CN113289005B

CN113289005B - Application of rhFGF-21 in preparation of medicine for treating xerophthalmia

Info

Publication number: CN113289005B
Application number: CN202110761686.6A
Authority: CN
Inventors: 王晓杰; 李校堃; 惠琦
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2021-07-06
Filing date: 2021-07-06
Publication date: 2022-05-31
Anticipated expiration: 2041-07-06
Also published as: CN113289005A

Abstract

The invention discloses an application of rhFGF-21 in preparing a medicine for treating xerophthalmia. The rhFGF-21 improves the basal tear secretion amount, BUT, corneal fluorescein sodium staining and corneal nerve sensitivity of a scopolamine combined dry environment induced xerophthalmia mouse in a dose-dependent mode, and develops a new method for treating xerophthalmia.

Description

Application of rhFGF-21 in preparation of medicine for treating xerophthalmia

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of rhFGF-21 in preparing a medicine for treating xerophthalmia.

Background

The eyeball is an important organ for feeling peripheral things, the healthy eye surface is covered with a tear film structure which is jointly formed by mucin, aqueous tears and grease secreted by accessory glandular organs of the eyeball, and the component plays a role in maintaining the normal microenvironment of the eye surface and fully lubricating and protecting the eyeball; dry eye is induced when the homeostasis of any of the components of the tear film covering the surface of the eyeball is disrupted and timely replenishment and self-repair are not obtained.

The most visible symptoms of dry eye are manifested by qualitative and quantitative changes in the tear film components constituting the ocular surface, which result from disease development or morphological functional structural changes in the corresponding accessory secretory glands of the eye. Thus, dry eye disease is classified into various diseases, but the most important disease is tear-deficient dry eye and water-deficient dry eye. In addition, autoimmune deficiency syndrome induced dry eye, diabetic dry eye are also part of the type of dry eye disease. At present, corresponding solution strategies are provided for different dry eye inducing factors, but the anti-inflammatory drug therapy for the surface of the eye still has a plurality of adverse reactions and other limiting factors.

Fibroblast growth factor-21, a member of the FGF-19 subfamily, has received much attention because of its ability to maintain lipid and glucose metabolic balance. FGF-21 inhibits the NF-. kappa.B signaling cascade in macrophages and decreases the expression of inflammatory factors, including IL-1. alpha., IL-6, and TNF-. alpha. FGF-21 promotes the activation of islet secretory cells by activating PI3K-AKT signaling pathway; regulating the PI3K-caspase3 signal pathway acts to inhibit apoptosis. In addition, FGF-21 has the effects of inducing sympathetic nerve activity and protecting nerves to a certain extent. When FGF-21 is applied to a drug for treating dry eye, dry eye can be fundamentally ameliorated.

Therefore, how to apply FGF-21 to the preparation of a medicament for treating dry eye is a problem to be urgently solved by those skilled in the art.

Disclosure of Invention

In view of the above, the invention applies rhFGF-21 to the preparation of the medicine for treating dry eye, and the rhFGF-21 improves the basal tear secretion amount, BUT, corneal fluorescein sodium staining and corneal nerve sensitivity of a scopolamine combined drying environment induced dry eye mouse in a dose-dependent manner, thereby developing a new method for treating dry eye.

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

application of rhFGF-21 in preparing medicine for treating xerophthalmia.

As a preferred embodiment of the present invention, rhFGF-21 down-regulates the expression levels of TNF α, MMP-3, and MMP-9 inflammatory factors induced by dry eye.

As the preferable technical proposal of the invention, the rhFGF-21 increases the basal tear secretion of the scopolamine combined dry environment induced xerophthalmia mouse.

As the preferable technical proposal of the invention, the rhFGF-21 improves the tear film rupture time of the scopolamine combined dry environment induced xerophthalmia mice.

As the preferable technical scheme of the invention, the rhFGF-21 improves the corneal fluorescein sodium staining score of the scopolamine combined dry environment induced xerophthalmia mice.

As the preferred technical scheme of the invention, the rhFGF-21 improves the corneal nerve sensitivity of the scopolamine combined dry environment induced xerophthalmia mouse.

The medicine for treating xerophthalmia comprises rhFGF-21, and the use concentration of the rhFGF-21 is 100 mu g/ml-800 mu g/ml.

As a preferred embodiment of the present invention, rhFGF-21 downregulates the dry eye-induced expression levels of TNF α, MMP-3, and MMP-9 from the intraorbital lacrimal glands.

As a preferred technical scheme of the invention, the rhFGF-21 can up-regulate the expression of mouse lacrimal gland FGFR 2.

As a preferred technical scheme of the invention, the rhFGF-21 up-regulates the expression of the intraorbital lacrimal gland PI3K-AKT signal pathway.

According to the technical scheme, compared with the prior art, the rhFGF-21 is applied to the preparation of the medicine for treating the xerophthalmia, the basic tear secretion, BUT, corneal fluorescein sodium staining and corneal nerve sensitivity of a scopolamine combined drying environment induced xerophthalmia mouse are improved in a dose-dependent mode by the rhFGF-21, and the rhFGF-21 can not only reduce the xerophthalmia signs of the mouse to the maximum extent, BUT also repair damaged corneal nerves; reduce inflammatory factors of MMP-3, MMP-9 and TNF alpha of lacrimal gland, promote Ki-67 expression, and recover the function of secreting tears.

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 embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the basal tear secretion of mice treated with 5mg/ml scopolamine hydrobromide solution;

FIG. 2 is a graph showing the change in BUT of mice treated with 5mg/ml scopolamine hydrobromide solution;

FIG. 3 is a graph showing corneal sensitivity readings of mice treated with 5mg/ml scopolamine hydrobromide solution;

FIG. 4 is a graph showing the staining of sodium keratectin in mice treated with 5mg/ml scopolamine hydrobromide solution;

FIG. 5 is a graph showing the staining scores of fluorescein sodium after treatment of mice with 5mg/ml scopolamine hydrobromide solution;

FIG. 6 is a graph showing HE staining of cornea in normal group and in mice with persistent induction of dry eye;

FIG. 7 is a graph showing the amount of tear secretion and the tear film rupture time before and after the treatment of a dry eye mouse, wherein A is a graph showing the detection lengths of the 14D, phenol red cotton threads of healthy mice, PBS treatment groups and rhFGF-21 instillation treatments with different concentrations; b is a statistical graph of tear secretion of healthy mice, PBS treatment group and rhFGF-21 treatment mice at different treatment time nodes within 14D of treatment. C. D represents the lacrimal secretion amount of the mice treated by the rhFGF-21 under the 10D and 14D respectively; e is the BUT statistical plot of different treatment time nodes within 14D, PBS treatment group and rhFGF-21 treatment mice; F. g represents BUT statistical plots for 10D and 14D, respectively, treated mice;

FIG. 8 is a graph showing the staining of sodium fluorescein and staining of sodium fluorescein in the cornea of animals treated with rhFGF-21; a is the staining pattern of 7D, 10D and 14D of the treated mice in PBS treatment group and medium and high dose rhFGF-21 treatment group with sodium fluorescein in the cornea; B. c, D represent the 7D, 10D and 14D treated, respectively, plots of the staining of mouse corneal sodium fluorescein;

FIG. 9 is a graph showing HE staining and corneal nerve staining of mouse cornea; a is corneal HE staining patterns of healthy and dry eye-induced 35D mice, and corneal HE staining patterns of treatment 7D and 14D, different groups of mice; b is the staining pattern of superficial nerve of cornea of mouse for treating 14D; C. d is treatment No. 7D and 14D respectively, and a contact tactile instrument detection mouse cornea sensitivity log graph;

FIG. 10 is a graph of HE staining and Ki-67 immunofluorescence staining of extra-orbital lacrimal glands in mice; a is HE staining pattern for treatment of extraorbital lacrimal glands in 7D and 14D mice; b is Ki-67 immunofluorescent staining pattern of mouse extraorbital lacrimal gland for treatment of 7D and 14D; C. d is a statistical result chart of treatment of 7D and 14D, mouse extraorbital lacrimal gland Ki-67, respectively;

FIG. 11 is a graph of HE staining and Ki-67 immunofluorescence staining of lacrimal glands in the orbit of a mouse eye; a is the HE staining pattern for treatment of the intraorbital lacrimal gland in mice 7D and 14D; b is (Ki-67 immunofluorescent staining pattern for treatment of lacrimal glands in mouse orbit at 7D and 14D; C, D is a statistical pattern for treatment of lacrimal glands Ki-67 in mouse orbit at 7D and 14D, respectively;

FIG. 12 is a graph showing the measurement of mouse basal tear secretion, BUT, and WB and IHC contents of FGF-21; wherein A, B are tear secretion and BUT profiles before lacrimal gland resection, after lacrimal gland resection and induction of dry eye, and treatment of 7D and 14D with PBS or 800 μ g/ml rhFGF-21, respectively; C. d is FGF-21 expression detection and corresponding statistical graphs of the intraorbital lacrimal gland and the extraorbital lacrimal gland of the normal healthy mouse respectively; E. f, detecting the intraorbital lacrimal gland FGF-21 expression patterns of a normal healthy mouse and a xerophthalmia mouse by WB and IHC methods respectively; g is a statistical result graph of E;

FIG. 13 is a graph showing immunohistochemical staining for TNF α, MMP-3, and MMP-9 and WB detection results; a is the immunohistochemical staining result of TNF alpha, MMP-3 and MMP-9 from lacrimal glands in the eye orbit on day 14 of treatment; b is a WB result plot of TNF α, MMP-3, and MMP-9 from lacrimal glands in the orbit on day 14 of treatment; C. d, E is a statistical chart of immunohistochemical staining results; F. g, H is the expression statistical analysis chart of WB;

fig. 14 is a graph of IHC staining and statistics of WB, FGFR2 of mouse intraorbital lacrimal gland FGFRs; a is WB results of FGFR1, FGFR2, FGFR3 and FGFR4 of lacrimal glands in eye orbits of mice in a normal control group, a PBS treatment group and a middle-high concentration rhFGF-21 treatment group on the 14 th day of treatment; (B, C, D, E is a statistical graph corresponding to A; F is the IHC staining of the intraorbital lacrimal FGFR2 on day 14 of treatment; G is a statistical graph of the staining of graph F;

FIG. 15 is a graph of IHC staining and WB results for the intraorbital lacrimal glands PI3K, P-AKT and AKT; wherein, A is an immunohistochemical staining pattern of lacrimal glands PI3K, P-AKT and AKT in an orbit of a mouse on the 14 th day of treatment; B. c, D is a statistical chart of immunohistochemical staining results of Panel A; e is a WB result plot of the intraorbital lacrimal glands PI3K, P-AKT and AKT of the mice on day 14 of treatment; F. g is a statistical chart of WB results;

FIG. 16 is a graph showing the basal tear secretion, BUT and staining of lacrimal gland HE, Ki-67 in mice; wherein A, B shows, on

days

7 and 14 of treatment, basal tear secretion and BUT detection maps of normal group, high dose rhFGF-21 treatment group and T5122, LY294002 mice, respectively; c is HE staining pattern of lacrimal glands in orbit of normal group, high dose rhFGF-21 treatment group and T5122 and LY294002 mice on treatment day 14; D. e represents the immunofluorescence staining results and corresponding statistical graphs of intraorbital lacrimal gland Ki-67 of the normal group, the high-dose rhFGF-21 treatment group and the T5122 and LY294002 mice on the 14 th day of treatment;

FIG. 17 is an IHC staining pattern of mouse intraorbital lacrimal gland FGFR2, PI3K, P-AKT and AKT; wherein, A is an immunohistochemical staining result graph of FGFR2, PI3K, P-AKT and AKT of intraorbital lacrimal glands of a normal group, a high-dose rhFGF-21 treatment group and T5122 and LY294002 mice on the 14 th day of treatment; B. c, D, E are statistical plots of IHC staining results for FGFR2, PI3K, P-AKT and AKT, respectively;

FIG. 18 is a graph of IHC staining of mouse intraorbital lacrimal glands TNF- α, MMP-3, and MMP-9; wherein A is an immunohistochemical staining result graph of TNF-alpha, MMP-3 and MMP-9 of intraorbital lacrimal glands of a normal group, a high-dose rhFGF-21 treatment group and T5122 and LY294002 mice on the 14 th day of treatment; B. c, D is a statistical chart of the IHC staining results for TNF- α, MMP-3, and MMP-9, respectively.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 modeling of Dry eye in mice

Experimental materials used in example 1:

6-8 week old female C57BL/6J mice, purchased from Zhejiang Wintonlihua laboratory animals Co., Ltd, clean grade, with a weight range of 18-20 g. During the study, all test mice were housed in the second phase animal breeding center of pilot test base of Wenzhou medical university. Feeding conditions are as follows: 5 mice are raised in each cage, each cage is provided with an independent ventilation device, the indoor temperature of the raised mice is kept at 20-26 ℃, the relative humidity is kept within 50-70%, 12h illumination/12 h darkness alternate, experimental animals can freely drink water and are fed with full-value solid particles, and the experimental animals can freely eat food in the period. Specifically, it states that: the experimental animal feeding and experimental operation are in accordance with the relevant scientific Research animal use specifications established by the Association for Research in Vision and Ophthalmology (ARVO).

The main instruments and supplies are shown in table 1:

TABLE 1

The main reagents and drugs are shown in table 2:

TABLE 2

Arrangement of the reagents involved

Scopolamine hydrobromide solution of 5mg/ml

Taking the individual package in an ampoule bottle, dissolving scopolamine hydrobromide freeze-dried powder with the content of 500mg in sterile normal saline, and diluting the solution to a final volume of 100ml by using the sterile normal saline, wherein the concentration of the scopolamine hydrobromide is 5 mg/ml. The solution was dispensed into brown EP tubes, labeled and stored briefly at-20 ℃ for future use.

Chloral hydrate 5%

An appropriate amount of chloral hydrate was weighed in a brown Ep tube, and 0.9% sterile physiological saline was added before use to prepare a 5% solution, which was mixed well and then immediately used.

0.01M PBS buffer

The bagged phosphate buffer powder was dissolved in 2L of purified water and mixed well to obtain 0.01M PBS buffer.

4% Paraformaldehyde

10g of paraformaldehyde powder was added to the PBS solution, diluted to 250ml, and stored in a closed container. Adding 1 piece of NaOH solid, fully stirring by using a magnetic stirrer, and storing at 4 ℃.

0.05% sodium fluorescein solution

Weighing appropriate amount of solid powder of fluorescein sodium in brown EP tube, preparing into 0.05% solution with sterile physiological saline, and storing at-4 deg.C for no more than 5 days.

(1) At 4-5m²Two dehumidifier are arranged in the fume hood, the airflow speed of the fume hood is adjusted to 1-3 m/s, and a liquid pipeline in the fume hood is closed. Meanwhile, anhydrous calcium chloride powder is placed in the fume hood to serve as a drying agent, so that the relative humidity of the environment in the fume hood is lower than 40% stably;

(2) the purchased C57BL/6J mice were kept on routine rearing for 1 week for acclimation, and some of the mice with non-smooth corneas and diseased eyes were screened and excluded by slit lamps before the mouse dry eye disease model was established. The mice were transferred to the inside of a dry fume hood, and each mouse was injected subcutaneously with scopolamine hydrobromide physiological saline solution at a concentration of 5mg/ml behind the mouse's bilateral ears at a fixed time point (17 th) per day. The daily single-side retroauricular subcutaneous injection dose of each mouse is 100 mu l, the daily injection dose of each mouse is 1mg, and the injection time is 35 days. During the period of inducing the mouse xerophthalmia by combining scopolamine with a dry environment, the mental state and the activity of the mouse are observed every day. Meanwhile, healthy mice were raised under normal environment as a normal control.

(3) The head of the mouse is fixed under the condition that the mouse is not anesthetized, and the mouse can naturally shoot instantly. On

days

0, 7, 14, 21, 28 and 35 of the model build, the vertical corneal surface was brought into contact with the mouse eyeball using a corneal contact tactile meter. The contact type tactile meter starts to measure from the maximum reading of 6cm, the scale is adjusted to be 0.5cm each time until 3 transient positive reactions of the eyeballs of the mouse are observed, and the reading is recorded;

(4) basal tear secretion measurement of mice: measurements were taken at fixed time points of each day on

days

0, 7, 14, 21, 28 and 35 of the model set-up using the Schirmer's test method, respectively. After anesthesia by intraperitoneal injection of a proper amount of chloral hydrate, phenol red cotton thread was placed at the lower eyelid outer canthus of the mice for 15 seconds. Measuring and reading the length of the phenol red cotton thread, and recording the measured length (accurate to 0.1mm), namely the basic tear secretion of the mouse;

(5) tear film Break Up Time (BUT) measurement in mice: on the 0 th, 7 th, 14 th, 21 th, 28 th and 35 th days of the model establishment, after the mice were anesthetized by injecting a proper amount of chloral hydrate to the abdominal cavity, 1 to 2 μ l of a sodium fluorescein normal saline solution with a concentration of 0.05% was dropped on the ocular surface of the mice. After the fluorescein sodium is uniformly distributed on the surface of the eyes of the mouse, the rupture time of the fluorescein sodium covering the surfaces of the eyeballs of the mouse is observed by using dispersed cobalt blue light through a slit lamp, and the rupture time is recorded, and the average value of each eyeball of the mouse is repeatedly measured for three times at the measuring time point and is taken as the rupture time (BUT) of the tear film of the mouse on the day;

(6) sodium corneal fluorescein staining photographs and ocular surface inflammation assessment in mice: after the tear film rupture time is measured, each mouse is rested for 2-3 minutes, the fluorescein sodium staining condition on the surfaces of the eyeballs of the mice is observed under the condition of dispersed cobalt blue light under a slit lamp, and the fluorescein sodium staining condition is shot under the slit lamp by selecting a proper angle and magnification. Mouse ocular surface fluorescein sodium staining evaluation method as follows: dividing the cornea into 4 quadrants, and respectively evaluating the staining score according to the previous 4 quadrants, namely, 4 quadrants of the fluorescein positive plaque; very dense punctate fluorescence, 3 points; dense punctate fluorescent patterns, 2 points; mild fluorescence was similar to that of sparse spots, 1 point; non-fluorescent, score 0. The scores for each eye were added and analyzed. All fluorescein staining was scored by single observer (LW) blindness;

basal tear secretion, tear break-up time (BUT) and corneal sensitivity readings for the mice are shown in figures 1-3; as can be seen from fig. 1, at induced 7D, the basal tear secretion of the mice was significantly lower than that of normal healthy mice (. p <0.01), at induced 28D and 35D, the basal tear secretion of the mice decreased to a certain level, and there was a statistical difference in tear secretion difference compared to healthy mice (. p < 0.001); from FIG. 2, it can be seen that there is a tendency for the tear film Break Up Time (BUT) to decrease progressively with increasing dry eye induction time, with the difference being statistically significant in BUT in model mice compared to healthy mice starting at induced 7D; after induced 28D, the model mice had a sustained decrease in BUT statistically different (×) from healthy mice (0.001); as can be seen from fig. 3, the sensitivity of the corneal nerves decreased from induced 21D with a significant difference compared to normal mice (. p <0.01), 28D and 35D, with an increase in corneal sensitivity (. p < 0.001).

Experimental sampling time and pretreatment

Mice were drawn on

days

7, 14, 21, 28 and 35 of the molding. 5% chloral hydrate is used for carrying out abdominal cavity over-injection for euthanasia, then eyeballs and eye appendages of mice are picked up and placed in pre-cooled 4% paraformaldehyde, paraffin embedding is prepared, and paraffin sections are prepared for HE staining.

Paraffin embedding and slicing

1) Fixing the eyeball and the eye appendage in a paraformaldehyde solution at 4 ℃ in a dark place for 4 hours, trimming, putting into a paraffin embedding box, marking, fixing a rope, washing with running water for 6 hours under tap water, and finally dehydrating in 70% alcohol at 4 ℃ overnight;

2) the following day, the tissue organs were dehydrated with an alcohol gradient: respectively dehydrating with 80%, 90% and 95% ethanol for 30min, respectively, dehydrating with 100% ethanol I for 15min, and dehydrating with 100% ethanol II for 15 min;

3) and (3) transparency: xylene I for 20min and xylene II for 15 min;

4) soaking in soft wax for 1.5h, and then soaking in hard wax for 1 h;

5) embedding: preheating an embedding mold, after dropping hard wax, horizontally placing eyeballs, lacrimal glands, conjunctiva and the like at the bottom of the mold, embedding an embedding box into the mold, filling the hard wax, then transferring the mold carrying the embedding box onto a cooling table, taking down the embedding box after complete solidification, connecting a tissue paraffin block chain with the embedding box, marking, storing in a refrigerator at 4 ℃, and waiting for paraffin slicing;

6) slicing: the cassette was mounted on a microtome and sectioned to 5 μm thickness near the center of the cornea.

HE staining

1) Baking and dewaxing: drying the paraffin sections at 65 ℃ for 3-4 hours, transferring the paraffin sections into a fume hood, soaking the paraffin sections in xylene I for 15 minutes, and then soaking the paraffin sections in xylene II for 15 minutes;

2) hydration: soaking in 100% I and 100% II ethanol for 5min, respectively, and soaking in 95%, 90%, 80% and 75% ethanol for 5 min;

3) soaking in purified water for 5min, and staining with hematoxylin for 3-5 min. Flushing with running water for 5 min;

4) soaking and washing with purified water for 1 min;

5) differentiating with 1% ethanol hydrochloride for 2 s;

6) soaking and washing with purified water for 30 s. Returning PBS to blue for 2 min;

7) soaking in 95% ethanol for 2s, and staining with 0.5% eosin for 3 s;

8) carrying out 3 times of soaking and washing on the purified water for 30s, and then carrying out gradient dehydration: dehydrating 75%, 80%, 90%, 95% ethanol for 1min, and dehydrating 100% I and 100% II ethanol for 30 s;

9) and (3) transparency: soaking in xylene I for 15min and xylene II for 10min in a fume hood;

10) sealing: the slides were removed and the xylene was wiped off the back and edge of the slide with a paper towel. Dropping 1 drop of neutral resin onto the tissue while the xylene is not dry, covering with a cover glass, airing the film at room temperature overnight, and taking the film the next day.

As a result, as shown in fig. 4 to 6, it is clear from fig. 4 that the cornea of the mouse at 7D showed slight staining with fluorescein sodium, 14D began to show a large amount of punctate staining with fluorescein sodium in the corneal epithelium, and 28D showed a large area of staining with fluorescein sodium in the form of a flake. Figure 5 shows the score for dry eye-induced sodium fluorescein staining in the cornea of healthy mice compared to healthy mice, with the score for sodium fluorescein staining in the cornea of mice statistically significant compared to healthy mice (p <0.01) starting at 7D for dry eye induction. As shown in FIG. 6, in the observation of HE staining of mouse cornea, 28D and 35D showed that the outermost cells of mouse corneal epithelium were nuclear-deficient and the extracellular matrix was tightly arranged (arrows).

EXAMPLE 2 Effect of rhFGF-21 treatment of Dry eye syndrome in mice

Laboratory animal

6-8 week old female C57BL/6J mice were purchased from Zhejiang Wintonlihua laboratory animals, Inc., clean grade, ranging in weight from 18-20 g. During the study, all test mice were housed in the second phase animal breeding center of pilot test base of Wenzhou medical university. Feeding conditions are as follows: feeding 5 mice per cage, wherein each cage of mice is provided with an independent ventilation device, the indoor temperature of the feeding is kept at 20-26 ℃, the relative humidity is kept in the range of 50-70%, 12h illumination/12 h darkness are alternated, experimental animals can freely drink water and feed with full-value solid particles, and the experimental animals can freely eat food in the period. Specifically, it states that: the experimental animal feeding and experimental operation are in accordance with the relevant scientific Research animal use specifications established by the Association for Research in Vision and Ophthalmology (ARVO).

The main instruments and articles are shown in table 3;

TABLE 3

The main reagents and drugs are shown in Table 4

TABLE 4

Arrangement of the reagents involved

rhFGF-21 solution

The freeze-dried powder of the rhFGF-21 is dissolved in sterile PBS solution, the solution with the concentration of 400 mu g/ml and the solution with the concentration of 800 mu g/ml are respectively prepared, and the solution is subpackaged, marked and stored in a refrigerator at the temperature of-20 ℃ for standby.

The preparation of 5mg/ml scopolamine hydrobromide solution, 5% chloral hydrate, 0.01M PBS buffer, 4% paraformaldehyde is given in example 1;

0.01M sodium citrate buffer solution

Dissolving bagged sodium citrate buffer salt powder in 2L of purified water, and mixing thoroughly to obtain 0.01M sodium citrate buffer solution;

0.05% fluorescein sodium solution

Weighing appropriate amount of fluorescein sodium solid powder in a brown EP tube, preparing into 0.05% solution with sterile normal saline, and storing at-4 deg.C for no more than 5 days;

5% goat serum blocking solution

Mu.l goat blocking serum was taken and dissolved in 950. mu.l PBS solution and stored at-20 ℃ until use.

The primary antibodies are shown in table 5;

TABLE 5

Experimental methods

rhFGF-21 eye drop treatment of xerophthalmia mice

A mouse dry eye model was established as in example 1, and at induced 35D, the mice were transferred to an environment with normal relative humidity (50-70%) and raised under normal conditions.

The treated animals were grouped as:

blank control group (normal healthy mice were spotted with PBS);

saline treated control group (PBS eyedrops for dry eye mice);

rhFGF-21 medium dose group (dry eye mice receiving 400. mu.g/ml rhFGF-21 treatment);

rhFGF-21 high dose group (dry eye mice receiving 800. mu.g/ml rhFGF-21 treatment);

in the following 14D, daily at fixed time points: 9. 15 and 21, respectively dripping 2 mul of PBS or 400 mug/ml and 800 mug/ml of rhFGF-21 into conjunctival sac at two sides of two eyes of the mouse; the eyeballs of the mouse are instantly ocular, and the liquid medicine is fully contacted and absorbed. 0-7D, 10D and 14D of eye drop treatment of the mice are respectively treated, a certain amount of 5% chloral hydrate solution is injected into the abdominal cavity of the mice, and the basal tear secretion amount, BUT, fluorescein sodium staining photographing of the ocular surface and fluorescein sodium staining score of the mice are detected, and the specific operation method is as in example 1. Corneal sensitivity was measured with a contact tactile meter at 7D and 14D of mouse treatment. The results are shown in FIGS. 7 and 8;

as can be seen from FIG. 7, FIG. 7A and FIG. 7B show that the secretion of lacrimal fluid of rhFGF-21 treated mice at 1W, 400 μ g/ml and 800 μ g/ml of rhFGF-21 treated mice is higher than that of PBS eye drop treated control group during the treatment period of continuous rhFGF-21 treatment, and the statistical difference is not obvious. But at 10D and 14D of treatment, there was a significant difference in the amount of tear secretion of the rhFGF-21-treated mice compared to the PBS control group at the medium and high doses (fig. 7C, 7D) (. p <0.01,. p < 0.001); meanwhile, the basal tear secretion amount of the rhFGF-21 treated mice with 400 mu g/ml and 800 mu g/ml is statistically different (p is less than 0.01). The amount of tear fluid secreted in the treated 14D, PBS treated mice and mice administered with different concentrations of rhFGF-21 is shown in fig. 7A. The burs of rhFGF-21 treated mice also showed statistical differences from the PBS treated group at 10D and 14D (fig. 7E, 7F, 7G) (. p <0.01,. p <0.001), with the burs of the rhFGF-21 treated group at 14D, 800 μ G/ml, higher than those of the healthy control group.

As can be seen from FIG. 8, 400. mu.g/ml and 800. mu.g/ml of rhFGF-21 had the effect of reducing the positive coloration of corneal fluorescein sodium caused by dry eye at each of the treated 7D, 10D and 14D, as compared with the PBS-treated group (FIG. 8A). The corneal sodium fluorescein staining score showed that rhFGF-21 at medium and high doses significantly down-regulated the sodium fluorescein staining score at the beginning of treatment at 7D, with the differences being statistically significant compared to the PBS group (fig. 8B, 8C, 8D,. p <0.01,. p < 0.001). The difference in sodium fluorescein staining score between the middle and high dose groups of rhFGF-21 treatment at 10D was statistically significant (fig. 8C) p < 0.01.

Experimental sampling time and pretreatment

Mice were drawn on

days

7 and 14 of treatment. 5% chloral hydrate was used for intraperitoneal over-injection for euthanasia, then the eyeballs and eye appendages of the mice were picked up and placed in pre-cooled 4% paraformaldehyde, and part of the fresh tissue organs were stored at-80 ℃ for subsequent operations.

Nerve staining of mouse cornea

At 14D of treatment, the eyes were removed after anesthetizing the mice, an incision was made behind the limbus to dissect the cornea including the scleral limbus, the cornea was fixed with 4% paraformaldehyde for 30min, and then washed 3 times with 1% Triton X-100(Triton)/PBS for 15min of penetration. Then blocked with 10% goat serum (BSA) overnight in 0.1% Triton/PBS, and then incubated overnight with 1% FBS containing β -III tubulin (1: 200). After overnight incubation, wash 3 times with 0.1% Triton/PBS for 30min, incubate with fluorescently labeled secondary antibody (1: 500) for 1.5h at 37 deg.C, stain with the nuclear dye 4', 6-diamino-2-phenylindole (DAPI), wash 3 times with 0.1% Triton/PBS for 30 min. The corneas were unfolded in PBS solution under dark room conditions and incisions were made on each cornea to obtain a flower-like integral scaffold (four quadrants) and mounted with anti-fluorescence quenching PVP mounting fluid. The images were taken with an upright fluorescence microscope. As a result, as shown in fig. 9, it was found that continuous dry eye induced keratinization of the corneal epithelium in mice. In HE staining observation of the cornea of the treated mice, it was found that, at the 7D and 14D of the treatment, rhFGF-21 at a high dose had the effect of improving corneal epithelial cell keratinization as compared with the PBS-only treated mice, as shown in FIG. 9A. In 14D, the corneal epithelium of the mice in the high-dose rhFGF-21-treated group exhibited a similar state to that of the cornea of healthy mice, and the corneal epithelium of the contemporary PBS-treated group also observed a keratinocyte layer. Corneal nerve sensitivity tests of mice showed that, at 7D of treatment, rhFGF-21 at medium and high doses improved corneal nerve sensitivity with significant differences compared to the PBS group, as shown in fig. 9C p <0.05, p < 0.001. In 14D of treatment, corneal sensitivity readings of the medium-high dose rhFGF-21 treated group were restored to healthy mouse levels, as shown in fig. 9D, × p < 0.01. As shown in FIG. 9B, the cornea of the 14D mouse was stained with superficial nerve, and as a result, it was found that when the mouse was treated with PBS alone, the corneal superficial nerve structure was abnormal, the nerve fiber structure was abnormal, the number of brush-like nerve fibers was reduced, and the "vortex" structure in the nerve center was disappeared, as compared with the normal mouse. Meanwhile, the rhFGF-21 has the function of restoring the corneal superficial neural network, and the high-dose rhFGF-21 enables the 'vortex' structure in the center of the corneal nerve to be restored again.

Paraffin embedding and slicing

The eyeballs and accessory lacrimal glands of the treated rhFGF-21 treatment groups of 7D and 14D, PBS control group and healthy control group mice were paraffin-embedded and sectioned. The specific procedure was as in example 2.

HE staining

HE staining procedure of mouse eyeball and lacrimal gland was as in example 2.

Fluorescent marker staining of immune tissue (IF)

1) Baking and dewaxing: after the paraffin sections were baked at 65 ℃ for 3-4 hours, they were transferred to a fume hood and soaked in xylene I for 15 minutes followed by another soaking in xylene II for 15 minutes.

2) Hydration: soaking in 100% I and 100% II ethanol for 5min respectively, and soaking in 95%, 90%, 80% and 75% ethanol for 5min respectively.

3) Wash 3 times with 0.01M PBS for 5min each.

4) Heating 0.01M sodium citrate buffer solution with pH of 6.0 by autoclave water bath method until the temperature of the buffer solution is higher than 95 deg.C, placing the rack with glass slides into the buffer solution, bubbling at high pressure for 2min, and taking out and naturally cooling at room temperature.

5) Wash 3 times with 0.01M PBS for 5min each.

6) The excess water of the glass slide is absorbed by the tissue paper, the tissue is placed in a wet box, 5% goat blocking serum prepared by 0.01M PBS is dripped on the tissue, and the tissue is placed in an environment at 37 ℃ for incubation for 1 h.

7) After the blocking solution was aspirated through the paper towel, primary antibody diluted with 1% goat blocking serum was added dropwise and incubated overnight at 4 ℃.

8) The next day, the wet box was taken out and allowed to rewarmed at room temperature for 1 hour.

9) Wash 3 times with 0.01M PBS for 5min each.

10) Excess water on the slide was removed with a piece of tissue paper, placed in a wet box, and a secondary antibody diluted with 1% goat blocking serum was added dropwise and incubated at 37 ℃ for 1 h.

11) In the dark, the plates were shaken and washed 4 times with 0.01M PBS for 5min each.

12) DAPI was stained for 5min at room temperature.

13) In the dark, on a shaker, wash 3 times with 0.01M PBS for 5min each.

14) In the dark, excess water was removed from the slides, a drop of anti-fluorescence quencher was placed on each tissue, and coverslipped.

15) And taking images of the Nikon by a confocal microscope.

The results are shown in fig. 10 and fig. 11, and fig. 10 shows that, in the HE staining observation of the extraorbital lacrimal glands of the treated 7D and 14D mice, there is no obvious difference in tissue morphology between the lacrimal glands of the PBS-treated mice and the lacrimal glands of the medium and high dose rhFGF-21-treated mice, as shown in fig. 10A. As shown in fig. 10B, 10C, 10D, immunofluorescence staining of extra-orbital lacrimal glands in treated 7D and 14D mice showed no significant statistical difference in Ki-67 expression between the PBS-treated group and the moderately high concentration rhFGF-21-treated group;

FIG. 11 shows that, in the observation of HE staining of lacrimal glands in the orbit of mice treated in the numbers 7D and 14D, rhFGF-21 in the middle-to-high dose group promoted the proliferation of lacrimal gland cells in the orbit of mice as compared with the PBS-treated group. In 7D, the number of lacrimal gland cells of the mice treated by the rhFGF-21 with the high dose is more than that of the mice treated by the PBS only, and simultaneously, the cells are plump in shape and have the trend of obviously increasing the number of the cells, and at 14D, the lacrimal gland cells in the orbit of the mice treated by the rhFGF-21 with the high dose are closely arranged and have plump shapes, as shown in fig. 11A. Meanwhile, the immunofluorescence staining results of the lacrimal gland Ki-67 in the orbit of the mouse also show the difference, at 7D and 14D, the expression of Ki-67 in the lacrimal gland tissue of the mice in the medium-high dose rhFGF-21 treatment group is increased compared with the PBS treatment group, and the difference has statistical significance compared with the PBS treatment group, p is less than 0.01, p is less than 0.001, and fig. 11B, 11C and 11D. Statistical analysis

In this experiment, GraphpadPrism 6.0 software was used to statistically analyze the data, which are expressed as mean ± SD, where n > is 6. ANOVA test was used between groups, and if p <0.05, the groups were considered to have significant statistical differences.

Example 4 mechanism of rhFGF-21 treatment of Dry eye syndrome in mice

The main instruments and articles are shown in table 6;

TABLE 6

The main reagents and drugs are shown in Table 7;

TABLE 7

Arrangement of the reagents involved

The formulation of rhFGF-21 solution, 5mg/ml scopolamine hydrobromide solution, 5% chloral hydrate, 0.01M PBS buffer, 4% paraformaldehyde, 0.05% fluorescein sodium solution, 5% goat blocking serum solution is given in example 1 and example 2

T5122 suspension for injection

Dissolving 5mg of the drug powder in 333.4. mu.l of DMSO solution, adding 1ml of olive oil, adding 70. mu.l of Tween-20, supplementing to a final volume of 7ml with physiological saline, vortexing, mixing, and packaging in EP tube at-20 deg.C. The single injection dose of the abdominal cavity in the animal body is 7.5 mg/kg.

LY294002 suspension for injection

Dissolving 10mg of the drug powder in 500. mu.l of DMSO solution, adding 1.3ml of olive oil, adding 100. mu.l of Tween-20, supplementing to the final volume of 10ml with physiological saline, vortexing, mixing, and packaging in EP tube at-20 deg.C. The single injection dosage of the abdominal cavity in the animal body is 10 mg/kg.

An appropriate amount of chloral hydrate was weighed in a brown Ep tube, and 0.9% physiological saline was added before use to prepare a 5% solution, which was thoroughly mixed and then immediately used.

0.01M sodium citrate buffer solution

The bagged sodium citrate buffer salt powder is dissolved in 2L of purified water and fully mixed to obtain 0.01M sodium citrate buffer solution.

10g of paraformaldehyde powder was added to the PBS solution, diluted to 250ml, and stored in a closed container. Adding 1 NaOH solid, stirring with magnetic stirrer, and storing at 4 deg.C.

3％H₂O₂Methanol solution

400mL of methanol was dissolved in 100mL of purified water to give an 80% methanol solution. 50mL of 30% H was taken₂O₂The solution was dissolved in 450mL of 80% methanol to give 3% H₂O₂And (3) solution.

TBST solution

Weighing 40g of sodium chloride, 1g of potassium chloride and Tris15g, adding pure water to 450ml, and adjusting the pH value of the solution to 7.4 by using concentrated hydrochloric acid; the final volume was then made up to 500ml with pure water, in this case 10X TBS was prepared. When in use, 50ml of 10XTBS is measured, pure water is added for dilution to 495ml, and 500 μ l of Tween-20, namely TBST solution, is added by a pipette.

5% skimmed milk

2g of skim milk powder was added to a 50ml centrifuge tube, 40ml of TBST solution was added, and the tube was placed on a horizontal shaker to dissolve it sufficiently for use.

The primary antibodies are shown in table 8;

TABLE 8

rhFGF-21 treatment of dry eye syndrome in mice

At induction 35D, mice were transferred to normal relative humidity (50-70%) environment and raised under normal conditions. The treated animals were grouped as: a blank control group (normal healthy mice were eyed with PBS), a saline-treated control group (PBS eyed with dry eye mice), a rhFGF-21 medium dose group (dry eye mice receiving 400. mu.g/ml of rhFGF-21 treatment), and a rhFGF-21 high dose group (dry eye mice receiving 800. mu.g/ml of rhFGF-21 treatment). In the following 14D, daily at fixed time points: 9. 15 and 21, respectively dripping 2 mul of PBS or 400 mug/ml and 800 mug/ml of rhFGF-21 into conjunctival sac at two sides of two eyes of the mouse; the eyeballs of the mouse are instantly ocular, and the liquid medicine is fully contacted and absorbed.

Extirpation, induction of dry eye and treatment of lacrimal gland outside orbit of mouse

Female B6 mice of 6-8 weeks old are bred conventionally, and after anesthesia by injecting a proper amount of chloral hydrate into the abdominal cavity, the skin part of the rear ear of the mice is cut open to the area of the connecting line of the lower jaw part by an operating blade. Muscle tissue is exposed, extra-orbital lacrimal glands are sought, lacrimal ducts connecting the orbits are found, the ducts are ligated and the entire extra-orbital lacrimal tissue is excised, followed by suturing. After 1 week of recovery, induction of the dry eye model was performed in the manner described in the first section. After induction, treatment was performed by instillation with 800. mu.g/ml rhFGF-21 using the treatment method described above, and a PBS control group was also included.

Xerophthalmia mouse eye-dropping rhFGF-21 combined injection inhibitor treatment

As described in example 2, the eyes of the mice after successfully inducing dry eye are dripped with 800 μ g/ml of rhFGF-21 for eye dropping treatment, and T5122 and LY294002 inhibitor groups are respectively arranged at the same time of treatment; the single injection dose of the T5122 suspension solution in the animal body abdominal cavity is 7.5mg/kg, and the single injection dose of the LY294002 suspension solution in the animal body abdominal cavity is 10 mg/kg; mice were dosed i.p. daily prior to instillation of rhFGF-21. A normal control group was also set. The mice were injected with an appropriate amount of 5% chloral hydrate solution intraperitoneally, and the specific procedures for measuring the basal tear secretion amount and BUT of the mice were as described in the first section. The detection times were 0D, 7D and 14D of treatment, respectively.

Experimental sampling time and pretreatment

Conventionally treated mice were drawn on

days

7 and 14 of treatment; lacrimal gland resected mice as well as rhFGF-21 combination inhibitor treated mice were euthanized at treatment 14D: 5% chloral hydrate was used for intraperitoneal over-injection for euthanasia, then the eyeballs and eye appendages of the mice were picked up and placed in pre-cooled 4% paraformaldehyde, and part of the fresh tissue organs were stored at-80 ℃ for subsequent operations.

Paraffin embedding and slicing

The eyeballs and accessory lacrimal glands of the treated rhFGF-21 treatment groups of 7D and 14D, PBS control group and healthy control group mice were paraffin-embedded and sectioned. The specific steps are as described in the first section.

HE staining

HE staining procedure of mouse eyeball and lacrimal gland was the same as in example 2;

immunohistofluorescent marker staining (IF) as in example 2;

immunohistochemical staining (IHC)

(1) Baking and dewaxing: after the paraffin sections were baked at 65 ℃ for 3-4 hours, they were transferred to a fume hood and soaked in xylene I for 15 minutes, followed by further soaking in xylene II for 15 minutes.

(2) Hydration: soaking in 100% I and 100% II ethanol for 5min respectively, and soaking in 95%, 90%, 80% and 75% ethanol for 5min respectively.

(3) Wash 3 times with 0.01M PBS for 5min each.

(4) Heating 0.01M sodium citrate buffer solution with pH of 6.0 by autoclave water bath method until the temperature of the buffer solution is higher than 95 deg.C, placing the rack with glass slides into the buffer solution, bubbling at high pressure for 2min, and taking out and naturally cooling at room temperature.

(5) Wash 3 times with 0.01M PBS for 5min each.

(6)3％H₂O₂The solution was allowed to stand at room temperature for 10 min.

(7) Wash 3 times with 0.01M PBS for 5min each.

(8) Excess water on the glass slide is absorbed by the tissue paper, the tissue is placed in a wet box, 5% goat blocking serum prepared by 0.01M PBS is dripped on the tissue, and then the tissue is incubated for 1h at 37 ℃.

(9) After the blocking solution was aspirated through the paper towel, primary antibody diluted with 1% goat blocking serum was added dropwise and incubated overnight at 4 ℃. And 0.01M PBS was used as a negative control.

(10) The next day, the wet box was removed and allowed to rewet at room temperature for 25 minutes.

(11)4 washes with 0.01M PBS for 5min each.

(12) Excess water on the slide was removed with a piece of tissue paper, placed in a wet box, and a secondary antibody diluted with 1% goat blocking serum was added dropwise and incubated at 37 ℃ for 1 h.

(12)4 washes with 0.01M PBS for 5min each.

(13) DAB developing solution (solution A: solution B is 1:1) is added dropwise, and the dyeing depth is observed under a microscope for about 1-20 min. The reaction was terminated by soaking and washing with purified water.

(14) Hematoxylin staining is carried out for 30-60 s. If the staining is too dark, 1% hydrochloric acid ethanol differentiates. If too light, the color is heavily dyed.

(15) Soaking and washing with PBS for 1 min.

(16) 70%, 80%, 90%, 95% ethanol respectively dehydrate for 2min, 100% ethanol dehydrate for 2 times each for 1 min.

(17) And the xylene I is transparent for 10min, and the xylene II is transparent for 10 min.

(18) Taking out the glass slide, absorbing xylene on the back and the edge of the glass slide by using a piece of tissue paper, dripping 1 drop of neutral resin on the tissue while the tissue is not dry, covering a cover glass, airing the glass slide at room temperature overnight, and taking a photograph of Nikon on a fluorescence microscope on the next day.

Tissue homogenate

(1) Taking out the tissue after the material is taken out from an ultralow temperature refrigerator at minus 80 ℃, and rewarming the tissue on ice for 10 to 20 minutes. The tube was filled in a 2ml EP tube and the balance weight was recorded. The tissue in the EP tube was minced with scissors until no large tissue mass was visible.

(2) Cell lysate + enzyme inhibitor (protease inhibitor and phosphate inhibitor added at 1: 100) was prepared, lysate and tissue weight were added at 10:1(v/w) ratio to EP tube containing minced tissue and lysed on ice for several minutes.

(3) The EP tube containing the tissue was placed under a well-established homogenizer and homogenized on ice until the tissue was completely disrupted.

(4) Centrifuging at 12000rpm for 10min with ultra-low temperature high speed centrifuge, and collecting homogenized tissue lysate.

(5) Protein concentration detection and sample processing

(6) According to the BCA protein concentration quantification kit instruction, the reagent A: reagent B was disposed at a ratio of 1: 50.

(7) The supernatant after tissue lysis was diluted. 10-fold dilution: 18 μ l PBS +2 μ l tissue supernatant +200 μ l BCA reagent.

(8) After being left in an incubator at 37 ℃ for 30 minutes, absorbance was measured at 562 nm.

(9) Calculating the protein concentration of the tissue lysate according to the BCA protein concentration standard curve, diluting the sample to an equal concentration by using pure water, and adding 5X loading.

(10) The mixed sample is subjected to 100 ℃ for 10 minutes to obtain an animal tissue protein sample. Then, the mixture was mixed and centrifuged, and the mixture was stored at-20 ℃ for a while and subjected to SDS-PAGE gel electrophoresis.

Western Blot(WB)

(1) SDS-PAGE gel electrophoresis: adding the prepared animal tissue protein sample into polyacrylamide gel, and separating the protein in the tissue sample in an electrophoresis apparatus of 80-120V.

(2) Electric conversion: a0.22 μm PVDF membrane of the corresponding size was cut off and placed in anhydrous methanol for activation for several seconds. The concentrated gum and unwanted areas are cut away. Soaking sponge and filter paper in the electrotransformation liquid, and sequentially placing into a black and white clamp according to requirements. And putting a red and black box into the electrophoresis tank, and then putting a black and white clip into the electrophoresis tank. Under the condition of ice bath, voltage is applied to perform electrotransformation.

(3) And (3) sealing: the 5% skim milk powder was blocked in a horizontal shaker at room temperature for 1.5 hours. The TBST solution was washed 3 times for 7 minutes each after blocking.

(4) Primary antibody incubation: after diluting the corresponding primary antibody with TBST solution according to the dilution ratio of the antibody specification, the primary antibody is added to the PVDF membrane after closed cleaning, and the PVDF membrane is incubated overnight in a horizontal shaking table under the environment of 4 ℃. After the next day primary antibody was recovered, the TBST solution was washed 3 times for 7 minutes each.

(5) And (3) secondary antibody incubation: according to the type of primary antibody, IgG (H + L) -HRP-labeled rabbit or mouse secondary antibody was selected and diluted with TBST solution at a dilution ratio of 1: 10000. Incubate with shaker for 1h at room temperature. Secondary antibodies were then recovered, washed with TBST, shaken 3 times for 7 minutes each.

(6) Exposure of gel imager: the exposed images were taken with a gel imaging system (Thermo, usa) and stored. The images were then processed and analyzed by Image-Lab.

As a result:

dry eye syndrome reduces FGF-21 expression of lacrimal gland in mouse eye orbit

The mice were excised from the extraorbital lacrimal glands, and dry eye was induced by scopolamine in combination with dry environment and treated with 800 μ g/ml rhFGF-21, as shown in fig. 12, high concentrations of rhFGF-21 improved BUT also BUT at 7D and 14D of treatment increased the basal tear secretion in the mice, with statistical differences compared to the contemporary PBS control group, fig. 12A, fig. 12B p <0.05, p < 0.01. The expression level of FGF-21 in intraorbital lacrimal glands and extraorbital lacrimal glands of normal healthy mice is detected by a WB method, and the result shows that the intraorbital lacrimal glands are tissues mainly expressing FGF-21, and the tissues are shown in FIG. 12C and FIG. 12D. The results of immunohistochemical staining (IHC) and WB methods jointly detecting the expression of lacrimal gland FGF-21 in the orbit of healthy mice and the dry eye-induced mice of 35D showed that the expression of lacrimal gland FGF-21 in the orbit of dry eye-induced mice was significantly reduced, and the difference was statistically significant compared to normal healthy mice, as shown in fig. 12E, fig. 12F, and fig. 12G.

rhFGF-21 downregulation of dry eye-induced intraorbital lacrimal gland TNF alpha, MMP-3, and MMP-9 expression

To further demonstrate the protective effect of rhFGF-21 on lacrimal glands in the orbit of mice. At 14D of the eye treatments, changes in the expression of the dry eye-related inflammatory markers TNF α, MMP-3, and MMP-9 were detected. The results are shown in fig. 13, immunohistochemistry and WB results in fig. 13A and 13B: compared with healthy mice, the expression of lacrimal glands TNF alpha, MMP-3 and MMP-9 in the eye socket of the mice in the PBS treatment group is obviously up-regulated, and the difference has statistical significance; the rhFGF-21 with medium and high concentration can remarkably reduce the expression of dry eye induced lacrimal gland TNF alpha, MMP-3 and MMP-9 in the orbit; of these, rhFGF-21 at 800 μ g/ml down-regulated dry eye-induced inflammatory factors more significantly, with statistical differences compared to the PBS treated group ([ p ] p <0.05, [ p ] p <0.01, [ p ] p < 0.001).

Expression of mouse lacrimal gland FGFR2 up-regulated by rhFGF-21

In order to further explore the specific mechanism of the protective effect of rhFGF-21 on the lacrimal gland in the orbit of the mouse. This example examined the expression of receptors corresponding to FGFs in day 14 lacrimal tissues, corresponding to FGFR1, FGFR2, FGFR3, and FGFR4, respectively. As shown in WB results of fig. 14, the difference in the expression levels of FGFR1, FGFR3, and FGFR4 in lacrimal gland tissue after the treatment with middle and high doses of rhFGF-21 did not have significant statistical significance compared to the PBS-treated group. It is noteworthy that the expression level of FGFR2 was down-regulated in the PBS-treated group and the expression level of FGFR2 was up-regulated in the medium-and high-dose rhFGF-21-treated group, with statistical significance ([ p <0.01, [ p <0.001 ]). Meanwhile, the staining results of immunohistochemistry were consistent with WB detection.

rhFGF-21 upregulates the intraorbital lacrimal gland PI3K-AKT signaling pathway

The PI3K-AKT signaling pathway is a classical signaling pathway for inhibiting apoptosis, and in order to further confirm the rhFGF-21 regulated related signaling pathway, protein expression of PI3K, P-AKT and total AKT in lacrimal glands in the eye orbit of mice was examined at the time of 14D treatment. As shown in fig. 15, the expression level of PI3K was significantly decreased in the intraorbital lacrimal gland of the PBS-treated mice compared with the healthy mice, and the difference was statistically significant. The expression of PI3K was increased in the lacrimal glands of mice treated with high and medium concentrations of rhFGF-21, and the differences were statistically significant compared to the PBS treated group (p <0.05, p < 0.01). Similarly, immunohistochemistry and WB results also showed similar differences in P-AKT expression between the different treatment groups: compared to the PBS treated group, healthy mice and mice in the medium and high dose rhFGF-21 treated group expressed P-AKT with statistical significance (. P <0.01,. P < 0.001).

3 inhibiting FGFR2 and PI3K-AKT and reducing the protective effect of rhFGF-21

To further demonstrate that rhFGF-21 regulates the action pathway of lacrimal gland in orbit, intraperitoneal injection of FGFR2 inhibitor T5122 and PI3K-AKT pathway inhibitor LY294002 to mice was set on the basis of using 800 μ g/ml of rhFGF-21, respectively. The results are shown in FIG. 16, and the difference between the basal tear secretion of mice using the LY294002 inhibitor at 7D and 14D was statistically lower than that of the group treated with rhFGF-21 alone at 800. mu.g/ml. However, the amount of T5122 inhibitor inhibited tear secretion showed a difference at 14D (fig. 16A × p < 0.01). Tear film break-up time measurements showed that at 14D of treatment, the difference was statistically significant in the group given the LY294002 formulation compared to the rhFGF-21 treated group alone (fig. 16B, <0.01 x p). HE staining of lacrimal glands revealed that the lacrimal glands of mice administered with T5122 and LY29004 were morphologically full and closely aligned to the degree of lacrimal gland cells within the same treatment period of 14D, rather than the lacrimal glands administered with rhFGF-21 alone (fig. 16C). Ki-67 immunofluorescent staining of lacrimal glands showed no statistical significance for Ki-67 expression differences between T5122 and LY29004 groups compared to rhFGF-21 group administered alone (FIG. 16E).

Inhibition of FGFR2 reduces lacrimal gland PI3K and P-AKT expression

The effect of FGFR2 and PI3K-AKT pathway inhibitors on the resistance to high doses of rhFGF-21 in the treatment of dry eye in mice is demonstrated. Changes in the expression of FGFR2, PI3K, P-AKT and AKT proteins corresponding to the lacrimal glands in the eye orbit of these mice were examined using the IHC method. The result is shown in fig. 17, the inhibitor T5122 can resist the high-dose rhFGF-21 to promote the expression up-regulation of FGR2 protein in lacrimal gland tissues, and the difference has statistical significance; meanwhile, the expression level of FGFR2 in the group using LY294002 was not statistically different from that in the group treated with high-dose rhFGF-21 (fig. 17A, 17B × P < 0.01). In addition, T5122 inhibited PI3K expression, which was statistically significant compared to the high dose rhFGF-21 treated group, whereas the LY294002 inhibitor did not significantly down-regulate PI3K protein, which was not statistically significant compared to the high dose rhFGF-21 group (fig. 17A, fig. 17C p < 0.05). It is noteworthy that both T5122 and LY294002 groups reduced rhFGF-21-induced high expression of P-AKT, with statistical differences (fig. 3-6A E P <0.05, P < 0.01).

Inhibiting FGFR2 and P-AKT, and resisting rhFGF-21 to reduce inflammation of lacrimal gland in orbit

To further validate the effect of inhibiting FGFR2 and PI3K-AKT pathway against rhFGF-21 in treating lacrimal glands in the orbit. The expression of the corresponding TNF-alpha, MMP-3 and MMP-9 proteins was detected by IHC staining. The results are shown in fig. 18, where inhibitors T5122 and LY294002 reversed the effect of high concentrations of rhFGF-21 in down-regulating TNF- α, MMP-3 and MMP-9, with statistical differences compared to the high dose rhFGF-21 treatment group (fig. 18A, 18B, 18C, 18D p <0.05, p < 0.01). Interestingly, however, there was no significant statistical difference in the effect of T5122 upregulation of TNF- α compared to high doses of rhFGF-21.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

Application of rhFGF-21 in preparing medicine for treating xerophthalmia.
2. The use of rhFGF-21 for the manufacture of a medicament for treating dry eye according to claim 1, wherein the rhFGF-21 down-regulates the expression levels of TNF α, MMP-3, and MMP-9 inflammatory factors induced by dry eye.
3. The use of rhFGF-21 for manufacturing a medicament for treating dry eye according to claim 1, wherein the rhFGF-21 increases basal tear secretion of a mouse with dry eye induced by scopolamine in combination with a dry environment.
4. The use of rhFGF-21 of claim 1 for preparing a medicament for treating dry eye, wherein the rhFGF-21 increases the tear film break-up time of scopolamine in combination with dry environment induced dry eye mice.
5. The use of rhFGF-21 for the preparation of a medicament for the treatment of dry eye according to claim 1, wherein rhFGF-21 is used to improve the Scopolamine-associated Dry Environment-induced staining score for sodium keratecholate in mice with dry eye.
6. The use of rhFGF-21 for the manufacture of a medicament for the treatment of dry eye according to claim 1, wherein rhFGF-21 enhances the corneal nerve sensitivity of scopolamine in combination with dry environment induced dry eye mice.