Application of OLA1 in preparation of sepsis-related drugs

Abstract

The invention provides application of OLA1 in preparation of sepsis-related drugs, relates to the technical field of biomedicine, and aims to solve the problems that in the prior art, sepsis pathogenesis is unknown and treatment means are deficient, an OLA1 gene knockout mouse model is constructed, and it is proved that the survival rate of sepsis mice can be remarkably increased and weight loss and abnormal body temperature can be improved by inhibiting OLA1 expression. Based on this, the invention proposes that the OLA1 inhibitor (including small molecule compounds, siRNA and the like) is used for preparing the sepsis treatment medicine, and a new treatment strategy is provided for clinic. According to the application, the direct association between the OLA1 gene and the pathologic process of sepsis is disclosed for the first time, the gene knockout experiment proves that the prognosis of sepsis can be obviously improved by inhibiting the OLA1, and a new direction is provided for developing targeted drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of OLA1 in the preparation of sepsis-related drugs. Background Art

[0002] Sepsis refers to life-threatening organ dysfunction caused by an imbalance in the body's response to infection, and is a common complication in clinical settings such as severe trauma, shock, infection, and major surgery. If left uncontrolled, the progression of the disease will lead to septic shock and multiple organ dysfunction syndrome, which is the main cause of death in critically ill patients. Typical clinical manifestations include chills, high fever, rapid breathing, increased heart rate, and changes in mental state. It has a high incidence, high mortality, and high economic burden, seriously threatening human health. For many survivors, there is still a continuous risk of death, as well as long-term cognitive and functional deficits after discharge.

[0003] In 2016, sepsis was defined as an infection accompanied by organ dysfunction. So far, the pathogenesis of sepsis is not yet clear. Current research believes that the pathogenesis of sepsis is complex, including inflammatory imbalance, endothelial dysfunction, coagulation dysfunction, immune dysfunction, mitochondrial damage, endoplasmic reticulum stress, autophagy, and the neuroendocrine immune network.

[0004] Current sepsis treatment mainly relies on antibiotics, fluid resuscitation, and organ support treatment, but specific drugs targeting the pathological mechanism are still lacking. Existing research shows that cytokine storms and endothelial barrier dysfunction are key factors in the deterioration of sepsis, yet the targets for regulating these processes have not been clearly identified. For example, TNF-α inhibitors can relieve the inflammatory response but may exacerbate immunosuppression; anticoagulant drugs such as activated protein C have been withdrawn from clinical use due to the risk of bleeding. Therefore, there is an urgent need to discover new therapeutic targets for the treatment of sepsis. Summary of the Invention

[0005] The purpose of the present invention is to solve the problems in the prior art that the pathogenesis of sepsis is not very clear and the treatment methods are lacking.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The application of OLA1 in the preparation of sepsis-related drugs.

[0008] Preferably, the drug is used to inhibit the expression of the OLA1 gene.

[0009] The present application also provides a drug for the treatment of sepsis, and the drug is used to inhibit the expression of the OLA1 gene.

[0010] Based on the above, the present application also provides a verification method for verifying the application of OLA1 in sepsis. The verification method includes constructing a mouse model with OLA1 gene knockout, and then constructing a severe sepsis model in the established mouse model with Ola1 gene knockout. In the sepsis model, observe the changes in body weight, anal temperature and survival rate of the mice after the operation to determine the impact of OLA1 on sepsis.

[0011] Preferably, the method for constructing the mouse model with OLA1 gene knockout is as follows: By using the Cre-loxp system, primers are designed to insert LoxP on both sides of the second exon of the OLA1 gene, and Tek Cre is selected as the endothelial cell-specific Cre to obtain the mouse with OLA1 gene knockout.

[0012] Beneficial effects:

[0013] In the present application, the direct association between the OLA1 gene and the pathological process of sepsis is revealed for the first time. Through gene knockout experiments, it is proved that inhibiting OLA1 can significantly improve the prognosis of sepsis, providing a new direction for the development of targeted drugs. In order to verify the effectiveness of the OLA1 gene as a therapeutic target, specific verification experiments were carried out in the present application. In the severe sepsis model, the mice with OLA1 gene knockout showed a higher 72-hour survival rate than the OLA1 mice. This result not only proves the key role of the OLA1 gene in the pathological process of sepsis, but also clearly supports its effectiveness as a therapeutic target. Further experimental results show that by inhibiting the OLA1 gene, the weight loss and abnormal body temperature caused by sepsis can be effectively reduced. The changes in these physiological indicators are important bases for evaluating the severity of the disease and the therapeutic effect, indicating that the inhibition of the OLA1 gene can have a positive impact on the systemic pathological changes caused by sepsis. More importantly, inhibiting the OLA1 gene can also significantly reduce the risk of organ bleeding. Organ bleeding is one of the serious complications caused by sepsis, which often leads to poor prognosis of patients. This finding means that the inhibition strategy targeting the OLA1 gene may provide a new means for preventing and treating sepsis-related organ damage. Based on the above results, the present application reveals the key role of the OLA1 gene in the pathological process of sepsis and confirms its potential clinical value as a therapeutic target. This finding provides a basis and a new direction for the development of new targeted drugs and the improvement of the therapeutic effect of sepsis. Description of the Drawings

[0014] Figure 1 It is the change of the body weight (g) of the mice after sepsis operation in an embodiment of the present invention; Specifically, the Figure 1Line graph showing the weight changes of OLA1fl / fl mice and TekCre OLA1fl / fl mice after sepsis surgery. The body weight of the mice was recorded starting from 0 h after the surgery. By comparison, it was found that the body weight of OLA1 mice decreased significantly at 31 h after sepsis surgery.

[0015] Figure 2 Graph showing the change in rectal temperature of mice after sepsis surgery in one embodiment of the present invention; specifically, the Figure 2 Line graph showing the change in rectal temperature of OLA1fl / fl mice and TekCre OLA1fl / fl mice after sepsis surgery;

[0016] Figure 3 Survival curve graph of mice after sepsis surgery in one embodiment of the present invention; specifically, the Figure 3 The red curve represents the OLA1fl / fl group, and the black curve represents the TekCre OLA1fl / fl group.

[0017] Figure 4 Graph of partial organs of mice at 10 h after sepsis surgery in one embodiment of the present invention; specifically, the Figure 4 The organs from left to right are lung, liver, spleen, heart, and kidney.

[0018] (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001) Specific embodiments

[0019] The following will further elaborate on the present invention in conjunction with specific embodiments.

[0020] Application of OLA1 in the preparation of drugs for preventing and treating sepsis.

[0021] The drug is used to inhibit the expression of the OLA1 gene.

[0022] The present application also provides a drug for treating sepsis, and the drug is used to inhibit the expression of the OLA1 gene.

[0023] To verify the application of OLA1 in sepsis, the present application also constructed an OLA1 gene knockout mouse model. Through the Cre-loxp system, primers were designed to insert LoxP on both sides of the second exon of the OLA1 gene. Tek Cre was selected as the endothelial cell-specific Cre to obtain OLA1 gene knockout mice.

[0024] In addition, based on the above OLA1 gene knockout mouse model, a verification method is provided in this application, and the steps are as follows: construct a severe sepsis (75% injury) model in the established OLA1 gene knockout mouse model, and in the sepsis model, observe the changes in the body weight, anal temperature and survival rate of the mice after the operation to determine the effect of OLA1 on sepsis.

[0025] The above content is elaborated below in combination with specific verification experiments:

[0026] Experimental materials and sources:

[0027] 1. The electrophoresis apparatus was purchased from Liuyi Instrument Factory in Beijing, China;

[0028] 2. The animal feed was purchased from the Animal Experiment Center of Nantong University;

[0029] 3. The electronic balance was purchased from Sartorius Company in Germany;

[0030] 4. The Milli-Q ultrapure water instrument was purchased from Merck Company in Germany;

[0031] 5. Absolute ethanol was purchased from Shanghai Zhenxing Chemical Factory No. 1;

[0032] 6. The mouse tail direct PCR kit was purchased from Bimake Company;

[0033] Experimental method: Cecal ligation and puncture - construction of a sepsis mouse model

[0034] Select 7 male Tek Cre OLA1fl / fl and OLA1fl / fl mice each aged 8 - 12 weeks and weigh them.

[0035] (1) Anesthetize the mice by intraperitoneal injection of 10% chloral hydrate (0.1 ml / 10 g).

[0036] (2) Depilate the abdomen of the mice and disinfect the area, wipe it with a 70% alcohol swab, and under sterile conditions, perform a midline laparotomy of 1 to 2 cm and expose the cecum and adjacent intestines.

[0037] (3) Ligate the cecum with a suture at the base below the ileocecal valve and puncture it once with a needle on the same side of the cecum. (The length of the ligated cecum, defined as the distance from the distal end of the cecum to the ligation point, will determine the severity. A distance > 1 cm can cause high - grade sepsis, and a distance ≤ 1 cm can produce moderate - to - low - grade sepsis.) In this experiment, the ligation was > 1 cm for severe sepsis.

[0038] (4) Gently squeeze the cecum to extrude a small amount of feces from the puncture site. Then put the cecum back into the abdominal cavity, rinse the abdominal cavity with normal saline, and then suture the peritoneum and skin layer by layer.

[0039] (5) The mice were resuscitated by subcutaneous injection of 1 ml of pre-warmed 0.9% saline solution using a 25G needle. (This fluid resuscitation measure will induce the hyperdynamic phase of sepsis.)

[0040] (6) The animals were returned to their cages and exposed to an infrared heating lamp of 150 W until they recovered from anesthesia.

[0041] (7) Food and water placed at the bottom of the cage were provided ad libitum.

[0042] Analysis of experimental results:

[0043] Please refer to Figure 1 , compared with the mice in the OLA1fl / fl group, the body weight change of the mice in the Tek Cre OLA1fl / fl group gradually tended to be stable 9 h to 31 h after cecal ligation and puncture surgery; it was shown that inhibiting the expression of OLA1 could reduce the impact of sepsis surgery on body weight.

[0044] As Figure 2 shown, the rectal temperature of the mice was recorded starting from 0 h after the end of the surgery. The rectal temperatures of the OLA1fl / fl mice and the Tek Cre OLA1fl / fl mice reached the peak at 3 h after cecal ligation and puncture surgery. At 31 h after the surgery, the rectal temperature of the OLA1 mice was lower than that of the Tek Cre OLA1fl / fl mice.

[0045] Please refer to Figure 3 , it can be seen from the figure that the survival probability of the OLA1fl / fl group rapidly decreased at about 10 h and further decreased at about 30 h. The survival probability of the Tek Cre OLA1fl / fl group began to decrease at about 30 h. By comparing with the Tek Cre OLA1fl / fl mice, it can be observed that the change in the survival rate of the OLA1fl / fl group fluctuated greatly.

[0046] As Figure 4 shown, compared with the Tek Cre OLA1fl / fl mice, the bleeding in the lungs, liver, spleen, heart, and kidneys of the OLA1fl / fl mice increased after sepsis surgery.

[0047] This application verified through in vivo experiments that the survival rate of the OLA1 knockout mice was higher and the organ bleeding was less after sepsis surgery.

[0048] In summary, through specific verification experiments in this application, it has been proven that in a severe sepsis model, OLA1 gene knockout mice showed a higher 72-hour survival rate than OLA1 mice. This result not only proves the key role of the OLA1 gene in the pathological process of sepsis, but also clearly supports its effectiveness as a therapeutic target. Further experimental results show that by inhibiting the OLA1 gene, the weight loss and abnormal body temperature caused by sepsis can be effectively alleviated. These changes in physiological indicators are important bases for evaluating the severity of the disease and the therapeutic effect, indicating that the inhibition of the OLA1 gene can have a positive impact on the systemic pathological changes caused by sepsis. More importantly, inhibiting the OLA1 gene can also significantly reduce the risk of organ bleeding. Organ bleeding is one of the serious complications caused by sepsis, often leading to poor prognosis of patients. This finding means that the inhibition strategy targeting the OLA1 gene may provide a new means for preventing and treating sepsis-related organ damage. Based on the above results, this application reveals the key role of the OLA1 gene in the pathological process of sepsis and confirms its potential clinical value as a therapeutic target. This finding provides a basis and a new direction for the development of new targeted drugs and the improvement of the therapeutic effect of sepsis.

Claims

1. Use of OLA1 in drugs for treating sepsis.

2. Use of OLA1 according to claim 1 in the preparation of a medicament for treating sepsis, characterized in that: The drug is used to inhibit the expression of the OLA1 gene.

3. A drug for treating sepsis, characterized in that: The drug is used to inhibit the expression of the OLA1 gene.

4. A drug for treating sepsis according to claim 3, characterized in that: The drug includes at least one of siRNA, CRISPR, and small molecule compounds for inhibiting the expression of the OLA1 gene.

5. A drug for treating sepsis according to claim 3, characterized in that: The drug contains a small molecule compound for inhibiting the expression of the OLA1 gene, and the small molecule compound is selected from RNA interference agents, antisense oligonucleotides, or OLA1 protein inhibitors.

6. A verification method, which is used to verify the application of OLA1 in sepsis, and is characterized in that: The verification method includes constructing an OLA1 gene knockout mouse model, then constructing a severe sepsis model in the established Ola1 gene knockout mouse model, and observing the changes in body weight, anal temperature, and survival rate of the mice after surgery in the sepsis model to determine the effect of OLA1 on sepsis.

7. The verification method according to claim 4, characterized in that: The method for constructing the OLA1 gene knockout mouse model is as follows: By using the Cre-loxp system, primers are designed to insert LoxP on both sides of the second exon of the OLA1 gene, and Tek Cre is selected as the endothelial cell-specific Cre to obtain OLA1 gene knockout mice.

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