A tlr9 polypeptide and uses thereof

Abstract

The application discloses a TLR9 polypeptide and application thereof. The amino acid sequence of the TLR9 polypeptide is shown as SEQ ID NO. 1. The TLR9 polypeptide is found to be capable of enhancing the phagocytosis and clearance function of liver macrophages on red blood cells, thereby effectively reducing the abnormal retention of red blood cells in liver tissues under a high-altitude hypoxic environment, reducing blood viscosity, and restoring the dynamic balance between red blood cell production and clearance. The polypeptide can be used for preparing a targeted drug for preventing or treating high-altitude polycythemia, and provides a novel, safe and effective potential scheme for solving the limitations of existing clinical treatment methods, and is particularly suitable for disease prevention and treatment of long-term high-altitude resident populations.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a TLR9 polypeptide and its applications. Background Technology

[0002] High altitude polycythemia (HAPC) is the most common and widespread type of chronic mountain sickness, prevalent among people who live at altitudes above 2500 m for extended periods. Its root cause lies in the body's compensatory mechanisms to adapt to the low-oxygen environment of high altitudes, leading to abnormally high hemoglobin concentrations and excessive red blood cell proliferation. However, once this compensation becomes unbalanced, it causes a significant increase in blood viscosity, slowed blood flow, and microcirculatory disturbances, further exacerbating tissue hypoxia and creating a vicious cycle. Patients often exhibit symptoms such as headache, fatigue, palpitations, and cyanosis, seriously threatening the health of soldiers stationed on high-altitude garrisons, mountaineers, high-altitude engineering workers, and long-term residents. Epidemiological surveys show that the prevalence of this disease increases significantly with altitude, reaching over 15% in migrant populations living above 5000 m, making it a prominent public health problem in high-altitude areas.

[0003] Currently, the most direct clinical treatment for high-altitude chronic paralysis (HAPC) is phlebotomy or recommending patients transfer to lower altitudes. However, both methods have significant limitations. Phlebotomy only treats the symptoms and cannot address the underlying pathological mechanism; repeated procedures pose risks such as infection and anemia, leading to poor patient compliance. Furthermore, for those permanently stationed or living at high altitudes, leaving the hypoxic environment often means leaving their jobs or homes, which is not practically feasible. Therefore, there is an urgent clinical need to develop a preventative or therapeutic drug that can intervene at the source of the pathological mechanism and is suitable for long-term high-altitude environments.

[0004] Recent studies have gradually revealed that the occurrence of HAPC is not only related to hypererythropoiesis, but also closely related to the homeostatic imbalance caused by impaired erythrocyte clearance. The spleen and liver are the main organs for clearing senescent and damaged erythrocytes, with resident macrophages (such as splenic erythropoiesis-macrophages and Kupffer cells) acting as "scavengers" to perform this function. Animal experimental evidence has shown that long-term exposure to high-altitude hypoxia can lead to a decrease in the number and function of splenic macrophages, resulting in cell loss through ferroptosis and other forms, thus reducing their ability to phagocytose and clear erythrocytes. This leads to the retention of abnormal erythrocytes in the blood and tissues, causing tissue hypoxia. Hypoxia further exacerbates erythropoiesis, thereby further aggravating hemorheological deterioration. When the spleen's erythropoiesis is impaired, the liver mainly plays a compensatory role. Therefore, targeting and enhancing the erythropoiesis function of hepatic macrophages to restore the dynamic balance between erythrocyte production and clearance is a potential new strategy for intervening in HAPC from a key pathophysiological link.

[0005] Toll-like receptor 9 (TLR9) is an important pattern recognition receptor in the innate immune system. TLR9 agonists (such as CpG ODN) are known to nonspecifically activate various immune cells, including macrophages, enhancing their phagocytic and killing capabilities, and have been studied in anti-infection and tumor immunotherapy. However, to date, no studies have reported or suggested that specific TLR9 ligands (especially short peptides with well-defined sequences) can specifically and effectively enhance the phagocytic clearance function of hepatic macrophages against erythrocytes, nor have they linked them to the treatment of high-altitude polycythemia. Currently, there is a lack of targeted therapeutic strategies that can safely and effectively restore erythrocyte homeostasis under high-altitude hypoxic environments through precise immune modulation pathways.

[0006] In summary, given the current lack of effective treatments and limited efficacy for high-altitude polycythemia, and considering the crucial link of impaired erythrocyte clearance in its underlying pathological mechanism, there is an urgent need in this field to develop a novel intervention method with a clearly defined mechanism. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a TLR9 peptide and its application. This TLR9 peptide can effectively restore erythrocyte homeostasis under high-altitude hypoxic conditions by specifically enhancing the erythrophagic function of liver macrophages. This provides a safe, effective, and targeted treatment strategy for the prevention and treatment of high-altitude polycythemia without requiring the patient to leave the high-altitude environment.

[0008] This invention is achieved through the following technical solution:

[0009] A TLR9 polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] Preferably, the polypeptide is prepared by solid-phase synthesis and has a purity of ≥95%.

[0011] A pharmaceutical composition comprising a therapeutically effective amount of the above-described TLR9 peptide and a pharmaceutically acceptable carrier.

[0012] Preferably, the pharmaceutically acceptable carrier is selected from at least one of liposomes, nanoparticles, sustained-release microspheres, injectable solvents, or lyophilization protectants.

[0013] The use of the above-mentioned TLR9 peptide, or the above-mentioned pharmaceutical composition, in the preparation of a drug for enhancing the function of hepatic macrophages in phagocytizing erythrocytes.

[0014] The use of the above-mentioned TLR9 peptide, or the above-mentioned pharmaceutical composition, in the preparation of a medicament for the prevention and / or treatment of high altitude polycythemia.

[0015] Preferably, the drug is used to reduce blood viscosity induced by high-altitude exposure, improve oxygen transport efficiency, or restore erythrocyte homeostasis.

[0016] Preferably, the drug is administered via intravenous injection, subcutaneous injection, or intramuscular injection.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) This invention creatively proposes and confirms that administration of TLR9 peptide can significantly enhance the phagocytic clearance function of hepatic macrophages on erythrocytes. This discovery breaks through the traditional thinking that mainly focuses on "reducing erythrocyte production" or "improving the properties of erythrocytes", and provides a new therapeutic target and pathway for the prevention and treatment of high-altitude polycythemia from the new perspective of "promoting the clearance of pathological erythrocytes".

[0019] (2) The TLR9 peptide of the present invention can specifically enhance the function of macrophages in the liver, the main organ where pathological erythrocyte retention occurs, and effectively reduce the abnormal retention of total erythrocytes and immature erythrocytes in the blood and tissues, thereby reducing whole blood viscosity and improving microcirculation and oxygen transport efficiency. This effect directly targets the core pathological link of "insufficient clearance" of HAPC, which helps to fundamentally restore the dynamic balance between erythrocyte production and clearance, and achieve etiological treatment of the disease.

[0020] (3) Compared with the current clinical routine bloodletting therapy (which treats the symptoms but not the root cause, is invasive and prone to recurrence) or "descent therapy" (which is impractical for people who live in high-altitude areas), the polypeptide drug regimen provided by this invention has the advantage of long-term repeated administration. Animal experiments have shown that this polypeptide intervention can significantly improve pathological indicators under high-altitude exposure, and no adverse reactions such as excessive systemic immune activation were observed, showing good efficacy and safety, and providing a new treatment option for high-altitude patients without leaving the environment.

[0021] (4) The polypeptides of the present invention can not only be used to treat existing HAPC, but are also suitable for early prevention of high-risk groups such as those entering high-altitude areas, mountaineering, and working in high-altitude areas, thereby reducing the incidence of chronic altitude sickness. The pharmaceutical composition can be made into various dosage forms (such as sustained-release preparations), which are easy to carry and use, and are particularly suitable for use in high-altitude areas with limited medical conditions. It has important social and economic value for protecting the health of high-altitude workers and residents and improving their work and living abilities.

[0022] (5) The core active ingredient of this invention—the TLR9 polypeptide—has a well-defined sequence and can be prepared in high purity and on a large scale using a mature solid-phase synthesis process. The quality is controllable and the production cost is relatively controllable, laying a solid technical foundation for subsequent drug development and commercial production. Attached Figure Description

[0023] Figure 1 The phagocytosis of PKH67-labeled red blood cells by Kupffer cells in the liver tissue of mice in each group in Example 2 is shown in Figure A: a representative scatter plot by flow cytometry; and a statistical chart of the absolute number of PKH67-positive Kupffer cells in each group of mice.

[0024] Figure 2 The following figures illustrate the red blood cell retention in the liver tissue of mice in each group in Example 3: A is an example of RBC retention in liver tissue; B is a statistical representation of the RBC percentage in each field of view in the liver after 7 days of high-altitude exposure in Figure A; C is a statistical representation of the RBC percentage in each field of view in the liver after 14 days of high-altitude exposure in Figure A.

[0025] Figure 3 For the flow cytometry analysis of changes in the percentage of erythrocytes in the liver tissue of mice in each group in Example 3: A is the flow cytometry result of single-cell suspension of liver tissue; B is the total percentage of erythrocytes in Figure A; C is the percentage of immature erythrocytes in Figure A; D is the percentage of mature erythrocytes in Figure A.

[0026] Figure 4 The following is an example of erythrocyte retention in the liver tissue of mice in each group after TLR9 peptide intervention in Example 3: A is an example of RBC retention in liver tissue; B is a statistical representation of the percentage of RBCs in each field of view in the liver after 7 days of high-altitude exposure in Figure A.

[0027] Figure 5 The following is a flow cytometry analysis of the changes in the percentage of erythrocytes in the liver tissue of mice in each group after TLR9 peptide intervention in Example 3: A is a flow cytometry result of single-cell suspension of liver tissue; B is the total percentage of erythrocytes in Figure A; C is the percentage of immature erythrocytes in Figure A; D is the percentage of mature erythrocytes in Figure A.

[0028] Figure 6 The effect of TLR9 peptide on the histology of major organs in mice exposed to high altitudes, as shown in Example 4. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0030] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] The experimental animals used in the following examples were C57BL / 6N mice, provided by the Animal Experiment Center of Nantong University.

[0033] Example 1 Synthesis and preparation of TLR9 peptide

[0034] 1. Sequence Information

[0035] The target polypeptide is the TLR9 polypeptide (SEQ ID NO.1), as detailed below:

[0036] Gln-Leu-Arg-Lys-Leu-Asn-Leu-Ser-Phe-Asn-Tyr-Gln-Lys-Arg-Val-Ser-Phe-Ala-His-Leu-Ser-Leu-Ala-Pro-Ser-Phe-Gly-Ser-Leu-Val.

[0037] 2. Synthesis Method

[0038] The polypeptide was prepared by Nanjing Peptide Valley Biotechnology Co., Ltd. using an in vitro synthesis method and the standard Fmoc (9-fluorenylmethoxycarbonyl) solid-phase polypeptide synthesis method.

[0039] 3. Quality Control

[0040] High-performance liquid chromatography (HPLC) analysis showed that the product purity was >95%. Mass spectrometry (MALDI-TOF MS) results were consistent with the theoretical molecular weight.

[0041] Example 2: In vitro verification of the TLR9 peptide's effect on the phagocytic function of mouse liver macrophages on erythrocytes

[0042] 1. Isolation and fluorescent labeling of red blood cells (RBCs)

[0043] (1) Red blood cell collection: Eight-week-old male C57BL / 6N mice were selected and acclimatized for one week. They were anesthetized with isoflurane inhalation and blood was collected from the posterior venous plexus of the eye. The blood was placed in an EDTA anticoagulant tube.

[0044] (2) Red blood cell washing: Mix the collected whole blood with an equal volume of PBS, centrifuge at 500 g for 5 min, and carefully discard the supernatant and white blood cell layer. Resuspend the precipitate in PBS and wash three times to obtain a pure red blood cell precipitate.

[0045] (3) PKH67 labeling: The concentration of washed red blood cells was adjusted to approximately 1×10⁻⁶ using PBS. 91 mL of red blood cell suspension was centrifuged at 500 g for 5 min, and the supernatant was discarded. 1 mL of PKH67 Green Fluorescent Cell Linker staining working solution was added for resuspending. An equal volume (2 mL) of PBS or fetal bovine serum containing 1% BSA was added to terminate staining, and the mixture was incubated at room temperature for 1 min. The cells were centrifuged at 400 g for 5 min at 25°C, and the supernatant was discarded. The precipitate was resuspended in complete culture medium. Washing was repeated twice to remove unbound dye. Finally, the labeled red blood cells were resuspended in PBS, and the concentration was adjusted to 1 × 10⁻⁶ cells / mL. 9 Quantity / mL, store at 4℃ protected from light for later use.

[0046] 2. Animal grouping and establishment of a high-altitude exposure model

[0047] (1) Animal grouping: 15 male C57BL / 6N mice aged 8 weeks were selected and acclimatized for 1 week. They were then randomly divided into 3 groups of 5 mice each according to their body weight.

[0048] Plain control group (plain group): normal pressure and normal oxygen environment;

[0049] High-altitude model group (high-altitude group): High-altitude hypoxia exposure;

[0050] High-altitude peptide treatment group (high-altitude peptide group): high-altitude hypoxia exposure + TLR9 peptide intervention.

[0051] (2) Establishment of a high-altitude exposure model

[0052] Mice in the plateau group and plateau peptide group were placed in an animal hypobaric oxygen chamber to simulate the plateau environment at an altitude of 6000 m. The chamber parameters were set as follows: pressure 54 kPa, oxygen partial pressure 11 kPa, temperature 26℃, humidity 40%, and ascent / descent speed 5 m / s. The light-dark cycle was 12 h:12 h. Sufficient drinking water and feed were provided in the chamber. The exposure time was 7 consecutive days.

[0053] The mice in the plain control group were placed in a normal pressure and oxygen chamber, maintaining the same temperature, humidity and light cycle.

[0054] 3. TLR9 peptide intervention and PKH67-labeled RBC injection

[0055] (1) PKH67-RBC injection: All three groups of mice were injected via tail vein with PKH67-labeled autologous red blood cell suspension (200 μL / mouse) before the start of high-altitude exposure (day 0).

[0056] (2) TLR9 peptide intervention

[0057] High-altitude polypeptide group: On the third day of high-altitude exposure, the TLR9 polypeptide (20 mg / kg) prepared in Example 1 was injected via tail vein.

[0058] High-altitude group and plain group: At the same time point, an equal volume of sterile saline was injected via the tail vein.

[0059] (3) Continued exposure: After injection, the high-altitude group and the high-altitude polypeptide group continued to be exposed in the hypobaric oxygen chamber until day 7; the plain group continued to be exposed in the normobaric oxygen chamber.

[0060] 4. Liver tissue sample collection and processing

[0061] (1) Perfusion treatment: After exposure, mice in each group were deeply anesthetized with isoflurane, the abdominal cavity was opened, and a cannula was inserted through the portal vein to perfuse the liver with preheated PBS to flush out residual blood for flow cytometry.

[0062] (2) Preparation of single-cell suspension

[0063] Take approximately 100 mg of fresh liver tissue, cut it into small pieces, and place it in a mixed working solution containing collagenase II (10 mg / mL) and collagenase I (10 mg / mL); digest at 37°C with shaking for 30 min; filter through a 70 μm cell sieve and collect the filtrate; centrifuge at 500 g for 5 min at 4°C and discard the supernatant; resuspend the precipitate in red blood cell lysis buffer and lyse on ice for 5 min; stop lysis with PBS, centrifuge at 500 g for 5 min and discard the supernatant; resuspend the precipitate in PBS containing 2% FBS and count the cells for later use.

[0064] 5. Detection of phagocytic function of liver macrophages (flow cytometry analysis)

[0065] Take the prepared liver single-cell suspension (approximately 1×10⁻⁶) 6 Add Fc receptor blocker to each cell and incubate at 4°C for 10 min; add PE-labeled anti-mouse F4 / 80 flow cytometry antibody (1:100 dilution) and incubate at 4°C in the dark for 30 min; wash with 2 mL PBS, centrifuge at 400 g for 5 min, and discard the supernatant; resuspend the cells in 500 μL PBS and analyze them using flow cytometry; gating strategy: first delineate the F4 / 80 positive cell population (macrophages), and then analyze the proportion of PKH67 positive cells in the cell population; collect at least 10,000 F4 / 80 positive cells for each sample.

[0066] 6. Experimental Results and Analysis

[0067] Representative scatter plots of flow cytometry are shown below. Figure 1 As shown in Figure A, the liver F4 / 80 of the plain control group + Among macrophages, the proportion of PKH67-positive cells (i.e., macrophages that have engulfed erythrocytes) was 25.6%, indicating that Kupffer cells in the liver have a basic erythrocyte clearance function under normal physiological conditions. After 7 days of exposure at an altitude of 6000 m in the plateau model group, the F4 / 80 ratio was [not specified].+ The proportion of PKH67-positive cells in macrophages increased to 31.6%, suggesting that the high-altitude hypoxic environment induced a compensatory enhancement of hepatic macrophage phagocytic function, which may be related to the body's attempt to clear increased or damaged red blood cells. In the high-altitude peptide treatment group, after intervention with TLR9 peptide (20 mg / kg) on ​​day 3 of high-altitude exposure, the F4 / 80 ratio was [not specified]. + The proportion of PKH67-positive cells in macrophages was significantly increased to 42.5%, which was significantly higher than that in the plateau model group, indicating that TLR9 peptide treatment can further synergistically enhance the phagocytic capacity of macrophages and promote the clearance of abnormal red blood cells.

[0068] To further quantify the enhancing effect of TLR9 peptide, the absolute number of PKH67-positive Kupffer cells in each of the five mice in each group was statistically analyzed. Figure 1 As shown in Figure B, the plateau model group PKH67 + KCs count was significantly higher in the plateau control group than in the plain control group, while PKH67 was higher in the plateau polypeptide treatment group. + KCs count was significantly higher than in the original model group. The above statistical results show that high-altitude exposure itself can increase the number of Kupffer cells phagocytosing erythrocytes by about 1.5 times (compared to the plain group), while TLR9 peptide intervention further increased the number of phagocytic cells to 2.6 times that of the plain group and 1.7 times that of the high-altitude group, indicating that the peptide can significantly enhance the clearance capacity of macrophages under high-altitude exposure.

[0069] Further analysis of the phagocytic activity of different macrophage subsets revealed that Kupffer cells (KCs, a high-expression F4 / 80 subset) were the main effector cells phagocytosing erythrocytes, and their phagocytic activity trend was similar to that of the overall F4 / 80 subset. + Cellular consistency: The phagocytic rate of KCs was increased in the plateau group, and further significantly increased in the peptide intervention group. The phagocytic rates of macrophages (tMs) and monocytes (Mos) in other tissues were low (both <4%), and there were no significant differences among the groups, suggesting that the TLR9 peptide mainly targets Kupffer cells in the liver.

[0070] The experimental results of this embodiment show that high-altitude hypoxia exposure can induce compensatory enhancement of the phagocytic function of Kupffer cells in the liver, and increase the number of phagocytic cells. This may be an adaptive response of the body to high-altitude stress. TLR9 peptide intervention can further significantly enhance the phagocytic activity of Kupffer cells and promote the clearance of abnormal red blood cells. The effect of TLR9 peptide is cell-specific, mainly activating the function of liver-resident macrophages (Kupffer cells).

[0071] Example 3: Therapeutic effect of TLR9 peptide in a mouse model of high-altitude polycythemia

[0072] 1. Construction and validation of a mouse model of high-altitude polycythemia

[0073] (1) Eight-week-old male C57BL / 6N mice were selected and randomly divided into two groups according to body weight after one week of acclimatization: a plain exposure group and a plateau exposure group, with 10 mice in each group. The plateau exposure treatment was carried out by the method in Example 2, with exposure for 7 days and 14 days respectively. The mice in the plain exposure group were placed in a normal pressure and normal oxygen chamber for 7 days and 14 days as a control.

[0074] (2) After exposure, liver tissues from two groups of mice were collected, and liver tissue single-cell suspensions were prepared according to the method in Example 2. The retention of erythrocytes in the liver tissues of the two groups of mice was then detected. Figure 2 Flow cytometry was used to analyze changes in total RBCs, the proportion of immature RBCs, and the percentage of mature RBCs. Figure 3 ).

[0075] (3) Model validation

[0076] like Figure 2 As shown in the AC, compared with the plain group, the retention of erythrocytes in the liver tissue of mice was significantly increased after 7 and 14 days of exposure at high altitude.

[0077] like Figure 3 As shown, compared with the plain group, the total erythrocyte retention in the liver tissue of mice was significantly increased after 7 days of exposure at high altitude ( Figure 3 (A, B) Significantly increased retention of immature erythrocytes ( Figure 3 (A, C) The retention of mature erythrocytes was significantly reduced ( Figure 3 (A, D)

[0078] The above experimental results indicate that the high-altitude exposure mouse model was successfully established and that high-altitude exposure significantly promotes the retention of total red blood cells and immature red blood cells in the liver of mice.

[0079] 2. The effect of TLR9 peptide on the improvement of high-altitude polycythemia model mice

[0080] (1) Eight-week-old male C57BL / 6N mice were selected and acclimatized for one week before being randomly divided into three groups according to body weight: plain exposure group (plain group), plateau exposure group given saline (plateau saline group), and plateau exposure group given TLR9 peptide (plateau peptide group). Each group consisted of five mice. The plain and plateau exposure treatments were the same as in Example 2.

[0081] (2) On the 3rd and 6th days of high-altitude hypoxia exposure, mice in the two groups exposed to high altitude were injected with equal amounts (20 mg / kg) of the TLR9 polypeptide prepared in Example 1 or physiological saline into their tail veins, and then placed in an animal hypobaric chamber for exposure treatment; mice in the plain control group were injected with physiological saline at the same time and then placed in a normal pressure normal oxygen chamber for exposure treatment.

[0082] (3) After exposure, liver tissues from the three groups of mice were collected, and liver tissue single-cell suspensions were prepared according to the method in Example 2. The retention of red blood cells in the liver tissues of the three groups of mice was then detected. Figure 4 Flow cytometry was used to analyze changes in total RBCs, the proportion of immature RBCs, and the percentage of mature RBCs. Figure 5 ).

[0083] (4) Experimental results and analysis

[0084] like Figure 4 As shown in Figures A and B, compared with the high-altitude saline group, the retention of erythrocytes in the liver tissue of mice in the high-altitude polypeptide group was significantly reduced.

[0085] like Figure 5 As shown, compared with the high-altitude saline group, the total erythrocyte retention in the liver tissue of mice in the high-altitude polypeptide group was reduced ( Figure 5 (A, B) Decreased retention of immature red blood cells ( Figure 5 (A, C) No significant changes were observed in the retention of mature erythrocytes. Figure 5 (A, D)

[0086] The experimental results of this embodiment show that TLR9 peptide intervention under high-altitude exposure can significantly reduce the retention of total erythrocytes and immature erythrocytes in the liver, which is beneficial to improving the liver's erythropoiesis function under high-altitude exposure, thereby improving blood viscosity and restoring erythrocyte homeostasis. This indicates that TLR9 peptide intervention can effectively alleviate the excessive erythrocyte proliferation and high blood viscosity caused by high-altitude hypoxia exposure, reduce the abnormal retention of erythrocytes in liver tissue, and has a clear therapeutic effect on high-altitude polycythemia.

[0087] Example 4: Safety evaluation of TLR9 peptide

[0088] 1. Experimental Methods

[0089] (1) Experimental animals: Mice were grouped into groups of 5 according to the method in Example 3. After the high-altitude exposure and drug treatment were completed, they were sacrificed on the 7th day after the exposure ended, and heart, liver, spleen, lung and kidney tissues were collected.

[0090] (2) Tissue fixation and dehydration: Freshly isolated mouse heart, liver, spleen, lung, and kidney tissues were immediately placed in 4% PFA and fixed at 4°C for at least 24 h. The fixed tissues were then removed and rinsed with running water for 30 min to remove excess fixative. They were then dehydrated in a gradient of ethanol: 30% ethanol, 50% ethanol, and 75% ethanol for 30 min each; 80% ethanol for 1 h; 95% ethanol for 1 h (repeated twice); 100% ethanol for 1 h; and 100% ethanol for 30 min.

[0091] (3) Clearing and paraffin infiltration: Anhydrous ethanol and recycled xylene are mixed at a ratio of 1:1 and dehydrated for 30 min; recycled xylene for 15 min; new xylene for 7 min; the tissue is transferred into melted paraffin and infiltrated for 3 h, and then embedded into a paraffin block.

[0092] (4) Sectioning and dewaxing / rehydration: The paraffin section was cut into 5 μm thick sections by a paraffin microtome, and the sections were mounted on glass slides and baked at 60℃ for 2 h. The sections were dewaxed and rehydrated in sequence: xylene recovery for 5 min; new xylene for 7 min; anhydrous ethanol for 2 min; 95% ethanol for 2 min; 80% ethanol for 2 min; 75% ethanol for 2 min; and rinsed with running water for 2 min.

[0093] (5) HE staining: stain with hematoxylin solution for 8 min, rinse with running water for 2 min; stain with eosin solution for 10 s, rinse with running water for 2 min.

[0094] (6) Dehydration, clearing and mounting: The slides were placed in a gradient of ethanol for dehydration in sequence: 75% ethanol for 3 min; 80% ethanol for 3 min; 95% ethanol for 3 min; anhydrous ethanol for 3 min. Xylene was recovered for 2 min; new xylene for 2 min; and the slides were mounted with neutral resin. After drying at room temperature, the slides were observed and photographed under an optical microscope.

[0095] 2. Experimental Results and Analysis

[0096] like Figure 6 As shown, the specific analysis is as follows:

[0097] (1) Heart: The myocardial fibers of mice in each group were neatly arranged with clear striations, and no myocardial cell edema, necrosis or inflammatory cell infiltration was observed. There was no significant difference in myocardial structure between the plateau polypeptide group and the plateau saline group and the plain group.

[0098] (2) Liver: The liver lobule structure was intact, the hepatic cords were neatly arranged, and the hepatocytes were normal in morphology, with no obvious hydropic degeneration, fatty degeneration, or necrosis. No obvious congestion or inflammatory cell infiltration was observed in the sinusoids of the liver in the high-altitude polypeptide group, and there were no obvious pathological changes compared with the high-altitude saline group.

[0099] (3) Spleen: The red pulp and white pulp are clearly demarcated, the splenic corpuscle structure is intact, and no obvious congestion or hemosiderin deposition is seen in the red pulp. The spleen structure of the high-altitude polypeptide group is normal, and no obvious abnormalities are seen.

[0100] (4) Lungs: The alveolar structure was clear, the alveolar walls were thin, and no obvious inflammatory cell infiltration, alveolar septal thickening, or edema fluid exudation was observed. The lung tissue of the high-altitude polypeptide group was similar to that of the plain group, and no obvious damage was observed.

[0101] (5) Kidneys: The glomerular structure was clear, the renal tubular epithelial cells were of normal morphology, no casts were seen in the lumen, and there was no inflammatory cell infiltration in the interstitium. The renal tissue structure of the high-altitude polypeptide group was normal, and there was no significant difference compared with the high-altitude saline group.

[0102] The experimental results of this embodiment show that, compared with the high-altitude saline group, no significant histopathological changes were observed in any organ of the high-altitude polypeptide group; the tissue structure remained intact, and the cell morphology was normal. This indicates that under the experimental conditions, the TLR9 polypeptide (20 mg / kg, tail vein injection) had no significant toxic effects on the major organs of mice and exhibited good in vivo safety, providing preliminary safety evidence for its further development and application.

[0103] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A TLR9 polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The TLR9 polypeptide according to claim 1, characterized in that, The polypeptide was prepared by solid-phase synthesis and has a purity of ≥95%.

3. A pharmaceutical composition, characterized in that, It contains a therapeutically effective amount of the TLR9 peptide as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutically acceptable carrier is selected from at least one of liposomes, nanoparticles, sustained-release microspheres, injectable solvents, or lyophilization protectants.

5. The use of the TLR9 peptide as described in claim 1 or 2, or the pharmaceutical composition as described in claim 3 or 4, in the preparation of a medicament for enhancing the phagocytic function of hepatic macrophages on erythrocytes.

6. The use of the TLR9 peptide as described in claim 1 or 2, or the pharmaceutical composition as described in claim 3 or 4, in the preparation of a medicament for the prevention and / or treatment of high altitude polycythemia.

7. The application according to claim 6, characterized in that, The drug is used to reduce blood viscosity induced by high-altitude exposure, improve oxygen transport efficiency, or restore erythrocyte homeostasis.

8. The application according to claim 6, characterized in that, The drug is administered via intravenous injection, subcutaneous injection, or intramuscular injection.

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