CN114452284B

CN114452284B - Application of STING inhibitor in preparation of medicine for treating conjunctival scarring of filter

Info

Publication number: CN114452284B
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: 2023-09-01
Anticipated expiration: 2042-01-20
Also published as: CN114452284A

Abstract

The application of the STING inhibitor in preparing medicines for resisting the treatment of the conjunctival scarring of the filterable tract is found that H151 can inhibit the fibrosis, proliferation and migration capacity of HTF cells induced by angiotensin II, further inhibit the activation of TBK1 and IRF3 mediated by STING and the transcriptional activity of transcription factor NF- κB, so as to relieve the fibrosis of conjunctival sac of the filterable tract and the scarring of the filterable tract, and improve the long-term effective rate after glaucoma is small Liang Shu.

Description

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

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of STING inhibitors in preparation of medicines for treating conjunctival scarring of a filter passage.

Background

Glaucoma is a disease that causes irreversible optic nerve degeneration due to pathological changes in ocular tension, and is the first irreversible blinding eye disease worldwide. Glaucoma trabeculectomy is the most classical and widely used surgical procedure for treating various types of glaucoma, and can effectively reduce elevated intraocular pressure of glaucoma. According to previous studies, early trabeculectomy has been found to have an early surgical success rate as high as 83% to 92%, but this ratio gradually drops to 65% -44% during long follow-up.

The main cause of failure after trabeculectomy is scarring of the conjunctiva of the filter, resulting in a decrease in the effective filtering function after surgery and a renewed rise in ocular pressure. At present, the pathogenesis of scar formation after glaucoma trabeculectomy is still not completely clear, and the occurrence and development of scar formation in a filtration passage cannot be completely and effectively prevented only by adopting traditional means such as filtration, infusion and needle-stick separation, laser broken wire or subconjunctival injection of cell proliferation inhibiting medicaments. Therefore, the generation mechanism of the conjunctiva scarring 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 inflammatory inhibitory activity in vitro and in vivo. H151 has effects of anti-inflammatory, suppressing excessive immune reaction, relieving skin injury, protecting kidney and liver, etc.

However, no treatment of trabeculectomy followed by scar formation of the filtrate was reported for H-151.

Disclosure of Invention

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

The conjunctival scarring of the filter tract is carried out after the glaucoma is small Liang Shu.

The STING inhibitor is a STING inhibitor H151.

The structure of STING inhibitor H151 is as follows:

。

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

The application of TBK1, IRF3 and inhibitors of transcription factor NF- κB signal path in preparing medicine for treating conjunctival scarring of filter path.

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

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

The beneficial effects of the invention are as follows: the invention provides an application of STING inhibitor in preparing medicines for treating conjunctival scarring of a filter, and discovers that H151 can inhibit fibrosis, proliferation and migration capacity of HTF cells induced by angiotensin II, further inhibit activation of TBK1 and IRF3 mediated by STING and transcriptional activity of transcription factor NF- κB, so as to relieve conjunctival sac fibrosis of the filter and scarring of the filter, and improve long-term effective rate after glaucoma is small Liang Shu.

Drawings

Fig. 1 is a graph of H151 stabilized mouse bleb morphology following trabeculectomy and relief of post-operative filter fibrosis.

FIG. 2 is a graph of H151 inhibiting the fibrosis response induced by Ang II in HTF cells.

Fig. 3 h151 inhibits activation of the STING downstream signaling pathway.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the invention, are within the scope of the invention based on the embodiments of the invention.

Example 1

The trabeculectomy postfiltration tract scarring model is successfully constructed, the trabeculectomy operation mode of the mice is referred to the previous literature report (Invest Ophthalmol Vis Sci.2017, 58 (9): 3432-3439), the treatment is carried out by injecting the STING specific small molecule inhibitor H151 into conjunctival sac after the operation for 2 weeks, and the condition of conjunctival bleb of the mice is observed by photographing under an operation microscope after the anesthesia for 2 weeks.

The test process comprises the following steps: male C57BL/6 mice were purchased from commercial animal experiment centers. Mice were kept in animal houses with a constant circadian 12-12h rhythm with standard rodent diet and water. Animals were grown for environmental adaptation one week prior to the start of the experiment. The H151 used in the experiment was prepared as a DMSO-soluble dosage form at a final concentration of 1mg/kg. A trabeculectomy filter scar mouse model was established, and mice were sacrificed 2 weeks later under anesthesia using a sham operation as a control group. Sham is a Sham surgery group; surry is the trabeculectomy group; the surgery+h151 group is a trabeculectomy combined H151 treatment group. (A) Pre-sacrificed mice trabeculectomy filter bleb conditions after anesthesia; (B) trabeculectomy followed by filtration lane HE staining; (C, D) immunofluorescence detection of mouse trabeculectomy filter αsma expression levels; (E) Real time qPCR detects mRNA levels of fibrinectin and CTGF. * P < 0.01, < p < 0.001, sham vs. Surgery; # p < 0.01, # p < 0.001, surgery vs Surgery +h151.

The surgical group showed that the bleb was significantly limited and scarred under the surgical microscope, the bleb surface was hyperemic, and the surgical combination H151 treatment group showed that blebs formed well with a pale surface, see fig. 1A. To investigate the effect of H151 on the intervention of changes in trabeculectomy morphology and fibrosis, we used HE staining and alpha SMA immunofluorescence observations, respectively. As shown in fig. 1B-D, HE staining showed a significant increase in collagen deposition and αsma expression in the mouse filter following trabeculectomy, while there was a significant relief after H151 treatment. The RT-qPCR results showed a significant increase in the levels of fibractin and CTGF mRNA in the trabecular conjunctival tissue of mice receiving the trabecular surgery compared to the sham surgery group, while the H151 treated group was decreased as shown in fig. 1E, F.

Example 2

HTF cells were pre-incubated with H151 for 1H, and then HTF cells were stimulated with Ang II (1 μm) for 6 or 24H, using fluorescent staining, fluorescent quantitation, RT-qPCR, western Blot, and scratch experiments, respectively, to examine the effect of H151 on Ang II.

The test process comprises the following steps: h151 (final concentration 1. Mu.g/ml) pre-incubated HTF cells for 1 hour, ang II (1. Mu.M) stimulated 6 or 24 hours. (a, B) immunofluorescence detection of mouse trabeculectomy filter αsma expression levels; (C) Western Blot detects protein levels of fibractin and CTGF; (D, E) Real time qPCR to detect mRNA levels of fibreonectin and CTGF; (F) a migration scratch test to detect HTF cell migration ability; (G) CCK8 detects the proliferation potency of the cells. * P < 0.01, < p < 0.001, 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 αsma expression by immunofluorescent staining. As can be seen in FIGS. 2C-E, the expression of the factor associated with Ang II induced pro-fibrosis, fibrinectin and CTGF, was increased at the protein and mRNA levels, whereas preincubation of H151 inhibited the expression of the factor associated with Ang II induced fibrosis. Meanwhile, the cell scratch experiment indicates that the migration of cells is obviously accelerated under the stimulation of Ang II, the migration acceleration of the pre-incubated H151 group is relieved (figure 2F), and H151 is observed to obviously inhibit the proliferation of HTF cells caused by Ang II in the cell proliferation experiment (figure 2G). The results show that the small molecule inhibitor H151 has better alleviation effect on HTF cell fibrosis, migration and proliferation caused by Ang II.

After 1 hour of pretreatment with H151, ang II was incubated for 2 hours, and changes in phosphorylation of the downstream signaling pathways TBK1, IRF3 and NF-. Kappa.B-p 65 were detected using Western Blot.

The test process comprises the following steps: h151 (final concentration 1. Mu.g/ml) pre-incubated HTF cells for 1 hour, ang II (1. Mu.M) was stimulated for 2h. Western Blot detects 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. Thus, the above results suggest that the effect of the small molecule inhibitor H151 on regulation of fibrosis is associated with TBK1, IRF3 and NF- κb signaling pathways.

The skilled person will know: while the invention has been described in terms of the foregoing embodiments, the inventive concepts are not limited to the invention, and any modifications that use the inventive concepts are intended to be within the scope of the appended 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 examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims

1. The application of STING inhibitor in preparing medicine for treating conjunctival scarring of filter is characterized in that the STING inhibitor is STING inhibitor H151.

2. The use according to claim 1, wherein the scarring of the conjunctiva of the bleb is performed after the glaucoma is small Liang Shu.

3. The use according to claim 1, wherein said STING inhibitor H151 has the structure:

。

4. the use according to claim 1, wherein said STING inhibitor H151 is present in a concentration of 1 μg/ml.

5. The application of the inhibitor of TBK1, IRF3 and transcription factor NF- κB signal path in preparing the medicine for treating conjunctival scarring of filter path is characterized in that the inhibitor of TBK1, IRF3 and transcription factor NF- κB signal path is H151.

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