桦褐孔菌乙醇提取物对脂多糖诱导肠损伤的缓解作用及机制

doi:10.13560/j.cnki.biotech.bull.1985.2024-0422

摘要/Abstract

摘要：

【目的】探究桦褐孔菌乙醇提取物（ethanol extract of Inonotus obliquus, EEIO）对脂多糖（lipopolysaccharides, LPS）诱导肠损伤的保护作用及分子机制。【方法】利用LPS诱导的小鼠小肠上皮细胞和C57BL/6小鼠，检测EEIO处理后LDH和NO水平；RT-qPCR和ELISA方法检测EEIO对炎症因子IL-1β和IL-18表达的影响；RT-qPCR和WB方法检测EEIO对MAPK和焦亡信号通路的影响。【结果】体内外结果一致表明，EEIO可有效缓解LPS导致的LDH和NO水平升高；EEIO处理后，IL-1β和IL-18表达明显降低。EEIO抑制MAPK（ERK、P38、JNK）信号通路，显著降低p-ERK/ERK、p-p38/p38和p-JNK/JNK的水平。同时，焦亡途径被抑制，ASC、Caspase-1、GSDMD和NLRP3水平显著降低。阳性药物白藜芦醇（resveratrol, RES）对各项检测指标的影响与EEIO相似，但效果不如EEIO。【结论】EEIO通过调控MAPK信号通路，抑制焦亡途径进而降低炎性因子的表达，缓解LPS诱导肠损伤。

关键词: 桦褐孔菌乙醇提取物, 脂多糖, 肠损伤, 炎症, 焦亡

Abstract:

【Objective】 To investigate the protective effect and molecular mechanism of ethanol extract of Inonotus obliquus（EEIO）on lipopolysaccharide（LPS）-induced intestinal injury. 【Method】 Using LPS to induce mouse small intestinal epithelial cells and C57BL/6 mice, LDH and NO levels were measured after EEIO treatment. RT qPCR and ELISA methods were to detect the effects of EEIO on the expressions of inflammatory factor IL-1β and IL-18. RT-qPCR and WB methods were used to detect the effects of EEIO on MAPK and pyroptosis signaling pathways. 【Result】 Both in vivo and in vitro results showed that EEIO effectively alleviated the increase in LDH and NO levels induced by LPS. After EEIO processing, the expressions of IL-1β and IL-18 were significantly reduced. EEIO inhibited the MAPK（ERK, P38 and JNK）signaling pathway, significantly reducing the levels of p-ERK/ERK, p-p38/p38 and p-JNK/JNK. Meanwhile, the pyroptosis pathway was inhibited, and the levels of ASC, Caspase-1, GSDMD and NLRP3 significantly reduced. The positive drug resveratrol（RES）had a similar effect on various detection indicators as EEIO, but its effect was not as good as EEIO. 【Conclusion】 EEIO regulates the MAPK signaling pathway, inhibits the pyroptosis pathway, and reduces the expression of inflammatory factors, thus alleviating LPS-induced intestinal injury.

Key words: ethanol extract of Inonotus obliquus, lipopolysaccharides, intestinal injury, inflammation, pyroptosis

马文澳, 杨微, 李迎春, 朱彦彬, 陈志宝, 刘娜. 桦褐孔菌乙醇提取物对脂多糖诱导肠损伤的缓解作用及机制[J]. 生物技术通报, 2024, 40(12): 299-308.

MA Wen-ao, YANG Wei, LI Ying-chun, ZHU Yan-bin, CHEN Zhi-bao, LIU Na. Alleviating Roles and Mechanisms of Ethanol Extract from Inonotus obliquus to Intestinal Injury Induced by Lipopolysaccharide[J]. Biotechnology Bulletin, 2024, 40(12): 299-308.

图/表 8

表1 基因名称和引物序列

Table 1 Gene names and primer sequences

基因Gene	引物序列Primer sequence（5'-3'）
IL-1β	F:CACTACAGGCTCCGAGATGAACAAC R:TGTCGTTGCTTGGTTCTCCTTGTAC
IL-18	F:ACAGGCCTGACATCTTCTGC R:ATTGTTCCTGGGCCAAGAGG
Caspase-1	F:TGCCCAGAGCACAAGACTTC
Caspase-1	R:TCCTTGTTTCTCTCCACGGC
GSDMD	F:ATGGCATGGCTTACACCACC
GSDMD	R:ATGGCATGGCTTACACCACC
NLRP3	F:GCCGTCTACGTCTTCTTCCTTTCC
NLRP3	R:CATCCGCAGCCAGTGAACAGAG
β-actin	F:TATGCTCTCCCTCACGCCATCC
β-actin	R:GTCACGCACGATTTCCCTCTCAG

图1 EEIO调节LPS诱导MODE-K细胞损伤时细胞存活率和LDH活性的表达与CON相比，*P<0.05、**P<0.01；与LPS相比，#P<0.05、##P<0.01，下同

Fig. 1 EEIO regulates the expressions of cell viability and LDH activity in LPS-induced MODE-K cell injury Compared with CON, * P< 0.05, ** P< 0.01. Compared with LPS, #P< 0.05, ##P< 0.01, the same below

图2 EEIO调节LPS诱导MODE-K细胞时NO、IL-1β和IL-18的表达

Fig. 2 EEIO regulates the expressions of NO, IL-1β and IL-18 in LPS-induced MODE-K cells

图3 EEIO调节LPS诱导MODE-K细胞时MAPK通路蛋白的表达

Fig. 3 EEIO regulates the expressions of MAPK pathway proteins in LPS-induced MODE-K cells

图4 EEIO调节LPS诱导MODE-K细胞时焦亡的表达

Fig. 4 EEIO regulates the expressions of the pyroptosis pathway during LPS-induced MODE-K cells

图5 EEIO调节LPS诱导小鼠时LDH释放及NO、IL-1β和IL-18的表达

Fig. 5 EEIO regulates LDH release and expression of NO, IL-1β and IL-18 in LPS-induced mice

图6 EEIO调节LPS诱导小鼠时肠组织MAPK通路蛋白的表达

Fig. 6 EEIO regulates the expressions of MAPK pathway proteins in intestinal tissues in LPS-induced mice

图7 EEIO调节LPS诱导小鼠时肠组织焦亡的表达

Fig. 7 EEIO regulates the expressions of the pyroptosis in intestinal tissues in LPS-induced mice

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