CN111214647A - Medicine for treating ankylosing spondylitis - Google Patents

Abstract

The invention discloses application of TRAF4 in preparing a medicament for treating ankylosing spondylitis. The invention also discloses application of the TRAF4 and Yisaipu in preparation of a medicament for treating ankylosing spondylitis. Compared with the existing medicine for treating AS, the medicine can obviously relieve chronic inflammation and subsequent pathological osteogenesis of AS patients, and has good effects on controlling the worsening progress of diseases and preventing deformity; the side effects of abnormal death or infection of the mice and the like are not seen in the treatment process of the medicine, which shows that the side effects are small.

Description

Medicine for treating ankylosing spondylitis

Technical Field

The invention relates to the technical field of treatment of spondylitis, in particular to a medicine for treating ankylosing spondylitis.

Background

Ankylosing Spondylitis (AS) is an autoimmune disease characterized by chronic inflammation and pathological osteogenesis. At present, the pathogenesis of AS is not clear, no radical treatment method is available, and the treatment methods can be roughly divided into three types: non-drug therapy, and surgical therapy. Non-drug treatments mainly include: educating patients and their family members about knowledge of diseases; long-term psychosocial and rehabilitation therapy is performed on the patient; persuade patients to reasonably and consistently perform physical exercise; when standing, the user should keep the posture of chest support, abdomen contraction and front vision of eyes as much as possible, and the user should keep the chest upright when sitting; administering the necessary physical therapy to the painful or inflammatory joint or soft tissue; the patient is advised to quit smoking.

The drug therapy is preferably non-steroidal anti-inflammatory drugs, and other common drugs also comprise tumor necrosis factor (TNF- α) antagonist and disease-improving antirheumatic drugs (DMARDs). for late AS patients, the narrow joint space, the rigidity and the deformity caused by hip joint involvement caused by pathological changes can be treated by artificial joint replacement surgery, for patients with serious deformity and limited activity of the spine, the spine osteotomy correction surgery can be carried out, and patients with combined vertebral body fracture need long-segment fixation, and usually 2 vertebral bodies are fixed on the upper part and the lower part of the injured vertebral body respectively to disperse the stress of each fixed point.

Although non-steroidal anti-inflammatory drugs can relieve inflammatory symptoms of most patients, long-term administration causes significant gastrointestinal reactions and cardiovascular risks; DMARDs only have an effect on peripheral symptoms of AS, but have no significant effect on central axis bone symptoms at the core of AS, while biological agents represented by pessimip, although having significant efficacy, are expensive and have significant side effects on the potential increase of infection and tumor risk.

Disclosure of Invention

Based on the above problems, the present invention aims to overcome the disadvantages of the prior art and provide a medicament for effectively treating ankylosing spondylitis, which is lower in cost.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

in one aspect, the invention provides the use of TRAF4 in the manufacture of a medicament for the treatment of ankylosing spondylitis.

In another aspect, the invention provides the use of TRAF4 in combination with pessulam in the manufacture of a medicament for the treatment of ankylosing spondylitis. Through multiple experiments, the inventor of the application finds that exogenous TRAF4 can obviously inhibit pathological spinal osteogenesis of an AS mouse animal model, and the effect of TRAF4 combined with Yisaipu on treating an AS mouse is obviously superior to the curative effect of singly applying Yisaipu or TRAF 4.

The invention also provides a medicine for treating ankylosing spondylitis, which comprises TRAF 4.

Preferably, the effective dose of TRAF4 is 0.1mg per gram of body weight.

More preferably, the medicament comprises TRAF4 and yi saipu.

Most preferably, the amounts of TRAF4 and gycept in the medicament are the same, both being 0.1mg per gram of body weight.

The invention also provides application of TRAF4 in preparation or screening of a medicament for treating ankylosing spondylitis as a target.

In conclusion, the beneficial effects of the invention are as follows:

compared with the existing medicine for treating AS, the medicine can obviously relieve chronic inflammation and subsequent pathological osteogenesis of AS patients, and has good effects on controlling the worsening progress of diseases and preventing deformity; the side effects of abnormal death or infection of the mice and the like are not seen in the treatment process of the medicine, which shows that the side effects are small.

Drawings

FIG. 1 is a graph showing the results of the determination of the TRAF4 content in the serum of normal persons and AS patients;

FIG. 2 is a graph showing the results of measuring the secretion level of TNF- α;

FIG. 3 is a schematic diagram of the experimental procedure of example 2.

Detailed Description

The present invention demonstrates for the first time that TRAF4 expression is reduced in patients with AS and is closely associated with chronic inflammation in patients with AS. The inventor of the application finds that the expression of TRAF4 in an AS patient is abnormally reduced, so that inflammatory reaction occurs in the AS patient, and the inventor discovers that the inflammation of an AS mouse animal model can be effectively relieved by carrying out tail vein injection on the AS mouse animal model by exogenous TRAF4, so that exogenous TRAF4 supplementation is a novel method for effectively treating AS, and TRAF4 can be applied to preparation or screening of a medicament for treating ankylosing spondylitis AS a target point.

In some embodiments, the invention provides a method for effectively relieving inflammation and subsequent spinal ossification of an AS mouse animal model by injecting exogenous TRAF4 into the AS mouse animal model through tail vein, which suggests that supplementing exogenous TRAF4 is a new method for effectively treating AS. Compared with the single use of exogenous TRAF4 or Yisaipu, the combination of TRAF4 and Yisaipu has obviously enhanced effect of treating ankylosing spondylitis.

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods in the present invention are all conventional methods.

Example 1

(1) Detection of TRAF4 levels in serum of AS patients

Serum from AS patients and those in the same period of physical examination were extracted, and the content of TRAF4 in the serum was measured according to the instructions of the reagents (SANTA CRUZBITECHNOLOGY, sc-390232).

As shown in FIG. 1, it can be seen from the results in FIG. 1 that the TRAF4 content in normal human serum was 1320. + -. 186.34 pg/mL; the TRAF4 content in the serum of AS patients is 456.8 +/-112.43 pg/mL, and p is less than 0.05, which shows that the level of TRAF4 in the serum of AS patients is obviously reduced compared with that of normal people.

(2) Effect of TRAF4 on TNF- α secretion by monocytes

Separating Peripheral Blood Mononuclear Cells (PBMC) from human peripheral blood by density gradient centrifugation, sorting with CD14 magnetic beads to obtain CD14+ mononuclear cells, and separating CD14+ mononuclear cells at 2 × 10⁵Inoculating into 24-well plate, selecting 10% serum-containing RPMI-1640 as culture medium, adding 0, and,1. 5, 10 and 20ng/mL of exogenous TRAF4, and supernatants were removed after 5 days of culture to test the secretion level of TNF- α by ELISA, as shown in FIG. 2.

According to the report in the literature, the level of TNF- α in AS patients is increased, and TNF- α is a marker of AS inflammatory response, and AS can be seen from figure 2, TRAF4 can obviously inhibit TNF- α secretion of mononuclear macrophages compared with a control group, which indicates that TRAF4 is reduced to cause inflammatory response in AS patients.

In conclusion, the expression level of TRAF4 in AS patients is remarkably reduced compared with that of normal people, so that the secretion of TNF- α by mononuclear macrophages is increased, and the inflammatory reaction in the patients can be caused.

Example 2 Effect of the drug of the present invention on the treatment of ankylosing spondylitis

The specific flow of this embodiment is shown in fig. 3, and includes the following steps:

(1) AS mouse animal model construction

Female BALB/c mice 24 weeks old were selected and administered with intraperitoneal injection of Proteoglycan (Proteoglycan) (1 injection for each of weeks 0, 3 and 6, 3 injections, 100. mu.g each).

(2) Group administration

The AS mouse animal model was constructed and divided into four groups, including a control group, a Yisaipu group (tumor necrosis factor inhibitor), an exogenous TRAF4 group, and a Yisaipu + TRAF4 combined group (hereinafter referred to AS combined group), and the four groups were administered with saline, 0.1mg/g of Yisaipu, 0.1mg/g of exogenous TRAF 40.1 mg/g, and 0.1mg/g of Yisaipu + TRAF4, respectively, in tail vein, once every 4 weeks, and 6 total injections were administered from week 10 to week 30. The Yisaipu adopted in this example was purchased from Shanghai China letter national medicine industry, Inc. (national drug Standard S20050058 and national drug Standard S20050059); exogenous TRAF4 was a human TRAF4 recombinant protein from NOVUS under the cat number H00009618-P01.

(3) Detection of inflammation in mice

Mice in the four groups were sacrificed by cervical dislocation from week 14, once every 4 weeks to week 34 for 6 times, and peripheral blood serum of the mice was isolated after the sacrifice to measure the levels of CRP, TNF- α, and IL-17A.

(4) Detection of pathological osteogenesis in mice

Previous studies have demonstrated that early effective intervention in inflammation can simultaneously significantly inhibit the progression of pathological osteogenesis, another central pathological feature of AS. Therefore, in this embodiment, the 3 rd to 5 th lumbar vertebra segments of the mouse spine are taken at the same time, sequentially fixed, decalcified, embedded in paraffin, and sectioned, and pathological osteogenesis conditions of the mouse spine are observed by HE staining and Masson staining, and the pathological osteogenesis scoring criteria are as follows: 0 minute: normal; 1 minute: slight hyperplasia of vertebral body edge; and 2, dividing: obvious osteophyte formation at the edge of the vertebral body; and 3, dividing: a bone bridge is formed at the edge of one side of the vertebral body; and 4, dividing: the edges of the vertebral bodies at both sides form a bone bridge and interbody fusion.

(5) Results and analysis

The results are shown in tables 1-4, compared with the control group, the Yisaipu group and the exogenous TRAF4 group can inhibit the in vivo inflammation level of the AS mouse animal model (CRP, TNF- α and IL17A are all significantly reduced), in addition, compared with the control group, the exogenous TRAF4 group can also significantly inhibit the pathological spinal osteogenesis of the AS mouse animal model, the effect of the combined group is obviously better than that of the single Yisaipu group or the TRAF4 group, and no side effects such AS abnormal death or infection of mice and the like are seen in the four groups.

TABLE 1C reactive protein (CRP) levels (ng/mL)

Time of day	Control group	Yisaipu group	Exogenous TRAF4 group	Combined group
					14 weeks	25.6±1.4	22.1±3.2	19.2±1.5	14.2±1.1
For 18 weeks	24.2±1.8	20.5±2.9	15.5±1.0	11.3±1.1
					22 weeks	23.3±2.2	20.2±1.0	18.9±1.2	9.7±0.9
For 26 weeks	20.5±0.9	16.7±0.8	14.5±0.8	7.3±0.8
					30 weeks	21.5±2.5	13.2±1.4	12.4±0.5	4.2±0.4
34 weeks	26.7±1.8	6.4±0.6	5.7±0.3	2.3±0.2

TABLE 2 TNF- α levels (pg/mL)

Time of day	Control group	Yisaipu group	Exogenous TRAF4 group	Combined group
					14 weeks	323.6±34.6	333.6±25.1	335.1±23.7	221.2±17.4
For 18 weeks	403.4±44.1	308.1±24.9	303.2±45.1	200.3±16.3
					22 weeks	394.7±34.9	267.5±22.3	294.7±15.7	165.8±15.9
For 26 weeks	405.6±25.7	221.3±19.8	215.6±18.8	120.5±13.2
					30 weeks	354.5±39.1	178.2±15.6	165.6±19.9	103.2±11.3
34 weeks	323.6±27.4	98.8±18.9	103.1±6.5	78.3±3.4

TABLE 3 Interleukin 17A levels (pg/mL)

Time of day	Control group	Yisaipu group	Exogenous TRAF4 group	Combined group
					14 weeks	189.7±23.4	205.1±24.2	198.7±18.9	134.8±21.3
For 18 weeks	196.5±14.7	167.2±21.1	156.9±12.3	121.3±16.9
					22 weeks	191.5±18.7	186.5±22.2	165.4±21.2	102.3±17.4
For 26 weeks	184.3±21.2	67.4±15.3	78.5±13.4	50.7±11.4
					30 weeks	193.2±14.2	56.7±10.6	66.4±6.5	34.6±10.3
34 weeks	203.5±21.3	50.3±8.7	46.7±5.6	24.6±8.8

TABLE 4 spinal pathological osteogenesis score

Time of day	Control group	Yisaipu group	Exogenous TRAF4 group	Combined group
					14 weeks	4.8±0.4	4.7±1.5	4.3±1.7	3.7±1.4
For 18 weeks	4.5±1.4	4.2±1.8	3.8±0.9	3.0±1.0
					22 weeks	5.3±1.3	4.5±1.3	3.2±1.3	2.4±0.8
For 26 weeks	5.5±1.8	4.9±2.5	2.7±1.5	2.1±0.6
					30 weeks	7.6±1.9	7.5±4.1	1.9±0.5	1.4±0.3
34 weeks	7.5±1.8	7.3±3.5	1.8±0.4	1.1±0.2

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (6)

1. Use of TRAF4 in the manufacture of a medicament for the treatment of ankylosing spondylitis.
2. The application of TRAF4 and Yisaipu in preparing a medicine for treating ankylosing spondylitis.
3. 3. A medicine for treating ankylosing spondylitis comprises TRAF 4.
4. 4. The medicament of claim 3 wherein the effective dose of TRAF4 is 0.1mg per gram of body weight.
5. 5. The medicament of claim 3 comprising TRAF4 and yi saipu.
6. 6. The medicament of claim 5 wherein TRAF4 and Yixepu are used in the same amount, both 0.1mg per gram of body weight.

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