Effects of Senescent Hepatocytes Exosomes on Inflammatory Chondrocytes Induced by LPS and Intervention Effect of Paeonia Lactiflora
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摘要: 目的 研究衰老表型的肝细胞外泌体对脂多糖(LPS)刺激软骨细胞的影响及白芍的干预效应。方法 提取小鼠肝原代细胞，CCK-8法筛选白芍水煎液干预肝细胞的最佳浓度。随后，肝细胞随机分为空白组、衰老组和白芍组，0.3 mmol·L-1过氧化氢诱导肝细胞衰老，检测肝细胞p16、p21、p53的基因和蛋白表达，观察肝细胞衰老及白芍的干预效应。分别提取各组肝细胞上清中的外泌体，通过电镜、粒径分析、四旋蛋白CD9、CD63、CD81鉴定外泌体。培养小鼠软骨细胞，LPS刺激以模拟KOA炎症环境，检测细胞上清液中TNF-α和IL-1β含量以验证造模成功。PHK67染色标记肝细胞外泌体并观察软骨细胞的摄入情况。将收集的各组肝细胞外泌体用于干预LPS致炎软骨细胞，检测软骨细胞基质降解合成标记物MMP3、MMP13、SOX9、ADAMTS5的基因和蛋白表达水平。结果 经CCK-8法筛选，100 μg·mL-1为白芍水煎液干预肝细胞的最佳浓度。过氧化氢干预后，衰老组中细胞衰老标记物p16、p21、p53的基因和蛋白表达均高于正常组(P < 0.05)，而在白芍组则较衰老组降低(P < 0.05)。各组肝细胞所收集外泌体均呈现双层膜结构，形态大小无差异，粒径富集均满足40~120 nm区间，代表性粒径富集于116.8 nm，丰度98%，外泌体标记物四旋蛋白CD9、CD63、CD81均为阳性表达。软骨细胞上清液中，促炎因子TNF-α、IL-1β的含量均在LPS刺激后较刺激前升高(P < 0.01)，PHK67染色试剂确认软骨细胞对各组肝细胞外泌体的摄入。衰老组肝细胞外泌体的干预下，软骨细胞基质降解合成标记物MMP3、MMP13、SOX9、ADAMTS5的基因和蛋白表达水平均较空白组肝细胞外泌体干预升高(P < 0.05)，而白芍组肝细胞外泌体干预则较衰老组肝细胞外泌体干预降低(P < 0.05)。结论 衰老表型肝细胞外泌体可加剧LPS致炎软骨细胞的退变进程，白芍水煎剂对此环节存在良性干预。Abstract: OBJECTIVE To explore the effects of senescence hepatocellular exosomes on LPS-induced chondrocytes and the intervention effect of Paeonia lactis.METHODS The primary hepatocytes of mice were extracted and the optimal concentration of the decoction of Paeonia lactis was selected by CCK-8. Subsequently, liver cells were randomly divided into the normal group, the senescence group and the Paeonia lactide group. The senescence of liver cells was induced by 0.3 mm hydrogen peroxide, and the gene and protein expressions of p16, p21 and p53 were detected to observe the senescence of liver cells and the intervention effect of Paeonia lactis. Exosomes were extracted from the supernatant of liver cells in each group, and identified by electron microscopy, particle size analysis, and tetrapod CD9, CD63, CD81. Chondrocytes were cultivated in mice, the content of TNF-α and IL-1β in cell supernatant were determined with or without LPS. The intake of chondrocytes were observed by PHK67 dye marker, and LPS-induced chondrocytes were treated with exosomes collected from the supernatant of each liver cell groups. Both gene and protein expression of chondrocyte matrix degradation-synthetic markers MMP3, MMP13, SOX9, ADAMTS5 were detected.RESULTS 100 μg·mL-1 was the optimal concentration of the decoction of Paeonia lactis. After hydrogen peroxide intervention, the gene and protein expression of cell senescence markers p16, p21 and p53 in the senescence group were all higher than those in the normal group (P < 0.05), but were lower in the Paeonia lactis group than those in the senescence group (P < 0.05). Exosomes collected by liver cells from each group presented a bistratal membranaceous structure, with no difference in morphology and size. The enrichment of the exosomes in particle sizes all met the range of 40-120 nm, the representative particle sizes were enriched at 116.8 nm, and the abundance was 98%. The exosome markers CD9, CD63, and CD81 were all positively expressed. The levels of pro-inflammatory cytokines TNF-α and IL-1β in chondrocytes supernatant increased after LPS stimulation compared with before (P < 0.01), and PHK67 staining reagent was used to confirm the uptake of chondrocyte exosomes in each group. The expression levels of genes and proteins of chondrocyte matrix degradation-synthetic markers MMP3, MMP13, SOX9 and ADAMTS5 in the senescence group were all higher than that in the normal group (P < 0.05), while the exosomes in the Paeonia lactis group were lower than that in the senescence group (P < 0.05).CONCLUSION Ageing phenotype hepatocyte exosomes can aggravate the LPS-induced degeneration of inflammatory chondrocytes, and the decoction of Paeonia lactiflora has a benign intervention in this process.
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Key words:
- knee osteoarthritis /
- chondrocytes /
- hepatocytes /
- cell senescence /
- exosomes
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图 1 白芍缓解H2O2诱导的肝细胞衰老
注：A.不同剂量的白芍水煎液对肝细胞存活率的影响，与10 μg·mL-1比较，*P < 0.05，**P < 0.01。B.各组肝细胞衰老相关基因p16、p21、p53的mRNA表达水平，与空白组比较，**P < 0.01;与衰老组比较, #P < 0.05，##P < 0.01。C.各组肝细胞衰老相关基因p16、p21、p53的代表性蛋白条带。D.各组肝细胞衰老相关基因p16、p21、p53的蛋白表达水平，与空白组比较，**P < 0.01；与衰老组比较，#P < 0.05，##P < 0.01。
图 5 各组软骨细胞合成降解相关基因的mRNA表达水平
注：与空白组比较, *P < 0.05，**P < 0.01；与衰老组比较，#P < 0.05，##P < 0.01。
表 1 目的基因序列
目的基因 前引（5'→3'） 后引（5'→3'） p16 GGTCGCAGGTTCTTGGTC CTCGCTGTCCTGGGTCTC p21 TGATCTGCTGCTGTTTTCC TACATTCCCTTCCAGTCCA p53 AACGCTTCGAGATGTTCC GTTTGGGCTTTGGTGGTT MMP3 GGGTGGATGCTGTCTTTG TGCCTTCCTTGGATCTCTT MMP13 GGGGAGCCACAGATGAG AACGCTCGCAGTGAAAG SOX9 CCACGGAACAGACTCACA GAGATTGCCCAGAGTGCT ADAMTS5 TGGTGAAGGTGGTGGTG GCTGGTTATGTTGGTGCTG GAPDH AAGAAGGTGGTGAAGCAGG GAAGGTGGAAGAGTGGGAGT 
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