Protective Mechanism Of Chlorogenic Acid On LPS-induced Intestinal Epithelial Injury Of Mouse

The dynamic balance between stem cell self-renewal and differentiation of progenitor cells at the bottom of the intestinal crypt is fundamental in maintaining the stability of the intestinal epithelial structure.Lineage tracing on mouse revealed that intestinal stem cells and progenitor cells having significant plasticity and make intestinal epithelial regeneration after injury.Chlorogenic acid(CGA)has been of great interest as a most promising plant-derived functional food ingredient.Based on cell and animal models,CGA has protective effects on damage of intestine(cell),but the mechanism is still unclear.In this study,LPS induced intestinal damage in mice and intestinal organoids were investigated by in vivo and in vitro models,using qPCR,Elisa,flow cytometry,stem cell in vitro 3D culture and other techniques to explore the CGA in the intestine The role of stem cells in intestinal epithelial regeneration under injury conditions revealed that CGA protects intestinal epithelium by regulating intestinal stem cell function.The main findings are as follows:(1)Compared with the control,CGA alleviated body weight loss in mice at the apoptotic phase(control group/CGA group lost 1.0/0.6 g,respectively)and shortened intestinal length(33.0 ± 0.9/36.0 ± 0.3 cm),the small intestine villus height decreased(712 ± 9/737 ± 18 ?m);promoted the increase of small intestine villus height(control group/CGA group: 665 ± 21/864 ± 20 ?m).In addition to body weight and intestinal structure,compared with control mice,CGA reduced the release of pro-inflammatory factors and expression in intestinal tissues,including IFN?(control group/CGA group: 121.6 ± 12.5/104.1 ± 7.6 pg/mL),IL-1?(692.3 ± 45.8/639.1 ± 40.4 pg/mL),IL-6(70686 ± 16421/52696 ± 14271 pg/mL)in serum and TNF?(21.65 ± 5.27/12.41 ± 1.13)in intestinal tissues.Moreover,CGA reduced the activation of apoptotic genes by reducing the expression of pro-apoptotic gene Bcl2ll1(control group/CGA group: 3.97 ± 0.73/1.60 ± 0.38)and the apoptotic gene Caspase-3(control group/CGA group: 6.07 ± 1.14/4.15 ± 0.87).The above results indicate that CGA may promote the activation of stem cells in the apoptotic phase by promoting LPS,the expression of active stem cells in the proliferative phase and alleviate intestinal damage.(2)Analysis of intestinal epithelial cell lineage showed that CGA promoted the expression of intestinal active stem cells in the intestinal apoptosis phase of LPS-induced intestinal injury in mice compared with the control group(control group/CGA group Lgr5 expression: 0.43 ± 0.19/0.019 ± 0.004;Olfm4 expression: 3.32 ± 0.59/2.19 ± 0.48),but significantly upregulated reserve stem cell expression(Bmi1 expression: 5.52 ± 2.69/8.59 ± 2.79).In the intestinal proliferation phase,CGA promoted the expression of intestinal active stem cells(Lgr5 expression in the control group/CGA group: 1.22 ± 0.07/2.62 ± 0.40;Olfm4: 0.775 ± 0.125/1.30 ± 0.11).Furthermore,flow cytometry analysis and EdU method analysis of the results of intestinal stem cell composition in the proliferative phase confirmed that CGA significantly increased the number of Lgr5 positive cells in the proliferative phase compared with the control mice(control group/CGA group: 8.92%/17.33%),the number of EdU positive cells in a single crypt(6.1 ± 0.4/11.0 ± 0.5),while reduced the migration distance of stem cells(71.4 ± 4.8/52.4 ± 3.3 ?m).The above results indicate that CGA may accelerate the loss of active stem cells and alleviate intestinal damage caused by the activation of reserve stem cells during the induction of apoptosis by LPS.(3)Analysis of intestinal tissue in the apoptotic and proliferative phases of LPS-induced intestinal injury showed that CGA significantly enhanced JAK signaling pathway gene JAK1 expression(control group/CGA group apoptosis phase: 1.37 ± 0.66/3.66 ± 0.38;proliferation phase: 1.57 ± 0.26/2.17 ± 0.12),and analysis based on intestinal organoids also showed that CGA enhanced LPS-induced JAK1 expression(control group /0.1 mM CGA/0.5 mM chlorogenic acid): 0.77 ± 0.04/1.93 ± 0.13 / 2.12 ± 0.12).Similar to the results in mice,CGA also enhanced the expression of intestinal active stem cells in the organoids of the LPS-induced proliferative phase(control group/CGA group: 0.83 ± 0.05/1.50 ± 0.22),but this effect was blocked by the JAK signaling pathway protein inhibitor Tofacitinib and showed a decrease in the expression of active stem cells(CGA group/inhibitor group: 1.50 ± 0.22/0.89 ± 0.14).The above results indicate that CGA activates the function of the intestinal stem cell bank by enhancing JAK signaling pathway activation.In conclusion,the change of stem cell function caused by CGA additive can be considered as an important parameter for the protection of intestinal damage,and the regulation of stem cell function by CGA is regulated by JAK pathway.The clarification of the mechanism provides a theoretical basis for the in-depth development of CGA in the food and pharmaceutical fields,and provides new ideas for the study of the efficacy of food active substances.