阅读说明：本技术 4-辛基衣康酸在制备治疗脓毒症急性肺损伤药物中的应用 (Application of 4-octyl itaconic acid in preparing medicine for treating acute lung injury caused by sepsis ) 是由 庞庆丰 刘钢 王志强 吴亚先 陈丹 于 2020-12-16 设计创作，主要内容包括：本发明属于脓毒症医药应用领域,涉及4-辛基衣康酸在制备治疗脓毒症急性肺损伤药物中的应用。本发明为制备治疗MRSA引起的急性肺损的药物提供了一个新的靶点,将4-OI应用到脓毒症急性肺损伤相关的药物开发过程中,以制备更好的治疗药物。本发明为目前治疗MRSA引起的急性肺损的药物提供了全新的思路,拓宽了治疗MRSA引起的急性肺损的选择领域,也为该技术领域的发展做出了贡献。(The invention belongs to the field of sepsis medicine application, and relates to application of 4-octyl itaconic acid in preparation of a medicine for treating sepsis acute lung injury. The invention provides a new target point for preparing the medicine for treating acute lung injury caused by MRSA, and the 4-OI is applied to the development process of the medicine related to acute lung injury of sepsis to prepare a better treatment medicine. The invention provides a brand new thought for the existing medicine for treating the acute lung injury caused by the MRSA, widens the selection field for treating the acute lung injury caused by the MRSA, and also makes a contribution to the development of the technical field.)

Application of 4-octyl itaconic acid in preparing medicine for treating acute lung injury caused by sepsis.

Technical Field

The invention belongs to the field of sepsis medicine application, and relates to application of 4-octyl itaconic acid in preparation of a medicine for treating sepsis acute lung injury.

Background

Pneumonia caused by gram-positive bacterial pathogens is a leading cause of Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) worldwide. Staphylococcus aureus (SA, s.aureus) is the most clinically common gram-positive bacterial pathogen. Staphylococcus aureus is the producer of many toxins that collectively contribute to their potential toxicity. Drug-resistant staphylococcus aureus is a hospital or community acquired organism that causes serious infections and is currently a major healthcare problem worldwide. MRSA can cause infectious diseases including bacteremia, osteomyelitis, skin and soft tissue, pleuropneumonia and other infectious diseases. MRSA bacteremia is a clinically important disease caused by MRSA, and can lead to seeding and consequent complications in almost any body part. In MRSA sepsis, the lung is the most common site of infection. Due to resistance and insensitivity to first-line antibiotics and the lack of equally effective alternatives, the lung injury complications associated with MRSA sepsis remain devoid of relevant effective drugs to treat. However, to date, there is no effective MRSA therapeutic drug or strategy.

It is obviously an urgent task to find new drug therapy targets and new therapeutic approaches.

Itaconic acid is an endogenous metabolite that has been shown to have anti-inflammatory and antioxidant effects. However, the use of itaconic acid is limited because its hydrophilic structure does not penetrate cell membranes well. The derivative of itaconic acid, 4-Octyl itaconic acid (4-OI, 4-Octyl Itaconate), has higher fat solubility than itaconic acid and can penetrate cell membrane more easily, playing an important role. No research reports about the effect of 4-OI in preparation of MRSA-resistant acute lung injury exist at present.

Disclosure of Invention

The invention aims to provide a new way for the technical field of the existing preparation of drugs for treating MRSA, and provides the function of 4-OI in the preparation of drugs for treating acute lung injury caused by MRSA.

In order to solve the technical problems, the technical scheme provided by the invention is to apply the 4-OI to the preparation of the medicine for treating the acute lung injury of the MRSA.

Research shows that after mice are injected with MRSA to induce sepsis, the mice are injected with 4-OI to relieve the damage caused by sepsis. The present study found that 4-OI can reduce the mortality of MRSA-induced sepsis; reducing the number of neutrophils in the alveoli and alveolar spaces; reducing expression of Myeloperoxidase (MPO), Malondialdehyde (MDA) in lung tissue; reducing the expression of lung tissue inflammatory factors (IL-1 beta, IL-6 and TNF alpha); increase the expression of antioxidant genes (Nrf2, HO-1, NQO 1); reducing MRSA bacterial load in lung tissue. The results show that the 4-OI can remarkably improve the sepsis acute lung injury caused by the MRSA by inhibiting inflammation and oxidative stress, can be developed as a novel MRSA-resistant medicament, and provides a novel means and way for treating the sepsis lung injury caused by the MRSA.

The invention has the beneficial effects that: 4-OI can reduce inflammation and oxidative stress injury in acute lung injury caused by MRSA sepsis. The invention provides a new target point for preparing the medicine for treating acute lung injury caused by MRSA, and the 4-OI is applied to the development process of the medicine related to acute lung injury of sepsis to prepare a better treatment medicine. The invention provides a brand new thought for the existing medicine for treating the acute lung injury caused by the MRSA, widens the selection field for treating the acute lung injury caused by the MRSA, and also makes a contribution to the development of the technical field.

Drawings

FIG. 1: 4-OI reduced mouse MRSA sepsis alveolar inflammatory cell infiltration and protein exudation, indicated by alveolar lavage fluid cell count, alveolar lavage fluid protein concentration and immunohistochemical staining (MPO), respectively.

FIG. 2: 4-OI improves the MRSA sepsis lung injury condition of the mice, and takes the inflammatory cell infiltration quantity in the alveolar space in HE staining and the lung injury score as indexes.

FIG. 3: the 4-OI relieves the MRSA sepsis lung tissue inflammation of the mouse, and takes the expression of proinflammatory factors (IL-1 beta, IL-6 and TNF alpha) mRNA as an index.

FIG. 4: the 4-OI can relieve MRSA sepsis oxidative stress of mice, and takes the activity of mouse serum Malondialdehyde (MDA) and the expression of lung tissue antioxidant gene (Nrf2, HO-1, NQO1) mRNA as indexes.

FIG. 5: the 4-OI reduces the mice from being invaded and damaged by MRSA, and takes the survival rate of the mice and the bacterial load of lung tissues as indexes.

Detailed Description

The invention is further described in the following description with reference to the figures and the examples, which are not intended to limit the invention in any way, except as specifically stated, reagents, methods and apparatus used in the practice of the invention are conventional in the art.

Example 1 establishment of MRSA sepsis model

Wild male C57 mice were randomly divided into three groups:

(1) saline group (saline): normal vein, mice were injected with a single bolus of saline in the abdominal cavity.

(2) MRSA mouse group (MRSA): a single intraperitoneal injection of MRSA (3x 108).

(3) MRSA +4-OI group (MRSA + 4-OI): 4-OI was injected intraperitoneally in a single injection, and 1 hour later MRSA was injected intraperitoneally.

The preparation method of the MRSA sepsis model comprises the following steps: wild male C57 mice were used to simulate a sepsis model by intraperitoneal injection of MRSA. The specific method is that the wild male C57 mouse is subjected to intraperitoneal single injection of MRSA (3x 10)⁸/only), normal saline (saline) was injected as a control.

Example 2RNA extraction and Real-time PCR

Lung tissue RNA was extracted by trizol (life technologies) method, and the specific procedures were performed as described in the specification. And detecting the content of RNA by using an ultraviolet spectrophotometer, and detecting the purity of the RNA according to the ratio of the absorbance at 260nm to the absorbance at 280 nm. The OD260/OD280 ratio of pure RNA should be close to 2.0 (reliable range between 1.9-2.1). Mu.g of total RNA was added with 2. mu.l of 5 XPrimeScript RT Master Mix (Takara), and the amount of the mixture was adjusted to 20. mu.l with deionized water for reverse transcription to synthesize cDNA; real-time PCR was performed using the set-up program with the primers listed below.

Mouse-HO-1:

Forward CAAGCCGAGAATGCTGAGTTCATG

Reverse GCAAGGGATGATTTCCTGCCAG

Mouse-Nrf2:

Forward TTCAGCCAGCCCAGCACATC

Reverse CGTAGCCGAAGAAACCTCATTGTC

Mouse-NQO-1:

Forward TTCTGTGGCTTCCAGGTCTT

Reverse AGGCTGCTTGGAGCAAAATA

Mouse-TNF-α:

Forward CATGAGCACAGAAAGCATGATCCG

Reverse AGCAGGAATGAGAAGAGGCTGAG

Mouse-IL-1β:

Forward GCAACTGTTCCTGAACTCAACT

Reverse ATCTTTTGGGGTCCGTCAACT

Mouse-IL-6:

Forward ACAACCACGGCCTTCCCTACTT

Reverse CACGATTTCCCAGAGAACATGTG

Mouse-GADPH:

Forward AGGTCGGTGTGAACGGATTTG

Reverse TGTAGACCATGTAGTTGAGGTCA

Example 3 protein and cellular acquisition of alveolar lavage fluid

Lung tissue was irrigated 3 times with 0.5mL sterile PBS solution and the solution was centrifuged at 1,000g for 10 minutes at 4 ℃. Cell-free supernatants were collected for total protein analysis using the BCA kit (Beyotime), and the cell pellets were then resuspended in PBS (100 μ Ι). We used flow cytometry (BD C6) to detect the number of resuspended cells.

Example 4 Lung serum Myeloperoxidase (MPO) and Malondialdehyde (MDA) Activity

The MDA content is an important parameter reflecting the potential oxidation resistance of an organism, can reflect the lipid peroxidation rate and strength of the organism, and can also indirectly reflect the tissue peroxidation damage degree. MPO and MDA activities in serum were measured using myeloperoxidase and malondialdehyde measurement kit (Nanjing Kangji Co.).

Example 5 pathological damage detection of Lung tissue

The lung tissue of a mouse is soaked in 4% paraformaldehyde for fixation, washed, dehydrated by gradient alcohol with the concentration from 70% to 100%, and then the tissue is transparent by 100% dimethylbenzene after dehydration, and then the tissue is placed in a wax jar for wax dipping, embedded and sliced by paraffin. After staining with HE staining kit (Nanjing construction Co.), photographs were taken by microscopic observation. Sections were antigen repaired using sodium citrate solution (Solarbio, beijing, china). Sections were blocked with avidin and biotin in sequence and then incubated with 1/1000 diluted anti-MPO antibody (Abcam) overnight at 4 ℃. After washing the primary antibody with PBS, the sections were incubated with biotinylated IgG for 30 minutes at 37 ℃. Staining was performed using the SABC (streptavidin-biotin complex) method, and photographs were taken by microscopic observation using diaminobenzidine as a staining substrate.

Example 6 mouse survival assay

Lethal dose of MRSA [ 3X10 ] by intraperitoneal injection⁸Colony Forming Unit (CFU)/mouse]All mice were challenged and treated with 4-OI (25mg/kg) intraperitoneally 1 hour earlier. Survival status was recorded for the next 7 days.

Example 7 pulmonary tissue bacterial load detection

Lungs from each group of mice were homogenized in 1ml sterile 0.9% NaCl. After serial 10-fold dilution in sterile 0.9% NaCl, lung tissue homogenates of three concentrations were seeded in 100. mu.l volumes on Luria-Bertani agar plates, respectively. After 18 hours of incubation at 37 ℃, CFU were counted and bacterial load was expressed as Log 10(CFU/g) per organ.

The experimental results are shown in the attached figures 1 to 5:

(1)4-OI reduces MRSA sepsis alveolar inflammatory cell infiltration and protein exudation in mice

MRSA infection caused the migration of inflammatory cells in mouse alveoli, so that the number of cells in BALF was significantly higher in MRSA group than in control group, while 4-OI treatment reduced the infiltration of inflammatory cells; the BALF protein content (an indicator of pulmonary vascular permeability) was significantly higher in the MRSA group than in the control group, and MRSA-induced increase in total protein was prevented by 4-OI intervention in the BARSA (see figure one).

Data are expressed as mean ± standard error (mean ± SE) with 5 samples per group. P < 0.05 is statistically different. Comparison with Saline, P<0.01; comparison with Saline, P<0.001；^#In contrast to MRSA, P<0.05。

(2)4-OI improves MRSA lung injury condition of mice

After the MRSA stimulation, the mouse alveolar volume is obviously increased, the number of inflammatory cells is increased, the alveolar wall is thickened, and the 4-OI can improve the change of the cases, which is shown in the fact that the alveolar structure damage is repaired, the inflammatory cells are reduced, and the 4-OI can improve the lung tissue damage of the MRSA mouse (shown in the figure II).

Data are expressed as mean ± standard error (mean ± SE) with 5 samples per group. P < 0.05 is statistically different. Comparison with Saline, P<0.001；^##In contrast to MRSA, P<0.01。

(3)4-OI relieves MRSA sepsis lung tissue inflammation of mice

The qPCR results showed that MRSA infection significantly increased the mRNA expression of TNF-. alpha.IL-1. beta. and IL-6 in lung tissue, while 4-OI treatment correspondingly decreased their mRNA expression (see FIG. three).

Data are expressed as mean ± standard error (mean ± SE) with 5 samples per group. P < 0.05 is statistically different. Comparison with Saline, P<0.05; comparison with Saline, P<0.01; comparison with Saline, P<0.001；^#In contrast to MRSA, P<0.05。

(4)4-OI improves MRSA mouse oxidative stress

The MDA content is an important parameter reflecting the potential oxidation resistance of an organism, can reflect the lipid peroxidation rate and strength of the organism, and can also indirectly reflect the tissue peroxidation damage degree. The MDA activity in the mouse serum induced by the MRSA stimulation is obviously improved, the expression of antioxidant genes (Nrf2, HO-1 and NQO1) in lung tissues is obviously reduced, the activity of MDA is obviously reduced by 4-OI, and the expression of the antioxidant genes is promoted (see figure four).

Data are expressed as mean ± standard error (mean ± SE) with 5 samples per group. P < 0.05 is statistically different. Comparison with Saline, P<0.05; comparison with Saline, P<0.01；^#In contrast to MRSA, P<0.05；^###In contrast to MRSA, P<0.001。

(5)4-OI reduction of mice from invasive injury by MRSA

Survival rate is the most important index reflecting the damage of MRSA to the organism. The MRSA stimulates the mice to cause the survival rate of the mice to be reduced, and the survival time and the survival rate of the mice are obviously improved after the 4-OI is applied. Colonization of MRSA is the most important cause of damage to the body by MRSA, and the bacterial load in lung tissue reflects the invasive damage of MRSA to the body. MRSA stimulation of mice resulted in an increased load of lung tissue bacteria, and 4-OI could significantly reduce lung tissue bacterial load (see figure five).

Data are expressed as mean ± standard error (mean ± SE) with 5 samples per group. P < 0.05 is statistically different. Comparison with Saline, P<0.001；^##In contrast to MRSA, P<0.01。