Indirubin's Anti-lipopolysaccharide-induced Inflammation Of Human Colonic Epithelial Cells And Its Mechanism

Object: The purpose of this study was to investigate the effects of indirubin on lipopolysaccharide(LPS)-induced human colonic epithelial cells(HCoEpiCs).Changes in the expression levels of pro-inflammatory cytokines and anti-inflammatory cytokines were detected to clarify the anti-inflammatory effect of indirubin and explore the anti-inflammatory mechanism and action target of indirubin.Material and method: In this study,we stimulate HCoEpiCs with lipopolysaccharide,then the concentration of lipopolysaccharide was determined accord to the expression levels of IL-6 to ensure that the model is built successfully,next we determine the concentration of indirubin which we need by MTT assay.Cells were randomly distributed in six groups :control group,model group,indirubin groups: high dose group(1?mol/L)and middle dose group(0.5?mol/L)and low dose group(0.25?mol/L)and combined group(0.5?mol/L+ PPM-18).The levels of TNF-? and IL-12 and IL-4 were measured by ELISA and the protein levels of PI3 K and AKT and p-AKT and NF-?B were measured by Western Blot,and the mRNA levels NF-?B was measured by QPCR.Results: 1.Comparison of cell growth at different timesThe OD mean value of three groups(12H and 24 H and 48H)were compared.Compared with the 12 H group,the mean value of OD of the 24 H group was significantly increased(P < 0.01),compared with the 12 H group,the mean value of OD of the 48 H group was increased(P < 0.05),the mean value of OD of the 24 H group showed no significant difference with 48 H group(P>0.05).2.The level of IL-6 with different concentration of LPSCompared with the control group,the level of IL-6 was significantly increased in the other groups(P < 0.01),compared with the 5?g/ml group the level of IL-6 has significantly increased in 10?g/ml group and 15?g/ml group(P < 0.01),Compared with the 10?g/ml group,the level of IL-6 of 15?g/ml group has significantly increased(P < 0.01).3.The survival rate of HCoEpiC with different concentrations of indirubinCompared with the control group,the cell survival rate of LPS group and 1?mol/L group decreased significantly(P < 0.01),compared with the control group the groups of 0.5?mol/L and 0.25?mol/L and 0.125?mol/L without significant difference(P > 0.05).Compared with the LPS group,the cell survival rate increased in the 1?mol/L group(P < 0.05).Compared with the LPS group,the survival rate of cells in the groups of 0.5 ?mol/L and 0.25 ?mol/L and 0.125?mol/L increased significantly(P < 0.01).Compared with the 1?mol/L group,the survival rate of cells in the 0.5?mol/L group increased(P < 0.05).Compared with the 1?mol/L group,the survival rate of cells in the 0.25?mol/L group and 0.125mol/L group increased significantly(P < 0.01).There was no significant difference in cell survival among the groups of 0.5?mol/L and 0.25?mol/L and 0.125?mol/L(P > 0.05).4.The survival rate of LPS-induced HCoEpiC with different concentrations of indirubinCompared with the normal group,the cell survival rate of the other groups decreased significantly(P<0.01).Compared with the LPS group,there was no significant difference between 25?mol/L group and 5?mol/L group(P > 0.05).Compared with the LPS group,the survival rate of cells of 1?mol/L group,0.5 ?mol/L group,0.25?mol/L group and 0.125?mol/L group increased significantly(P < 0.01).Compared with 25?mol/L group,there was no significant difference between 5 ?mol/L group and 1?mol/L group(P > 0.05).Compared with 25?mol/L group,the cell survival rate of 0.5?mol/L group and 0.25?mol/L group increased significantly(P < 0.01).Compared with the 25?mol/L group,the survival rate of cells in the 0.125?mol/L group increased(P < 0.05).Compared with the 5?mol/L group the survival rate of cells increased in the 1?mol/L and 0.25?mol/L group(P < 0.05).Compared with the 5 ?mol/L group,the survival rate of cells in the 0.5 ?mol/L group and 0.25 ?mol/L group increased significantly(P < 0.01).There was no significant difference among 1 ?mol/L group,0.5?mol/L group,0.25?mol/L group and 0.125?mol/L group(P > 0.05).5.The level of TNF-? in different groupsCompared with the control group,the level of TNF-? was significantly increased in other groups(P < 0.01).Compared with the model group,the level of TNF-? in high dose group medium dose group,low dose group and combined group were significantly decreased(P < 0.01).The expression of TNF-? was significantly different among high dose group,medium dose group and low dose group(P < 0.01),and the expression of medium dose group was the lowest.There was no significant difference between medium dose group and combined group(P > 0.05).Compared with the low dose group the level of TNF-? was significantly decreased in medium dose group and the combined group(P < 0.01).6.The level of IL-12 in different groupsCompared with the control group,the level of IL-12 was significantly increased in other groups(P < 0.01).Compared with the model group,the level of IL-12 in the high dose group,medium dose group,low dose group and combined group were significantly decreased(P < 0.01).Compared with the high dose group,the level of IL-12 in medium dose group decreased(P < 0.05),and the level of IL-12 was significantly increased in low dose group and combined group(P < 0.01).Compared with the medium dose group,the level of IL-12 in low dose group increased significantly(P < 0.01),and the level of IL-12 in combined group was significantly decreased(P < 0.01).Compared with the low dose group,the level of IL-12 was significantly decreased in combined group(P < 0.01).7.The level of IL-4 in different groupsCompared with the control group,the level of IL-4 was significantly decreased in other groups(P < 0.01).Compared with the model group,the level of IL-4 in the high and medium and low dose group and the combined group were significantly increased(P < 0.01).There was no significant difference between high dose group and medium dose group(P > 0.05),and the level of IL-4 was significantly decreased in the low dose group and combined indirubin group(P < 0.01).Compared with medium dose group,the level of IL-4 in low dose group was significantly decreased(P < 0.01).Compared with the high dose group,the level of IL-4 in low dose group and combined group were significantly decreased(P < 0.01).Compared with combined group,there was no difference between medium dose group and low dose group(P > 0.05).8.The level of NF-?B protein in different groupsCompared with the control group,the levels of NF-?B protein in the model group and high dose group,medium dose group and low dose group were significantly increased(P < 0.01),and the level of NF-?B protein in combined group was increased(P < 0.05).Compared with the model group,the level of NF-?B protein in the high dose group,medium dose group and combined group were significantly decreased(P < 0.01),while there was no difference between low dose group and model group(P > 0.05).Compared with the high dose group,the level of NF-?B protein in low dose group was increased(P < 0.05),and the level of NF-?B protein in combined group was significantly decreased(P < 0.01).There was no difference between medium dose group and high dose group(P > 0.05).Compared with the high dose group,the level of NF-?B protein was decreased in combined group(P < 0.05).Compared with the low dose group,the level of NF-?B protein were significantly decreased in high dose group and combined group(P < 0.01).9.The level of NF-?B mRNA in different groupsCompared with the control group,the levels of NF-?B mRNA in model group,high dose group,medium dose group and low dose group were significantly increased(P < 0.01),and the level of NF-?B mRNA in combined group were increased(P < 0.05).Compared with the model group,the level of NF-?B mRNA in high dose group,medium dose group,low dose group and combined group were significantly decreased(P < 0.01).Compared with the high dose group,the level of NF-?B mRNA of low-dose group was significantly increased(P < 0.01).There was no significant difference among high and medium and combined groups(P>0.05).Compared with the low dose indirubin group,the level of NF-?B mRNA were significantly decreased in the high dose group and the combined group(P < 0.01).10.The level of PI3 K protein in different groupsCompared with the control group,the level of PI3 K protein was significantly increased in other groups(P < 0.01).Compared with the model group,the level of PI3 K protein of high dose group,medium dose group and combined group were significantly decreased(P < 0.01),while there was no difference between low dose group and the model group(P>0.05).Compared with the high dose group,PI3 K protein expression was significantly increased in the low dose group(P < 0.01).Compared with the high dose group,there was no difference between medium dose group and combined group(P > 0.05).Compared with medium dose group,PI3 K protein expression in low dose group increased significantly(P < 0.01),while there was no difference between medium dose group and indirubin combined group(P > 0.05).Compared with the low dose group,the PI3 K protein expression in combined group was significantly decreased(P < 0.01).11.The level of p-AKT protein in different groupsThere was no difference among all the groups(P > 0.05).12.The level of AKT protein in different groupsCompared with the control group,the level of p-AKT protein in model group,high dose group,medium dose group and low dose group were significantly increased(P < 0.01),and the level of p-AKT protein in combined group was increased(P < 0.05).Compared with the model group,p-AKT protein expression was significantly decreased in the high dose group,medium dose group and combined group(p < 0.01),while p-AKT protein expression was decreased in low dose group and the model group(p < 0.05).Compared with the high dose group,the level of p-AKT protein expression in the low dose group was significantly increased(p < 0.01),while the level of p-AKT protein expression in combined group was significantly decreased(p < 0.01).There was no difference between the high dose group and the medium dose group(p > 0.05).Compared with the medium dose group,the level of p-AKT protein was significantly increased in the low dose group(P < 0.01),while the level of p-AKT protein expression was significantly decreased in combined group(P < 0.01).Compared with the low dose group,the level of p-AKT protein expression was significantly decreased in combined group(P < 0.01).Conclusion: 1.LPS induced HCoEpiCs could establish UC modelStimulate HCoEpiC cells by LPS can induce inflammation and release pro-inflammatory cytokines,we can simulate inflammation of human colon tissue by this way.2.Indirubin affects the release of various inflammatory factorsIndirubin could reduce the expression of pro-inflammatory cytokines TNF-?,IL-12 and IL-6,and increase the expression of anti-inflammatory cytokine IL-4,so it could treat UC from anti-inflammatory and immune regulation aspects.In this study,the combined group's IL-12 release was significantly reduced due to the decreased activity of the NF-?B signaling pathway,suggesting that the expression of IL-12 may be more correlated with the NF-?B signaling pathway,and the regulation of the expression of TNF-? and IL-4 by indirubin may also be related to other signaling pathways.3.Safety and efficacy of indirubinThe appropriate concentration of indirubin has minimal effect on cell activity,and indirubin can also reduce the release of pro-inflammatory cytokine IL-6,so indirubin has high safety and obvious therapeutic effect.4.Indirubin inhibits the activity of NF-?B signaling pathway and controls the development of inflammationNF-?B signaling pathway is related to inflammation closely,this test results show that indirubin has good anti-inflammatory effects,the treatment group compared with model group can effectively restrain the activity of NF-?B signaling pathway,reduce the release of downstream pro-inflammatory factors to treat UC.5.Indirubin inhibits the activity of PI3K/AKT signaling pathway and reduce canceration ratePI3K/AKT signaling pathway has a direct relationship with UC and the occurrence of cancer.The results of this study showed that compared with the model group,all treatment groups can effectively inhibit the activity of PI3K/AKT signaling pathway,thus inhibiting PI3 K phosphorylation and down-regulating the activation of AKT.Therefore,indirubin may inhibit the activity of PI3K/AKT signaling pathway to reducing the probability of UC canceration.6.Indirubin can act on multiple therapeutic targetsIndirubin can simultaneously inhibit the activity of PI3K/AKT and NF-?B signaling pathways,and inhibit the up-regulation of PI3K/AKT signaling pathway to inhibit NF-?B signaling pathway,at the same time,it can reduce the reactivation of PI3K/AKT signaling pathway by proinflammatory cytokines produced by activation of NF-?B signaling pathway,thus playing an anti-inflammatory role and preventing UC canceration.