TXM-CB13 Improves the Intestinal Mucosal Barrier and Alleviates Colitis by Inhibiting the ROS/TXNIP/TRX/NLRP3 and TLR4/MyD88/NF-κB/NLRP3 Pathways

Abstract

The activation of inflammasomes (NLRP3 and NLRP1) is central to the pathogenesis of inflammatory bowel disease (IBD). Here we examined the protective effects of a thioredoxin-mimetic peptide CB13 (TXM-CB13), known for its antioxidative stress and anti-inflammatory properties. We examined the effects of TXM-CB13 on dextran sulfate sodium (DSS)-induced colitis and lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation in RAW264.7 macrophages. TXM-CB13 appeared to alleviate symptoms of DSS-induced colitis and to significantly suppress the protein and mRNA levels of NLRP3, Mlck, and IL-1β in colonic tissues. Additionally, TXM-CB13 treatment increased the levels of the intestinal barrier proteins Occludin, ZO-1, and NLRP1, as shown through immunohistochemistry and Western blot analysis. In vitro, TXM-CB13 inhibited LPS-induced TLR4 signaling, reducing MyD88 levels and consequently attenuating the activation of the NF-κB pathways, including p-IκB-α/IκB-α and p-NF-κB-p65/NF-κB-p65. This inhibition further reduced the activation of the NLRP3 inflammasome components, NLRP3, ASC, Caspase-1, GSDMD, and IL-1β. In addition, TXM-CB13 prevented the ROS-mediated dissociation of TXNIP from TRX, inhibiting NLRP3 activation. These findings suggest that TXM-CB13 is a potential therapeutic candidate for IBD through its modulation of the TLR4/MyD88/NF-κB/NLRP3 and ROS/TXNIP/TRX/NLRP3 pathways.

Keywords: Inflammatory bowel disease; NF-κB; NLRP3; TXM-CB13; TXNIP.

Fig. 1

TXM-CB13 treatment alleviates DSS-induced colitis in mice. A The chemical structure of TXM-CB13 (Acetyl-Cys-Met-Lys-Cys-amide). TXM-CB13, derived from the canonical- C-x-x-C- motif of the TRX-active site, called the thioredoxin mimetic (TXM) peptide, reversed inflammatory and oxidative stress. B Experimental design of acute DSS-induced colitis. The mice were divided into three groups:, water-SPSS, DSS-SPSS, and DSS-TXM-CB13. Drug administration was 11 for days, and animals euthanized on the 12th day. C) Mouse weight loss was recorded daily from the 1 to11th day. The average body weight in each group was calculated and compared with that in the DSS-SPSS group, the average loss of body weight was significantly greater after nine days in the DSS-TXM-CB13 group. The values are the means ± SEMs of six mice per group, Student’s t test *p < 0.05. D, E Changes in colon length. A representative colon from each group on the 12th day is shown. D The length of the colon was measured, and the average length was analyzed. E The change in colon length was significantly larger in the TXM-CB13-treated group. The data are expressed as the means ± SEMs of six mice per group, Student’s t test (***p < 0.001). F Representative images of colon tissue stained with H&E. Compared with DSS-SPSS, TXM-CB13 suppressed the pathological progression of colon tissue, the in glandular structure, reduction in goblet cell and in neutrophil infiltration. The upper set of images was photographed by a low-power microscope (Bar, 200 µm). The lower set of images is the enlargement of the tissues in the box of the above group (100 µm). (n = 6 per group). G Histological injury scores were calculated from inflammatory cell infiltration and tissue damage data. The values are the means ± SEMs of three mice per group Student’s t test; (****p < 0.0001)

Fig. 2

TXM-CB13 reduced NLRP3 inflammasome activation, the expression of NLRP3 inflammasome-related factors, and the secretion of IL-1β in colonic tissues from DSS-induced mice. A–E The mRNA expression levels of NLRP3, ASC, Caspase-1, GSDMD, and IL-1βin DSS-induced colon tissues were detected via qRT‒PCR. The results revealed that the mRNA expression levels of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β were significantly inhibited after TXM-CB13 treatment. The values are the means ± SEMs of six mice per group Student’s t test; ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05. F–K Densitometric analysis was used to quantify the protein expression levels of NLRP3 inflammasome activation-related proteins in colon tissue.). The values are the means ± SEMs of six mice per group. Student’s t test; ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05. M, N A Western blot analysis detecting the expression levels of NLRP3, ASC, procaspase-1, Caspase-1 (p20), GSDMD, Pro-IL-1β, and IL-1β (p17) in DSS-induced colon tissues treated with TXM-CB13. TXM-CB13 significantly decreased the activation and expression of NLRP3, the expression of ASC, Caspase-1 (p20), and IL-1β (p17); and increased the expression of Occludin. (n = 6 per group). O Immunohistochemistry detecting the expression of NLRP3, IL-1β, and Occludin in colon tissue sections. Expression of NLRP3 and IL-1β was inhibited by TXM-CB13 and Occludin. ZO-1 expression increased by TXM-CB13 (bars: 100 μm, n = 6 per group)

Fig. 3

TXM-CB13 promoted the expression of NLRP1 in colon tissue and RAW264.7 cells. A, B Densitometric analysis of NLRP1 expression levels in colon tissues. (A) The values are presented as the means ± SEMs of six mice per group (Student’s t test), and of macrophages. B Values are shown as the mean ± SEM of three experiments (Student’s t test). (****p < 0.0001 and **p < 0.01). C Western blot analysis of NLRP1 protein expression levels in LPS-induced RAW264.7 macrophages. NLRP1 after TXM-CB13 treatment (upper image). Western blot analysis of NLRP1 protein expression levels in DSS-induced colon tissues (lower image). D Immunohistochemistry was used to detect NLRP1 expression in colon tissue. The upper set of images was photographed with a low-power microscope (Bar, 200 µm). The lower set of images shows the enlargement of the tissues in the box of the above group (100 µm, n = 6 per group)

Fig. 4

TXM-CB13 inhibited the expression of IL-1β and IL-18 in LPS-induced RAW264.7 macrophages. A Densitometric analysis of IL-1β (p17) and Pro-IL-1β in the LPS-induced RAW264.7 macrophages. The values are shown as the means ± SEMs of three experiments. Student’s t test; (**p < 0.01). B, D qRT‒PCR was used to detect the expression levels of IL-1β and IL-18 mRNAs in LPS-induced RAW264.7 macrophages treated with TXM-CB13. The values are shown as the means ± SEMs of three experiments. Student’s t test (***p < 0.001, and ****p < 0.0001). C Western blotting of IL-1β (p17) and Pro-IL-1β expression levels in LPS-induced RAW264.7 macrophages treated with TXM-CB13

Fig. 5

TXM-CB13 reduced NLRP3 inflammasome activation by modulating the TLR4/MyD88/NF-κB pathway in LPS-induced RAW264.7 macrophages and THP-1 cells. A-E Densitometric analysis of the expression levels of proteins related to NLRP3 inflammasome activation in RAW264.7 macrophages. Testing the expression of NLRP3, ASC, caspase-1 (p20)/ pro-caspase-1, GSDMD-N/GSDMD, and Il-β (p17)/pro-IL-1β. (*p < 0.05, **p < 0.01 with or without TXMCB13. The values are shown as the means ± SEMs of three experiments Student’s t test; (***p < 0.001, and ****p < 0.0001). F Western blotting of the protein expression levels of NLRP3, ASC, pro-caspase-1, Caspase-1 (p20), GSDMD, GSDMD-N, Pro-IL-1β, and IL-1β (p17) in LPS-induced RAW264.7 macrophages treated with TXM-CB13. G-J Densitometric analysis of the expression levels of proteins associated with the TLR4/MyD88/NF-κB signaling pathway in RAW264.7 macrophages. The expression of TLR4, MyD88, IκB/pIκB, and p-NF-κB (p65)/NF-κB (p65). The values are shown as the means ± SEMs of three experiments Student’s t test; (*p < 0.05, **p < 0.01, and ****p < 0.0001). K Western blotting of TLR4, MyD88, IκB, pIκB, NF-κB (p65), and p-NF-κB (p65) expression in LPS-induced RAW264.7 macrophages treated with TXM-CB13. L Western blotting was used to detect NLRP3, Myd88, and IL-1β expression in LPS-induced THP-1 cells. M‒O Densitometric analysis of the protein expression levels of NLRP3, MyD88 and IL-1β. The values are shown as the means ± SEMs of three experiments. Student’s t test; (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Fig. 6

TXM-CB13 decreased the production of ROS induced by LPS and blocked the separation of TXNIP from TRX and the activation of NLRP3 inflammatory bodies. A, B ROS content in LPS-induced RAW264.7 macrophages after TXM-CB13 treatment (Bar, 200 µm). C The ROS content TXM-CB13 in LPS-induced RAW264.7 macrophages. The values are shown as the means ± SEMs of three experiments Student’s t test; (**p < 0.01, and ****p < 0.0001). D, E qRT‒PCR of mRNA expression levels of TRX and TXNIP in LPS-induced RAW264.7 macrophages treated with TXM-CB13. The values are shown as the means ± SEMs of three experiments. Student’s t test **p < 0.01, ***p < 0.001, and ****p < 0.0001). F, G Densitometric analysis of protein expression levels of TRX and TXNIP.. The values are shown as the means ± SEMs of three experiments. Student’s t test; (**p < 0.01, ***p < 0.001, and ****p < 0.0001). H Western of protein expression levels of TXNIP and TRX in LPS-induced RAW264.7 macrophages treated with TXM-CB13