Application of MSR1 gene as target spot in screening of sepsis prevention and treatment drugs

Abstract

The invention relates to the technical field of biology, in particular to application of an MSR1 gene as a target spot to screening of sepsis prevention and treatment drugs. The invention aims to provide an application of a preparation with MSR1 gene expression knocked out in preparation of a medicine for treating immune function disorder and excessive inflammation caused by sepsis, and the immune function disorder and excessive inflammation reaction caused by sepsis can be regulated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of MSR1 gene as a target in screening drugs for preventing and treating sepsis. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by the body's dysregulated response to infection. It is a serious disease with a high mortality rate. Every year, 48.9 million people suffer from sepsis worldwide, and the number of deaths from sepsis is as high as 11 million. Sepsis is an important cause of mortality and morbidity in the global population. Sepsis can rapidly activate the body's innate and adaptive immune systems, causing severe systemic inflammatory responses and immune hyperfunction, followed by long-term immune suppression due to the massive consumption of cytokines and immune cells. This is the key mechanism for repeated infections and multiple organ dysfunction syndrome (MODS) in patients with sepsis.

[0003] Currently, sepsis, especially severe sepsis and septic shock, often relies on anti-infection and supportive treatments, which are passive symptomatic supportive treatments that only treat the symptoms but not the root cause. In addition, with the increase in drug resistance rates, the effectiveness is limited when facing some refractory septic shock, which makes the treatment of sepsis more difficult.

[0004] MSR1, also known as scavenger receptor A (SR-A) or cluster of differentiation 204 (CD204), was first described by Brownn and Goldstein in 1979. MSR1 is mainly expressed on macrophages and DCs, and is also present on the surface of lymphocytes. It may be involved in the pathogenesis of asthma and chronic obstructive pulmonary disease. Since macrophages mainly clear pathogens through phagocytosis and cytokine production, MSR1 plays an important role in the process of clearing foreign pathogens. LPS is an important endogenous toxin in sepsis, and its main ligand is TLR4. The synergistic effect of MSR1 and TLR4 can activate the phagocytosis of Gram-negative bacteria and promote the synthesis of inflammatory cytokines by activating NF-κB and downstream signaling pathways that enter the cell nucleus and bind to DNA. Although MSR1 is active throughout the disease spectrum, it can trigger different signaling pathways depending on the recognition receptor, thereby exerting different or even opposite biological functions. Therefore, studying the different functions and regulatory mechanisms of MSR1 is of great significance for using it to treat specific diseases. Summary of the invention

[0005] In order to solve the problems in the above-mentioned background technology, the present invention provides the use of MSR1 gene as a target in screening drugs for preventing and treating sepsis, so as to improve the survival rate of mice with sepsis.

[0006] The purpose of the present application is to provide an application of a preparation for knocking out the expression of the MSR1 gene in the preparation of a drug for treating immune dysfunction and excessive inflammation caused by sepsis, wherein the drug is gRNA-B1 or gRNA-B2 for knocking out the expression of the MSR1 gene; the sequence of the gRNA-B1 is: GGCCTTTGCACGTGAAGAGG-AGG, as shown in SAQ ID NO.1; the sequence of the gRNA-B2 is: ACTTGGGGAAATAAGGTAC-AGG, as shown in SAQ ID NO.2.

[0007] Furthermore, agents that inhibit or knock out the expression of the MSR1 gene can inhibit the decrease in the number of CD3+T cells and CD3+CD4+T cells in septic mice.

[0008] Furthermore, the preparation that inhibits or knocks out the expression of the MSR1 gene can inhibit the increase in the level of pro-inflammatory cytokines in the serum of septic mice, and the markers of the pro-inflammatory cytokines are IFN-γ, IL-2, IL-4 and IL-12.

[0009] The present application regulates the immune response of refractory sepsis by knocking out the expression of MSR1 gene, thereby helping to alleviate immune dysfunction and excessive inflammatory response.

[0010] In summary, the beneficial effects of the present invention are as follows: knocking out the MSR1 gene can regulate the immune dysfunction and excessive inflammatory response caused by sepsis, improve the reduction in the number of T cells and cytokine storm caused by sepsis, maintain the function of important organs, reduce tissue damage, and improve the survival rate of septic mice.

[0011] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :Effect of conditional knockout of MSR1 gene on the proportion of CD3+T cells in peripheral blood of septic mice;

[0013] Figure 2 :Effect of conditional knockout of MSR1 gene on the proportion of CD3+CD4+T cells in peripheral blood of septic mice;

[0014] Figure 3:Effect of conditional knockout of MSR1 gene on the proportion of CD3+Foxp3+Tregs in peripheral blood of septic mice;

[0015] Figure 4 :Effects of conditional knockout of MSR1 gene on the levels of inflammatory cytokines in the serum of septic mice;

[0016] Figure 5 :Effects of conditional knockout of MSR1 gene on damage to important organs and tissues in septic mice;

[0017] Figure 6 : Detect Msr1 fl / fl Mouse and Cd11c cre Msr1 fl / fl Levels of important organ damage markers in the serum of mice after CLP modeling and MSC-Exo treatment;

[0018] Figure 7 :Effect of conditional knockout of MSR1 gene on 7-day survival rate of septic mice after CLP surgery. DETAILED DESCRIPTION

[0019] In order to make the content of the present invention more clearly understood, the present invention is further described below based on specific embodiments in conjunction with the accompanying drawings.

[0020] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0021] WT male C57BL / 6J mice were used in the following experiments, each weighing about 20-25 g, provided by Huafukang Biotechnology Co., Ltd. The mice were kept in an SPF animal room with an ambient temperature of 22°C and an ambient humidity of 65%, with good ventilation, drinking water at any time, and adaptive feeding for 1 week.

[0022] Before the experiment, 24 normal Msr1 fl / fl Mice and 24 mice with gRNA-B1 or gRNA-B2 knocking out Msr1 gene expression; the sequence of gRNA-B1 is: GGCCTTTGCACGTGAAGAGG-AGG; the sequence of gRNA-B2 is: ACTTGGGGAAATAAGGTAC-AGG. Cd11c cre Msr1 fl / flThe mice were randomly divided into 3 groups according to the random number table method, with 8 mice in each group. One group was opened and the cecum was turned over before closing the abdomen, which was the sham operation group (Sham); one group was subjected to CLP modeling, which was the model group (CLP); and one group was injected with MSC-exo through the tail vein immediately after the CLP mouse sepsis model was established, with a dose of 100 μg / mouse, which was the treatment group (CLP+MSC-exo). The sham operation group, model group, and treatment group were sampled 24 hours after modeling, and the eyeballs were removed after anesthesia to collect blood. After blood collection, the mice were killed by cervical dislocation for sampling.

[0023] Among them, the steps of CLP to establish a mouse sepsis model are as follows:

[0024] S1, mice were fasted for 12 h before surgery, but were not allowed to drink water. Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate. The anesthetic dose was calculated as 10 mL / kg. After anesthesia, the mice were fixed on the operating board in the supine position.

[0025] S2, disinfect the mouse abdominal skin with iodine, and make incisions layer by layer along the midline of the abdomen with a length of 1 cm;

[0026] S3, after opening the abdomen, quickly find the cecum, use sterile No. 4 thread to ligate it 1.0 cm away from the end of the cecum, use a 22G needle to penetrate the cecum once at the distal end of the ligature, squeeze the ligated intestine to make some intestinal contents overflow, return the cecum to its original position in the abdominal cavity, suture it layer by layer, and close the abdomen;

[0027] S4, inject 1 mL of 0.9% saline subcutaneously behind the neck of the mice, transfer them to the feeding cage, and take measures to keep them warm. After waking up, the mice can eat and drink freely.

[0028] Experiment 1: Analysis of the proportion of immune cells in mouse peripheral blood by flow cytometry

[0029] (1) Experimental procedures

[0030] S1, peripheral blood mononuclear cells of each group of mice were isolated and counted, and collected in flow tubes at 3×105 cells / tube;

[0031] S2, add 1 mL of sterile phosphate buffered saline (PBS) to each tube, then place the flow tube in a centrifuge and centrifuge at room temperature at a speed of 1500 rpm for 5 min. Discard the supernatant and resuspend with 1 mL of PBS and centrifuge again. Repeat twice, and finally resuspend the cell pellet with 200 μL PBS.

[0032] S3, add 1 μL CD3 flow cytometry antibody and 1 μL CD4 flow cytometry antibody to each tube, set up blank tube, CD3 single stain tube and CD4 single stain tube at the same time, incubate at room temperature in the dark for 30 min after adding antibodies;

[0033] S4, after the incubation is completed, 1 mL of PBS is added to each tube for resuspending, and then the flow tube is placed in a centrifuge for centrifugation at room temperature, with a centrifugal speed of 1500 rpm and a centrifugal time of 5 min;

[0034] S5, prepare FOXP3 fixation / permeabilization buffer (1X) and FOXP3 permeabilization buffer (1X); mix FOXP3 fixation / permeabilization buffer (4X) and fixation / permeabilization diluent at a ratio of 1:3 to obtain FOXP3 fixation / permeabilization buffer (1X); mix FOXP3 permeabilization buffer (10X) and deionized water at a ratio of 1:9 to obtain FOXP3 permeabilization buffer (1X);

[0035] S6, after the flow tubes were centrifuged in step S4, the supernatant was discarded, and 500 μL of OXP3 fixation / permeabilization buffer (1X) was added to each flow tube and incubated at 4°C for 14 h;

[0036] S7, after the membrane incubation is completed, wash the cells twice with membrane permeabilization buffer, resuspend the cell pellet with 200 μL membrane permeabilization buffer, blow evenly, add Foxp3 flow cytometry antibody at 1.5 μL / tube, and incubate at room temperature in the dark for 1 h;

[0037] S8. After incubation, resuspend the cells with 1 mL of membrane permeabilization buffer, then place the flow tube in a centrifuge and centrifuge at room temperature at a speed of 1500 rpm for 5 min. Discard the supernatant and resuspend the cells with 300 μL of PBS for testing. Use FlowJo V 10.0 software to analyze the proportions of CD3+T cells, CD3+CD4+T cells, and CD4+Foxp3+Tregs cells in the peripheral blood of mice in different groups.

[0038] (2) Experimental results

[0039] like Figure 1 , Figure 2 and Figure 3 As shown, compared with the sham operation group, Msr1 fl / fl Mouse and Cd11c cre Msr1 fl / fl The proportion of CD3+T cells in PBMCs in mouse peripheral blood (Msr1 fl / fl :P<0.0001;Cd11ccreMsr1 fl / fl :P<0.0001) and the proportion of CD3+CD4+T cells to CD3+T cells (Msr1 fl / fl :P<0.0001;Cd11c cre Msr1 fl / fl:P<0.01) were significantly decreased, but Cd11c cre Msr1 fl / fl Levels in septic mice were significantly higher than Msr1 fl / fl Septic mice (CD3+T: P<0.0001; CD3+CD4+T: P<0.0001).

[0040] Msr1 after MSC-Exo treatment fl / fl The proportion of CD3+T cells (P<0.0001) and CD3+CD4+T cells (P<0.0001) in the peripheral blood of mice were significantly higher than those in the sepsis group. cre Msr1 fl / fl The proportion of CD3+T cells in the treatment group mice increased significantly (P<0.0001), while the proportion of CD3+CD4+T cells did not change significantly.

[0041] Msr1 fl / fl Mouse and Cd11c cre Msr1 fl / fl The proportion of Treg cells in the peripheral blood of mice after CLP modeling increased significantly (Msr1 fl / fl :P<0.001;Cd11c cre Msr1 fl / fl :P<0.0001), Cd11c cre Msr1 fl / f The levels in mice were still significantly higher than those in Msr1 fl / fl After MSC-Exo treatment, Msr1 fl / fl Mouse and Cd11c cre Msr1 fl / fl The proportion of Treg in the peripheral blood of mice increased significantly (Msr1 fl / fl :P<0.0001;Cd11c cre Msr1 fl / fl :P<0.001).

[0042] The results showed that conditional knockout of the MSR1 gene could improve the reduction in the number of CD3+T cells and CD3+CD4+T cells caused by sepsis, indicating that conditional knockout of the MSR1 gene can alleviate the immune dysfunction caused by sepsis.

[0043] Experiment 2: ELISA to detect cytokine levels

[0044] (1) Experimental procedures

[0045] S1, sample processing method: remove the eyeballs of each group of mice to collect blood, let the blood coagulate naturally at room temperature for 30 minutes, then place the blood in a centrifuge and centrifuge at room temperature, the centrifugal speed is 3000rpm, the centrifugal time is 20 minutes, and then collect the serum and retain it. Detect the cytokine levels in the cell culture supernatant or serum using an ELISA kit;

[0046] S2, take out the pre-coated and sealed 96-well plate, equilibrate to room temperature, add standard and specimen universal diluent to the blank wells, add 100 μL of diluted cell culture supernatant or serum from each group to the remaining wells, seal the reaction wells with sealing tape, place in a 37°C incubator, and incubate in the dark for 90 min;

[0047] S3, after the incubation is completed, the reaction solution in the reaction well is aspirated, and the biotinylated antibody working solution is prepared 20 minutes in advance, and 100 μL of the biotin-labeled antibody diluted at 1:100 is added to the reaction well, and then the reaction well is sealed with sealing tape, and the well is placed in a 37°C incubator and incubated for 60 minutes in the dark;

[0048] S4, wash the plate with washing buffer, soak for 1 min each time, repeat 5 times;

[0049] S5, after washing the well plate, add 100 μL of diluted avidin labeled with peroxidase into the reaction well, where the dilution ratio of avidin is 1:100, then seal the reaction well with sealing tape, place in a 37°C incubator, and incubate in the dark for 30 min;

[0050] S6, wash the plate with washing buffer, soak for 90 seconds each time, repeat 5 times;

[0051] S7, add 90 μL of chromogenic substrate to the reaction well, place in a 37°C incubator, and incubate in the dark for 20 min;

[0052] S8. After incubation, add 100 μL of reaction stop solution to the reaction wells and mix gently. Then, within 3 minutes of adding the reaction stop solution, place the well plate in a microplate reader to detect the OD value of each well at a wavelength of 450 nm, and calculate the levels of pro-inflammatory cytokines such as IFN-γ, IL-2, IL-4 and IL-12, as well as the concentrations of anti-inflammatory cytokines such as IL-10 and TGF-β.

[0053] (2) Experimental results

[0054] The results are as follows Figure 4 As shown, compared with the sham-operated group, Msr1 fl / fl Mouse and Cd11c cre Msr1 fl / flAfter CLP modeling, the pro-inflammatory cytokines (IL-2, IL-4, IL-12, and IFN-γ) and anti-inflammatory cytokines (TGF-β) in the serum of mice were significantly upregulated. After MSC-Exo treatment, Msr1 fl / fl Mouse and Cd11c cre Msr1 fl / fl The levels of pro-inflammatory cytokines in the serum of mice were significantly reduced, while the levels of anti-inflammatory cytokines were significantly increased.

[0055] In addition, Cd11c cre Msr1 fl / fl The levels of proinflammatory cytokines such as IFN-γ (53.86682316 vs 123.3084397, P < 0.0001), IL-4 (76.79720214 vs 140.3172192, P < 0.0001) and IL-12 (77.72535024 vs 123.715057, P < 0.05) in mice 24 hours after CLP modeling were significantly lower than those in Msr1 fl / fl The level of anti-inflammatory cytokine IL-10 (99.3495874 vs 204.8831152, P < 0.01) was also significantly lower than that of Msr1 fl / fl Septic mice.

[0056] The results showed that conditional knockout of the MSR1 gene could effectively reduce the levels of pro-inflammatory cytokines such as IFN-γ, IL-2, IL-4 and IL-12 in the serum of septic mice, while the levels of anti-inflammatory cytokines such as IL-10 and TGF-β did not show obvious compensatory increases.

[0057] Experiment 3: Hematoxylin and eosin (HE) staining to assess tissue damage

[0058] (1) Experimental procedures

[0059] S1, after the mice in each group were killed, the heart, liver, lung, and kidney were obtained and fixed in 4% paraformaldehyde;

[0060] S2, gradient dehydration, the order is 75% alcohol → 85% alcohol → 95% alcohol → 100% alcohol;

[0061] S3, sectioned after paraffin embedding, section thickness approximately 4 μm;

[0062] S4, dewaxing the paraffin sections until they are hydrated, in the following order: dewaxing with environmentally friendly dewaxing solution I for 20 min → dewaxing with environmentally friendly dewaxing solution II for 20 min → dewaxing with anhydrous ethanol I for 5 min → dewaxing with anhydrous ethanol II for 5 min → dewaxing with 75% alcohol for 5 min, and then rinsing with running water after completion;

[0063] S5, after dewaxing, use hematoxylin staining solution to stain for 4 minutes, and rinse the slices with tap water after staining; use differentiation solution for differentiation, and rinse the slices with tap water after completion; use bluing solution to blue the slices, and rinse the slices with tap water after completion;

[0064] S6, dehydrate the slices in a gradient manner, in the order of 85% alcohol dehydration for 5 min → 95% alcohol dehydration for 5 min, and then place them in HE staining solution for 5 min;

[0065] S7, after staining, the sections were immersed in the following solutions and operated in order: ethanol I for 5 min → ethanol II for 5 min → ethanol III for 5 min → xylene I for 5 min → xylene II for 5 min until transparent, and then sealed with neutral gum;

[0066] S8, after sealing, observe the pathological morphology of each organ under a microscope and collect images.

[0067] (2) Experimental results

[0068] The results are as follows Figure 5 As shown, sepsis induces Msr1 fl / fl The mouse lung, heart, liver and kidney were damaged, but Cd11c cre Msr1 fl / fl There was no obvious tissue damage in these organs after CLP modeling in mice.

[0069] The results showed that conditional knockout of the MSR1 gene could significantly improve tissue damage caused by sepsis.

[0070] Experiment 3: Biochemical index test

[0071] (1) Experimental procedures

[0072] Blood was collected from the eyeballs of each group of mice and allowed to coagulate naturally at room temperature for 30 minutes. The blood was then placed in a centrifuge and centrifuged at room temperature at a speed of 3000 rpm for 20 minutes. The serum was then collected and stored. After configuring the working reagents and setting the parameters of the fully automatic biochemical analyzer, the sample was loaded at a volume of 100 μL background serum + 10 μL / index. The fully automatic biochemical analyzer automatically determined and detected Msr1 fl / fl Mouse and Cd11c cre Msr1 fl / fl Levels of important organ damage markers in the serum of mice after CLP modeling and MSC-Exo treatment.

[0073] (2) Experimental results

[0074] like Figure 6 As shown, compared with the sham-operated group, Msr1 fl / fl The levels of organ damage markers such as ALT (P < 0.0001), AST (P < 0.001), BUN (P < 0.001), CREA (P < 0.0001) and CK (P < 0.001) were significantly increased in mice after CLP modeling, and these indicators were significantly downregulated after MSC-Exo treatment (ALT: P < 0.01; AST: P < 0.05; BUN: P < 0.05; CREA: P < 0.01; CK: P < 0.01). fl / fl Compared with septic mice, Cd11c cre Msr1 fl / fl The above indicators were significantly decreased in mice after CLP modeling (ALT: P < 0.01; AST: P < 0.01; BUN: P < 0.01; CREA: P < 0.01; CK: P < 0.05).

[0075] The results showed that conditional knockout of the MSR1 gene could significantly protect the functions of important organs.

[0076] Experiment 4: Statistical analysis of mouse survival in each group

[0077] The Kaplan-Meier method was used for survival analysis of each group of mice, and the Log-rank (Mantel-Cox) Test was used to calculate statistical differences.

[0078] like Figure 7 As shown, Msr1 fl / fl The 7-day survival rates of mice in the sham operation group, sepsis group and treatment group were 100%, 20% and 70%, respectively. There was a significant difference in the survival rates between the sepsis group and the treatment group (P<0.05). cre Msr1 fl / fl The 7-day survival rates of mice in the sham operation group, sepsis group, and treatment group were 100%, 60%, and 80%, respectively. cre Msr1 fl / fl Sepsis mice and Msr1 fl / fl There was a significant difference in the survival rate of mice with sepsis (P<0.05).

[0079] The above experiments were performed using Graphpad Prism 9.0 software for statistical graph drawing and data statistical analysis. The quantitative data were expressed as mean ± standard deviation (x ± s). When the data of each group conformed to normal distribution and variance was equal, ANOVA was used for multiple group analysis, and Tukey post hoc test was used for pairwise comparison. P < 0.05 was considered to be significantly different, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The experimental results were repeated more than three times.

[0080] The embodiments described above are only preferred implementation modes of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantial changes and modifications made by those skilled in the art on the basis of the invention shall fall within the protection scope of the present invention.

Claims

1. Use of a preparation for knocking out the expression of the MSR1 gene in the preparation of a drug for treating immune dysfunction and excessive inflammation caused by sepsis, wherein the drug is gRNA-B1 or gRNA-B2 for knocking out the expression of the MSR1 gene; the sequence of the gRNA-B1 is: GGCCTTTGCACGTGAA GAGG-AGG; the sequence of the gRNA-B2 is: ACTTGGGGAAATAAGGTAC-AG G.

2. The use according to claim 1, characterized in that: Preparations that inhibit or knock out the expression of the MSR1 gene can inhibit the decrease in the number of CD3+T cells and CD3+CD4+T cells in septic mice.

3. The use according to claim 1, characterized in that: The preparation for inhibiting or knocking out the expression of MSR1 gene can inhibit the increase of the level of pro-inflammatory cytokines in the serum of septic mice, and the markers of the pro-inflammatory cytokines are IFN-γ, IL-2, IL-4 and IL-12.

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