine whether KB can affect LPS level in peripheral blood.Then organ homogenate of heart,liver,spleen,lung and kidney were obtained at the same time points and fluorescence intensity of FITC-LPS was detected to confirm the distribution of free LPS or KB/LPS complexes in various organs.2.Study on the main cell types involved in the uptake of KB/LPS complex in liver2.1 FITC-LPS or KB/FITC-LPS was injected in C57 BL/6 mice via tail vein,liver tissue was obtained 1 hour after injection and frozen sections were prepared.The hepatocytes(HC),Kupffer cells(KC),liver sinusoidal endothelial cells(LSEC),and hepatic stellate cells(HSC),were labeled by antibodies for Cy3-cytokeration18,Cy3-F4/80,Cy3-CD31 and Cy3-GFAP respectively,the co-localization of the main cell and FITC-LPS were detected by laser scanning confocal microscope(LSCM).2.2 A mice model of KC depletion was prepared by tail vein injection of Clophosome.A.Clodronate liposomes.Liver tissue was obtained after 48 h and frozen tissue sections were prepared.KC or HC were labeled by Cy3-F4/80 or Cy3-cytokeration 18 antibody respectively and detected by LSCM.KB/FITC-LPS complex was injected in KC depletion mice or C57BL/6 mice via tail vein injection.Liver tissue was obtained after 1h and frozen tissue sections were prepared.KC or HC were labeled by Cy3-F4/80 or Cy3-cytokeration 18 antibody respectively,the mean fluorescence intensity of FITC-LPS and co-localization of FITC-LPS and KC or HC in liver were detected by LSCM and confirmed whether KC was involved in the uptake of KB/FITC-LPS complex in liver.2.3 The primary HC and KC were isolated from C57 BL/6 mice via situ liver lavage,density gradient centrifugation and immunomagnetic beads separation.Human liver cancer cells(Hep G2)or mouse macropahge like RAW264.7 cells were used as cell line controls.Each type of cells was treated with FITC-LPS or KB/FITC-LPS complex for 1 hour,The uptake of FITC-LPS was detected by FCM.Comparative analysis was made for the uptake of free LPS and KB/LPS complex in HC or KC.3.Study on the receptor type involved in the uptake of KB/LPS complex in HC.3.1 The effect of KB on the expression of endocytosis receptor mRNA in HC was detected by RT-PCR.Primary HC was treated with LPS(1?g/ml),KB(15?M)or KB/LPS complex for 4 hour,the mRNA expression of ASGPR,SR-B1,SR-A,TRFC,MARCO,LOX-1 and TLR4 were detected by RT-PCR,and analyzed the receptor types involved in the uptake of KB or KB/LPS complex in HC.3.2 The relationship between ASGPR receptor and the uptake of KB/LPS complex in HC was analyzed by Western blotting(WB)and FCM.(1)Primary HC were treated with KB(15?M)for 1,2 and 4 hours,the protein level of ASGPR receptor was detected by WB,compared with control and analyzed the effect of KB on the expression of ASGPR receptor.(2)KB(3.6mg/kg)as injected in C57 BL/6 mice by tail vein injection,the liver tissue was obtained after 1,2 and 4 hours,the protein expression of ASGPR receptor was detected by WB,compared with untreated mice and analyzed the effect of KB on the protein expression of liver ASGPR receptor.(3)ASGPR receptor of Hep G2 was down-regulated by siRNA transfection,then treated Hep G2 cells with KB/FITC-LPS complex for 1h,the fluorescence intensity of FITC-LPS in Hep G2 was detected by FCM,analyzed the effect of ASGPR receptor to the uptake of KB/LPS complex in HC and confirmed that whether ASGPR receptors involved in uptake of KB/LPS complexes in HC.3.3 The co-localization of KB and ASGPR receptor by LSCM,and combining the competitive uptake experiments,molecular simulation of docking,to confirm whether the uptake of KB/LPS complex was mediated by ASGPR receptor.(1)The primary HC of C57BL/6 mice were treated with FITC-KB(15??)for 30 min,ASGPR receptor was labeled by Cy3-ASGPR,the co-localization of KB and ASGPR receptor was detected by LSCM.(2)The primary HC of C57BL/6 mice were treated with FITC-KB(15??)or together with Ga LNAc(0.25,0.5 and 1.0mM)for 1 hour,the uptake of FITC-KB was detected by LSCM,confirmed that whether competitive uptake of GaLNAc and KB in HC.(3)Binding sites and binding force of KB and the H1 subunit of ASGPR receptor was simulated by Auto Dock Vina,in the H1 subunit space which size was 20 x 18 x 20,at 6.46 x-4.47 x 10.41 points as the center,contains carbon compounds identify region as the conformation research object,analyzed and compared its possible binding sites and binding force of H1 subunit and KB by AutoDockTools GUI,and analyzed the results by AutoDock Tools and Lig Plot+.4.Molecular mechanism research of degradation and removal of KB/LPS complex in HC.Labeled KB/LPS complex by FITC-LPS or FITC-KB in primary HC of TLR4-/-mice,treated TLR4-/-HC with KB/LPS complex for 1 hour,early endosome,late endosome,lysosome and ASGPR receptor were labeled by Cy3-EEA1 antibody,Cy3-Rab7 antibody,Cy3-LAMP1 antibody and Alexa Fluor 647-ASGPR antibody respectively.The co-localization of KB/LPS complex and ASGPR receptor,endosome or lysosome was detected By LSCM,confirmed the possible pathway of degradation and removal of KB/LPS complex in HC.Results:1.The study of KB/LPS Complex formed effect LPS distributionPlasma detection results,after injected FITC-LPS or KB/FITC-LPS complex 1,4,8 or 24 h,KB+LPS group FITC-LPS detection value showed significant decreases than that in LPS group(P<0.01),showed that KB/LPS Complex accelerated the peripheral blood LPS elimination;Viscera tissues homogenate detection results,in 1,4,8 and 24 h,demonstrated that LPS mostly distributed on liver,less in heart,spleen,lung and kidney.These results indicate that liver is the major LPS metabolic organ and KB/LPS complex promotes the LPS accumulate in liver.2.The main cell type of KB/LPS Complex uptake in liver.2.1 As demonstrated in LSCM detection,LPS is mainly distributed in HC and KC in liver section while HSC and LSEC didn't have obvious LPS uptake.Therefore,HC and KC were the main cell type of KB/LPS Complex uptake in liver.2.2 After tail vein injection of the Clophosome.A.Clodronate liposomes,Cy3-F4/80)positive cell(HC)obviously decreased in the liver section.However,the number of Cy3-cytokeration 18 positive cell(HC)remained unaltered.Therefore,KC was selectively exhaused.In KC exhausation group,KB/FITC-LPS complex was still absorbed by liver,suggesting that KC lost didn't affect the liver uptake of the KB/LPS Complex.Moreover,KC might not be the main cell take part in the KB/LPS Complex uptake.2.3 The FCM detection showed that KB/LPS Complex formation could obviously promote C57 BL/6 mice HC and Hep G2 cell to uptake LPS(P<0.05),while significantly inhibited C57 BL/6 mice KC and RAW264.7 to uptake LPS(P<0.05).These data suggest that HC mainly participates in conjugated LPS uptake while KC is mainly involved in free LPS uptake in liver.3.Type of receptor for uptake of the KB/LPS Complex3.1 RT-PCR detection results demonstrated that KB and KB/LPS Complex could both up-regulate ASGPR receptor mRNA in HC(P<0.05,P<0.01).3.2 Western blot showed that the HC untreated by KB for 1,2 and 4h displayed decrease of ASGPR receptor gradually.In KB treated group,HC expression of ASGPR receptor increased gradually in 1 and 2h but decreased obviously in 4h.Injection of KB to mice liver resulted in gradual increase of ASGPR receptor while the control group got into weaken expression.These results indicate that KB could significantly up-regulate ASGPR receptor expression.FCM relative intensity of fluorescence showed that Hep G2 and ASGPR receptor down-regulated could significant inhibited HepG2 uptake LPS(P<0.05,vs NC-siRNA treatment group),suggested that ASGPR receptor is required to mediate the uptakeof the KB/LPS Complex in liver.3.3 LSCM detection image indicated that KB(green)colocalize with ASGPR receptor(red)on HC cell membrane,illustrating KB and ASGPR receptor had space colocalization,suggested that KB combined the ASGPR receptor.With the increase of Ga LNAc injection,HC phagocytosed less FITC-KB(green).FITC-KB average flourescence intensity showed that Ga LNAc significant inhibited HC taking in the KB(P<0.01).Furthermore,Ga LNAc and KB had common Mediated internalization pathway on HC membrane?The Molecular docking results showed that KB had hydrogen bond with the ASGPR receptor getting through the Asp241?Gln239?Glu252?Asn264?Asn208 amino acid residue.Moreover,KB showed hydrophobic interaction with Tyr272,Asp266,Arg270,Trp243,Asn264,Arg236 and Thr258 amino acid residues on ASGPR receptor.These all suggested KB directly binds to the ASGPR receptor.4.HC degradation and KB/LPS Complex eliminate pathwayLSCM detection image indicated that after KB/LPS Complex got into HC,FITC-KB(green)or FITC-LPS(green)existed in Cy3 mark EEA1(red)or Cy3 mark Rab7(red)or Cy3 mark LAMP1(red),and these coincide with 647-ASGPR(yellow),illustrated all three had space colocalization.Indicate that KB/LPS Complex might eliminated by ASGPR receptor related lysosome pathway when it got into HC by ASGPR receptor.Conclusions:1.The formation of LPS/KB complex accelerates the peripheral clearance of LPS in mice,which may be attributed to the fact that KB enhances the accumulation of LPS in liver.2.The uptake of LPS/KB complex in liver occurs mainly in hepatocytes.The ASGPR receptor which is mainly expressed in membrane of hepatocytes mediates uptake of the complex by recognizing KB directly.3.The internalized LPS/KB complex enters endosomes and underwent degradation in the endosome-lysosome pathway. |
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