LXRα通过下调IRF3-GRIP1抑制LPS诱导的Kupffer细胞活化
发布时间：2018-08-14 10:03
【摘要】： 目的核受体肝X受体(liver X receptor, LXR)α是在肝脏以及巨噬细胞中呈高表达的核受体,它作为诸多基因的转录因子在机体发挥着重要作用。许多实验表明LXRα作为一个转录开关,除了诱导许多参与胆固醇逆转动、肝糖原代谢以及脂肪酸合成的基因外,另外LXRα还抑制了一系列由细菌感染、LPS、TNF-α或IL-1β等因子诱导的炎症基因。但LXRα负性调控炎性基因的表达的具体机制尚不明确。本实验旨在通过观察LXRα激动剂对LPS刺激后Kupffer细胞IRF3、GRIP1、LXRα及炎症因子IFNβ表达的影响,探讨LXRα抑制LPS诱导的Kupffer细胞(KCs)激活效应的可能机制。 方法采用密度梯度离心法结合选择性贴壁法分离雄性KM小鼠肝脏中的Kupffer细胞,分离出的Kupffer细胞用含20%FCS的RPMI1640培养液24h后随机分为四组:(1)正常对照组:以RPMI1640完全培养液置孵箱培养30h;(2)LPS处理组:倒掉原培养液,以RPMI1640完全培养液培养24h后,再用含1μg/ml LPS的RPMI1640完全培养液培养6h;(3)T0901317组:倒掉原培养液,用含5μg/ml T0901317的RPMI1640完全培养液培养24h,再以RPMI1640完全培养液培养6h;(4)LPS+T0901317组:倒掉原培养液,以含5μg/ml T0901317的RPMI1640完全培养液培养24h后,再改为含1μg/ml LPS的RPMI1640完全培养液,置孵箱培养6h。收集培养上清液及细胞,采用免疫细胞化学(SABC法)及Western blot法检测Kupffer细胞的LXRα、GRIP1及IRF3蛋白表达水平;SYBR Green I嵌合荧光法Real-Time PCR测定LXRα、GRIP1及IRF3 mRNA表达水平;酶联免疫吸附法(ELISA)检测Kupffer细胞培养上清液中IFNβ含量。 结果(1)免疫细胞化学结果:LXRα、IRF3及GRIP1阳性表达均位于核内,阳性被染成棕色。IRF3蛋白在LPS处理组染色最深,联合处理组次之,在正常对照组及LXR激动剂几乎未着色;GRIP1的染色情况与IRF3大致相同;LXRα阳性染色在LXRα激动剂组表达最高,在联合处理组明显降低,在LPS处理组最低。(2)以各指标的log cDNA/logβ-actin比值的x±s表示各指标mRNA平均表达水平。在LPS组中IRF3及GRIP1 mRNA表达量最高(分别为1.089±0.0074、0.8922±0.0095),联合处理组中其表达明显降低(分别为0.7234±0.0072、0.6905±0.0042),两组间均数差异均有显著意义(P0.05);在LPS组及联合处理组中,IRF3及GRIP1 mRNA水平均高于对照组(分别为0.3558±0.0051、0.3842±0.0083 )及LXRα激动剂组(分别为0.333±0.0054、0.2778±0.0091),差异具有统计学意义(P0.05);LXRα在LPS组中(0.2722±0.0038)的表达明显低于其它三组(对照组:0.3953±0.0051,联合处理组:0.7963±0.0075,LXRα激动剂组:0.9912±0.0098),差异具有统计学意义(P0.05);LXRα在LXRα激动剂组的表达最高,与其它三组相比具有统计学意义(P0.05);联合处理组中,LXRα表达明显低于LXRα激动剂组(P0.05),但又显著高于其它两组(P0.05)。(3)Western blot结果:用内参照对待测物灰度值进行标准校正。IRF3及GRIP1蛋白表达量在LPS组最高(分别为0.388±0.018, 0.276±0.015),联合处理组表达明显降低(分别为0.318±0.014, 0.224±0.017),两组间均数差异均有显著意义(P0.05);在LPS组及联合处理组中,IRF3及GRIP1蛋白表达量均高于对照组(分别为0.268±0.025、0.162±0.013)及LXRα激动剂组(分别为0.213±0.017、0.133±0.013),差别具有统计学意义(P0.05); LPS组LXRα表达最低(其值为0.534±0.014),明显低于其它三组(对照组:1.03±0.024,联合处理组:1.224±0.027,LXRα激动剂组:1.74±0.034) ,差别具有统计学意义(P0.05);LXRα在LXRα激动剂组的表达最高,与其它三组相比具有统计学意义(P0.05);联合处理组中,LXRα表达明显低于LXRα激动剂组(P0.05),但又显著高于其它两组(P0.05)。(4)ELISA结果表明:IFNβ在LPS处理组的含量(329.5±35)显著高于对照组(129.6±17)及LXRα激动剂组(112.8±24),差异具有统计学意义(P0.05);联合处理组IFNβ含量(224.4±33)比LPS处理组明显降低,差异有统计学意义(P0.05);IFNβ表达LXRα激动剂组表达最低。 结论本实验结果表明,在应用LPS处理之前预防性应用LXRα激动剂,能明显抑制Kupffer细胞的IRF3及GRIP1表达,且在应用LXRα激动剂后LPS所诱生的炎症因子IFNβ大大降低,进一步说明了LXRα对IRF3通路的抑制。此外,相对于对照组及LXRα激动剂组,在经过LPS处理后,LXRα的表达明显降低,IFNβ表达明显上升。因些,LXRα与IRF3存在相互抑制作用,LXRα能够通过抑制IRF3、GRIP1的表达而发挥抗炎效应,从而抑制LPS所诱导的Kupffer细胞活化。
[Abstract]:Objective Liver X receptor (LXR) alpha is a highly expressed nuclear receptor in the liver and macrophages. It plays an important role as a transcription factor for many genes in the body. In addition, LXRalpha inhibited a series of inflammatory genes induced by bacterial infection, LPS, TNF-alpha or IL-1bet. However, the mechanism of LXRalpha negatively regulating the expression of inflammatory genes remains unclear. To explore the possible mechanism of LXR alpha inhibiting LPS induced Kupffer cell (KCs) activation.
Methods Kupffer cells were isolated from the liver of male KM mice by density gradient centrifugation combined with selective adherence method. The isolated Kupffer cells were randomly divided into four groups after 24 hours in RPMI 1640 medium containing 20% FCS: (1) normal control group: incubated in RPMI 1640 incubator for 30 hours; (2) LPS treatment group: the original culture medium was poured out and RPMI 1640 was used to complete the incubation. T0901317 group: the original medium was removed, the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 24 hours, and then the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 6 hours; (4) LPS+T0901317 group: the original medium was poured out and the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 6 hours; (4) LPS+T0901317 group: the original medium was poured out and the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured. The culture supernatant and cells were collected and the expression levels of LXR alpha, GRIP1 and IRF3 were detected by immunocytochemistry (SABC) and Western blot. The expression levels of LXR alpha, GRIP1 and IRF3 mRNA in Kupffer cells were detected by SYBR Green I chimeric fluorescence Real-Time PCR. Enzyme linked immunosorbent assay (ELISA) was used to detect the content of IFN beta in Kupffer cell supernatant.
Results (1) Immunocytochemical staining showed that LXRalpha, IRF3 and GRIP1 were positively expressed in the nucleus, and the positive staining was brown. IRF3 protein stained the deepest in the LPS treatment group, followed by the combined treatment group, in the normal control group and LXR agonist almost no staining; GRIP1 staining was approximately the same as IRF3; LXRalpha positive staining was the most expressed in the LXRalpha agonist group. The expression of IRF3 and GRIP1 mRNA was the highest in LPS group (1.089.0074, 0.8922.0095, respectively). The expression of IRF3 and GRIP1 mRNA was significantly decreased in LPS group (0.7234.0072, 0.690, respectively). The levels of IRF3 and GRIP1 mRNA in LPS group and combined treatment group were higher than those in control group (0.3558.0051, 0.3842.0083) and LXR alpha agonist group (0.333.0054, 0.2778.0091, respectively), the difference was statistically significant (P 0.05). The expression of LXRalpha was significantly lower than that of the other three groups (control group: 0.3953 + 0.0051, combined treatment group: 0.7963 + 0.0075, LXRalpha agonist group: 0.9912 + 0.0098), the difference was statistically significant (P 0.05); the expression of LXRalpha in LXRalpha agonist group was the highest, compared with the other three groups was statistically significant (P 0.05); the expression of LXRalpha in combined treatment group was significantly lower than that in LXRalpha agonist group (P 0.05). The expression of IRF3 and GRIP1 protein was the highest in LPS group (0.388 [0.018] and 0.276 [0.015] respectively. The expression of IRF3 and GRIP1 protein was significantly lower in combined treatment group (0.318 [0.014] and 0.224 [0.017] respectively). The expression of IRF3 and GRIP1 protein in LPS group and combined treatment group were higher than those in control group (0.268.025, 0.162.013) and LXR alpha agonist group (0.213.017, 0.133.013, respectively), the difference was statistically significant (P 0.05); the expression of LXR alpha in LPS group was the lowest (0.534.014), significantly lower than that in control group (0.014). The expression of LXRalpha in the three groups (control group: 1.03 + 0.024, combined treatment group: 1.224 + 0.027, LXRalpha agonist group: 1.74 + 0.034) was statistically significant (P 0.05); the expression of LXRalpha in the LXRalpha agonist group was the highest, compared with the other three groups was statistically significant (P 0.05); the expression of LXRalpha in the combined treatment group was significantly lower than that in the LXRalpha agonist group (P 0.05). The results of ELISA showed that the content of IFN-beta in LPS group (329.5+35) was significantly higher than that in control group (129.6+17) and LXR-alpha agonist group (112.8+24), the difference was statistically significant (P 0.05); the content of IFN-beta in combined treatment group (224.4+33) was significantly lower than that in LPS group (P 0.05); The agonist group showed the lowest expression.
Conclusion LXR-alpha agonist can significantly inhibit the expression of IRF3 and GRIP1 in Kupffer cells before LPS treatment, and the expression of IFN-beta induced by LPS is significantly decreased after LXR-alpha agonist treatment, which further illustrates the inhibition of LXR-alpha on IRF3 pathway. After LPS treatment, the expression of LXRalpha was significantly decreased and the expression of IFNbeta was significantly increased. Therefore, LXRalpha and IRF3 were mutually inhibited. LXRalpha could exert anti-inflammatory effects by inhibiting the expression of IRF3 and GRIP1, thereby inhibiting the activation of Kupffer cells induced by LPS.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
本文编号：2182511
[Abstract]:Objective Liver X receptor (LXR) alpha is a highly expressed nuclear receptor in the liver and macrophages. It plays an important role as a transcription factor for many genes in the body. In addition, LXRalpha inhibited a series of inflammatory genes induced by bacterial infection, LPS, TNF-alpha or IL-1bet. However, the mechanism of LXRalpha negatively regulating the expression of inflammatory genes remains unclear. To explore the possible mechanism of LXR alpha inhibiting LPS induced Kupffer cell (KCs) activation.
Methods Kupffer cells were isolated from the liver of male KM mice by density gradient centrifugation combined with selective adherence method. The isolated Kupffer cells were randomly divided into four groups after 24 hours in RPMI 1640 medium containing 20% FCS: (1) normal control group: incubated in RPMI 1640 incubator for 30 hours; (2) LPS treatment group: the original culture medium was poured out and RPMI 1640 was used to complete the incubation. T0901317 group: the original medium was removed, the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 24 hours, and then the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 6 hours; (4) LPS+T0901317 group: the original medium was poured out and the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 6 hours; (4) LPS+T0901317 group: the original medium was poured out and the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured. The culture supernatant and cells were collected and the expression levels of LXR alpha, GRIP1 and IRF3 were detected by immunocytochemistry (SABC) and Western blot. The expression levels of LXR alpha, GRIP1 and IRF3 mRNA in Kupffer cells were detected by SYBR Green I chimeric fluorescence Real-Time PCR. Enzyme linked immunosorbent assay (ELISA) was used to detect the content of IFN beta in Kupffer cell supernatant.
Results (1) Immunocytochemical staining showed that LXRalpha, IRF3 and GRIP1 were positively expressed in the nucleus, and the positive staining was brown. IRF3 protein stained the deepest in the LPS treatment group, followed by the combined treatment group, in the normal control group and LXR agonist almost no staining; GRIP1 staining was approximately the same as IRF3; LXRalpha positive staining was the most expressed in the LXRalpha agonist group. The expression of IRF3 and GRIP1 mRNA was the highest in LPS group (1.089.0074, 0.8922.0095, respectively). The expression of IRF3 and GRIP1 mRNA was significantly decreased in LPS group (0.7234.0072, 0.690, respectively). The levels of IRF3 and GRIP1 mRNA in LPS group and combined treatment group were higher than those in control group (0.3558.0051, 0.3842.0083) and LXR alpha agonist group (0.333.0054, 0.2778.0091, respectively), the difference was statistically significant (P 0.05). The expression of LXRalpha was significantly lower than that of the other three groups (control group: 0.3953 + 0.0051, combined treatment group: 0.7963 + 0.0075, LXRalpha agonist group: 0.9912 + 0.0098), the difference was statistically significant (P 0.05); the expression of LXRalpha in LXRalpha agonist group was the highest, compared with the other three groups was statistically significant (P 0.05); the expression of LXRalpha in combined treatment group was significantly lower than that in LXRalpha agonist group (P 0.05). The expression of IRF3 and GRIP1 protein was the highest in LPS group (0.388 [0.018] and 0.276 [0.015] respectively. The expression of IRF3 and GRIP1 protein was significantly lower in combined treatment group (0.318 [0.014] and 0.224 [0.017] respectively). The expression of IRF3 and GRIP1 protein in LPS group and combined treatment group were higher than those in control group (0.268.025, 0.162.013) and LXR alpha agonist group (0.213.017, 0.133.013, respectively), the difference was statistically significant (P 0.05); the expression of LXR alpha in LPS group was the lowest (0.534.014), significantly lower than that in control group (0.014). The expression of LXRalpha in the three groups (control group: 1.03 + 0.024, combined treatment group: 1.224 + 0.027, LXRalpha agonist group: 1.74 + 0.034) was statistically significant (P 0.05); the expression of LXRalpha in the LXRalpha agonist group was the highest, compared with the other three groups was statistically significant (P 0.05); the expression of LXRalpha in the combined treatment group was significantly lower than that in the LXRalpha agonist group (P 0.05). The results of ELISA showed that the content of IFN-beta in LPS group (329.5+35) was significantly higher than that in control group (129.6+17) and LXR-alpha agonist group (112.8+24), the difference was statistically significant (P 0.05); the content of IFN-beta in combined treatment group (224.4+33) was significantly lower than that in LPS group (P 0.05); The agonist group showed the lowest expression.
Conclusion LXR-alpha agonist can significantly inhibit the expression of IRF3 and GRIP1 in Kupffer cells before LPS treatment, and the expression of IFN-beta induced by LPS is significantly decreased after LXR-alpha agonist treatment, which further illustrates the inhibition of LXR-alpha on IRF3 pathway. After LPS treatment, the expression of LXRalpha was significantly decreased and the expression of IFNbeta was significantly increased. Therefore, LXRalpha and IRF3 were mutually inhibited. LXRalpha could exert anti-inflammatory effects by inhibiting the expression of IRF3 and GRIP1, thereby inhibiting the activation of Kupffer cells induced by LPS.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
【共引文献】
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相关硕士学位论文 前4条
1 戴卓娅;肝X受体-α对炎症的调节与神经元衍生孤核受体-1的关联[D];重庆医科大学;2011年
2 欧志兵;LXRα通过IRF3途径负性调控LPS诱导的Kupffer细胞激活的机制研究[D];重庆医科大学;2009年
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