| IBD inflammatory bowl disease(IBD)is a chronic,recurrent inflammatory disease of unknown etiology.Its pathogenesis may be related to genetic,environmental,immune and gut microbes,which combine to result in immune abnormalities and impaired mucosal barrier function.The immune response of IBD is closely related to T lymphocytes,B lymphocytes,and cytokines produced by immune response.At present,therapeutic drugs for IBD are still under development.Glycyrrhiza uralensis Fisch.is a traditional Chinese herbal medicine with low toxic side effects.It has pharmacological activities such as anti-inflammatory,immunomodulating,and antifibrotic and is considered a potential immunomodulator.Licorice extracts such as glycyrrhizic acid and flavonoids have been used in the researches of IBD.Glycyrrhiza uralensis Fisch.is the most widely distributed licorice in China,with the highest content of flavonoids and special components such as ural glycyrrhizin,which has certain research value.In this study,a TNBS one-time enema method was used to create a rat colitis model.Glycyrrhiza uralensis Fisch.extract is then administered by gavage.The therapeutic effect and mechanism of Ural licorice extract were investigated through using methods involving clinical and pathological observation,flow cytometry,transcriptome sequencing and quantitative PCR.Ural liquorice extract was provided by the School of Pharmacy of Shihezi University.The TOF-MS test results showed that the content of glycyrrhizin and glycyrrhizic acid in the extract met the requirements of the Pharmacopoeia of the People’s Republic of China.This study is divided into three parts.Part Ⅰ: the efficacy evaluation of Glycyrrhiza uralensis Fisch.extract in colitis.A total of 144 male SD rats(SPF grade)were divided into 6 groups,includes control group(K group),TNBS-induced group(M group),high-dose group of Glycyrrhiza uralensis Fisch.extract groups(G group,200 mg/kg),medium-dose group(Z group,100 mg/kg),low-dose group(D group,50 mg/kg),and positive control group(sulfasalazine,Y group).Except for the K group,the remaining 5 groups of rats were modeled with TNBS one-time enema on day 8.Gastric gavage administration was started on the fourth day of modeling for7 consecutive days.During the whole feeding period,rats in the control group were active,with shiny coats,normal growth in feeding and weight,and normal fecal properties.The rats with colitis appeared sauce-red liquid on the day of modeling,diarrhea and bloody stool appeared on the second day,and the rats had reduced mobility,dull coat color,decreased appetite,decreased weight,and increased DAI score.After administration,rats began to gradually recover,with more activity than those in the M group.They have increased appetite,glossy coat color and gradually recovered body weight,and decreased DAI score.In the M group,the colon of rats had edema,hyperemia and eye-visible ulcers.And an increased colonic weight and length shortening of colons were observed.Besides,the CMDI score is increased.After administration,the degree of lesions was reduced,and the CMDI score was reduced.General observation of the colon showed that TNBS segmented injection was beneficial to create a more stable colitis model.The results of HE and Masson stains showed that the rats in the M group had severe epithelial injury in the colon.We observed epithelial structural disorders and a large number of inflammatory cell infiltrates,and a large number of collagen fibers appear at the ulcer site.The pathological changes are reduced after administration,and fibrotic deposition is reduced.At the same time,rats with colitis had obvious spleen congestion and the expanded germinal center of lymphoid follicles,and the spleen lesions were relieved after administration.Preliminary results showed that TNBS enema caused colonic epithelial damage,resulting in an immune response in rats,which was relieved after administration.It is suggested that Glycyrrhiza uralensis Fisch.extract can alleviate colitis in rats,which may be due to the effect of immune response on colitis in rats.Part Ⅱ: the effect of Glycyrrhiza uralensis Fisch.extract on the number of immune cells in rats with colitis.Routine blood tests results show an increase in the number of white blood cells and platelets,a decrease in the number of red blood cells,and these changes were reversed after administration.The flow cytometry results showed that the blood of rats with colitis had a decrease in T lymphocytes,B lymphocytes and NK cells,and an increase in macrophages.The proportion of T,B,NK and macrophages in the spleen and colon was increased.The immune system of the rats with IBD was unbalanced,resulting in different changes of T lymphocytes,B lymphocytes,NK cells and macrophages in the peripheral blood,spleen and colon.Glycyrrhiza uralensis Fisch.extract can reduce the inflammatory response by influencing changes in immune-related cells and reducing immune cell imbalance.Then the MPO content were detected by Elisa.The MPO in the M group was higher than that in the K group,and it was reduced after administration,indicating that neutrophil activation in serum in colitis rats,and administration could reduce neutrophil activation.Part Ⅲ: the cellular immune balance mechanism of Glycyrrhiza uralensis Fisch.extract on TNBS-induced colitis rats.The transcriptome results showed that the Glycyrrhiza uralensis Fisch.extract was less toxic.There are 608 genes of rats in the model group were changed compared with those in the control group.After the administration of Glycyrrhiza uralensis Fisch.extract,440 genes were reversed which account for 70% of the total differential genes.And 439 genes were identified as genes that function as Glycyrrhiza uralensis Fisch.GO and KEGG enrichment analysis showed that Glycyrrhiza uralensis Fisch.extrac has a good regulatory effect on the related function of differential genes in IBD rats and the biochemical metabolic pathways and signal transduction pathways.The immune-related pathways of licorice involved TNF and NF-kappa B signaling pathways,in which IL-6,IL-β and Nod2 received corresponding regulation.The m RNA expression of Nod2,RIP2,TNF-α,NF-κB,IKK,IL-β and IL-6 in the colon was measured by q PCR.The results showed that the expression of m RNA of Nod2,RIP2,TNF-α,NF-κB,IKK,IL-β and IL-6 increased in the colonic tissues of colitis rats,and the expression of genes could be down-regulated after administration.The resultes of Nod2,IL-β and IL-6 were consistent with those of the reference transcriptome.It suggests that Glycyrrhiza uralensis Fisch.extract may alleviate colitis by regulating the Nod2 gene to act on the TNF/ NF-κB signaling pathway.In summary,Glycyrrhiza uralensis Fisch.extract has a therapeutic effect on the clinical symptoms and specific pathological changes of colitis in IBD rats,and 100mg/kg is the optimal dose.The study shows that Glycyrrhiza uralensis Fisch extract can act on cellular immune balance,affecting the activation of T,B,NK,macrophages and neutrophils in systemic immunity and local immunity,and its mechanism of action may be related to the Nod2/ NF-κB /TNF signaling pathway. |
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