CN112716938A

CN112716938A - Application of ellagic acid in preparing medicine for relieving ocular tissue pathological changes

Info

Publication number: CN112716938A
Application number: CN202110211278.3A
Authority: CN
Inventors: 张之; 张燕; 黄琦敏; 李倩; 祝颖玲; 加娜尔·依布拉阿洪; 王青; 古丽海夏; 古丽米拉
Original assignee: Xinjiang Medical University
Current assignee: Xinjiang Medical University
Priority date: 2021-02-25
Filing date: 2021-02-25
Publication date: 2021-04-30

Abstract

The invention relates to the technical field of treating ocular tissue pathological changes, and particularly discloses application of ellagic acid in preparing a medicine for relieving ocular tissue pathological changes, wherein the ellagic acid can regulate and control the increase of LC3b/a protein expression level and the decrease of p62 protein expression level in eyeball tissues of diabetic mice, improve autophagy disorder of the diabetic ocular tissues, relieve pathological damage of the ocular tissues of the diabetic mice, inhibit ocular tissue inflammation NLRP3 and IL-1 beta protein expression, inhibit ocular tissue apoptosis protein c-caspase1 expression, and inhibit the expression of mouse ocular tissue apoptosis proteins GSDME, GSDME-N and GSDMDM-N, so that GSDMD and GSDME mediated cell apoptosis are inhibited, and an anti-inflammatory protection effect is generated on cells. The ellagic acid provided by the invention can relieve pathological damage of eye tissues of diabetic mice and has a protective effect on retinas of the diabetic mice.

Description

Application of ellagic acid in preparing medicine for relieving ocular tissue pathological changes

Technical Field

The invention relates to the technical field of treating eye tissue pathological changes, in particular to application of ellagic acid in preparing a medicine for relieving eye tissue pathological changes.

Background

Diabetes is a common endocrine disease, the greatest harm is that the diabetes can cause a plurality of chronic complications, and ocular diseases are one of the most common chronic complications of diabetes, and are called diabetic eye diseases. Among them, Diabetic Retinopathy (DR) is one of serious microvascular and neurological complications, and glycolipid metabolic disorders cause damage to diabetic ocular tissue cells, which may lead to ocular tissue structural and functional abnormalities. The cell autophagy can timely eliminate damaged proteins or organelles in cells, restore cell homeostasis and reduce apoptosis of eye tissues. Apoptosis is a newly discovered mode of programmed cell death. After the inflammasome is activated, caspase-1/4/5/11 is cut, the cut mature cysteine acts on gasdermin D (dermolide D, GSDMD), the formed GSDMD-N end forms a hole on a cell membrane through oligomerization, water inflow is generated, ion gradients inside and outside the cell membrane disappear, and the cell is swollen, osmotically dissolved and finally cracked to die. Ellagic acid is a natural plant polyphenol component, and natural antioxidant, and has effects of activating autophagy and inhibiting lung cancer cell proliferation. Ellagic acid is widely present in natural polyphenols of various soft fruit nuts such as pomegranate, blueberry, grape, red raspberry and the like, and is also an important active ingredient in traditional Chinese medicines such as phyllanthus emblica, myrobalan, gallnut and the like. According to the domestic and foreign literature reports: high carbohydrate induced lysosomal damage and autophagy dysfunction are early events that occur during the pathological process of DR, ultimately leading to abnormalities in ocular tissue structure and function. At present, western medicines are mainly used for expanding and dredging stasis blood vessels of diabetic eye patients through chemical components, belong to the category of treatment symptoms and cannot fundamentally solve diabetic eye diseases, and meanwhile, the long-term hyperglycemia of the diabetic eye patients causes damage to organs such as heart, liver and kidney, and the western medicines further aggravate damage to cause adverse reactions such as cardiovascular and cerebrovascular diseases, renal failure and the like. In the prior art, natural substances with few adverse reactions specially used for improving diabetic eye tissue lesions are few, and whether the ellagic acid can improve the diabetic eye tissue lesions (including retinopathy) is not reported.

Disclosure of Invention

In order to solve the technical problems, the invention provides the application of ellagic acid in preparing a medicine for relieving eye tissue pathological changes.

Further, the ellagic acid is used for preparing a medicament for relieving diabetic ocular tissue pathology.

Furthermore, the ellagic acid is used for preparing a medicament for relieving pathological damage of eye tissues of diabetic mice.

Further, the ellagic acid is used for preparing a medicament for regulating and controlling the reduction of p62 protein expression in eyeball tissues of diabetic mice.

Further, the ellagic acid is used for preparing a medicament for regulating and controlling the increase of LC3b/a protein expression in eyeball tissues of diabetic mice.

Further, the ellagic acid is used for preparing a medicament for inhibiting the expression of mouse eye tissue inflammatory proteins NLRP3 and IL-1 beta.

Further, the ellagic acid is used for preparing a medicament for inhibiting the expression of mouse eye tissue apoptosis protein clear-caspase 1.

Further, the ellagic acid is used for preparing a medicament for inhibiting the expression of mouse eye tissue apoptosis proteins GSDME, GSDME-N and GSDMDM-N.

Further, the ellagic acid is used for preparing a medicament for relieving diabetic retinopathy.

Further, the ellagic acid is used for preparing a medicament for relieving mouse diabetic retinopathy induced by the ureaplasma urealyticum.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides application of ellagic acid in preparing a medicament for preventing, treating and relieving ocular tissue pathological changes;

2. the ellagic acid disclosed by the invention can be used for preventing, treating and relieving pathological injury of eye tissues of diabetic mice, reducing edema, inflammation and bleeding of the eye tissues, reducing capillary congestion of the eye tissues, reducing retinal hemorrhage and congestion, and has a protection effect on the eye tissues of the diabetic mice;

3. the ellagic acid can regulate and control the p62 protein expression level in an eyeball tissue to be obviously reduced, simultaneously promote the LC3b/a protein expression level in the eyeball tissue to be obviously increased, regulate and control the expression quantity of mouse eye tissue inflammatory protein NLRP3 and IL-1 beta to be reduced, regulate and control the expression quantity of mouse eye tissue apoptosis protein cleared-caspase 1 to be reduced, thereby playing a role in relieving pathological damage of the eye tissue of a diabetic mouse;

4. the ellagic acid can regulate and control GSDME, GSDME-N and GSDMDM-N protein expression in eyeball tissues to be obviously reduced, so that cell apoptosis mediated by GSDMD and GSDME is inhibited, and an anti-inflammatory protection effect is generated on cells;

5. the ellagic acid disclosed by the invention can be used for improving autophagy flow disorder of diabetic eye tissues, promoting removal of damaged proteins or organelles, reducing apoptosis of diabetic eye tissue cells and reducing inflammation of the diabetic eye tissues;

6. the invention also provides the application of the ellagic acid in preparing the medicine for relieving diabetic eye tissue lesion;

7. the invention also provides application of the ellagic acid in preparing a medicament for relieving mouse diabetic eye tissue lesion induced by the ureabamectin.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the effect of ellagic acid on the pathologic morphological structure of the eye tissue of a KM diabetic model mouse;

wherein, FIG. a1 shows a microphotograph of pathological morphology of mouse eye tissue in a normal control group (group C) under HE X100;

FIG. a2 shows a photomicrograph of the pathologic morphology of mouse eye tissue of a normal control group (group C) under HE X200;

FIG. b1 shows a microphotograph of pathologic morphology of mouse eye tissue in the diabetic eye disease group (group M) under HE X100;

FIG. b2 shows a photomicrograph of the pathologic morphology of mouse eye tissue in the diabetic retinopathy group (group M) under HE X200;

FIG. c1 shows a microphotograph of the pathologic morphology of mouse eye tissue in the ellagic acid group (EA group) under HE X100;

FIG. c2 shows a microphotograph of the pathologic morphology of mouse eye tissue in the ellagic acid group (EA group) under HE X200;

FIG. 2 shows the effect of ellagic acid on the expression of the KM diabetes model mouse eye tissue marker LC3b/a protein;

wherein, FIG. 2a shows the expression level of the KM diabetes model mouse eye tissue marker LC3b/a protein under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 2b shows a gel diagram of KM diabetic model mouse eye tissue marker LC3b/a protein under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 3 shows the effect of ellagic acid on the expression of the KM diabetes model mouse eye tissue marker p62 protein;

wherein, FIG. 3a shows the expression level of p62 protein in KM diabetes model mouse eye tissue marker under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 3b shows a gel diagram of KM diabetes model mouse eye tissue marker p62 protein under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 4 shows the effect of ellagic acid on the expression of the mouse ocular tissue inflammatory protein NLRP3 and IL-1 β in a model of diabetes;

wherein, FIG. 4a shows the relative expression level differences of the mouse ocular tissue inflammatory proteins NLRP3 and IL-1 beta under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 4b shows graphs of the eye tissue inflammatory protein NLRP3 and IL-1. beta. protein glue of mice under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 5 shows the effect of ellagic acid on the expression of the eye tissue apoptotic proteins caspase1 and clear-caspase 1 in mice model diabetes;

wherein, FIG. 5a shows the relative expression level differences of mouse eye tissue apoptotic proteins caspase1 and clear-caspase 1 under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 5b shows a protein glue pattern of the apoptotic proteins caspase1 and clear-caspase 1 of mouse eye tissue under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 6 shows the effect of ellagic acid on the expression of the eye tissue apoptosis proteins GSDME, GSDME-N, GSDMD and GSDMDM-N in mice model for diabetes;

wherein, FIG. 6a shows the relative expression levels of GSDME, GSDME-N, GSDMD and GSDMDM-N in eye tissue of mice under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group);

FIG. 6b shows the protein glue pattern of mouse eye tissue apoptosis proteins GSDME, GSDME-N, GSDMD and GSDMDM-N under three treatments (group C, normal control group; group M, model group; group EA, ellagic acid group).

Detailed Description

The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific 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. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.

Example 1:

this example 1 provides the use of ellagic acid in the preparation of a medicament for alleviating ocular tissue pathologies.

The improvement effect of ellagic acid intervention on streptozotocin-induced diabetic mouse retinopathy was studied as follows:

materials and instruments

Glucometer, test paper, electronic scale, electronic balance, ellagic acid, Streptozotocin (STZ);

secondly, the specific experimental operation process is as follows:

1. animal grouping and model establishment

30 KM mice were prepared and the environment was controlled at 22 + -1 deg.C and 30-40% relative humidity in a clean environment with a relatively constant temperature and humidity. Randomly drawing 10 normal control groups (group C) and feeding the normal control groups with common feed;

the other mice were randomly and equally divided into diabetic ocular disease group (group M) and diabetic ocular disease + ellagic acid group (group EA), both groups were fed with high fat diet, after 6 weeks, each group was administered with Streptozotocin (STZ) for intraperitoneal injection to induce diabetes model, STZ was dissolved in 0.1mol/L citric acid buffer solution of pH 4.2 before use to prepare 1% STZ solution, and the concentration was 85 mg/kg^-1Injecting for 2 times, taking tail blood after 72h to measure blood sugar and blood sugar>11.1mmol/L indicates successful molding.

2. Pharmaceutical intervention

Ellagic acid group (EA group) in an amount of 50 mg/kg^-1·d^-1Ellagic acid was administered by gavage for 8 weeks, and distilled water was administered in equal amounts by gavage to a normal control group (group C) and a diabetic eye disease group (group M).

3. Study of the Effect of Ellagic Acid (EA) on Water intake and body weight in diabetic mice

And (5) fasting for 6 hours without water prohibition at the end of 1, 3 and 5 weeks, selecting mice, weighing the mice and drinking water.

4. Preparation of eyeball specimen and HE staining

After the ellagic acid intervention is performed for 8 weeks, the mice are fasted and are not forbidden to be water for 6 hours, after the mice are anesthetized by 10% chloral hydrate, fresh tissues of double eyeballs of the mice are taken and placed in a neutral formaldehyde solution with the volume fraction of 10% for fixation, after paraffin embedding, the mice are sliced into 5 mu m slices, HE staining is performed, and pathological morphological structure changes of retina tissues are observed under an optical microscope.

5. Ocular tissue protein expression assay

After blood collection is finished, the mice are sacrificed, eye tissues are immediately taken out, protein is extracted, and each group of samples are added into 12% polyacrylamide gel for electrophoresis at 80V constant voltage for about 125 min; when the bromophenol blue reaches the bottom of the glass plate, the electrophoresis is stopped, then a gel is taken out to cut a mesh strip zone, the gel is transferred to a PVDF membrane, 5% of skimmed milk powder is added under a 37C shaking table for sealing for 2h, beta-uctin (1: 1000) is added for incubation at 4C overnight, the membrane is washed for 3 times and 5 min/time in TBST on the next day, a secondary antibody (1:5000) is added for incubation at 37C for 2h, and protein strip images are analyzed by Image lab software.

6. Statistical analysis

Statistical analysis is carried out by adopting SPSS 22.0 statistical software, the measured data is expressed by (x +/-s), single-factor variance analysis is adopted according with normal distribution and homogeneity of variance, and ISD test is adopted for pairwise comparison; the uniformity of the distribution and the variance which do not conform to the normal distribution adopts the rank sum test, and the difference with P less than or equal to 0.05 has statistical significance.

The detection results of the above examples are as follows:

TABLE 1 Effect of EA on body weight in diabetic mice

Group/weight (g)	Week 1	Week 3	Week 5
				Group C	34.45±3.60	37.67±5.28	39.00±5.76
M groups	32.43±3.69	27.14±3.13*	26.57±2.64**
				EA group	33.63±3.70	21.25±13.22	27.40±2.30

As can be seen from table 1, the body weight of the model group (M group) mice decreased significantly both at the third and fifth weeks (P < 0.05P <0.01), with no statistical significance remaining.

TABLE 2 Effect of EA on Water intake in diabetic mice (mL)

group/Water intake (mL)	Week 1	Week 3	Week 5
				Group C	8.46±0.68	5.67±1.57	7.73±2.13
M groups	23.49±3.03**	37.80±7.62**	41.60±0.15**
				EA group	22.40±2.19	31.43±5.22^#	35.65±0.13^#

As can be seen from table 2, the diabetic model mice in group M had significantly increased water intake (. about.p) compared to the normal control group (group C)<0.01), mice of the ellagic acid group (EA group) were significantly decreased in water intake at the third and fifth weeks compared to the model group (M group) ((M group)^#P<0.05)。

TABLE 3 Effect of EA on the pathologic indices of the eye tissue of mice in diabetic model

As shown in table 3, the ocular histopathological indices of the mice in the model group (group M) compared with those in the normal control group (group C): disordered cellular structure, looseness, edema and moderate hyperplasia of each layer; the dilation of the capillary vessel wall and the microvasculature is accompanied by intravascular congestion; severe retinal hemorrhage, congestion; disorder of thickening of the inner boundary layer structure with moderate bleeding;

in the EA group, the anacardic acid stem prognosis, the pathological indexes of the eye tissues of the diabetic mice are more completely arranged than the pathological indexes of each layer of cells in the model group, and endothelial cells slightly proliferate; slight dilatation of capillaries and microvessels; retinal extravasated blood hemorrhage is mild; the inner boundary layer is moderately thickened, and edema is rare; inflammatory reaction is rare, and as shown in figure 1, pathological damage of eye tissues of diabetic mice is obviously relieved after the ellagic acid is dried.

TABLE 4 Effect of EA on the expression of autophagy markers in eye tissues of mice in a model of diabetes

Group of	LC3b/a	p62
			Group C	1.00±0.20	1.00±0.40
M groups	0.14±0.08**	3.28±0.70**
			EA group	0.52±0.10^##	1.61±0.50^##

Note: compared with normal control group^*P<0.05，^**P<0.01; compared with model group^#P<0.05，^##P<0.01; compared with EA group^&&P<0.01

As shown in table 4, compared to the normal control group (group C), the expression level of P62 protein was significantly increased (P <0.01) and the expression level of LC3b/a protein was significantly decreased (P <0.01) in the eyeball tissue of the model group (group M);

compared with the model group (group M), the P62 protein expression level in the eyeball tissue of the ellagic acid group (group EA) is remarkably reduced (P <0.01), and the LC3b/a protein expression level is remarkably increased (P <0.01), which shows that the ellagic acid can inhibit and delay the damage effect of hyperglycemia on the normal autophagy flow of diabetic eye tissues, and is shown in figure 2 and figure 3.

TABLE 5 Effect of EA on the expression of inflammatory proteins in the eye tissue of mice in a diabetes model

Group of	NLRP3	IL-1β
			Group C	1.00±0.16	1.00±0.18
M groups	7.28±0.58^aa	4.74±0.47^aa
			EA group	2.93±0.37^bb	2.05±0.42^bb

Note: compared with the group C, the method has the advantages that,^aP<0.05，^aaP<0.01; in comparison with the set of M,^bP<0.05，^bbP<0.01

as shown in table 5 and fig. 4, the expression of NLRP3 and IL-1 β protein was significantly increased in the eye tissue of the model group (group M) mice (P <0.01) compared to the normal control group (group C); compared with the model group, the expression of NLRP3 and IL-1 beta protein in the eye tissue of the mice in the ellagic acid group (EA group) is remarkably reduced (P < 0.01).

TABLE 6 Effect of EA on the expression of apoptotic proteins in the eye tissue of diabetic mice (X. + -. s)

Group of	caspase1	cleaved-caspase1
			Group C	1.00±0.09	1.00±0.15
M groups	1.05±0.08	5.61±0.32^aa
			EA group	1.05±0.11	2.40±0.36^bb

Note: aP <0.05, aaP <0.01 compared to group C; bP <0.05, bbP <0.01, compared to group M

As shown in Table 6 and FIG. 5, compared with the normal control group (group C), the clear-caspase 1 protein expression was significantly increased (P <0.01) and no significant difference was observed in caspase1 (P > 0.05) in the eye tissue of the mice in the model group (group M); compared with the model group, the expression of clear-caspase 1 protein in the eye tissues of the mice in the ellagic acid group (EA group) is obviously reduced (P <0.01), and caspase1 has no obvious difference (P > 0.05); the ellagic acid can also relieve the inflammation of eye tissues, thereby playing a role in protecting the eye tissues.

TABLE 7 Effect of EA on the expression of focal death protein in eye tissue of diabetic mice (X. + -. s)

Group of	GSDME	GSDME-N	GSDMD	GSDMD-N
					Group C	1.00±0.14	1.00±0.12	1.00±0.16	1.00±0.10
M groups	4.72±0.12^aa	6.86±0.15^aa	1.67±0.09^a	4.78±0.23^aa
					EA group	1.85±0.20^bb	2.64±0.17^bb	1.32±0.21^b	2.24±0.13^bb

as shown in table 7 and fig. 6, the eye tissues of the model group (group M) mice showed significantly increased expression of GSDME, GSDME-N and GSDMD-N proteins (P <0.01) and increased expression of GSDMD protein (P <0.05) compared to the normal control group (group C); the GSDME, GSDME-N and GSDMDM-N protein expression in the eye tissues of the mice in the ellagic acid group (EA group) is obviously reduced (P <0.01) and the GSDMDM protein expression is reduced (P <0.05) compared with the model group; the ellagic acid can inhibit GSDMD and GSDME mediated cell apoptosis in a diabetes model mouse, so that an anti-inflammatory protection effect is generated on cells.

In conclusion, the ellagic acid EA can protect eye tissues of diabetic mice, reduce eye tissue damage caused by glycolipid metabolic disturbance, reduce the expression of eye tissue inflammatory proteins (NLRP3 and IL-1 beta), reduce the expression of eye tissue apoptosis-related proteins (caspase1 and clear-caspase 1) and reduce the expression of eye tissue focal death proteins (GSDME, GSDME-N, GSDMD and GSDMDM-N), so that inflammation is relieved, eye tissue apoptosis is reduced, cell focal death is inhibited, eye tissues are protected, and a basic scientific basis is provided for developing an action mechanism of a medicament for treating diabetic eye tissue pathological changes.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. Use of ellagic acid in the preparation of a medicament for alleviating ocular tissue pathologies.

2. Use of ellagic acid according to claim 1 in the preparation of a medicament for alleviating ocular tissue pathologies, wherein said ellagic acid is used in the preparation of a medicament for alleviating diabetic ocular tissue pathologies.

3. Use of ellagic acid in the manufacture of a medicament for alleviating pathological damage to ocular tissue according to claim 2, wherein said ellagic acid is used in the manufacture of a medicament for alleviating pathological damage to ocular tissue in diabetic mice.

4. Use of ellagic acid in the manufacture of a medicament for alleviating ocular tissue pathologies according to claim 3, for the preparation of a medicament for modulating the reduction of the expression of the p62 protein in the ocular globe tissue of diabetic mice.

5. Use of ellagic acid in the manufacture of a medicament for alleviating ocular tissue pathology according to claim 4, wherein said ellagic acid is used in the manufacture of a medicament for modulating elevated expression of LC3b/a protein in the eyeball tissue of diabetic mice.

6. Use of ellagic acid in the manufacture of a medicament for alleviating ocular tissue pathologies according to claim 2, for the preparation of a medicament for inhibiting the expression of the mouse ocular tissue inflammatory proteins NLRP3 and IL-1 β.

7. Use of ellagic acid in the manufacture of a medicament for alleviating ocular tissue pathologies according to claim 2, wherein said ellagic acid is used in the manufacture of a medicament for inhibiting the expression of the mouse ocular tissue apoptotic protein clear-caspase 1.

8. Use of ellagic acid in the manufacture of a medicament for alleviating ocular tissue pathology according to claim 2, wherein said ellagic acid is used in the manufacture of a medicament for inhibiting the expression of mouse ocular tissue apoptosis proteins GSDME, GSDME-N and GSDMD-N.

9. Use of ellagic acid according to claim 8 in the manufacture of a medicament for alleviating ocular tissue pathologies, wherein said ellagic acid is used in the manufacture of a medicament for alleviating diabetic retinopathy.

10. Use of ellagic acid in the manufacture of a medicament for alleviating ocular tissue pathology according to claim 9, wherein said ellagic acid is used in the manufacture of a medicament for the slowing of ureaplasma urealyticum-induced diabetic retinopathy in mice.