目的 通过调控半胱氨酸蛋白酶-1（Caspase-1）水平探讨核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）/Caspase-1通路对成骨细胞焦亡的影响机制。方法 培养小鼠胚胎MC3T3-E1成骨细胞,分为A组（高糖处理）、B组（高糖+Caspase-1抑制剂处理）、C组（高糖+Caspase-1 siRNA处理）和对照组。采用吸光法检测成骨细胞乳酸脱氢酶（LDH）释放量,采用Hoechst33342/PI染色法检测成骨细胞焦亡情况,分别采用RT-PCR技术和Western blot技术检测成骨细胞中NLRP3、Caspase-1、白细胞介素-1β（IL-1β）和IL-18相对表达水平。结果 A组、B组和C组MC3T3-E1成骨细胞焦亡比例和LDH活性均显著高于对照组,B组、C组焦亡比例和LDH活性显著低于A组（P<0.05）。A组、B组和C组NLRP3、Caspase-1、IL-1β、IL-18 mRNA和蛋白相对表达水平均显著高于对照组,B组、C组Caspase-1、IL-1β、IL-18 mRNA和蛋白相对表达水平显著低于A组（P<0.05）。A组、B组和C组NLRP3 mRNA和蛋白相对表达水平比较差异无统计学意义（P>0.05）。结论 通过抑制Caspase-1能够抑制高糖引起的成骨细胞NLRP3升高导致的IL-1β、IL-18升高,进而抑制成骨细胞焦亡作用。
Objective To explore the mechanism of nucleotide binding oligomerization domain-like receptor protein 3 （NLRP3）/cysteine aspartate specific proteinase-1（Caspase-1） pathway on osteoblast pyroptosis by regulating the level of Caspase-1. Methods Mouse embryonic MC3T3-E1 osteoblasts were cultured and divided into group A with high sugar treatment, group B with high sugar + Caspase-1 inhibitor treatment, group C with high sugar + Caspase-1 siRNA treatment, and control group. The level of lactate dehydrogenase （LDH） released from osteoblasts was detected by light absorption method, the osteoblast pyroptosis was detected by Hoechst33342/PI staining, and the NLRP3, Caspase-1, interleukin-1β （IL-1β）, and IL-18 of osteoblasts were detected by RT-PCR and Western blot respectively. Results The pyroptosis ratio and LDH activity of MC3T3-E1 osteoblasts in group A, B, and C were significantly higher than those in the control group, while those in group B and C were significantly lower than those in group A （P<0.05）. The relative expression levels of NLRP3, Caspase-1, IL-1 β, and IL-18 mRNA and protein in group A, B, and C were higher than those in the control group, while the relative expression levels of Caspase-1, IL-1 β, and IL-18 mRNA and protein in group B and C were significantly lower than those in group A （P<0.05）. There was no significant difference in the relative expression levels of NLRP3 mRNA and protein among group A, B, and C （P>0.05）. Conclusion Inhibition of Caspase-1 inhibits the elevation of IL-1β and IL-18 caused by the elevation of NLRP3 in osteoblasts due to high glucose, thus inhibiting osteoblast pyroptosis.
[1] 林贤灿,吴建军,黄宏兴,等.我国基层骨质疏松症防治的问题及策略[J]. 中国骨质疏松杂志,2022,28(11):1678-1682.
[2] Komori T.Cell death in chondrocytes, osteoblasts, and osteocytes[J]. Int J Mol Sci, 2016, 17(12): 2045.
[3] Shi J, Gao W, Shao F.Pyroptosis: gasdermin-mediated programmed necrotic cell death[J]. Trends Biochem Sci, 2017, 42(4): 245-254.
[4] Tsuchiya K, Nakajima S, Hosojima S, et al.Caspase-1 initiates apoptosis in the absence of gasdermin D[J]. Nat Commun, 2019, 10(1): 2091.
[5] Wang L, Heckmann BL, Yang X, et al.Osteoblast autophagy in glucocorticoid-induced osteoporosis[J]. J Cell Physiol, 2019, 234(4): 3207-3215.
[6] 朱洁云,高敏,宋秋韵,等.中国老年人骨质疏松症患病率的Meta分析[J]. 中国全科医学,2022,25(3):346-353.
[7] 王剑虹,董万涛,宋敏,等.“缺氧-高糖”轴对糖尿病性骨质疏松症的影响机制[J]. 中国骨质疏松杂志,2022,28(11): 1634-1639.
[8] 倪会芳,李隽,丁源,等.中性粒细胞/淋巴细胞比值和单核细胞/高密度脂蛋白胆固醇比值预测绝经后2型糖尿病患者发生骨质疏松症的价值研究[J]. 中国全科医学,2022,25(18): 2207-2214.
[9] Yang L, Liu J, Shan Q, et al.High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis[J]. Biochem Biophys Res Commun, 2020, 522(2): 471-478.
[10] Sendler M, Mayerle J, Lerch MM. Necrosis, apoptosis, necroptosis, pyroptosis:it matters how acinar cells die during pancreatitis[J]. Cell Mol Gastroenterol Hepatol, 2016,31, 2(4): 407-408.
[11] Behera J, Ison J, Voor MJ, et al.Exercise-linked skeletal irisin ameliorates diabetes-associated osteoporosis by inhibiting the oxidative damage-dependent mir-150-fndc5/pyroptosis axis[J]. Diabetes, 2022, 71(12): 2777-2792.
[12] Man SM, Karki R, Kanneganti TD.Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases[J]. Immunol Rev, 2017, 277(1): 61-75.
[13] Komuczki J, Tuzlak S, Friebel E, et al.Fate-Mapping of GM-CSF expression identifies a discrete subset of inflammation-driving T helper cells regulated by cytokines IL-23 and IL-1β[J]. Immunity, 2019, 50(5): 1289-1304.
[14] Blaschke M, Koepp R, Cortis J, et al.IL-6, IL-1β, and TNF-α only in combination influence the osteoporotic phenotype in Crohn's patients via bone formation and bone resorption[J]. Adv Clin Exp Med, 2018, 27(1): 45-56.
[15] An Y, Zhang H, Wang C, et al.Activation of ROS/MAPKs/NF-κB/NLRP3 and inhibition of efferocytosis in osteoclast-mediated diabetic osteoporosis[J]. FASEB J, 2019, 33(11): 12515-12527.
[16] Amarasekara DS, Kim S, Rho J.Regulation of osteoblast differentiation by cytokine networks[J]. Int J Mol Sci, 2021, 22(6): 2851.
[17] Li JR, Xu HZ, Nie S, et al.Fluoxetine-enhanced autophagy ameliorates early brain injury via inhibition of NLRP3 inflammasome activation following subrachnoid hemorrhage in rats[J]. J Neuroinflammation, 2017, 14(1): 186.
