Study On The Effect And Mechanism Of Eriocalyxin B On TNBS-induced Colitis In Mice

Objective: To study the effect and mechanism of Erocalyxin B(Eri B)on 2,4,6-trinitrobenzene sulfonic acid(TNBS)induced colitis in mice.Method: 1.C57BL/6 mice aged 6-8 weeks were selected as the study subjects.The experimental settings were normal control group(Control),model group(TNBS),low dose group of Eri B(5 mg/kg),medium dose group of Eri B(10 mg/kg),high dose group of Eri B(20 mg/kg)and 5-aminosalicylic acid(5-ASA)groups,with 8mice in each group.The model mice were constructed by injecting 2.5% TNBS ethanol solution into the anus.Normal control group and model group were given normal saline for 7 days;Eri B low,middle and high dose groups were given Eri B5 mg/kg,10 mg/kg and 20 mg/kg,respectively,for 7 consecutive days.The disease activity index was calculated by observing and recording the weight change,blood in stool and diarrhea of mice in each group.The length of mouse colon was measured by ruler.The inflammatory score of each group was calculated by HE staining results of mouse colon sections.The expression level of IL-6,TNF-α and IL-1β in colon tissues was detected by ELISA.The ratio of M1 macrophage and M2 macrophage in the lamina propria of mouse mucosa was detected by flow cytometry.The m RNA levels of M1 macrophag markers(i NOS,CD86),M2 macrophage markers(CD206,Arg1)and intestinal barrier associated proteins(ZO-1,occludin,claudin-1)were detected by q RTPCR.2.The effect of Eri B on the viability of BMDMs was detected by CCK-8 assay.LPS and IFN-γ were used to induce M1 macrophage;IL-4 was used to induce M2 macrophage.The effect of Eri B on the polarization of BMDMs was detected by flow cytometry;The m RNA levels of M1 macrophage markers(i NOS,CD86)and M2 macrophage markers(CD206,Arg1)were detected by q RT-PCR.The expression level of IL-6,TNF-α,IL-1β,IL-10 and TGF-β in supernatants was detected by ELISA.3.Molecular docking was used to verify the docking of Eri B and JAK2.Western blotting was used to detect the effect of Eri B on the M1 polarization signal pathway JAK2/STAT1 and its phosphorylated protein of BMDMs.Western blotting was used to detect the expression of JAK2/STAT1 and its phosphorylated protein in mice colon tissues.Result: 1.Compared with TNBS group,Eri B(10,20 mg/kg)and 5-ASA(50 mg/kg)alleviated weight loss,DAI score,colon shortening and pathological damage in mice,and made the expression of inflammatory factors IL-6,TNF-α and IL-1β decreased.Flow cytometry showed that Eri B(10,20 mg/kg)and 5-ASA(50 mg/kg)treatment inhibited the proportion of M1 macrophages in the lamina propria of the colon,but had no effect on the proportion of M2 macrophages.The results of q RT-PCR showed that Eri B(10,20mg/kg)and 5-ASA(50 mg/kg)downregulated the expression of M1 macrophage markers(i NOS and CD86),but had no effect on the expression of M2 macrophage markers(CD206,Arg1).In addition,q RT-PCR results showed that Eri B treatment had no effect on the expression of intestinal barrier associated proteins(ZO-1,occludin,claudin-1).2.CCK-8 showed Eri B(2.5,5,10 μM)had no toxicity to BMDMs.The flow cytometry results show that Eri B(5,10 μM)Treatment inhibited the polarization of BMDMs to M1 type macrophages,but had no significant effect on the polarization of BMDMs to M2 type macrophages.The results of q RT-PCR showed that Eri B(5,10 μM)downregulated the expression of M1 macrophage markers(i NOS and CD86),but had no effect on the expression of M2 macrophage markers(CD206,Arg1).ELISA results showed that Eri B(5,10 μM)inhibited the secretion of M1 macrophage related cytokines(IL-6,TNF-α and IL-1β)from BMDMs while had no effect on M2 macrophage related cytokines(IL-10 and TGF-β).3.Molecular docking results showed that Eri B formed hydrogen bonds with the four amino acid residues of JAK2(LYS-607,GLU-791,ASP-789,and TYR-790).Western blotting results showed that Eri B can reduce the phosphorylation of JAK2 and STAT1.Conclusion: Eri B can alleviate TNBS-induced colitis by inhibiting the polarization of macrophage in colon tissue,which may be related to Eri B’s antagonism to JAK2/STAT1 signal pathway.