CN114452284A

CN114452284A - Application of STING inhibitor in preparation of medicine for treating conjunctival scarring of filter tract

Info

Publication number: CN114452284A
Application number: CN202210063512.7A
Authority: CN
Inventors: 乐融融; 叶慧芳; 邵旭; 王庆; 陆欣蕾
Original assignee: Eye Hospital of Wenzhou Medical University
Current assignee: Eye Hospital of Wenzhou Medical University
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-05-10
Anticipated expiration: 2042-01-20
Also published as: CN114452284B

Abstract

An application of a STING inhibitor in preparation of a medicine for treating filtering channel conjunctival scarring finds that H151 can inhibit fibrosis, proliferation and migration capabilities of HTF cells induced by angiotensin II, further inhibits activation of TBK1 and IRF3 mediated by STING and transcriptional activity of a transcription factor NF-kappa B, relieves filtering channel conjunctival sac fibrosis and filtering channel scarring, and improves long-term effective rate after glaucoma trabecular operation.

Description

Application of STING inhibitor in preparation of medicine for treating conjunctival scarring of filter tract

Technical Field

The invention relates to the technical field of biological medicines, and in particular relates to application of a STING inhibitor in preparation of a therapeutic drug for resisting conjunctival scarring of a filter channel.

Background

Glaucoma is a disease caused by irreversible optic nerve degenerative change due to pathological intraocular pressure change, and is the first irreversible blindness-causing eye disease in the world. Glaucoma trabeculectomy is the most classical and widely used surgical method for treating various types of glaucoma at present, and can effectively reduce the elevated intraocular pressure of the glaucoma. According to previous researches, the early surgical success rate of trabeculectomy is as high as 83-92%, but the ratio is gradually reduced to 65-44% in the long-term follow-up process.

The main reason for failure after trabeculectomy is scarring of the conjunctiva of the filtering tract, resulting in reduction of effective filtering function and re-increase of intraocular pressure after the operation. At present, the pathogenesis of scar formation after glaucoma trabeculectomy is still not completely clear, and the traditional means of filtration bubble acupuncture separation, laser thread breakage or subconjunctival injection of cytostatic drugs and the like can only be adopted for treatment in the past, but the occurrence and the development of filter aisle scarring cannot be completely and effectively prevented. Therefore, the generation mechanism of the scar formation of the conjunctiva of the filtering passage after the trabeculectomy is clarified, a new drug target is searched, the development of targeted therapeutic drugs is facilitated, and the method has important scientific significance and clinical application value.

H151 is a potent, selective and covalent STING antagonist with significant anti-inflammatory activity in vitro and in vivo. H151 has the effects of resisting inflammation, inhibiting excessive immune response, relieving skin injury, protecting kidney and liver, and the like.

But scarring of the filter corridor after H-151 treatment for glaucoma trabeculectomy has not been reported.

Disclosure of Invention

In order to solve the technical defects in the prior art, the invention provides application of the STING inhibitor in preparing a medicament for treating the conjunctival scarring of the filtering channel.

The filtering channel conjunctiva scarring is glaucoma trabecular postoperative filtering channel conjunctiva scarring.

The STING inhibitor is STING inhibitor H151.

The structure of the STING inhibitor H151 is as follows:

。

the concentration of the STING inhibitor H151 is 1 mug/ml.

The application of inhibitors of TBK1, IRF3 and transcription factor NF-kB signal channels in preparing medicaments for resisting filter channel conjunctiva scarring.

The inhibitor of the signal path of TBK1, IRF3 and transcription factor NF-kB is H151.

Use of STING inhibitor H151 for the preparation of a medicament for alleviating fibrosis, proliferation and migration of HTF cells.

The invention has the beneficial effects that: the invention provides application of a STING inhibitor in preparation of a medicine for treating filtering channel conjunctival scarring, and finds that H151 can inhibit fibrosis, proliferation and migration capabilities of HTF cells induced by angiotensin II, further inhibits activation of STING-mediated TBK1 and IRF3 and transcriptional activity of a transcription factor NF-kappa B, relieves filtering channel conjunctival sac fibrosis and filtering channel scarring, and improves long-term effective rate after glaucoma trabecular surgery.

Drawings

Figure 1 is H151 stabilizing post-trabeculectomy bleb morphology and alleviating post-operative filter tract fibrosis in mice.

Fig. 2 is a graph showing that H151 inhibits the fibrotic response induced by Ang II in HTF cells.

Fig. 3H 151 inhibits activation of the STING downstream signal path.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Successfully constructs a filtering channel scarring model after trabeculectomy, the mode of trabeculectomy is referred to the prior literature report (Invest Ophthalmol Vis Sci, 2017, 58(9): 3432. 3439), the STING specific small molecule inhibitor H151 is injected into the conjunctiva sac after the operation for 2 weeks, and the photographing is carried out under an operation microscope after anesthesia for observing the conjunctiva bleb condition after the operation for 2 weeks.

The test process comprises the following steps: male C57BL/6 mice were obtained from a commercial animal laboratory center. Mice were housed in a constant temperature animal house with a circadian rhythm of 12-12h with standard rodent chow and water. Animals were grown environmentally adaptive one week prior to the start of the experiment. H151 used in the experiments was prepared as a DMSO-soluble formulation at a final concentration of 1 mg/kg. A trabeculectomy filter tract scarring mouse model was established, sham surgery was used as a control group, and the mice were sacrificed under anesthesia 2 weeks later. Sham is a Sham group; surgery is the trabeculectomy group; the Surgery + H151 group was a trabeculectomy in combination with H151 treatment group. (A) Filtration bleb status of mice by trabeculectomy after anesthesia before sacrifice; (B) post trabeculectomy filter aisle HE staining; (C, D) detecting the expression level of the alpha SMA in the filter channel of the mouse trabeculectomy by immunofluorescence; (E) real time qPCR detects mRNA levels of fibrinectin and CTGF. P < 0.01, Sham vs Surgery; # p < 0.01, # p < 0.001, and Surgery vs Surgery + H151.

The operation group can be seen under an operation microscope to show that the filtration bleb is limited and scar, the surface of the filtration bleb is congested, and the result of the operation combined with the H151 treatment group shows that the filtration bleb is formed well and the surface is pale, which is shown in figure 1A. To investigate the interfering effects of H151 on filter morphology change and fibrosis after trabeculectomy, we used HE staining and alpha SMA immunofluorescence observations, respectively. As shown in fig. 1B-D, HE staining showed significant increases in collagen deposition and α SMA expression in the filtered pathway of mice after trabeculectomy, with significant relief after H151 treatment. The RT-qPCR results showed that the levels of fibrinectin and CTGF mRNA were significantly increased in the filter membrane tissue of mice receiving trabecular surgery compared to the sham group, while the H151 treated group was decreased, as shown in fig. 1E, F.

Example 2

HTF cells are preincubated for 1H by using H151, and then the HTF cells are stimulated by Ang II (1 mu M) for 6 or 24 hours, and the effect of the H151 on Ang II resistance is detected by respectively adopting fluorescent staining, fluorescent quantitation, RT-qPCR, Western Blot, scratch experiment and the like.

The test process comprises the following steps: h151 (final concentration 1. mu.g/ml) preincubated HTF cells for 1 hour, Ang II (1. mu.M) stimulated for 6 or 24 hours. (A, B) detecting the expression level of alpha SMA in a filter channel of a mouse trabeculectomy by immunofluorescence; (C) western Blot for detecting the protein levels of fibrinectin and CTGF; (D, E) Real time qPCR to detect the mRNA levels of Fibronectin and CTGF; (F) detecting the migration capacity of the HTF cells by a migration scratch test; (G) CCK8 measures cell proliferative capacity. P < 0.01, Ctrl vs Ang II; # p < 0.01, # # # p < 0.001, Ang II vs Ang II + H151.

As shown in fig. 2A, B, H151 was found to inhibit Ang II-induced α SMA expression by immunofluorescence staining. As can be seen from FIGS. 2C-E, Ang II induced increased expression of Fibronectin and CTGF, which are factors involved in promotion of fibrosis, at protein and mRNA levels, whereas pre-incubation of H151 inhibited the expression of the factors involved in fibrosis induced by Ang II. Meanwhile, the cell scratch experiment suggests that the migration of the cells is remarkably accelerated under the stimulation of Ang II, the accelerated migration of the pre-incubated H151 group is relieved (fig. 2F), and the H151 can remarkably inhibit the proliferation of HTF cells caused by Ang II in the cell proliferation experiment (fig. 2G). The results show that the small molecule inhibitor H151 has a good relieving effect on the fibrosis, migration and proliferation of HTF cells caused by Ang II.

We used H151 pretreatment 1 hours later, then Ang II incubation 2 hours, using Western Blot detection of STING downstream signaling pathway TBK1, IRF3 and NF-. kappa.B-p 65 phosphorylation changes.

The test process comprises the following steps: h151 (final concentration 1. mu.g/ml) preincubated HTF cells for 1 hour, and Ang II (1. mu.M) for 2 hours. Western Blot was used to detect protein levels of p-TBK1, p-IRF3 and p-NF- κ B p 65.

As shown in FIG. 3, H151 significantly inhibited TBK1, IRF3 and NF-. kappa.B-p 65 phosphorylation. Therefore, the results suggest that the function of small molecule inhibitor H151 in regulating fibrosis is related to TBK1, IRF3 and NF- κ B signaling pathways.

The skilled person should understand that: although the invention has been described in terms of the above specific embodiments, the inventive concept is not limited thereto and any modification applying the inventive concept is intended to be included within the scope of the patent claims.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims

Application of STING inhibitor in preparation of medicine for treating conjunctival scarring of filter tract.
2. The use of claim 1, wherein the filtering conjunctiva scarring is glaucoma trabecular post-surgery filtering conjunctival scarring.
3. The use of claim 1, wherein the STING inhibitor is STING inhibitor H151.
4. The use according to claim 1, wherein the STING inhibitor H151 has the structure:

。
5. the use according to claim 1, wherein the concentration of STING inhibitor H151 is 1 μ g/ml.
The application of inhibitors of TBK1, IRF3 and transcription factor NF-kB signal pathways in preparing medicaments for resisting filter channel conjunctiva scarring.
7. The use of claim 6, wherein the inhibitor of the signaling pathway of TBK1, IRF3 and the transcription factor NF-. kappa.B is H151.
Use of STING inhibitor H151 for the preparation of a medicament for alleviating fibrosis, proliferation and migration of HTF cells.