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IL-1β作为炎性细胞因子,在椎间盘退变的过程中起着至关重要的作用。本研究的主要目的是观察评价桃仁-红花药对含药血清在体外对IL-1β诱导退变的椎间盘软骨终板细胞的作用,并探讨其机制。桃仁和红花作为药对灌胃给大鼠,收集、准备相应的含药血清。经大鼠椎间盘分离、鉴定的第三代软骨终板细胞作为实验细胞,随机分组为正常组(Group NC)、IL-1β诱导组(Group IL)、桃仁红花药对含药血清组(Group TRHH),正常组仅用标准的培养基培养,IL-1β诱导组加用IL-1β,桃仁-红花药对含药血清组加用IL-1β、桃仁-红花药对含药血清。CCK-8法检测细胞增殖,流式检测细胞凋亡,Real-time PCR(RT-PCR)检测Aggrecan,Col2α1,Col10α1,IL-6和SOX9 mRNA的表达。细胞免疫组化检测II型胶原、X型胶原的表达。臧红-O染色用于鉴定各组细胞中细胞外基质的表达。与正常组相比较,IL-1β诱导组软骨终板细胞增殖降低,细胞凋亡增加(P<0.05),下调Aggrecan,Col2α1和SOX9 mRNA的表达,上调Col10α1和IL-6 mRNA的表达(P<0.05);同时免疫组化和臧红-O染色示II型胶原蛋白的表达降低、X型胶原蛋白的表达升高。与IL-1β诱导组相比较,桃仁-红花药对含药血清组在抑制软骨终板细胞凋亡的同时促进软骨终板细胞的增殖(P<0.05),上调Aggrecan,Col2α1和SOX9mRNA表达的同时下调Col10α1和IL-6 mRNA的表达(P<0.05);促进II型胶原蛋白的表达增强、X型胶原蛋白的表达降低。桃仁-红花药对含药血清能够抑制IL-1β诱导椎间盘软骨终板细胞发生退变。
IL-1β, as an inflammatory cytokine, plays a crucial role in the process of disc degeneration. The main purpose of this study is to observe and evaluate the effect of Peach-safflower on disc-induced disc degeneration of disc cartilage end-plate cells in vitro and its mechanism. Peach kernel and safflower as gavage to rats, collect, prepare the corresponding drug-containing serum. The third-generation cartilage end-plate cells isolated and identified by rat disc were randomly divided into Group NC, Group IL and Peach Anthurium for group TRHH). The normal group was cultured in standard medium only. The IL-1βinduced group was treated with IL-1β. Peach kernel-safflower was added to the drug-containing serum group with IL-1β and peach kernel-safflower drug-containing serum. Cell proliferation was detected by CCK-8 assay. Apoptosis was detected by flow cytometry. The expression of Aggrecan, Col2α1, Col10α1, IL-6 and SOX9 mRNA was detected by Real-time PCR (RT-PCR). Cell immunohistochemistry was used to detect the expression of type II collagen and type X collagen. Zang-O-O staining was used to identify the expression of extracellular matrix in each group of cells. Compared with normal group, IL-1βinduced decreased proliferation and apoptosis of cartilage end-plate cells (P <0.05), down-regulated the expression of Aggrecan, Col2α1 and SOX9 mRNA and up-regulated the expression of Col10α1 and IL-6 mRNA (P < 0.05). Meanwhile, immunohistochemistry and Zang-O-staining showed that the expression of type II collagen decreased and the expression of type X collagen increased. Compared with IL-1βinduced group, peach kernel-safflower could inhibit the proliferation of cartilage end-plate cells (P <0.05) and up-regulate the expressions of Aggrecan, Col2α1 and SOX9 mRNA while inhibiting the apoptosis of cartilage end-plate cells At the same time, the expressions of Col10α1 and IL-6 mRNA were down-regulated (P <0.05), and the expression of type II collagen was enhanced and the expression of type X collagen was decreased. Peach kernel-safflower could inhibit IL-1β-induced degeneration of disc cartilage end-plate cells.