| Objectives:To investigate test the expression of NOD2 in mice lung of invasive pulmonary aspergillosis,and whether NOD2 is involved in the intracellular recognition of aspergillus fumigatus in mice macrophages.Methods:(1) The neutropenic mice model was made by intraperitoneal administration of cyclophosphamide,then were instilled intranasally aspergillus fumigatus conidia to induced invasive pulmonary asperguiilosis.The mRNA expression of NOD2 and RIP2 in lung tissue were examined by real-time PCR;(2) Three different siRNA sequences and a negative control sequence were synthesized,and were transfected into RAW264.7,checked their silence abilities by real-time PCR and western blot;(3) Transfected RAW264.7 with the most efficiency siRAN sequence,took the normal RAW264.7 as control,stimulated them with MDP,aspergillus fumigatus conidia,or both MDP and aspergillus fumigatus conidia separately,collected the cells and supernatant at 24h,48h,72h,examined the expression of NOD2 and RIP2 with real-time PCR for mRNA and western-blot for proteins,also tested the expression of NF-κB with western-blot and TNF-α/IL-8 with ELISA.RESULTS:(1) After inoculation of aspergillus fumigatus,there were obviously features of congestion, edema,infiltration of PMN in lung tissue section stained with HE,and many focus surrounded with PMN,conidia germinated and a lot of hyphae in clones could be observed in GMA stain,the most impressive changes occurred during 18-48h,much less severe changes appeared in immunocompetent mice without any hyphae or conidia were observed;(2) the mRNA expression of NOD2 in the lung tissue of both groups of mice showed an obviously increase after conidia inoculation(P＜0.05),and reach the peaks at 72h,those of ICH group were much more than control group at 24h and 48h(P＜0.05);same performances were observed in the expression of RIP2(P＜0.05),but there were no obvious dfference between the ICH group and the control group at any time;(3)Three different sequences of siRNA aimed to NOD2 were synthesized and transfected into RAW264.7,after 24h,the mRNA and proteins of NOD2 were examined,the suppression ratios of the third sequence were 96%in mRNA and 80%in proetein separately(P＜0.05);(4) the mRNA expression of NOD2 were suppressed at 24h,48h,and 72h after transfection(compared with the normal control cells,P＜0.05);(5) the expression of NOD2(both in mRNA and protein), RIP2(both in mRNA and protein),NF-κB in normal cells and IL-8/TNF-αin culture supernatant increased remarkably after stimulated with MDP,Af and MDP+Af (compared with the nomal control cells,P＜0.05),especially those stimulated with both MDP and Af(P＜0.05);(6) The mRNA expression of NOD2 and RIP2 in NOD2-RNAi cells were higher after stimulated with Af and MDP+Af than before (P＜0.05),but no obvious changes occurred when stimulated only by MDP;The mRNA expression of NOD2 and RIP2 were remarkably knocked down than those of normal cells received the same stimulaiton(P＜0.05);(7) the expression of NF-κB in NOD2-RNAi RA2W264.7 given stimulations showed no obvious changes than those without any stimulations;(8) the expression of TNF-αand IL-8 in NOD2-RNAi RAW264.7 were much lower than those of normal cells at any time(P＜0.05),and increased after stimulation were given than those cells with siRNA transfection were not given stimulations(P＜0.05).CONCLUSIONS:In vivo,NOD2 mRNA and RIP2 mRNA increased in mice lung after aspergillus pulmonary infection.In vitro,NOD2 expression in RAW264.7 increased remarkably after MDP,Af,MDP+Af stimulation, so did the expression of RIP2/NF-κB/TNF-α/IL-8,but the elevation could be knocked down after siRAN interfenence of NOD2,indicated that NOD2 play an important role in the defense of aspergillus fumigatus,and the possible mechanism is depend on the activation of RIP2 and NF-κB.
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