Use of itih4 in the preparation of a medicament for the treatment of intestinal inflammation

Abstract

The present application finds that ITIH4 secreted by macrophages can promote the viability and repair of intestinal epithelial cells, promote the expression of intestinal tight junction protein ZO-1, and inhibit the expression of macrophage inflammatory factors, and is an effective molecule for treating intestinal inflammation. Based on this, the present application provides the use of ITIH4 in the preparation of a medicament for treating intestinal inflammation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of ITIH4 in the preparation of drugs for treating enteritis. Background Technology

[0002] Enteritis occurs when the intestinal barrier is compromised, allowing pathogens to invade the intestinal tissue and triggering an immune response to eliminate them. However, an excessive immune response can cause tissue damage and persistent inflammation. Severe enteritis can lead to complications such as bleeding, intestinal perforation, and toxic megacolon, even becoming life-threatening. Therefore, finding targeted drugs to treat intestinal inflammation, thereby optimizing treatment strategies, enhancing treatment efficacy, and reducing the occurrence of complications, is crucial.

[0003] The intestinal barrier comprises the intestinal epithelial barrier, mucus barrier, immune barrier, and microbial barrier. Macrophages, as the first line of defense in intestinal immunity, play a crucial role in the development and progression of enteritis. Uncontrolled pro-inflammatory polarization of macrophages significantly exacerbates the progression of intestinal inflammation. Intestinal epithelial cells play a key role in maintaining intestinal health, immune defense, and barrier function. Therefore, targeting intestinal macrophage inflammation and promoting epithelial repair has become a promising therapeutic strategy. However, how to precisely regulate this process remains a key scientific question in current research.

[0004] Studies have reported that ITIH4 (Inter-alpha-trypsin inhibitor heavy chain 4), an acute-phase protein, is expressed at increased levels in various inflammatory diseases, such as infection, tissue damage, and inflammation. However, the role of ITIH4 in the intestinal barrier during intestinal inflammation remains unclear and requires further investigation. Summary of the Invention

[0005] The inventors of this invention constructed macrophage cell lines that overexpressed or knocked down ITIH4 using cell editing technology, and analyzed the protective effect of ITIH4 on the intestinal barrier during intestinal inflammation. The results showed that ITIH4 secreted by macrophages can promote the vitality and repair of intestinal epithelial cells, promote the expression of intestinal tight junction protein ZO-1, and inhibit the expression of macrophage inflammatory factors.

[0006] Based on the above findings, this invention proposes the application of ITIH4 in the preparation of drugs for treating enteritis.

[0007] Specifically, the present invention provides the use of ITIH4 in the preparation of a medicament for treating enteritis.

[0008] In the application of ITIH4 provided by this invention in the preparation of a drug for treating enteritis, it may also have the following characteristics: wherein ITIH4 promotes the healing of damaged intestinal epithelial cells, promotes the expression of intestinal tight junction protein ZO-1, and inhibits the expression of macrophage inflammatory factors, thereby repairing the intestinal barrier.

[0009] The present invention also provides the use of a substance overexpressing ITIH4 in the preparation of a medicament for treating enteritis.

[0010] The present invention also provides the use of cells overexpressing ITIH4 in the preparation of drugs for treating enteritis.

[0011] In the application of the ITIH4-overexpressing cells provided in this invention in the preparation of drugs for treating enteritis, the cells may also have the following characteristics: the cells are macrophages.

[0012] The present invention also provides the use of drugs that promote ITIH4 overexpression in the preparation of drugs for treating enteritis.

[0013] In the above-described applications provided by the present invention, the following feature may also be included: wherein the enteritis is congenital megacolon-associated enterocolitis.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] The application of ITIH4 provided by the present invention in the preparation of a drug for treating enteritis is effective in treating intestinal inflammation because ITIH4 can promote the vitality and repair of intestinal epithelial cells, promote the expression of intestinal tight junction protein ZO-1, and inhibit the expression of macrophage inflammatory factors. Attached Figure Description

[0016] Figure 1 This is a real-time quantitative PCR image of ITIH4 regulating the expression of inflammatory factors in macrophages in Example 1 of the present invention, wherein... Figure 1 A is a comparison of the mRNA expression levels of inflammatory factors in macrophages overexpressing ITIH4 and the control group; Figure 1 B is a comparison of the mRNA expression levels of inflammatory factors in ITIH4-knocked macrophages and the control group.

[0017] Figure 2 These are scratch assay images and cell viability detection images of ITIH4 regulating intestinal epithelial cells in Embodiment 2 of the present invention. Figure 2 A shows a scratch assay diagram of ITIH4 regulating intestinal epithelial cells; Figure 2 Figure B shows the effect of ITIH4 on the healing of damaged intestinal epithelium compared to the control group. Figure 2 Figure C shows the effect of ITIH4 on intestinal epithelial cell viability compared to the control group.

[0018] Figure 3 This is an immunoblotting analysis diagram of ITIH4 regulating intestinal epithelial cells in Embodiment 3 of the present invention, wherein... Figure 3 Figure A shows the effect of ITIH4 on the protein expression level of ZO-1 in intestinal epithelial cells. Figure 3 Figure B shows the effect of macrophages overexpressing ITIH4 on the protein expression levels of ZO-1 in serum and intestinal epithelial cells of a mouse model of enteritis. Detailed Implementation

[0019] To facilitate understanding of the technical means, inventive features, objectives, and effects of this invention, the following embodiments, in conjunction with the accompanying drawings, specifically illustrate the application of ITIH4 in the preparation of a medicament for treating enteritis. In the following embodiments, reagents and materials not explicitly stated were obtained through common commercial channels, and experimental procedures and conditions not described were performed in accordance with conventional procedures and conditions in the art.

[0020] This invention utilizes lentiviral plasmids and shRNA technology to construct macrophages that overexpress and knock down ITIH4. Unless otherwise specified, in this invention, gene-edited cells and intestinal epithelial cells are co-cultured to analyze the effects of ITIH4 on macrophages and intestinal epithelial cells.

[0021] Example 1

[0022] This embodiment examines the effect of ITIH4 on macrophage inflammatory response.

[0023] The experimental method in this embodiment is as follows:

[0024] Real-time quantitative PCR (qRT-PCR): Raw264.7 macrophages successfully overexpressing and knocking down ITIH4 were seeded into 6-well plates. After the cells grew into a monolayer, total RNA was extracted from the cells using a cell / tissue total RNA extraction kit. The concentration and quality of the obtained total RNA were determined using a NanoDrop 2000c spectrophotometer. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit. Next, qRT-PCR was performed using a Sybr Green Master Mix kit, with GAPDH as an internal control, and the expression level was calculated according to 2-ΔΔCt.

[0025] Figure 1 This is a real-time quantitative PCR image of ITIH4 regulating the expression of inflammatory factors in macrophages in Example 1 of the present invention, wherein... Figure 1A is a comparison of the mRNA expression levels of inflammatory factors in macrophages overexpressing ITIH4 and the control group; Figure 1 B is a comparison of the mRNA expression levels of inflammatory factors in ITIH4-knocked macrophages and the control group.

[0026] like Figure 1 As shown in Figure A, macrophages overexpressing ITIH4 exhibited a significantly decreased expression level of the inflammatory cytokine IL-1β compared to the control group. Figure 1 As shown in Figure B, compared with the control group, macrophages with ITIH4 knockdown showed a significant increase in the expression level of the inflammatory factor IL-1β.

[0027] Experimental results showed that overexpression of ITIH4 significantly reduced the expression of the inflammatory cytokine IL-1β, while knockdown of ITIH4 had the opposite effect, i.e., ITIH4 could inhibit the expression of macrophage inflammatory factors, thereby inhibiting the macrophage inflammatory response.

[0028] Example 2

[0029] This embodiment examines the effect of ITIH4 on the healing of damaged intestinal epithelium.

[0030] The experimental method in this embodiment is as follows:

[0031] 1.1 Cell Scratch Assay: CT26 intestinal epithelial cells were digested, passaged, and seeded in 6-well plates. After the intestinal epithelial cells reached 100% confluence, scratches were gently created by slicing the cells with a pipette tip, and the scratch width was recorded using microscopy. CT26 intestinal epithelial cells were incubated with the supernatant of Raw264.7 macrophages overexpressing ITIH4 (ITIH4 medium, IM) as the IM group, while CT26 intestinal epithelial cells without other treatment served as the Blank control group. The scratch width was recorded by microscopy at 0, 12, and 24 hours of incubation. The scratch healing rate was calculated using the following formula:

[0032] Wound closure rate (%) = 1 − (wound area at TX after treatment / initial wound area at T0) × 100

[0033] 1.2 Cell Viability Assay: CCK-8 assay was used to determine cell viability. Intestinal epithelial cells were digested, resuspended, and counted. Cells were seeded at a density of 2000 cells / well in 96-well plates, with 100 μL of culture medium added to each well. CT26 intestinal epithelial cells were incubated with ITIH4-overexpressing macrophage supernatant (ITIH4 medium, IM) as the IM group, while untreated CT26 intestinal epithelial cells served as the Blank control group. At 0 and 24 h, CCK-8 solution was added to both the IM and control groups, and incubation was continued at 37°C for 2 hours. Absorbance values ​​(450 nm) were measured using a microplate reader.

[0034] Figure 2 These are scratch assay images and cell viability detection images of ITIH4 regulating intestinal epithelial cells in Embodiment 2 of the present invention. Figure 2 A shows a scratch assay diagram of ITIH4 regulating intestinal epithelial cells; Figure 2 Figure B shows the effect of ITIH4 on the healing of damaged intestinal epithelium compared to the control group. Figure 2 Figure C shows the effect of ITIH4 on intestinal epithelial cell viability compared to the control group.

[0035] like Figure 2 As shown in Figures A and 2B, the IM group exhibited smaller scratch widths at 12 and 24 hours compared to the control group. At 12 hours, the scratch healing rate in the control group was approximately 0.2%, while the scratch healing rate in the IM group was approximately 0.4%, significantly higher than the control group. At 24 hours, the scratch healing rate in the control group was approximately 0.3%, while the scratch healing rate in the IM group was approximately 0.5%, still significantly higher than the control group. These results indicate that intestinal epithelial cells incubated with IM showed significantly faster scratch healing rates at 12 and 24 hours, suggesting that ITIH4 can promote the healing of damaged intestinal epithelium.

[0036] like Figure 2 As shown in Figure C, at 0 h, there was no significant difference in OD450 between the control group and the IM group, meaning that the intestinal epithelial cell viability was essentially the same in both groups at 0 h. At 24 h, the OD450 of the control group was approximately 0.23, while the OD450 of the IM group was approximately 0.28, significantly higher than that of the control group. These results indicate that intestinal epithelial cells incubated with IM showed higher cell viability after 24 hours, with a highly significant difference compared to the control group, suggesting that ITIH4 can improve intestinal epithelial cell viability.

[0037] Experimental results showed that ITIH4 can promote the healing of damaged intestinal epithelium and improve the vitality of intestinal epithelial cells.

[0038] Example 3

[0039] This embodiment investigates the effect of ITIH4 on the expression of ZO-1 protein in intestinal epithelial cells. ZO-1 is an intestinal tight junction protein involved in maintaining the structure and function of intercellular connections.

[0040] The experimental method in this embodiment is as follows:

[0041] 1.1 Intestinal epithelial cells CT26 were incubated with the supernatant of macrophages Raw264.7 overexpressing ITIH4 (ITIH4 medium, IM), denoted as the treatment group +, and intestinal epithelial cells CT26 without other treatments were denoted as the untreated group -.

[0042] 1.2 Animal Model Construction: Ednrb gene knockout mice, a classic model of congenital megacolon-associated enterocolitis, were constructed using CRISPR / Cas9 gene editing technology. Raw264.7 macrophages overexpressing ITIH4 were injected intraperitoneally into the mice. Three days later, the mice were euthanized, and colonic epithelial cells (CECs) and serum were collected. These were designated as the "treated group" (+). Intestinal epithelial cells and serum collected from untreated enterocolitis mouse models were designated as the "untreated group" (-).

[0043] 1.3 Western blot assay: Cells or tissues are lysed using RIPA buffer, and proteins are denatured using 4xSDS loading buffer and heating at 100°C. After electrophoresis, transfer, blocking, incubation with primary and secondary antibodies, and exposure for development.

[0044] Figure 3 This is an immunoblotting analysis diagram of ITIH4 regulating intestinal epithelial cells in Embodiment 3 of the present invention, wherein... Figure 3 Figure A shows the effect of ITIH4 on the protein expression level of ZO-1 in intestinal epithelial cells. Figure 3 Figure B shows the effect of macrophages overexpressing ITIH4 on the protein expression levels of ZO-1 in serum and intestinal epithelial cells of a mouse model of enteritis.

[0045] like Figure 3 As shown in Figure A, compared to the untreated group, the treated group showed increased ZO-1 protein expression in intestinal epithelial cells, indicating that ITIH4 secreted by macrophages can promote ZO-1 protein expression in intestinal epithelial cells.

[0046] like Figure 3As shown in Figure B, compared to the untreated group, the treated group showed increased serum ITIH4 protein expression and increased ZO-1 protein expression in intestinal epithelial cells. This indicates that intraperitoneal injection of ITIH4-overexpressing macrophages (Raw264.7) into a mouse model of enteritis can increase the expression level of ITIH4 in mouse serum and promote the expression level of ZO-1 in mouse intestinal epithelial cells. This demonstrates that exogenously supplemented ITIH4 can achieve an effective concentration in systemic circulation (serum) and ultimately achieve a therapeutic effect of increasing ZO-1 protein expression in the diseased intestine, indicating the feasibility and effectiveness of ITIH4 in treating complex disease environments.

[0047] Experimental results showed that ITIH4 can promote ZO-1 expression in intestinal epithelial cells.

[0048] Based on the experimental results of Examples 1-3 above, it can be concluded that: overexpression of ITIH4 in macrophages shows that ITIH4 secreted by macrophages can promote the healing of damaged intestinal epithelial cells, promote the expression of intestinal tight junction protein ZO-1, and inhibit the expression of macrophage inflammatory factors, thus verifying that ITIH4 is an effective molecule for treating intestinal inflammation.

[0049] Based on this, ITIH4 derived from macrophages has a protective effect on the intestinal barrier and can be used to prepare drugs for treating enteritis.

[0050] In addition, since ITIH4 is derived from macrophages, macrophages that overexpress ITIH4 can be constructed using cell editing technology to play a role in the treatment of enteritis. Macrophages that overexpress ITIH4 can also be used to prepare drugs for the treatment of enteritis.

[0051] Furthermore, since ITIH4 has been proven to be an effective molecule for treating intestinal inflammation, namely, by promoting the overexpression of ITIH4 in vivo, it can play a role in treating enteritis. Therefore, drugs that promote the overexpression of ITIH4 can also be used to prepare drugs for treating enteritis.

[0052] Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. The application of cells overexpressing ITIH4 in the preparation of drugs for treating enteritis, characterized in that: in, The cells in question are macrophages.

2. The application according to claim 1, characterized in that: in, The enteritis described is congenital megacolon-associated enterocolitis.

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