藤黄健骨胶囊通过SIRT1/NF-κB/NLRP3信号通路调节绝经后骨质疏松大鼠破骨细胞分化

Tenghuang-Jiangu capsule regulates osteoclast differentiation in rats with postmenopausal osteoporotic via SIRT1/NF-κB/NLRP3 signaling pathway

目的:探讨藤黄健骨胶囊(TJC)对绝经后骨质疏松大鼠沉默信息调节因子1(SIRT1)/核因子κB(NF-κB)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)信号通路关键蛋白表达的影响及分子机制。方法:采用随机数字表法将SD雌性大鼠分为假手术组,模型组[卵巢切除术(OVX)组],SIRT1抑制剂组(EX-527+OVX组),阳性药物戊酸雌二醇(ESV)对照组(ESV+OVX组),以及高、中、低剂量TJC处理组(TJC-H+OVX、TJC-M+OVX和TJCL+OVX组),每组10只。OVX建模,建模成功后给予TJC或ESV干预,并根据实验设计给予EX-527干预,8周后取材。ELISA法检测相关炎症因子水平;双重免疫荧光染色分别检测SIRT1、NF-κB p65和NLRP3蛋白;RT-qPCR和Western blot分别检测SIRT1/NF-κB/NLRP3信号通路相关分子和破骨分化标志分子的mRNA和蛋白水平。结果:与假手术组相比,OVX组和EX-527+OVX组大鼠血清中白细胞介素1β(IL-1β)、IL-18和肿瘤坏死因子α(TNF-α)含量显著升高(P<0.01),破骨细胞NF-κB p65及NLRP3表达荧光面积显著增大(P<0.01),股骨组织NF-κB p65、NLRP3和活化T细胞核因子胞浆1型(NFATc1)mRNA及蛋白水平显著升高(P<0.01),而破骨细胞SIRT1表达荧光面积显著减小(P<0.01);与OVX组相比,高剂量TJC处理组大鼠血清IL-1β含量和股骨组织NLRP3蛋白表达水平均显著降低,中、高剂量TJC处理组大鼠血清IL-18含量及股骨组织NF-κB p65和NFATc1蛋白表达水平均显著降低,低、中、高剂量TJC处理组大鼠血清TNF-α含量、破骨细胞NF-κB p65和NLRP3表达荧光面积、股骨组织NF-κB p65、NLRP3及NFATc1 mRNA水平均显著降低,而破骨细胞SIRT1表达荧光面积显著增大(P<0.05);与EX-527+OVX组相比,高剂量TJC处理组大鼠股骨组织NLRP3蛋白表达水平显著降低,中、高剂量TJC处理组大鼠血清IL-1β及IL-18含量、股骨组织NF-κB p65和NFATc1蛋白表达水平均显著降低,低、中、高剂量TJC处理组血清TNF-α含量、破骨细胞NF-κB p65和NLRP3表达荧光面积、股骨组织NF-κB p65、NLRP3及NFATc1 mRNA水平均显著降低,而破骨细胞SIRT1表达荧光面积显著增大(P<0.05)。结论:TJC能够抑制绝经后骨质疏松模型大鼠破骨细胞分化,其作用机制可能与抑制SIRT1/NF-κB/NLRP3信号通路激活、减轻炎症反应有关。

AIM:To investigate the effect of Tenghuang-Jiangu capsule(TJC)on the expression of silent information regulator 1(SIRT1)/nuclear factorκB(NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway in postmenopausal rats with osteoporosis and its molecular mechanism.METHODS:Female Sprague-Dawley rats were randomly divided into sham group,model group[ovariectomy(OVX)group],SIRT1 inhibitor(EX-527)+OVX group,positive control estradiol valerate(ESV)+OVX group,and high-,medium-and low-dose TJC groups(TJC-H+OVX,TJC-M+OVX and TJC-M+OVX groups),with 10 rats in each group.The TJC,ESV or EX-527 intervention after OVX was performed.Double immunofluorescence staining was used to detect SIRT1,NF-κB p65 and NLRP3 proteins,and ELISA was performed to detect inflammatory factors.RT-qPCR and Western blot were used to detect the mRNA and protein expression of SIRT1/NF-κB/NLRP3 signaling pathway-related molecules and osteoclast dif-ferentiation markers.RESULTS:Compared with sham group,serum levels of interleukin-1β(IL-1β),IL-18,and tumor necrosis factor-α(TNF-α)increased significantly in the OVX model group and in SIRT1 inhibitor EX-527 group(P<0.01).Similarly,the fluorescence area of NF-κB p65 and NLRP3 in osteoclast(P<0.01),and the mRNA and protein ex-pression levels of NF-κB p65,NLRP3,and activated T cell c1 nuclear factor cytoplasmic 1(NFATc1)in the femur also increased significantly(P<0.01).However,the fluorescence area of SIRT1 in osteoclast decreased significantly in the OVX model group and in SIRT1 inhibitor EX-527 group(P<0.01).Compared with OVX group,serum IL-1βcontent and NLRP3 protein expression level in the femur in the TJC-H treatment group decreased significantly.A similar decrease was observed for the serum IL-18 content,NF-κB p65 and NFATc1 protein expression levels in the femur in the TJC-H and TJC-M group rats.Serum TNF-αcontent,NF-κB p65 and NLRP3 expression fluorescence area in osteoclast,NF-κB p65,NLRP3,and NFATc1 mRNA expression levels in the femur decreased significantly in the TJC-H,TJC-M,and TJC-L groups.In contrast,the fluorescence area of SIRT1 in osteoclast increased significantly(P<0.05).Compared with EX-527+OVX group,NLRP3 protein expression level in the TJC-H treatment group decreased significantly.Serum IL-1βcon-tent in the TJC-H and TJC-M treatment group decreased significantly.A similar decrease was observed for the serum IL-18 content,NF-κB p65 and NFATc1 protein expression levels in the femur in the TJC-H and TJC-M groups.Serum TNF-αcontent,NF-κB p65 and NLRP3 expression fluorescence area in osteoclast,NF-κB p65,NLRP3,and NFATc1 mRNA ex-pression levels in the femur decreased significantly in the TJC-H,TJC-M,and TJC-L treatment groups,in contrast,the fluorescence area of SIRT1 in osteoclast increased significantly(P<0.05).CONCLUSION:Tenghuang-Jiangu capsule can inhibit osteoclast differentiation in postmenopausal osteoporosis model rats.The mechanism may be related to the inhi-bition of SIRT1/NF-κB/NLRP3 signaling pathway activation and alleviation of inflammatory response.