【作者】 丁杰；

【导师】 胡伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2022， 硕士

【摘要】 目的骨关节炎(Osteoarthritis,OA)是一种常见的慢性退行性关节疾病,其特点是关节软骨退化和软骨细胞衰老。酸敏感离子通道1a(ASIC1a)是一种质子激活的阳离子通道,在软骨细胞中含量丰富,可感知和调节关节腔的p H值。我们之前的研究表明,ASIC1a参与了酸诱导的大鼠关节软骨细胞衰老,但其机制基础仍不清楚。在本研究中,我们探讨了ASIC1a在软骨细胞衰老和OA中的作用机制。结果表明,在p H 6.0组,衰老相关-β-半乳糖苷酶、衰老相关标志物(p53和p21)和自噬相关蛋白Beclin-1增加,但发现Lamin B1蛋白水平明显降低。这些作用分别被软骨细胞中的ASIC1a特异性阻断剂psalmotoxin-1或ASIC1a-短发夹RNA抑制。此外,沉默Lamin B1增强了ASIC1a介导的软骨细胞衰老,这种效应被Lamin B1的过表达逆转,表明Lamin B1参与了ASIC1a介导的软骨细胞衰老。此外,ASIC1a的阻断抑制了酸诱导的自噬小体和Beclin-1蛋白水平的增加,表明ASIC1a参与了酸诱导的软骨细胞自噬。在酸诱导的软骨细胞衰老中,用氯喹阻断自噬能够抑制Beclin-1并增加Lamin B1蛋白水平。我们进一步证明ASIC1a介导的Lamin B1表达减少是由自噬依赖性蛋白质降解引起的。最后,在大鼠OA模型中,阻断ASIC1a保护了软骨组织,恢复了Lamin B1蛋白水平并抑制了软骨细胞衰老。总之,这些结果表明,ASIC1a的阻断通过抑制自噬依赖性Lamin B1蛋白降解来抑制OA软骨细胞衰老。对此,我们将采用培养大鼠原代软骨细胞构建体外模型,并将一部分重要的结果在人正常软骨细胞系(C28/I2)上进行重复性验证,以及构建OA大鼠体内模型,展开如下研究:方法1.沉默ASIC1a对胞外酸化诱导的大鼠关节软骨细胞衰老的影响:处理分为NC对照组、p H 6.0+NC组、ASIC1a沉默组、p H 6.0+ASIC1a沉默组,β-半乳糖苷酶染色用于检测关节软骨细胞衰老;Western blot方法检测ASIC1a、p21、p53、Lamin B1和Beclin-1蛋白的表达;细胞免疫荧光法检测Lamin B1的表达水平;RT-q PCR法检测ASIC1a基因的表达。2.阻断蛋白合成途径、自噬溶酶体途径和蛋白酶体途径对胞外酸化诱导的软骨细胞衰老的影响:处理分为正常组、p H 6.0组、工具药组、p H 6.0+工具药组。使用Western blot方法检测Beclin-1、p21、p53和Lamin B1蛋白的表达;RT-q PCR检测Lamin B1基因的表达;细胞免疫荧光法检测Lamin B1和γ-H2A.X的表达;透射电镜用于观察细胞内自噬小体、线粒体等超微结构;β-半乳糖苷酶染色用于检测关节软骨细胞衰老。3.沉默/过表达Lamin B1对胞外酸化诱导的软骨细胞衰老的影响:处理分为NC对照组、p H 6.0+NC组、沉默/过表达Lamin B1组、p H 6.0+沉默/过表达Lamin B1组,β-半乳糖苷酶染色用于检测关节软骨细胞衰老;Western blot法检测p21、p53、Lamin B1和Beclin-1蛋白的表达;细胞免疫荧光法检测Lamin B1和γ-H2A.X的表达;RT-q PCR法检测Lamin B1基因的表达。4.阻断ASIC1a对OA大鼠衰老的影响及对OA的保护作用:雄性SD大鼠6-8周龄,200-220g左右,随机分为正常组、模型组和给药组。其中,正常组和模型组各8只,给药组:Pc Tx1组、透明质酸(HA,1%)组每组8只。HE染色、番红O-固绿和甲苯胺蓝染色用于观察大鼠膝关节病理改变;免疫组织化学用于检测膝关软骨Lamin B1、p21和p53蛋白的表达;电子摄片用于分析关节软骨表面破坏程度及Pelletier评分。结果1.沉默ASIC1a抑制胞外酸化诱导的软骨细胞衰老:与p H 6.0组相比,ASIC1a沉默组在酸化诱导后明显降低了衰老细胞的阳性率。并且能够增加Lamin B1蛋白的表达,抑制衰老相关蛋白p53和p21以及自噬相关蛋白Beclin-1的表达。2.Lamin B1的沉默促进了ASIC1a介导的软骨细胞衰老:β-半乳糖苷酶染色结果显示,与正常组相比,Lamin B1沉默组的衰老细胞阳性率显著增加,衰老相关蛋白p53和p21的表达明显增加。3.ASIC1a通过自噬介导Lamin B1蛋白降解:在p H 6.0组中加入自噬抑制剂CQ(20μM)可以逆转胞外酸化诱导的Lamin B1蛋白表达的降低,并且能够抑制衰老相关蛋白p53和p21以及自噬相关蛋白Beclin-1的表达。4.ASIC1a的阻断能够保护OA大鼠模型的软骨细胞:软骨特异性染色表明,用Pc Tx1阻断ASIC1a可以显着减轻OA大鼠软骨结构的破坏。降低衰老相关蛋白p21和p53的表达,显著恢复Lamin B1蛋白的表达。结论综上所述,胞外酸化的微环境能够激活ASIC1a,通过自噬途径降解Lamin B1蛋白,破坏细胞核的结构和功能,加速OA的进程。为OA治疗和软骨细胞衰老提供了新机制。更多还原

【Abstract】 Objective Osteoarthritis(OA)is a common and debilitating chronic joint disease,which is characterized by degeneration of articular cartilage and the aging of chondrocytes.Acidsensitive ion channel 1a(ASIC1a)is a proton-activated cationic channel abundant in chondrocytes,which senses and regulates joint cavity p H.Our previous study demonstrated that ASIC1 a was involved in acid-induced rat articular chondrocyte senescence,but the mechanistic basis remained unclear.In this study,we explored the mechanism of ASIC1 a in chondrocyte senescence and OA.The results showed that senescence-related-β-galactosidase,senescence-related markers(p53 and p21)and the autophagy-related protein Beclin-1 were found to be increased,but Lamin B1 was found to be reduced with acid(p H 6.0)treatment.These effects were inhibited by ASIC1 aspecific blocker psalmotoxin-1 or ASIC1a-short hairpin RNA respectively in chondrocytes.Moreover,Silencing of Lamin B1 enhanced ASIC1a-mediated chondrocyte senescence,this effect was reversed by overexpression of Lamin B1,indicating that Lamin B1 was involved in ASIC1a-mediated chondrocyte senescence.Further,blockade of ASIC1 a inhibits acid-induced autophagosomes and Beclin-1 protein expression,suggesting that ASIC1 a is involved in acid-induced chondrocyte autophagy.Blocking autophagy with chloroquine inhibited Beclin-1 and increased Lamin B1 in acidinduced chondrocyte senescence.We further demonstrated that ASIC1a-mediated reduction of Lamin B1 expression was caused by autophagy pathway-dependent protein degradation.Finally,blocking ASIC1 a protected cartilage tissue,restored Lamin B1 levels and inhibited chondrocyte senescence in a rat OA model.In summary,these results indicate that blockade of ASIC1 a inhibits osteoarthritis chondrocyte senescence by promoting autophagy pathway-dependent Lamin B1 protein degradation.In this regard,we will use cultured rat primary chondrocyte to construct an in vitro model,and perform repetitive verification of some important results on Human cartilage C28/I2 cell lines,and construct an in vivo OA rat model.The following studies are carried out:Methods1.The effect of silencing ASIC1 a on the senescence of rat articular chondrocyte induced by extracellular acidification: The treatment was divided into NC control group,p H6.0+NC group,ASIC1 a silent group,p H 6.0+ASIC1a silent group,and β-galactosidase staining to detect articular chondrocyte senescence;Western blot method to detect ASIC1 a,p21,p53,Lamin B1 and Beclin-1 protein expression;Cellular immunofluorescence to detect Lamin B1 expression in rat primary chondrocyte;RTq PCR to detect ASIC1 a gene expression.2.The effect of blocking protein synthesis pathway,autophagy lysosome pathway and proteasome pathway on the senescence of chondrocyte induced by extracellular acidification: Treatment was divided into normal group,p H 6.0 group,tool drug group,and p H 6.0+ tool drug group.Western blot method was used to detect the expression of Beclin-1,p21,p53 and Lamin B1;RT-q PCR was used to detect the expression of Lamin B1 gene;cell immunofluorescence was used to detect the expression of Lamin B1 and γ-H2 A.X in primary rat chondrocyte;Transmission electron microscope was used to observe the ultrastructure of autophagosomes;β-galactosidase staining was used to detect the senescence of articular chondrocyte.3.The effect of silent/overexpression Lamin B1 on the senescence of chondrocytes induced by extracellular acidification: Treatment was divided into NC control group,p H 6.0 group,silence/overexpression Lamin B1 group,p H 6.0+silence/overexpression Lamin B1 group,β-galactosidase staining was used to detect the senescence of articular chondrocyte;Western blot method was used to detect the expression of p21,p53,Lamin B1 and Beclin-1 protein;cell immunofluorescence was used to detect the expression of Lamin B1 and γ-H2 A.X in chondrocytes;RT-q PCR to detect the expression of Lamin B1 gene.4.Blocking the effect of ASIC1 a on the aging of OA rats and its protective effect on OA:Male SD rats were 6-8 weeks,about 200-220 g,randomly divided into normal group,model group and treatment groups.There were 8 rats in each of the normal group and the model group,and 8 rats in each of the treatment groups: Pc Tx1 group and hyaluronic acid(HA,1%)group.HE staining,Safranin O-fast green and toluidine blue staining were used to observe the pathological changes of the knee joint;immunohistochemistry was used to detect the expression of Lamin B1,p21 and p53 proteins in the knee cartilage;electronic imaging of articular cartilage observation and Pelletier score.Results1.Silencing ASIC1 a inhibits chondrocyte senescence induced by extracellular acidification: Compared with the p H 6.0 group,the ASIC1 a silence group significantly reduced the positive rate of senescent cells after acidification induction.And it could increase the expression of Lamin B1 protein,inhibited the expression of senescencerelated proteins p53 and p21 and autophagy-related protein Beclin-1.2.The silencing of Lamin B1 promoted ASIC1a-mediated chondrocyte senescence: β-galactosidase staining showed that compared with the normal group,the positive rate of senescent cells was significantly increased in the Lamin B1 silenced group,and the expression of senescence-related proteins p53 and p21 obviously increased.3.ASIC1a-mediated Lamin B1 protein degradation by autophagy: In p H 6.0 group adding CQ(20 μM)could reverse the decrease in Lamin B1 protein expression induced by extracellular acidification,and inhibited senescence-related proteins p53 and p21 and autophagy-related protein Beclin-1.4.Blockade of ASIC1 a protected chondrocyte on the rat OA model: Cartilage-specific staining showed that blocking ASIC1 a with Pc Tx1 significantly reduced the destruction of cartilage structure in OA rats.Blockade of ASIC1 a significantly restored Lamin B1,whereas downregulated the expression of p21 and p53 in OA cartilage.ConclusionIn summary,the micro-environment of extracellular acidification activated ASIC1 a,degraded Lamin B1 protein through the autophagy pathway,destroy the structure and function of the nucleus,and accelerated the process of osteoarthritis.It provides a new mechanism for osteoarthritis treatment and cartilage chondrocyte senescence.更多还原