Application of REG gamma

Abstract

The invention relates to application of a preparation for detecting REG gamma expression level in preparation of a reagent for diagnosing sepsis. The invention further relates to application of the preparation for degrading Bim in preparation of drugs for treating sepsis. The invention proves that REG gamma has an inhibiting effect on pyroptosis, and excessive pyroptosis is regulated and controlled by degrading BCL-2 family pro-apoptosis protein Bim, so that sepsis is relieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of REGγ. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection that can rapidly progress to serious conditions such as septic shock and multiple organ dysfunction syndrome. Sepsis is usually caused by bacterial infection but can also be the result of viral, fungal, or other microbial infections. Symptoms may include fever, chills, rapid heartbeat, rapid breathing, low blood pressure, and confusion. Treatment of sepsis typically involves antibiotic therapy, supportive care measures, and sometimes intensive care.

[0003] Pyroptosis is a form of programmed cell death that primarily occurs in immune cells, particularly macrophages and dendritic cells. Pyroptosis is mediated by inflammasomes, protein complexes that recognize pathogen-associated and damage-associated molecular patterns, thereby activating Caspase-1. Caspase-1 further promotes the maturation of inflammatory cytokines IL-1β and IL-18, leading to cell membrane rupture and the release of these cytokines and other substances, which in turn triggers an inflammatory response. However, the inflammatory cytokine storm caused by excessive pyroptosis is a common cause of death in sepsis.

[0004] Sepsis not only involves pyroptosis but also apoptosis regulated by the BCL-2 family. The BCL-2 family is a group of key proteins that regulate apoptosis (programmed cell death). These proteins are divided into several subgroups: anti-apoptotic proteins (such as Bcl-2 and Bcl-XL) and pro-apoptotic proteins (such as Bax and Bak). BCL-2 family members control the process of apoptosis by regulating the permeability of the mitochondrial outer membrane. When pro-apoptotic proteins dominate, mitochondria release cytochrome c and other pro-apoptotic factors, initiating the apoptotic pathway. The BCL-2 family's regulation of pyroptosis has also been widely reported, and multiple forms of death intersect in sepsis.

[0005] REGγ is a proteasome activator belonging to the REG (regulator of calcineurin) family. Proteasomes are macromolecular complexes within cells responsible for degrading damaged or unwanted proteins. REGγ can enhance proteasome activity, particularly during ATP-independent degradation processes. The regulatory role of REGγ in various cancers makes it a potential cancer biomarker for early diagnosis and prognosis prediction. Knockout of REGγ not only inhibits cancer cell proliferation but also helps reduce cancer cell resistance to chemotherapeutic drugs, such as increasing sensitivity to sorafenib in renal cancer and cisplatin in breast cancer. These studies provide evidence for the feasibility of REGγ-targeted therapeutic approaches. However, REGγ has primarily been used in tumor treatment. Although its role in autoimmunity has been confirmed, there have been no reports attempting to apply REGγ to immune diseases. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide an application of REGγ.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A first aspect of the present invention is to provide a use of a preparation for detecting REGγ expression levels in the preparation of a reagent for diagnosing sepsis.

[0009] The second aspect of the present invention is to provide a use of a preparation for degrading Bim in the preparation of a drug for treating sepsis.

[0010] Preferably, the agent that degrades Bim is REGγ or an agonist of REGγ.

[0011] Preferably, the REGγ agonist is Nrf2 or an agonist of Nrf2.

[0012] Preferably, the Nrf2 agonist is Oltipraz.

[0013] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0014] The present invention demonstrates the inhibitory effect of REGγ on cell pyroptosis, and regulates excessive cell pyroptosis by degrading the BCL-2 family pro-apoptotic protein Bim, thereby alleviating sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure A shows the expression of REGγ in peripheral blood samples of healthy patients and patients with sepsis (raw data were converted by Log function and plotted); Figure B shows the expression of REGγ in PBMCs of healthy patients and patients with sepsis;

[0016] Figure 2 In the figure, Panel A shows H&E staining of mouse lung tissue (scale bar is 100 μm); Panel B shows the expression of REGγ detected by Western blotting; Panel C shows immunofluorescence of mouse lung tissue (REGγ is red fluorescence, DAPI is blue fluorescence, scale bar is 100 μm);

[0017] Figure 3 Figure A is a schematic diagram of cecal ligation and puncture in mice; Figure B shows the edema of the main organ tissues in mice; Figure C shows H&E staining of organ damage in mice; Figure D shows the blood biochemistry of mouse blood specimens (n=5-12 for CK, n=7-9 for ALT, n=8-11 for BUN); Figure E shows the serum cytokine levels in mice (n=4-6); Figure F shows the bacterial load in mouse blood;

[0018] Figure 4 Figure 3, Panel A shows the expression of REGγ in tissues of septic mice detected by Western blotting; Panel B shows the immunofluorescence of lung tissues of septic mice (REGγ is red fluorescence, DAPI is blue fluorescence, scale bar is 100 μm); Panel C shows the protein expression of REGγ in BMDMs of septic mice detected by Western blotting; Panel D shows the mRNA level of REGγ in BMDMs of septic mice detected by qPCR (repeated 3 times independently);

[0019] Figure 5 Figure A shows the expression levels of each protein in the BCL-2 family in HeLa cells; Figure B shows the protein level of Bim in HeLa cells; Figure C shows the differential expression bar graph of Bim in HeLa shN and HeLa shREGγ; Figure D shows the mRNA levels of each protein in the BCL-2 family in HeLa cells; Figure E shows the mRNA level of Bim in HeLa cells;

[0020] Figure 6 Figure A shows the concentration of REGγ protein after in vitro purification and concentration; Figure B shows the concentration of 20S proteasome after in vitro purification and concentration; Figure C shows p21, a known substrate of REGγ, which is degraded in vitro; Figure D shows the expression of Bim protein after in vitro degradation detected by WB; Figure E is a statistical graph of the in vitro degradation experiment (assuming that the expression level of Bim in the untreated state is 1, and the bar graph is drawn based on this normalization process);

[0021] Figure 7 WT mice, REGγ-KO mice, and REGγ - / - Bim Lysm-cre H&E staining of the main organ tissues of mice;

[0022] Figure 8The expression of pyroptosis protein GSDMD in BMDMs after treatment with Nrf2 agonist Oltipraz;

[0023] Among them, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns: no significant difference (No Significant). DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention will be described in detail below.

[0025] Unless otherwise defined, technical or scientific terms used in the claims and the specification shall have the same general meaning as understood by persons having ordinary skills in the technical field to which the present invention belongs.

[0026] The words "include" or similar used in the patent application specification and claims of the present invention mean that the items before "include" include the items listed after "include" or their equivalents, and do not exclude other items.

[0027] The numerical values ​​mentioned in the present invention include all numerical values ​​that increase by one unit from the lowest to the highest, assuming that there is at least two units between any lower value and the higher value. For example, if a component or a physical quantity is said to be from 1 to 100, preferably from 10 to 90, and most preferably from 20 to 80, it is intended that values ​​such as 5 to 95, 14 to 76, 23 to 67, 32 to 58, and 41 to 49 are clearly listed in this specification; for values ​​less than 1, 0.0001, 0.001, 0.01, or 0.1 are considered to be more appropriate units. The above examples are for illustrative purposes only. In fact, all numerical combinations between the lowest value and the highest value listed are considered to be clearly listed in this specification in a similar manner.

[0028] Unless otherwise specified, the reagents, consumables, and instruments involved in the present invention are all commercially available products.

[0029] Example 1

[0030] In the NCBI (National Center for Biotechnology Information (nih.gov)) database, select the GEO DataSets option to search Sepsis, select the entry that uploaded the RNAseq data of blood samples from clinical patients with sepsis, obtain the gene expression difference table, and analyze the expression changes of REGγ. Finally, the three databases GSE69063, GSE100159, and GSE32707 were selectively analyzed. The three databases used blood samples from healthy patients as controls and compared them with blood samples from sepsis patients to obtain the expression difference map of REGγ at the transcriptional level ( Figure 1 A).

[0031] In GSE69063, blood samples were collected from confirmed patients at the first time and labeled T0. A second blood sample was collected 1 hour after admission and labeled T1. A third blood sample was collected 3 hours after admission and labeled T3. RNAseq analysis was performed along with blood samples from healthy patients. REGγ expression levels were extracted and analyzed, revealing that REGγ expression levels were essentially consistent at T0, T1, and T2, but significantly downregulated compared to the healthy control group. In GSE32707, blood samples were similarly collected on the first day of admission and labeled Day0, and on the seventh day of admission and labeled Day7. These samples were compared with the healthy control group, yielding consistent results showing downregulation of REGγ expression in patients with sepsis. In GSE100159, samples from patients with sepsis were divided into those with Malleoidosis infection (SS-M) and those without Malleoidosis infection (SS-O), as well as two recovered patients (recovery). Compared with the healthy control group, REGγ expression was downregulated in sepsis regardless of whether it was caused by Malleoidosis infection or not. Interestingly, in the two patients who recovered after treatment, REGγ expression returned to the level of healthy patients. In summary, REGγ transcriptional levels are downregulated in clinical patients.

[0032] In collaboration with Shanghai Fifth People's Hospital affiliated to Fudan University, after a clinical physician diagnoses sepsis, with the patient's consent, about 2-4 mL of fresh blood is collected from the clinical patient immediately after diagnosis in a purple anticoagulant tube and transported to the laboratory. The blood is diluted with an equal amount of PBS, and separation fluid is added to a 15 mL centrifuge tube in advance. The diluted blood is slowly spread on top of the separation fluid. If the blood volume is less than 3 mL, 3 mL of separation fluid is used. If the blood volume is greater than 3 mL, an equal amount of separation fluid is used, and the total volume does not exceed 10 mL. Centrifuge at 800-1200 rpm for 20-30 minutes. The liquid in the centrifuge tube is divided into a plasma layer, a white layer, a separation fluid layer, and a red blood cell layer from top to bottom. Carefully aspirate the middle white layer into a new centrifuge tube and wash it once with PBS. After centrifugation at 1000 rpm for 5 minutes, the precipitate is peripheral blood mononuclear cells (PBMCs). The cells are cultured at 37°C and 5% CO2 using 1640 culture medium. The expression of REGγ in PBMCs was detected using Western blotting ( Figure 1 B) The results showed that REGγ expression was significantly reduced in patients with sepsis, even becoming difficult to detect. The above results indicate that REGγ expression was significantly downregulated in patients with sepsis, both at the RNA and protein levels.

[0033] Example 2

[0034] Wild-type C57BL / 6 mice (WT mice) were provided by the Animal Center of East China Normal University. Mice were housed by trained personnel in an SPF environment. The temperature in the enclosure was maintained at approximately 22–28°C, and the relative humidity was maintained at approximately 40%–60%. The temperature inside the cage was slightly higher than the ambient temperature by 1–2°C, and the humidity was slightly higher by 5–10%. The light / dark cycle was 12 / 12, with ventilation rates of 10–20 times per hour, noise levels <60 dB, and ammonia concentrations below 20 ppm. Sterile, complete nutritional pellets were used for feed, and sunflower seeds were fed regularly. The drinking water was autoclaved, bottled purified water, and the bedding was autoclaved corn cobs. During the feeding process, sufficient drinking water and feed were maintained in the cages, and the cages were kept dry and clean. The number of mice per cage did not exceed six.

[0035] Wild-type mice were intraperitoneally injected with two concentrations of Pseudomonas aeruginosa (PA), the higher concentration of which was 1×10 6 CFU / mL (High), the lower concentration is 2×10 5 CFU / mL (Low), and the sham-operated group was intraperitoneally injected with sterile saline.

[0036] The mice were killed, and the complete lung tissues of the mice were collected. After fixation with 4% paraformaldehyde for 12-24 hours, tissue wax blocks were obtained according to the normal dehydration and embedding process. The dehydration time of the lung tissue was 1 hour, and the immersion time in xylene was 30 minutes. The wax immersion time was the same, 1 hour. After embedding, the slices were sliced, and the wax blocks needed to be ice-bathed in advance. The slices were 5 mm thick and stored at room temperature after baking at 42°C overnight. Before the experiment, the slices were baked again at 65°C for 2-3 hours, and the routine H&E staining steps were performed. The slices were photographed under a microscope, and the scale and other information were recorded. H&E staining of the lung tissue showed obvious thickening of the alveolar wall and infiltration of inflammatory cells, indicating that the lung damage was severe and PA induction was successful ( Figure 2 A).

[0037] After the mice were killed, lung and colon tissues were extracted, and rice-sized tissue blocks were cut out and placed in a grinding tube. 3-4 ceramic beads for grinding were added to the tube. About 250 μL of pre-cooled RIPA lysis buffer was added and ground in a tissue grinder at a frequency of 60 Hz. After grinding for 60 seconds and then standing for 30 seconds, the grinding was repeated until the tissue blocks were completely ground (no large pieces of tissue remained). The homogenate was placed on ice and allowed to stand for 30 minutes, then centrifuged at 12000 rpm for 10 minutes. The supernatant was added with 6× Loading (Loading was diluted to 1× with PBS) for WB detection. Western Blot detection showed that REGγ was significantly downregulated in the lung tissue of PA-infected mice, while this phenomenon was not seen in the colon tissue of the control group ( Figure 2 B), Suppression of REGγ expression occurs in specific lung injury caused by PA-induced pneumonia.

[0038] Immunofluorescence experiments were performed on mouse lung tissue. Mouse tissue sections were operated according to the H&E procedure until EDTA fixation. The subsequent steps were to soak in a punching solution (0.1% Triton X100) for 10 minutes and then quench with hydrogen peroxide. Use 3% BSA to block at room temperature for 1 hour, wash 3 times with PBS, and then add freshly prepared primary antibody solution to the tissue and incubate overnight at 4°C. To prevent a small amount of primary antibody from volatilizing, the tissue can be placed in a wet box. Wash with PBS 3 times the next day, incubate with secondary antibody in the dark for 1 hour, wash 3 times with PBS, and then perform DAPI staining. After staining, wash twice with distilled water, wipe off excess water, use anti-fluorescence quenching sealing medium, and store the prepared slides at 4°C in the dark. Use a fluorescence microscope to take pictures, record the scale and other information, and use ImageJ to merge multiple fluorescence channel pictures. REGγ downregulation was observed. It can be seen that the red fluorescence indicating REGγ was significantly weaker in PA-infected mice than in the sham operation group ( Figure 2 C), the above data demonstrate that REGγ expression is downregulated in mouse tissues after P. aeruginosa infection.

[0039] Subsequently, a sepsis model was established in wild-type C57BL / 6 male mice using cecal ligation and puncture (CLP) surgery. Figure 3 A) 6-10 week old C57BL / 6 male mice were fasted for 12 hours beforehand. Under sterile conditions, mice were anesthetized with 20 mg / mL tribromoethanol (approximately 200 μL per 10 g mouse, which can be increased or decreased as needed). After mice were fully anesthetized (approximately 5 minutes), their abdomens were shaved, disinfected with alcohol cotton pads, and placed on the operating table with the abdomen facing upward. The abdomen was incised layer by layer, approximately 1 cm in size, along the left side of the midline to expose the cecum. A No. 5 suture was used to ligate the mid-section of the cecum. A single or double puncture was performed with a 20G needle. A small amount of feces was squeezed out of the puncture site to ensure circulation. After carefully retracting the cecum, the cecum was then closed layer by layer with 5-0 sutures. The surgery was completed within 10 minutes. 1 mL of sterile saline was injected subcutaneously, and the mice were placed on a 37°C incubator to warm up until awake. Mice with raised dorsal hair, slowed movements, and loss of awareness after CLP surgery were designated as sepsis models (CLP group). The sham group underwent the same procedure, but after exposing the cecum, no ligation or puncture was performed. The cecum was directly retracted into the abdomen, and the wound was sutured. Postoperative observation of the mice revealed raised, fluffy, and matted fur on their backs, decreased self-awareness, slowed or absent activity, slight tremors, and curled-up posture. Their responsiveness to external stimuli was reduced or almost nonexistent, and significant eye discharge was observed.

[0040] The mice were killed, and the intact heart, liver, spleen, lung, kidney and other major organ tissues of the mice were collected and the initial weight was recorded, which was recorded as wet weight. After drying each tissue in a 65°C oven for 24 hours, the weight was recorded again, which was recorded as dry weight. The wet / dry weight ratio was obtained by comparing the two weighings before and after, and the values ​​of the Sham group and the CLP group were compared. GraphPad Prism 9.0 was used to make a bar graph, and the wet / dry weight ratio data of the two groups were subjected to Two-way ANOVA statistical analysis. The results showed that compared with the control group (Sham group), the liver tissue and lung tissue of the septic mice (CLP group) showed obvious edema ( Figure 3 B), indicating a damaged endothelial barrier.

[0041] Mice were sacrificed, and intact tissues of major organs, including the heart, liver, spleen, lung, and kidney, were collected. After fixation with 4% paraformaldehyde for 12-24 hours, tissue wax blocks were obtained according to the normal dehydration and embedding procedures. Lung tissue was dehydrated for 1 hour and immersed in xylene for 30 minutes; heart and kidney tissues were dehydrated for 30 minutes and immersed in xylene for 15 minutes; spleen and liver tissues were dehydrated for 20 minutes and immersed in xylene for 15 minutes. The wax immersion time was the same, 1 hour. After embedding, sections were sliced. The sections were placed in an ice bath in advance, with a thickness of 5 mm. The sections were baked at 42°C overnight and then stored at room temperature. Prior to the experiment, the sections were baked again at 65°C for 2-3 hours and subjected to the standard H&E staining procedure. The sections were photographed under a microscope, and information such as the scale was recorded. The liver cells of septic mice showed obvious swelling, cell morphology changed, blurred liver lobule structure, obvious spleen damage, diffuse splenic bodies, and a large number of extramedullary blood cell infiltration in the red pulp. The lung tissue showed thickening of the alveolar wall and hemorrhage in the alveolar cavity. The glomerular capsule in the kidney tissue was narrowed, and the renal tubular epithelial cells were edematous and deformed, bulging into the tubular lumen. There was a large infiltration of blood cells and inflammatory cells in many tissues ( Figure 3 C).

[0042] 24 hours after surgery, use a small amount of anesthetic to anesthetize the mice, and quickly use the eyeball removal method to obtain mouse blood, about 400-700 μL / mouse. After standing for 2-3 hours, centrifuge at low temperature and centrifuge at 3000rpm for 5 minutes. Collect the upper serum sample in a new EP tube, about 100-350 μL / mouse. Be careful not to suck up the red precipitate at the bottom. If the sample is not tested immediately, store it at -80°C and test it within one month. The sample was sent to Sevier Biotechnology Co., Ltd. for biochemical testing, including creatine kinase (CK, indicating myocardial damage), alanine aminotransferase (ALT, indicating liver damage), and blood urea nitrogen (BUN, indicating kidney damage). In the serum samples of septic mice, indicators such as CK, ALT, and BUN, which indicate heart, liver, and kidney dysfunction, respectively, increased by about 2-4 times ( Figure 3 D).

[0043] Mouse serum samples were collected and sent to Sevier Biotechnology Co., Ltd. for detection of cytokines such as IL-1β, IL-6, and TNFα. Proinflammatory cytokines such as IL-1β, IL-6, and TNFα were significantly upregulated ( Figure 3 E).

[0044] 24 hours after surgery, 50 μL of mouse blood was collected and immediately diluted with LB medium without resistance in a clean bench. The dilution gradient was 1:10→1:100→1:1000. The dilution ratio can be adjusted according to the amount of bacteria. The appropriate dilution concentration can be selected to facilitate observation and counting. The diluted blood was evenly spread on LB (Luria-Bertani) solid medium and cultured in an incubator for 12-16 hours before observation. A large number of colonies also appeared in the blood of CLP mice, indicating that after surgery, enterobacteria flowed into the abdominal cavity through the puncture hole and passed through the peritoneum into the blood circulation, causing pathogen infection in the blood ( Figure 3 F), whereas no colony growth was observed in the sham-operated group.

[0045] It can be determined that after the CLP mouse model was constructed, multiple organ damage and dysfunction occurred, inflammatory cytokines were released, and blood pathogen infection occurred, which is consistent with the symptoms of sepsis.

[0046] The lung, colon, and small intestine tissues of the Sham and CLP groups were extracted (the method was the same as above). After detection, it was found that REGγ was significantly downregulated in the lung tissue of the CLP group, but no expression difference was found in the colon and small intestine tissues ( Figure 4 A). Immunofluorescence experiments on mouse lung tissue (same method as above) further confirmed the downregulation of REGγ. The red fluorescence indicating REGγ in CLP mice was significantly weaker than that in the sham-operated group. At the same time, alveolar wall thickening was observed ( Figure 4 B) shows that REGγ expression is suppressed in damaged lung tissue of septic mice.

[0047] A mouse model was established, and mice were killed 12h and 24h after surgery, respectively. Bone marrow macrophages were extracted for experiments:

[0048] a. Select 6- to 10-week-old male WT mice and sacrifice them by cervical dislocation. Place them flat on the experimental table with their abdomen facing up. Secure the mice and disinfect the surface of the mice with 75% alcohol.

[0049] b. Using sterilized dissection equipment, remove the skin from the hind legs, separate the hind limbs, including the femur and tibia, remove the feet, disinfect in 75% alcohol for several minutes, and transfer to a clean bench for subsequent procedures.

[0050] c. Wash the bone three times with PBS and then remove the muscle attached to the bone surface in a clean bench. Remove the femoral and tibial epiphyses and make cuts on both sides. Flush out the bone marrow cavity contents with pre-chilled PBS until the bone is translucent white. Transfer the contents to a 15 mL centrifuge tube.

[0051] d. Centrifuge at 1000 rpm for 5 minutes, resuspend the cell pellet in 1640 complete medium, and culture overnight at 37°C, 5% CO2 for 12 hours. The next day, remove the supernatant and re-inoculate the cells into a new culture dish. Add the cytokine M-CSF (final concentration of 10 ng / mL) and allow the cells to adhere until confluent. During this time, wash thoroughly with PBS to remove any foreign matter. The adherent cells are BMDMs. Complete the experiment within one week.

[0052] (BMDMs were cultured in a 37°C, 5% CO2 incubator in RPMI Medium 1640 containing 10% serum and 1% penicillin-streptomycin solution, supplemented with the cytokine M-CSF.)

[0053] The expression of REGγ was detected at both the protein and transcript levels (the method was the same as above. To extract RNA samples, 1 mL of RNAiso Plus lysis buffer was added to the tissue block and ground in the same way. After grinding, it was allowed to stand for 30 minutes before proceeding to the conventional chloroform-isopropanol RNA extraction step.). It can be seen that it was significantly downregulated during the course of sepsis ( Figure 4 C). GSDMD-NT, a GSDMD splice, was detected in BMDMs of septic mice, indicating that REGγ downregulation is accompanied by GSDMD-mediated pyroptosis during sepsis. Figure 4 D).

[0054] REGγ expression is inhibited during the development of sepsis. Using CLP surgery, it was demonstrated at the animal level that REGγ expression is downregulated in the tissues of septic mice, and the downregulation is most obvious in the lung tissue. The same conclusion was reached after specific induction of lung injury by the strain Pseudomonas aeruginosa. This specific downregulation in lung tissue indicates that the expression difference of REGγ in the course of sepsis is not widespread, which also suggests that REGγ may play a certain role in the study of sepsis-related dysfunction of specific organs. It is worth noting that the downregulation of REGγ is also accompanied by the splicing activation of GSDMD, a key protein in cell pyroptosis.

[0055] Example 3

[0056] Western blotting was used to detect the protein levels of the BCL-2 family in REGγ knockout cell lines. The BCL-2 family is divided into anti-apoptotic members, pro-apoptotic members, and effector proteins Bax / Bak. The results showed that in HeLa cells after REGγ knockout, the expression of almost all pro-apoptotic members of the BCL-2 family was affected. In contrast, there was no obvious difference in the protein expression of anti-apoptotic Bcl-XL and MCL-1. REGγ deficiency induced a significant upregulation of pro-apoptotic proteins, and the protein level of Bim was even increased by more than 2 times ( Figure 5AC). The mRNA levels of various proteins were detected. Bid was the protein most significantly affected by REGγ deficiency, but its transcription level was also significantly upregulated, while the transcription levels of Bad and Puma were decreased. The effector protein Bax was not regulated by REGγ at both the protein and transcription levels, while Bak and Bim not only had their protein expressions significantly upregulated, but their transcription levels were also unaffected ( Figure 5 DE). In addition, Bim is located upstream and regulates the activity of Bak.

[0057] It was also found that REGγ and Bim had a negative regulatory relationship at the protein level in BMDMs and mouse tissues, but REGγ defective expression did not affect the transcription level of Bim.

[0058] Multiple experiments have shown that REGγ not only binds to the three isoforms of Bim (BimEL, BimL and BimS), but also promotes the degradation of Bim.

[0059] In vitro purified REGγ protein and 20S proteasome ( Figure 6 AB), and in vitro translated HA-BimEL and the reported REGγ substrate protein p21, and incubated with REGγ and 20S, as Figure 6 As shown in C, p21 can be degraded, proving that the in vitro degradation system is effective. Further attempts to degrade Bim in vitro showed that Bim could not be degraded when REGγ and 20S were present alone. Only when both were present could Bim degradation be observed ( Figure 6 DE). This confirms that REGγ-20S proteasome can directly degrade Bim, independent of ubiquitin and ATP, and that Bim is a substrate protein of REGγ.

[0060] Example 4

[0061] Wild-type C57BL / 6 mice (WT mice) were treated as before, and C57BL / 6 mice with systemic REGγ knockout (REGγ-KO or REGγ - / - ) were bred and provided by themselves. Bim conditional knockout mice were purchased from Jicui Yaokang Biotechnology Co., Ltd., Lysm-Cre tool mice were provided by themselves, and WT mice with macrophage-specific Bim knockout (Bim Lysm-cre )Bim flox / flox The mice were housed together with Lysm-Cre mice. The breeding method was the same as above.

[0062] Wild-type C57BL / 6 mice (WT), C57BL / 6 mice with systemic REGγ knockout (REGγ-KO), and REGγ-KO mice with macrophage-specific Bim knockout (REGγ - / - Bim Lysm-cre)Construct a CLP-induced sepsis mouse model (same method as before).

[0063] The mice were killed within 18-24 hours, and the intact liver, lung, kidney and other major organ tissues were collected for H&E staining (the same method as before) to assess organ damage. The results showed that REGγ-KO mice had more severe tissue damage than WT mice. After macrophage-specific knockout of Bim, the tissue damage in REGγ-KO mice was alleviated ( Figure 7 ).

[0064] Mouse bone marrow macrophages were isolated (using the same method as above) and cultured. Prior to the experiment, they were pretreated with the Nrf2 agonist Oltipraz for 16 hours. Subsequently, the cells were treated with 1 μg / ml LPS for a specified period of time. Before sampling, 5 mM ATP was added and the cells were harvested with 1× loading buffer for standard Western blot analysis. The results showed that Oltipraz affected REGγ expression. In BMDMs pretreated with Oltipraz, the cleavage of the pyroptotic protein GSDMD was inhibited in response to LPS and ATP stimulation. Figure 8 ).

[0065] In summary, the present invention demonstrates the inhibitory effect of REGγ on cell pyroptosis, which regulates excessive cell pyroptosis by degrading the BCL-2 family pro-apoptotic protein Bim, thereby alleviating sepsis.

[0066] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a preparation for detecting REGγ expression level in the preparation of a reagent for diagnosing sepsis.

2. Application of a preparation for degrading Bim in the preparation of a drug for treating sepsis.

3. The use according to claim 2, characterized in that: The agent for degrading Bim is REGγ or an agonist of REGγ.

4. The use according to claim 3, characterized in that: The REGγ agonist is Nrf2 or an agonist of Nrf2.

5. The use according to claim 3, characterized in that: The Nrf2 agonist is Oltipraz.

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