Use of an agent that inhibits expression of chrm3 receptors in the preparation of a medicament for treating a condition associated with lipopolysaccharide

Abstract

The application discloses application of a reagent for inhibiting Chrm3 receptor expression in preparation of a medicine for treating diseases related to lipopolysaccharide, and belongs to the field of biological medicine. The application specifically reduces the expression of Chrm3 through the inhibitor 4-damp and si-Chrm3, effectively reduces the inflammation of the body caused by LPS, and reversely verifies the role of Chrm3 in LPS-induced inflammation through overexpression lentivirus, for the first time finds that LPS can combine with Chrm3 receptors and transmit inflammatory signals through the MAPK pathway, and provides a new treatment target and a new treatment mechanism for inflammation caused by LPS, especially for liver injury induced by sepsis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of reagents that inhibit Chrm3 receptor expression in the preparation of medicaments for treating diseases caused by lipopolysaccharide. Background Technology

[0002] Lipopolysaccharide (LPS), a toxic component of endotoxins, is primarily found in the outer membrane of Gram-negative bacteria (GNB). When GNB dies and lyses, LPS is released from the outer membrane. Once inside the body, LPS can trigger a systemic inflammatory response. LPS exerts its effects mainly by binding to lipopolysaccharide-binding protein (LBP). LBP, an acute-phase protein, primarily functions to bind to the bacterial endotoxin LPS. It transports LPS to the CD14 receptor on the cell surface, forming an LPS-LBP-CD14 ternary complex, thereby activating the inflammatory response.

[0003] Studies have shown that knocking out the LBP gene can improve cell damage induced by LPS stimulation. Previous studies have used LBP... - / - Using primary mouse hepatocytes as the research subject, it was verified that angiotensin II receptor type 1a (Agtr1a) and adrenoceptor alpha 1A (Adra1a) are involved in LPS-induced inflammatory transmission. While using inhibitors and siRNA to specifically reduce Agtr1a and Adra1a expression did not completely block the LPS-induced inflammatory response, the degree of reduction in inflammation and damage needs further improvement. This suggests that other transmission pathways of LPS-stimulated signals exist after LBP gene knockout. Therefore, in LBP... - / - The discovery of an effective compensatory pathway for transmitting LPS-induced inflammatory signals in mice can provide important experimental evidence and new treatment ideas for the clinical treatment of organ damage or other severe inflammatory responses caused by endotoxin infection.

[0004] The cholinergic receptor muscarinic 3 (Chrm3) is a member of the G protein-coupled receptor family. It selectively binds to Gq protein and activates the Gq-PLC-DAG / IP3 signaling pathway to release Ca2+. 2+It can also activate the MAPK signaling pathway. Studies have shown that blocking M3R can inhibit inflammatory factors, enhance anti-tumor immune responses, thereby reducing tumor growth and improving the immune response to cancer. Furthermore, many studies have observed that administration via muscarinic receptors can reduce inflammation in vivo, indicating that muscarinic receptors have a pro-inflammatory effect. However, whether Chrm3 is involved in the occurrence and development of LPS-induced inflammation, or in the transmission pathway of LPS-induced inflammatory signals, has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide the application of reagents that inhibit Chrm3 receptor expression in the preparation of drugs for treating diseases related to lipopolysaccharide (LPS), thereby addressing the problems existing in the prior art. This invention is the first to discover that LPS can interact with Chrm3 receptors and transmit inflammatory signals through the MAPK pathway, providing a new therapeutic target and mechanism for LPS-induced inflammation, especially sepsis-induced liver injury.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of a reagent that inhibits Chrm3 receptor expression in the preparation of medicaments for treating diseases caused by lipopolysaccharide.

[0008] Optionally, the related diseases include inflammation-related diseases.

[0009] Optionally, the inflammation-related diseases include sepsis, hepatitis, liver dysfunction, and liver injury.

[0010] Optionally, the reagents for inhibiting Chrm3 receptor expression include Chrm3 receptor antagonists and interfering RNA that inhibits Chrm3 receptor expression.

[0011] Optionally, the Chrm3 receptor antagonist includes 4-damp.

[0012] Optionally, the drug may also include other active ingredients with anti-inflammatory and cell-protective effects.

[0013] Optionally, the active ingredient may include ulinastatin.

[0014] The present invention discloses the following technical effects:

[0015] This invention effectively reduces LPS-induced inflammation by specifically lowering Chrm3 expression through the inhibitors 4-damp and si-Chrm3. Furthermore, the role of Chrm3 in LPS-induced inflammation was verified by overexpressing lentiviruses. For the first time, it was discovered that LPS can bind to the Chrm3 receptor and transmit inflammatory signals through the MAPK pathway, providing a new therapeutic target and mechanism for LPS-induced inflammation, especially sepsis-induced liver injury. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The expression of Chrm3 receptor protein in primary mouse hepatocytes under LPS stimulation is shown in Figure 1. A represents the change in Chrm3 receptor protein in primary WT mouse hepatocytes under LPS stimulation; B represents the expression of Lbp receptor protein. - / - Changes in Chrm3 protein in primary mouse hepatocytes after LPS stimulation with the addition of the inhibitor 4-damp; C represents Lbp. - / - Changes in Chrm3 protein in primary mouse hepatocytes after LPS stimulation and the addition of si-Chrm3; D represents Lbp. - / - Changes in Chrm3 protein in primary mouse hepatocytes after LPS stimulation;

[0018] Figure 2 The expression of Chrm3 receptor protein in mouse peritoneal macrophages under LPS stimulation is shown in Figure 1. A represents the change in Chrm3 receptor protein in WT mouse peritoneal macrophages under LPS stimulation; B represents the expression of Lbp receptor protein. - / - Changes in Chrm3 protein in mouse peritoneal macrophages after LPS stimulation with the addition of the inhibitor 4-damp; C represents Lbp. - / - Changes in Chrm3 protein in mouse peritoneal macrophages after LPS stimulation and the addition of si-Chrm3; D represents Lbp. - / - Changes in the protein content of mouse peritoneal macrophages after overexpression of Chrm3 under LPS stimulation;

[0019] Figure 3 This diagram shows the changes in hepatocyte inflammatory factors after Chrm3 receptor inhibition, interference, and overexpression; where A through C represent Lbp levels after 4-damp inhibition, respectively. - / -Expression of inflammatory factors IL-1β, IL-6, and TNF-α in primary mouse hepatocytes; D to F represent Lbp levels after si-Chrm3 interference. - / - Expression of inflammatory factors IL-1β, IL-6, and TNF-α in primary mouse hepatocytes; G to I represent Lbp levels after Chrm3 overexpression. - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in primary mouse hepatocytes;

[0020] Figure 4 This diagram illustrates the changes in inflammatory factors in peritoneal macrophages after Chrm3 receptor inhibition, interference, and overexpression; where A through C represent Lbp levels after 4-damp inhibition, respectively. - / - Expression of inflammatory factors IL-1β, IL-6, and TNF-α in mouse peritoneal macrophages; D to F represent Lbp levels after si-Chrm3 interference. - / - Expression of inflammatory factors IL-1β, IL-6, and TNF-α in mouse peritoneal macrophages; G to I represent Lbp levels after Chrm3 overexpression. - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in mouse peritoneal macrophages;

[0021] Figure 5 This chart shows the changes in hepatocyte survival rate after Chrm3 receptor inhibition, interference, and overexpression; where A through C represent Lbp. - / - Changes in cell viability of primary mouse hepatocytes under LPS stimulation with the addition of inhibitor 4-damp, si-Chrm3, and Chrm3 overexpression.

[0022] Figure 6 This chart shows the changes in peritoneal macrophage survival rate after Chrm3 receptor inhibition, interference, and overexpression; where A through C represent Lbp. - / - Changes in the survival rate of mouse peritoneal macrophages after LPS stimulation with the addition of the inhibitor 4-damp, the addition of si-Chrm3, and overexpression of Chrm3;

[0023] Figure 7 This diagram illustrates the changes in the MAPK pathway in primary hepatocytes after Chrm3 receptor inhibition, interference, and overexpression; where A through C represent Lbp levels, respectively. - / - The phosphorylation of ERK, p38, and JNK proteins in mouse primary hepatocytes after LPS stimulation with the addition of the inhibitor 4-damp; D-F represent Lbp in sequence. - / - The phosphorylation of ERK, p38, and JNK proteins in primary mouse hepatocytes after LPS stimulation and the addition of si-Chrm3; G to I, in descending order of Lbp. - / -Phosphorylation of ERK, p38, and JNK proteins in mouse primary hepatocytes after overexpression of Chrm3 under LPS stimulation;

[0024] Figure 8 This diagram illustrates the changes in the MAPK pathway in peritoneal macrophages after Chrm3 receptor inhibition, interference, and overexpression; where A through C represent Lbp levels, respectively. - / - The phosphorylation of ERK, p38, and JNK proteins in mouse peritoneal macrophages after LPS stimulation and the addition of the inhibitor 4-damp; D-F represent Lbp in sequence. - / - The phosphorylation of ERK, p38, and JNK proteins in mouse peritoneal macrophages after LPS stimulation and the addition of si-Chrm3; G to I, in descending order of Lbp. - / - Phosphorylation of ERK, p38, and JNK proteins in mouse peritoneal macrophages after overexpression of Chrm3 under LPS stimulation;

[0025] Figure 9 This is a graph showing the results of the immunoprecipitation assay.

[0026] Figure 10 This diagram shows the changes in inflammatory factors in liver tissue after Chrm3 receptor inhibition and overexpression in vivo; where A through C represent Lbp levels after 4-damp inhibition, respectively. - / - Expression of inflammatory factors IL-1β, IL-6, and TNF-α in mouse liver tissue; D-F represent Lbp levels after Chrm3 overexpression, in descending order. - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in liver tissue of mice;

[0027] Figure 11 This chart shows the changes in inflammatory factors in peritoneal macrophages after Chrm3 receptor inhibition and overexpression in vivo; where A through C represent Lbp levels after 4-damp inhibition, respectively. - / - Expression of inflammatory factors IL-1β, IL-6, and TNF-α in peritoneal macrophages of mice; D-F represent Lbp levels after Chrm3 overexpression. - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in peritoneal macrophages in mice;

[0028] Figure 12 This diagram illustrates the changes in the MAPK pathway in liver tissue after Chrm3 receptor inhibition and overexpression in vivo; where A through C represent the changes in Lbp levels after the addition of the inhibitor 4-damp under LPS stimulation. - / - Phosphorylation of ERK, p38, and JNK proteins in the liver of mice; D-F represent Lbp levels after overexpression of Chrm3 under LPS stimulation. - / - Phosphorylation of ERK, p38, and JNK proteins in the liver of mice;

[0029] Figure 13 This diagram illustrates the changes in the MAPK pathway in peritoneal macrophages after Chrm3 receptor inhibition and overexpression in vivo; where A through C represent the Lbp levels after the addition of the inhibitor 4-damp under LPS stimulation. - / - Phosphorylation of ERK, p38, and JNK proteins in mouse peritoneal macrophages; D-F represent Lbp levels after overexpression of Chrm3 under LPS stimulation. - / - Phosphorylation of ERK, p38, and JNK proteins in peritoneal macrophages of mice;

[0030] Figure 14 The effects of Chrm3 receptor inhibition and overexpression on liver biochemical indicators in vivo; where A to C represent Lbp levels after 4-damp inhibition, respectively. - / - Changes in serum ALT, AST, and liver MPO in mice; D-F represent Lbp levels after Chrm3 overexpression, respectively. - / - Changes in serum ALT, AST, and liver MPO in mice;

[0031] Figure 15 The image shows the effect of in vivo Chrm3 alterations on the histological changes of LPS-induced liver injury. A represents a representative H&E staining image of the liver in the control group; B represents a representative H&E staining image of the liver in the LPS group; C represents a representative H&E staining image of the liver in the inhibitor group; and D represents a representative H&E staining image of the liver in the overexpression group. Arrows indicate hemorrhage, triangles indicate liver structural changes, squares indicate inflammatory infiltration, and pentagrams indicate hepatocyte degeneration.

[0032] Figure 16 The study investigated the effect of Chrm3 receptor inhibition on liver injury in WT mice. A through C represent the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the liver tissue of WT mice after drug inhibition, respectively. D through F represent the changes in serum ALT, AST, and hepatic MPO in the liver tissue of WT mice after drug inhibition, respectively. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] 1. Experimental animals used in this invention:

[0039] The inventors' team independently constructed and preserved 20 LBPs with an SPF rating. - / - Female mice weighing 20-22g at 6 weeks of age (the construction method has been disclosed in the literature "Li Sidi et al., Construction of lipopolysaccharide-binding protein gene knockout mice using CRISPR / Cas9 technology [J]. Experimental Animals and Comparative Medicine, 2022.42(04):294-300"), 20 female C57BL / 6J mice weighing 20-22g at 6 weeks of age with SPF grade [SCXK(Lu)2023-0002]. All experimental mice were raised at Shandong First Medical University under standard conditions of 12h / 12h light / dark cycle, temperature (22-25℃), and humidity (55%-60%) [SYXK(Lu)2023-0012]. This invention was carried out with the approval of the Shandong Provincial Experimental Animal Center (LS002024056). The use of experimental mice followed the 3R principle.

[0040] 2. Experimental cells of the present invention

[0041] Primary mouse hepatocytes were obtained using the collagenase IV method and were called Lbp. - / - Primary hepatocytes from mice and WT mice; mouse peritoneal macrophages were obtained by peritoneal lavage using Lbp. - / - Peritoneal macrophages of mice and WT mice.

[0042] The inhibitor 4-damp (4-dimethylaminophenol, 4-DAMP methiodide) used in this invention is a selective muscarinic M1 and M3 receptor antagonist, CAS number 1952-15-4. Unless otherwise specified, all reagents used in this invention can be obtained through conventional commercial channels.

[0043] Example 1 In vitro experiment

[0044] 1. Preparation of a cellular inflammation model

[0045] To find suitable LPS stimulation conditions, the stimulation time of LPS was divided into 0h, 6h, 8h, 12h, 16h, and 24h, and Lbp was stimulated under the same conditions. - / - Mouse primary hepatocytes and peritoneal macrophages were used to detect Chrm3 mRNA expression levels using RT-PCR to determine the optimal LPS stimulation time. Results showed that the Chrm3 receptor in Lbp... - / - The mRNA level in primary mouse hepatocytes increased over time, reaching its maximum at 12 hours; similarly, in Lbp... - / - The expression level of Chrm3 in mouse peritoneal macrophages also reached its maximum at 12 hours. These results indicate that the optimal stimulation time for LPS is 12 hours. A cellular inflammation model was constructed using LPS stimulation for 12 hours.

[0046] 2. Screening of usage conditions for the inhibitor 4-damp

[0047] 4-damp was added 0.5 h before LPS treatment at a dose of 1 × 10⁻⁶ per well. 4 Cells were seeded into 96-well plates at a predetermined number of cells. After determining the LPS stimulation time, the concentration of 4-damp was screened. Concentration gradients of 4-damp were established for grouping into control group (blank control), LPS group, 1 nM 4-damp + LPS group, 5 nM 4-damp + LPS group, 10 nM 4-damp + LPS group, and 20 nM 4-damp + LPS group. Lbp was measured using a CCK-8 assay. - / -The study investigated the cell viability of primary mouse hepatocytes and peritoneal macrophages after 12 hours of LPS treatment to determine the optimal concentration of 4-damp. Results showed that the optimal concentration of 4-damp for primary hepatocytes was 5 nM, and for peritoneal macrophages, it was 10 nM.

[0048] 3. Screening of si-Chrm3 usage conditions

[0049] 1×10 per hole 5 Cells were seeded into 24-well plates. Before transfection, cells were seeded in DMEM medium without antibiotics. After complete cell adhesion, transfection was performed. si-RNA was diluted with 50 μL of Opti-MEM to a final concentration of 50 nM in each well, and gently pipetted 3-5 times. Transfection reagent was diluted with 50 μL of Opti-MEM, gently pipetted, and incubated at room temperature for 5 min. The diluted si-RNA and transfection reagent were then mixed to form a transfection mixture, and the mixture was incubated at room temperature for 15 min. 100 μL of the transfection mixture was then evenly added to a 24-well plate containing 400 μL of antibiotic-free medium. Cells were incubated at 37°C in a 5% CO2 incubator for 4-6 hours, after which the medium was changed.

[0050] To achieve optimal gene blocking effect, the concentration of si-Chrm3 was determined to be 50 nM according to the standard usage conditions of siRNA, and the interference time gradient of si-Chrm3 was set at 8 h, 12 h, and 16 h. Lbp was detected by RT-PCR. - / - The mRNA expression levels of Chrm3 in mouse primary hepatocytes and peritoneal macrophages were investigated. Results showed that Chrm3 expression was significantly reduced in primary hepatocytes at 8, 12, and 16 hours after interference. Chrm3 expression was also significantly reduced in peritoneal macrophages at 8, 12, and 16 hours after interference. Since the receptor mRNA expression level of si-Chrm3 was significantly reduced after 12 hours of si-Chrm3 addition, and the reduction at 16 hours was not significant compared to 12 hours, the interference time for si-Chrm3 was determined to be 12 hours.

[0051] The target sequence for si-Chrm3 is GGATCTATAAGGAAACTGA (SEQ ID NO.1), with the forward sequence being 5'-GGAUCUAUAAGGAAACUGA(dT)(dT)-3' (SEQ ID NO.2) and the reverse sequence being 5'-UCAGUUUC CUUAUAGAUCC(dT)(dT)-3' (SEQ ID NO.3).

[0052] 4. Construct Chrm3-overexpressing lentivirus to transfect cells

[0053] The Chrm3 overexpression sequence (SEQ ID NO.4) was designed, and the Chrm3 overexpression lentivirus (MOI = 5) was synthesized by Beijing Qingke Biotechnology Co., Ltd. The empty vector lentivirus was Lentivirus (ZsGreen-Puro) / CMV-NC, and the overexpression lentivirus was Lentivirus (ZsGreen-Puro) / CMV-Chrm3. 1 × 10⁻⁶ cells were used per well. 5 Cells were seeded into 24-well plates and divided into four groups: control group (blank control), empty vector group (empty vector), overexpression-12h group (overexpression for 12 hours), overexpression-24h group (overexpression for 24 hours), and overexpression-36h group (overexpression for 36 hours). The mRNA expression level of Chrm3 was detected by RT-PCR to determine the appropriate overexpression conditions. Results showed that, compared with the empty vector group, the overexpression-24h group showed significantly higher Chrm3 expression levels in both primary hepatocytes and peritoneal macrophages, exhibiting the best effect. Therefore, the transfection time for lentivirus overexpression was determined to be 24 hours.

[0054] SEQ ID NO.4:

[0055]

[0056] 5. Cell grouping

[0057] (1) LPS stimulation experiment of peritoneal macrophages and primary hepatocytes in WT mice: control group (blank control) and LPS group (LPS stimulation for 12h).

[0058] (2)Lbp - / - Chrm3 inhibition experiment of mouse peritoneal macrophages and primary hepatocytes: control group (blank control), LPS group (LPS stimulation for 12h), inhibitor group (4-damp inhibition for 1h followed by LPS stimulation for 12h).

[0059] (3)Lbp - / - Chrm3 interference experiment of mouse peritoneal macrophages and primary hepatocytes: control group (blank control), LPS group (LPS stimulation for 12h), negative control group (si-Negative interference for 12h followed by LPS stimulation for 12h), and interference group (si-Chrm3 interference for 12h followed by LPS stimulation for 12h).

[0060] (4)Lbp - / - Chrm3 overexpression experiment in mouse peritoneal macrophages and primary hepatocytes: control group (blank control), LPS group (LPS stimulation for 12 h), negative control group (LPS stimulation for 12 h after transfection with empty vector lentivirus for 24 h), and overexpression group (LPS stimulation for 12 h after transfection with overexpressing lentivirus Chrm3 for 24 h).

[0061] 6. Expression of Chrm3 receptor protein

[0062] First, Western blotting (WB) was used to verify the expression of the Chrm3 receptor under LPS stimulation. The results showed that the protein expression level of Chrm3 in wild-type mouse primary hepatocytes did not change significantly under LPS stimulation. Figure 1 A; while Lbp - / - Primary mouse hepatocytes showed significantly increased protein expression levels after LPS stimulation (P < 0.001), and significantly decreased Chrm3 protein expression levels after the addition of an inhibitor (P < 0.05). Figure 1 B; After the addition of interfering RNA, the expression level of Chrm3 protein was significantly reduced (P < 0.001), such as Figure 1 The C; conversely, after the addition of overexpressing lentivirus, the expression level of Chrm3 protein increased significantly (P < 0.01), such as Figure 1 D.

[0063] Similarly, the Chrm3 receptor did not show significant changes in peritoneal macrophages of WT mice (P < 0.05), such as Figure 2A; while Lbp - / - Mouse peritoneal macrophages showed significantly increased protein expression levels after LPS stimulation (P < 0.001), and significantly decreased Chrm3 protein expression levels after the addition of an inhibitor (P < 0.05). Figure 2 B; After the addition of interfering RNA, the expression level of Chrm3 protein was significantly reduced (P < 0.01), such as Figure 2 The C; conversely, after the addition of overexpressing lentivirus, the expression level of Chrm3 protein increased significantly (P < 0.001), such as Figure 2 D.

[0064] 7. Effects of receptor inhibition, interference, and overexpression on inflammatory factors

[0065] The effects of inhibiting, interfering with, and overexpressing the Chrm3 receptor on the expression of inflammatory factors were observed by RT-PCR to verify the role of Chrm3 under LPS stimulation.

[0066] The results showed that Lbp under LPS stimulation - / - The expression levels of relevant inflammatory factors (IL-1β, IL-6, TNF-α) in primary mouse hepatocytes were significantly increased (P < 0.0001). When Chrm3 receptor expression was inhibited using 4-damp, the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α were significantly decreased (P < 0.01). Figure 3 A through C; After knocking down the expression of Chrm3 receptor using siRNA, the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α also decreased significantly (P < 0.0001, P < 0.0001, P < 0.001), such as Figure 3 The expression levels of D-F were significantly increased after overexpression of Chrm3 receptor; conversely, the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α were significantly increased after overexpression of Chrm3 receptor (P < 0.001, P < 0.0001, P < 0.01). Figure 3 The G~I. Therefore, it can be concluded that downregulating Chrm3 receptor expression can effectively reduce Lbp. - / - The expression of inflammatory factors in mouse primary hepatocytes was increased, and upregulation of Chrm3 expression exacerbated the expression of inflammatory factors in primary hepatocytes.

[0067] In Lbp - / - Chrm3 receptors also regulated LPS-induced Lbp in mouse peritoneal macrophages. - / - Expression of inflammatory factors in mice. For example... Figure 4 As shown in Figures A through C, the inhibitor 4-damp reduced the changes in inflammatory factors in peritoneal macrophages induced by LPS (P < 0.0001, P < 0.001, P < 0.01); Figure 4As shown in Figures D-F, the elimination of Chrm3 by interfering RNA significantly reduced the expression of IL-1β, IL-6, and TNF-α (P < 0.0001, P < 0.001, P < 0.0001); while treatment with overexpressing lentivirus resulted in a significant increase in inflammatory factors (P < 0.001, P < 0.01, P < 0.01). Figure 4 G~I.

[0068] The above results indicate that Chrm3 receptors mediate Lbp stimulation under LPS stimulation. - / - Expression of inflammatory factors in mouse cells.

[0069] 8. Effects of receptor inhibition, interference, and overexpression on cell survival

[0070] We used CCK-8 to verify whether changes in the Chrm3 receptor under LPS stimulation would affect cell viability.

[0071] The results showed that, compared with the control group, LPS treatment of Lbp - / - Primary mouse hepatocytes showed a significant decrease in cell viability (P < 0.0001), while inhibition of Chrm3 expression with 4-damp resulted in a slight increase in cell viability (P < 0.05). Figure 5 A; using si-Chrm3 to downregulate Chrm3 expression, it was observed that the cell survival rate was significantly increased compared to the LPS group (P < 0.01). Figure 5 B; subsequently, changes in cell viability were observed by overexpressing Chrm3. The cell viability of the overexpression group was significantly lower than that of the LPS group (P < 0.05). Figure 5 C.

[0072] For Lbp - / - In mice, LPS stimulation significantly decreased the cell viability of peritoneal macrophages (P < 0.01), while 4-damp inhibition of Chrm3 expression slightly increased cell viability (P < 0.05). Figure 6 A; after downregulating Chrm3 expression via si-Chrm3, cell viability was also significantly increased (P < 0.05), such as Figure 6 B; however, as the expression of Chrm3 receptor increased, the cell survival rate in the overexpression group decreased significantly (P < 0.05), such as Figure 6 C.

[0073] Therefore, changes in Chrm3 receptors regulate Lbp under LPS stimulation. - / - In mice, downregulating Chrm3 increases cell survival rate, while upregulating Chrm3 decreases cell survival rate.

[0074] 9. Effects of receptor inhibition, interference, and overexpression on the cellular MAPK signaling pathway

[0075] The MAPK signaling pathway plays a crucial role in normal cell growth. ERK proteins primarily regulate the expression of inflammatory factors (such as IL-1β, IL-6, and TNF-α), p38 proteins are involved in regulating cell proliferation and apoptosis, and JNK proteins are related to oxidative stress responses. Studies have shown that Chrm3 receptors can activate the MAPK signaling pathway and affect downstream responses; therefore, this invention utilizes Lbp... - / - The phosphorylation of ERK, p38, and JNK proteins in the MAPK pathway was investigated by Western blotting after inhibiting, interfering with, and overexpressing Chrm3 in mouse primary hepatocytes and peritoneal macrophages.

[0076] The results showed that reducing Chrm3 levels using inhibitors and interfering RNA effectively reduced LPS-induced phosphorylation of ERK, p38, and JNK proteins (P < 0.01). Figure 7 The values ​​of A to F. However, after transfection with overexpressing lentivirus to upregulate Chrm3, the phosphorylation levels of ERK, p38, and JNK proteins in the MAPK pathway were further increased (P < 0.001, P < 0.01, P < 0.01), such as... Figure 7 G~I.

[0077] In Lbp - / - Chrm3 also plays a similar role in mouse peritoneal macrophages. For example... Figure 8 As shown in Figures A through C, the protein levels of p-ERK, p-p38, and p-JNK in the MAPK pathway were significantly decreased after Chrm3 was inhibited by the inhibitor 4-damp (P < 0.01, P < 0.01, P < 0.001); similarly, as shown in Figures C... Figure 8 As shown in Figures D-F, interference with the Chrm3 receptor significantly reduced the protein levels of p-ERK, p-p38, and p-JNK (P < 0.01, P < 0.001, P < 0.01); while overexpression of Chrm3 significantly increased the protein expression of p-ERK, p-p38, and p-JNK (P < 0.01, P < 0.01, P < 0.001). Figure 8 G~I.

[0078] Therefore, it can be determined that Chrm3 receptors are involved in LPS stimulation of Lbp. - / - The MAPK signaling pathway in mouse cells mediates the occurrence and development of inflammatory responses.

[0079] 10. Immunoprecipitation

[0080] To determine how LPS transmits signals through Chrm3, this invention uses immunoprecipitation to verify the relationship between LPS and Chrm3.

[0081] Protein was extracted using cell lysis buffer. A / G magnetic beads were washed 5 times with 1×TBST, and 5 μg of antibody was added. The mixture was incubated at 4°C for 3-4 hours by rotation. The beads were then washed 5 times with 1×TBST, the supernatant was discarded, and the magnetic beads (antibody-magnetic bead conjugate) were harvested. A / G magnetic beads were washed 5 times with 1×TBST, and protein solution was added. The mixture was incubated at 4°C for 3-4 hours by rotation. The supernatant was collected. The previously collected magnetic beads were combined with the supernatant and incubated overnight at 4°C by rotation. The protein-antibody-magnetic bead conjugate was washed 5 times with 1×TBST, the supernatant was discarded, and the magnetic beads (protein-antibody-magnetic bead conjugate) were harvested. The protein-antibody-magnetic bead conjugate was then incubated with 1×loading buffer at room temperature for 20 minutes by rotation. The supernatant was collected and denatured at 100°C for 15 minutes. The denatured protein was subjected to SDS-PAGE electrophoresis to detect the binding of Chrm3 protein and LPS. The results are as follows: Figure 9 As shown, when the LPS is pulled down by the ferrite bead, the Chrm3 bonded to it is also pulled down, indicating that the LPS and Chrm3 are bonded together to transmit signals.

[0082] Example 2 In vivo experiment

[0083] 1. Preparation of an inflammatory mouse model

[0084] To further investigate the role of Chrm3 receptors in mice, an inflammation model was induced in mice via intraperitoneal injection of LPS (10 mg / kg). The optimal stimulation time was then screened using a time gradient method. This was applied to wild-type mice and Lbp... - / - Mice were simultaneously injected with the same dose of LPS, and the expression of inflammatory factors in their livers was measured. The results showed that although the expression levels of inflammatory factors gradually decreased over time, WT mice and Lbp mice showed a significant increase. - / - The differences between mice were also narrowing. Therefore, a validation mouse model was constructed 6 hours after LPS stimulation for further research.

[0085] 2. Screening of usage conditions for the inhibitor 4-damp

[0086] Mice were pre-injected with the inhibitor 4-damp at concentration gradients of 1 mg / kg, 5 mg / kg, and 10 mg / kg, followed by stimulation with LPS. The optimal concentration of 4-damp was screened using the expression of inflammatory cytokines as an indicator. Results showed that the expression of inflammatory cytokines decreased with increasing inhibitor concentration; therefore, a concentration of 10 mg / kg was selected as the optimal concentration for in vivo inhibition.

[0087] 3. Infecting mice with overexpressed lentivirus

[0088] The previously synthesized overexpression lentivirus was processed at a ratio of 1×102 7 TU was injected into mice at various concentrations, and the mice were grouped according to time gradients: control group (blank control), empty vector group (injected with empty vector lentivirus), overexpression-24h group (injected with overexpressed lentivirus for 24 hours), overexpression-48h group (injected with overexpressed lentivirus for 48 hours), and overexpression-72h group (injected with overexpressed lentivirus for 72 hours). The expression level of Chrm3 mRNA was then detected by RT-PCR to determine the appropriate overexpression time. The results showed that the expression level of the Chrm3 receptor reached a very high level 72 hours after injection; therefore, the 72h overexpression time point was chosen as the basis for subsequent experiments.

[0089] 4. Animal grouping

[0090] (1) WT mice and Lbp - / - Mouse LPS stimulation time gradient experiment: control group (blank control), LPS-6h group (LPS stimulation for 6h), LPS-8h group (LPS stimulation for 8h), LPS-12h group (LPS stimulation for 12h), LPS-16h group (LPS stimulation for 16h), LPS-24h group (LPS stimulation for 24h).

[0091] (2)Lbp - / - Chrm3 inhibition experiment in mouse peritoneal macrophages and hepatocytes: control group (blank control), LPS group (LPS stimulation for 6 h), inhibitor group (4-damp inhibition for 0.5 h followed by LPS stimulation for 6 h).

[0092] (3)Lbp - / - In vivo overexpression of Chrm3 in mouse peritoneal macrophages and hepatocytes: control group (blank control), LPS group (LPS stimulation for 6 h), negative control group (LPS stimulation for 6 h 3 days after transfection with empty vector lentivirus), and overexpression group (LPS stimulation for 6 h 3 days after transfection with overexpressing lentivirus Chrm3).

[0093] (4) WT mouse liver injury experiment: control group (blank control), LPS group (LPS stimulation for 6 h), UTI group (LPS stimulation for 1 h followed by injection of ulinastatin 5×10 4 U / kg), 4-damp group (4-damp 10mg / kg injected 1 hour after LPS stimulation), combined inhibition group (ulinastatin 5×10g injected 1 hour after LPS stimulation). 4 U / kg and 4-damp 10mg / kg).

[0094] 5. Effects of Chrm3 receptor inhibition and overexpression on inflammatory factors in vivo

[0095] RT-PCR testing showed that after Lbp was administered... - / - In mice, LPS injection significantly increased the expression levels of liver inflammatory factors IL-1β, IL-6, and TNF-α (P < 0.0001). Pretreatment with the inhibitor 4-damp significantly decreased the expression levels of these inflammatory factors (P < 0.0001, P < 0.001, P < 0.0001). Figure 10 The expression of Chrm3 was increased by using an overexpressing lentivirus, and the inflammatory factors in the mouse liver were also significantly increased (P < 0.0001, P < 0.001, P < 0.001). Figure 10 D~F.

[0096] Lbp - / - The same results were obtained in mouse peritoneal macrophages. Figure 11 As shown in Figures A through C, pre-injection of inhibitors to block Chrm3 can reduce the increase in inflammatory factors caused by LPS stimulation (P < 0.001, P < 0.01, P < 0.01); while... Figure 11 As shown in D to F, overexpression of Chrm3 receptor had the opposite effect, namely, a significant increase in the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α (P < 0.001, P < 0.0001, P < 0.0001).

[0097] Therefore, it can be determined that the Chrm3 receptor is located in Lbp. - / - It also plays a role in mediating the release of inflammatory factors in mice.

[0098] 6. Effects of Chrm3 receptor inhibition and overexpression on the MAPK signaling pathway in vivo

[0099] To further verify at the protein level whether Chrm3 regulates Lbp - / - The expression of MAPK pathway-related factors in mice was investigated by first examining the phosphorylation levels of ERK, p38, and JNK proteins in the liver. The results are as follows: Figure 12 As shown, compared with the control group, the protein phosphorylation level in the LPS group was significantly increased (P < 0.001). Compared with the LPS group, the protein phosphorylation levels of ERK, p38, and JNK in the inhibitor group were significantly decreased (P < 0.01, P < 0.01, P < 0.05), while the phosphorylation levels of related proteins in the overexpression group were significantly increased (P < 0.05, P < 0.01, P < 0.001). This indicates that in Lbp... - / - Chrm3 regulates LPS-induced cell necrosis through the MAPK pathway in mouse liver injury.

[0100] After that, at Lbp - / - The phosphorylation levels of ERK, p38, and JNK proteins were also examined in mouse peritoneal macrophages. The same results were obtained: LPS-induced protein phosphorylation levels were significantly reduced after Chrm3 expression was inhibited (P < 0.01, P < 0.01, P < 0.05). Figure 13 The levels of protein phosphorylation in the MAPK pathway were further increased after Chrm3 overexpression (P < 0.05, P < 0.01, P < 0.01), such as... Figure 13 D~F.

[0101] 7. Effects of Chrm3 receptor inhibition and overexpression on liver damage in vivo

[0102] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are normally present in the liver. However, when liver cells are damaged, these enzymes are released into the bloodstream, and thus they are often used as indicators of liver damage. Myeloperoxidase (MPO), as an indicator of neutrophil surface activity, plays a crucial role in oxidative stress and tissue damage that induce inflammation. In LPS-induced LBP... - / - The effect of Chrm3 on the activity of the above enzymes in mouse liver injury is as follows: Figure 14 As shown, by Figure 14 As shown in Figures A through C, compared with the control group, the activities of ALT, AST, and MPO in the LPS group were significantly increased (P < 0.0001, P < 0.001, P < 0.001), while their enzyme activities were significantly decreased after Chrm3 inhibition (P < 0.01, P < 0.01, P < 0.05). Figure 14 As shown in D-F, after Chrm3 overexpression, the enzyme activities of ALT, AST, and MPO were significantly increased compared with LPS (P<0.01, P<0.01, P<0.01).

[0103] To investigate the role of Chrm3 in liver injury, assessing histological changes in the liver after LPS stimulation provides the most direct evidence. Figure 15 As shown, inflammatory infiltration, hepatocyte degeneration, hemorrhage, and hepatospinal cord dissociation appeared in the livers of mice treated with LPS. The addition of the inhibitor effectively alleviated hepatic hemorrhage and inflammatory infiltration, and restored the structure of the liver lobules. Furthermore, compared with the LPS group, the overexpression group further exacerbated the histopathological damage to the liver, increasing the incidence of hemorrhage and structural changes.

[0104] 8. Effects of the combined action of the inhibitor 4-damp and ulinastatin on liver injury in WT mice

[0105] Ulinastatin (UTI) is a serine protease inhibitor with protective effects in multiple organs. UTIs exhibit potent anti-inflammatory and cytoprotective effects in various cell and animal models, modulating innate immunity and pro-inflammatory responses. This invention involved intervention with LPS-treated WT mice using UTI, 4-damp, and a combination of UTI and 4-damp. The results showed that the combined treatment groups had significantly lower levels of inflammatory factors compared to other groups, such as... Figure 16 As shown in A to C. This invention also detected liver injury indicators, finding that the combined treatment group showed significantly better therapeutic effects on liver injury indicators than other groups, such as... Figure 16 As shown in D~F.

[0106] This invention utilizes Lbp - / - LPS stimulation in mice significantly increased Chrm3 protein expression, followed by a significant increase in the phosphorylation of MAPK signaling pathway-related proteins. Specific inhibition of Chrm3 protein expression by the inhibitor 4-damp significantly reduced the phosphorylation levels of MAPK pathway-related proteins, leading to a decrease in the expression of downstream inflammatory factors, improved liver histopathological damage, and reduced biochemical indicators. Conversely, injection of a Chrm3-overexpressing lentivirus exacerbated liver damage. We then confirmed that LPS can bind to the Chrm3 receptor. Therefore, we hypothesize that LPS activates MAPK signaling pathway transduction via Chrm3 protein, thereby inducing the production of inflammatory factors and leading to liver damage, suggesting that Chrm3 may play a compensatory role against LBP protein.

[0107] In summary, this invention discovers a novel pathway for LPS signaling, which can exacerbate Lbp through interaction with Chrm3 receptors and the MAPK pathway. - / - Sepsis-induced liver dysfunction and damage in mice. The results of this invention suggest that Chrm3 may be a promising therapeutic target for sepsis-induced liver injury.

[0108] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a reagent that inhibits Chrm3 receptor expression in the preparation of a medicament for treating lipopolysaccharide-induced liver injury in the presence of LBP protein dysfunction, characterized in that, The reagent for inhibiting Chrm3 receptor expression is a Chrm3 receptor antagonist and an interfering RNA that inhibits Chrm3 receptor expression; the target sequence of the interfering RNA is shown in SEQ ID NO.1; the Chrm3 receptor antagonist is 4-damp.

2. The application according to claim 1, characterized in that, The drug also includes other active ingredients with anti-inflammatory and cell-protective effects.

3. The application according to claim 2, characterized in that, The active ingredient includes ulinastatin.

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