基于Caspase-1研究NLRP3/Caspase-1通路对成骨细胞焦亡作用机制

Study on the mechanism of NLRP3/Caspase-1 pathway on osteoblast pyroptosis based on Caspase-1

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NLRP3mRNA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-1β,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-1β,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-1βand IL-18 caused by the elevation of NLRP3 in osteoblasts due to high glucose,thus inhibiting osteoblast pyroptosis.