Preparation method and application of escherichia coli bacterial wall carbon dots

Abstract

The invention discloses a preparation method and application of escherichia coli bacterial wall carbon dots, and relates to a nanoscale carbon material.The preparation method comprises the steps that escherichia coli bacterial walls collected after competent cells of escherichia coli DH5 alpha with a special biological structure are broken and centrifuged serve as raw materials, and the escherichia coli bacterial wall carbon dots are obtained after heating reaction, filtration, dialysis and drying. Preparing escherichia coli bacterial wall carbon dots; the application of the escherichia coli bacterial wall carbon dots provides a nano treatment means for treatment of sepsis, and comprises the following steps: injecting 0.5-5mg / kg escherichia coli bacterial wall carbon dots into an abdominal cavity to improve inflammatory response and organ injury caused by sepsis; the escherichia coli wall carbon dots with the concentration of 250-500 mu g / mL and LPS are competitively combined with LBP protein, and TLR4 is promoted to be degraded through lysosome; the RAW264.7 cells are treated by using the escherichia coli bacterial wall carbon dots with the concentration of 250-1000 [mu] g / mL, so that mitochondrial DNA leakage is reduced, and excessive activation of a cGAS-STING pathway is inhibited. According to the invention, the technical blank that in the prior art, a specific structure of a microorganism, such as a cell wall, is not used as a carbon dot precursor to prepare the carbon dot is filled up, and the defect that in the prior art, a sepsis treatment strategy cannot achieve a satisfactory treatment effect is overcome.

Description

Technical Field

[0001] The technical solution of the present invention relates to nano-scale carbon materials, specifically a preparation method of Escherichia coli bacterial wall carbon dots and application thereof. Background Art

[0002] Carbon dots (CDs) are a type of fluorescent nanoparticles that have attracted much attention in the field of nanomaterials in recent years. They are widely used in bioimaging, drug delivery, sensors, and optoelectronic devices due to their small particle size, high optical stability, adjustable fluorescence, excellent biocompatibility, and low toxicity. Existing carbon dot preparation technologies are mainly concentrated in chemical and physical methods, including hydrothermal method, laser ablation method, etc. These methods usually use simple molecules or polymers as precursor materials during the synthesis process. Although the preparation technology of the above methods is relatively mature, researchers have been exploring new precursor materials for the preparation of carbon dots in order to develop carbon dots with specific functions and higher efficiency. Most of the existing publicly reported carbon dot preparation technology research still focuses on the use of chemical reagents or the preparation of carbon dots from natural plant extracts. Although there are a few studies that use microorganisms as a whole as raw materials for carbon dot preparation, there are no reports on the use of microorganisms and their specific structures, such as cell walls, as carbon dot precursors to prepare carbon dots. Escherichia coli is a model bacteria widely studied and applied in microbiology. Its cell wall is mainly composed of lipopolysaccharide and peptidoglycan. However, there has been no report on the use of Escherichia coli cell wall to prepare carbon dots.

[0003] Sepsis is a systemic inflammatory response syndrome caused by infection. Its pathological mechanism is complex and involves the interaction between the host immune system and pathogens. In the early stages of sepsis, pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS), trigger immune responses by binding to pattern recognition receptors (PRRs) on the surface of host cells. The loss of control of this process will lead to inflammatory cytokine storms and multi-organ dysfunction, thus threatening the patient's life. Although existing sepsis treatment strategies include antibiotic therapy, anti-inflammatory drugs and supportive therapy, due to the emergence of multidrug-resistant strains and the complexity of the pathological mechanism of sepsis, existing technical therapies cannot achieve satisfactory therapeutic effects, and new sepsis treatment methods are urgently needed. Summary of the invention

[0004] The technical problem to be solved by the present invention is: to provide a preparation method of Escherichia coli bacterial wall carbon dots and application thereof, wherein the preparation method of Escherichia coli bacterial wall carbon dots uses Escherichia coli bacterial wall with a special biological structure as a raw material to prepare a kind of Escherichia coli bacterial wall carbon dots; the application of the Escherichia coli bacterial wall carbon dots provides a new nano-therapy means for the treatment of sepsis, fills the technical gap in the prior art that the specific structure of microorganisms such as cell walls are not used as carbon dot precursors to prepare carbon dots, and overcomes the defect that the treatment strategy for sepsis in the prior art cannot achieve a satisfactory therapeutic effect.

[0005] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing carbon dots on the bacterial wall of Escherichia coli, the specific steps are as follows: In the first step, take 10 μL of DH5α competent cells from the -80°C refrigerator and place on ice. Operate in a clean bench and take the 10 μL DH5α competent cells and inoculate them in 200 mL In LB medium, culture at 37° C. for 8 to 16 hours until the logarithmic growth phase, then collect the LB medium, set the centrifuge to 5000 g, centrifuge the collected LB medium for 10 seconds, add 10 mL of ultrapure water to the collected Escherichia coli DH5α competent cells, use an ultrasonic disruptor to perform ultrasonic disruption at an energy setting of 10%, stop for 10 seconds after each ultrasonic disruption for 10 seconds, and the total ultrasonic time is 1.5 hours, and all the obtained Escherichia coli DH5α competent cells are disrupted, then set the centrifuge to 8000 g, and centrifuge for 20 seconds until the Escherichia coli cell wall and cytoplasm components are separated, then wash the collected Escherichia coli cell wall 3 times with 4° C. precooled PBS detergent until impurities in the Escherichia coli and incompletely lysed Escherichia coli are removed, then set the centrifuge to 8000 g, centrifuge the washed Escherichia coli cell wall for 20 seconds, and collect all the washed Escherichia coli cell wall precipitates; In the second step, the Escherichia coli cell wall precipitate collected in the first step is immersed in 20 mL of deionized water, and then placed in a high-pressure reactor lined with polytetrafluoroethylene, and heated at 180°C to 220°C for 8 to 16 hours. After the system is naturally cooled to room temperature, the resulting liquid is filtered through a filter membrane with a pore size of 0.22 μm, and then the filtrate is dialyzed for 24 hours using a dialysis membrane with a molecular weight cutoff of 10 kDa to obtain purified Escherichia coli cell wall carbon dots, whose English abbreviation is ECW-CDs. Finally, the Escherichia coli cell wall carbon dot product is dried to a powder by freeze-drying for later use.

[0006] In the above-mentioned method for preparing Escherichia coli cell wall carbon dots, the first step of "culturing at 37°C for 8 to 16 hours to the logarithmic growth phase" is preferably "culturing at 37°C for 12 hours to the logarithmic growth phase".

[0007] In the above-mentioned method for preparing Escherichia coli cell wall carbon dots, in the second step, "immerse the Escherichia coli cell wall precipitate collected in the above-mentioned first step in 20 mL of deionized water, then place it in a high-pressure reactor lined with polytetrafluoroethylene, and heat it at 180°C to 220°C for 8 to 16 hours", preferably "heat it at 200°C for 12 hours".

[0008] The above-mentioned method for preparing Escherichia coli cell wall carbon dots, wherein the equipment, raw materials, reagents and operation processes involved are all well known to those skilled in the art.

[0009] The application of the above-mentioned Escherichia coli cell wall carbon dots provides a nanotherapy for the treatment of sepsis, including: intraperitoneal injection of 0.5-5 mg / kg concentration of Escherichia coli cell wall carbon dots to improve the inflammatory response and organ damage caused by sepsis; 250-500 μg / mL concentration of Escherichia coli cell wall carbon dots compete with LPS to bind to LBP protein and promote TLR4 degradation through lysosomes; 250-1000 μg / mL concentration of Escherichia coli cell wall carbon dots are used to treat RAW264.7 cells to reduce mitochondrial DNA leakage and inhibit the over-activation of the cGAS-STING pathway.

[0010] In the following text, Escherichia coli cell wall carbon dots are referred to as ECW-CDs.

[0011] The beneficial effects of the present invention are as follows: compared with the prior art, the present invention has the following outstanding substantive features and significant improvements: Through creative work, the research team of the present invention discovered that the E. coli bacterial wall as a raw material can form nanomaterials with special structures and functions in the process of preparing carbon dots, revealing for the first time the unique advantages of the E. coli bacterial wall as a source of carbon dot preparation and expanding the selection range of carbon dot precursor materials. The present invention uses the E. coli bacterial wall with a special biological structure as a raw material to prepare a kind of E. coli bacterial wall carbon dot, filling the technical gap in the prior art that has not used the specific structure of microorganisms such as cell walls as carbon dot precursors to prepare carbon dots; Existing treatment strategies for sepsis include antibiotic therapy, anti-inflammatory drugs and supportive therapy, but due to the emergence of multidrug-resistant strains and the complexity of the pathological mechanism of sepsis, existing technical therapies have not yet achieved satisfactory therapeutic effects. Through creative work, the research team of the present invention has provided a new nanomedicine strategy for the treatment of sepsis. Specifically, ECW-CDs can target and induce the tandem silencing of two key innate immune pathways, TLR4 and cGAS-STING, effectively reducing the over-activation of the immune system and cell damage, thereby reducing the cellular inflammatory factor storm and multi-organ damage caused by sepsis, thereby providing a new method for the treatment of sepsis and other infectious diseases, showing potential clinical application value and research significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0013] Figure 1 This is a schematic diagram of the therapeutic effect of ECW-CDs on septic mice, where: Figure 1 A is a graph showing the survival rate and body temperature changes in CLP-induced septic mice after treatment with ECW-CDs.

[0014] Figure 1 B is a graph showing the survival rate and body temperature changes in mice with LPS-induced sepsis after treatment with ECW-CDs.

[0015] Figure 1 C: Peritoneal view of mice treated with different doses of ECW-CDs for CLP-induced sepsis.

[0016] Figure 1 D is a graph showing the levels of TNF-α and IL-1β in the serum of mice with CLP-induced sepsis treated with different doses of ECW-CDs.

[0017] Figure 1 E is a graph showing the levels of TNF-α and IL-1β in the serum of mice with LPS-induced sepsis treated with different doses of ECW-CDs.

[0018] Figure 1 F is the HE-stained sections of the lung, liver, and kidney tissues of mice with CLP-induced sepsis treated with different doses of ECW-CDs.

[0019] Figure 2 Schematic diagram of ECW-CDs competing with LPS to bind to LBP protein and promote TLR4 degradation through lysosomes, where: Figure 2 A is the micro-thermophoresis (MST) analysis of the interaction between LPS and LBP by ECW-CDs.

[0020] Figure 2 B is the in situ proximity ligation assay (i.e., PLA) of LPS and LBP in LPS-induced RAW264.7 cells after ECW-CDs treatment.

[0021] Figure 2 C is the flow cytometric detection of TLR4 in LPS-induced RAW264.7 cells after 3 hours of treatment with different doses of ECW-CDs.

[0022] Figure 2 D is the flow cytometric detection of TLR4 in LPS-induced RAW264.7 cells after 6 hours of treatment with different doses of ECW-CDs.

[0023] Figure 2 E is the fluorescence confocal co-localization image and the Ellson coefficient of TLR4 and lysosomes in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs.

[0024] Figure 2 F is a Western Blot analysis of the TLR4 protein content in the cytoplasm of LPS-induced RAW264.7 cells after ECW-CDs were treated with chloroquine, a lysosomal inhibitor at a concentration of 100 μM.

[0025] Figure 2 G is a Western Blot analysis of the protein expression levels of TLR4, p-NF-κB, NF-κB, p-IRF3, and IRF3 in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs.

[0026] Figure 3 Schematic diagram of ECW-CDs reducing mitochondrial DNA leakage and inhibiting overactivation of the STING3 pathway, wherein: Figure 3 A is the GO enrichment analysis of LPS-induced RAW264.7 cells after ECW-CDs treatment.

[0027] Figure 3 B is the GSEA analysis of LPS-induced RAW264.7 cells after ECW-CDs treatment.

[0028] Figure 3 C is a Western Blot analysis of the protein expression levels of cGAS, STING, TBK1, and p-TBK1 in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs.

[0029] Figure 3D is the RT-qPCR analysis of ISG genes in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs.

[0030] Figure 3 E is the RT-qPCR analysis of the mRNA expression levels of TOMM20, VDAC1 and TFAM in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs.

[0031] Figure 3 F is the Western Blot analysis of the protein expression levels of TOMM20, VDAC1 and TFAM in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs.

[0032] Figure 3 G is the RT-qPCR analysis of mitochondrial DNA expression levels in the cytoplasm of LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs.

[0033] Figure 3 H is the co-localization fluorescence image of dsDNA (green) and mitochondria (red) in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs.

[0034] Figure 3 I is the analysis of mitochondrial damage in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs for 24 hours.

[0035] Figure 3 J is the in vitro analysis of DPPH scavenging and SOD of ECW-CDs.

[0036] Figure 3 K is the fluorescence analysis of ROS in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs. DETAILED DESCRIPTION

[0037] Example 1 A method for preparing ECW-CDs, the specific steps are as follows: In the first step, take 10 μL of DH5α competent cells from the -80°C refrigerator and place on ice. Operate in a clean bench and take the 10 μL DH5α competent cells and inoculate them in 200 mL In LB medium, culture at 37°C for 12 hours to the logarithmic growth phase, then collect the LB medium, set the centrifuge to 5000g, centrifuge the collected LB medium for 10 seconds, add 10mL of ultrapure water to the collected Escherichia coli DH5α competent cells, use an ultrasonic disruptor to perform ultrasonic disruption at an energy setting of 10%, stop for 10 seconds after each ultrasonic disruption for 10 seconds, and the total ultrasonic time is 1.5 hours, and all the obtained Escherichia coli DH5α competent cells are disrupted, then set the centrifuge to 8000g, and centrifuge for 20 seconds until the Escherichia coli bacterial wall and cytoplasmic components are separated, and then wash the collected Escherichia coli bacterial wall 3 times with 4°C precooled PBS detergent until impurities in the Escherichia coli and incompletely lysed Escherichia coli are removed, then set the centrifuge to 8000g, centrifuge the Escherichia coli bacterial wall collected after washing for 20 seconds, and collect all the washed Escherichia coli bacterial wall precipitation; In the second step, the E. coli cell wall precipitate collected in the first step is immersed in 20 mL of deionized water, and then placed in a high-pressure reactor lined with polytetrafluoroethylene, and heated at 200°C for 12 hours. After the system is naturally cooled to room temperature, the resulting liquid is filtered through a filter membrane with a pore size of 0.22 μm, and then the filtrate is dialyzed for 24 hours using a dialysis membrane with a molecular weight cutoff of 10 kDa, thereby obtaining purified E. coli cell wall carbon dots, whose English abbreviation is ECW-CDs. Finally, the E. coli cell wall carbon dot product is dried to a powder by freeze-drying for later use.

[0038] Example 2 A method for preparing ECW-CDs, the specific steps are as follows: In the first step, take 10 μL of DH5α competent cells from the -80°C refrigerator and place on ice. Operate in a clean bench and take the 10 μL DH5α competent cells and inoculate them in 200 mL In LB medium, culture at 37° C. for 8 hours to the logarithmic growth phase, then collect the LB medium, set the centrifuge to 5000 g, centrifuge the collected LB medium for 10 seconds, add 10 mL of ultrapure water to the collected Escherichia coli DH5α competent cells, use an ultrasonic disruptor to perform ultrasonic disruption at an energy setting of 10%, stop for 10 seconds after each ultrasonic disruption for 10 seconds, and the total ultrasonic time is 1.5 hours, and all the obtained Escherichia coli DH5α competent cells are disrupted, then set the centrifuge to 8000 g, and centrifuge for 20 seconds until the Escherichia coli bacterial wall and cytoplasmic components are separated, and then wash the collected Escherichia coli bacterial wall 3 times with 4° C. precooled PBS detergent until impurities in the Escherichia coli and incompletely lysed Escherichia coli are removed, then set the centrifuge to 8000 g, centrifuge the Escherichia coli bacterial wall collected after washing for 20 seconds, and collect all the washed Escherichia coli bacterial wall precipitations; In the second step, the E. coli cell wall precipitate collected in the first step is immersed in 20 mL of deionized water, and then placed in a high-pressure reactor lined with polytetrafluoroethylene, and heated at 220°C for 8 hours. After the system is naturally cooled to room temperature, the resulting liquid is filtered through a filter membrane with a pore size of 0.22 μm, and then the filtrate is dialyzed for 24 hours using a dialysis membrane with a molecular weight cutoff of 10 kDa to obtain purified E. coli cell wall carbon dots, whose English abbreviation is ECW-CDs. Finally, the E. coli cell wall carbon dot product is dried to a powder by freeze-drying for later use.

[0039] Example 3 A method for preparing ECW-CDs, the specific steps are as follows: In the first step, take 10 μL of DH5α competent cells from the -80°C refrigerator and place on ice. Operate in a clean bench and take the 10 μL DH5α competent cells and inoculate them in 200 mL In LB medium, culture at 37° C. for 16 hours to the logarithmic growth phase, then collect the LB medium, set the centrifuge to 5000 g, centrifuge the collected LB medium for 10 seconds, add 10 mL of ultrapure water to the collected Escherichia coli DH5α competent cells, use an ultrasonic disruptor to perform ultrasonic disruption at an energy setting of 10%, stop for 10 seconds after each ultrasonic disruption for 10 seconds, and the total ultrasonic time is 1.5 hours, and all the obtained Escherichia coli DH5α competent cells are disrupted, then set the centrifuge to 8000 g, and centrifuge for 20 seconds until the Escherichia coli cell wall and cytoplasm components are separated, and then wash the collected Escherichia coli cell wall 3 times with 4° C. precooled PBS detergent until impurities in the Escherichia coli and incompletely lysed Escherichia coli are removed, then set the centrifuge to 8000 g, centrifuge the washed Escherichia coli cell wall for 20 seconds, and collect all the washed Escherichia coli cell wall precipitates; In the second step, the E. coli cell wall precipitate collected in the first step is immersed in 20 mL of deionized water, and then placed in a high-pressure reactor lined with polytetrafluoroethylene, and heated at 180°C for 16 hours. After the system is naturally cooled to room temperature, the resulting liquid is filtered through a filter membrane with a pore size of 0.22 μm, and then the filtrate is dialyzed for 24 hours using a dialysis membrane with a molecular weight cutoff of 10 kDa to obtain purified E. coli cell wall carbon dots, whose English abbreviation is ECW-CDs. Finally, the E. coli cell wall carbon dot product is dried to powder by freeze-drying for later use.

[0040] Example 4 The application of the Escherichia coli cell wall carbon dots of the present invention is used for intraperitoneal injection of 0.5-5 mg / kg ECW-CDs to improve the inflammatory response and organ damage caused by sepsis. The specific method steps are as follows: The sepsis model was established by cecal ligation and puncture (CLP), as follows: male mice aged 6 to 8 weeks were used, randomly divided into groups, anesthetized, and the abdomen was shaved to prepare for surgery. Under sterile conditions, a 1.5-2 cm incision was made in the abdomen of the mouse with a scalpel, and a 4-0 suture was used to ligate approximately 75% of the cecum, and a 21G needle was used to puncture, and a small amount of fecal droplets were gently squeezed out. Subsequently, the cecum was returned to the abdominal cavity and the wound was sutured. After the operation, 1 mL of normal saline was injected subcutaneously for fluid resuscitation. There were 6 mice in each group. The mice in the drug-treated group were injected intraperitoneally with ECW-CDs once every 24 hours. The concentration of ECW-CDs in the low-dose group was 0.5 mg / kg, and the concentration of ECW-CDs in the high-dose group was 5 mg / kg, for a total of three injections. The vital signs of the mice were monitored every 6 hours for 7 days. The results are shown in Figure 1 The schematic diagram of the therapeutic effect of ECW-CDs on septic mice is shown as follows: Figure 1 A is a graph showing the survival rate and body temperature changes of CLP-induced septic mice after ECW-CDs treatment. The graph shows that the decrease in body temperature of mice in the ECW-CDs treatment group was effectively alleviated, and the survival rate was significantly improved compared with that in the model group. Figure 1 B is a graph showing changes in body temperature 24 hours after ECW-CDs were administered to treat the cynomolgus monkey sepsis model, which shows that a similar therapeutic effect was observed in the sepsis model induced by intraperitoneal injection of 25 mg / kg LPS. Figure 1 C is a view of the abdominal cavity of mice with CLP-induced sepsis treated with different doses of ECW-CDs. The figure shows that the suppurative inflammation in the abdominal cavity of the ECW-CDs treatment group was alleviated, the pus was lighter in color, and the swelling of the intestinal wall was significantly improved.

[0041] To further evaluate the protective effect of ECW-CDs on septic mice, serum was collected 24 hours after the establishment of the mouse model, and the concentrations of key inflammatory factors TNF-α and IL-1β in the serum of mice in the model group and the treatment group were measured by enzyme-linked immunosorbent assay (ELISA). Figure 1 D is the level of TNF-α and IL-1β in the serum of mice with CLP-induced sepsis treated with different doses of ECW-CDs. Figure 1 E shows the levels of TNF-α and IL-1β in the serum of mice with LPS-induced sepsis treated with different doses of ECW-CDs. The figure shows that ECW-CDs treatment can significantly reduce the levels of these inflammatory factors in both models.

[0042] Collect the liver, lung and kidney of mice and fix them in 10% formalin, embed and slice them for HE staining, drop hematoxylin on the tissue loaded on the slide, wait for about five minutes, quickly wash with water for 3 to 5 seconds, add eosin to the tissue slice for staining for 10 to 30 seconds, rinse with color enhancer 1 to 2 times, dry with filter paper or wait for natural drying, and add neutral resin to carefully seal the slide. The results are as follows Figure 1 F is the HE-stained sections of the lung, liver, and kidney tissues of mice with CLP-induced sepsis treated with different doses of ECW-CDs. The mice in the model group had alveolar hemorrhage, increased inflammatory cell infiltration, disordered hepatic cord structure, multiple diffuse coagulation, glomerular swelling, and cavitation of renal tubular epithelial cells, while the ECW-CDs treatment group effectively alleviated the damage to the lung, liver, and kidney.

[0043] Example 5 The application of the Escherichia coli cell wall carbon dots of the present invention is used for competitive binding of 250-500 μg / mL ECW-CDs with LPS to LBP protein and promote TLR4 degradation through lysosomes. The specific method steps are as follows: Using LBP purified in vitro and fluorescently labeled, the binding ability of LBP to LPS and ECW-CDs at a concentration of 250-500 μg / mL was detected by MST (microscale thermophoresis). Figure 2 A is the MST analysis of the interaction between LPS and LBP by ECW-CDs. The addition of ECW-CDs significantly interfered with the interaction between LPS and LBP, thereby blocking the original binding between the two. RAW264.7 cells were treated with 1 μg / mL LPS or LPS combined with ECW-CDs at a concentration of 250 μg / mL for low-dose group and 500 μg / mL for high-dose group for ECW-CDs for 6 hours, and then fixed with 4% formaldehyde, blocked with Duolink Blocking Solution for one hour, and then permeabilized with 0.1% Triton X-100. The cells were incubated with primary antibody dilutions of different species against LPS and LBP overnight. Subsequently, Duolink in situ PLA probe anti-mouse MINUS (Sigma, USA) and Duolink in situ PLA probe anti-rabbit PLUS (Sigma, USA) were incubated with cells, and when the PLA probe was tightly bound, ligase was added to form a circular DNA chain, and polymerase and oligonucleotides were added to allow rolling circle amplification. The fluorescently labeled probe was complementary to the sequence of the rolling circle amplification product to achieve amplification of the corresponding fluorescent signal. Images were acquired using an LSM800 confocal microscope. The results showed that in cells not treated with ECW-CDs, LPS and LBP had significant co-localization signals, while under the intervention of ECW-CDs, the co-localization positive signal points were significantly reduced in a dose-dependent manner (see Figure 2 B is an in situ proximity ligation assay (PLA) of LPS and LBP in LPS-induced RAW264.7 cells after ECW-CDs treatment).

[0044] Similarly, RAW264.7 cells treated with the same method in this example were used to detect TLR4 by flow cytometry. The results are as follows: Figure 2 C is the flow cytometric detection of TLR4 in LPS-induced RAW264.7 cells after 3 hours of treatment with different doses of ECW-CDs. Figure 2 D is a flow cytometric detection diagram of TLR4 in LPS-induced RAW264.7 cells after 6 hours of treatment with different doses of ECW-CDs. It was found that the model group treated with LPS could significantly increase the number of TLR4 on the membrane surface, and the expression level of TLR4 on the membrane surface gradually increased over time. After treatment with ECW-CDs, the expression level of TLR4 on the membrane surface decreased in a dose-dependent manner. And after treatment with ECW-CDs, the expression level of TLR4 protein decreased in a dose-dependent manner. Then, the co-localization of TLR4 and lysosomes was observed by immunofluorescence experiments. The results showed that after the high-dose group ECW-CDs and LPS co-treated RAW264.7 cells for one hour, the Pearson co-localization coefficient of TLR4 and lysosomes (p = 0.73 ± 0.0088, n=3) was compared with the Pearson co-localization coefficient of the model group (p = 0.2368 ± 0.0350, n = 3) significantly increased (see Figure 2 E is the fluorescence confocal co-localization image and the Eisenberg coefficient of TLR4 and lysosomes in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs. This indicates that ECW-CDs guide TLR4 to approach lysosomes and degrade it through the lysosomal pathway. Similarly, RAW264.7 cells were co-treated with LPS and ECW-CDs for 6 hours in the presence or absence of 100 μM chloroquine, a lysosomal inhibitor, and then cytoplasmic proteins were isolated. The results are shown in Figure 2. Figure 2 F is a Western Blot analysis of the TLR4 protein content in the cytoplasm of LPS-induced RAW264.7 cells after ECW-CDs treatment with 100 μM chloroquine, a lysosomal inhibitor. The results show that the content of TLR4 in the cytoplasm decreased in a dose-dependent manner with the increase of ECW-CDs concentration without chloroquine treatment. In contrast, there was no significant difference in the expression level of cytoplasmic TLR4 between the ECW-CDs treatment group and the model group after treatment with 100 μM chloroquine, which also indicates that ECW-CDs can promote the migration and degradation of TLR4 to lysosomes. Western blot experiments were used to further detect sites downstream of the TLR4 pathway. The results showed that after 3 hours, LPS treatment significantly increased the expression level of p-NF-κB p65 to 1.7 times that of the control group. Compared with the model group, the expression level of p-NF-κB p65 was reduced by 23.5% after treatment with low-dose ECW-CDs at a concentration of 250 μg / mL; the expression level of p-NF-κB p65 was reduced by 52.9% after treatment with high-dose ECW-CDs at a concentration of 500 μg / mL; after 6 hours, LPS treatment increased the expression level of p-NF-κB p65 to 3.7 times that of the control group. Compared with the model group, the expression level of p-NF-κB p65 was reduced by 29.7% after treatment with low-dose ECW-CDs and by 51.4% after treatment with high-dose ECW-CDs. Similarly, LPS treatment caused the p-IRF3 level to increase to 1.3 and 2.3 times of the control group after 3 and 6 hours, respectively, while after high-dose ECW-CDs treatment, the expression level of p-IRF3 decreased by 65.2% after 6 hours compared with the model group. The above results indicate that ECW-CDs can reduce TLR4-NFκB cascade signal transduction by competing with LPS for binding to LBP; ECW-CDs at a concentration of 250-500 μg / mL compete with LPS for binding to LBP protein and promote TLR4 degradation through lysosomes, thereby reducing the activation of TLR4-IRF3 cascade signaling pathway.

[0045] Example 6 The application of the Escherichia coli bacterial wall carbon dots of the present invention is used to treat RAW264.7 cells with 250-1000 μg / mL concentration of ECW-CDs to reduce mitochondrial DNA leakage and inhibit excessive activation of the cGAS-STING pathway. The specific method steps are as follows: RAW264.7 cells were treated according to the procedure in Example 5, and transcriptome sequencing (i.e., RNA-seq) was performed, followed by GO enrichment analysis. The results are as follows: Figure 3 A is a GO enrichment analysis diagram of LPS-induced RAW264.7 cells after ECW-CDs treatment, showing the effects of ECW-CDs on mitochondrial membrane, mitochondrial respiration and other molecular functions under inflammatory conditions. Gene set enrichment analysis (GSEA) data also demonstrated that ECW-CDs alleviated the over-activation of innate immunity of RAW264.7 cells after LPS stimulation and upregulated the activity of mitochondrial respiratory chain complex I. ECW-CDs may have improved mitochondrial function and enhanced the ability of RAW264.7 to resist oxidative stress. Figure 3 B is the GSEA analysis of LPS-induced RAW264.7 cells after ECW-CDs treatment. Western Blot experiments were performed on RAW264.7 cells treated with ECW-CDs to evaluate the activation of the cGAS-STING pathway. The results showed that the activation of the cGAS-STING pathway was systematically evaluated. Figure 3 C is a Western Blot analysis of the protein expression levels of cGAS, STING, TBK1, and p-TBK1 in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs. LPS significantly upregulated the expression levels of cGAS, STING, and phosphorylated TBK1 proteins in RAW264.7 cells, while under the intervention of ECW-CDs treatment, the expression levels of these proteins were dose-dependently downregulated. In addition, RT-qPCR experiments showed that ECW-CDs treatment greatly reduced the expression levels of interferon-stimulated genes (ISGs) such as Ifit1, Ifit3, and Ifi44 in RAW264.7 cells induced by LPS (see Figure 3 D is the RT-qPCR analysis of ISG genes in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs). Mitochondrial damage markers were detected by RT-qPCR and Western Blot experiments. Figure 3 E is the RT-qPCR analysis of the mRNA expression levels of TOMM20, VDAC1 and TFAM in RAW264.7 cells induced by LPS after treatment with different doses of ECW-CDs. Figure 3F is a Western Blot analysis of the protein expression levels of TOMM20, VDAC1 and TFAM in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs. The graph shows that the RNA and protein levels of mitochondrial membrane proteins TOMM20, VDAC1 and mitochondrial DNA-binding protein transcription factor A (TFAM) were significantly downregulated in RAW264.7 cells after LPS stimulation. After treatment with ECW-CDs, the expression levels of these proteins were significantly restored. Since mitochondrial membrane damage is often accompanied by the leakage of mtDNA, RAW264.7 cells were stained with double-stranded DNA (dsDNA) antibodies and mitochondrial probes (MitoTracker Red) to detect the leakage of mtDNA. The results showed that after LPS stimulation of RAW264.7 cells, a large amount of dsDNA was transported from mitochondria to the cytoplasm. However, after treatment with ECW-CDs, this transport phenomenon was significantly improved (see Figure 3 H is a co-localized fluorescence image of dsDNA (green) and mitochondria (red) in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs). In order to confirm whether mtDNA is released into the cytoplasm, the cytoplasmic mtDNA in RAW264.7 cells with or without E-CDs treatment after LPS stimulation was quantified. The results showed that after LPS stimulation, the cell membrane mtDNA in RAW264.7 cells was significantly enriched, and its level was 6 times that of the control group. After treatment with different doses of ECW-CDs, the enrichment level of cell membrane mtDNA in the cytoplasm decreased by 25.7% and 58.3% respectively compared with the model group (see Figure 3 G is the RT-qPCR analysis of mitochondrial DNA expression levels in the cytoplasm of RAW264.7 cells induced by LPS after treatment with different doses of ECW-CDs. After LPS stimulation, RAW264.7 cells lost mitochondrial membrane potential and increased mitochondrial mass (see Figure 3 Figure 1 shows the analysis of mitochondrial damage in LPS-induced RAW264.7 cells after 24 hours of treatment with different doses of ECW-CDs, as determined by flow cytometry analysis of JC-1 and Mitotracker Green staining, respectively. Subsequently, the mechanism by which ECW-CDs alleviate mitochondrial membrane damage was studied. Its antioxidant capacity was tested in vitro, and the results are shown in Figure 2. Figure 3J is an in vitro analysis of DPPH scavenging and SOD analysis of ECW-CDs. ECW-CDs have DPPH scavenging ability and superoxide dismutase (SOD)-like activity. DCFH-DA probe staining showed that after LPS stimulation, the positive signal of reactive oxygen species (ROS) in RAW264.7 cells increased significantly, but after treatment with ECW-CDs, the number of ROS-positive cells decreased in a dose-dependent manner (see Figure 3 K is the fluorescence analysis of ROS in LPS-induced RAW264.7 cells after treatment with different doses of ECW-CDs), which also shows that it has significant antioxidant capacity.

[0046] "DH5α competent cells" are a type of Escherichia coli strain used for efficient transformation of conventional plasmids, so the DH5α competent cells in this article are often referred to as "bacteria" in the technical field.

[0047] In the above embodiments, the equipment, raw materials, reagents and operating processes involved are all well known to those skilled in the art.

Claims

1. A method for preparing Escherichia coli cell wall carbon dots, characterized in that , the specific steps are as follows: In the first step, take 10 μL of DH5α competent cells from the -80°C refrigerator and place on ice. Operate in a clean bench and take the 10 μL DH5α competent cells and inoculate them in 200 mL In LB medium, culture at 37° C. for 8 to 16 hours until the logarithmic growth phase, then collect the LB medium, set the centrifuge to 5000 g, centrifuge the collected LB medium for 10 seconds, add 10 mL of ultrapure water to the collected Escherichia coli DH5α competent cells, use an ultrasonic disruptor to perform ultrasonic disruption at an energy setting of 10%, stop for 10 seconds after each ultrasonic disruption for 10 seconds, and the total ultrasonic time is 1.5 hours, and all the obtained Escherichia coli DH5α competent cells are disrupted, then set the centrifuge to 8000 g, and centrifuge for 20 seconds until the Escherichia coli cell wall and cytoplasm components are separated, then wash the collected Escherichia coli cell wall 3 times with 4° C. precooled PBS detergent until impurities in the Escherichia coli and incompletely lysed Escherichia coli are removed, then set the centrifuge to 8000 g, centrifuge the washed Escherichia coli cell wall for 20 seconds, and collect all the washed Escherichia coli cell wall precipitates; In the second step, the Escherichia coli cell wall precipitate collected in the first step is immersed in 20 mL of deionized water, and then placed in a high-pressure reactor lined with polytetrafluoroethylene, and heated at 180°C to 220°C for 8 to 16 hours. After the system is naturally cooled to room temperature, the resulting liquid is filtered through a filter membrane with a pore size of 0.22 μm, and then the filtrate is dialyzed for 24 hours using a dialysis membrane with a molecular weight cutoff of 10 kDa to obtain purified Escherichia coli cell wall carbon dots, whose English abbreviation is ECW-CDs. Finally, the Escherichia coli cell wall carbon dot product is dried to a powder by freeze-drying for later use.

2. The method for preparing Escherichia coli cell wall carbon dots according to claim 1, characterized in that: In the first step, "culturing at 37° C. for 8 to 16 hours until the logarithmic growth phase" is preferably "culturing at 37° C. for 12 hours until the logarithmic growth phase".

3. The method for preparing Escherichia coli cell wall carbon dots according to claim 1, characterized in that: In the second step, "immerse the Escherichia coli cell wall precipitate collected in the first step in 20 mL of deionized water, then place it in a high-pressure reactor lined with polytetrafluoroethylene, and heat it at 180°C to 220°C for 8 to 16 hours", wherein "heat it at 200°C for 12 hours" is preferred.

4. The use of the Escherichia coli cell wall carbon dots according to claim 1, characterized in that It provides a nanotherapy for the treatment of sepsis, including: intraperitoneal injection of 0.5-5 mg / kg concentration of Escherichia coli wall carbon dots to improve the inflammatory response and organ damage caused by sepsis; 250-500 μg / mL concentration of Escherichia coli wall carbon dots compete with LPS for binding to LBP protein and promote TLR4 degradation through lysosomes; treatment of RAW264.7 cells with 250-1000 μg / mL concentration of Escherichia coli wall carbon dots to reduce mitochondrial DNA leakage and inhibit overactivation of the cGAS-STING pathway.

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