Use of il-17d in scar prevention and treatment

Abstract

The application discloses application of a cytokine interleukin-17D (IL-17D) in preparation of a medicine for preventing and / or treating scars. IL-17D is highly expressed in fibroblasts, but is down-regulated in myofibroblasts in skin scars and keloids. Low expression of IL-17D makes IL-17D unable to inhibit expression of fibrosis-related genes, thus unable to inhibit differentiation of fibroblasts into myofibroblasts, thereby causing generation of scars. Overexpression of IL-17D or injection of recombinant IL-17D protein in a mouse full-thickness skin wound can significantly inhibit formation of scars; after IL-17D gene of the mouse is knocked out, expression of fibrosis-related genes in the skin wound is up-regulated, and scars after wound healing are obviously aggravated. Therefore, the application discloses that IL-17D can be used as a medicine for diagnosing and treating skin scars or keloids.

Description

Technical Field

[0001] This invention relates to the fields of biochemistry, molecular biology, immunology, and dermatology, and specifically to the application of IL-17D in scar prevention and treatment. Background Technology

[0002] Scars often form when the skin is injured, such as by burns, trauma, surgery, or infection. It is estimated that nearly 100 million people worldwide develop scars each year due to surgery or trauma, and 15% of these individuals develop hypertrophic scars or even keloids on top of existing wounds. 1 Extensive scarring affects appearance, and the accompanying symptoms such as pain, itching, hardening, and scar contracture significantly impact a patient's quality of life and mental well-being. 1 Therefore, in-depth research into the molecular mechanisms of scar formation will provide new methods and targets for the treatment of scars and keloids.

[0003] From a cellular and molecular perspective, the overactivation of myofibroblasts is the primary cause of pathological scar formation. Myofibroblasts possess the dual characteristics of both fibroblasts and smooth muscle cells, capable of both secreting collagen and exhibiting contractile abilities. 2 Myofibroblasts originate from three pathways: differentiation from bone marrow-derived fibroblasts; transdifferentiation of pericytes, epithelial cells, and endothelial cells; and activation of resting resident fibroblasts. Among these, activation of resting resident fibroblasts is the primary source of myofibroblasts. 3 During myofibroblast activation, transforming growth factor-β (TGF-β) and mechanical stress at the wound site are the dominant factors. 4 During wound healing, various immune cells, such as macrophages and lymphocytes, secrete TGF-β to activate the Smad2 / 3 (Drosophila mothers against decapentaplegic protein 2 / 3) complex, which then binds to Smad4 and enters the nucleus to regulate the expression of α-smooth muscle actin (α-SMA), type I collagen, and tissue inhibitor of metalloproteinase (TIMP). This, in turn, promotes the activation of myofibroblasts and increases the mechanical stress at the scar tissue site. 5 The high mechanical tension in scar tissue further induces the activation of myofibroblasts, releasing more collagen and increasing the stiffness of the scar tissue, creating a vicious cycle. 4Furthermore, TGF-β-activated Smad3 can recruit the histone acetyltransferase CBP / p300 to regulate the histone acetylation levels of pro-fibrosis genes, thereby promoting their expression. In addition to the classic Smad signaling pathway, TGF-β also activates other non-Smad-dependent signaling pathways such as the MAPK (Mitogen-activated protein kinase) signaling pathway, the PI3K (Phosphatidylinositol 3-kinase) signaling pathway, and the Rho (Ras homolog)-coupled GTPase (Guanosine triphosphatase) signaling pathway to promote myofibroblast activation. 6 .

[0004] Besides TGF-β, other cytokines also participate in regulating myofibroblast activation. For example, cytokines such as IL-6, IL-1β, IL-1α, and IL-8 are highly expressed in keloid fibroblasts. The inflammatory microenvironment created by these cytokines makes scars more sensitive to external stimuli, leading to the continuous expansion of scars and the formation of keloids. 7 In addition, IL-4 and IL-13 have also been found to exacerbate scar formation by upregulating the expression of periostin (Postn) to activate the TGF-β signaling pathway. 8 These findings demonstrate that inflammatory cytokine-mediated inflammatory responses play a crucial role in scar formation and exacerbation. Therefore, in clinical practice, interferon (IFN) injections are used to inhibit TGF-β-induced myofibroblast activation. 9 Unfortunately, the above methods cannot completely cure scars because they fail to block the differentiation of fibroblasts into myofibroblasts. Therefore, identifying factors that inhibit the differentiation of fibroblasts into myofibroblasts during skin damage is crucial for the clinical treatment of scars and keloids. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes the cytokine interleukin-17D (IL-17D), which can be used as a target for preparing diagnostic reagents for scar occurrence and / or severity, or for preparing drugs for scar treatment, or for preparing reagents for predicting scar occurrence and / or severity, or for preparing reagents for scar prognostic evaluation. This invention proposes a novel method and strategy for targeting IL-17D to treat scars, thereby effectively and specifically inhibiting the expression of pro-fibrosis-related genes in fibroblasts, thus inhibiting fibroblast differentiation into myofibroblasts and ultimately inhibiting scar formation.

[0006] IL-17D is highly expressed in fibroblasts, but its expression is downregulated in myofibroblasts of skin scars and keloids. Low IL-17D expression prevents it from inhibiting the expression of pro-fibrosis-related genes, thus failing to suppress fibroblast differentiation into myofibroblasts, leading to skin scarring. This invention demonstrates that overexpression of IL-17D or injection of recombinant IL-17D protein (R&D, Cat. No. 2274-ML) in full-thickness skin wounds in mice significantly inhibits scar formation; however, knocking out the IL-17D gene in mice upregulates the expression of pro-fibrosis-related genes in skin wounds, and significantly worsens scarring after wound healing. Therefore, this invention proposes that IL-17D can be used as a drug for the diagnosis and treatment of skin scars or keloids.

[0007] The term "IL-17D" used in this invention refers to the cytokine interleukin-17D, which belongs to the IL-17 family of cytokines and is one of the members of the IL-17 family.

[0008] This invention proposes the use of IL-17D in the preparation of medicaments for the prevention and / or treatment of scars.

[0009] The scars mentioned include, but are not limited to, skin scars, keloids, hypertrophic scars, etc.

[0010] The IL-17D mentioned above is highly expressed in dermal fibroblasts, but its expression is downregulated in skin scars, keloids, and hypertrophic scars.

[0011] The IL-17D mentioned above is significantly downregulated during the differentiation of fibroblasts into myofibroblasts.

[0012] The IL-17D inhibits the expression of profibrosis-related genes in fibroblasts, thereby inhibiting the differentiation of fibroblasts into myofibroblasts and thus inhibiting scar formation.

[0013] The genes mentioned include, but are not limited to, Acta2, Col3a1, En-1, Lrrc15, Cxcl12, Col1a1, Postn, Adam12, and actin α-SMA.

[0014] The present invention also proposes that the absence of IL-17D in mice promotes the expression of type I collagen (Col1a1), actin α-SMA, and Lrrc15, thereby aggravating the formation of full-thickness skin wound scars in mice.

[0015] The present invention also proposes that the IL-17D protein, when injected into a full-thickness skin wound in mice, can inhibit the expression of type I collagen and Lrrc15, thereby inhibiting the formation of skin scars.

[0016] The present invention also proposes that overexpression of IL-17D in mouse skin can inhibit the expression of Lrrc15, Acta2, Col1a1, Postn, En-1, and Adam12, thereby inhibiting the formation of skin scars.

[0017] The present invention also provides a detection reagent / kit containing IL-17D as described above.

[0018] The present invention also provides the use of the detection reagent / kit described above in the preparation of medicaments for the prevention and / or treatment of scar formation.

[0019] In the applications described in this invention, the detection reagent / kit can be used alone or in combination with other drugs.

[0020] In the applications described in this invention, the scars include, but are not limited to, skin scars, keloids, hypertrophic scars, etc.

[0021] The present invention also provides a method for screening candidate drugs for the prevention and / or treatment of scars, the method comprising detecting the effect of the candidate drug on the biological effects of IL-17D as described above in a subject or a sample obtained from the subject.

[0022] If the biological function of IL-17D is improved after using the candidate drug, it indicates that the candidate drug has the effect of preventing and / or treating scars; the improved biological function of IL-17D includes an increase in the expression level of IL-17D.

[0023] The present invention also provides a method for preventing and / or treating scars in subjects in need, or a method for assessing the severity of scars in patients with scars, or a method for predicting or assessing whether the patient is suitable for IL-17D treatment, the method comprising: determining the IL-17D level from a scar tissue sample of the patient or subject, wherein the level is used to prevent or treat the scar in the subject, or to assess the severity of a patient with scars, or to predict or assess whether the patient is suitable for IL-17D-targeted treatment.

[0024] The beneficial effects of this invention include: by identifying the factor that inhibits the differentiation of fibroblasts into myofibroblasts during skin damage, this invention discovers that the factor is the cytokine interleukin-17D. The IL-17D inhibits the expression of pro-fibrosis-related genes in fibroblasts, thereby inhibiting the differentiation of fibroblasts into myofibroblasts and thus inhibiting the formation of scars. This brings new hope for the clinical treatment of scars and keloids and has broad application prospects. Attached Figure Description

[0025] Figure 1 The results show that IL-17D expression was significantly reduced in keloids and myofibroblasts. Figure A shows the expression of IL-17D in normal skin and keloids; Figure B shows the expression of IL-17D in fibroblasts and myofibroblasts.

[0026] Figure 2 The results show that the expression of IL-17D gradually decreases during the differentiation of fibroblasts into myofibroblasts. Figure A shows the expression of IL-17D mRNA during the differentiation of fibroblasts into myofibroblasts, and Figure B shows the expression of IL-17D protein during the differentiation of fibroblasts into myofibroblasts.

[0027] Figure 3 This indicates that IL-17D expression is significantly reduced during the differentiation of fibroblasts into myofibroblasts.

[0028] Figure 4 This indicates that IL-17D inhibits the expression of fibrosis-related genes Acta2, Col3a1, En-1, Lrrc15, Cxcl12, Col1a1, Postn, and Adam12 in fibroblasts and suppresses fibroblast differentiation into myofibroblasts. Figure A shows the expression of pro-fibrosis-related genes in fibroblasts from Il17d-deficient and WT mice using RNA-Seq detection; Figure B shows the expression of pro-fibrosis-related genes in fibroblasts from Il17d-deficient and WT mice using RT-PCR detection; Figure C shows the differentiation of Il17d-deficient and WT mouse fibroblasts into myofibroblasts induced by TGFβ; Figure D shows the immunofluorescence staining of α-SMA after TGFβ-induced differentiation of Il17d-deficient and WT mouse fibroblasts into myofibroblasts, with the filamentous fiber structure in the cells being the result of α-SMA staining.

[0029] Figure 5 The results show that Il17d deletion exacerbates skin scarring in mice and increases the expression of pro-fibrosis-related genes type I collagen (Col1a1), actin α-SMA, and Lrrc15. Figure A shows photographs of skin scars from Il17d-deficient mice and WT mice; Figure B shows the mRNA expression of pro-fibrosis-related genes Tgfβ1, actin α-SMA (Acta2), type I collagen (Col1a1), and Lrrc15 in skin scar tissue from Il17d-deficient mice and WT mice; Figure C shows the protein expression of pro-fibrosis-related genes actin α-SMA, type I collagen (Col1a1), and Lrrc15 in skin scar tissue from Il17d-deficient mice and WT mice.

[0030] Figure 6The results indicate that injecting IL-17D protein into skin wounds can inhibit the expression of profibrosis-related genes type I collagen Col1a1 and Lrrc15 and the formation of scars. Figure A shows the skin scars after injection of PBS and IL-17D into Il17d-deficient mice and WT mice, respectively; Figure B shows the mRNA expression of type I collagen (Col1a1) and Lrrc15 in skin scar tissue after injection of PBS and IL-17D into Il17d-deficient mice and WT mice, respectively.

[0031] Figure 7 The results show that overexpression of IL-17D in skin wounds can inhibit the expression of profibrosis-related genes Lrrc15, Acta2, Col1a1, Postn, En-1, and Adam12, as well as scar formation. Figure A shows a photograph of skin scars after IL-17D overexpression in WT mice; Figure B shows the mRNA expression of Lrrc15, Acta2, Col1a1, Postn, En-1, and Adam12 in skin scar tissue after IL-17D overexpression in WT mice. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the following embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for carrying out the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particularly limiting content.

[0033] Example 1: Expression of IL-17D in keloids and myofibroblasts

[0034] Analysis of the RNA-Seq dataset GSE158395 from dermal fibroblasts of keloid patients in the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE158395) revealed that IL-17D expression was downregulated in keloid patients (e.g., Figure 1 (As shown in A). Furthermore, after inducing dermal fibroblasts from WT newborn mice to differentiate into myofibroblasts using TGFβ, RNA was extracted, and IL-17D mRNA expression was detected by RT-PCR. The results showed that IL-17D expression in fibroblasts was significantly higher than that in myofibroblasts (e.g., as shown in A). Figure 1 (as shown in B).

[0035] Example 2: Expression of IL-17D in fibroblasts and differentiated myofibroblasts

[0036] Dermal fibroblasts were isolated from WT newborn mice. Once the cell density reached 85%, the cells were digested, passaged, and cultured from p0 to p5. RNA and protein were collected from each passage. RNA was used for RT-PCR to detect IL-17D mRNA expression, and protein was used for Western blot to detect IL-17D protein expression. Figure 2 As shown in Figure A, IL-17D mRNA expression decreased with increasing cell passage number, while IL-17D protein expression, consistent with mRNA levels, also decreased with increasing passage number (e.g., ...). Figure 2 (as shown in B).

[0037] Example 3: Expression of IL-17D during the differentiation of fibroblasts into myofibroblasts

[0038] Dermal fibroblasts were isolated from WT neonatal mice. When the cell density reached 50%, 10 ng / mL TGFβ was added to stimulate the cells for 0, 6, 12, 18, 24, and 30 hours. Protein samples were then collected. Western blot analysis revealed that IL-17D protein levels were downregulated as fibroblasts differentiated into myofibroblasts (e.g., ...). Figure 3 (As shown).

[0039] Example 4: IL-17D inhibits the differentiation of fibroblasts into myofibroblasts

[0040] Separate WT and Il17d – / – Fibroblasts from knockout newborn mice were collected for RNA analysis when the cell density reached 90%. The sequencing results showed upregulated expression levels of scar-related factors such as Postn, En1, Adam12, and Lrrc15; and upregulated expression levels of scar-related transcription factors such as En1, Ewsr1, MyoD, and Myogenin. Figure 4 As shown in A). RT-PCR further confirmed that IL-17D deletion significantly promoted the expression of Acta2, Col3a1, En-1, Lrrc15, Cxcl12, Col1a1, Postn, and Adam12 (as shown in A). Figure 4 (as shown in B).

[0041] Next, WT and Il17d were stimulated with 10 ng / mL TGFβ, respectively. – / – Fibroblasts were observed under a microscope 24 hours later to examine cell morphological changes. Figure 4 As shown in C, TGFβ stimulation in Il17d – / – The differentiation of fibroblasts was significantly stronger than that of WT fibroblasts stimulated by TGFβ.

[0042] Finally, the cell slides were treated with 75% ethanol, drained, and then plated into 24-well plates. WT and Il17d cells were then added. – / – Fibroblasts were seeded into 24-well plates. When the cell density reached 40%, they were induced with 10 ng / mL LTFβ for 12 or 24 hours. The culture medium was removed, and the cells were washed three times with PBS. After fixation with 4% PFA for 15 minutes, the fixative was discarded, and the cells were washed three times with PBS. They were then incubated with 0.4% Triton X-100 permeabilization buffer for 10 minutes, washed three times with PBS, blocked with 3% BSA for 1 hour, and incubated overnight at 4°C with primary antibody α-SMA. After recovering the primary antibody, the cells were washed three times with PBST solution, incubated with secondary antibody at room temperature in the dark for 1 hour, washed three times with PBST solution, stained with phalloidin, and incubated at room temperature in the dark for 30 minutes. The cells were washed twice with PBS solution, and 10 μL of DAPI was added to a glass slide. The cell slide was then inverted onto the slide, stained for 15 minutes, mounted with nail polish, and photographed using a fluorescence microscope. Figure 4 As shown in Figure D, IL-17D knockout fibroblasts differentiated into significantly more myofibroblasts under TGFβ induction than WT fibroblasts.

[0043] In summary, these experimental results all demonstrate that IL-17D can inhibit fibroblast differentiation into myofibroblasts. Example 5: IL-17D deficiency promotes the expression of fibrosis-related genes in mouse skin scars and exacerbates scar formation.

[0044] WT and Il17d at 7-8 weeks old – / – After shaving the back of mice, a 6mm diameter incision was made and a silicone ring was fixed in place. The scar size was recorded by photographing 28 days later. Figure 5 As shown in A, Il17d – / – The dorsal skin scars of the mice were significantly more severe than those of the WT mice. Furthermore, RNA was extracted from tissue near the scars after the mice were sacrificed, and the expression of scar-related genes was detected, such as... Figure 5 As shown in B, Il17d – / – The expression of Acta2, Col1a1, Lrrc15, and Tgfrβ1 in mouse scar tissue was significantly higher than that in WT mouse scar tissue. Furthermore, protein samples collected from scar tissue were analyzed by Western blot, revealing that Il17d... – / – The expression levels of type I collagen (Col1a1), actin (α-SMA), and Lrrc15 in mouse scar tissue were higher than those in WT mouse scar tissue (e.g., ...). Figure 5 (as shown in C).

[0045] Example 6: Injection of IL-17D protein into skin wounds can inhibit the expression of profibrosis-related genes and skin scar formation.

[0046] WT and Il17d at 7-8 weeks old – / – After shaving the skin on the back of the mice, a 6mm diameter incision was made and a silicone ring was fixed in place. Two additional experimental groups were also set up: WT and 117d mice. – / – Four equal sections were placed around the wound on the back of mice, and 100 μL of purified recombinant IL-17D protein (1 μg) was injected subcutaneously into each section. The size of the scars was recorded by photograph after 28 days. Figure 6 As shown in A, Il17d – / – The dorsal skin scars of the mice were significantly more severe than those of the WT mice, and injection of IL-17D significantly inhibited the WT and IL-17D effects. – / – The formation of scars on the back of mice. Furthermore, RNA was extracted from the four groups of scarred skin tissues mentioned above, and the expression of scar-related genes was quantitatively detected. Figure 6 As shown in Figure B, IL-17D injection significantly inhibited the expression of Lrrc15, Acta2, Col1a1, Postn, En-1, and Adam12.

[0047] Example 7: Overexpression of IL-17D in skin wounds inhibits the formation of full-thickness skin scars in mice.

[0048] C57BL / 6 mice aged 7-8 weeks were used. After removing hair from their backs, 6 mm of skin was removed. 100 μL of ZsGreen empty vector virus and IL-17D-ZsGreen virus were thoroughly mixed with 100 μL of hydrogel, and then dripped onto the wound. The mixture was then irradiated with ultraviolet light for 30 seconds to rapidly coagulate the virus around the wound. The size of the scar was recorded by photography after 28 days. Figure 7 As shown in Figure A, IL-17D overexpression significantly inhibited the formation of dorsal skin scars in C57BL / 6 mice. RNA was then collected from the scar tissue, and the expression of IL-17D, as well as scar-related genes Col1a1 and Lrrc15, was quantitatively detected. Figure 7 As shown in Figure B, overexpression of IL-17D in the skin significantly inhibited the expression of Col1a1 and Lrrc15.

[0049] References

[0050] 1.Monstrey S,Middelkoop E,Vranckx JJ,Bassetto F,Ziegler UE,Meaume S,Téot L.Updated scar management practical guidelines:non-invasive and invasivemeasures.J Plast Reconstr Aesthet Surg.2014 Aug;67(8):1017-25.

[0051] 2.Macarak EJ,Wermuth PJ,Rosenbloom J,Uitto J.Keloid disorder:Fibroblast differentiation and gene expression profile in fibrotic skindiseases.Exp Dermatol.2021 Jan;30(1):132-145.

[0052] 3.Hinz B.Myofibroblasts.Exp Eye Res.2016 Jan;142:56-70.

[0053] 4.Hinz B.Tissue stiffness,latent TGF-beta1 activation,and mechanicalsignal transduction: implications for the pathogenesis and treatment offibrosis.Curr Rheumatol Rep.2009 Apr;11(2):120-6.

[0054] 5.Meng XM,Nikolic-Paterson DJ,Lan HY.TGF-β:the master regulator offibrosis.Nat Rev Nephrol.2016 Jun;12(6):325-38.

[0055] 6.Zhang YE.Non-Smad Signaling Pathways of the TGF-βFamily.Cold SpringHarb Perspect Biol. 2017 Feb1;9(2):a022129.

[0056] 7.Ogawa R.Keloid and Hypertrophic Scars Are the Result of ChronicInflammation in the Reticular Dermis.Int J Mol Sci.2017 Mar 10;18(3):606.

[0057] 8.Maeda D,Kubo T,Kiya K,Kawai K,Matsuzaki S,Kobayashi D,Fujiwara T,Katayama T, Hosokawa K.Periostin is induced by IL-4 / IL-13 in dermalfibroblasts and promotes RhoA / ROCK pathway-mediated TGF-β1 secretion inabnormal scar formation.J Plast Surg Hand Surg.2019 Oct;53(5):288-294.

[0058] 9.A.M.Loordhuswamy,and S.Elango,"Interferon Therapy for HypertrophicScars and Keloids", in Recent Advances in Wound Healing.London,UnitedKingdom:IntechOpen,2021[Online]。

Claims

1. Use of the cytokine interleukin-17D in the preparation of a medicament for preventing scarring.

2. Use according to claim 1, wherein The cytokine interleukin-17D inhibits the expression of pro-fibrotic related genes in fibroblasts, thereby inhibiting the differentiation of fibroblasts into myofibroblasts, and further inhibiting the formation of scars.

3. Use according to claim 2, wherein the compound is ###0002### The genes include Lrrc15, Acta2, Col1a1, Postn, En-1, Adam12, Col3a1, Cxcl12, actin alpha-SMA.

4. The use according to claim 1, wherein The scars include skin scars, keloids, hypertrophic scars.

Images