自体富血小板血浆对骨关节炎模型兔软骨细胞凋亡及NLRP3/IL-1β通路的影响

Effects of platelet-rich plasma on chondrocyte apoptosis and NLRP3/IL-1β signaling pathway in rabbits with osteoarthritis

目的:探究富血小板血浆(PRP)对兔骨关节炎的作用及可能的机制。方法:制备膝骨关节炎兔模型,分为模型组、玻璃酸钠(SH)组和PRP组,另设假手术组,每组6只兔。观察兔软骨大体形态,进行半定量评分;HE染色观察软骨病理形态并进行半定量评分;TUNEL染色观察软骨组织细胞凋亡情况;免疫组化法检测核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/白细胞介素1β(IL-1β)通路相关蛋白的表达;Western blot法检测caspase-3、Bcl-2和Bax蛋白表达情况;分离软骨细胞,分组处理后采用ELISA测定细胞NLRP3和IL-1β水平。结果:与假手术组相比,模型组的Pelletier评分,Mankin评分,软骨细胞凋亡率,NLRP3、含caspase募集结构域的凋亡相关斑点样蛋白(ASC)、caspase-1和IL-1β蛋白阳性表达率,以及caspase-3和Bax蛋白水平均显著升高(P<0. 05),Bcl-2蛋白水平显著降低(P<0. 05)。与模型组相比,SH组和PRP组的Pelletier评分,Mankin评分,软骨细胞凋亡率,NLRP3、ASC、caspase-1和IL-1β蛋白阳性表达率,以及caspase-3和Bax蛋白水平均显著降低(P<0. 05),Bcl-2蛋白水平显著升高(P<0. 05)。PRP组的Pelletier评分,Mankin评分,软骨细胞凋亡率,NLRP3、ASC、caspase-1和IL-1β蛋白阳性表达率,以及caspase-3和Bax蛋白水平均低于SH组,Bcl-2蛋白水平高于SH组(P<0. 05)。细胞培养实验中,与对照组相比,NLRP3抑制剂MCC950组NLRP3及IL-1β表达显著降低(P<0. 05),IL-1β抑制剂eucalyptol组NLRP3表达无明显变化(P>0. 05),IL-1β表达显著降低(P<0. 05)。结论:富血小板血浆能够促进骨关节炎模型兔受损软骨的修复,效果优于SH,其机制可能与抑制NLRP3/IL-1β通路并减少软骨细胞凋亡有关。

AIM:To explore the effect of platelet-rich plasma(PRP)on rabbit osteoarthritis and its possible mechanism.METHODS:The rabbits with knee osteoarthritis were prepared and then divided into model group,sodium hyaluronate(SH)group and PRP group,and another sham operation group was set up,with 6 rabbits in each group.The gross morphological changes of rabbit cartilage were observed.HE staining was used to evaluate the pathomorphological changes of the cartilage.TUNEL staining was used to detect the apoptosis of chondrocytes.The expression of nucleotidebinding oligomerization domain-like receptor protein 3(NLRP3)/interleukin-1β(IL-1β)signaling pathway-related molecules was observed by immunohistochemical staining,and the protein levels of caspase-3,Bcl-2 and Bax were determined by Western blot.Chondrocytes were isolated and processed according to grouping,and the NLRP3 and IL-1βlevels of the cells were measured by ELISA.RESULTS:Compared with sham operation group,Pelletier score,Mankin score,chondrocyte apoptotic rate,the positive protein expression rates of NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),caspase-1 and IL-1β,and the protein levels of caspase-3 and Bax in model group were increased significantly(P<0.05),while the protein expression of Bcl-2 was decreased significantly(P<0.05).Compared with model group,Pelletier score,Mankin score,the apoptotic rate of chondrocytes,the positive protein expression rates of NLRP3,ASC,caspase-1 and IL-1β,and the protein levels of caspase-3 and Bax in SH group and PRP group were decreased significantly(P<0.05),while the protein expression of Bcl-2 was increased significantly(P<0.05).In PRP group,Pelletier score,Mankin score,the apoptotic rate of chondrocytes,the positive protein expression rates of NLRP3,ASC,caspase-1 and IL-1β,and the protein levels of caspase-3 and Bax were lower than those in SH group,while the protein expression of Bcl-2 was higher than that in SH group(P<0.05).Compared with control group,the expression of NLRP3 and IL-1βin MCC950(NLRP3 ihibitor)group were significantly reduced(P<0.05),the expression of NLRP3 in eucalyptol(IL-1βinhibitor)group was not significantly changed(P>0.05),and the expression of IL-1βwas significantly reduced(P<0.05).CONCLUSION:Platelet-rich plasma promotes the repair of cartilage in osteoarthritis rabbits,which has better effect than SH.The mechanism may be related to the inhibition of NLRP3/IL-1βpathway and the reduction of chondrocyte apoptosis.