CN116115605A

CN116115605A - Application of wedelolactone in preparation of medicines for treating inflammatory response in pseudomonas aeruginosa keratitis

Info

Publication number: CN116115605A
Application number: CN202211624567.7A
Authority: CN
Inventors: 徐烁; 刘文慧
Original assignee: Wuxi No 2 Peoples Hospital
Current assignee: Wuxi No 2 Peoples Hospital
Priority date: 2022-12-16
Filing date: 2022-12-16
Publication date: 2023-05-16

Abstract

The application of wedelolactone in preparing the medicine for treating inflammatory reaction in pseudomonas aeruginosa keratitis is proved by experiments, the wedelolactone can reduce the inflammation in pseudomonas aeruginosa keratitis and improve prognosis by inhibiting caspase-4/5/11/GSDMD mediated non-classical pyrosis, and new medicines are also developed. Only subject researches show that the expression of non-classical pyroapoptosis in clinical samples and in-vivo and in-vitro models of the pseudomonas aeruginosa keratitis is increased for the first time, and wedelolactone can play an anti-inflammatory role in the pseudomonas aeruginosa keratitis by regulating and controlling the pyroapoptosis pathway, so that a novel therapeutic target and a novel medicament are provided for the anti-inflammatory treatment of the clinical infectious keratitis.

Description

Application of wedelolactone in preparation of medicines for treating inflammatory response in pseudomonas aeruginosa keratitis

Technical Field

The invention relates to the technical field of medicines, in particular to application of wedelolactone in preparing an inflammatory response medicine for treating pseudomonas aeruginosa keratitis.

Background

Pseudomonas aeruginosa (P.aeromonas) is the primary gram-negative pathogen responsible for bacterial keratitis, associated with the use of contact lenses. Pseudomonas aeruginosa infection, if not timely and properly treated, can lead to corneal perforation within 48-96 hours after infection. Although antibiotics are effective if initially used for treatment, the infected eye is still harmed by the inflammatory response caused by the pathogen, and proteases such as pseudomonas IV and pseudomonas aeruginosa small proteases are thought to damage the basement membrane and weaken the corneal stroma. In this case, proper control of the body inflammatory response plays an important role in maintaining the patient's vision.

In the host innate immune defenses against bacterial infections, there is increasing evidence that programmed forms of cell death may play an important role. The type of cell death that occurs during bacterial infection depends on the state of the host inflammatory response, as well as the type of tissue and cells that are infected, due to the potential mechanism of programmed cell death. Pyrodeath was originally introduced early in the 21 st century to describe a form of programmed cell death of salmonella typhimurium infected mouse macrophages. Currently, focal death is divided into two types, the classical pathway and the atypical pathway. Uncontrolled focal death can lead to uncontrolled inflammation, resulting in corneal perforation. Whereas the subject group studies of the applicant demonstrated that the focal death of atypical pathways was involved in the pathogenesis of pseudomonas aeruginosa-induced keratitis.

Wedelolactone (WDL) is the main active ingredient of wedelolactone and eclipta alba. Many studies have shown that WDL has a variety of biological properties, such as snake venom antidotes, liver protection, anti-cancer, anti-oxidant and anti-inflammatory activity. In sepsis-related encephalopathy, WDL inhibits LPS and Adenosine Triphosphate (ATP) -induced non-classical focal death pathways and cell death, and studies have also demonstrated that WDL can directly inhibit IKK complex, inhibiting lipopolysaccharide-induced caspase-11 expression. These results confirm the anti-inflammatory effect of wedelolactone. However, wedelolactone (WDL) has not been reported to be used for the treatment of keratitis and corneal injury induced by Pseudomonas aeruginosa.

Disclosure of Invention

Therefore, based on the background, the Wedelolactone (WDL) is applied to the treatment of keratitis and cornea injury induced by pseudomonas aeruginosa, especially to the improvement of inflammatory response in pseudomonas aeruginosa keratitis, and therefore the application of the wedelolactone in the preparation of the inflammatory response medicine for treating pseudomonas aeruginosa keratitis is developed.

The technical scheme provided by the invention is as follows:

Further, the wedelolactone reduces inflammatory response in pseudomonas aeruginosa keratitis and improves prognosis thereof by inhibiting caspase-4 (caspase-4), caspase-5 (caspase-5), caspase-11 (caspase-11), GSDMD mediated non-classical focal death pathway.

Further, the medicine comprises wedelolactone and pharmaceutic adjuvant.

Further, the medicament further comprises an antibiotic.

Further, the antibiotic is ciprofloxacin.

Further, the pharmaceutical excipients comprise a diluent.

Further, the medicine is eye drop.

The beneficial effects achieved by the invention are as follows:

the application of wedelolactone in preparing the medicine for treating inflammatory reaction in pseudomonas aeruginosa keratitis is proved by experiments, the wedelolactone can reduce the inflammation in pseudomonas aeruginosa keratitis and improve prognosis by inhibiting caspase-4/5/11/GSDMD mediated non-classical pyrosis, and new medicines are also developed. The subject research discovers that the expression of a non-classical focal death pathway in a clinical sample and an in-vivo model of the pseudomonas aeruginosa keratitis is increased for the first time, and wedelolactone can play an anti-inflammatory role in the pseudomonas aeruginosa keratitis by regulating and controlling the non-classical focal death pathway, so that a novel therapeutic target and a novel medicament are provided for the anti-inflammatory treatment of the clinical infectious keratitis.

Drawings

FIG. 1 shows the clinical characteristics and over-expression results of non-classical focal death related factors of keratitis patients;

wherein FIG. 1A is a photograph of a control normal cornea and 3 cases of clinical Pseudomonas aeruginosa keratitis;

FIGS. 1B-E are the results of Western blot analysis of caspase-4, caspase-5, and GSDMD, respectively;

FIGS. 1F-H are qRT-PCR results for caspase-4, caspase-5, and GSDMD, respectively, with GAPDH as an internal control group, where P <0.05, P <0.01, P <0.001, P <0.0001, compared to the control group; ns: nonsensical; n=3.

FIG. 2 is an in vivo expression of atypical focal death-related factors in the group of Pseudomonas aeruginosa keratitis;

wherein fig. 2A is a representative slit lamp image of a control group, an injury-only group, and a keratitis group;

FIGS. 2B-C are immunoblot analysis of caspase-11 and GSDMD;

FIG. 2D is a qRT-PCR analysis of caspase-11 and GSDMD; using GAPDH as an internal control, comparing P <0.05, P <0.01, P <0.001, P < 0.0001; ns: nonsensical; n=3.

FIG. 3 is an in vitro non-classical focal death of LPS-induced piHCKs;

wherein FIG. 3A shows the results of the detection of cell proliferation by CCK-8 kit;

FIG. 3B shows the LDH results of the release of piHCKs after 48 hours of treatment with LPS (0,0.25,0.5,1and 2. Mu.g/ml), respectively;

FIGS. 3C-F show the results of Western blot detection of caspase-4, caspase-5, GSDMD;

FIG. 3G-I shows qRT-PCR results of caspase-4, caspase-5, GSDMD;

FIG. 3J is an immunofluorescence picture of piHCKs after treatment with 0 μg/ml (control group) and 2 μg/ml LPS, respectively; blue: nuclear staining (DAPI); red: caspase-4 and caspase-5 staining; GAPDH was used as an internal control group; comparing with the control group, P <0.05, P <0.01, P <0.001, P < 0.0001; ns: nonsensical; n=3.

FIG. 4 shows the protection of wedelolactone against LPS-induced atypical pyrosis piHCKs;

wherein FIG. 4A shows the cell viability of piHCKs treated with wedelolactone at different concentrations for 2 hours as detected by CCK-8 kit;

FIG. 4B shows piHCKs pretreated with wedelolactone at various concentrations prior to induction with 2. Mu.g/ml LPS;

FIGS. 4C-F show the results of Western blot detection of caspase-4, caspase-5, GSDMD;

FIG. 4G-I shows qRT-PCR results of caspase-4, caspase-5, GSDMD;

FIG. 4J-O shows the levels of caspase-4, caspase-5, IL-1. Beta. And IL-18 expression and Western blot detection of LPS-induced piHCKs pre-treated with 200. Mu.M Ac-YVAD-cmk or 4. Mu.M WDL or 200. Mu.M Ac-YVAD-cmk in combination with 4. Mu.M WDL, respectively, for 2 h;

FIG. 4P-Q shows the results of Elisa assay for IL-1. Beta. And IL-18 in cell culture supernatants; compared with the control group, the # P is less than 0.05, the # P is less than 0.01,

# # P <0.001, P < 0.05P <0.01, P <0.001, P <0.0001, ns: nonsensical; n=3.

FIG. 5 shows the reduction of Pseudomonas aeruginosa keratitis and atypical pyrosis in wedelolactone in vivo;

FIG. 5A is a slit lamp photograph of a control, keratitis + ciprofloxacin + DMSO, keratitis + ciprofloxacin + WDL group, respectively, at day 5 post-infection;

FIG. 5B shows HE staining results for different groups; high resolution (40×) and low resolution (100×), respectively;

FIG. 5C shows the results of immunohistochemical analysis in caspase-11 at high resolution (40X) and low resolution (100X), respectively;

FIG. 6 is activation of Pseudomonas aeruginosa keratitis lesions and atypical pyrosis in vivo;

FIGS. 6A-B are protein blot analysis of wespase-11 and GSDMD;

FIG. 6C is the effect of cell apoptosis;

FIGS. 6D-G are Western blotting and qRT-PCR detection results.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Accordingly, it is intended that the present invention cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention will be disclosed in or be apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention. The invention is further illustrated below with reference to examples.

Example 1: the invention researches, tests and verifies the process

1. Materials and methods

2.1. Human tissue sample

3 cases were obtained from patients who had undergone cornea transplantation in need of Pseudomonas aeruginosa keratitis from the ophthalmic hospital of the first affiliated hospital of Harbin medical university in China. 4 donors were derived from the eye store of the Heilongjiang province, and samples were immediately stored at low temperature until further experiments. The eye tissue of the patient is observed by a slit lamp biological microscope before operation and is stained by fluorescent sodium. Photographs of the cornea of the control group were taken from healthy persons on the ocular surface. All patients and donors signed informed consent. The study was conducted according to the declaration of Helsinki by the world medical Association and was approved by the university of medical university research ethics Committee of Harbin (approval No. 2020119).

2.2. Establishment and treatment of keratitis animal model

All animal experiments strictly followed the harbine university of medical university animal survey committee (2020046). In this example, a total of 70 male spray-Dawley rats (6 weeks; 100-120 g) were used for animal experiments.

First, male SD rats were randomly divided into three groups (control group, injury-only group and keratitis group, n=10 each), and the activation and effect of atypical focal death was studied.

Control group, no injury or infection treatment was performed.

For the cornea-only injury group, after general anesthesia with sodium pentobarbital and local anesthesia with 1 drop of oxybuprocaine hydrochloride (benoxicil), the epithelium layer 3mm in diameter was scraped from the cornea surface with a scalpel, and then the stroma was injured with a25 gauge sterile needle (3 1mm incisions).

For the keratitis-only group, 10 μl of Pseudomonas aeruginosa suspension containing 1×106CFU was used for scar cornea.

Only the cornea damaged group was treated with 10 μl of 0.3% ciprofloxacin eye drops, avoiding postoperative infection. On day 1 after modeling, rats were sacrificed for cervical dislocation under pentobarbital sodium-induced anesthesia and corneas were removed.

Next, male SD rats were randomly divided into 4 groups (normal, keratitis, keratitis+ciprofloxacin+dmso, keratitis+ciprofloxacin+wdl group, n=10 per group), and the treatment effect of WDL on pseudomonas aeruginosa keratitis was studied.

After Pseudomonas aeruginosa infection, 10 μl of 0.3% ciprofloxacin eye drops were used in combination with WDL (Sai chemical; 15 μM) or DMSO (Beyotidme; shanghai, china) for injection into the cornea of infected rats. Starting at 18h post infection, 3 times per day for 5 consecutive days. As previously described, on day 5 after modeling, slit lamp photography (Guo et al 2020) was used between the different groups, the inflammatory response was confirmed and recorded using slit lamp photography, and then rats were sacrificed under pentobarbital sodium-induced anesthesia for cervical dislocation and corneal tissue was obtained.

2.3. Hematoxylin-eosin (HE) and immunohistochemical staining

For HE and immunohistochemical staining, cornea samples were fixed with 4% paraformaldehyde, paraffin embedded, dewaxed, rehydrated, and cut into 5- μm sections. After sectioning, HE was routinely stained and caspase-11 was immunohistochemically stained. The image acquisition uses a microscope.

2.4. Primary culture and treatment of human corneal keratocytes

After removal of the corneal epithelium and endothelial layer, the corneal stroma was cut into small pieces and cultured in a medium of DMEM/F12 comprising 10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin at 5% CO ₂ 37 ℃; the experiments were performed on cultured 3 rd and 10 th generation cells.

To assess atypical pyrodeath, piHCKs were cultured in LPS (0, 0.25,0.5,1and 2 μg/ml) medium for the indicated times. To verify the effect of wedelolactone, piHCKs were premixed with 1 μM, 2 μM and 4 μM Wedelolactone (WDL) for 2 hours, respectively, followed by treatment with 2 μg/ml LPS (pseudomonas aeruginosa 10,Sigma Aldrich L9143) for 24 hours. Treatment was performed for 2 hours with the Caspase-1 inhibitor Ac-YVADcmk ((200. Mu.M, cayman chemical 10014) before treatment with 2. Mu.g/ml LPS.

2.5. Real-time fluorescent quantitative PCR

Gene expression analysis was performed using TRIzol reagent (Invitrogen, carlsbad, calif., USA) to extract collected cornea samples and LPS-induced total RNA of piHCK within 24 hours, followed by real-time fluorescent quantitative PCR to normalize target gene expression to GAPDH and by 2 ^-ΔΔCT Three independent replicates of the assay were performed. The amplification primer sequences are shown in Table 1.

Table 1: amplification primer sequence for PCR

2.6. Western blotting

RIPA buffer+1% protease inhibitor (Biyunshii Biotechnology; shanghai, china) and BCA are used ^TM The total protein extracted from the collected cornea samples and LPS-induced piHCKs within 48 hours were extracted and quantified using a protein assay kit (bi yunshan biotechnology; shanghai, china). Western blotting was then performed according to conventional methods, and the main antibodies were directed against caspase-4 ((Eboltag, catalog A6495), caspase-5 (Boston Biotechnology, catalog)BM 4577), caspase-11 (Stokes Biotechnology, catalog No. sc-374615), IL-1 beta (Ab-carbomer, catalog No. ab 9722), IL-18 (Boston Biotechnology, cata-Log No. A06457), GSDMD (cell signaling technology, catalog No. 93709S), IL-6 (Abcam, catalog No. 9324), IL-8 (Aibotag, catalog No. A2541), TNF-alpha (Aibotag, catalog No. A11534) and GAPDH (Aibotag, catalog No. A19056) were performed in 3 independent replicates per sample.

2.7 immunofluorescence analysis

Immunofluorescent staining was performed on paraformaldehyde-fixed cells. Cells were stained with caspase-4 (1:200) and caspase-5 (1:200), nuclei were stained with DAPI (Biyunshan Biotechnology, shanghai, china), and images were acquired by fluorescence microscopy (Nikon 80i, japan).

2.8. Cell proliferation assay

piHCKs at 10 ⁴ Cell/well density was seeded into 96-well plates. After the treatment, the cell viability was monitored using CCK-8 kit (CCK-8, co., ltd., japan). The absorbance (A) at 450nm was measured using a spectrophotometer microplate reader.

2.9. Lactate Dehydrogenase (LDH) assay

piHCKs at 10 ⁴ Cell/well density was seeded into 96-well plates. piHCKs at 10 ⁴ Cell/well density was seeded into 96-well plates. Adopts an LDH kit (the mixture is prepared

Non-radioactive cytotoxicity detection Co, promega, USA) absorbance at 0nm was measured with a spectrophotometer microplate reader. The percentage of LDH release was calculated as 100× (experimental LDH-spontaneous LDH)/(maximum LDH-spontaneous LDH).

2.10 ELISA experiments for IL-1 beta and IL-18

After exposure of piHCKs to LPS (2. Mu.g/ml) (4. Mu.M) with or without WDL and caspase-1 inhibitor Ac-YVAD-cmk (200. Mu.M), cell culture supernatants were collected, centrifuged at 1000 Xg for 20min, and then assayed for secretion of IL-1. Beta. And IL-18 in the cell supernatants using an enzyme-Linked immunosorbent assay (ELISA) kit (WH. Yi Rate Biotechnology Co., china).

2.11 statistical analysis

Statistical analysis of data was performed using GraphPad Prism version 7. Data are expressed as mean ± Standard Deviation (SD). Differences between the two groups were analyzed using unpaired T-test data. And analyzing the differences among the multiple groups by adopting single-factor analysis of variance. P < value of 0.05 was considered to have significant statistical differences.

3. Results

3.1 non-classical focal death-related factors are overexpressed in the cornea of clinical keratitis patients

As can be seen from the photograph in fig. 1A, three cases of patients with pseudomonas aeruginosa keratitis exhibited corneal epithelial defects in the cornea, purulent stroma infiltration with obvious mucopurulent exudates, neovascularization, hypotonic, and conjunctival congestion, as compared to normal, healthy cornea.

Cornea samples from patients with Pseudomonas aeruginosa keratitis and normal control cornea samples were collected and analyzed for protein expression of Caspase-4, hemi-Caspase-5 and GSDMD-N by immunoblotting (FIGS. 1B-E). Protein levels of these factors are significantly elevated in monal keratitis. Cornea samples of Pseudomonas aeruginosa keratitis were higher than those of the control group (active Caspase-4, caspase-5: P <0.01; GSDMD-N: P < 0.05). mRNA levels controlling caspase-4, caspase-5 and GSDMD were further confirmed in three cases of Pseudomonas aeruginosa keratitis patients using qRT-PCR. As shown in FIGS. 1F-H, mRNA expression levels associated with factors such as Caspase-4, caspase-5 and GSDMD were significantly elevated in the cornea of Pseudomonas aeruginosa keratitis compared to the control cornea samples (Caspase-4, caspase-5: P <0.05; GSDMD-N: P < 0.00). That is, compared with a normal cornea sample, the cornea expression level of atypical scorch related factors in the pseudomonas aeruginosa keratitis is obviously increased, so that the pathogenesis of the pseudomonas aeruginosa keratitis can be shown.

3.2 high expression of non-classical Coke death-related factors in the cornea of Pseudomonas aeruginosa infected rats

To further confirm the high expression of non-classical focal death-related factors in pseudomonas aeruginosa keratitis, the present experiment was conducted in animal experiments to further verify. Fig. 2A is a photograph of an established rat model of pseudomonas aeruginosa keratitis. During the pilot experiments, the expression of related factors other than classical focal death was significantly increased in the infected eyes after 1 day of molding, so that the next experiments were chosen to be performed 1 day after infection. Caspase-11 (a homologue of Caspase-4/5) is a cysteine protease identified as a specific molecular marker of non-classical pyrodeath.

This experiment first verifies that Pseudomonas aeruginosa infection triggers caspase-11 mediated apoptosis.

FIGS. 2B-C show that protein expression levels of active caspase11 and GSDMD-N in keratitis were analyzed by immunoblotting, and that the results were higher in both infected corneas than in the control group. (caspase-11: P <0.01, and GSDMD: P < 0.05). qRT-PCR results also showed that the levels of caspase-11 and GSDMD were higher in the Pseudomonas aeruginosa keratitis group than in the control group (see FIG. 2D; caspase-11 and GSDMD: P < 0.05). Meanwhile, in order to demonstrate that activation of atypical pyrosis is caused by corneal infection, not by corneal injury alone, a "injury-only" control group was also established. The results showed that there was no significant increase in caspase-11 and GSDMD expression in the "injury only" group compared to the control group, indicating that injury alone did not activate atypical pyrosis.

3.3LPS (lipopolysaccharide) induced non-classical pyro-apoptosis of cultured primary human corneal machinery cells (piHCKs) in vitro

Corneal stromal fibroblasts (active stromal cells) promote entry of inflammatory cells into the cornea under inflammatory conditions. Thus, the experiment selects LPS-induced primary human corneal fibroblasts as a cell model. The viability of piHCKs after 24h exposure at various concentrations of LPS (0, 0.25,0.5,1and 2. Mu.g/ml) is shown by the results of the CCK-8 assay (see FIG. 3A). LPS inhibits proliferation of piHCKs in a concentration-dependent manner; at concentrations of 0.25,0.5,1and 2 μg/ml LPS, there was a significant difference in inhibition (P < 0.0001). After 48h of stimulation with LPS, the release of cytoplasmic Lactate Dehydrogenase (LDH) in the supernatant was used as an indicator of cell lysis during cell apoptosis.

LDH levels in the culture supernatants of the LPS group increased significantly with increasing LPS concentration (P < 0.001) compared to the control group (see figure 3B). Western blot analysis (FIG. 3C-F) showed that after 48h of LPS treatment, relative to Western blot analysis (FIG. 3C-F) showed that after 48h of LPS treatment, relative levels of active caspase-4, active caspase-5 and GSDMD-N proteins increased significantly, although the differences were only statistically significant (P < 0.01) at LPS 1and 2. Mu.g/ml. qRT-PCR results (see FIG. 3G-I) showed significant increases in relative expression levels of mRNA associated with caspase-4, caspase-5 and GSDMD after 24 hours of LPS 1and 2 μg/ml treatment. Immunofluorescence images (FIG. 3J) showed a significant increase in fluorescence intensity of caspase-4/5 (homology of caspase-11 in humans) in piHCK. Immunofluorescence images (FIG. 3J) showed a significant increase in fluorescence intensity of caspase-4/5 after treatment with 2. Mu.g/ml LPS in piHCKs compared to control. Thus, an increase in atypical pyro-related factors in LPS-treated piHCKs suggests that LPS may induce atypical pyro-death, thereby participating in keratitis lesions of P.aeruginosa keratitis.

3.4 inhibition of LPS-induced atypical pyrosis of piHCKs by wedelolactone and protection thereof

In order to verify the inhibition of wedelolactone, wedelolactone was used in this experiment to treat piHCKs for 2h in advance, and then LPS was used to stimulate cells to induce apoptosis. The safe concentration of wedelolactone on piHCKs was first determined using the CCK-8 kit, as shown in FIG. 4A, wedelolactone at a concentration of 1. Mu.M to 10. Mu.M induced an improvement in cell viability compared to untreated cells, and thus, wedelolactone at concentrations of 1, 2, 4. Mu.M was selected for subsequent experiments. Wedelolactone also reversed improvement in dose-dependent manner of induced cell proliferation inhibition with 2.0 μg/ml LPS compared to control group (as shown in figure 4B).

This experiment demonstrates that non-classical focal death-related factors are significantly elevated under LPS induction, but with increasing wedelolactone concentration, the expression of all atypical focal death-related factors is reduced (see fig. 4C-F). Further analysis, as shown in FIGS. 4G-I, showed the same trend for expression of mRNAs associated with caspase-4, active caspase-5 and GSDMD. Therefore, the results show that wedelolactone can target the related factors of atypical pyrodeath and can reduce the activity of the atypical pyrodeath factors in vitro.

To further verify the effect of classical and non-classical apoptosis on pseudomonas aeruginosa keratitis, the experiment continued with pretreatment of piHCKs with wedelolactone (4 μm) or caspase-1 inhibitor Ac-YVAD-cmk (200 μm) or wedelolactone (4 μm) in combination with Ac-YVAD-cmk (200 μm) for 2h, followed by stimulation of cells with LPS, western blot detection results are shown in fig. 4J-O, which inhibited activation of IL-1 β/18 and GSDMD, in comparison to LPS-treated groups, although Ac-YVAD-cmk pretreatment did not reduce caspase-4/5 expression. In addition, the expression of IL-1 beta/18 and GSDMD can be reduced by combining wedelolactone (4. Mu.M) with Ac-YVAD-cmk (200. Mu.M) with wedelolactone alone or with Ac-YVAD-cmk alone. These results all indicate that wedelolactone is involved in the pathological changes of pseudomonas aeruginosa keratitis as an atypical focal death inhibitor.

To further verify the effect of wedelolactone and Ac-YVAD-cmk on cell apoptosis, IL-1β/18 (IL-1β and IL-18) in cell supernatants was tested by ELISA, only that IL-1β/18 was significantly increased after LPS treatment, although the expression level of IL-1β/18 was significantly lower in wedelolactone-only and Ac-YVAD-cmk-only groups than in wedelolactone-only and Ac-YVAD-cmk-only groups, the wedelolactone-4 μm-Ac-YVAD-cmk-only (200 μm) -combined groups were significantly reduced compared to wedelolactone-only and Ac-YVAD-cmk-only treatments.

3.5 Wedeliolactone attenuation experiment in adjuvant treatment of Pseudomonas aeruginosa keratitis injury and atypical focal death activation

As shown in fig. 5A, photographs of the different groups of infected corneas were taken with a slit lamp to show the symptoms after day 5 of infection. Figure 5A shows that treatment with ciprofloxacin + DMSO (dimethyl sulfoxide) can be improved, exhibiting dense turbidity and partial or complete coverage of the pupil on day 5 of infection. The infected cornea group treated with ciprofloxacin+wdl (wedelolactone) showed more improvement than the other group with only slight central haze.

HE staining results (see fig. 5B) further confirm the observed symptom response. After infection by Pseudomonas aeruginosa, the stroma edema thickens and the anterior chamber of the eye is infiltrated with a large number of inflammatory cells. However, although treatment with ciprofloxacin + DMSO appears to improve the severity of the disease, matrix degradation is less, but the matrix still appears to be edematous and is accompanied by inflammatory cell infiltration. After ciprofloxacin + WDL treatment, there was only a small amount of stromal edema and inflammatory cell infiltration was significantly reduced. Immunohistochemical staining of caspase-11 confirmed activation of atypical pyrodeath. Positive staining of caspase-11 in keratitis had very high expression levels compared to ciprofloxacin+wdl group (see fig. 5C). After scraping the epithelium and damaging the stromal layer of the cornea, the normal structure of the cornea is destroyed. Based on the immunohistochemical results, the foremost layer with increased caspase-11 expression was the ulcerative corneal stroma layer, not the corneal epithelium layer.

To further investigate the effect of WDL on apoptosis, western blotting and qRT-PCR were used to detect caspase-11 and GSDMD expression levels. As shown in FIGS. 6A-B, caspase-11 and GSDMD protein levels were significantly higher in P.aeruginosa keratitis cornea compared to the normal group, and all protein levels were significantly reduced after WDL treatment. P was detected by qRT-PCR. Significant expression levels of mRNA associated with caspase-11 and GSDMD were reversed in corneal Pseudomonas aeruginosa keratitis after WDL treatment in the cornea after Pseudomonas aeruginosa infection (as shown in FIG. 6C).

Considering that pseudomonas aeruginosa keratitis is associated with inflammatory response, the levels of pro-inflammatory cytokines in the different groups were examined. The results demonstrate that the secretion of the pro-inflammatory cytokines IL-6, IL-8 and TNF- α is significantly increased in the keratitis group compared to the control group. However, the WDL treated group was able to prevent a dramatic increase in the inflammatory response in rats (see fig. 6D-G.) these results indicate that WDL reduced pseudomonas aeruginosa-induced keratitis damage by inhibiting atypical pyrosis.

Pseudomonas aeruginosa can cause vision-threatening keratitis. Moderate inflammatory responses help to address infection by promoting leukocyte migration, but uncontrolled inflammatory responses lead to breakdown of corneal tissue, ultimately leading to corneal ulcers and perforation (hazett, 2004). caspase-4/5/11 (caspase-4, caspase-5, caspase-11) mediated non-classical apoptosis has been shown to play a key role in regulating inflammatory responses, but its role in P.aeruginosa has not been studied. In the present invention, studies in the subject group of applicant found that caspase-4/5/11/GSDMD expression was up-regulated in corneal tissue, pseudomonas aeruginosa keratitis rats and lipopolysaccharide-induced primary cultured human keratocytes (piHCKs) in patients with Pseudomonas aeruginosa keratitis. And it is verified that WDL can inhibit caspase-4/5/11/GSDMD mediated focal death of non-classical focal death pathway, so WDL can be applied in treatment of Pseudomonas aeruginosa keratitis, thereby providing new direction and thought for Pseudomonas aeruginosa induced keratitis and cornea damage thereof.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

Claims

1. Application of wedelolactone in preparing medicine for treating inflammatory reaction of pseudomonas aeruginosa keratitis.

2. Use according to claim 1, characterized in that wedelolactone reduces the inflammatory response in pseudomonas aeruginosa keratitis and improves prognosis by inhibiting caspase-4, caspase-5, caspase-11, GSDMD mediated non-classical pyrodeath.

3. The use according to claim 1, wherein the medicament comprises wedelolactone and a pharmaceutically acceptable adjuvant.

4. The use according to claim 3, wherein the medicament further comprises an antibiotic.

5. The use according to claim 4, wherein the antibiotic is ciprofloxacin.

6. The use according to claim 3, wherein the pharmaceutical adjuvant comprises a diluent.

7. The use according to claim 6, wherein the medicament is an eye drop.