石榴皮鞣质对慢性结肠炎小鼠保护作用机制研究

Protective effect and mechanism of Tannis in Pomegranate Peel on chronic colitis mice model

目的研究石榴皮鞣质对葡聚糖硫酸钠(DSS)诱导的慢性结肠炎模型小鼠的保护作用及对c-Jun氨基末端激酶(JNK)信号通路的影响。方法用序贯法(第1~5天、第11~15天和第21~25天)灌胃3%DSS溶液0.3 mL建立慢性结肠炎模型。按照体重将小鼠随机分为5组:正常组、模型组、5-氨基水杨酸(5-ASA)对照组和低、中、高3个剂量实验组,每组12只。低、中、高3个剂量实验组(石榴皮鞣质:20,40,80 mg·kg^(-1)·d^(-1))灌肠,5-ASA对照组给予5-ASA 10.4 mg·d^(-1),1次/天,连续10 d。酶联免疫吸附法检测结肠组织中白细胞介素^(-1)β(IL^(-1)β)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)及干扰素因子-γ(INF-γ)含量。实时荧光定量-PCR法和蛋白质印迹法分别检测c-Jun、JNK基因和蛋白的表达水平。结果正常组、模型组、低、中、高3个剂量实验组和5-ASA对照组的IL^(-1)β含量分别为(3.17±1.11),(5.16±0.54),(4.94±0.47),(3.53±0.64),(2.95±0.60),(4.11±0.71)μmoL·L^(-1);这6组的IL-8含量分别为(110.76±26.47),(159.72±10.98),(130.17±15.83),(110.56±16.73),(96.82±22.92),(150.78±7.61)μmoL·L^(-1);这6组的TNF-α含量分别为(96.46±6.18),(116.95±21.15),(84.54±5.27),(78.15±6.55),(74.51±5.17),(56.85±6.66)μmoL·L^(-1);这6组的IFN-γ含量分别为(186.02±36.74),(265.63±18.82),(142.35±46.14),(114.35±21.25),(54.74±14.52),(35.29±24.24)μmoL·L^(-1),模型组与正常组相比,上述指标的差异均有统计学意义(P<0.05,P<0.01);低、中、高3个剂量实验组与模型组相比,上述指标的差异均有统计学意义(P<0.05,P<0.01)。正常组、模型组、高剂量实验组和5-ASA对照组的c-Jun基因水平分别为1.00±0.22,7.85±1.94,0.62±0.11和1.03±0.35;这4组的JNK基因水平分别为0.68±0.17,3.27±0.63,0.54±0.13和0.97±0.22,模型组与正常组相比,或高剂量实验组与模型组相比,上述指标的差异均有统计学意义(均P<0.05)。蛋白结果的趋势与基因一致。结论石榴皮鞣质通过抑制c-Jun/JNK通路,抑制炎症发生发展,发挥对慢性结肠炎小鼠的保护作用。

Objective To study the protective effect of Tannis in Pomegranate Peel(TPP) on dextran sulfate sodium(DSS)-induced chronic colitis model mice and the effect on c-Jun N-terminal kinase/c-Jun(JNK/c-Jun) pathway. Methods The chronic colitis model were established by sequential method(day 1-5, day 11-15, and day 21-25) and intragastric administration of 0. 3 mL 3% dextran sodium sulfate( DSS) solution. The mice were divided into 5 groups according to weight: normal group,model group,5-aminosalicylic acid( 5-ASA) control group,and high,medium and low doses experimental groups,12 mice in each group. The high,medium and low doses experimental groups were enema( 80,40,20 mg· kg^(-1)· d^(-1) TPP),5-ASA group were enema 5-ASA 10. 4 mg·d^(-1),once a day for 10 consecutive days. The content of interleukin^(-1)β( IL-1β),interleukin-8( IL-8),tumor necrosis factor-α( TNF-α) and interference factor-γ( INF-γ) in the colon tissue homogenate were detected by enzyme linked immunosorbent assay. Real-time fluorescence quantification-PCR and Western blot were used to detect the mRNA and protein levels of c-Jun and JNK. Results The levels of IL-1 in the normal group,model group,high,medium and low doses experimental groups and 5-ASA control group were( 3. 17 ± 1. 11),( 5. 16 ± 0. 54),( 4. 94 ± 0. 47),( 3. 53 ± 0. 64),( 2. 95 ± 0. 60) and( 4. 11 ± 0. 71) μmoL·L^(-1),respectively;the levels of IL-8 in the 6 groups were( 110. 76 ± 26. 47),( 159. 72 ± 10. 98),( 130. 17 ± 15. 83),( 110. 56 ± 16. 73),( 96. 82 ± 22. 92),( 150. 78 ± 7. 61) μmoL · L^(-1),respectively;the contents of TNF-α in the 6 groups were( 96. 46 ± 6. 18),( 116. 95 ± 21. 15),( 84. 54 ± 5. 27),( 78. 15 ± 6. 55),( 74. 51 ± 5. 17) and( 56. 85 ± 6. 66) μmoL · L^(-1),respectively;the levels of IFN-γ in the 6 groups were( 186. 02 ± 36. 74),( 265. 63 ± 18. 82),( 142. 35 ± 46. 14),( 114. 35 ± 21. 25),( 54. 74 ± 14. 52),( 35. 29 ± 24. 24) μmoL·L^(-1),respectively. The difference of the above indexes between the model group and the normal group was statistically significant( P < 0. 05,P < 0. 01);the difference between the low,middle and high dose group and the model group was statistically significant( P < 0. 05,P < 0. 01). The levels of c-Jun mRNA in normal group,model group,high-dose experimental group and 5-ASA control group were1. 00 ± 0. 22,7. 85 ± 1. 94,0. 62 ± 0. 11 and 1. 03 ± 0. 35,respectively;the levels of JNK mRNA were 0. 68 ± 0. 17,3. 27 ± 0. 63,0. 54 ± 0. 13 and 0. 97 ± 0. 22. The difference of the above indexes between the model group and the normal group was statistically significant( all P < 0. 05);the difference of the above indexes between high dose experimental group and model group was statistically significant( all P < 0. 05). The trend of protein results is consistent with that of gene. Conclusion TPP inhibits the c-Jun/JNK pathway by down-regulating the expression of c-Jun and JNK mRNA and protein in colon tissue. In a dose-dependent manner,it can reduce the content of IL-1β,IL-6,IL-8,TNF-α and IFN-γ in tissue homogenate,inhibit the occurrence and development of inflammation,and play a protective role in mice with chronic colitis.