Effect Of Geniposide On NF-?B/IAP In LPS-Induced Intestinal Damage In Mice

In recent years,people have paid more and more attention to the effect of natural food substances or Chinese herbal medicines as feed additives on intestinal health.Gardenoside is the main medicinal active ingredient extracted from gardenia,which has the effect of antagonizing lipopolysaccharide.In view of this situation,this study carried out the detection of related indexes of geniposide on intestinal inflammation in mice and explored the effect of geniposide as a feed additive on intestinal health.Pro vide a theoretical basis to prevent intestinal diseases such as intestinal mucosal damage.50 male healthy ICR(22-25g)mice were randomly divided into 5 groups.The pre-feeding period is one week.The mice were free to eat and drink during the test period.The mice in the control group were injected with physiological saline,while those test groups with LPS concentrations of 2.5,5,10,and 20 mg/kg respectively.Blood samples were collected from the eyeballs after 6 h,and serum was used to detect(IL)-1?,IL-4,IL-6,IL-8,TNF-?,IAP levels by ELISA.At the same time,specimens of duodenum,jejunum and ileum were collected for histological section with HE staining,and the height of villi and the depth of crypt were measured.In the second part,35 male healthy ICR mice were similar weight and randomly divided into 7 groups with 5 mice in each group.The mice were killed after injiected 0,6,12,24,48,72 h,and specimens of duodenum,jejunum and ileum were collected for the detection of IAP,NF-?B P65 mRNA and protein expression and co-immunoprecipitation detection.After building a model of LPS-induced enteritis,25 male healthy ICR mice were similar weight and randomly divided into 5 groups with 5 mice in each group.The mice in the control group were fed normally without LPS and geniposide treatment.The mice in the experimental group were intraperitoneally injected with 5 mg/kg LPS.After 6 hours,intragastric administration of different concentrations of geniposide and the concentration of geniposide in this study was 0,0.25,0.5,1 g/mL.The mice were killed,the duodenum,jejunum,and ileum were collected for the detection of IAP,NF-?B P65 mRNA and protein expression by RT-qPCR and Western blot.The main results were as follows:1)Effects of different LPS concentrations on immune function in miceThe results of the serum test showed that the IAP,(IL)-1?,IL-4,IL-6,IL-8,TNF-? levels reached the maximums at a LPS concentration of 5 mg/kg,with no further increase with the increasing LPS concentration2)Effects of different LPS concentrations on tissue morphology in miceAfter the abdominal cavity of the control group was opened,the shape was normal and there was no bleeding.The mice treated with 5 mg/kg LPS had obvious bleeding symptoms after dissection,and the intestine was damaged or swollen.The results of villi height and crypt depth showed that compared with the control group,the ratio of villi height,villi height and crypt depth in duodenum,jejunum and ileum of mice decreased with increasing LPS concentration.The depth of the crypt gradually increased with each concentration gradient3)Effects of different LPS induction time on intestinal IAP and NF-?B P65 mRNAThe results of duodenum,jejunum,and ileum are as follows:compared with the control group,the IAP mRNA gradually increases with time.The relative expression of IAP mRNA reached a peak at 6 h,and then gradually decreased with time delay.Compared with the control group,the relative expression of NF-?B P65 mRNA reached a peak when the inflammation time of LPS was 24 h(p<0.05).The results of duodenum,jejunum,and ileum are as follows:compared with the control group,the IAP mRNA gradually increases with time.The relative expression of IAP mRNA reached a peak at 6 h,and then gradually decreased with time delay.Compared with the control group,the relative expression of NF-?B P65 mRNA reached a peak when the inflammation time of LPS was 24 h4)Effects of different LPS induction time on small intestine IAP and NF-?B P65 proteinThe relative expression levels of IAP and NF-?B P65 protein in the intestine of mice in the experimental group reached a peak at the 6th hour of LPS injection,and they all declined to some extent with time,until they returned to normal levels at 72 h5)Interaction between IAP and NF-?B in mouse ileum induced by LPSImmunoprecipitation results showed that NF-?B P65 protein was detected in IAP immunoprecipitation.IAP protein was also detected in the immunoprecipitation of NF-?B P656)Effects of different concentrations of geniposide on IAP and NF-?B P65 mRNA in small intestine of inflamed miceCompared with the control group,intragastric administration of geniposide 1 g/mL can inhibit the relative expression of IAP and NF-?B P65 mRNA in the duodenum,jejunum,and ileum of mice7)Effects of different concentrations of geniposide on IAP and NF-?B P65 protein in small intestine of inflammatory miceCompared with the control group,the relative expression of IAP and NF-?B P65 protein in the small intestine was the lowest by gavage of geniposide lg/mL.As the concentration decreased,the relative expression of IAP and NF-?B P65 proteins in the small intestine increasedIn summary,when mice are injected with 5 mg/kg LPS,they can induce intestinal mucosal damage,VH value of each segment was reduced,CD value was increased,and the concentration of each cytokine reaches the highest,can be used as a suitable induction dose for subsequent inflammatory bowel disease;When LPS is used to induce inflammation,IAP and NF-?B P65 interact in the ileum of mice,Indicates that the two proteins are related.1 g/ml geniposide can be used as a suitable concentration for future research on geniposide on LPS-induced intestinal injury.