阅读说明：本技术 褪黑素在制备治疗外伤性白内障药物中的应用 (Application of melatonin in preparation of medicine for treating traumatic cataract ) 是由 周健 郑超 吴桐 于 2021-10-28 设计创作，主要内容包括：本发明公开了褪黑素在制备治疗外伤性白内障药物中的应用。褪黑素在应激的晶状体上皮细胞中,对ATF6的上调具有显著的抑制作用,腹腔注射褪黑素能够显著抑制小鼠晶状体在针刺诱导后EMT的发生,进而抑制晶状体的混浊,实验结果表明褪黑素可以达到治疗外伤性白内障的目的。(The invention discloses an application of melatonin in preparing a medicament for treating traumatic cataract. The melatonin has a remarkable inhibiting effect on the up-regulation of ATF6 in stressed lens epithelial cells, the generation of EMT (acute myocardial infarction) of mouse lenses after needle stick induction can be remarkably inhibited by intraperitoneal injection of the melatonin, and further the opacity of the lenses is inhibited, and experimental results show that the melatonin can achieve the purpose of treating traumatic cataract.)

1. Use of an inhibitor of unfolded protein response in the manufacture of a medicament for the treatment of traumatic cataract.

2. Use according to claim 1, characterized in that: the unfolded protein response inhibitor is selected from the group consisting of activating transcription factor 6 inhibitors.

3. Use according to claim 1 or 2, characterized in that: the unfolded protein response inhibitor is selected from melatonin or CeapinA 7.

4. Use of melatonin in the preparation of a medicament for the treatment of traumatic cataract.

5. Use according to claim 4, characterized in that: the melatonin inhibits unfolded protein response dependent epithelial-mesenchymal transition.

6. Use according to claim 4 or 5, characterized in that: the melatonin down-regulates expression of markers associated with the occurrence of epithelial-mesenchymal transition.

7. Use according to claim 6, characterized in that: the marker is selected from the transcription factor Snail 1.

Use of CeapinA7 in the manufacture of a medicament for the treatment of traumatic cataract.

9. Use according to claim 1, 4 or 8, characterized in that: the traumatic cataract is caused by perforation injury or contusion of eyeball.

10. Use according to claim 1, 4 or 8, characterized in that: the route of administration of the drug is intramuscular, subcutaneous, intravenous, intracameral, vitreous or peribulbar injection.

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of melatonin (melatonin) in preparation of a medicine for treating traumatic cataract.

Background

Traumatic cataract refers to clouding of the lens caused by disruption of lens integrity and induction of fibrosis or scar repair of the lens. Traumatic cataract is one of the common complications after perforation injury or contusion of eyeball, is commonly seen in children and young people, and has a blindness rate of 47.5%. Surgical removal of the crystalline lens is a well-established method for treating traumatic cataract, but there is increasing evidence that there are many risks of surgery and strong vitreoretinal adhesions due to postoperative complications, intraocular inflammation. At present, except for symptomatic treatment of relieving inflammation, resisting infection and the like, no medicine for intervening the pathological process of lenticular opacity caused by trauma is clinically available, so that the traumatic cataract has a disease clinical management gap which is not overlooked between conservative treatment and operative treatment.

The early studies prove that EMT is the main cause of traumatic cataract, namely, the level of various cytokines and growth factors is increased in the aqueous humor after trauma, stimulation or operation, Lens Epithelial Cell (LEC) proliferation is stimulated and the epithelial phenotype is changed into the mesenchymal phenotype, and a mouse disease model of traumatic cataract is established as the research basis.

Melatonin is an indole heterocyclic compound secreted by pineal body, the production and release of which are regulated by sympathetic nerves, can regulate the circadian rhythm and seasonal reproduction of organisms, and has remarkable anti-inflammatory, antioxidant and anti-aging properties. It has been demonstrated to have certain therapeutic effects in age-related cataracts and diabetic cataracts. In the research of age-related cataract, melatonin is used as an indirect antioxidant, which can reduce the lipid peroxidation level of rats, increase the production of glutathione and increase the activity of antioxidant enzyme of cataractous rats; in the diabetic rat cataract induced by STZ, melatonin can reduce aldose reductase activity and slow down the occurrence of diabetic related cataract; in vitro culture of rabbit lenses under high sugar conditions can inhibit cataract formation by melatonin intervention (quhong, experimental study of melatonin on inhibition of high sugar-induced cataract formation). Recent studies found that melatonin can also play a protective role as an ATF6 inhibitor in brain injury secondary to cerebral hemorrhage (dawn yang, protective role of melatonin/ATF 6/CHOP pathway in brain injury secondary to cerebral hemorrhage and mechanism studies thereof).

Chinese patents CN111153846A, CN111153850A report pyrrole, indole compounds that can be used for treating related diseases (including cataract) with ID01 mediated pathological features of the metabolic pathway of fist-urea-ammonia acid. However, the cataract mentioned in these patents is caused by the bad differentiation and apoptosis of lens fiber cells caused by abnormal generation of amino acids, and has obvious difference in the type and pathogenesis of cataract, and the treatment target point from traumatic cataract (radial opacity is formed by the liquid flowing between lens fiber and lens fiber plate layer due to the structural damage of lens fiber and lens fiber (Y-shaped seam), or opacity is formed due to tissue damage, lens fiber degeneration and scar formation).

Although studies report that there is a self-limited natural history of lens capsule rupture without developing progressive cataract, suggesting that non-surgical conservative drug intervention is feasible, the drug efficacy developed for cataract remains unclear, and traumatic cataract is more rapid and is not yet a drug management strategy at present. Therefore, the search for effective pathogenic targets of traumatic cataract and the development of reasonable drug intervention strategies are urgent needs for clinical disease management.

Disclosure of Invention

The invention aims to provide application of melatonin in preparing a medicine for treating traumatic cataract.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to the discovered pathogenesis of the traumatic cataract, namely, the Unfolded Protein Response (UPR) dependent epithelial-mesenchymal transition (EMT), the Unfolded Protein Response (UPR) inhibitor is given in an in-vitro stress model of lens epithelial cells for intervention, for example, 100-200 mu M melatonin is given in the model, and 5-20 mg/kg/d melatonin is given in a disease model of the traumatic cataract caused by mouse acupuncture, so that the up-regulation of an activating transcription factor 6(ATF6) can be obviously inhibited, the UPR mediated by ATF6 is blocked, the EMT dependent by the UPR is inhibited, and the aim of treating the traumatic cataract is finally achieved.

The invention has the beneficial effects that:

the in vivo and in vitro pharmacological experiments prove that the medicament for inhibiting the up-regulation of ATF6 can effectively inhibit the lenticular opacity caused by stimulation factors such as perforation injury or contusion of eyeballs. The experimental result shows that UPR inhibitors (such as a non-specific ATF6 inhibitor melatonin and a specific ATF6 inhibitor CeapinA7) can be developed into clinical treatment drugs for traumatic cataract.

Drawings

FIG. 1 is a schematic representation of a traumatic cataract mouse model; wherein: (A) mouse lens acupuncture model modeling (LECs: lens epithelial cells); (B) postoperative observation of scar under anterior capsule and lenticular opacity.

FIG. 2 shows the results of the morphological changes, histopathology and molecular marker detection of the traumatic cataract mouse model; wherein: (A) performing lens morphology observation after 7d of modeling operation (the upper part of the figure is photographed by a living slit lamp, the lower part of the figure is photographed by an in vitro lens), (B) performing HE staining observation on morphological characteristics of modeling parts (the lower part of the figure is a magnified result of a boxed area on the upper part of the figure), (C) performing immunofluorescence staining detection on the expression of EMT markers alpha SMA and Fibrinectin (FN) of the modeling parts at plaques under the capsule.

Fig. 3 shows the result of the toxicity test of CCK8 drug (melatonin).

FIG. 4 shows the results of detecting the expression of UPR and EMT-associated markers in each stable transgenic cell line; wherein: (A) representative wb (western blot) results for ATF6 and Snail1 protein expression; (B) RT-qPCR results of ATF6, Bip, CHOP, Snail1, fibrinectin, alpha SMA, N-cadherin mRNA expression,^*P<0.05、^**P<0.01。

fig. 5 shows the in vivo intervention outcome of melatonin on traumatic cataract; wherein: (A) morphological observation of crystalline lens, (B) expression of EMT marker of anterior lens capsule (slide immunofluorescence staining), (C) morphological feature of modeling part observed by HE staining, and (D) immunofluorescence staining of frozen section of crystalline lens.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

Pathogenesis of traumatic cataract

It was experimentally found that the mouse disease model of traumatic cataract activated Unfolded Protein Response (UPR) on the endoplasmic reticulum and confirmed that UPR-dependent epithelial-mesenchymal transition (EMT) is the main pathogenesis of traumatic cataract, i.e. mechanical injury stresses the lens epithelium, activates to activate transcription factor 6(ATF6) -mediated UPR, ATF6 further transcriptionally activates the EMT important transcription factor SNAIL1, drives lens epithelial cell proliferation and transitions from epithelial to mesenchymal phenotype, and mediates fiber repair by secreting large amounts of extracellular matrix proteins (e.g. collagen, fibronectin, etc.) eventually leading to clear lens opacification. Thus, targeted inhibition of UPR using drugs may be an effective intervention strategy for traumatic cataracts.

(II) establishment of model of traumatic cataract caused by acupuncture

Referring to FIG. 1, C57BL/6J mice were selected for the left eye as the experimental eye and the right eye as the control eye. The mice were anesthetized with 1% pentobarbital sodium by a conventional method, then mydriasis was performed with compound tropicamide eye drops, the cornea (punch cornea) was punctured at the central position beside the cornea of the experimental eye with a 32G sterile syringe needle (needle) under a body microscope, the needle was further inserted until the needle point of 1/3 puncture the anterior capsule, gatifloxacin gel was dropped on the surface of the cornea after the operation, and the degree of opacity of the anterior capsule scar (subensular plane) and the lens (lens) was observed 7 days later.

(III) lens morphology, histopathology, molecular marker detection

3.1 Observation of lens morphology

Carrying out cataract phenotype observation and photographing by using a slit lamp after mydriasis of the mouse; taking a picture under a retrobulbar microscope of the mouse, and observing the damage repair condition and the anterior chamber environment; the lens of the mouse eyeball was isolated and photographed under an inverted microscope using transillumination.

3.2 pathological staining of the lens

Sacrificing a mouse in the drawing time, picking out eyeballs, adding a proper amount of Davidson stationary liquid, fixing the whole eyes at room temperature overnight, washing a specimen by PBS, dehydrating by 30% sucrose, and slicing after OCT embedding; after the frozen section is rewarming, washing off the embedding medium by PBS, carrying out conventional hematoxylin-eosin (HE) staining, and observing the forming range, the form and the cell components of the lens fiber plaque at the rupture and damage part of the capsular sac.

3.3 immunofluorescence in situ detection of UPR and EMT molecular markers

Taking the lens material and preparing a frozen sheet as same as 3.2, carrying out immunofluorescence staining by using an ATF6 antibody, detecting the expression change of ATF6 protein, and determining the relationship between traumatic cataract and UPR activation; the same 3.2 is used for obtaining the crystalline lens and freezing the section, the alpha-SMA and the fibrinectin antibody are respectively used for immunofluorescence staining, the expression of the alpha-SMA and the fibrinectin is detected, and the repairing mode of the damaged part is determined.

Day 7 post needle induction, lens was completely cloudy (fig. 2A); scarring of the needle puncture site (FIG. 2B) and the development of EMT (FIG. 2C) were observed on the cryosections.

(IV) melatonin cytotoxicity test

4.1 lens epithelial cell line culture

Human lens epithelial cell line (HLE-B3) was cultured in low sugar DMEM (containing 10% fetal bovine serum, 100IU/mL cyan/streptomycin).

4.2CCK8 cell proliferation assay

Preparing single cell suspension, inoculating a 96-well plate according to the density of 2000 cells/well after counting, arranging 5 multiple wells in each group, respectively adding melatonin with the concentration gradient of 0 (a control group), 5, 10, 20, 50, 100 and 200 mu M for incubation for 24 hours after the cells are attached to the wall 2 hours, respectively adding 10 mu L of CCK8 cell counting solution into each well for uniformly mixing, incubating for 1 hour in an incubator, measuring the absorbance OD (OD) values of 5 wells and blank wells (only containing cell culture medium and CCK8 reagent) in each group (different melatonin concentrations among groups) at the wavelength of 450nm by using an enzyme reader, and calculating the cell proliferation activity of each group:

proliferation activity ═ 100% (mean OD value in experimental group-mean OD value in blank group)/(mean OD value in control group-mean OD value in blank group) ×

After three independent repeated experiments, the difference of proliferation activity among cell groups of each well is compared. The experimental result shows that melatonin with the concentration of 5-200 mu M has no influence on the vitality of lens epithelial cells (figure 3).

(V) in vitro stress model and intervention experiment of lens epithelial cells

5.1 in vitro Experimental groups

The experimental groups of UPR agonist (Tunicamycin, TM) administered in combination with shRNA-ATF6 (i.e., shRNA expression vector for silencing ATF6 gene, shATF6), ATF6 inhibitor (melatonin), positive control drug (CeapinA7) in vitro were as follows: control group (normal cultured cells only, Ctrl group), TM + PBS group, TM + shATF6 group, TM + cerapin a7 group, and TM + melatonin group.

Preparing single cell suspension, counting, and weighing at 2 × 10⁵Inoculating 6-pore plates at the density of each pore, setting 3 multiple pores in each group, adding TM and corresponding experimental medicaments into each group (except Ctrl group) 2h after the cells adhere to the wall, incubating for 24h, removing the culture medium, and extracting total protein and mRNA to be detected by using the kit. In the experiment, TM was added to each well to a final concentration of 4. mu.g/mL, shATF6 MOI value of 50, and CeapinA7 administration concentration of 10. mu. M, melatonin administration concentration of 100. mu.M.

5.2 detection of UPR-dependent changes in EMT cell levels

(1) Western blot

Pouring 12% separation gel and 6% concentrated gel into electrophoresis buffer (1X), wherein the loading amount of the protein sample is 40 μ g, and 5 μ L of pre-dyed protein standard is added; protein separation is completed by using concentrated gel of 90V and separation gel of 110V and electrophoresis time of about 110 min; taking out the gel, putting the gel into a transfer buffer solution (1 x), performing constant current of 400mA for 2h, electrically transferring the protein on the gel to a PVDF membrane, sealing skim milk for 2h, soaking a target strip in a 1:1000 diluted ATF6, Snail1 and GAPDH primary antibody solution, and standing overnight at 4 ℃; PVDF membrane was washed 3 times for 10 minutes each using 1 × TBST; after incubation for 1 hour at 37 ℃ with the corresponding secondary antibody, the PVDF membrane was washed 3 times for 10 minutes each using 1 XTSST; the hypersensitivity ECL luminescence solution is dropped on PVDF membrane in dark room and exposed, the strip gray value is calculated by using ImageJ software, and the statistical analysis is carried out by using GraphPad Prism6 software.

(2)RT-qPCR

And (3) measuring the absorbance of the extracted cell total RNA at 260nm and 280nm by using an ultraviolet spectrophotometer, and calculating the content and purity of the sample to be detected. Reverse transcription reaction (20. mu.L reaction system) was carried out using 2. mu.g of the extracted total RNA, and the specific procedure was described with reference to the reverse transcription reaction kit. PCR was carried out using the cDNA obtained by reverse transcription, and the reaction system (10. mu.L of cDNA 1. mu. L, SYBR probe, 1. mu.L of forward primer, 1. mu.L of reverse primer, and 7. mu.L of RNase-free water) was mixed and amplified by machine. Reaction conditions are as follows: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 15s for 45 cycles. Wherein, the positive primer and the reverse primer are respectively designed according to the fact that the substances to be detected are ATF6, Bip, CHOP, Snail1, alpha SMA, N-cadherin and fibrinectin mRNA (namely target genes), and after the reaction is finished, the expression level of each target gene is analyzed by taking beta-actin as an internal reference.

Upon examination of the UPR and EMT markers (fig. 4), it was found that ATF6 and the EMT key transcription factor Snail1 still exhibited significant synergistic upregulation in the TM + PBS group, but that the upregulation of Snail1 by UPR agonists was abolished after melatonin action (i.e. in the TM + melatonin group) or cepaina 7 action (i.e. in the TM + cepaina 7 group).

(VI) disease model of traumatic cataract caused by mouse acupuncture and intervention experiment

6.1 mouse dosing strategy

An 8-week C57BL/6J mouse was used to establish a needle-induced traumatic cataract model and was randomized into two groups:

melatonin group: dissolving melatonin (melatonin) powder in corn oil containing 5% DMSO, performing ultrasonic treatment until completely dissolved, and performing intraperitoneal injection of melatonin (n-3) to C57 mice at 8mg/kg dose 10 o' clock every day later;

control group: another mouse from the same model (n ═ 3) was intraperitoneally injected daily with the same volume of solvent (corn oil, 5% DMSO).

Two groups of mice were photographed in the anterior segment of the living body on day 7 after the induction of acupuncture, and the effect of melatonin on the traumatic cataract phenotype (lenticular opacity) was observed by taking the material and pathologically staining.

6.2 melatonin in vivo significantly improved traumatic cataract phenotype

On the basis of a mouse needle-induced traumatic cataract model, melatonin (8mg/kg/D) is injected into the abdominal cavity continuously, and as a result, the degree of lenticular opacity of melatonin mice is reduced after 7 days (fig. 5A), the expression of ATF6 and the expression of the EMB marker fibrinectin is reduced by immunofluorescence staining of anterior lens capsule membranes (fig. 5B), the morphological characteristics of the molding part of the melatonin group are approximately disappeared by HE staining (fig. 5C), and the expression of ATF6 of the molding part is reduced by immunofluorescence staining of frozen lens sections (fig. 5D).

On the basis of a mouse model with traumatic cataract caused by acupuncture, a solvent is continuously injected into the abdominal cavity, and as a result, obvious turbidity can be seen after 7 days of slit lamp photographing of a control group, local plaques are formed on lesions visible by anterior lens capsule film tablet paving of the control group, ATF6 and the expression of an EMT marker fibrinectin are formed, and obvious scar plaques are formed by capsule film curling and breaking repair visible by HE staining of the control group.

The above results demonstrate that melatonin can significantly inhibit EMT from occurring, thereby inhibiting opacity of the lens.

In conclusion, melatonin has a remarkable inhibiting effect on the up-regulation of ATF6 in stressed lens epithelial cells, and intraperitoneal injection of melatonin can remarkably inhibit the occurrence of EMT of mouse lenses after acupuncture induction, so that opacity of the lenses is inhibited. Therefore, the UPR inhibitors such as melatonin, CeapinA7 and the like are feasible to treat the traumatic cataract, the action target point is consistent with the research and discovery of experimental theories, and the urgent clinical requirement on the drug treatment of the traumatic cataract at present can be met.