Keratin 1 modulates intestinal barrier and immune response via kallikrein kinin system in ulcerative colitis

Abstract

BACKGROUND

External factors in ulcerative colitis (UC) exacerbate colonic epithelial permeability and inflammatory responses. Keratin 1 (KRT1) is crucial in regulating these alterations, but its specific role in the progression of UC remains to be fully elucidated.

AIM

To explore the role and mechanisms of KRT1 in the regulation of colonic epithelial permeability and inflammation in UC.

METHODS

A KRT1 antibody concentration gradient test, along with a dextran sulfate sodium (DSS)-induced animal model, was implemented to investigate the role of KRT1 in modulating the activation of the kallikrein kinin system (KKS) and the cleavage of bradykinin (BK)/high molecular weight kininogen (HK) in UC.

RESULTS

Treatment with KRT1 antibody in Caco-2 cells suppressed cell proliferation, induced apoptosis, reduced HK expression, and increased BK expression. It further downregulated intestinal barrier proteins, including occludin, zonula occludens-1, and claudin, and negatively impacted the coagulation factor XII. These changes led to enhanced activation of BK and HK cleavage, thereby intensifying KKS-mediated inflammation in UC. In the DSS-induced mouse model, administration of KRT1 antibody mitigated colonic injury, increased colon length, alleviated weight loss, and suppressed inflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor-α. It also facilitated repair of the intestinal barrier, reducing DSS-induced injury.

CONCLUSION

KRT1 inhibits BK expression, suppresses inflammatory cytokines, and enhances markers of intestinal barrier function, thus ameliorating colonic damage and maintaining barrier integrity. KRT1 is a viable therapeutic target for UC.

Key Words: Keratin 1; Ulcerative colitis; Kallikrein kinin system; Digestive system diseases; Bradykinin; High molecular weight kininogen; Intestinal barrier function; Inflammatory cytokines

Core Tip: Keratin 1 (KRT1) plays a protective role in ulcerative colitis by modulating the kallikrein kinin system and reducing inflammation. KRT1 enhances intestinal barrier function, inhibits inflammatory cytokines, and preserves colon integrity, positioning it as a potential therapeutic target for ulcerative colitis.

INTRODUCTION

Ulcerative colitis (UC) is a chronic inflammatory disorder primarily affecting the mucosal layer of the large intestine, beginning in the rectum[1-4]. The disease’s pathogenesis involves a complex interplay of genetic predisposition, environmental triggers, gut microbiome imbalances, and dysregulated immune responses[5-8]. Although these factors are well-established, a comprehensive understanding of UC’s pathophysiology remains elusive, which hinders the development of effective therapeutic strategies. The unclear nature of key components of UC’s immunopathogenesis poses significant challenges to advancing treatment options.

The kallikrein kinin system (KKS) is a multifaceted cascade of plasma proteins that plays a critical role in both physiological and pathological processes. This system comprises serine proteases, such as coagulation factor XII (FXIIα) and prekallikrein, along with high molecular weight kininogen (HK), an enzymatic cofactor that generates bioactive peptides like bradykinin (BK) and kallidin upon activation[9,10]. Activation of the KKS is triggered by the binding of FXIIα and HK to negatively charged surfaces, initiating enzymatic reactions that release these potent vasoactive peptides[11,12]. While traditionally known for its protective roles in cardiovascular and cerebrovascular health, including the blood pressure regulation, vascular permeability, and tissue homeostasis, the KKS also plays roles in inflammation, pain modulation, and tissue repair by facilitating the release of BK and kallidin via the kinin B1 and B2 receptors[13-15]. Notably, KKS activation is implicated in inflammatory conditions such as inflammatory bowel disease (IBD) and acute colitis, where it exacerbates inflammation and tissue damage[16,17]. Studies have demonstrated that intravenous HK injection elevates interleukin (IL)-1 levels in colon tissue and BK levels in plasma, highlighting its significance in IBD pathogenesis[18]. Furthermore, BK-induced activation of mitogen-activated protein kinases has been shown to elevate pro-inflammatory cytokines such as IL-6, IL-1β, IL-8, and tumor necrosis factor (TNF)-α[19-21]. Beyond its inflammatory role, the KKS contributes to cardiovascular stability and protection against ischemic injuries, underscoring its dual role as both a protective and pathogenic mediator[22]. Understanding the involvement of the KKS in inflammatory diseases like IBD provides critical insights into its physiological relevance and therapeutic potential.

Keratin 1 (KRT1) plays a role in regulating and counteracting the enhanced permeability and inflammatory response of colonic epithelial cells induced by external stimuli, as well as in restoring their motility[23,24]. The mechanism of action involve the modulation of zonula occludens-1 (ZO-1)[23,25,26]. The cellular localization of KRT1 appears to be contingent on its expression levels, though the specific molecular mechanisms involved remain unclear. Some studies suggest that KRT1 binds to HK at specific sites, thereby enhancing the membrane localization of HK’s and influencing the activation of prekallikrein[27-29].

Interestingly, KRT1 is downregulated in UC, though the underlying causes of this reduction remain inadequately understood[25,30]. In this study, we assessed the protective effect of KRT1 against UC-induced colonic damage employing a concentration gradient of KRT1 antibody in Caco-2 cells and an in vivo dextran sulfate sodium (DSS)-induced animal model. Additionally, this study explored the mechanisms by which KRT1 sustains colonic barrier integrity, providing new insights into its potential therapeutic applications.

MATERIALS AND METHODS

Cell culture

Dulbecco’s Modified Eagle Medium (DMEM) was selected for culturing Caco-2 cells due to its nutrient-rich composition, which supports their growth, proliferation, and differentiation into enterocyte-like models. DMEM provides essential nutrients, amino acids, vitamins, and high glucose levels to meet the energy demands of Caco-2 cells, thus promoting rapid growth and facilitating the formation of tight junctions and other functional characteristics. This medium is particularly suitable for studies on intestinal permeability and transport. Additionally, its widespread acceptance, cost-effectiveness, and compatibility with standard supplements ensure reliable and reproducible results, affirming it as the preferred medium for culturing this cell line[31,32]. Caco-2 cells (ATCC, United States) were cultured as monolayers in DMEM supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, United States) and 1% penicillin-streptomycin (Thermo Fisher Scientific, United States). The cultures were housed in a humidified incubator at 37 °C with 5% carbon dioxide (Thermo Fisher Scientific, 3111, United States). After five passages, Caco-2 cells adhered within 48 hours and entered the logarithmic growth phase, achieving 80%-90% confluence within 3-4 days post-passage. Once reaching 80%-90% confluence, cells were dissociated using a trypsin-ethylene diamine tetraacetic acid (EDTA) solution (Thermo Fisher Scientific, United States) and seeded into 12-well plates at a density of 5 × 10⁴ cells per well. Each well contained 1 mL of DMEM supplemented with 20% FBS, and the cells were incubated at 37 °C for 48 hours. Subsequently, the KRT1 antibody (Abcam, United Kingdom) was administered to the Caco-2 cells at concentrations of 1 ng/mL, 5 ng/mL, and 10 ng/mL for 24 hours. The control group was treated with an equal volume of phosphate-buffered saline (PBS). Cell proliferation and apoptosis were assessed, and additional analyses including immunofluorescence, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (RT-qPCR), and Western blotting were performed to assess the effects of KRT1 antibody treatment.

Animals and DSS-induced colitis model

A total of 18 eight-week-old healthy C57BL/6J mice (average weight: 21.84 ± 0.75 g) were acquired from Yunnan Best Biotechnology Co., Ltd. [license No. SCXK (Dian) K2020-0006]. The mice were accommodated in a controlled environment with a 12-hour light/dark cycle, a stable temperature of 22 ± 0.5 °C, and unrestricted access to a standard laboratory diet (Hunan SJA Laboratory Animal Co., Ltd., Changsha, Hunan Province, China) and water. All experiments were operated based on the guidelines approved by the Ethics Review Committee for Animal Experimentation of Kunming Medical University, Approval No. SCXK (Dian) K2020-0006, date of approval: February 9, 2021, and performed according to the Guiding Principles of the Care and Use of Animals approved by the American Physiological Society.

Following a 1-week acclimatization period, all mice were randomly divided into three groups: DSS group, treatment group, and control group, with 6 mice in each group and a 1:1 male-to-female ratio. Except for the control group, the other two groups were given 2.5% (weight/volume) DSS (MP Biomedicals, Santa Ana, United States) in drinking water for 8 consecutive days to induce colitis[33]. Meanwhile, mice in the treatment group received daily intraperitoneal injections of recombinant mouse KRT1 protein (P04264, Huamei Bio, Shanghai, China) at a dose of 15 μg/kg during the DSS treatment period. Mice in the control group were given sterile water and intraperitoneally injected with 1% bovine serum albumin solution (ST023, Beyotime, Shanghai, China). The DSS solution intake was consistent across all groups. During the study, body weight, stool consistency, and fecal occult blood were monitored daily, and the disease activity index (DAI) score was assessed based on the criteria outlined in Table 1[34]. After three rounds of intervention, no mice died, and on Day 45, the mice were euthanized, the colon length was measured, and the colons were longitudinally opened to collect tissues for further studies.

Table 1 Disease activity index scoring criteria.

Score	Weight loss	Fecal consistency	Fecal occult blood
0	None	Normal	Normal
1	1%-5%	-	-
2	5%-10%	Mucoid stools	Haemoccult +
3	10%-20%	-	-
4	> 20%	Diarrhea	Gross bleeding

Proliferation detection

The BeyoClick™ 5-ethynyl-2’-deoxyuridine (EdU)-594 cell proliferation detection kit (Beyotime, C0078S, China) was used to assess Caco-2 cell proliferation. Cells were cultured overnight into 6-well plates before drug treatments were applied. A 2 × EdU working solution was prepared and administered equally across wells to achieve a final concentration of 10 μM EdU. The cells were incubated with the EdU solution for 2 hours, followed by fixation with 1 mL of fixative for 15 minutes at room temperature. After three 5-minute washes with washing solution, cells were permeabilized at room temperature for 10-15 minutes. Post two more washes, cells were stained with 1 mL of 1 × Hoechst 33342 solution in the dark at room temperature for 10 minutes. Following three additional washes, cells were examined under fluorescence microscopy, displaying blue nuclei and red cytoplasm. Flow cytometry was then employed for further analysis of the labeled cells (Biosciences, C6 Plus, United States).

Apoptotic detection

Apoptosis in Caco-2 cells was detected using the annexin V-fluorescein 5-isothiocyanate (FITC) apoptosis detection kit (Beyotime, C1062S, China). Initially, adherent cells were washed with PBS and detached using trypsin-EDTA solution. The cell suspension was gently pipetted, and the trypsinized cells were incubated at room temperature briefly before centrifugation at 1000 g for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in PBS and counted. Next, 50000-100000 cells were prepared and subjected to a second centrifugation. After removing the supernatant, the pellet was gently resuspended in 195 μL of Annexin V-FITC binding solution, followed by the addition of 5 μL of Annexin V-FITC and 10 μL of propidium iodide staining solution. The mixture was incubated at room temperature (20-25 °C) in the dark for 10-20 minutes in an ice bath, wrapped in aluminum foil to protect from light. During incubation, cells were gently resuspended 2-3 times to ensure uniform staining.

Immunofluorescence

Paraffin-embedded colon tissue sections were initially deparaffinized by incubating in xylene over three 5-minute intervals. The sections were then rehydrated through a sequence of graded ethanol solutions (100%, 95%, and 70%) for 2 minutes at each concentration, followed by a 5-minute wash in PBS. Antigen retrieval involved heating the sections in a citrate-EDTA buffer (pH 6.0) within a boiling water bath for 10-15 minutes, followed by cooling to room temperature in PBS. To permeabilize the tissues, the sections were treated with 0.3% triton X-100 in PBS for 10 minutes at room temperature, then blocked with 5% goat serum in PBS for 30 minutes. After blocking, the sections were incubated overnight at 4 °C with the primary antibody, rabbit anti-kininogen (ERP6097, Abcam, United Kingdom, 1:200 in 1% goat serum/PBS). After three 5-minute washes in PBS, sections were exposed to the secondary antibody, Goat Anti-Rabbit IgG (HL)-Alexa Fluor 594 (RS3611-100, Immunoway, United States, 1:1000 in PBS), for 1 hour at room temperature in the dark. Following three additional washes in PBS, sections were mounted using an antifade mounting reagent containing 4’,6-diamidino-2-phenylindole for nuclear counterstaining and allowed to set for 5-10 minutes before analysis under a fluorescence microscope.

ELISA

BK activity was measured using the BK ELISA kit (Biological Engineering, China). For Caco-2 cells, the culture medium was transferred to a sterile tube, centrifuged at 1000 g for 20 minutes at 4 °C to remove debris, and the supernatant was collected. For colon tissue, pre-weighed tissue was rinsed with cold PBS, minced, and homogenized in a 1:9 PBS solution, then centrifuged at 5000 g for 5 minutes to collect the supernatant.

Prior to analysis, reagents and kit were equilibrated to room temperature for 30 minutes. A volume of 50 μL of both standards and samples were added in duplicate to a 96-well plate, followed by 50 μL of biotinylated anti-BK antibody. The plate was sealed and incubated at 37 °C for 45 minutes. The wells were washed four times with 350 μL of wash buffer, followed by the addition of 100 μL of horseradish peroxidase (HRP)-conjugated streptavidin to each well and incubated at 37 °C for 30 minutes. After four washes with 300 μL of wash buffer, 90 μL of chromogenic substrate was added to each well and incubate at 37 °C in the dark for 15 minutes. The reaction was terminated by adding 50 μL stop solution. Optical density was measured at 450 nm using a microplate reader within 5 minutes, and BK concentrations were calculated from a standard curve using the four-parameter model.

Western blot

Murine colonic tissues were homogenized on ice for 30 minutes in a lysis buffer containing 1% protease inhibitors (Sigma-Aldrich, United States). The protein concentrations were determined using a protein assay kit (Bio-Rad Inc, United States) according to the manufacturer’s instructions. Equal amounts of protein (typically 30 μg per lane) were loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and proteins were separated based on their molecular weight. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, United States) using a semi-dry transfer system at 100 V for 1 hour. The membrane was then blocked with 5% non-fat dry milk in tris-buffered saline (TBST) (with 0.1% tween-20) for 1 hour at room temperature to prevent non-specific binding. Following overnight incubation at 4 °C with primary antibodies: KRT1 (1: 2000, LifeSpan BioSciences, United States), Claudin-2 (1:500, Santa Cruz, TX, United States), occludin (1:1000, Santa Cruz, TX, United States), ZO-1 (1:1000, Santa Cruz, TX, United States), actin (1:5000, Santa Cruz, TX, United States). After washing the membrane with TBST (3 × 10 minutes), the membrane was incubated with HRP-conjugated secondary antibodies (1:5000, Santa Cruz, TX, United States) for 1 hour at room temperature. After three additional washes with TBST, protein bands were visualized using enhanced chemiluminescence substrate (Thermo Scientific, United States), and the signals were detected with a chemiluminescence imaging system (Bio-Rad, United States). The grayscale values of the protein bands were analyzed using ImageJ software. Actin served as a loading control, with protein expression levels normalized to Actin to ascertain relative protein levels in the samples.

RNA extraction and RT-qPCR

Total RNA was extracted from mouse colonic tissues using the TRIzol reagent (Thermo Fisher, United States) and employed as the template for cDNA synthesis using a single-strand cDNA synthesis kit (Takara Inc., China), adhering to the manufacturer’s protocol. A SYBR Green-based qPCR kit (Roche, Basel, Switzerland) was used to measure mRNA expression levels on a LightCycler^® 480 System (Roche Applied Science, Switzerland). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functioned as the internal control. The primers used were as follows: occludin, 5’- GACTTCAGGCAGCCTCGTTAC-3’ (F), 5’- GCCAGTTGTGTAGTCTGTCTCA-3’ (R); ZO-1, 5’-ACCAGTAAGTCGTCCTGATCC-3’ (F), 5’-TCGGCCAAATCTTCTCACTCC-3’ (R); Claudin, 5’-CCTCCTGGGAGTGATAGCAAT-3’ (F), 5’-GGCAACTAAAATAGCCAGACCT-3’ (R); and GAPDH, 5’-CTGGGCTACACTGAGCACC-3’ (F), 5’-AAGTGGTCGTTGAGGGCAATG-3’ (R)[35-38]. The qPCR reaction system consisted of 10 μL of 2X ChamQ SYBR color qPCR master mix, 0.8 μL of each forward and reverse primer, 10 μL of 50X ROX reference dye, 6 μL of double distilled water, and 2 μL of diluted cDNA. The RT-qPCR conditions were set as follows: Initial denaturation at 95 °C for 5 minutes, followed by 40 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 40 seconds[39]. The relative expression of target genes was calculated using the comparative computed tomography (CT) method (relative expression = 2^-ΔΔCT)[40].

Hematoxylin-eosin staining

Colon samples were fixed in 4% paraformaldehyde for 24 hours at room temperature, sequentially dehydrated in a graded ethanol series (70%, 80%, 90%, 100%) for 1 hour and cleared in xylene, each step lasting one hour. The samples were then embedded in paraffin, and paraffin blocks were sectioned into 5-μm thick slices using a microtome. The paraffin sections underwent deparaffinization in xylene (2 × 10 minutes) and rehydration in a descending ethanol (100%, 95%, 70%, 50%, 30%, each for 5 minutes). Staining involved hematoxylin solution for 2-5 minutes, followed by rinsing with running tap water for 5 minutes. The sections were then stained with eosin solution for 1-3 minutes. Following staining, the slides underwent dehydrated in graded ethanol (70%, 90%, 100%) for 5 minutes each, followed by clarification in xylene for 10 minutes. Finally, the sections were mounted using a neutral resin. Images of the hematoxylin-eosin (HE)-stained sections were captured randomly using brightfield microscopy. The entire staining process typically required approximately 2-3 hours.

Statistical analysis

Data analysis was performed using statistical product and service solutions 21.0 and GraphPad Prism 6.0. measurement data were presented as mean ± SEM. Homogeneity of variance was assessed, and where it was confirmed, one-way analysis of variance was employed for statistical analysis; otherwise, the Kruskal-Wallis test was used. Statistical significance was established at a P value < 0.05. For inter-group comparisons, ^aP value < 0.05, ^bP < 0.01 and ^cP < 0.001.

RESULTS

KRT1 antibody inhibits cell proliferation and promotes apoptosis in Caco-2 cells

The influence of KRT1 on intestinal epithelial cells was examined by assessing its effects on cellular proliferation and apoptosis using antagonist assays at concentrations of 1, 5, and 10 ng/mL. The results showed that the KRT1 antibody inhibited cellular proliferation in a dose-dependent manner, with greater suppression evident at higher concentrations (Figure 1A and B, P < 0.001). At the lower concentration of 1 ng/mL, there was no significant effect on cellular apoptosis. However, apoptotic levels significantly increased at higher antibody concentrations. Notably, when the KRT1 antibody concentration exceeded 10 ng/mL, a marked enhancement of cellular apoptosis was observed (Figure 1C, P < 0.001).

Figure 1 Effect of different concentrations of keratin 1 antibody on Caco-2 cell proliferation and apoptosis. A and B: Keratin 1 (KRT1) antibody inhibited cellular proliferation; C: KRT1 antibody induced apoptosis. ^cP < 0.001. P value vs control group. Control refers to untreated Caco-2 cells; Treatment 1: Keratin 1 (KRT1) antibody at 1 ng/mL; Treatment 2: KRT1 antibody at 5 ng/mL; Treatment 3: KRT1 antibody at 10 ng/mL. Same for subsequent figures.

KRT1 antibody promotes HK and BK expression in Caco-2 cells

To evaluate the effect of KRT1 on the expression of HK and BK in the KKS, the levels of HK and BK were assessed at various KRT1 antibody concentrations (1 ng/mL, 5 ng/mL, and 10 ng/mL) using immunofluorescence and ELISA. Immunofluorescence analysis revealed a reduction in HK expression in Caco-2 cells treated with the KRT1 antibody (Figure 2A). In contrast, ELISA analysis demonstrated that the KRT1 antibody significantly increased BK expression in Caco-2 cells (Figure 2B, P < 0.001), with this effect observed even at the lowest antibody concentration (1 ng/mL).

Figure 2 Effect of different concentrations of keratin 1 antibody on the expression levels of high molecular weight kininogen and bradykinin in Caco-2 cells. A: Immunofluorescence analysis shows a reduction in high molecular weight kininogen expression in Caco-2 cells treated with different concentrations of keratin 1 (KRT1) antibody; B: Enzyme-linked immunosorbent assay results demonstrate an increase in bradykinin expression in Caco-2 cells treated with various concentrations of KRT1 antibody. ^aP < 0.05. ^cP < 0.001. P value vs control group. Treatment 1: Keratin 1 (KRT1) antibody at 1 ng/mL; Treatment 2: KRT1 antibody at 5 ng/mL; Treatment 3: KRT1 antibody at 10 ng/mL. ELISA: Enzyme-linked immunosorbent assay.

KRT1 antibody inhibits the expression of intestinal epithelial cells and promotes the expression of intestinal barrier factor FXIIα in Caco-2 cells

The intestinal mechanical barrier plays a critical role in the pathophysiology of UC[41,42]. To assess the impact of the KRT1 antibody on genes associated with the intestinal mechanical barrier in Caco-2 cells, mRNA and protein expression levels were analyzed. The results showed that at a low concentration (1 ng/mL), the KRT1 antibody does not significantly impact the expression of marker genes for the intestinal mechanical barrier. However, at medium and high concentrations, the KRT1 antibody significantly reduced the mRNA and protein levels of occludin (Figure 3A-C, P < 0.001), ZO-1 (Figure 3C-E, P < 0.001), and claudin (Figure 3C, F and G, P < 0.001). Additionally, there was a marked increase in the expression of the negative regulator FXIIα (Figure 3C and H, P < 0.001).

Figure 3 Effects of different concentrations of the keratin 1 antibody on intestinal mechanical barrier marker genes in Caco-2 cells. A: Real-time polymerase chain reaction (RT-qPCR) results showed a reduction in the expression levels of occludin treated with varying concentrations of the keratin 1 (KRT1) antibody; B: Western blot quantification revealed decreased expression of occludin in Caco-2 cells treated with the KRT1 antibody; C: Western blot analysis of occludin, zonula occludens-1, claudin, and coagulation factor XII in Caco-2 cells under different treatment conditions; D: RT-qPCR results showed a reduction in the expression levels of zonula occludens-1 treated with varying concentrations of the KRT1 antibody; E: Western blot quantification revealed decreased expression of zonula occludens-1 in Caco-2 cells treated with the KRT1 antibody; F: RT-qPCR results showed a reduction in the expression levels of claudin in Caco-2 cells treated with varying concentrations of the KRT1 antibody; G: Western blot quantification revealed decreased expression of claudin in Caco-2 cells treated with the KRT1 antibody; H: Western blot quantification increased expression of coagulation factor XII, in Caco-2 cells treated with the KRT1 antibody. ^cP < 0.001. P value vs control group. Treatment 1: Keratin 1 (KRT1) antibody at 1 ng/mL; Treatment 2: KRT1 antibody at 5 ng/mL; Treatment 3: KRT1 antibody at 10 ng/mL. ZO-1: Zonula occludens-1; FXIIα: Coagulation factor XII; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

KRT1 protein blocks the inflammatory response in vivo

We assessed classic indicators of colitis using a mouse model of colitis, established by administering drinking water containing 5% DSS for 8 consecutive days. Compared to the control group, mice in the DSS group compared significant weight loss, which was notably alleviated by KRT1 treatment (Figure 4A-C, P < 0.001). The therapy also significantly reversed DSS-induced shortening of colon length (Figure 4D and E). The DAI, which quantifies the severity of intestinal inflammation based on weight loss, stool consistency, and rectal bleeding, was significantly higher in the DSS group but was substantially reduced following KRT1 treatment (Figure 4F). Further exploration of the anti-inflammatory effects of KRT1 protein in DSS-induced colitis showed significant reductions in BK levels and activity, leading to a marked decrease in pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, compared to the DSS group (Figure 4G-J, P < 0.001). The reduction in these cytokines helped alleviate the intestinal inflammatory response and promote the repair of colonic damage. Overall, these findings suggest that KRT1 mitigates inflammation by suppressing BK activation, which in turn downregulates pro-inflammatory factors and alleviates the inflammatory response.

Figure 4 Anti-inflammatory effects of keratin 1 protein in mice with dextran sulfate sodium-induced colitis. A-C: Relative body weight changes; D: Representative images of colons; E: Colon length measurements for each group; F: Disease activity index score; G: Enzyme-linked immunosorbent assay (ELISA) quantitative of bradykinin; H: ELISA quantitative of interleukin-1β; I: ELISA quantitative of interleukin-6; J: ELISA quantitative of tumor necrosis factor-α. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. P value vs control group. DSS: Dextran sulfate sodium; DAI: Disease activity index; ELISA: Enzyme-linked immunosorbent assay; BK: Bradykinin; IL: Interleukin; TNF: Tumor necrosis factor.

KRT1 protein treatment in DSS-induced mice significantly alleviates pathological symptoms and restores the integrity of the intestinal epithelial barrier

Finally, to evaluate the impact of KRT1 protein on the intestinal mechanical barrier function of the intestine in mice with DSS-induced colitis, we conducted assessments using HE staining. This revealed significant damage to the mucosal lining and the intestinal epithelial cell barrier in the DSS group compared to the control group. However, this injury was notably reversed in the KRT1 treatment group (Figure 5A). KRT1 significantly upregulated the expression of key tight junction proteins, including occludin (Figure 5B-D), ZO-1 (Figure 5C, E and F), and claudin (Figure 5C, G and H) in the intestinal epithelial cells of DSS-treated mice, as confirmed at both the mRNA and protein levels (P < 0.001). In contrast, the expression of the intestinal mechanical barrier factor FXIIα was significantly reduced (Figure 5C and I). These changes indicate that KRT1 contributes to the repair of intestinal damage and reduces intestinal permeability. By promoting the expression of tight junction proteins in intestinal epithelial cells and suppressing the expression of the negative regulator FXIIα, KRT1 effectively restores the intestinal barrier, thereby alleviating the inflammatory response.

Figure 5 Intestinal changes and alterations in intestinal mechanical barrier marker gene expression in different treatment groups. A: Hematoxylin-eosin staining of intestinal cross-sections from different treatment groups (100 ×); B: Real-time polymerase chain reaction (RT-qPCR) detection results of occludin expression in different groups; C: Western blot results of occludin, zonula occludens-1, claudin, and coagulation factor XII in different groups; D: Grayscale quantification of Western blot data for occludin in different groups; E: RT-qPCR detection results of zonula occludens-1 in different groups; F: Grayscale quantification of Western blot data for zonula occludens-1 in different groups; G: RT-qPCR detection results of claudin expression in different groups; H: Grayscale quantification of Western blot data for claudin in different groups; I: Grayscale quantification of Western blot data for coagulation factor XII in different groups. DSS: Dextran sulfate sodium; ZO-1: Zonula occludens-1; FXIIα: Coagulation factor XII; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

DISCUSSION

The prevalence of UC is increasing in Eastern countries, and the disease has become a global concern[43-45]. Dysfunction of the mucosal barrier and the dysregulation of gut immune responses play a crucial role in the pathophysiology of UC, though the detailed mechanisms remain incompletely understood[46,47]. In line with our previous findings, KRT1 appears to have a downregulatory effect in UC, although its precise mechanism of action is yet to be elucidated[24]. In this study, it is demonstrated that KRT1 influences the intestinal barrier and immune response in UC through the KKS.

The KKS plays a critical role in regulating inflammation, particularly in response to acute injuries, by mediating the recruitment of proteases, acute-phase proteins, and neutrophils via kallikreins and kinins[48-50]. However, in conditions of severe inflammation, these mechanisms can exacerbate the inflammatory cascade, contributing to tissue damage and chronic inflammation[51,52]. HK, a key protein in this system, is cleaved by FXIIa (activated FXII) to produce BK, a potent mediator of inflammation[53-55]. In Caco-2 cells, the interaction between KRT1 and FXIIα enhances the activation of FXIIα, triggering the KKS and releasing BK[56,57]. This leads to increased vascular permeability, recruitment of immune cells, such as neutrophils, and upregulation of pro-inflammatory cytokines like TNF-α and IL-1β, which amplify the inflammatory response[58]. These processes contribute to the disruption of the intestinal barrier and the progression of conditions such as IBD[59].

Our study confirms that the KRT1 antibody significantly impacts cell proliferation, apoptosis, and intestinal barrier function in Caco-2 cells, highlighting the pivotal role of KRT1 in regulating intestinal inflammation and barrier integrity. These findings align with prior studies indicating that BK plays a critical role in intestinal inflammation, particularly under conditions such as UC, by increasing vascular permeability and facilitating inflammatory responses[60,61]. Specifically, integrin beta-1 (ITβ1) interacts with the activated KRT1 antibody to modulate protein kinase C/SRC activity, thereby influencing the binding of ITβ1 to protein kinase C, which in turn affects cell proliferation and apoptosis[62-64]. Moreover, our results also show that the KRT1 antibody enhances BK expression and significantly upregulates the expression of coagulation factor FXIIα, suggesting that KRT1 contributes to the promotion of inflammatory responses and disruption of intestinal barrier function through the regulation of these factors. This corroborates previous research concerning KRT1’s role in inflammation, particularly its interactions with coagulation factors and vascular permeability regulation[23].

Furthermore, it was observed that the KRT1 antibody significantly suppressed the expression of key tight junction proteins, including occludin, ZO-1, and claudin, leading to impaired intestinal barrier functionality. This observation aligns with earlier studies that the integrity of the intestinal barrier is reliant on the proper expression of tight junction proteins, which are often downregulated in the context of intestinal inflammation[65,66]. Notably, it has been demonstrated that in patients with IBD, a reduction in tight junction proteins levels correlates with increased intestinal permeability and intensified inflammation[67]. These findings support the idea that KRT1 antibodies may exacerbate damage to the intestinal barrier and promote intestinal inflammation by reducing the expression of these tight junction proteins. Consequently, KRT1 antibodies play a crucial role in modulating the proliferation of intestinal epithelial cells in UC, driving inflammatory responses by enhancing FXIIα expression and inhibiting the expression of tight junction proteins.

KRT1 is essential for maintaining skin integrity and is also involved in the inflammatory response. It plays a significant role in the repair and carcinogenesis of UC through mechanisms involving the inflammasome, IL-22, and keratin 8[24,68]. In UC patients, various inflammatory factors heighten intestinal permeability, exacerbate inflammation, and perpetuate a vicious cycle. This process further heightens intestinal permeability by promoting the secretion of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, leading to more severe intestinal damage[69]. Our study demonstrates that in the DSS-induced mouse model, KRT1 treatment significantly inhibited the release of pro-inflammatory cytokines by blocking BK activation. Moreover, it facilitated the growth of intestinal epithelial cells and inhibited FXIIα expression, contributing to the restoration of the intestinal mechanical barrier, preservation of colon integrity, and mitigation of the inflammatory response (Figures 4 and 5). These findings are consistent with previous research, which indicates that KRT1 can significantly inhibit the activation of the nuclear factor kappa-B signaling pathway, thereby reducing the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and alleviating intestinal inflammation[70]. Furthermore, KRT1 promotes the regeneration and repair of intestinal epithelial cells and enhances intestinal barrier function by modulating the Wnt/β-catenin signaling pathway[71]. thereby alleviating UC by facilitating epithelial cell recovery and enhancing intestinal barrier function through the inhibition of the KKS.

While our study confirms that KRT1 treatment has a significant protective effect on DSS-induced colitis in mice, some limitations remain. The long-term effects of KRT1 treatment on chronic colitis remain unclear, and the potential side effects of prolonged KRT1 therapy need further evaluation.

Additionally, it is important to acknowledge that while the KKS has historically been viewed as a protective system for cardiovascular and cerebrovascular health, it also plays a role in modulating inflammation. The KKS releases not only BK but also other kinins from kininogens, which further amplify the inflammatory response[72]. Kinins, including BK, act by activating B1 and B2 receptors, influencing vascular permeability, pain perception, and immune cell recruitment[73]. In the context of UC, these effects can exacerbate both the exacerbation of inflammation and impair the intestinal barrier, suggesting that the KKS functions as a double-edged sword in inflammatory processes. Understanding the nuanced balance between the protective and inflammatory roles of the KKS could provide novel therapeutic insights for managing UC and other inflammatory diseases.

CONCLUSION

In summary, KRT1 regulates the KKS by promoting the expression of HK and inhibiting BK production, thereby contributing to the maintenance of colon integrity. It achieves this protective effect by suppressing key inflammatory mediators, such as IL-1β, IL-6, and TNF-α, and by facilitating the repair of intestinal barrier proteins, including occludin, ZO-1, and claudin. These mechanisms collectively mitigate DSS-induced colonic damage. Our findings provide a compelling therapeutic rationale for targeting KRT1 in the treatment of UC.