CN107050429B - Application of human fibroblast growth factor 21 in preparing medicine for treating cerebral apoplexy - Google Patents

Abstract

The invention discloses application of human fibroblast growth factor 21(FGF21) in preparation of a medicine for treating cerebral apoplexy. The effect of the medicine on the cerebral apoplexy is mainly shown in the aspects of protecting the integrity of a blood brain barrier, inhibiting neuroinflammation in the brain, promoting the remodeling of blood vessels and white matter in the brain, improving the recovery of the nerve function after the cerebral apoplexy and greatly improving the brain injury of patients with the cerebral apoplexy and the patients with diabetes and cerebral apoplexy.

Description

Application of human fibroblast growth factor 21 in preparing medicine for treating cerebral apoplexy

Technical Field

The invention belongs to the technical field of medicines, and relates to a new application of human fibroblast growth factor 21(FGF21), in particular to an application of human FGF21 in preparation of a medicine for treating stroke.

Background

Stroke is commonly called as stroke, has the characteristics of high morbidity, high disability rate, high recurrence rate and high mortality rate, is one of the most important lethal diseases in the world, and is the first disability and lethal disease in China. Cerebral apoplexy is mainly divided into cerebral arterial thrombosis and hemorrhagic stroke, wherein about 80 percent of cerebral arterial thrombosis is cerebral arterial thrombosis. Ischemic stroke is caused by a transient or persistent reduction in blood flow in cerebral arteries. Brain protection drugs are currently the focus of research in the neurology community, and the only drug currently proven to be effective and recommended as a conventional treatment is aspirin. r-tPA is used within 3 hours after ischemic stroke starts, and urokinase is used before artery within 6 hours, which is proved to have certain curative effect. However, the complexity and pleiotropy of stroke still greatly limits the effective number of cerebroprotective drugs in clinical practice.

It is noted that ischemic stroke is one of many complications of diabetes, and is a brain-related lesion that occurs when diabetes progresses to a later stage (i.e., a complication stage). Diabetics are statistically 2-6 times more sensitive to ischemic stroke, with about 30% of stroke patients suffering from diabetes and 90% being type II diabetes (T2D). Compared with non-diabetic people, the death rate of diabetes combined with ischemic stroke is improved to two times, the disability rate and the recurrence rate are high, the recovery of nerve function damage is slow, the nerve regeneration capability is weakened, and the standard medicament r-tPA treatment effect of the ischemic stroke is reduced due to higher hemorrhagic transformation. At present, no medicine with better treatment effect on diabetes combined with cerebral apoplexy is discovered or published.

With the improvement of living standard and the increasing aging degree, the number of diabetics is increased, the risk of ischemic stroke is increased by diabetes, and the diabetes and the ischemic stroke become huge factors threatening public health. However, despite numerous reports of diabetic renal and retinal microvascular complications, little is known about the mechanisms of diabetic neuropathy (e.g., stroke). Patients with T2D often have hyperglycemia, dyslipidemia, and insulin resistance, which exacerbate their long-term loss of function following stroke and also limit the brain's inherent neural repair and reconstruction ability. Studies have shown that insulin glycemic control alone after stroke in T2D patients does not reverse their functional impairment after stroke.

Therefore, the search for effective target drugs for treating stroke, even diabetes complicated with stroke, is an urgent problem to be solved at present.

Disclosure of Invention

The present inventors have found through extensive studies that symptoms of stroke patients and T2D stroke patients can be improved by human fibroblast growth factor 21, and the mechanism of improving the symptoms is elucidated, thereby completing the present invention.

The invention aims to provide application of human fibroblast growth factor 21 in preparing a medicament for treating cerebral apoplexy.

Another object of the present invention is to provide a medicament for treating stroke, which comprises a therapeutically effective amount of human fibroblast growth factor 21.

The application of the human fibroblast growth factor 21 in preparing the medicine for treating the cerebral apoplexy has the following beneficial effects:

(1) the invention discovers that human FGF21 can: 1. protection of the Blood Brain Barrier (BBB) by activating PPAR γ; 2. reducing neuroinflammation by inhibiting expression of NF κ B protein; 3. promotes the reconstruction of blood vessels/white matter regulated by AMPK/Nrf2, thereby promoting nerve regeneration after cerebral apoplexy, so that the compound can be used as an effective component of various dosage form medicaments, and has great significance for treating cerebral apoplexy and diabetes combined with cerebral apoplexy.

(2) FGF21 is the only mitogenic gene currently found in the FGF family, thereby greatly reducing the risk of clinical medication.

(3) The treatment mechanism disclosed by the invention has guiding significance for the subsequent research of medicines for treating cerebral apoplexy and diabetes combined cerebral apoplexy.

Drawings

FIG. 1 is an electrophoretogram of purified FGF 21;

FIG. 2 is a high performance liquid chromatogram of FGF21 after purification;

FIG. 3 shows the results of a paste removal test;

FIG. 4 shows the results of an forelimb stepping-off experiment;

FIG. 5 shows the results of grip strength tests;

FIG. 6 shows the results of the Y maze test;

FIG. 7 is a graph of infarct volume size and quantification for each group;

FIG. 8 is the expression of FGF21, p-FGFR1 and FGFR1- β -klotho complex in mouse brain;

FIG. 9 is a graph of the effect of FGF21 treatment on high glucose plus IL-1 β in combination with induction of claudin expression damaging brain vascular endothelial cells;

FIG. 10 is a graph showing the effect of FGF21 treatment on hyperglycemia and IL-1 β in combination inducing adhesion factor expression in injured cerebral vascular endothelial cells;

FIG. 11 is a graph showing the effect of FGF21 treatment on hyperglycemia and IL-1 β in combination with induction of permeability to injured cerebral vascular endothelial cells;

figure 12 is the effect of FGF21 on expression of phosphorylated FGFR 1;

FIG. 13 is a graph of the effect of FGF21 on PPAR γ expression;

FIG. 14 is a graph of the effect of PPAR γ on vascular endothelial cell permeability;

FIG. 15 is a graph of the effect of FGF21 on anti-inflammatory factor M2 microglia;

FIG. 16 is a graph of the effect of FGF21 on inflammatory factors in LPS-induced cells;

FIG. 17 is a graph of the effect of FGF21 on LPS-induced phosphorylation of FGFR1, NF κ B activity, and PPAR γ expression levels in cells;

FIG. 18 is a graph of the effect of FGF21 on axonal damage and myelin loss;

FIG. 19 is a graph of the effect of FGF21 on oligodendrocyte regeneration;

FIG. 20 is a graph of the effect of FGF21 on cerebral vascular density in the brain;

FIG. 21 is a graph of the effect of FGF21 on AMPK/Nrf2 activity in the brain;

FIG. 22 is a graph showing the effect of FGF 21-treated conditioned medium of cerebrovascular endothelial cells on the proliferation of OPC cells;

FIG. 23 is a graph of the blood glucose regulation of type II diabetes mellitus in mice with stroke by FGF 21.

Detailed Description

The present invention is further described below in terms of specific embodiments, and features and advantages of the present invention will become apparent as the description proceeds.

Fibroblast growth factor 21(FGF21) is a newly discovered factor related to glycolipid metabolism, can improve the function of islet beta cells and promote glucose absorption in adipose tissues, is the only gene without mitogenesis currently found in FGF family, and therefore, the clinical medication risk is greatly reduced. It is mainly expressed in liver, and has a small amount of expression in brain regions and glial cells. Among them, human FGF21(MW:19.5kD) has 181 amino acids and is 75% identical to the peptide chain of murine FGF 21.

Research has shown that under some pathological conditions, FGF21 functions to repair tissue damage. Recombinant FGF21 has been shown to have therapeutic efficacy in a variety of animal models, such as liver disease, diabetic nephropathy, atherosclerotic plaque formation, myocardial infarction, and diabetic cardiomyopathy. Although the signaling pathway mediated by FGF21 remains unclear, its therapeutic mechanism of action may involve vascular protection and remodeling, anti-inflammation, anti-oxidative stress, inhibition of formation of advanced glycation end products, and promotion of tissue repair.

At present, FGF21 has not been reported on brain metabolism, brain protection or cognition of stroke or diabetes combined with stroke, and has not been systematically researched.

The inventor finds that exogenous recombinant human fibroblast growth factor 21(rFGF21) can activate FGFR 1-beta-klotho compound through a large number of experiments, and (1) the Blood Brain Barrier (BBB) is protected by activating PPAR gamma; (2) inhibition of NF κ B-mediated neuroinflammation; (3) promoting vascular/white matter remodeling mediated by AMPK/Nrf2 activation; improves the recovery of nerve function after stroke, and improves the symptoms of patients with stroke and T2D patients with stroke.

Therefore, in one aspect of the present invention, there is provided a use of human fibroblast growth factor 21 in the preparation of a medicament for treating stroke.

In the present invention, the stroke includes diabetes combined with stroke, including type II diabetes combined with stroke (i.e. T2D stroke). Clinical and experimental data indicate that patients with T2D often have hyperglycemia, dyslipidemia, and insulin resistance, which factors exacerbate permeability to the blood-brain barrier and neuroinflammation, exacerbate long-term neurological deficits following stroke, and limit the brain's intrinsic neurological repair and reconstruction ability, which plays an important role in restoring normal after stroke.

In order to realize the application of the human FGF21 in preparing the medicines for treating cerebral apoplexy, the invention obtains the exogenous recombinant human FGF21 by a microbial fermentation method. A corresponding DNA sequence is optimally designed according to a protein sequence of human FGF21(NP _061986.1) on GenBank, a proper expression vector is constructed, the expression vector is transferred to a prokaryotic host cell to obtain a strain with high protein expression level, the strain is activated and amplified, FGF21 is induced and expressed, and high-purity FGF21 is obtained through multi-step separation and purification (as shown in figure 1 and figure 2).

In a preferred embodiment, the nucleotide sequence encoding human FGF21 protein is shown in SEQ ID NO.1, and has NdeI cleavage site at the 5 'end and BamHI cleavage site at the 3' end.

In a further preferred embodiment, the coding sequence of human FGF21 is inserted into the expression vector, preferably the expression vector is a prokaryotic cell expression vector, such as plasmid pET3c containing an IPTG inducible promoter, which is suitable for prokaryotic cells, in particular for inducible expression in e.

In a further preferred embodiment, the host cell is a prokaryotic cell, preferably E.coli, such as E.coli BL21(DE 3). Coli, in particular escherichia coli BL21(DE3), is suitable for the expression of expression vectors, in particular the plasmid pET3 c.

The inventor finds that FGF21 has a protective effect on the integrity of the Blood Brain Barrier (BBB) in T2D cerebral apoplexy through experiments.

Central nervous system diseases often cause drastic changes of the structure and function of the blood brain barrier, the permeability of the blood brain barrier is obviously improved, so that macromolecular substances such as plasma albumin can pass through the barrier, and the serious damage of the blood brain barrier is caused by serious brain injury. By performing nerve function and cognitive function tests on a T2D cerebral apoplexy model before and after FGF21 treatment, namely db/db mice subjected to peripheral middle cerebral artery occlusion surgery, FGF21 treatment can reduce nerve function damage of the db/db mice after cerebral apoplexy (fig. 3-6). Meanwhile, comparing the volume of cerebral infarction of mice, the volume of cerebral infarction of db/db cerebral apoplexy mice treated by FGF21 is obviously reduced compared with that of mice treated by FGF (figure 7). The above in vivo experiments all show that FGF21 has a protective effect on the integrity of the blood brain barrier in T2D stroke, and reduces secondary damage related to the blood brain barrier.

The increase in blood brain barrier permeability is directly due to the opening of tight junctions between cerebrovascular endothelial cells. Diabetes combined with cerebral stroke exacerbates blood brain barrier breakdown, where reduced expression of claudin is an important factor. Endothelial adhesion factor plays a crucial role in leukocyte penetration into the brain after stroke, which causes inflammation of the brain and destruction of the blood-brain barrier.

Thus, further, using high levels of glucose and the proinflammatory cytokine interleukin 1 β (IL-1 β) in combination with injured cerebrovascular endothelial cells to mimic the acute phase db/db T2D mouse in vitro model for cerebral stroke, the effect of FGF21 on the blood-brain barrier was further determined by the effect on the permeability of cerebrovascular endothelial cells, endothelial cell adhesion factors, and expression of claudin before and after FGF21 treatment.

It was found that the combination of high glucose plus IL-1 β levels and induced injury decreased the expression of brain vascular endothelial cell fibronectin (FIG. 9), increased adhesion factors in endothelial cells (FIG. 10), and increased brain vascular endothelial cell permeability (FIG. 11), suggesting that the blood brain barrier may be disrupted. FGF21 treatment can increase the expression of tight junction protein after injury, inhibit the expression of adhesion factors and reduce the permeability of cerebrovascular endothelial cells. From in vitro experiments, the protective effect of FGF21 on the blood brain barrier was further determined.

FGF21 signaling requires activation of FGF receptors (FGFRs), particularly FGFR1 and its co-receptor β -Klotho. By subcutaneous injection of FGF21 to db/db T2D brain stroke mice, increased expression of FGF21 and phosphorylated FGFR (p-FGFR1) was found in the brain, and at the same time, a complex of FGFR 1-beta-klotho was generated in the brain, and the expression was significant (FIG. 8). It was determined that FGF21, unlike other FGF family members, due to its very low affinity for heparin, enables FGF21 to activate the FGFR1- β -klotho complex by simple diffusion across the blood brain barrier, enabling signaling.

It was found that activation of PPAR γ (peroxisome proliferator-activated receptor γ) may alter the expression of claudin, and protection of blood brain barrier permeability by FGF21 may be associated with its activation. In vitro experiments can determine that FGF21 activates an FGFR 1-beta-klotho compound, so that downstream PPAR gamma is activated to protect a blood brain barrier.

In vitro experiments, high levels of glucose and IL-1 β were used in conjunction with injury-induced cerebral vascular endothelial cells for validation. Compared with cerebral vascular endothelial cells not treated by FGF21, the FGF21 treatment can increase the expression of FGFR1 phosphorylation (FIG. 12) and promote the expression level and activity of PPAR gamma protein (FIG. 13). Inhibition of PPAR γ expression, increased permeability of brain vascular endothelial cells, and failure of the protective effect of FGF21 (fig. 14).

Further, the present inventors found that FGF21 can inhibit neuroinflammation.

T2D cerebral stroke produced significant brain inflammation, and in vivo experiments showed that FGF21 was able to increase the number of M2 type microglia as anti-inflammatory cells in the brain after cerebral stroke in db/db T2D mice (FIG. 15).

Through in vitro experiments, FGF21 can inhibit the expression of M1 proinflammatory factors iNOS and TNF-alpha mRNA in rat microglia cells (OPC) which are primarily cultured and stimulated by Lipopolysaccharide (LPS), and simultaneously increase the expression of M2 anti-inflammatory factors Arg1 and IGF-1mRNA, thereby participating in the repair after injury (figure 16).

Among the microglia, PPAR γ is the most prominent among PPARs of the same type and is also a key "information transmitter" transcription factor that controls the transcription process by affecting the phenotype of microglia and macrophages and inflammatory factors released from activated microglia and macrophages. NF κ B is an early key factor in the regulation of inflammation. Increased expression of NF κ B following stroke activates microglia and macrophages, increasing the release of harmful proinflammatory factors.

In vitro experiments showed that FGF21 significantly decreased NF κ B activity induced by LPS and increased PPAR γ levels in microglia nuclei (FIG. 17). FGF21 acts on microglia, increasing the expression of its FGFR1 phosphorylation, whereas FGFR inhibitor administration inhibits the phosphorylation of FGFR 1. FGF21 can also reduce the activity of NF kappa B in primary rat microglia cell nucleus induced by LPS, and increase the expression of cell nucleus transcription factor PPAR gamma in cell nucleus protein, and after the FGFR inhibitor is added, the FGF21 can inhibit the increase of PPAR gamma expression and the reduction of NF kappa B activity. Therefore, after the FGF21 activates an FGFR 1-beta-klotho complex, the activity of downstream NF kappa B can be reduced, and neuroinflammation can be inhibited.

The inventors have also found that FGF21 promotes vascular and white matter remodeling.

Type II diabetes mellitus can exacerbate vascular and white matter damage and attenuate its remodeling function following ischemic stroke. The research of the invention shows that FGF21 has potential effect on the protection and reconstruction of cerebral vessels in vascular endothelial cells cultured in vivo and in vitro.

In comparison to db/db T2D cerebral stroke mice before and after FGF21 treatment, FGF21 increased the levels of pro-angiogenic factor (VEGF-A, IGF-1 and eNOS) mRNA in isolated cerebral microvascular fragments (Table 1). FGF21 reduced axonal damage and loss of myelin following stroke in db/db T2D mice (fig. 18); increasing vascular density and oligodendrocyte regeneration in the perilesional area (fig. 20 and 19). Conditioned medium of brain vascular endothelial cells treated with FGF21 promoted proliferation of oligodendrocyte cells in vitro (fig. 22).

In the brain of type II diabetes models, early inflammatory and oxidative stress interferes with metabolism and cellular homeostasis, resulting in loss of function associated with AMPK and Nrf2 signaling pathways, leading to cerebrovascular and degenerative diseases. Experiments show that the phosphorylation of AMPK in brain and the activity of Nrf2 in cell nucleus can be obviously increased by FGF21 treatment (figure 21), and a part of action mechanism of FGF21 is determined that the AMPK and Nrf2 activities are up-regulated by FGF21, and the increase of the AMPK and Nrf2 activities after stroke can promote neuronal repair and recovery of nerve functions.

FGF21 was also able to reduce hyperglycemia following stroke, in addition to being effective in the treatment of T2D stroke-complicated brain (fig. 23). Studies have shown that, compared to untreated diabetes-associated stroke models, FGF21 treatment significantly reduces the concentration of glucose in the blood, while FGF21 also significantly reduces the level of glycosylated hemoglobin (Hb A1 c). These results indicate that FGF21 can regulate the metabolic function of T2D on sugar after stroke.

In the invention, the stroke has the same meaning as the stroke, and the T2D stroke has the same meaning as the T2D stroke.

In a second aspect, the present invention provides a medicament for treating stroke, particularly type II diabetes combined with stroke, comprising a therapeutically effective amount of human fibroblast growth factor 21.

The medicine also comprises other pharmaceutically acceptable carriers or auxiliary materials, such as pharmaceutically allowable excipients, fillers, absorption enhancers, surfactants, adsorption carriers, synergists or other additives. The administration form of the medicine can be injection. The preparation form of the medicament can be obtained by a common preparation method well known to those skilled in the art. The administration route of the medicament can be subcutaneous injection, intravenous injection, intramuscular injection or other effective injection forms.

The effect of the medicine on stroke, particularly diabetes combined stroke, is mainly shown in (1) the integrity of blood brain barrier is protected; (2) inhibiting neuroinflammation in the brain; (3) promoting vascular and white matter remodeling in the brain.

Among them, the integrity of protecting the blood-brain barrier is mainly reflected in:

(1a) FGF21 reduced impairment of sensory and cognitive function following cerebral stroke;

(1b) FGF21 reduced the size of cerebral infarction after cerebral stroke;

(1c) FGF21 increases the expression of vascular endothelial cell claudin ZO-1 and VE-cadherin;

(1d) FGF21 inhibits the expression of the adhesion factors VCAM-1 and E-selectin in brain vascular endothelial cells;

(1e) FGF21 reduces cerebrovascular endothelial cell permeability;

(1f) FGF21 increased FGFR1 phosphorylation expression in brain and/or promoted PPAR γ expression.

Inhibition of neuroinflammation in the brain is mainly manifested by:

(2a) FGF21 increases the number of M2 type microglia in anti-inflammatory cells in the brain;

(2b) FGF21 reduces the expression level of pro-inflammatory factors iNOS mRNA and TNF-alpha mRNA in brain;

(2c) FGF21 increasing the expression levels of anti-inflammatory factor Arg1mRNA and IGF-1mRNA in the brain;

(2d) FGF21 reduces the expression level of the inflammatory factor IL-1 β mRNA in the brain;

(2e) FGF21 decreased NF κ B activity in the brain.

The promotion of vascular and white matter remodeling in the brain is mainly reflected in:

(3a) FGF21 reduces axonal damage and loss of myelin;

(3b) FGF21 promotes regeneration of oligodendrocytes;

(3c) FGF21 increased cerebral vascular density;

(3d) FGF21 increases the expression levels of the pro-angiogenic factors VEGF mRNA, eNOS mRNA, and IGF-1mRNA in the cerebrovascular segment.

Examples

The invention is further illustrated by the following specific preferred examples. These examples are illustrative only and should not be construed as limiting the invention.

EXAMPLE 1 obtaining exogenous recombinant human fibroblast growth factor 21

Example 1.1 construction of high expression Strain

Firstly, a corresponding DNA sequence is optimally designed according to the protein sequence of human FGF21(NP _061986.1) on GenBank, and is shown as SEQ ID NO. 1. Subsequently, a corresponding DNA fragment was artificially synthesized, which had Nde I cleavage site at the 5 'end and BamH I cleavage site at the 3' end. The DNA of FGF21 thus obtained was digested with Nde I and BamH I, and the expression empty vector pET3c was treated with the same enzyme. The two fragments were ligated with T4DNA ligase, transformed into competent E.coli DH5 α, Ampicillin resistance-screening positive clones, and subjected to DNA sequence analysis to ensure that the resulting cDNA sequence of FGF21 was correct. The resulting recombinant plasmid was designated as pET3c-FGF 21. The recombinant plasmid pET3c-FGF21 was transformed into E.coli BL21(DE3), and transformants were selected for resistance to Ampicillin. Finally obtaining the strain with high expression of FGF 21.

EXAMPLE 1.2 activation and amplification of the Strain (FGF21 fermentation)

Inoculating qualified strains (the inoculation amount is 10%) into a first-generation culture medium, culturing for 4h at 37 ℃, inoculating strains (the inoculation amount is 10%) into a second-generation culture medium, and culturing for 10-12 h at 37 ℃ for inoculation of a fermentation tank.

The fermentation culture medium is filled into a fermentation tank with the volume of 200L (the fermentation tank is pre-subjected to air digestion at 115 ℃ for 30min), then actual digestion sterilization is carried out (at 115 ℃ for 30min), then a proper amount of the second-generation seed liquid obtained by the culture is inoculated (with the inoculation amount of 10%) into the fermentation tank, a high-density fermentation culture process is adopted, the DO value of the culture liquid is controlled to be not less than 30% by controlling the feeding rate, the ventilation amount, the stirring rate and the like of glucose, and when the OD600 of the thalli reaches about 15-18%, IPTG is added for induction expression. After 1h of induction, a fed-batch culture medium is added, and the fermentation is stopped after 4h of induction.

After the fermentation is finished, separating the thalli in the fermentation liquor by using a tubular centrifuge (16000r/min) and collecting the thalli.

EXAMPLE 1.3 FGF21 purification

1) Preparation of Inclusion bodies

The wet cells were suspended in a 10-fold volume (w/v) of a buffer containing 25mmol/L Tris (pH8.0), 150mmol/L NaCl, 10mmol/L EDTA-2 Na; according to the required FGF21 wet bacterial cells, the ratio of 1:1000(w/w), 1: 10000(w/w) of lysozyme and DNA enzyme are weighed, dissolved by lysate and added into the thallus suspension, the mixture is stirred and lysed overnight at room temperature, the bacteria liquid is examined by a microscope until no complete thallus exists in the visual field, and then a high-speed refrigerated centrifuge (9000r/min, 4 ℃) is used for separating and collecting precipitates.

The precipitate obtained in the previous step was suspended in washing solution I containing 25mmol/L Tris (pH8.0), 150mmol/L NaCl, 10mmol/L EDTA-2Na, and 0.2% sodium deoxycholate in a volume (w/v) 15 times that of the wet cells, and after thoroughly stirring, the mixture was dissolved and mixed well, and the precipitate was separated and collected by a high-speed refrigerated centrifuge (9000r/min, 4 ℃).

Suspending the precipitate in the last step in washing liquid II containing 25mmol/L Tris (pH8.0), 150mmol/L NaCl, 10mmol/L EDTA-2Na and 0.2% Triton-X100 in 20 times the volume (w/v) of the wet thallus, stirring, dissolving and mixing, separating in a high speed freezing centrifuge (9000r/min at 4 deg.c), and collecting the precipitate as FGF21 inclusion body.

2) Renaturation of inclusion bodies

The inclusion bodies were suspended in 15 volumes (w/v) of a denaturing solution containing 25mmol/L Tris (pH8.9), 2mmol/L EDTA-2Na, and 8mol/L urea, and after thoroughly stirring and dissolving at 4 ℃ and mixing, they were added to a dialysis bag (cut-off molecular weight of 7000 kd). The FGF 21-denatured solution was dialyzed overnight at 4 ℃ against 200 volumes (w/v) of a renaturation solution containing 25mmol/L Tris (pH8.9) and 2mmol/L EDTA-2 Na. After the dialysis, the supernatant was separated and collected by a high-speed refrigerated centrifuge (9000r/min, 4 ℃).

3) Purification of

The crude FGF21 supernatant obtained above was first passed through a DEAE Sepharose Fast Flow ion exchange column, equilibrated with a 25mmol/L Tris (pH8.9), 10mmol/L EDTA-2Na, and 0.03mol/L NaCl equilibrium solution to remove proteins, and then eluted with an eluent of 25mmol/L Tris (pH8.9), 10mmol/L EDTA-2Na, and 0.1mol/L NaCl to collect the desired protein peak.

And (3) allowing the target protein collection liquid in the previous step to pass through a hydrophobic column, balancing by using a buffer solution of 25mmol/L Tris (pH8.9) and 2mol/L NaCl, removing protein, adjusting the NaCl concentration of the sample solution to 2mol/L, then loading, eluting by using an eluent of 25mmol/L Tris (pH8.9) and 0.1mol/L NaCl, and collecting a target protein peak. Concentrating the collected target protein solution, passing through a Sephadex G25Coarse gel filtration chromatographic column, eluting with 20mmol/L PB (pH 7.4) and 50mM NaCl buffer solution, collecting an active peak, and detecting by SDS-PAGE and high performance liquid chromatography to obtain FGF21 protein with the purity of more than 95%, namely exogenous recombinant human fibroblast growth factor 21(rFGF21), which is shown as rFGF21 in the attached drawing of the specification and is abbreviated as FGF21 in the specification, as shown in FIG. 1 and FIG. 2. In the figure, 1 is a map obtained by performing SDS-PAGE on three purified FGF21 protein samples ( samples 1, 2 and 3); FIG. 2 is a high performance liquid chromatogram of FGF 21.

Example 2 Effect of FGF21 on blood brain Barrier integrity in T2D strokes

Example 2.1 animal experiments (in vivo experiments)

Type II diabetes model (T2D model): in the study of obesity-induced type II diabetes, the most widely used animal models are rodent models of innate leptin deficiency (ob/ob) and leptin receptor deficiency (db/db). C57BLKS-Leprdb type II diabetic mice (db/db T2D mice, Jackson laboratory inventory # 000642, 12-13 weeks old) were used in the present invention. This model has been widely used in the study of new mechanisms of carbohydrate metabolism and complications, and the db/db T2D model of stroke reflects the destruction of the BBB, neuroinflammation, vascular/white matter damage.

Ischemic stroke model: since the mortality rate of Middle Cerebral Artery Occlusion (MCAO) in rodent models of type II diabetes is up to 70%, we selected mice with type II diabetes and middle cerebral artery occlusion as models of ischemic stroke.

The steps for obtaining the cerebral arterial thrombosis model are as follows: anesthetizing a type II diabetic mouse, fixing the mouse in a supine position, under an operating microscope, incising the skin along the center of the anterior cervical part, separating bilateral common carotid arteries, and passing through the mouse with a suture without ligation; fixing the right lateral position of the mouse, making a longitudinal incision at the connecting line of the inner canthus and the external auditory canal, separating the temporal muscle, and drawing the temporal muscle to two sides by using a suture to expose the temporal bone; drilling with a cranial drill, removing bone flap, stripping off dura mater, and exposing middle cerebral artery; carefully hooking the middle cerebral artery, coagulating with an electrocoagulator, and flushing with normal saline to reduce the temperature; the mice were again placed supine and the bilateral common carotid arteries were occluded with vascular clamps and the wound was closed, 60min later the arterial clamps were loosened and the skin was sutured.

All data from FGF 21-treated db/db T2D stroke mice were compared to two non-treated stroke control groups, namely the normoglycemic control group (db/+) and db/db T2D.

Animal experiments: c57BLKS-Leprdb db db/db T2D mice (Jackson laboratories, male, 12 weeks old) were injected subcutaneously once every 12h at a daily dose of 3.0mg/kg for a total of 14 days using 1.5mg/kg of exogenous recombinant human fibroblast growth factor 21(rFGF2, shown in the drawing as rFGF21, abbreviated as FGF21 in the specification). Groups 3 were tested and compared: normal blood sugar db/+ mice group (db/+ group for short, non-treatment group), db/db T2D mice group (db/db group for short, non-treatment group) and FGF21 treatment adult male db/db T2D mice group (db/db + FGF21 group for short, treatment group), each group consisting of 12 mice. After the cerebral apoplexy, the cerebral apoplexy is db/+ cerebral apoplexy group, db/db + FGF21 cerebral apoplexy group.

Data processing: for the measurement of parameters and continuous variables, such as lesion size, blood brain barrier permeability, mRNA and protein expression levels, immunohistochemistry, biochemical analysis, Tukey-Kramer Post-hoc tests analysis of variance was used. For non-parametric ordinal data (e.g., the results of neurobehavioral), we used the non-parametric rank sum test, Post-hoc Mann-Whitney tests. P <0.05 was statistically significant.

Example 2.1.1 FGF21 reduces impairment of nerve function after stroke in the brain

Sensory function was assessed by a stick-removal test, a forelimb step-off test, and a grip test before and 1, 3, 5, 7, and 14 days after stroke, and cognitive function was tested by the Y maze 28 days after stroke. The test is a test means which is conventional in the art and is not particularly limited herein. The test results are shown in FIGS. 3 to 6, wherein P<0.01, compared to db/+ cerebral stroke group;^#P<0.01, compared to db/db stroke group.

As shown in FIG. 3, the patch removal experiments indicated that the time spent in the db/db stroke group was increased compared to the db/+ stroke group, whereas FGF21 decreased the time.

As shown in FIG. 4, the forelimb stepping-out experiment showed that the percentage of footstep errors in db/db stroke group was increased compared to db/+ stroke group, while the footstep errors were decreased from day 3 to day 14 of FGF21 administration.

As shown in FIG. 5, grip strength experiments showed that grip strength was not significantly changed in db/db stroke group compared to db/+ stroke group, whereas increased grip strength was obtained on days 3, 5, 7 and 14 of FGF21 administration.

As shown in FIG. 6, in the Y maze experiment, the percentage of correct alternating selection was significantly decreased in the db/db stroke group compared to the db/+ stroke group, whereas FGF21 was increased by day 14.

From the above test results, FGF21 reduced the impairment of neural function in db/db mice after cerebral stroke.

Example 2.1.2 FGF21 decreases post-stroke cerebral infarction size

Mice were sacrificed after the above-described behavioral experimental tests, and brain tissue was taken for determination of cerebral infarction size.

The detection results are shown in fig. 7, the left graph is a graph of the size of the HE stained cerebral infarction, and the right graph is a quantitative graph of the infarct volume of each group. (n-6). P<0.05, compared to db/+ stroke group;^#P<0.05, compared to db/db stroke group.

HE staining and quantitative analysis results show that the cerebral infarction volume of db/db cerebral apoplexy mice is obviously increased compared with that of db/+ cerebral apoplexy groups, and FGF21 obviously reduces the cerebral infarction volume of db/db cerebral apoplexy mice 14 days after administration.

Example 2.1.3 FGF21 can activate FGFR1 in the brain via the BBB to exert neuroprotective effects (mechanism)

FGF21 signaling requires activation of FGFR, particularly FGFR1 and its co-receptor β -Klotho complex. C57BLKS-Leprdb db db db/db T2D mice were injected subcutaneously with 2mg/kg FGF21, the brains were harvested after 2h, the expression of FGF21 and phosphorylated FGFR1(p-FGFR1) in the mouse brains was determined by Western Blot (FIG. 8A), FGFR1- β -klotho complex formation was detected by co-immunoprecipitation (FIG. 8C), and C57BLKS-Leprdb db db db db db db db db/db T2D mice which were not treated with FGF21 were used as a control group.

As can be seen from fig. 8A and 8B, FGF21 levels were increased in the brain of mice in the group of FGF21 injected, and accordingly, expression of p-FGFR1 was significantly increased, compared to mice in the group of FGF21 non-injected.

As can be seen from fig. 8C, FGFR1- β -klotho complex appeared in the brain of the mice injected with FGF21 compared to the mice not injected with FGF 21. Fig. 8A-C illustrate that FGF21 can cross the BBB to activate FGFR1 and FGFR1- β -klotho complex in the brain to achieve signaling after subcutaneous injection of FGF 21.

Example 2.2 cell experiments

Due to the pathophysiological heterogeneity of T2D stroke, we could not get an ideal model of a similar in vivo environment in vitro. Hyperglycemia and inflammatory cytokines play important roles in the integrity distribution of the early BBB, and also contribute to secondary brain damage from T2D stroke. Therefore, we will use an in vitro model of High Glucose (HG) plus IL-1 β in combination with impaired vascular endothelial cell integrity to mimic the damage of T2D stroke BBB.

Cell experiments: an in vitro model of acute phase db/db T2D mouse cerebral stroke was simulated using 25mmol/L D-glucose plus 30ng/L IL-1 β in combination with injured cerebral vascular endothelial cells.

Example 2.2.1 FGF21 increasing expression of brain vascular endothelial cell Claudin

Diabetes combined with stroke exacerbates the disruption of the blood brain barrier, where reduced expression of claudin is an important factor.

The cerebrovascular endothelial cells were treated with High Glucose (HG) plus IL-1 β combined with the lesion and then with 50nmol/L FGF21 to give HG-IL-1 β + FGF21 group. It was compared to the control group: and comparing the normal cerebral vascular endothelial cells with the cerebral vascular endothelial cells after HG-IL-1 beta combined injury treatment. The expression of zonulin ZO-1 and tenascin VE-cadherin was analyzed by RT-PCR and Western Blot, and the results are shown in FIG. 9.

Western blot detection of the expression of tight junction protein of brain vascular endothelial cells. Quantitative plot of ZO-1. Quantitative graph of VE-Cadherin. (n-5). P<0.01, compared to a normal control group;^#P<0.01, compared to HG-IL-1 β + FGF21 group. P<0.01, compared to a normal control group;^#P<0.01, compared to the HG-IL-1 β group.

As can be seen in FIG. 9, the combination of high sugar plus IL-1 β induced injury reduced the expression of claudin by brain vascular endothelial cells, whereas FGF21 increased the expression of claudin after injury.

Example 2.2.2 FGF21 inhibition of adhesion factor expression in brain vascular endothelial cells

The adhesion factor of the endothelial cells of the cerebral vessels plays a crucial role in the infiltration of leukocytes into the brain after stroke, which causes inflammation of the brain and destruction of the blood-brain barrier.

After treatment of high glucose (high glucose, HG) plus IL-1 β combined injury, cerebrovascular endothelial cells were treated with 50nmol/L FGF21 to give HG-IL-1 β + FGF 21. It was compared to the control group: and comparing the normal cerebral vascular endothelial cells with the cerebral vascular endothelial cells after HG-IL-1 beta combined injury treatment. The expression of vascular adhesion factors VCAM-1 and E-selectin was detected by RT-PCR and Western Blot, and the results are shown in FIG. 10

Western Blot is used for detecting the expression of VCAM-1 and E-Selectin after 24h of brain vascular endothelial cell injury by high glucose and IL-1 beta. Quantitative graph of VCAM-1. C.E-quantitative graph of Selectins. (n-5). P<0.01, compared to a normal control group;^#P<0.01, compared to the HG-IL-1 β group.

As can be seen in FIG. 10, the increase in the adhesion factors VCAM-1 and E-selectin in cerebrovascular endothelial cells following treatment with high glucose plus IL-1 β injury suggests that the blood brain barrier may be disrupted. The level decreased after FGF21 was added, indicating that FGF21 can inhibit the expression of the adhesion factors VCAM-1 and E-Selectin in endothelial cells induced by high-sugar plus IL-1 beta.

Example 2.2.3 FGF21 reduction of permeability of cerebrovascular endothelial cells

The cerebrovascular endothelial cells were treated with high-glucose plus IL-1 β combined injury and then treated with 50nmol/L FGF21 to give HG-IL-1 β + FGF21 group. It was compared to the control group: and comparing the normal cerebral vascular endothelial cells with the cerebral vascular endothelial cells after HG-IL-1 beta combined injury treatment. The vascular endothelial cell permeation after 24h of administration was detected by 70kD FITC-dextran, and the detection results are shown in FIG. 11. Wherein, P<0.01, compared to a normal control group;^#P<0.01, compared to the HG-IL-1 β group.

As can be seen from fig. 11, the permeability of the cerebrovascular endothelial cells after the damage treatment of the hyperglycosemia IL-1 β is significantly increased compared to the untreated normal cells, and the permeability is restored after the administration of FGF21, indicating that FGF21 can reduce the permeability of the cerebrovascular endothelial cells caused by the damage associated with hyperglycosemia IL-1 β.

Example 2.2.4 FGF21 increased expression of FGFR1 phosphorylation (mechanism)

Treating cerebral vascular endothelial cells with high sugar and IL-1 beta combined injury, adding 50nmol/L FGF21, 10nmol/L FGFR1 inhibitor (PD173074), or both, and the effect of FGF21 on FGFR1 phosphorylation expression. FIG. 12 shows phosphorylation expression of FGFR 12h after Western Blot assay. (n-5). P<0.01, compared to a normal control group;^#P<0.01, compared to the HG-IL-1 β group.

As can be seen from FIG. 12, compared with the HG-IL-1 β group, the FGFR1 level in the HG-IL-1 β + FGF21 group was significantly improved, and FGF21 was ineffective after the addition of the FGFR1 inhibitor, indicating that the phosphorylation expression of FGFR1 can be increased by the addition of FGF 21. Since FGF21 signaling requires activation of FGFRs, particularly FGFR1 and its co-receptor β -Klotho complex, an increase in the expression of phosphorylation of FGFR1 may further evidence a protective effect of FGF21 on cerebral vascular endothelial cells.

Example 2.2.5 FGF21 promotion of PPAR γ expression by activating FGFR1 (mechanism)

The method comprises the steps of respectively treating cerebral vascular endothelial cells with High Glucose (HG) and IL-1 beta combined injury, treating with FGF21 at a concentration of 50nmol/L after injury, treating with FGF21 and an FGFR1 inhibitor (PD173074, 10nmol/L) after injury, and measuring the expression level and activity of PPAR gamma in the cells so as to determine the influence of FGF21 on PPAR gamma expression and the relation between FGFR1 phosphorylation and PPAR gamma activation.

The measurement results are shown in fig. 13, wherein, a graph is that the expression level of PPAR gamma protein in the brain vascular endothelial cell nucleus is detected by Western Blot, and P is less than 0.05, compared with a normal control group; # P <0.05, compared to FGF21 group. Panel B shows the activity of PPAR γ in the nucleus.

As can be seen from FIG. 13, the addition of HG and IL-1. beta. decreased the activity of PPAR γ protein, although it did not significantly decrease the expression level of PPAR γ protein. After FGF21 is added, the expression level and activity of PPAR gamma protein in injured cerebrovascular endothelial cells are obviously increased; and after the FGFR1 inhibitor PD173074 is added, the expression level and the activity of PPAR gamma protein are reduced. As demonstrated by examples 2.2.4 and 2.2.5, FGF21 significantly promoted nuclear PPAR γ levels and activity positively correlated with FGFR1 expression.

Example 2.2.6 activation of PPAR γ by FGF21 protects permeability of brain vascular endothelial cells

The brain vascular endothelial cells are respectively treated by High Glucose (HG) and IL-1 beta combined injury, 50nmol/L FGF21 after injury, 10nmol/L PPAR gamma antagonist GW9662 after injury and FGF21 and PPAR gamma antagonist GW9662 after injury in a combined way, the permeability of the vascular endothelial cells is detected by 70KD FITC-glucan, the influence of PPAR gamma on the permeability of the vascular endothelial cells is determined, the detection result is shown in figure 14, and the P is less than 0.05, compared with a normal control group.

Figure 14 shows that FGF21 reduces permeability of cerebrovascular endothelial cells and that protection by FGF21 is inhibited following administration of the PPAR γ inhibitor GW 9662. In combination with examples 2.2.4 to 2.2.5, it was demonstrated that FGF21 can activate FGFR1 (or FGFR1- β -klotho complex) and further activate PPAR γ to protect vascular endothelial cell permeability.

Example 3 Effect of FGF21 on T2D cerebral Stroke neuroinflammation

Example 3.1 animal experiments

The type II diabetes model (T2D model), the construction of ischemic stroke model, the administration and grouping of animal experiments (db/+, db/db and db/db + FGF21 group), and the data processing were the same as example 2.1.

Example 3.1.1 FGF21 increasing the number of anti-inflammatory cells-type M2 microglia

Immunohistochemistry for CD206+ marker for M2 type microglia in brain was determined by immunohistochemistry method and CD206+ positive cell number was quantified. The detection result is shown in FIG. 15, and graph A is CD206⁺DAPI immunohistochemistry (where, red shows CD206+ positive cells); b is a CD206⁺Quantitative plot of the number of positive cells. (n-6). P<0.05, compared to db/+ stroke group;^#P<0.05, compared to db/db stroke group.

The immunohistochemical method comprises the following steps: mice were sacrificed 14 days after stroke, and brains were removed and assayed using conventional immunohistochemical methods.

CD206+ positive cell quantification: quantification was performed using the image analysis software Imagepro Plus.

As can be seen in FIG. 15, FGF21 increased the number of anti-inflammatory factor-M2 type microglia (CD206+ positive cells) in the brain following brain stroke in db/db T2D mice.

Example 3.2 cell experiments

Culturing primary rat microglia by separating cortex tissue of 0-2day rat, digesting with 0.25% pancreatin at 37 deg.C for 15-20min, adding DMEM medium containing 10% FBS, and blowing and beating to plant in 75cm²The culture flask of (1) is changed every two days. After culturing for 8-12day, shake at 218rpm on a constant temperature shaker at 37 ℃ for 2 h. The supernatant was then centrifuged at 1000g for 5min and the cells were plated on plates or dishes for the experiment. The cells were divided into a normal control group, an LPS group and an LPS + FGF21 group. The concentration of Lipopolysaccharide (LPS) was 50ng/mL, and the concentration of FGF21 was 50 nmol/L.

Example 3.2.1 FGF21 reduction of the inflammatory response of microglia

RT-PCR was used to detect mRNA expression of mRNA for iNOS and TNF-alpha, which are proinflammatory factors of type M1 microglia, and mRNA expression of Arg1 and IGF-1, which are anti-inflammatory factors of type M2 microglia. The results are shown in fig. 16, where a.rt-PCR determined that FGF21 acted on LPS to stimulate the expression of mRNA of each factor of primary rat microglia cells,. P <0.01, compared to the LPS group; RT-PCR was used to determine the expression of FGF21 on mRNA of various factors of resting primary rat microglia. (n-5); (P < 0.01), compared to normal control group.

As can be seen from fig. 16, FGF21 was able to inhibit the expression of pro-inflammatory factor gene M1 in rat microglia cells in primary culture stimulated by LPS, while increasing the expression of anti-inflammatory factor gene M2. The expression result of mRNA of each factor determined by RT-PCR shows that mRNA of an M1 type marker iNOS is obviously reduced, and simultaneously expression of a proinflammatory factor TNF-alpha mRNA is also obviously reduced, which indicates that FGF21 can inhibit the transformation of primary rat microglia induced by LPS to M1 type.

FGF21 acted on primary rat microglia for 8h, and the result shows that the expression of an Arg1mRNA marker of M2 type is increased, and the expression of an IGF-1mRNA of an anti-inflammatory factor is also increased, which indicates that FGF21 can promote the conversion of the microglia in a resting state to an M2 type with a neuroprotective effect.

Example 3.2.2 phosphorylation of FGFR1 by FGF21 inhibits NF κ B activity (mechanism)

The phosphorylation level of FGFR1, nfkb activity and PPAR γ expression in LPS-induced primary rat microglia cells were determined by control experiments in the normal cell group, LPS + FGF21 group, and LPS + FGF21+ FGFR inhibitor PD173074(10nmol/L) group.

The detection results are shown in fig. 17, wherein p-FGFR1 expression was measured by western blot; western blot assay of nuclear transcription factor PPAR γ expression in nucleoprotein<0.01 compared to control group;^#P<0.01 compared to the LPS + FGF21 group. B. The kit is used for measuring the expression of the activity of the NF kappa B; p<0.01 compared to control group;^#P<0.01 compared to the LPS + FGF21+ inhibitor group.

Fig. 17 shows that FGF21 acts on FGFR primary rat microglia for 2h, enabling increased expression of FGFR1 phosphorylation, whereas phosphorylation of FGFR1 is inhibited when the FGFR inhibitor PD173074 is administered. Meanwhile, FGF21 can also reduce the activity of NF kappa B in primary rat microglia cell nucleus induced by LPS, increase the expression of cell nucleus transcription factor PPAR gamma in cell nucleus protein, and after the addition of FGFR inhibitor PD173074, the effects of FGF21 on the expression of PPAR gamma and the activity of NF kappa B are reduced.

It is known that FGF21 can inhibit the activity of NF κ B induced by LPS by promoting phosphorylation of FGFR1, and increase the expression level of PPAR γ in the microglial cell nucleus.

Example 4 Effect of FGF21 on vascular and white matter remodeling in T2D stroke

Example 4.1 animal experiments

The type II diabetes model (T2D model), the construction of ischemic stroke model, the administration and grouping of animal experiments (db/+, db/db and db/db + FGF21 group), and the data processing were the same as example 2.1.

Example 4.1.1 FGF21 reduction of axonal damage and loss of myelin sheath

Brain tissue from three groups of mice was sampled 14 days after stroke to determine the effect of FGF21 on axonal damage and myelin loss. Axons were identified by SMI32 immunohistochemistry and myelin was detected by MBP immunohistochemistry.

The results are shown in fig. 18, where the upper panel is an immunohistochemistry map of marker SMI32 for axonal injury on the left (where the marker SMI32 is shown in red) and its quantification map on the right. Lower panel left is immunohistochemistry for marker MBP of myelin sheath (where, green shows marker MBP), rightThe side is its quantitative map. (n-6). P<0.05, compared to db/+ stroke group;^#P<0.05, compared to db/db stroke group.

As can be seen in FIG. 18, db/db stroke aggravated axonal damage and myelin loss compared to db/+ stroke. After FGF21 treatment, axonal injury in db/db cerebral apoplexy mice does not reach the level of db/+ cerebral apoplexy group, but is obviously improved before untreated; while myelin levels were restored to the same levels as db/+ stroke mice. Suggesting that FGF21 can greatly reduce axonal damage and myelin loss.

Example 4.1.2 FGF21 promotion of oligodendrocyte regeneration

Three groups of mouse brain tissue were sampled 14 days after stroke and NG2 was detected immunohistochemically in the same brain area to identify oligodendrocyte (OPC cell) regeneration.

The results are shown in fig. 19, where the left side is an immunohistochemistry map of the marker NG2 for OPC cells (where the green color shows the marker NG2) and the right side is a quantification map thereof. (n-6) P<0.05, compared to db/+ stroke group;^#P<0.05, compared to db/db stroke group.

As can be seen from FIG. 19, after FGF21 treatment, the glial cells of db/db stroke mice were significantly higher than db/db stroke mice without FGF21 treatment, although they were lower than db/+ stroke mice. Indicating that FGF21 promotes oligodendrocyte regeneration.

Example 4.1.3 FGF21 increasing cerebrovascular Density

Three groups of mouse brain tissue were sampled 14 days after stroke to determine the effect of FGF21 on mouse cerebral vascular density. Cerebrovascular density was analyzed by immunohistochemistry to detect and quantify CD31 positive cells.

The results of the assay are shown in fig. 20, where the left side is an immunohistochemistry graph of the endothelial cell marker CD31 (where the red color shows the marker CD31) and the right side is a quantification graph thereof. (n-6) of a compound represented by formula (I),^#P<0.05, compared to db/db stroke group.

As can be seen from FIG. 20, after FGF21 treatment, the cerebral vascular density of db/db stroke mice was higher than that of db/+ stroke mice, and significantly higher than that of db/db stroke mice not treated with FGF 21. It is shown that FGF21 has obvious effect in increasing cerebral apoplexy of normal blood sugar and cerebral vascular density of type II diabetes.

Example 4.1.4 FGF21 increases the expression of pro-angiogenic factor mRNA in cerebrovascular fragments

Three groups of mouse brain tissue were sampled 14 days after stroke to determine the effect of FGF21 on mouse cerebral vessel regeneration. The mRNA expression of the relevant factor was determined by RT-PCR and the results are shown in Table 1. Wherein, P<0.05, compared to db/+ stroke group;^#P<0.05, n is 6 compared to db/db stroke group.

TABLE 1mRNA levels in mouse brain microvascular fragments

Genes	db/+	db/db	db/db+FGF21
				VEGFA
	1	0.35±0.05*	0.78±0.12#
				eNOS	1	1.05±0.09	2.19±0.25#
IGF-1	1	0.50±0.06*	2.75±0.18#
				IL-1β	1	1.91±0.17*	1.17±0.14#

The RT-PCR results in Table 1 show that FGF21 increases the expression of VEGFA, eNOS and IGF-1mRNA, which are pro-angiogenic factors, and decreases the expression of IL-1 beta mRNA, 14 days after FGF21 administration of db/db T2D mice had a cerebral stroke.

Example 4.1.5 FGF21 increases the activity of AMPK/Nrf2 in the brain, thereby promoting the remodeling (mechanism) of cerebral and white blood vessels

After 7 days from stroke, brain tissue from three groups of mice was sampled and brain activity of AMPK, p-AMPK and Nrf2 was measured by Western Blot and activity kit, and the results are shown in FIG. 21. Western blot is used for measuring the expression of p-AMPK/AMPK; b.p-quantitative graph of AMPK/AMPK; C. activity of Nrf2 in nucleoprotein. (n-5). P<0.01, compared to db/+ group;^#P<0.01, compared to db/db group.

As can be seen in FIG. 21, p-AMPK/AMPK expression and Nrf2 activity were significantly reduced after stroke in db/db T2D as compared to db/+ stroke. db/db T2D mice administered FGF21 at a dose of 3.0mg/kg/day for 7 days showed a significant increase in brain AMPK phosphorylation and nuclear Nrf2 activity.

Example 4.2 cell experiments: FGF 21-treated conditioned medium of cerebrovascular endothelial cells for promoting proliferation of Oligodendrocytes (OPC)

Collecting additive solution or endothelial cell conditioned medium: (1) a cell-free medium group; (2) cell-free medium + FGF21(50 nmol/L); (3) a normal endothelial cell culture group; (4) normal endothelial cell culture + FGF21(50nmol/L) group: after 50nmol/L FGF21 was added to the endothelial cell culture medium for 6 hours, the medium was replaced with new one, the culture was continued for 24 hours, and the endothelial cell conditioned medium was collected. Transferring the additive solution or the endothelial cell conditioned medium into rat primary OPC cells, wherein the endothelial cell conditioned medium in (3) and (4) groups is mixed with the OPC cell culture solution at a ratio of 1:1, and is transferred to OPC cells, and FGF21 is added to the group (2) at a level equivalent to that of the group (4).

The results of measuring the proliferation of each group of cells by the MTT method are shown in FIG. 22. Wherein, (n-6),. P<0.01, compared to cell-free medium;^#P<0.01, compared to endothelial cell conditioned medium group.

As can be seen from fig. 22, the conditioned medium of cerebrovascular endothelial cells treated with FGF21 significantly increased the proliferation of OPC cells compared to the other groups, and after that, when the conditioned medium of normal cerebrovascular endothelial cells not treated with FGF21 was used to culture OPC cells, the proliferation of OPC cells was still significantly increased compared to the groups (1), (2), and (3). Suggesting that FGF 21-treated conditioned culture medium of cerebrovascular endothelial cells might promote OPC proliferation.

Example 5 Regulation of blood glucose by FGF21 in type II diabetic stroke mice

The type II diabetes model (T2D model), the construction of ischemic stroke model, the administration and grouping of animal experiments (db/+, db/db and db/db + FGF21 group), and the data processing were the same as example 2.1.

The body weight, blood glucose and glycated hemoglobin (Hb A1c) levels of the groups of mice before and after stroke were monitored. The results of the measurements are shown in fig. 23, which is a quantitative graph of body weight (left), blood glucose (middle), and glycated hemoglobin (Hb A1c) (right) after stroke. (n-12). P<0.01, compared to db/+ cerebral stroke group;^#P<0.01, compared to db/db stroke group.

As can be seen in FIG. 23, FGF21 was able to reduce hyperglycemia in db/db T2D mice following brain stroke. Body weight was slightly reduced but not significantly changed compared to the control group. But the concentration of glucose in blood decreased significantly from day 3 to day 14. At the same time, FGF21 also significantly reduced the level of glycosylated hemoglobin. These data indicate that FGF21 can modulate carbohydrate metabolism in db/db T2D mice following brain stroke.

SEQUENCE LISTING

<110> cooperative innovation center of biological medicine in Wenzhou city

General Hospital of Massachusetts (Massachusetts General Hospital)

Application of <120> human fibroblast growth factor 21 in preparing medicine for treating cerebral apoplexy

<130> 2010

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 561

<212> DNA

<213> Artificial sequence

<400> 1

catatgcatc cgattccgga tagcagcccg ctgctgcagt ttggtggtca ggtgcgtcag 60

cgttatctgt ataccgatga tgcgcagcag accgaagcgc atctggaaat tcgtgaagat 120

ggtaccgtgg gtggtgcggc ggatcagagc ccggaaagcc tgctgcagct gaaagcgctg 180

aaaccgggtg tgattcagat tctgggtgtg aaaaccagcc gttttctgtg ccagcgtccg 240

gatggtgcgc tgtatggtag cctgcatttt gatccggaag cgtgcagctt tcgtgaactg 300

ctgctggaag atggttataa tgtgtatcag agcgaagcgc atggtctgcc gctgcatctg 360

ccgggtaata aaagcccgca tcgtgatccg gcgccgcgtg gtccggcgcg ttttctgccg 420

ctgccgggtc tgccgccggc gctgccggaa ccgccgggta ttctggcgcc gcagccgccg 480

gatgtgggta gcagcgatcc gctgagcatg gtgggtccga gccagggtcg tagcccgagc 540

tatgcgagct aatgaggatc c 561

Claims (2)

1. Use of human fibroblast growth factor 21 in the manufacture of a medicament for increasing cerebral vascular density in type II diabetes mellitus and in mice with occluded middle cerebral artery.

2. The use according to claim 1, wherein the nucleotide sequence encoding human fibroblast growth factor 21 is as shown in SEQ ID No. 1.

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