Activity And Mechanism Of Koumine On Intestinal Porcine Epithelial Cells

Gelsemium elegans Benth is a kind of evergreen vine belongs to the genus Gelsemium.,As well know to us,the plant has the effect of anticancer,sedative,anti-inflammatory and other effects.It can promote the growth and rejuvenation of animals,and has the development potential in animal husbandry.Objective:in the study of the growth promoting effect of Gelsemium,it was found that Gelsemium extract can play its role by affecting the intestinal activity in piglets.In this experiment,Koumine was used as the research drug,intestinal porcine epithelial cells（IPEC-J2）was used as the research object to explore the regulative effect and related mechanism of Gelsemium on intestinal physiological state of pigs.To provide theoretical basis for the development of Gelsemium as feed additive.Methods:To explore the relationship between Koumine and physiological state of pig intestinal epithelial cells by molecular biology experiment.MTT method,q PCR,flow cytometry,wound healing,WB was used to detect the effect of gelsemin on the expression of intestinal function related genes,cell migration rate,intracellular calcium concentration and expression of calcium-transporter proteins in ipec-j2,Results:12.5-100μM Koumine could enhance the activity of IPEC-J2 cells,which was significantly different from that of the control group（P<0.05）.Compared with the control group,200μM Koumine could up regulate the gene expression of histamine receptor（H1HR）in IPEC-J2,with a significant difference（P<0.01）;100μM Koumine and 200μM Koumine could up regulate the gene expression of 5-HT4R,with a significant difference（P<0.01）;Koumine had no significant effect on the gene expression of Muscarinic Receptor M3（CHRM3）.50μM,100μM and 200μM Koumine could increase the intracellular Ca²⁺concentration of IPEC-J2,which was significantly different from that of the control group（P<0.01）.At 6h,12h and 24h after the start of the cell scratch experiment,the wound healing degree of the experimental group was higher than that of the control group,and the cell migration rate of the experimental group was higher than that of the control group.After acting on IPEC-J2,the expression of calbindin protein was up-regulated by Koumine.Compared with the control group,there was a significant difference in the 100μM dose group（P<0.05）,and there was a significant difference in the 200μM dose group（P<0.01）;50,100μM Koumine significantly upregulated the expression of IP3R protein（P<0.05）,and 200μM Koumine significantly upregulated the expression of IP3R protein（P<0.01）;50,100,200μM Koumine could significantly up regulate the expression of TRPV4（P<0.05）.Conclusion:Koumine can increase the expression of H1HR and 5-HT4R genes in IPEC-J2,and affect the intestinal mucosal repair,electrolyte transport and neurotransmitter by regulating the expression of H1HR and 5-HT4R in intestinal epithelium.Koumine can promote the migration of IPEC-J2 cells and maintain the integrity of intestinal physical barrier.Koumine increased the concentration of free Ca²⁺in IPEC-J2 and upregulated the expression of calbindin,IP3R and TRPV4.It can regulate Ca²⁺transport and absorption,activate voltage dependent Ca²⁺channel in intestinal epithelium,activate intracellular Ca²⁺release channel,and release Ca²⁺and extracellular Ca²⁺influx from sarcoplasmic reticulum.So as to affect the intestinal movement and absorption capacity of intestinal wall.This study can provide a way to explore the effect of Koumine on porcine small intestinal epithelial cells and related mechanisms.