Construction of mouse intestinal organoid inflammation model

doi:10.11958/20231511

Abstract

Abstract:

Objective To establish in vitro the small intestinal organoid culture system and to investigate the effect of lipopolysaccharide (LPS) on the growth of small intestinal organoids and the secretion of inflammatory factors. Methods In vitro, the small intestinal crypt cell mass of C57BL/6 mice was aseptically isolated, collected and embedded in organoid matrix. Under the support of complete medium, the small intestinal organoids with three-dimensional multi-leaf structure with small intestinal epithelioid structure were formed. The small intestinal organoids were subcultured after 5-7 d culture. On the third day after passage, the small intestinal organoids were randomly divided into different mass concentrations of LPS groups (0, 150, 175, 200, 225, 250, 275 and 300 mg/L). After 24 h and 48 h of LPS induction, morphological changes of small intestinal organoid growth and differentiation were observed. CCK-8 method was used to detect the effect of different time points and mass concentrations of LPS on the proliferative activity of small intestinal organoids after induction of inflammation. The effects of four different mass concentrations of LPS (0, 175, 200 and 225 mg/L) on expression levels of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6 and IL-10 in organoid culture supernatant at different times were detected by enzyme-linked immunosorbent assay (ELISA). Results The mouse small intestinal organoid culture system was preliminarily constructed. After different time and mass concentration of LPS induced inflammation of small intestinal organoids, it was observed by morphology that small intestinal organoids would have different degrees of expansion and apoptosis in lumen. The proliferation, differentiation and budding of damaged intestinal epithelial crypts or intestinal stem cells were also inhibited to varying degrees, indicating that the growth of small intestinal organoids would be limited to varying degrees after induced inflammation. The proliferation activity of small intestinal organoids decreased to varying degrees after 24 h and 48 h of LPS induction at 175-225 mg/L (P<0.05), but the cell viability was still greater than 50%. The levels of IL-1α, IL-6 and GM-CSF partially increased after induction with 200 mg/L and 225 mg/L LPS for 24 h and 48 h (P<0.05). The level of IL-10 decreased after induction with 200 mg/L LPS for 24 h and 48 h (P<0.05). Conclusion In this study, a model of intestinal inflammatory injury in vitro induced by LPS with different mass concentrations and time points is preliminarily constructed, which provides a more reliable research platform for the mechanism research of intestinal diseases and the screening of effective drugs in the future.

Key words: intestine, small, organoids, lipopolysaccharides, biological factors, crypt

CLC Number:

R574.5

Figures/Tables 4

Fig.1 Changes of growth morphology of small intestinal organs in mice after primary and post-passage

Fig.2 Effects of different time points and concentrations of LPS on the growth and differentiation of intestinal organoids

Fig.3 Effects of different time points and concentrations of LPS on the proliferation activity of small intestinal organoids

Fig.4 The expression of inflammatory factors in small intestinal organoids treated by four concentrations of LPS

References 22

[1]	GARRETA E, KAMM R D, CHUVA DE SOUSA LOPES S M, et al. Rethinking organoid technology through bioengineering[J]. Nat Mater, 2021, 20(2):145-155. doi:10.1038/s41563-020-00804-4.
[2]	HOU Q, HUANG J, AYANSOLA H, et al. Intestinal stem cells and immune cell relationships: potential therapeutic targets for inflammatory bowel diseases[J]. Front Immunol, 2020, 11:623691. doi:10.3389/fimmu.2020.623691.
[3]	HAN Y, YANG L, LACKO L A, et al. Human organoid models to study SARS-CoV-2 infection[J]. Nat Methods, 2022, 19(4):418-428. doi:10.1038/s41592-022-01453.
[4]	ZHANG X, MA Z, SONG E, et al. Islet organoid as a promising model for diabetes[J]. Protein Cell, 2022, 13(4):239-257. doi:10.1007/s13238-021-00831-0.
[5]	MARTINEZ-GURYN K, HUBERT N, FRAZIER K, et al. Small intestine microbiota regulate host digestive and absorptive adaptive responses to dietary lipids[J]. Cell Host Microbe, 2018, 23(4):458-469.e5. doi:10.1016/j.chom.2018.03.011.
[6]	NEAL J T, LI X, ZHU J, et al. Organoid modeling of the tumor immune microenvironment[J]. Cell, 2018, 175(7):1972-1988. doi:10.1016/j.cell.2018.11.021.
[7]	LANCASTER M A, KNOBLICH J A. Organogenesis in a dish:modeling development and disease using organoid technologies[J]. Science, 2014, 345(6194):1247125. doi:10.1126/science.1247125.
[8]	ANDREWS M G, KRIEGSTEIN A R. Challenges of organoid research[J]. Annu Rev Neurosci, 2022, 45:23-39. doi:10.1146/annurev-neuro-111020-090812.
[9]	PRIOR N, INACIO P, HUCH M. Liver organoids:from basic research to therapeutic applications[J]. Gut, 2019, 68(12):2228-2237. doi:10.1136/gutjnl-2019-319256.
[10]	HUANG S, ZHANG S, CHEN L, et al. Lipopolysaccharide induced intestinal epithelial injury: a novel organoids-based model for sepsis in vitro[J]. Chin Med J (Engl), 2022, 135(18):2232-2239. doi:10.1097/CM9.0000000000002348.
[11]	余应嘉, 叶淑芳, 邓燕芳, 等. 大黄素甲醚对LPS诱导肠上皮细胞损伤自噬与缝隙连接蛋白的作用影响[J/OL]. 中国医院药学杂志, 2023. http://kns.cnki.net/kcms/detail/42.1204.R.20230728.1723.006.html.
	YU Y J, YE S F, DENG Y F, et al. Effect of physcion on autophagy and gap junction protein in LPS-induced intestinal epithelial cell injury model[J/OL]. Chinese Journal of Hospital Pharmacy, 2023. http://kns.cnki.net/kcms/detail/42.1204.R.20230728.1723.006.html.
[12]	郭子涵. 吲哚-3-甲醇对肉兔生长发育和LPS诱导的小肠炎症的影响及机制[D]. 重庆: 西南大学, 2022.
	GUO Z H. Effects and mechanism of indole-3-carbinol on growth and development and LPS induced intestinal inflammation in rabbits[D]. Chongqing: Southwest University, 2022. doi:10.27684/d.cnki.gxndx.2022.000176.
[13]	OKAMURA T, HASHIMOTO Y, MAJIMA S, et al. Trans fatty acid intake induces intestinal inflammation and impaired glucose tolerance[J]. Front Immunol, 2021, 12:669672. doi:10.3389/fimmu.2021.669672.
[14]	SAHOO D K, BORCHERDING D C, CHANDRA L, et al. Differential transcriptomic profiles following stimulation with lipopolysaccharide in intestinal organoids from dogs with inflammatory bowel disease and intestinal mast cell tumor[J]. Cancers, 2022, 14(14):3525. doi:10.3390/cancers14143525.
[15]	PARK B S, LEE J O. Recognition of lipopolysaccharide pattern by TLR4 complexes[J]. Exp Mol Med, 2013, 45(12):e66. doi:10.1038/emm.2013.97.
[16]	ANDREWS C, MCLEAN M H, DURUM S K. Cytokine tuning of intestinal epithelial function[J]. Front Immunol, 2018, 9:1270. doi:10.3389/fimmu.2018.01270.
[17]	王稣嫱. 绿原酸对LPS诱导小鼠肠上皮损伤的保护机制研究[D]. 杭州: 浙江工商大学, 2020.
	WANG S Q. Protective mechanism of chlorogenic acid on LPS-induced intestinal epithelial injury of mouse[D]. Hangzhou: Zhejiang Gongshang University, 2020. doi:10.27462/d.cnki.ghzhc.2020.000452.
[18]	GOOD D W, GEORGE T, WATTS B A 3rd. Toll-like receptor 2 is required for LPS-induced Toll-like receptor 4 signaling and inhibition of ion transport in renal thick ascending limb[J]. J Biol Chem, 2012, 287(24):20208-20220. doi:10.1074/jbc.M111.336255.
[19]	FUKUDA M, MIZUTANI T, MOCHIZUKI W, et al. Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon[J]. Genes Dev, 2014, 28(16):1752-1757. doi:10.1101/gad.245233.114.
[20]	WATSON C L, MAHE M M, MÚNERA J, et al. An in vivo model of human small intestine using pluripotent stem cells[J]. Nat Med, 2014, 20(11):1310-1314. doi:10.1038/nm.3737.
[21]	GUO S, NIGHOT M, AL-SADI R, et al. Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR4 signal transduction pathway activation of FAK and MyD88[J]. J Immunol, 2015, 195(10):4999-5010. doi:10.4049/jimmunol.140259.
[22]	GVIRTZ R, OGEN-SHTERN N, COHEN G. Kinetic cytokine secretion profile of LPS-induced inflammation in the human skin organ culture[J]. Pharmaceutics, 2020, 12(4):299. doi:10.3390/pharmaceutics12040299.