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Abstract：
BACKGROUND: There are currently no effective drugs to mitigate the ischemia/reperfusion injury caused by fluid resuscitation after hemorrhagic shock (HS). The aim of this study was to explore the potential of the histone deacetylase 6 (HDAC6)-specific inhibitor tubastatin A (TubA) to suppress nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation in macrophages under hypoxia/reoxygenation (H/R) conditions.
METHODS: The viability of RAW264.7 cells subjected to H/R after treatment with different concentrations of TubA was assessed using a cell-counting kit-8 (CCK8) assay. Briefly, 2.5 μmol/L TubA was used with RAW264.7 cells under H/R condition. RAW264.7 cells were divided into three groups, namely the control, H/R, and TubA groups. The levels of reactive oxygen species (ROS) in the cells were detected using fluorescence microscopy. The protein expression of HDAC6, heat shock protein 90 (Hsp90), inducible nitric oxide synthase (iNOS), NLRP3, gasdermin-D (GSDMD), Caspase-1, GSDMD-N, and Caspase-1 p20 was detected by western blotting. The levels of interleukin-1β (IL-1β) and IL-18 in the supernatants were detected using enzyme-linked immunosorbent assay (ELISA).
RESULTS: HDAC6, Hsp90, and iNOS expression levels were significantly higher (P<0.01) in the H/R group than in the control group, but lower in the TubA group than in the H/R group (P<0.05). When comparing the H/R group to the control group, ROS levels were significantly higher (P<0.01), but significantly reduced in the TubA group (P<0.05). The H/R group had higher NLRP3, GSDMD, Caspase-1, GSDMD-N, and Caspase-1 p20 expression levels than the control group (P<0.05), however, the TubA group had significantly lower expression levels than the H/R group (P<0.05). IL-1β and IL-18 levels in the supernatants were significantly higher in the H/R group compared to the control group (P<0.01), but significantly lower in the TubA group compared to the H/R group (P<0.01).
CONCLUSIONS: TubA inhibited the expression of HDAC6, Hsp90, and iNOS in macrophages subjected to H/R. This inhibition led to a decrease in the content of ROS in cells, which subsequently inhibited the activation of the NLRP3 inflammasome and the secretion of IL-1β and IL-18.
METHODS: The viability of RAW264.7 cells subjected to H/R after treatment with different concentrations of TubA was assessed using a cell-counting kit-8 (CCK8) assay. Briefly, 2.5 μmol/L TubA was used with RAW264.7 cells under H/R condition. RAW264.7 cells were divided into three groups, namely the control, H/R, and TubA groups. The levels of reactive oxygen species (ROS) in the cells were detected using fluorescence microscopy. The protein expression of HDAC6, heat shock protein 90 (Hsp90), inducible nitric oxide synthase (iNOS), NLRP3, gasdermin-D (GSDMD), Caspase-1, GSDMD-N, and Caspase-1 p20 was detected by western blotting. The levels of interleukin-1β (IL-1β) and IL-18 in the supernatants were detected using enzyme-linked immunosorbent assay (ELISA).
RESULTS: HDAC6, Hsp90, and iNOS expression levels were significantly higher (P<0.01) in the H/R group than in the control group, but lower in the TubA group than in the H/R group (P<0.05). When comparing the H/R group to the control group, ROS levels were significantly higher (P<0.01), but significantly reduced in the TubA group (P<0.05). The H/R group had higher NLRP3, GSDMD, Caspase-1, GSDMD-N, and Caspase-1 p20 expression levels than the control group (P<0.05), however, the TubA group had significantly lower expression levels than the H/R group (P<0.05). IL-1β and IL-18 levels in the supernatants were significantly higher in the H/R group compared to the control group (P<0.01), but significantly lower in the TubA group compared to the H/R group (P<0.01).
CONCLUSIONS: TubA inhibited the expression of HDAC6, Hsp90, and iNOS in macrophages subjected to H/R. This inhibition led to a decrease in the content of ROS in cells, which subsequently inhibited the activation of the NLRP3 inflammasome and the secretion of IL-1β and IL-18.
摘要：
背景：目前尚无有效的药物来减轻失血性休克（HS）后液体复苏引起的缺血/再灌注损伤。本研究的目的是探索组蛋白去乙酰化酶6（HDAC6）特异性抑制剂tubastatinA（TubA）在缺氧/复氧（H/R）条件下抑制巨噬细胞中核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎性体活化的潜力。
方法:使用细胞计数试剂盒-8(CCK8)测定法评估用不同浓度的TubA处理后经受H/R的RAW264.7细胞的活力。简而言之，在H/R条件下，将2.5μmol/L的TubA与RAW264.7细胞一起使用。RAW264.7细胞分为三组，即控制，H/R,和TubA组。使用荧光显微镜检测细胞中的活性氧(ROS)的水平。HDAC6、热休克蛋白90(Hsp90)、诱导型一氧化氮合酶（iNOS），NLRP3,gasdermin-D(GSDMD),胱天蛋白酶-1,GSDMD-N,蛋白质印迹法检测Caspase-1p20。采用酶联免疫吸附试验(ELISA)检测上清液中白细胞介素-1β(IL-1β)和IL-18水平。
结果:H/R组HDAC6、Hsp90、iNOS表达水平明显高于对照组(P<0.01),但TubA组低于H/R组(P<0.05)。当比较H/R组与对照组时，ROS水平显著升高(P<0.01),但TubA组显著降低(P<0.05)。H/R组NLRP3、GSDMD、胱天蛋白酶-1,GSDMD-N,而Caspase-1p20表达水平高于对照组(P<0.05),然而,TubA组的表达水平显著低于H/R组(P<0.05)。H/R组上清液中IL-1β和IL-18水平明显高于对照组(P<0.01),但TubA组显著低于H/R组(P<0.01)。
结论：TubA抑制H/R巨噬细胞中HDAC6、Hsp90和iNOS的表达。这种抑制导致细胞中ROS含量的降低，随后抑制NLRP3炎性体的激活以及IL-1β和IL-18的分泌。
方法:使用细胞计数试剂盒-8(CCK8)测定法评估用不同浓度的TubA处理后经受H/R的RAW264.7细胞的活力。简而言之，在H/R条件下，将2.5μmol/L的TubA与RAW264.7细胞一起使用。RAW264.7细胞分为三组，即控制，H/R,和TubA组。使用荧光显微镜检测细胞中的活性氧(ROS)的水平。HDAC6、热休克蛋白90(Hsp90)、诱导型一氧化氮合酶（iNOS），NLRP3,gasdermin-D(GSDMD),胱天蛋白酶-1,GSDMD-N,蛋白质印迹法检测Caspase-1p20。采用酶联免疫吸附试验(ELISA)检测上清液中白细胞介素-1β(IL-1β)和IL-18水平。
结果:H/R组HDAC6、Hsp90、iNOS表达水平明显高于对照组(P<0.01),但TubA组低于H/R组(P<0.05)。当比较H/R组与对照组时，ROS水平显著升高(P<0.01),但TubA组显著降低(P<0.05)。H/R组NLRP3、GSDMD、胱天蛋白酶-1,GSDMD-N,而Caspase-1p20表达水平高于对照组(P<0.05),然而,TubA组的表达水平显著低于H/R组(P<0.05)。H/R组上清液中IL-1β和IL-18水平明显高于对照组(P<0.01),但TubA组显著低于H/R组(P<0.01)。
结论：TubA抑制H/R巨噬细胞中HDAC6、Hsp90和iNOS的表达。这种抑制导致细胞中ROS含量的降低，随后抑制NLRP3炎性体的激活以及IL-1β和IL-18的分泌。
