Alleviating Roles and Mechanisms of Ethanol Extract from Inonotus obliquus to Intestinal Injury Induced by Lipopolysaccharide

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

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

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

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

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

References 38

[1]	Viladomiu M, Metz ML, Lima SF, et al. Adherent-invasive E. coli metabolism of propanediol in Crohn's disease regulates phagocytes to drive intestinal inflammation[J]. Cell Host Microbe, 2021, 29(4): 607-619.e8. doi: 10.1016/j.chom.2021.01.002 pmid: 33539767
[2]	Rathinam VAK, Zhao Y, Shao F. Innate immunity to intracellular LPS[J]. Nat Immunol, 2019, 20: 527-533. doi: 10.1038/s41590-019-0368-3 pmid: 30962589
[3]	Ciesielska A, Matyjek M, Kwiatkowska K. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling[J]. Cell Mol Life Sci, 2021, 78(4): 1233-1261. doi: 10.1007/s00018-020-03656-y pmid: 33057840
[4]	Cui Y, Qu YY, Yin K, et al. Selenomethionine ameliorates LPS-induced intestinal immune dysfunction in chicken jejunum[J]. Metallomics, 2021, 13(3): mfab003.
[5]	Deitch EA. The role of intestinal barrier failure and bacterial translocation in the development of systemic infection and multiple organ failure[J]. Arch Surg, 1990, 125(3): 403-404.
[6]	常轶聪. 二氢杨梅素通过调节ROS/NLRP3炎症小体干预LPS诱导鸡肠损伤的机制研究[D]. 哈尔滨: 东北农业大学, 2021.
	Chang YC. Mechanism of dihydromyricetin intervening LPS-induced intestinal injury in chickens by regulating ROS/NLRP3 inflammatory corpuscles[D]. Harbin:Northeast Agricultural University, 2021.
[7]	马伟平, 侯睿. 桦褐孔菌抗肿瘤作用的研究进展[J]. 医学研究生学报, 2017, 30(4): 440-443.
	Ma WP, Hou R. Research progress of inonotus obliquus in anti tumor[J]. J Med Postgrad, 2017, 30(4): 440-443.
[8]	王蔚, 周忠光, 刘旭, 等. 桦褐孔菌醇提物对几种消化系统恶性肿瘤的影响[J]. 中医药信息, 2018, 35(1): 12-15.
	WangW/Y, Zhou ZG, Liu X, et al. Effects of alcohol extracts of Inonotus obliquus on several malignant tumors of digestive system[J]. Inf Tradit Chin Med, 2018, 35(1): 12-15.
[9]	曾海. 桦褐孔菌对2型糖尿病患者硝化酪氨酸和血红素加氧酶-1水平的影响[D]. 延吉: 延边大学, 2014.
	Zeng H. Effect of Inonotus obliquus on the levels of nitrotyrosine and heme oxygenase-1 in patients with type 2 diabetes mellitus[D]. Yanji: Yanbian University, 2014.
[10]	延光海, 金光玉, 李良昌, 等. 桦褐孔菌乙醇提取物在小鼠哮喘模型中对p38MAPK信号通路的影响[J]. 中国中药杂志, 2011, 36(8): 1067-1070.
	Yan GH, Jin GY, Li LC, et al. Protective effects and mechanism of Inonotus obliquus on asthmatic mice[J]. China J Chin Mater Med, 2011, 36(8): 1067-1070.
[11]	李建华. 桦褐孔菌的药理作用研究进展[J]. 科学技术创新, 2019,(36):45-46.
	Li JH. Research progress on the pharmacological effects of Betula brownii fungus[J]. Science and Technology Innovation, 2019(36): 45-46.
[12]	陈盛宇, 田缘, 马俊秀, 等. 桦褐孔菌多糖研究现状与展望[J]. 食品研究与开发, 2022, 43(22): 215-224.
	Chen SY, Tian Y, Ma JX, et al. Research status and prospects of polysaccharides from Inonotus obliquus[J]. Food Res Dev, 2022, 43(22): 215-224.
[13]	Zhao YX, Zheng WF. Deciphering the antitumoral potential of the bioactive metabolites from medicinal mushroom Inonotus obliquus[J]. J Ethnopharmacol, 2021, 265: 113321.
[14]	李毅, 李建宽, 杨红, 等. 桦褐孔菌提取物对阿托伐他汀致小鼠肝损伤的保护作用[J]. 中国兽医杂志, 2024, 60(2): 18-26.
	Li Y, Li JK, Yang H, et al. Protective effect of Fusarium obliquus extract on atorvastatin induced liver injury in mice[J]. Chinese Journal of Veterinary Medicine, 2019, 60(2): 18-26.
[15]	乔羽, 项楠, 周忠光, 等. 桦褐孔菌醇提物对胃癌模型裸鼠和BGC-823胃癌细胞Ras/MAPK信号通路的影响[J]. 中医药信息, 2023, 40(11): 7-12.
	Qiao Y, Xiang N, Zhou ZG, et al. Effects of ethanol extract from Inonotus obliquus on ras/MAPK signaling pathway of gastric cancer model nude mice and BGC-823 gastric cancer cells[J]. Inf Tradit Chin Med, 2023, 40(11): 7-12.
[16]	杨微, 陈志宝, 陈操, 等. 桦褐孔菌乙醇粗提物对朊病毒复制的抑制作用[J]. 现代食品科技, 2021, 37(7): 8-13, 7.
	Yang W, Chen ZB, Chen C, et al. Inhibition on prion replication by ethanol crude extracts from Inonotus obliquus[J]. Mod Food Sci Technol, 2021, 37(7): 8-13, 7.
[17]	张松. 内毒素对畜禽机体产生的影响[J]. 畜牧兽医科学, 2022,(1): 124-125.
	Zhang S. The effects of endotoxins on the body of livestock and poultry[J]. Animal Husbandry and Veterinary Science, 2022,(1): 124-125.
[18]	Kesavardhana S, Malireddi RKS, Kanneganti TD. Caspases in cell death, inflammation, and pyroptosis[J]. Annu Rev Immunol, 2020, 38: 567-595. doi: 10.1146/annurev-immunol-073119-095439 pmid: 32017655
[19]	Ronkina N, Gaestel M. MAPK-activated protein kinases: servant or partner?[J]. Annu Rev Biochem, 2022, 91: 505-540. doi: 10.1146/annurev-biochem-081720-114505 pmid: 35303787
[20]	Fu YJ, Xu B, Huang SW, et al. Mechanistic insights into the attenuation of intestinal inflammation and modulation of the gut microbiome by krill oil using in vitro and in vivo models[J]. Acta Pharmacol Sin, 2021, 42(1): 88-96.
[21]	Liu F, Smith AD, Solano-Aguilar G, et al. Mechanistic insights into the attenuation of intestinal inflammation and modulation of the gut microbiome by krill oil using in vitro and in vivo models[J]. Microbiome, 2020, 8(1): 83.
[22]	Fan JJ, Liu ST, Ai ZY, et al. Fermented ginseng attenuates lipopolysaccharide-induced inflammatory responses by activating the TLR4/MAPK signaling pathway and remediating gut barrier[J]. Food Funct, 2021, 12(2): 852-861. doi: 10.1039/d0fo02404j pmid: 33404578
[23]	Liao XT, Zhang WK, Dai HJ, et al. Neutrophil-derived IL-17 promotes ventilator-induced lung injury via p38 MAPK/MCP-1 pathway activation[J]. Front Immunol, 2021, 12: 768813.
[24]	Kumar S, Boehm J, Lee JC. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases[J]. Nat Rev Drug Discov, 2003, 2(9): 717-726. doi: 10.1038/nrd1177 pmid: 12951578
[25]	Izadparast F, Riahi-Zajani B, Yarmohammadi F, et al. Protective effect of berberine against LPS-induced injury in the intestine: a review[J]. Cell Cycle, 2022, 21(22): 2365-2378.
[26]	曹文娟. 沙眼衣原体pORF5质粒蛋白通过激活NALP3炎性复合体诱导THP-1细胞分泌IL-1β和IL-18[D]. 衡阳: 南华大学, 2014.
	Cao WJ. Chlamydia trachomatis pORF5 plasmid protein induces THP-1 cells to secrete IL-1β and IL-18 by activating NALP3 inflammatory complex[D]. Hengyang: University of South China, 2014.
[27]	郑学森. IL-18通过共生菌调控肠道免疫系统稳态作用和机制的探究[D]. 合肥: 中国科学技术大学, 2021.
	Zheng XS. Study on the role and mechanism of IL-18 in regulating intestinal immune system homeostasis through symbiotic bacteria[D]. Hefei: University of Science and Technology of China, 2021.
[28]	Ramli FF, Ali A, Ibrahim N. Molecular-signaling pathways of ginsenosides Rb in myocardial ischemia-reperfusion injury: a mini review[J]. Int J Med Sci, 2022, 19(1): 65-73. doi: 10.7150/ijms.64984 pmid: 34975299
[29]	崔贺铭. 桦褐孔菌醇提物的成分分析及其对大鼠心肌缺血再灌注损伤的保护作用研究[D]. 长春: 吉林大学, 2022.
	Cui HM. Composition analysis of alcohol extract from Inonotus obliquus and its protective effect on myocardial ischemia-reperfusion injury in rats[D]. Changchun: Jilin University, 2022.
[30]	Wold CW, Gerwick WH, Wangensteen H, et al. Bioactive triterpenoids and water-soluble melanin from Inonotus obliquus(Chaga)with immunomodulatory activity[J]. J Funct Foods, 2020, 71: 104025.
[31]	Kou RW, Han R, Gao YQ, et al. Anti-neuroinflammatory polyoxygenated lanostanoids from Chaga mushroom Inonotus obliquus[J]. Phytochemistry, 2021, 184: 112647.
[32]	Yan KX, Zhou HY, Wang M, et al. Inhibitory effects of Inonotus obliquus polysaccharide on inflammatory response in Toxoplasma gondii- infected RAW_264.7 macrophages[J]. Evid Based Complement Alternat Med, 2021, 2021: 2245496.
[33]	肖昆. 桦褐孔菌提取物缓解溃疡性结肠炎作用及机制研究[D]. 无锡: 江南大学, 2022.
	Xiao K. Study on the effect and mechanism of Inonotus obliquus extract in relieving ulcerative colitis[D]. Wuxi: Jiangnan University, 2022.
[34]	Kim J, Lee HJ, Park SK, et al. Donepezil regulates LPS and aβ-stimulated neuroinflammation through MAPK/NLRP3 inflammasome/STAT3 signaling[J]. Int J Mol Sci, 2021, 22(19): 10637.
[35]	Yu P, Zhang X, Liu N, et al. Pyroptosis: mechanisms and diseases[J]. Signal Transduct Target Ther, 2021, 6(1): 128.
[36]	赵冠宇, 辛蕊华, 仇正英, 等. 基于NLRP3/Caspase-1/GSDMD信号通路研究乌梅丸对溃疡性结肠炎小鼠结肠上皮细胞焦亡的作用机制[J]. 中草药, 2023, 54(24): 8086-8093.
	Zhao GY, Xin RH, Qiu ZY, et al. Mechanism of Wumei Wan on colonic epithelial cell pyroptosis in mice with ulcerative colitis based on NLRP3/Caspase-1/GSDMD signaling pathway[J]. Chin Tradit Herb Drugs, 2023, 54(24): 8086-8093.
[37]	Xu YT, Tang XH, Fang AN, et al. HucMSC-Ex carrying miR-203a-3p.2 ameliorates colitis through the suppression of caspase11/4-induced macrophage pyroptosis[J]. Int Immunopharmacol, 2022, 110: 108925.
[38]	Wang YP, Gao WQ, Shi XY, et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin[J]. Nature, 2017, 547(7661): 99-103.