A yak spleen small molecule polypeptide, a preparation method and application thereof

Abstract

The application discloses yak spleen small molecule polypeptide, and a preparation method and application thereof. An amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The yak spleen small molecule polypeptide can effectively reduce red blood cell deformation caused by highland exposure, significantly reduce the stickiness of red blood cells, and directly cope with hypoxia symptoms caused by red blood cell deformation and stickiness. The yak spleen small molecule polypeptide has significant beneficial effects in reducing red blood cell deformation, reducing red blood cell stickiness and improving oxygen transport efficiency, and has important clinical values and social significance for improving physiological functions of human bodies in highlands or other hypoxic environments.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a yak spleen small molecule polypeptide as well as a preparation method and application thereof. BACKGROUND

[0002] As a special red blood cell morphology, acanthocytes have needle-like protrusions on their surface. The spacing of these protrusions is irregular, and the length and width vary. This morphology of red blood cells can occur in various disease states, especially in patients with beta-lipoprotein deficiency. In addition, the proportion of acanthocytes may also increase in pathological conditions such as alcoholic liver disease after splenectomy. The morphology of red blood cells is closely related to their oxygen carrying function. Normal biconcave disc morphology and low adhesion are essential for red blood cells to maintain efficient oxygen transport.

[0003] However, the increase of acanthocytes will have an adverse effect on the function of red blood cells. It will promote the adhesion between red blood cells, leading to reduced flowability of red blood cells in blood vessels, and thus shortening the life span of red blood cells. More importantly, this change in morphology will eventually lead to a significant decrease in oxygen transport efficiency, affecting the normal physiological functions of the body.

[0004] Although the influence of acanthocytes on red blood cell function has been widely recognized, there is still a lack of effective products or reagents on the market to inhibit the acanthocyte change of red blood cells, thereby alleviating the problem of reduced oxygen transport efficiency. Therefore, the development of products or reagents that can inhibit the formation of acanthocytes and improve the oxygen carrying efficiency of red blood cells has important clinical value and social significance for improving the quality of life of patients with related diseases. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a yak spleen small molecule polypeptide as well as a preparation method and application thereof. The polypeptide can adsorb free nucleic acids in blood, reduce the acanthocyte change of red blood cells, reduce the adhesion of red blood cells, prolong their life span, and improve the oxygen transport efficiency.

[0006] The present application is achieved by the following technical solutions:

[0007] A yak spleen small molecule polypeptide, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1.

[0008] Preferably, the polypeptide is extracted from the yak spleen or obtained by in vitro synthesis.

[0009] A preparation method of a yak spleen small molecule polypeptide, wherein the preparation method is extraction from the yak spleen, and comprises the following steps:

[0010] Step 1) Collecting the yak spleen tissue, washing the fresh or frozen yak spleen tissue with normal saline at room temperature, adding 2 times volume of sterile normal saline, and homogenizing with a homogenizer at 4℃;

[0011] Step 2) Adding 2 times volume of sterile normal saline and ultrasonic homogenization, and then repeatedly freezing and thawing in liquid nitrogen for 3 times;

[0012] Step 3) Taking the supernatant after freezing and centrifuging the homogenate at 12000 rpm for 15 min;

[0013] Step 4) Adding serine protease, sulfhydryl protease, aspartic acid protease, and metalloprotease to the supernatant, stirring and hydrolyzing at 37℃ for 30 min, centrifuging at 12000 rpm for 15 min, taking the supernatant, boiling, and then ultrafiltrating to remove substances with a molecular weight less than 3000 Da to obtain a filtrate, which is then sterilized, freeze-dried, and obtained.

[0014] Preferably, the molar ratio of the serine protease, sulfhydryl protease, aspartic acid protease, and metalloprotease in step 4) is 3:1:1:1.

[0015] The polypeptide is used for preparing a medicine for preventing and / or treating the change of spiky red blood cells caused by a disease.

[0016] Preferably, the disease is β-lipoprotein deficiency, high altitude reaction, or alcoholism-induced liver disease after splenectomy.

[0017] The polypeptide is used for preparing a medicine for preventing and / or treating high altitude reaction.

[0018] A medicine composition for preventing and / or treating high altitude reaction, comprising the polypeptide.

[0019] Preferably, it further comprises a pharmaceutically acceptable carrier.

[0020] The beneficial effects of the present application are as follows:

[0021] (1) The yak spleen small molecule polypeptide of the present application can effectively reduce the red blood cell deformation caused by high altitude exposure. In the high altitude environment, due to hypoxia and other environmental pressures, red blood cells often change shape from the normal double-concave butterfly shape to spiky or other abnormal shapes, which seriously affects the oxygen carrying capacity of red blood cells. However, by taking the yak spleen small molecule polypeptide of the present application, this deformation can be effectively inhibited, so that the red blood cells maintain the best shape, thereby maintaining their high oxygen transport function.

[0022] (2) The yak spleen small molecule polypeptide of the present application can significantly reduce the adhesion of red blood cells. The adhesion between red blood cells can cause the decrease of blood flowability and affect the transportation of oxygen and nutrients. The polypeptide of the present application can reduce the adhesion between red blood cells through a specific mechanism, so that the blood flows more smoothly in the blood vessels, thereby improving the transportation efficiency of oxygen.

[0023] (3) The yak spleen small molecule polypeptide of the present application can directly cope with the hypoxia symptoms caused by the deformation and adhesion of red blood cells. In the plateau or other hypoxic environment, the increase of red blood cell deformation and adhesion can cause hypoxia of the body, causing a series of symptoms such as fatigue, dizziness, etc. The polypeptide of the present application can effectively improve the morphology and adhesion of red blood cells, thereby improving the utilization efficiency of oxygen by the body, relieving the hypoxia symptoms, and improving the adaptability and life quality of the human body. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The red blood cell electron microscope shooting graph (A) and the spicule shape rate statistics (B) of each group of mice in Test Example 1 are shown in the following table:

[0025] Figure 2 The red blood cell bright field shooting graph (A) and the deformation rate statistics (B) of each group of mice in Test Example 2 are shown in the following table:

[0026] Figure 3 The P50 value of each group of mice in Test Example 2 is shown in the following table. DETAILED DESCRIPTION

[0027] The present application will be further described in detail below in combination with the drawings and specific examples.

[0028] Example 1

[0029] A yak spleen small molecule polypeptide, the amino acid sequence of which is abbreviated as LSFNYQKRVS (SEQ ID NO. 1), specifically Leu-Ser-Phe-Asn-Tyr-Gln-Lys-Arg-Val-Ser.

[0030] The polypeptide is prepared by in vitro synthesis, which is generally completed by a polypeptide synthesis company, and the general steps are as follows:

[0031] Various single pure amino acid raw materials are prepared, and then according to the yak spleen polypeptide sequence Leu-Ser-Phe-Asn-Tyr-Gln-Lys-Arg-Val-Ser, Leu and Ser are weighed and dissolved respectively, and a peptide bond is formed in vitro by dehydration condensation (one OH and H - and H +The first amino acid (Gly) is added to the Rink Amide resin, and then the next amino acid (Phe) is added by dehydration condensation (the two amino acids combine to form H2O, and a peptide bond is formed), and so on, until the last amino acid is added, and then the product is purified and dried.

[0032] Example 2

[0033] The method for extracting the yak spleen small molecular polypeptide described in Example 1 from yak spleen is as follows:

[0034] The yak spleen tissue is collected, and the fresh or frozen yak spleen tissue is washed with physiological saline at room temperature, 2 times the volume of sterile physiological saline is added, and homogenized at 4°C with a homogenizer; 2 times the volume of sterile physiological saline is added and homogenized again, and then placed in liquid nitrogen and repeatedly frozen and thawed 3 times; the frozen homogenate is centrifuged at 12000 rpm for 15 min, and the supernatant is taken; serine protease, sulfhydryl protease, aspartic acid protease, and metalloprotease (molar ratio 3:1:1:1) are added to the supernatant, and stirred and hydrolyzed at 37°C for 30 min, centrifuged at 12000 rpm for 15 min, and the supernatant is boiled and ultrafiltered to remove substances with a molecular weight less than 3000 Da, and the filtrate is sterilized, frozen and dried to obtain the product.

[0035] Example 1 and Example 2 are compared in terms of preparation method, and the purity of the yak spleen small molecular polypeptide obtained by in vitro synthesis in Example 1 is generally 98%, which is much higher than the purity obtained from yak spleen in Example 2.

[0036] Comparative Example 1

[0037] A polypeptide is prepared by in vitro synthesis (high purity), which is a long polypeptide containing the sequence of the yak spleen small molecular polypeptide in Example 1, and the sequence is abbreviated as LTQLRKLNLSFNYQKRVSFAHLSL (SEQ ID NO. 2), and specifically Leu-Thr-Gln-Leu-Arg-Lys-Leu-Asn-Leu-Ser-Phe-Asn-Tyr-Gln-Lys-Arg-Val-Ser-Phe-Ala-His-Leu-Ser-Leu.

[0038] Comparative Example 2

[0039] A polypeptide is prepared by in vitro synthesis (high purity), which contains all the amino acids of the yak spleen small molecular polypeptide in Example 1, but the sequence is randomly shuffled before synthesis, and the sequence is abbreviated as NKVFSRLQSY (SEQ ID NO. 3), and specifically Asn-Lys-Val-Phe-Ser-Arg-Leu-Gln-Ser-Tyr.

[0040] Test Example 1: Acanthocytosis Induction Experiment

[0041] 1. Experimental Procedure

[0042] (1) Eight-week-old male C57BL / 6N mice (provided by the Animal Experiment Center of Nantong University) were anesthetized, and blood was collected from their eyes into EDTA anticoagulant tubes. The blood was centrifuged at 500g for 5 min to separate plasma and red blood cells. The upper plasma layer was discarded, and the lower red blood cells were washed three times with isotonic PBS. The red blood cells were counted using a red blood cell counter.

[0043] (2) The experiment was divided into 6 groups, with approximately 1×10⁻⁶ samples taken from each group. 6 The experiment was conducted using 100 red blood cells. Before the experiment, equal amounts (1 μM) of the yak spleen small molecule polypeptides prepared in Examples 1 and 2 and the polypeptides prepared in Comparative Examples 1 and 2 were added to four groups, respectively. Then, 1 μM of CpG ODN sequence was added to induce acanthocytosis. In the remaining two groups, the group that only added 1 μM of CpG ODN sequence was the positive control group, and the group that only added isotonic physiological saline was the negative control group.

[0044] (3) After the experiment, the morphological changes of red blood cells in each group were detected, and the results are as follows: Figure 1 As shown.

[0045] 2. Experimental Results

[0046] Erythrocyte spinomorphism easily leads to erythrocyte adhesion and retention in the microvessels of various tissues, which is detrimental to oxygen transport. Figure 1 As can be seen, compared with the negative control group, incubation with CpG ODN alone can promote the spinous changes and increased viscosity of erythrocytes (positive control group). However, erythrocytes pre-incubated with the yak spleen small molecule polypeptides prepared in Examples 1 and 2 showed significantly better erythrocyte morphology and adhesion than those in the positive control group and Comparative Examples 1 and 2. This indicates that incubation with yak spleen small molecule polypeptides can inhibit CpG ODN-induced erythrocyte deformation and adhesion, and this treatment is beneficial to improving oxygen transport efficiency.

[0047] Test Example 2: Mouse Plateau Exposure Experiment

[0048] To further demonstrate that inhibiting erythrocyte deformation can effectively improve oxygen transport efficiency, this experiment was conducted, as follows:

[0049] 1. Experimental Procedure

[0050] (1) Select 8-week-old male C57BL / 6N mice (provided by the Animal Experiment Center of Nantong University) and adaptively feed for 1 week, then randomly divide them into 6 groups according to body weight, 10 in each group, of which 5 groups are exposed to simulated high altitude 6000m atmospheric environment for 3 days in an animal low pressure oxygen chamber to establish a high altitude hypoxia exposure model, and the remaining 1 group is placed in a normal pressure and normal oxygen chamber for 3 days as a negative control.

[0051] (2) Before the experiment, the mice in the high altitude hypoxia exposure model were injected with equal amounts (20mg / kg) of the yak spleen small molecule polypeptides prepared in Examples 1 and 2 and the polypeptides prepared in Comparative Examples 1 and 2 into the tail vein, and then placed in a low pressure and low oxygen animal chamber for exposure treatment; the remaining 1 group of mice in the high altitude hypoxia exposure model was used as a positive control group, injected with normal saline, and then placed in a low pressure and low oxygen animal chamber for exposure treatment; the mice in the negative control group were injected with normal saline and then placed in a normal pressure and normal oxygen chamber for exposure treatment.

[0052] (3) After the exposure, the red blood cell morphology and oxygen affinity (P50) of the 6 groups of mice were detected, and the results are shown in Figure 2 and Figure 3 .

[0053] 2. Experimental results

[0054] Red blood cell spiny shape change is easy to cause red blood cell stickiness and retention in each tissue microvascular, which is not conducive to oxygen transport. P50 is the half-saturation oxygen partial pressure, which is the oxygen partial pressure when 50% of hemoglobin is saturated, and it reflects the blood oxygen transport capacity and the affinity of hemoglobin for oxygen. The higher the P50, the lower the affinity for oxygen.

[0055] As can be seen from Figure 2 , compared with the negative control group, the red blood cell deformation rate and stickiness of the positive control group (simple high altitude hypoxia exposure) were significantly increased. The red blood cell morphology and stickiness of the mice injected with the yak spleen small molecule polypeptides of Examples 1 and 2 before high altitude hypoxia exposure were better than those of the positive control group mice, close to the level of the negative control group.

[0056] As can be seen from Figure 3 , compared with the negative control group, the oxygen binding capacity of the positive control group was significantly reduced. Compared with the positive control group, the oxygen binding capacity of the red blood cells of the mice injected with the yak spleen small molecule polypeptides of Examples 1 and 2 before high altitude hypoxia exposure was significantly lower than that of the positive control group mice. It shows that the red blood cell deformation of the mice injected with the yak spleen small molecule polypeptides under high altitude hypoxia exposure is reduced, the stickiness is reduced, and the oxygen carrying function is improved.

[0057] As shown in Figure 2 , 3As shown, the red blood cell deformability and P50 of the mice injected with the polypeptide of Example 1 are lower than those of Example 2, indicating that the high-purity yak spleen small molecule polypeptide has a better effect on improving the red blood cell morphology, stickiness and oxygen carrying function under high altitude exposure.

[0058] However, the red blood cells of the mice injected with the polypeptides of Comparative Examples 1 and 2 have more deformation, higher stickiness and lower oxygen carrying, which is basically close to the positive control group, indicating that the lack of yak spleen small molecule polypeptide cannot improve the higher red blood cell deformability and stickiness and lower oxygen carrying function under high altitude exposure, and cannot improve the oxygen transport efficiency of red blood cells, and the effect is obviously not as good as that of Examples 1 and 2.

[0059] The improvement of the red blood cell morphology, stickiness and oxygen carrying function of the mice in Examples 1 and 2 is mainly due to the protection of the yak spleen small molecule polypeptide on the red blood cell deformation, stickiness and oxygen carrying function.

[0060] The above is only the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application. The components not explicitly described in the present embodiment can be realized by the existing technology.

Claims

1. A small molecule polypeptide of yak spleen, characterized in that, The amino acid sequence of the polypeptide is shown as SEQ ID NO.

1.

2. The small polypeptide of yak spleen according to claim 1, characterized in that, The polypeptide is extracted from the yak spleen or obtained by in vitro synthesis.

3. The method for preparing small polypeptide of yak spleen according to claim 2, characterized in that, The preparation method is extraction from the yak spleen, comprising the following steps: Step 1) Collect the yak spleen tissue, wash the fresh or frozen yak spleen tissue with physiological saline at room temperature, add 2 times the volume of sterile physiological saline, and use a homogenizer to make a homogenate at 4℃; Step 2) Add 2 times the volume of sterile physiological saline and ultrasonic homogenization, and then freeze-thaw repeatedly in liquid nitrogen for 3 times; Step 3) Freeze the homogenate at 12000 rpm and centrifuge for 15 min, and then take the supernatant; Step 4) Add serine protease, sulfhydryl protease, aspartic acid protease, and metalloprotease to the supernatant, stir and hydrolyze at 37℃ for 30 min, centrifuge at 12000 rpm for 15 min, take the supernatant, boil, and then perform ultrafiltration to remove substances with a molecular weight less than 3000 Da, obtain the filtrate, sterilize, freeze-dry, and then obtain the polypeptide.

4. The method for preparing a small molecule polypeptide from yak spleen according to claim 3, characterized in that, Step 4) The molar ratio of the serine protease, sulfhydryl protease, aspartic acid protease, and metalloprotease is 3:1:1:

1.

5. Use of the polypeptide of claim 1 or 2 in the preparation of a medicine for preventing and / or treating high altitude reaction.

6. A pharmaceutical composition for preventing and / or treating altitude sickness, characterized by, The polypeptide of claim 1 or 2.

7. The pharmaceutical composition of claim 6, wherein, Also included is a pharmaceutically acceptable carrier.

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