Use of an ier3 recombinant protein in the preparation of a medicament for treating sepsis

Abstract

The application discloses application of IER3 recombinant protein in preparation of a medicine for treating sepsis, and the medicine is an injection preparation, and the infusion mode of the injection preparation is intravenous infusion. The IER3 recombinant protein can reduce the mortality of CLP sepsis mice, reduce the abdominal cavity bacterial load, the blood and abdominal cavity inflammatory factor level and the degree of tissue organ damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of a recombinant IER3 protein in the preparation of drugs for treating sepsis. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulation of the host's immune response to pathogenic microbial infection. It is a common and serious complication and leading cause of death in critically ill patients with infections, burns / trauma, shock, and other acute conditions. Currently, there is no specific drug that can effectively cure sepsis. Sepsis is characterized by high morbidity, high mortality, high economic burden, and poor prognosis. Numerous drug development studies have been conducted to treat sepsis, but none have achieved satisfactory results. Early prevention, early detection, and early intervention are crucial to reducing morbidity and improving survival rates. Treatment options for sepsis mainly focus on fluid resuscitation, antibiotic therapy, and immunotherapy. Although these treatments can significantly reduce short-term mortality from sepsis, the long-term mortality rate remains high.

[0003] IER3, also known as IEX-1, PRG1, DIF-2, and GLY96, is a stress-induced glycosylated protein molecule. It is a new member of the early response gene family discovered by Professor Charles in mouse fibroblasts in 1993. Under various stress stimuli such as pathogen infection, radiation exposure, growth factors, and cytokines, it is highly expressed in different tissues and cells, including the trachea, gastrointestinal tract, pancreas, urogenital system, skin epithelial cells, and vascular endothelial cells. The IER3 gene is an evolutionarily highly conserved gene for immediate early stress response, widely present in various species such as humans, chimpanzees, monkeys, rats, and mice. The human IER3 gene is located on the short arm of chromosome 6 (6p21.33), encoding 156 amino acids, with a protein size of approximately 17 kDa. Studies have found that IER3 plays different regulatory roles under different cellular environments and stimuli, positively or negatively regulating cell growth and apoptosis during cell growth. When IER3 enters the mitochondria, it can promote the degradation of F1F0 complex inhibitor (IF1), enhance ATPase activity, and maintain the inner mitochondrial membrane. Phosphorylation levels inhibit intracellular reactive oxygen species (ROS) levels, thus exerting an anti-apoptotic effect. It can also enter the nucleus and interact with the NF-κB subunit RelA / p65 to regulate the expression of the downstream anti-apoptotic molecule Bcl-2, thereby promoting apoptosis. Furthermore, IER3 has been found to affect cell proliferation, differentiation, and migration through its own phosphorylation and via the MAPK / ERK pathway. In a mouse model of intraperitoneal endotoxin injection, IER3 knockout induced the production of mitochondrial reactive oxygen species (mROS) in mouse granulocytes and exacerbated liver and kidney dysfunction, increasing the risk of death. Pretreatment with the mitochondrial-specific antioxidant MitoQ reduced mortality and improved organ function, suggesting that IER3 knockout may increase the risk of sepsis death by increasing mROS production, and targeting the IER3-mROS pathway may be an effective strategy to alleviate sepsis and its organ dysfunction. Further investigation in a mouse skin Leishmaniasis infection model revealed that IER3 knockout aggravated skin infection, increased IL-17A and decreased TNF-α expression, and caused an imbalance in Th1 and Th2-mediated immune responses, indicating the regulatory role of IER3 in the body's immune inflammatory response.

[0004] The applicant's previous Chinese patent (authorization number CN112011603B) disclosed the application of IER3 as a biomarker in the early prediction of sepsis. The application showed that the expression level of IER3 in patients who developed sepsis after trauma was significantly higher than that in patients who did not develop sepsis. IER3 can be used as a new biomarker to predict the occurrence of post-traumatic sepsis. Animal experiments found that IER3 expression was increased in various tissues, organs and plasma of septic mice, indicating that IER3 is involved in the regulation of the occurrence and development of sepsis. IER3 knockout can increase the mortality rate of CLP septic mice, aggravate organ dysfunction and systemic inflammation. However, it is unclear whether supplementing IER3 protein has a therapeutic effect on sepsis. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an application of recombinant IER3 protein in the preparation of drugs for treating sepsis. The recombinant IER3 protein can reduce the mortality rate of CLP-positive sepsis mice, decrease intraperitoneal bacterial load, blood and intraperitoneal inflammatory factor levels, and alleviate the degree of tissue and organ damage.

[0006] The technical solution of the present invention is as follows: Application of IER3 recombinant protein in the preparation of drugs for treating sepsis.

[0007] The drug reduces the bacterial load in the peritoneal cavity of patients with CLP sepsis.

[0008] The drug improves LPS-induced sepsis-induced organ damage.

[0009] The organs mentioned are the liver, heart, and lungs.

[0010] The drug is administered by injection at a dosage of 0.5 ug / kg to 50 ug / kg, preferably 5 ug / kg.

[0011] The nucleic acid sequence of the recombinant IER3 protein is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0012] The amino acid sequence of the IER3 recombinant protein is shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] Animal experiments of this invention show that the recombinant IER3 protein, when injected into CLP-infected mice, can reduce the degree of systemic inflammation, reduce the bacterial load, alleviate organ damage, and save mice from death, thus demonstrating its ability to treat sepsis.

[0014] The recombinant IER3 protein described in this invention can effectively treat septic mice, improve their survival rate, reduce intraperitoneal bacterial load, blood and intraperitoneal inflammatory factor levels, and alleviate tissue and organ damage. Attached Figure Description

[0015] Figure 1 A schematic diagram demonstrating that IER3 can effectively treat CLP sepsis mice, in which... Figure 1 A represents the timeline for drug administration and observation in mice, using intravenous injection. IER3 protein was injected via the tail vein 2 hours prior to administration at a dose of 5 μg / kg to establish a CLP sepsis model. The mice were then administered the drug once daily, and their condition and mortality were observed and recorded every 12 hours until the observation endpoint (168 hours). The mice were sacrificed 7 days after model establishment. Figure 1 B is a schematic diagram illustrating the effect of IER3 on the survival rate of normal mice. Figure 1 C is a schematic diagram illustrating the effect of IER3 on the survival rate of CLP septic mice.

[0016] Figure 2 A schematic diagram demonstrating that IER3 can significantly reduce the levels of inflammatory factors in the blood and peritoneum of CLP-secreting mice, wherein, Figure 2 AB is a schematic diagram showing the effect of IER3 on inflammatory factors in plasma and peritoneal lavage fluid of CLP septic mice.

[0017] Figure 3 A schematic diagram demonstrating that IER3 can significantly reduce the bacterial load in the peritoneal cavity of CLP septic mice.

[0018] Figure 4A schematic diagram to demonstrate that IER3 can effectively improve LPS-induced organ damage in septic mice. In this diagram, AC represents changes in liver function (AST), cardiac function (LDH), and lung function (PEFb, TVb), and D represents the microscopic images of lung, liver, and kidney tissues stained with H&E and the corresponding pathological damage scores. Detailed Implementation

[0019] The following describes a preferred embodiment, with reference to the appendix. Figure 1-4 The present invention will be further described below. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0020] The recombinant IER3 protein was obtained by FineTest Biotechnology Co., Ltd. using an E. coli prokaryotic expression system for protein expression, extraction, and purification. The recombinant IER3 protein is the C-terminal extracellular domain of the IER3 protein, with an amino acid sequence of 1-85 amino acids, as shown in SEQ ID NO. 1 and SEQ ID NO. 2 for its nucleotide sequence and SEQ ID NO. 3 and SEQ ID NO. 4 for its amino acid sequence. Because the amino acid sequences of the recombinant IER3 protein in humans and mice are highly conserved, and their experimental results are similar, the mouse amino acid sequence (nucleotide sequence as shown in SEQ ID NO. 1 and amino acid sequence as shown in SEQ ID NO. 3) was used in this application.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available; and all experimental methods used in the following examples are conventional methods unless otherwise specified.

[0022] Example 1: The therapeutic effect of IER3 on sepsis The mice used in this embodiment were all C57BL / 6J specific pathogen-free mice, purchased from Chongqing Enswell Biotechnology Co., Ltd. The mice were divided into a normal control group (n=10), a Saline+CLP group (n=29), and an IER3+CLP group (n=29). The normal control group consisted of mice that were fed under normal conditions without any special treatment.

[0023] The mouse rearing conditions were as follows: room temperature 23-25℃, humidity 40%-60%, day-night cycle every 12 hours, free access to food and water, and a density of 3-5 mice per cage. All experiments used 8-10 week old mice, with an equal number of males and females. Mice were given an acclimatization period of one week with unrestricted food and water before the experiments. All animal experiments were conducted strictly in accordance with the guidelines for laboratory animal care and use.

[0024] During the experiment, 24 hours after CLP modeling, mice in the normal control group exhibited normal activity, dispersed around the cage, had glossy fur, could eat normally, and were sensitive to external stimuli. Mice in the Saline+CLP group huddled in a corner of the cage, had limited activity, bristled fur, significantly reduced eating frequency, white discharge from the eyes, and a weakened response to external stimuli. Mice in the IER3+CLP group showed normal activity in the cage, dispersed around the cage, had glossy fur, could eat normally, had no white discharge from the eyes, and were sensitive to external stimuli. Figure 1 As shown in Figure B, IER3 treatment did not cause mouse death during the 7-day observation period, indicating that the dose of 5 ug / kg used in this example is a safe dose. Figure 4 As shown in Figure C, the mortality rate of LPS group mice within 7 days was 82.76%, which was significantly different from that of the normal control group (P<0.0001); IER3 significantly reduced the mortality rate of CLP septic mice (41.38%) (P=0.002).

[0025] Example 2: Effects of IER3 on the expression of inflammatory factors in CLP septic mice Mice were divided into two groups: the Saline+CLP group (n=10) and the IER3+CLP group (n=10). In this embodiment, the drug was administered via tail vein injection. The IER3+CLP group received a tail vein injection of recombinant IER3 protein 2 hours prior to administration at a dose of 5 μg / kg / day. The Saline+CLP group received a tail vein injection of the same volume of sterile saline (saline) 2 hours prior to administration. A CLP sepsis model was then established. Mice were sacrificed 24 hours after modeling, and peripheral blood and peritoneal lavage fluid were collected. Plasma and peritoneal lavage fluid supernatant were separated, and the levels of cytokines TNF-α, IL-1β, IL-6, and IL-10 in plasma and peritoneal lavage fluid were detected by enzyme-linked immunosorbent assay (ELISA).

[0026] The results are as follows Figure 2 As shown, compared with the Saline+CLP group, IER3 treatment significantly reduced the levels of TNF-α, IL-1β, IL-6 and IL-10 proteins in plasma and peritoneal lavage fluid (P<0.05).

[0027] Example 3: Effects of IER3 on bacterial load and organ damage in CLP septic mice Twenty-four hours after CLP modeling, respiratory function (tidal volume (TVb) and peak expiratory flow (PEFb) in awake mice was assessed using the FinePointe non-invasive detection system. After anesthetizing the mice, the peritoneal cavity was lavaged with pre-cooled PBS, and the peritoneal lavage fluid was collected. Bacterial colony-forming units (CFU) in the peritoneal lavage fluid were counted using the plate spread method. The results are as follows: Figure 3As shown, compared with the Saline+CLP group, IER3 treatment significantly reduced the bacterial load in peritoneal lavage fluid (P=0.008), indicating that IER3 can effectively reduce the bacterial load in the peritoneal cavity of CLP septic mice.

[0028] The mice were then dissected, and blood and plasma were separated. Liver function (AST) and cardiac function (LDH) were measured using a small animal biochemical analyzer. Lung, liver, and kidney tissues were obtained and prepared into sections for hematoxylin-eosin (HE) staining and tissue damage scoring. The results are as follows: Figure 4 As shown, from Figure 4 The AC results showed that, compared with the Saline+CLP group, IER3 treatment significantly reduced liver function AST and cardiac function LDH levels, and increased respiratory function PEFb and TVb levels; from Figure 4 As shown in Figure D, in mouse lung tissue, the Saline+LPS group exhibited disordered alveolar structure, lack of reticular arrangement, significant inflammatory cell infiltration, and alveolar wall thickening; the IER3+CLP group showed restoration of reticular alveolar structure, and improved inflammatory cell infiltration and alveolar wall thickening. In mouse liver tissue, the Saline+CLP group showed irregular arrangement near the central vein, with pathological changes such as cell swelling and inflammatory cell infiltration; the IER3+CLP group showed a restored radial arrangement centered on the central vein, and reduced cell swelling. In mouse kidney tissue, the Saline+CLP group showed no gap between the glomeruli and Bowman's capsule, glomerular swelling, and lymphocyte infiltration; the IER3+CLP group showed restored gap between the glomeruli and Bowman's capsule, and reduced glomerular swelling. The three histopathological damage scores in the IER3-treated groups were also significantly reduced (P<0.01).

[0029] In conclusion, IER3 can effectively improve organ damage in CLP septic mice.

[0030] This invention demonstrates through animal experiments that the recombinant IER3 protein can reduce systemic inflammation, decrease bacterial load, alleviate organ damage, and save mice from death. Therefore, the IER3 protein described in this invention has the ability to treat sepsis.

[0031] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that various variations and modifications can be made without departing from the inventive concept. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations and modifications derived therefrom remain within the scope of protection of this invention.

[0032] sequence list SEQ ID NO.1 human nucleotide Atgtgtcactctcgcagctgccacccgaccatgaccatcctgcaggccccgaccccggccccctccaccatcccgggaccccggcggggctccggtcctgagatcttcaccttcgaccctctcccggagcccgcagcggcccctgccgggcgccccagcgcctctcgcgggcaccgaaagcgcagccgcagggttctctaccctcgagtggtccggcgccagctgccagtcgaggaaccgaacccagccaaaagg SEQ ID NO.2 mouse nucleotide Atgtgccactcgcgcaaccatctccacaccatgactggcctgagggccccttctccagctccctccaccggcccggaactccggcggggctctggtcccgaaattttcaccttcgaccctctcccggagcgggccgtggtgtccaccgcgcgtttgaacacttctcgcgggcaccgaaaacgcagccgaagggtgctctaccctcgagtggtccggcgccagctaccaaccgaggaacccaacattgccaagagg SEQ ID NO.3 human amino acid MCHSRSCHPTMTILQAPTPAPSTIPGPRRGSGPEIFTFDPLPEPAAAPAGRPSASRGHRKRSRRVLYPRVVRRQLPVEEPNPAKR SEQ ID NO.4 mouse amino acid MCHSRNHLHTMTGLRAPSPAPSTGPELRRGSGPEIFTFDPLPERAVVSTARLNTSRGHRKRSRRVLYPRVVRRQLPTEEPNIAKR.

Claims

1. Application of recombinant IER3 protein in the preparation of drugs for treating sepsis.

2. The application according to claim 1, wherein the drug reduces the intraperitoneal bacterial load in CLP sepsis.

3. The application according to claim 1, wherein the drug improves LPS-induced septic organ damage.

4. The application according to claim 3, wherein the organ is the liver, heart, or lung.

5. The application according to claim 1, wherein the drug is administered by injection at a dosage of 0.5 ug / kg to 50 ug / kg.

6. The application according to claim 5, wherein the dosage is 5 μg / kg.

7. The application according to claim 1, wherein the nucleic acid sequence of the recombinant IER3 protein is shown in SEQ ID NO.1 and SEQ ID NO.

2.

8. The application according to claim 1, wherein the amino acid sequence of the recombinant IER3 protein is shown in SEQ ID NO.3 and SEQ ID NO.4.

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