血管活性肠肽对骨关节炎和关节软骨细胞体外培养的影响及其作用机制

Effects of vasoactive intestinal peptide on osteoarthritis in vivo and the cultured chondrocyte in vitroand the underlying mechanism

目的观察血管活性肠肽（VIP）对骨关节炎（OA）治疗效果和OA软骨细胞体外培养影响，并探讨其相关作用机制。 方法通过Hulth造模法建立SD大鼠膝关节OA模型大鼠，构建重组pcDNA3.1＋/VIP质粒，并通过关节腔连续1个月注射VIP质粒，通过苏木素-伊红（HE）染色观察OA大鼠膝关节病理变化，采用酶联免疫吸附试验（ELISA）试剂盒检测血清细胞因子[肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1、IL-2和IL-6]水平。同时，体外培养OA软骨细胞，VIP质粒处理后，通过细胞计数试剂盒（CCK-8）检测软骨细胞增殖能力，通过Western blot法检测软骨细胞的Ⅰ型胶原（CollagenⅠ）、Ⅱ型胶原（CollagenⅡ）、核因子（NF）-κB蛋白表达水平。通过反转录-聚合酶链反应（RT-PCR）检测软骨细胞的基质金属蛋白酶（MMP）-3和基质金属蛋白酶抑制剂（TIMP）-1的mRNA表达水平。 结果VIP可显著改善OA大鼠膝关节病理状态，此外，VIP质粒作用前，OA大鼠血清TNF-α、IL-1、IL-2、IL-6分别为（185.3±9.1）、（391.7±20.6）、（169.8±10.8）、（143.7±15.2） pg/ml，VIP质粒作用后OA大鼠血清TNF-α、IL-1、IL-2、IL-6分别为（112.8±10.2）、（298.2±15.9）、（131.4±13.4）、（81.6±13.4） pg/ml，有效降低了OA大鼠血清TNF-α、IL-1、IL-2、IL-6水平。同时，经CCK-8检测发现，OA组和VIP质粒处理组细胞测定的吸光度（A450 nm）值分别为0.75±0.12、1.38±0.14、即在VIP质粒处理下，软骨细胞增殖能力明显增强。此外，OA组和VIP质粒处理组软骨细胞的MMP-3的mRNA相对表达量分别为2.48±0.24、1.35±0.16；TIMP-1的mRNA相对表达量分别为0.56±0.11、0.85±0.09，即在VIP质粒作用下，MMP-3表达明显下调，TIMP-1表达显著上调。Western blot法检测CollagenⅠ、CollagenⅡ、NF-κB蛋白表达发现，在VIP质粒作用下，软骨细胞的CollagenⅠ和NF-κB表达降低，CollagenⅡ表达升高。 结论VIP质粒在一定程度上可通过抑制NF-κB信号通路治疗OA。

ObjectiveTo investigate the treating-effect of vasoactive intestinal peptide （VIP） on osteoarthritis （OA） in vivo and the improving-effect on OA chondrocyte in vitro, then clarify the underlying mechanism. MethodsThe OA model on the SD rat knee was established by Hulth method, and the recombinant pcDNA3.1＋ /VIP plasmid was also constructed. After the VIP plasmid were injected intra-articularly into OA rats for 1 month, the pathological changes of OA knee joint were observed by Hematoxylin-eosin （HE） staining. The levels of serum cytokines [tumor necrosis factor-α （TNF-α）, interleukin （IL）-1, IL-2 and IL-6] were measured by enzyme linked immunosorbent assay （ELISA） kits. Meanwhile, OA chondrocyte were cultured in vitro, and treated by plasmid VIP. The proliferation of chondrocyte was determined by cell counting kit-8 （CCK-8） kits. The protein expressions of collagen type Ⅰ （Collagen Ⅰ）, collagen type Ⅱ （Collagen Ⅱ） and nuclear factor （NF）-κB were evaluated by Western blotting. The mRNA expressions of matrix-degrading enzyme matrix metalloproteinase （MMP）-3 and matrix-degrading enzyme inhibitor tissue inhibitorof metalloproteinase （TIMP）-1 were determined by reverse transcriptase-polymerase chain reaction （RT-PCR）. ResultsVIP plasmid could effectively improve the pathological state of the OA ratskneejoint. In addition, before the OA rats treated by VIP plasmid, the level of serum TNF-α, IL-1, IL-2 and IL-6 were （185.3±9.1）, （391.7±20.6）, （169.8±10.8）, （143.7±15.2） pg/ml; then treated by VIP plasmid the level of serum TNF-α, IL-1, IL-2 and IL-6 were （112.8±10.2）, （298.2±15.9）, （131.4±13.4）, （81.6±13.4） pg/ml, the levels of serum cytokinesweresignificantly decreased. At the same time, the absorbance （A450 nm） of chondrocytein OA group and VIP-treat groupdeterminedby CCK-8 kit were 0.75±0.12, 1.38±0.14, respectively, suggesting that after the OA chondrocytes treated by VIP plasmid, the proliferation was obviously increased. The MMP-3 mRNA expression of chondrocytesin OA group and VIP-treat group were 2.48±0.24, 1.35±0.16, respectively; the TIMP-1 mRNA expression were 0.56±0.11, 0.85±0.09, respectively, suggesting that the MMP-3 expression was remarkably downregulated, and TIMP-1 expression was obviously upregulated by VIP plasmid. After the the protein expression of Collagen Ⅰ, Collagen Ⅱ and NF-κB in were evaluated by Western blotting, we found that the expression of Collagen Ⅰ and NF-κB in chondrocyteswere decreased, and the expression of Collagen Ⅱ in chondrocyteswas increased by VIP plasmid. ConclusionVIP could treat osteoarthritisto some extent via inhibiting NF-κB signal pathway.