| 1.Background and PurposeEndotoxemia is an acute inflammatory disease caused by endotoxin(main component is lipopolysaccharide,LPS)entering blood.LPS can activate macrophages and dendritic cells in the blood,which release massive pro-inflammatory cytokines and lead to abnormal activation of complement system and coagulation system.Endotoxemia can lead to multiple organ failure,diffuse intravascular coagulation,shock and even death.Interleukin-10(IL-10)is an important immunoregulatory cytokine,which can inhibit excessive immune response and reduce inflammatory injury in a variety of inflammatory diseases and autoimmune diseases.In mice model of endotoxemia,IL-10 can inhibit inflammation and improve the survival rate of mice.Traditional Chinese medicine Liangmianzhen is the dry root of Zanthoxylum nitidum,which is used in the treatment of bruise,gingivitis and rheumatism.Previous studies in our lab found that Nitidine chloride(NC),the bioactive ingredient in Liangmianzhen,could enhance IL-10 expression in dendritic cells.Here,we determined the characteristics of NC regulating IL-10,investigated its target and mechanism,and assessed its anti-inflammatory activity in endotoxemia mice.2.Experimental Approach(1)Study on the Characteristic of NC Regulating IL-10 expressionRAW264.7 cells,THP-1 cells,bone marrow-derived dendritic cells(BMDCs),and peritoneal macrophages were involved in this study.Cytotoxicity was determined by CCK8 assay,lactate dehydrogenase assay and Annexin V-PI staining.Time-dependent and dose-dependent IL-10 expression were assessed with non-toxic dose of NC.IL-10 mRNA level was determined by quantitative polymerase chain reaction(qPCR),and the secretion of IL-10 protein was assessed by enzyme-linked immunosorbent assay(ELISA).(2)NC enhancing IL-10 production by inhibiting TOP1At first,topoisomerase I(TOP1)and superhelix DNA were used to determine the inhibitory effect of NC on TOP1 enzyme activity.Next,the cytotoxicity of TOP1 inhibitors,topotecan(TPT)and active metabolite of irinotecan(SN-38),was determined by CCK8 assay and LDH assay.IL-10 mRNA levels and IL-10 protein levels was evaluated in myeloid immune cells treated with TOP1 inhibitor and LPS.Then,THP-1 cells were transfected with lentiviruses containing TOP1 interfering shRNA and the knockdown effect was determined by western blotting and qPCR.At last,we investigated how TOP1 knockdown influenced NC,SN-38 and TPT regulating IL-10 expression.(3)Effects of NC and TOP1 inhibitors in mice endotoxemiaFirstly,mice model of endotoxemia was established by i.p.injection of LPS into Balb/c male mice,and the influence of NC and TOP1 inhibitors CPT-11 and TPT on the mice survival were investigated.Then,the concentrations of IL-10 and proinflammatory cytokines TNF-? and IL-6 in mice serum were determined by ELISA,and the mRNA levels of IL-10,TNF-? and IL-6 in liver and lung tissues were determined by qPCR.Next,to evaluate the importance of IL-10 in the antiinflammatory action of NC and TOP1 inhibitors,IL-10 deficient mice and anti-IL-10 antibody were used.Under these conditions,we investigated the mice survival and serum pro-inflammatory cytokines.Lastly,the significance of IL-10 in vitro was evaluated in IL-10 deficient PMs and anti-IL-10 treated RAW264.7 cells.(4)Mechanism Study of NC and TOP1 inhibitors promoting IL-10 expressionFirstly,influence of NC and TOP1 inhibitors on IL-10 mRNA stability was evaluated by mRNA degradation assay.Then,the activation of PI3K-Akt pathway by NC and TOP1 inhibitors was investigated by western blotting,and the role of this pathway in the regulation of IL-10 by NC and TOP1 inhibitors was evaluated by pathway inhibitors.Next,wash out assay was used to examine the reversibility of NC and TOP1 inhibitors regulating IL-10 expression.Comet assay and phosphorylation of H2 AX,ATM,ATR and Akt were investigated to assess the DNA damage response.Lastly,we used ATM and ATR inhibitors to block DNA damage response,and determined the effects on IL-10 expression regulation by NC and TOP1 inhibitors.3.Results(1)Non-toxic dose of NC(1 ~ 4 ?M)could enhance IL-10 expression in LPSstimulated RAW264.7 macrophages,THP-1 monocytes,bone marrow-derived dendritic cells and peritoneal macrophages in a concentration-dependent and timedependent manner.NC could not enhance IL-10 expression when its pretreatment time was less than 9 hours,nor could it promote IL-10 production when cells were treated alone with NC without LPS stimulation.(2)NC could completely inhibit TOP1 activity at 10-50 ?M in TOP1 enzyme activity test.TOP1 inhibitors SN-38 and TPT could significantly increase IL-10 mRNA level and IL-10 protein secretion in LPS-stimulated RAW264.7 macrophages,THP-1 monocytes,bone marrow-derived dendritic cells and peritoneal macrophages in a nontoxic dose range(for SN-38,25-100 nM;for TPT,50-200 nM),which was similar to NC.By screening,we found that TOP1 shRNA(5'-GCCGAAGAAAGAAGAGGA ACA-3')could effectively knockdown TOP1 expression.Then we found that TOP1 knockdown by shRNA could enhance the expression of IL-10 gene,and TOP1 knockdown blocked the up-regulation of IL-10 expression by NC,SN-38 and TPT.(3)In endotoxemia mice,nontoxic dose of NC(5 mg/kg),CPT-11(10 mg/kg)and TPT(0.5 mg/kg)could significantly increase the mice survival rate to 70-80%.Organ damages,diarrhea and convulsions caused by endotoxemia were also alleviated by NC and TOP1 inhibitors.The production of pro-inflammatory cytokines and antiinflammatory IL-10 in serum,liver and lung were both elevated in endotoxemia mice compared with control mice.NC or TOP1 inhibitor treatment could further increase IL-10 production,while decreased the levels of pro-inflammatory TNF-? and IL-6.In IL-10 deficient mice,NC and CPT-11 could no longer improve the mice survival rate,and their inhibition on pro-inflammatory cytokines was attenuated.Similar phenomenon was observed in normal mice treated with anti-IL-10 antibody,which indicated that IL-10 is necessary to the anti-inflammatory effect of NC and TOP1 inhibitor.(4)In LPS-stimulated RAW264.7 cells,NC and TOP1 inhibitors did not affect the stability of IL-10 mRNA.By screening of pathway inhibitors,we found that PI3K-Akt pathway plays an important role in the up-regulation of IL-10 expression in NC.NC and TOP1 inhibitors promoted the activation of Akt and phosphorylation of downstream GSK3? and CREB in a concentration-dependent manner,thereby promoting IL-10 expression,while PI3 K and Akt inhibitors could block this process.In endotoxemia mice,the effects of NC on enhancing IL-10 production and elevating survival rate were also mediated by Akt.Nontoxic dose of NC and TOP1 inhibitors could induce DNA damage,enhance H2 AX phosphorylation,and activate DNA damage response,which promoted ATM,ATR activation,DNA repair,and Akt phosphorylation.Blocking DNA damage response by ATM and ATR inhibitor impeded the IL-10 regulating effect,and increased the cytotoxicity of NC and TOP1 inhibitors.PI3 K and Akt inhibitor.4.ConclusionsNatural product NC can promote IL-10 expression in LPS-stimulated myeloid immune cells by inhibiting TOP1.Similar effect can be found in medicinal TOP1 inhibitors SN-38 and TPT.By enhancing IL-10 production in mice endotoxemia,NC and TOP1 inhibitors could reduce the production of pro-inflammatory cytokines TNF-? and IL-6,alleviate the inflammation damage in liver and lung,and elevate the survival rate of mice.Mechanistically,NC and TOP1 inhibitors induced accumulated DNA damage and activated DNA damage response including ATR/ATM phosphorylation and subsequent AKT activation,which promoted LPS-induced IL-10 production.Blocking DNA damage response by ATM,ATR or Akt inhibitor prevented NC and TOP1 inhibitor from upregulating IL-10 expression.These data help to understand the mechanisms underlying anti-inflammatory effects of NC and TOP1 inhibitors,and provide experimental basis for their potential use in the treatment of endotoxemia. |
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