Effects And Mechanism Of CD38/MAPK Pathway On Pulmonary Injury In Mice With Sepsis Induced By Escherichia Coli

Objective:Mitogen activated protein kinase（MAPK）is a member of serine-threonine protein kinase family,acting as a signal transduction enzyme participating in immune response,cell migration,cell maturation and especially sepsis.The MAPK signaling pathway consists of three main components:extracellular regulated protein kinase（ERK）1/2,p38 and c-Jun N-terminal kinase（JNK）.Lipopolysaccharide（LPS）can induce phosphorylation of ERK1/2,p38 and JNK,as well as further activation of downstream transcription factors,leading to the production and release of various cytokines.CD38（cluster of differentiation 38）is a type II transmembrane protein with a molecular weight of 45 k Da,which can catalyze related reactions as cyclase and hydrolase,as well as a membrane receptor.Previous results of our laboratory showed that,after LPS stimulation,the pathological damage of kidney is aggravated in CD38^-/-sepsis mice,accompanied with up-regulation of pro-inflammatory factors（such as IL-1β,TNF-α,etc.）,and is associated with activation of TLR4/NF-κB signaling pathway.However,the effect of CD38 deficiency on MAPK signaling pathway and the role and mechanism of MAPK pathway in aggravated pulmonary injury and inflammatory response of CD38^-/-sepsis mice have not been reported.Therefore,we intend to use E.coli to induce sepsis mice model to illuminate the potential role and mechanism of CD38/MAPK pathway in pulmonary injury induced by E.coli.The results will broaden our understanding towards CD38 as a regulatory molecule,providing a new theoretical basis for the regulation of MAPK signaling pathway,as well as a new target for treatment of clinical sepsis.Methods:1.Eight-week old male wild type（WT）C57BL/6 mice were selected.Standard strains of E.coli ATCC25922 were diluted into suspensions with different concentrations with PBS.Mice were injected with suspensions with different concentrations intraperitoneally,and mice with injectiojn of equal amount of PBS were prepared for comparison.The lung tissues of mice were taken and H&E staining was used to observe the pathological changes in lung tissues.The optimal concentration and injection time of E.coli were determined according to the degree of pathological changes.2.The standard strains of E.coli ATCC25922 were prepared into a suspension with a concentration of 3×10⁸cfu/m L with PBS.Sepsis model was established by injecting 0.5m L E.coli suspensions into eight-week old male WT mice,and 0.5m L PBS was injected into other male WT mice as control.Mice were dissected 3 hours after the injection,the serums and lung tissues were isolated.3.H&E staining was used to compare the pathological differences of the lung tissues between mice injected with E.coli or PBS.Bacterial culture and gram staining were used to determine whether E.coli had successfully infected mouse lung tissues.4.Reverse transcription-polymerase chain reaction（RT-PCR）and real-time quantitative PCR were used to measure the mRNA expressions of pro-inflammatory factors（IL-1β,TNF-α,IL-6,and i NOS）,chemokine MCP-1 and chemokine receptor CCR2 in lung tissues of E.coli or PBS injected mice.5.Eight-week old male WT mice,CD38^-/-C3H/He N(CD38^-/-mice)mice and CD38^-/-C3H/HeJ(CD38^-/-TLR4^mutmice)mice were selected.Sepsis mice models were established by injection of 0.5m L 3×10⁸cfu/m L E.coli suspensions separately.Serums and lung tissues were isolated 3 hours after intraperitoneal injection.6.H&E staining was used to compare the histological differences of lung tissues of E.coli injected WT,CD38^-/-and CD38^-/-TLR4^mutmice.7.RT-PCR and real-time quantitative PCR were used to measure the mRNA expressions of pro-inflammatory factors（IL-1β,TNF-α,IL-6 and i NOS）,chemokine MCP-1 and chemokine receptor CCR2 in lung tissues of E.coli injected WT,CD38^-/-and CD38^-/-TLR4^mutmice.8.Western blotting was used to detect protein expression levels of MAPKs,NF-κB,phosphorylated MAPKs,phosphorylated NF-κB,pro-inflammatory factors（IL-1β,TNF-αand i NOS）,chemokine MCP-1 and chemokine receptor CCR2 in lung tissues of E.coli injected WT,CD38^-/-and CD38^-/-TLR4^mutmice.9.Enzyme-linked immunosorbent assay（ELISA）was used to detect the concentrations of MCP-1 in serum of E.coli injected WT,CD38^-/-and CD38^-/-TLR4^mutmice and PBS injected WT mice.Results:1.H&E staining results showed that,3 hours after intraperitoneal injection of3×10⁸cfu/m L E.coli suspensions could successfully induce sepsis mice model.2.Bacterial culture results showed no suspicious colony was observed in the plate of lung tissues of WT mice injected with PBS,while a suspicious colony was observed in the plate of lung tissues of WT mice injected with E.coli,which was identified as typical gram-negative bacilli by gram staining.3.H&E staining results showed that,compared with WT mice injected with PBS,WT mice injected with E.coli showed obvious lung tissue pathological damage.4.Compared with WT mice injected with PBS,mRNA expressions of pro-inflammatory factors（IL-1β,TNF-α,and i NOS）,chemokine MCP-1 and chemokine receptor CCR2 were significantly increased in the lung tissues of WT mice injected with E.coli.5.According to H&E staining,compared with WT mice injected with E.coli,CD38^-/-mice injected with E.coli had more severe pulmonary injury.Compared with CD38^-/-mice injected with E.coli,pulmonary injury of CD38^-/-TLR4^mutmice injected with E.coli was alleviated obviously.6.Compared with WT mice injected with E.coli,mRNA levels of pro-inflammatory cytokines（IL-1β,TNF-α,and i NOS）,chemokine MCP-1 and chemokine receptor CCR2 were increased markedly in the lung tissues of CD38^-/-mice injected with E.coli.7.Compared with CD38^-/-mice injected with E.coli,mRNA levels of inflammatory factors（IL-1β,TNF-αand i NOS）,chemokine MCP-1 and chemokine receptor CCR2 were significantly decreased in the lung tissues of CD38^-/-TLR4^mutmice injected with E.coli.8.Compared with WT mice injected with E.coli,CD38^-/-mice injected with E.coli showed increased levels of phosphorylated MAPK ERK1/2,phosphorylated MAPK p38,phosphorylated NF-κB p65,IL-1β,i NOS and MCP-1 in lung tissues,while the levels of phosphorylated MAPK JNK,phosphorylated NF-κB p105,MAPK ERK1/2,MAPK p38,MAPK JNK,NF-κB p65,NF-κB p105,TNF-αand CCR2 showed no significant change.9.Compared with CD38^-/-mice injected with E.coli,lung tissues of CD38^-/-TLR4^mutmice injected with E.coli showed decreased levels of phosphorylated MAPK ERK1/2,phosphorylated NF-κB p65,IL-1βand MCP-1,while the levels of phosphorylated MAPK p38,phosphorylated MAPK JNK,phosphorylated NF-κB p105,MAPK ERK1/2,MAPK p38,MAPK JNK,NF-κB p65,NF-κB p105,TNF-α,i NOS and CCR2 showed no significant change.10.Compared with WT mice injected with PBS,serum concentrations of chemokine MCP-1 in WT mice injected with E.coli were notably increased.Compared with WT mice injected with E.coli,the serum concentrations of MCP-1in CD38^-/-mice injected with E.coli were obviously increased.And compared with the CD38^-/-mice injected with E.coli,the serum concentrations of MCP-1 in CD38^-/-TLR4^mutmice injected with E.coli were significantly decreased.Conclusions:1.The sepsis mice model induced by the standard strain of E.coli ATCC25922was established successfully,which ensured the reliability of the experiments.2.After the injection of E.coli,the lung tissues of WT mice showed obvious tissue damage and increased expressions of inflammatory cytokines,chemokine and chemokine receptor.3.CD38 deficiency aggravates pulmonary injury,increases the expressions of pro-inflammatory cytokines IL-1β,i NOS and chemokine MCP-1 by activating the MAPK/NF-κB signaling pathways,while the expression of chemokine receptor CCR2 has no significant change.4.TLR4 mutation can alleviate pulmonary injuries in sepsis mice by down-regulating the activity of MAPK/NF-κB signaling pathways and inhibiting the enhanced expressions of inflammatory cytokine IL-1βand chemokine MCP-1induced by CD38 deficiency.