增强自噬的可注射水凝胶微球改善软骨微环境抗软骨细胞衰老

Injectable hydrogel microspheres that enhance autophagy can improve cartilage microenvironment and resist chondrocyte senescence

背景:细胞衰老是骨关节炎的重大危险因素之一,目前尚无广泛认可的针对衰老细胞的抗骨关节炎治疗策略。目的:制定可行的针对骨关节炎中衰老细胞的治疗策略。方法:采用薄膜分散法制备包封雷帕霉素的阳离子脂质体RAPA@Lipo,合成甲基丙烯酰化透明质酸水凝胶,将RAPA@Lipo加入甲基丙烯酰化透明质酸水凝胶水相液中,利用微流控设备制成水凝胶微球,再于紫光光照下交联成固体水凝胶微球(RAPA@Lipo@MS)。将人原代软骨细胞分别与RAPA@Lipo、 RAPA@Lipo@MS共培养,采用CCK-8法、活/死染色评价材料的生物相容性。将大鼠原代软骨细胞分4组培养:正常对照组常规培养48 h,造模组加入H2O2刺激24 h建立衰老细胞模型,RAPA@Lipo组、RAPA@Lipo@MS组建立衰老细胞模型后分别加入RAPA@Lipo、RAPA@Lipo@MS培养24 h,培养结束后,采用免疫荧光观察p62及Ⅱ型胶原蛋白的表达,RT-PCR检测白细胞介素6、基质金属蛋白酶13、Ⅱ型胶原、聚集蛋白聚糖及ADAMTS-5的m RNA表达。结果与结论:(1)CCK-8检测、活/死染色结果显示,RAPA@Lipo与RAPA@Lipo@MS均具有良好的生物相容性;(2)与正常对照组相比,造模组细胞p62蛋白表达升高(P <0.05),Ⅱ型胶原蛋白表达降低(P <0.05),白细胞介素6、基质金属蛋白酶13及ADAMTS-5的m RNA表达升高(P <0.05),Ⅱ型胶原及聚集蛋白聚糖m RNA表达降低(P <0.05);与造模组比较,RAPA@Lipo@MS组p62蛋白表达降低(P <0.05),Ⅱ型胶原蛋白表达升高(P <0.05),白细胞介素6、基质金属蛋白酶13及ADAMTS-5的m RNA表达降低(P <0.05),Ⅱ型胶原及聚集蛋白聚糖m RNA表达升高(P <0.05);(3)结果表明,RAPA@Lipo@MS通过增强自噬控制体内细胞质量、减少体内的衰老细胞,局部清除骨关节炎中的衰老细胞和衰老相关分泌表型因子,以此减缓骨关节炎的进展,创造一个促再生的软骨微环境。

BACKGROUND:Cell senescence is one of the major risk factors for osteoarthritis,but there is no widely accepted anti-osteoarthritis therapy targeting senescent cells.OBJECTIVE:To develop a feasible treatment strategy targeting senescent cells in osteoarthritis.METHODS:The cationic liposome containing rapamycin,RAPA@Lipo,was prepared by thin film dispersion method.Methylallylated hyaluronic acid hydrogel was synthesized,and RAPA@Lipo was added to the methylallylated hyaluronic acid hydrogel aqueous phase solution.The hydrogel microspheres were prepared by microfluidic equipment.Solid hydrogel microspheres(RAPA@Lipo@MS)were crosslinked under violet light.Primary human chondrocytes were co-cultured with RAPA@Lipo and RAPA@Lipo@MS,respectively.The biocompatibility of the materials was evaluated by CCK-8 assay and live/dead staining.Primary rat chondrocytes were cultured in four groups.Normal control group was cultured for 48 hours.The model group was stimulated with H2O2 for 24 hours to establish senescent cell model.RAPA@Lipo group and RAPA@Lipo@MS group were cultured for 24 hours after establishing senescent cell model with RAPA@Lipo and RAPA@Lipo@MS,respectively.After culture,immunofluorescence was used to observe the expression of p62 and type II collagen.RT-PCR was used to detect the mRNA expression of interleukin 6,matrix metalloproteinase 13,type II collagen,aggrecan,and ADAMTS-5.RESULTS AND CONCLUSION:(1)The results of CCK-8 assay and live/dead staining showed that RAPA@Lipo and RAPA@Lipo@MS had good biocompatibility.(2)Compared with the normal control group,the protein expression of p62 was increased(P<0.05);the expression of type II collagen was decreased(P<0.05),and the mRNA expression levels of interleukin 6,matrix metalloproteinase 13,and ADAMTS-5 were increased(P<0.05);mRNA expression levels of type II collagen and aggrecan were decreased(P<0.05)in the model group.Compared with the model group,the expression of p62 protein was decreased(P<0.05);the expression of type II collagen was increased(P<0.05),and the mRNA expression levels of interleukin 6,matrix metalloproteinase 13,and ADAMTS-5 were decreased(P<0.05);mRNA expression of type II collagen and aggrecan increased(P<0.05)in the RAPA@Lipo@MS group.(3)These findings indicate that RAPA@Lipo@MS can control the quality of cells in vivo by enhancing autophagy,reduce senescent cells in vivo,and locally eliminate senescent cells and senescence-associated secretory phenotype factors in osteoarthritis,thereby slowing the progression of osteoarthritis and creating a cartilage microenvironment that promotes regeneration.