Difference between Effects of the Main Terpenoids from Euphorbiae Ebracteolatae Radix before and after Simulated Vinegar-Processing on Inflammatory Toxicity on Macrophages and Regulation of the Expression of AQPs in Renal Cells
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摘要:
目的 探究狼毒效应部位中主要萜类成分模拟醋制前后毒、效作用变化，阐释狼毒醋制解毒存效的机制, 探寻狼毒醋品质量标志物。 方法 将狼毒萜类成分Eupractenoid A(EA)、Jolkinolide B(JNB)、Fischeria A(FA)采用模拟醋制法在160 ℃加6%冰醋酸加热40 min, 以Western blot法分析3种萜类成分醋制前后对巨噬细胞RAW264.7中TNF-α和IL-1β蛋白表达水平影响的差异; 以Western blot法分析3种萜类成分醋制前后对肾小管上皮细胞HK-2中AQP1及肾集合管上皮细胞mIMCD3中AQP2、3、4蛋白表达水平影响的差异。 结果 EA、JNB、FA可诱导巨噬细胞炎症因子TNF-α和IL-1β蛋白表达水平显著增高(P < 0.05)。EA、JNB、FA醋制后致炎毒性均显著下降。EA主要转化产物Euphebracteolatin A(EHTA)无明显促炎毒性。药效评价显示, EA及其模拟醋制产物可显著抑制AQP1、3、4蛋白表达(P < 0.05), EA醋制后对AQP1、2、4蛋白表达的抑制作用显著增强(P < 0.05)。EHTA对肾细胞AQP1、2、3蛋白表达的抑制作用显著强于EA(P < 0.05), 对AQP4蛋白表达的抑制作用与EA相比无显著性差异, 表明EA醋制可增强药效。JNB可显著抑制AQP1、3蛋白表达(P < 0.05)。其模拟醋制产物可显著抑制AQP1、2、3、4蛋白表达, 且JNB醋制后对AQP2、3、4蛋白表达的抑制作用显著增强(P < 0.05), 表明JNB醋制后药效增强。FA醋制后对AQPs调节作用显著降低(P < 0.05), 但高剂量组仍起作用。 结论 狼毒主要萜类效应成分经模拟醋制后致炎毒性减弱, 但醋制后药效总体呈现增强或保留。同时EA转化产物EHTA无明显促炎毒性, 药效作用强于其转化前体EA, 可作为狼毒醋制品质量控制指标。 Abstract:OBJECTIVE To explore the effect change of the main terpenoid components of active fraction in Euphorbiae ebracteolatae Radix (EER) during the vinegar processing to clarify the mechanism of detoxification and efficacy retention by vinegar processing of EER, and to explore the quality marker of EER stir-baked with vinegar. METHOD The terpenoids Eupractenoid A (EA), Jolkinolide B (JNB), Fischeria A (FA) were heated in a constant temperature heater (160 ℃) with 6% acetic acid for 40 min. The effects of the three terpenoids before and after simulated vinegar-processing on the protein expression levels of TNF-α and IL-β in macrophages (RAW264.7 cells), AQP1 in HK-2 cells and AQP2, 3, 4 in mIMCD3 cells were analyzed by Western blot. RESULT EA, JNB, FA induced expression enhancement of TNF-α and IL-β in macrophages (P < 0.05). Compared to EA, JNB, FA, the inflammatory toxicity of their simulated vinegar-processing's products were significantly reduced. EHTA, a main simulated vinegar-processing transformation product of EA, had no pro-inflammatory effect. EA and its processing product inhibited the protein expression of AQP1, 3, 4(P < 0.05). EA's processing product had stronger inhibition on the expression of AQP1, 2, 4 than EA (P < 0.05). EHTA had stronger inhibition effects on AQP1, 2, 3 than EA (P < 0.05), and in terms of influence on AQP4, EA and EHTA have no significant difference. The results showed that EA had stronger regulation effect on AQPs after processing. JNB inhibited the protein expression of AQP1, 3 (P < 0.05). Its processing product inhibited the expression of AQP1, 2, 3, 4 and had stronger inhibition effects on AQP2, 3, 4 than JNB (P < 0.05). The results showed that JNB had stronger regulation effect on AQPs after processing. FA's regulation effect on AQPs expression was decreased after simulated vinegar-processing (P < 0.05), but high dose of FA's processing product stiu had these effects. CONCLUSION The proinflammatory toxicity of these terpenoids in EER is reduced after processing. Terpenoids generally shows the results of enhancing or retaining the efficacy after vinegar processing. EHAT, the terpenoid components of EER and the main simulated vinegar-processing transformation product of EA, has non-sinificant proinflammatory toxicity and stronger efficacy than EA. EHTA can be an index of quality control standards of EER stir-baked with vinegar. -
图 1 EA模拟醋制前后对巨噬细胞炎症因子表达水平的影响(x±s, n=3)
注: 与空白组比较, *P < 0.05;与EA相同剂量比较, #P < 0.05。
Figure 1. The effects of EA and its simulated vinegar-processing product on the expression levels of inflammatory factors in macrophages (x±s, n=3)
图 2 EA与EHTA对巨噬细胞炎症因子表达水平的影响(x±s, n=3)
注: 与空白组比较, *P < 0.05;与EA相同剂量比较, #P < 0.05。
Figure 2. The effects of EA and EHTA on the expression levels of inflammatory factors in macrophages (x±s, n=3)
图 3 JNB模拟醋制前后对巨噬细胞炎症因子表达水平的影响(x±s, n=3)
注: 与空白组比较, *P < 0.05;与JNB相同剂量比较, #P < 0.05。
Figure 3. The effects of JNB and its simulated vinegar-processing product on the expression levels of inflammatory factors in macrophages (x±s, n=3)
图 4 FA模拟醋制前后对巨噬细胞炎症因子表达水平的影响(x±s, n=3)
注: 与空白组比较, *P < 0.05;与JNB相同剂量比较, #P < 0.05。
Figure 4. The effects of FA and its simulated vinegar-processing products on the expression levels of inflammatory factors in macrophages (x±s, n=3)
图 5 EA模拟醋制前后对肾脏AQPs蛋白表达水平的影响(x±s, n=3)
注: 与空白组比较, *P < 0.05;与EA相同剂量比较, #P < 0.05。
Figure 5. The effects of EA and its simulated vinegar-processing product on the expression levels of AQPs in renal cells (x±s, n=3)
图 6 EA与模拟醋制产物EHTA对肾脏细胞AQPs蛋白表达水平的影响(x±s, n=3)
注: 与空白组比较, *P < 0.05;与EA相同剂量比较, #P < 0.05。
Figure 6. The effects of EA and EHTA on the expression levels of AQPs in renal cells (x±s, n=3)
图 7 JNB模拟醋制前后对肾脏细胞AQPs蛋白表达水平的影响(x±s, n=3)
注: 与空白组比较, *P < 0.05;与JNB相同剂量比较, #P < 0.05。
Figure 7. The effects of JNB and its simulated vinegar-processing product on the expression levels of AQPs in renal cells (x±s, n=3)
图 8 FA模拟醋制前后对肾脏细胞AQPs蛋白表达水平的影响(x±s, n=3)
注: 与空白组比较, *P < 0.05;与FA相同剂量比较, #P < 0.05。
Figure 8. The effects of FA and its simulated vinegar-processing product on the expression levels of AQPs in renal cells (x±s, n=3)
表 1 4种化合物对3种细胞的毒性评价结果
Table 1. Toxicity evaluation results of 4 compounds on 3 cells
细胞 化合物 OD值(抑制率) 空白组 50 μmol·L-1 25 μmol·L-1 10 μmol·L-1 5 μmol·L-1 RAW264.7 EA 0.287±0.02 0.217±0.03
(24.42%)0.266±0.03
(7.54%)0.276±0.02
(4.12%)0.283±0.03
(1.45%)EHTA 0.287±0.02 0.016±0
(94.32%)0.125±0.03
(56.55%)0.203±0.01
(29.47%)0.265±0.04
(7.66%)JNB 0.268±0.02 0.241±0.02
(10.13%)0.248±0.02
(7.56%)0.256±0.03
(4.54%)0.266±0.01
(0.73%)FA 0.243±0.02 0.191±0.03
(21.52%)0.212±0.01
(13.02%)0.213±0.01
(12.47%)0.25±0.02
(11.65%)HK-2 EA 0.322±0.04 0.261±0.04
(19.06%)0.306±0.06
(4.97%)0.309±0.05
(3.94%)0.320±0.06
(0.73%)EHTA 0.322±0.04 0.026±0.01
(91.97%)0.052±0.01
(83.74%)0.180±0.06
(44.23%)0.288±0.07
(10.51%)JNB 0.549±0.06 0.488±0.13
(11.13%)0.507±0.07
(7.68%)0.519±0.08
(5.58%)0.519±0.06
(5.46%)FA 0.549±0.06 0.355±0.07
(35.35%)0.489±0.07
(10.98%)0.499±0.07
(9.25%)0.510±0.10
(7.13%)mIMCD3 EA 0.655±0.06 0.548±0.05
(16.22%)0.586±0.10
(10.54%)0.594±0.05
(9.27%)0.616±0.07
(5.83%)EHTA 0.655±0.06 0.008±0
(98.73%)0.198±0.05
(69.75%)0.432±0.04
(34.02%)0.593±0.06
(9.42%)JNB 0.941±0.07 0.890±0.19
(5.42%)0.949±0.10
(-0.83%)0.921±0.13
(2.145%)0.955±0.08
(-1.49%)FA 0.827±0.03 0.666±0.07
(19.40%)0.769±0.06
(7.00%)0.778±0.06
(5.85%)0.785±0.09
(4.98%)
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