| 汪灿锋,叶正从,沈钦荣,韩雷,曹国平.五福饮含药血清对肿瘤坏死因子-α诱导凋亡软骨细胞活性及基质金属蛋白酶表达的影响[J].浙江中西医结合杂志,2019,29(9): |
| 五福饮含药血清对肿瘤坏死因子-α诱导凋亡软骨细胞活性及基质金属蛋白酶表达的影响 |
| Effect of serum containing Wufu yin（五福饮） on the activity of apoptotic chondrocyte induced by Tumor Necrosis Factor -alpha and the expression of matrix metalloproteinase |
| 投稿时间：2019-04-01 修订日期：2019-08-15 |
| DOI： |
| 中文关键词: 骨关节炎 五福饮 软骨细胞 肿瘤坏死因子-α 基质金属蛋白酶 |
| 英文关键词:Osteoarthritis Wufu yin chondrocytes tumor necrosis factor-α matrix metalloproteinase |
| 基金项目:萧山区重大科技项目(NO.2017213) |
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| 摘 要 目的 观察五福饮含药血清对肿瘤坏死因子-α诱导凋亡软骨细胞活性、胶原蛋白（collagen）II、基质金属蛋白酶（matrix metalloproteinase，MMP）-3、基质金属蛋白酶-9、基质金属蛋白酶-13表达的影响，探讨其防治骨性关节炎的机制。方法 应用随机数字表法将2月龄大鼠分为3组，分别为生理盐水组（生理盐水灌胃）、硫酸氨基葡萄糖组（硫酸氨基葡萄糖灌胃）、五福饮组（五福饮灌胃），经干预后取得相应血清。用Ⅱ型胶原酶消化法获取2月龄SD 大鼠膝关节软骨细胞，应用随机数字表法将软骨细胞分为：空白组：采用肿瘤坏死因子（Tumor Necrosis Factor，TNF）-α0μg/l+10％生理盐水组血清；模型组：采用TNF-α20μg/l+10％生理盐水组血清，C：对照组，采用TNF-α20μg/l+10％硫酸氨基葡萄糖组血清；实验组：采用TNF-α20μg/l+10％五福饮组血清。干预48 h后检测软骨细胞collagen II的表达、软骨细胞凋亡增殖、MMP-3、-9、-13mRNA的表达。结果 collagen II的平均荧光表达值：空白组（70.92±3.72），模型组（43.39±1.52），对照组（62.92±5.32），实验组（53.33±2.33）。模型组、对照组和实验组显著低于空白组（p<0.01）；对照组和实验组高于模型组（p<0.05, p<0.01）。对照组和实验组比较无统计学差异（ p>0.05）。软骨细胞凋亡：模型组凋亡率最高，对照组与实验组凋亡率接近，低于模型组，高于空白组。软骨细胞增殖能力吸光度（OD）值：给药24h后，空白组（0.396±0.017），模型组（0.297±0.014），对照组（0.342±0.021），实验组（0.316±0.028），三组对比空白存在差异（p<0.01）。给药48h后，空白组（0.470±0.018），模型组（0.333±0.021），对照组（0.414±0.029），实验组（0.394±0.028），三组均显著低于空白组（p<0.05，p<0.01）；与模型组相比，对照组和实验组细胞OD值显著升高，有统计学差异（p<0.05, p<0.01）。给药72h后，空白组（0.570±0.031），模型组（0.361±0.014），对照组（0.510±0.035），实验组（0.493±0.030）。三组均显著低于空白组（p<0.05，p<0.01）；与模型组相比，对照组和实验组细胞OD值显著升高，有统计学差异（ p<0.01）；三个时间点实验组与对照组比较无统计学差异（ p>0.05）。MMP-3、MMP-9、MMP-13 mRNA表达：与空白组比较，模型组、实验组中MMP-3、MMP-9、MMP-13，对照组中MMP-9水平显著升高（P<0.01）。与模型组比较，对照组和实验组中MMP-3、MMP-9、MMP-13的mRNA水平显著下降（P<0.01）。与对照组比较，实验组中MMP-3升高显著（P<0.01），MMP-9、MMP-13表达无差异（p>0.05）。结论 五福饮能延缓肿瘤坏死因子-α诱导引起的软骨的细胞凋亡，其机制可能与抑制MMP-3、MMP-9、MMP-13等基质金属蛋白酶表达相关。 |
| 英文摘要: |
| ABSTRACT Objective To observe the effect of Wufu yin（五福饮） containing serum on tumor necrosis factor-α induced apoptosis of chondrocytes, collagen II, matrix metalloproteinase (MMP)-3, matrix metalloproteinase-9 and matrix metalloproteinase-13 The impact of its mechanism to prevent and treat osteoarthritis. Methods Two-month-old rats were divided into three groups by random number table method: saline group (saline gastric lavage), glucosamine sulfate group (glucosamine sulfate gastric lavage), and Wufuyin group (Wufuyin gastric lavage). After intervention, the corresponding serum was obtained. Chondrocytes of knee joint of two-month-old SD rats were obtained by type II collagenase digestion. Chondrocytes were divided into blank group: serum of Tumor Necrosis Factor (TNF) - alpha 0 ug/l + 10% saline group; serum of model group: serum of TNF-alpha 20 ug/l + 10% saline group; serum of C: control group, serum of TNF-alpha 20 ug/l + 10% saline group; serum of control group, serum of TNF-alpha 20 ug/l + 10% glucosamine sulfate group. Serum of sugar group and experimental group: serum of TNF-alpha 20 ug/l+10% Wufu Yin group were used. The expression of collagen II, chondrocyte apoptosis and proliferation, and the expression of MMP-3, -9, -13 mRNA were detected 48 hours after intervention. Results Average fluorescence expression value of collagen II: blank group (70.92 ±3.72).The model group (43.39± 1.52) and the control group (62.92±5.32) and the experimental group (53.33±2.33). Model group, control group and experimental group were significantly lower than blank group (p < 0.01); control group and experimental group were higher than model group (p < 0.05, P < 0.01). There was no significant difference between the control group and the experimental group (p > 0.05). Chondrocyte apoptosis: The apoptotic rate of the model group was the highest. The apoptotic rate of the control group was close to that of the experimental group, lower than that of the model group and higher than that of the blank group. The absorbance (OD) of chondrocyte proliferation ability: 24 hours after administration, the blank group (0.396 + 0.017), the model group (0.297 + 0.014), the control group (0.342 + 0.021), the experimental group (0.316 + 0.028), there were differences in the blank of the three groups (p < 0.01). 48 hours after administration, the OD values of the blank group (0.470 (+0.018), the model group (0.333 (+0.021), the control group (0.414 (+0.029), the experimental group (0.394 (+0.028), the three groups were significantly lower than those of the blank group (p < 0.05, P < 0.01); compared with the model group, the OD values of the control group and the experimental group were significantly higher (p < 0.05, P < 0.01). 72 hours after administration, the blank group (0.570 < 0.031), the model group (0.361 < 0.014), the control group (0.510 < 0.035), and the experimental group (0.493 < 0.030). Compared with the model group, the OD value of the control group and the experimental group increased significantly (p < 0.01), and there was no significant difference between the experimental group and the control group at three time points (p > 0.05). MMP-3, MMP-9, MMP-13 mRNA expression: Compared with the blank group, MMP-3, MMP-9, MMP-13 in the model group and experimental group, MMP-9 level in the control group was significantly increased (P< 0.01). Compared with the model group, the mRNA levels of MMP-3, MMP-9 and MMP-13 in the control group and the experimental group were significantly decreased (P<0.01). Compared with the control group, MMP-3 was significantly increased in the experimental group (P<0.01), and there was no difference in the expression of MMP-9 and MMP-13 (p>0.05). Conclusions Wufuyin can delay the apoptosis of cartilage cells induced by TNF-alpha, and its mechanism may be related to the inhibition of MMP-3, MMP-9, MMP-13 and other matrix metalloproteinases. |
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