Application of reagent for inhibiting expression of Chrm3 receptor in preparation of medicine for treating related diseases caused by lipopolysaccharide

Abstract

The invention discloses application of a reagent for inhibiting Chrm3 receptor expression in preparation of a medicine for treating related diseases caused by lipopolysaccharide, and belongs to the field of biological medicine. Expression of Chrm < 3 > is specifically reduced through inhibitors 4-damp and si-Chrm < 3 >, body inflammation caused by LPS is effectively reduced, the effect of Chrm < 3 > in LPS-induced inflammation is reversely verified through overexpression lentivirus, it is found for the first time that LPS can be combined with a Chrm < 3 > receptor and transfer of inflammation signals through an MAPK pathway, and the LPS can be used for treating the inflammation caused by LPS. The invention provides a new therapeutic target and a new therapeutic mechanism for liver injury, especially sepsis-induced liver injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of an agent for inhibiting the expression of Chrm3 receptors in the preparation of medicines for treating related diseases caused by lipopolysaccharide. Background Art

[0002] Lipopolysaccharide (LPS), as a toxic component of endotoxin, is mainly present in the outer membrane of gram-negative bacteria (GNB). When gram-negative bacteria die and lyse, LPS will be released from the outer membrane of the cell. Once LPS enters the body, it may trigger a systemic inflammatory response. After entering the body, LPS mainly exerts its effect by binding to lipopolysaccharide-binding protein (LBP). As an acute response protein, LBP's main function is to bind to the bacterial endotoxin lipopolysaccharide (LPS). It can transport LPS to the CD14 receptor on the cell surface to form an LPS-LBP-CD14 ternary complex, thereby activating an inflammatory response.

[0003] Studies have shown that knocking out the LBP gene can improve cell damage caused by LPS stimulation. - / - When primary mouse hepatocytes were used as the research object, it was verified that angiotensin II receptor type 1a (Agtr1a angiotensin II receptor, type 1a, Agtr1a) and adrenoceptor alpha 1A receptor (Adrenoceptor alpha 1A, Adra1a) were involved in LPS-induced inflammatory transmission. When inhibitors and siRNA were used to specifically reduce the expression of Agtr1aAdra1a, the LPS-induced inflammatory response was not completely blocked, and the degree of reduction of inflammation and damage needs to be improved. Therefore, it is speculated that there are other transmission pathways for LPS stimulation signals after LBP gene knockout. Therefore, in Lbp - / - Finding an effective compensatory pathway for transmitting LPS-induced inflammatory signals in mice can provide important experimental basis and new treatment ideas for the clinical treatment of body damage or other severe inflammatory responses caused by endotoxin infection.

[0004] Cholinergic receptor, muscarinic 3, cardiac, Chrm3 is a member of the G protein-coupled receptor family that can selectively bind to Gq protein and activate the Gq-PLC-DAG / IP3 signaling pathway to release Ca 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 immune responses to cancer. In addition, many studies have also observed that the administration of muscarinic receptors can reduce inflammation in the body, which also shows that muscarinic receptors have pro-inflammatory effects. However, whether Chrm3 is involved in the occurrence and development of LPS-induced inflammation and whether it is involved in the pathway of transmitting LPS-induced inflammatory signals has not been reported yet. Summary of the invention

[0005] The purpose of the present invention is to provide an agent for inhibiting the expression of Chrm3 receptors for use in the preparation of a drug for treating lipopolysaccharide-induced diseases, so as to solve the problems of the prior art. The present invention first discovered that LPS can bind to Chrm3 receptors and transmit inflammatory signals through the MAPK pathway, providing a new therapeutic target and a new therapeutic mechanism for LPS-induced inflammation, especially sepsis-induced liver damage.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides application of a reagent for inhibiting Chrm3 receptor expression in preparing medicine for treating related 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 the expression of Chrm3 receptor include Chrm3 receptor antagonists and interfering RNA that inhibits the expression of Chrm3 receptor.

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

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

[0013] Optionally, the active ingredient includes ulinastatin.

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

[0015] The present invention specifically reduces the expression of Chrm3 through the inhibitors 4-damp and si-Chrm3, effectively reducing the body inflammation caused by LPS, and reversely verifies the role of Chrm3 in LPS-induced inflammation by overexpressing lentivirus. It is the first time to discover that LPS can bind to the Chrm3 receptor and transmit inflammatory signals through the MAPK pathway, providing a new therapeutic target and a new therapeutic mechanism for LPS-induced inflammation, especially sepsis-induced liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

[0019] Figure 3 The changes of hepatocyte inflammatory factors after Chrm3 receptor inhibition, interference and overexpression; A to C are Lbp after 4-damp inhibition - / -Expression of inflammatory factors IL-1β, IL-6 and TNF-α in primary mouse hepatocytes; D to F are Lbp after si-Chrm3 interference - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in primary mouse hepatocytes; G to I are Lbp after overexpression of Chrm3 - / - Expression of inflammatory factors IL-1β, IL-6, and TNF-α in primary mouse hepatocytes;

[0020] Figure 4 The changes of inflammatory factors in peritoneal macrophages after Chrm3 receptor inhibition, interference and overexpression; A to C are Lbp after 4-damp inhibition - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in mouse peritoneal macrophages; D to F are Lbp after si-Chrm3 interference - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in mouse peritoneal macrophages; G to I are Lbp after overexpression of Chrm3 - / - The expression of inflammatory factors IL-1β, IL-6, and TNF-α in mouse peritoneal macrophages;

[0021] Figure 5 The changes in the survival rate of hepatocytes after Chrm3 receptor inhibition, interference and overexpression; A to C are Lbp - / - Changes in cell survival rate of mouse primary hepatocytes after addition of inhibitor 4-damp, addition of si-Chrm3, and overexpression of Chrm3 under LPS stimulation;

[0022] Figure 6 The changes in the survival rate of peritoneal macrophages after Chrm3 receptor inhibition, interference and overexpression; A to C are Lbp - / - Changes in cell survival rate of mouse peritoneal macrophages after addition of inhibitor 4-damp, addition of si-Chrm3, and overexpression of Chrm3 under LPS stimulation;

[0023] Figure 7 The changes of MAPK pathway in primary hepatocytes after Chrm3 receptor inhibition, interference and overexpression; A to C are Lbp - / - Phosphorylation of ERK, p38, and JNK proteins in mouse primary hepatocytes after adding the inhibitor 4-damp under LPS stimulation; D to F are Lbp - / - Phosphorylation of ERK, p38, and JNK proteins in mouse primary hepatocytes after addition of si-Chrm3 under LPS stimulation; G to I are Lbp - / -Phosphorylation of ERK, p38, and JNK proteins in mouse primary hepatocytes after overexpression of Chrm3 under LPS stimulation;

[0024] Figure 8 The changes of MAPK pathway in peritoneal macrophages after Chrm3 receptor inhibition, interference and overexpression; A to C are Lbp - / - Phosphorylation of ERK, p38, and JNK proteins in mouse peritoneal macrophages after adding the inhibitor 4-damp under LPS stimulation; D to F are Lbp - / - Phosphorylation of ERK, p38, and JNK proteins in mouse peritoneal macrophages after addition of si-Chrm3 under LPS stimulation; G to I are Lbp - / - Phosphorylation of ERK, p38, and JNK proteins in mouse peritoneal macrophages after overexpression of Chrm3 under LPS stimulation;

[0025] Fig. 9 Figure 2 is the result of co-immunoprecipitation;

[0026] Fig.10 The changes of inflammatory factors in liver tissue after Chrm3 receptor inhibition and overexpression in vivo; A to C are Lbp after 4-damp inhibition - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in liver tissue of mice; D to F are Lbp after overexpression of Chrm3 - / - The expression of inflammatory factors IL-1β, IL-6, and TNF-α in liver tissue of mice;

[0027] Fig.11 The changes of inflammatory factors in peritoneal macrophages after Chrm3 receptor inhibition and overexpression in vivo; A to C are Lbp after 4-damp inhibition - / - Expression of inflammatory factors IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice; D to F are Lbp after overexpression of Chrm3 - / - The expression of inflammatory factors IL-1β, IL-6, and TNF-α in peritoneal macrophages of mice;

[0028] Fig.12 Figure 2 is the change of MAPK pathway in liver tissue after Chrm3 receptor inhibition and overexpression in vivo; A to C are Lbp after adding inhibitor 4-damp under LPS stimulation. - / - Phosphorylation of ERK, p38, and JNK proteins in the liver of mice; D to F are respectively Lbp after overexpression of Chrm3 under LPS stimulation - / - Phosphorylation of ERK, p38, and JNK proteins in the liver of mice;

[0029] Fig.13 Figure 2 is the change of MAPK pathway in peritoneal macrophages after Chrm3 receptor inhibition and overexpression in vivo; A to C are Lbp after adding inhibitor 4-damp under LPS stimulation. - / - Phosphorylation of ERK, p38, and JNK proteins in peritoneal macrophages in mice; D to F are respectively Lbp after overexpression of Chrm3 under LPS stimulation - / - Phosphorylation of ERK, p38, and JNK proteins in peritoneal macrophages in mice;

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

[0031] Fig.15 The effect of in vivo Chrm3 changes on histological changes of liver injury induced by LPS; A is a representative image of H&E staining of the liver in the control group; B is a representative image of H&E staining of the liver in the LPS group; C is a representative image of H&E staining of the liver in the inhibitor group; D is a representative image of H&E staining of the liver in the overexpression group; arrows indicate hemorrhage, triangles indicate changes in liver structure, squares indicate inflammatory infiltration, and stars indicate hepatocyte degeneration;

[0032] Fig.16 The effects of Chrm3 receptor inhibition on liver damage in WT mice in vivo; A to C are the expressions of inflammatory factors IL-1β, IL-6 and TNF-α in the liver tissue of WT mice after drug inhibition; D to F are the changes in serum ALT, AST and liver MPO in the liver tissue of WT mice after drug inhibition. DETAILED DESCRIPTION

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

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

[0038] 1. Experimental animals of the present invention:

[0039] The inventor team built and preserved 20 Lbps with SPF level - / - Female mice weighing 20-22 g at 6 weeks of age (the construction method has been disclosed in the document "Li Sidi et al., Construction of lipopolysaccharide binding protein gene knockout mice using CRISPR / Cas9 technology [J]. Laboratory Animals and Comparative Medicine, 2022.42(04):294-300"), 20 SPF-grade C57BL / 6J female mice weighing 20-22 g at 6 weeks of age [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°C), and humidity (55%-60%) [SYXK(Lu)2023-0012]. The present invention was carried out under the ethical approval of Shandong Provincial Experimental Animal Center (LS002024056), and the use of experimental mice followed the 3R principle.

[0040] 2. Experimental cells of the present invention

[0041] Mouse primary hepatocytes were used to extract Lbp using collagenase IV method - / - Primary hepatocytes from mice and WT mice; mouse peritoneal macrophages were Lbp extracted by peritoneal lavage - / - Peritoneal macrophages from mice and WT mice.

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

[0043] Example 1 In vitro experiment

[0044] 1. Preparation of Cellular Inflammation Model

[0045] In order to find the appropriate LPS stimulation conditions, the LPS stimulation time was divided into 0h, 6h, 8h, 12h, 16h, and 24h. - / - Mouse primary hepatocytes and peritoneal macrophages were cultured and the mRNA expression of Chrm3 was detected by RT-PCR to determine the optimal stimulation time of LPS. - / - Its mRNA level in mouse primary hepatocytes increased with time and reached the maximum at 12h. - / - The expression of Chrm3 in mouse peritoneal macrophages also reached the maximum at 12h. From the above results, it can be concluded that the optimal stimulation time of LPS is 12h. The cell inflammation model was constructed by stimulating LPS for 12h.

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

[0047] 4-damp was added 0.5 h before LPS treatment at 1 × 10 4 The cells were inoculated into 96-well plates. After determining the stimulation time of LPS, the concentration of 4-damp was screened. The concentration gradient of 4-damp was set to control group (blank control), LPS group, 1nM 4-damp+LPS group, 5nM 4-damp+LPS group, 10nM 4-damp+LPS group, 20nM4-damp+LPS group, and Lbp was detected by CCK-8. - / -The cell survival rate of mouse primary hepatocytes and peritoneal macrophages after 12 hours of LPS treatment was determined to determine the optimal concentration of 4-damp. The results showed that the optimal concentration of 4-damp for primary hepatocytes was 5nM. The optimal concentration of 4-damp for peritoneal macrophages was 10nM.

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

[0049] 1×10 per well 5 The cells were inoculated in a 24-well plate. Before the transfection experiment, the cells were inoculated in DMEM medium without double antibodies. The transfection experiment was performed after the cells were completely attached. Dilute si-RNA with 50μL Opti-MEM in each well to make the final concentration of 50nM, and gently blow and mix 3-5 times; dilute 1μL transfection reagent with 50μL Opti-MEM, gently blow and mix, and let stand at room temperature for 5min; mix the diluted si-RNA and transfection reagent to form a transfection mixture, blow and mix, and let stand at room temperature for 15min. 100μL of transfection mixture was evenly dropped into a 24-well plate containing 400μL of double antibody-free medium, and the cells were cultured in a 37℃, 5% CO2 incubator. The medium can be changed after 4-6h.

[0050] In order to achieve the best gene blocking effect, according to the conventional use conditions of siRNA, the concentration of si-Chrm3 was determined to be 50nM, and the interference time gradient of si-Chrm3 was set to 8h, 12h, and 16h. - / - The mRNA expression level of Chrm3 in mouse primary hepatocytes and peritoneal macrophages. The results showed that the expression of Chrm3 in primary hepatocytes was significantly reduced at 8, 12, and 16 hours of interference. The expression of Chrm3 in peritoneal macrophages was significantly reduced at 8, 12, and 16 hours of interference. Since the mRNA expression of the receptor was significantly reduced after 12 hours of si-Chrm3 addition, and the reduction was not obvious at 16 hours compared with 12 hours, the interference time of si-Chrm3 was 12 hours.

[0051] The targeting sequence of si-Chrm3 is GGATCTATAAGGAAACTGA (SEQ ID NO. 1), the forward sequence is: 5'-GGAUCUAUAAGGAAACUGA (dT) (dT)-3' (SEQ ID NO. 2); the reverse sequence is: 5'-UCAGUUUC CUUAUAGAUCC (dT) (dT)-3' (SEQ ID NO. 3).

[0052] 4. Construction of Chrm3 overexpression lentiviral transfected cells

[0053] The Chrm3 overexpression sequence (SEQ ID NO.4) was designed, and the Chrm3 overexpression lentivirus (virus infection multiplicity 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 5 The cells were inoculated in 24-well plates and divided into control group (blank control), empty vector group (empty vector), overexpression-12h group (overexpression 12h), overexpression-24h group (overexpression 24h), overexpression-36h group (overexpression 36h). The mRNA expression of Chrm3 was detected by RT-PCR to determine the appropriate overexpression conditions. The results showed that compared with the empty vector group, the Chrm3 expression in the overexpression-24h group was significantly increased in both primary hepatocytes and peritoneal macrophages, and the effect was the best. Therefore, the transfection time of the overexpression lentivirus was determined to be 24h.

[0054] SEQ ID NO.4:

[0055]

[0056] 5. Cell Grouping

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

[0058] (2) Lbp - / - Chrm3 inhibition experiment in 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 on mouse peritoneal macrophages and primary hepatocytes: control group (blank control), LPS group (LPS stimulation for 12 hours), negative control group (si-Negtive interference for 12 hours and then LPS stimulation for 12 hours), interference group (si-Chrm3 interference for 12 hours and then LPS stimulation for 12 hours).

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

[0061] 6. Expression of Chrm3 receptor protein

[0062] First, we used WB to verify the expression of Chrm3 receptor under LPS stimulation. The results of Western Blot experiments showed that the protein expression of Chrm3 in primary hepatocytes of wild-type mice did not change significantly under LPS stimulation. Figure 1 A; and Lbp - / - The protein expression of mouse primary hepatocytes increased significantly after LPS stimulation (P < 0.001), and the expression of Chrm3 protein decreased significantly after adding the inhibitor (P < 0.05). Figure 1 B; After adding interfering RNA, the expression of Chrm3 protein was significantly reduced (P < 0.001). Figure 1 C; on the contrary, after adding the overexpression lentivirus, the expression of Chrm3 protein increased significantly (P < 0.01). Figure 1 D.

[0063] Similarly, the expression of Chrm3 receptor in peritoneal macrophages of WT mice was not significantly changed (P < 0.05). Figure 2A; and Lbp - / - The protein expression of mouse peritoneal macrophages increased significantly after LPS stimulation (P < 0.001), and the expression of Chrm3 protein decreased significantly after adding the inhibitor (P < 0.05). Figure 2 B; After adding interfering RNA, the expression of Chrm3 protein was significantly reduced (P<0.01). Figure 2 C; on the contrary, after adding the overexpression lentivirus, the expression of Chrm3 protein increased significantly (P < 0.001). Figure 2 D.

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

[0065] RT-PCR was used to observe the effects of inhibition, interference and overexpression of Chrm3 receptor on the expression of inflammatory factors to verify the role of Chrm3 under LPS stimulation.

[0066] The results showed that under LPS stimulation, Lbp - / - The expression levels of related inflammatory factors (IL-1β, IL-6, TNF-α) in primary mouse hepatocytes were significantly increased (P < 0.0001). When 4-damp was used to inhibit the expression of Chrm3 receptor, the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α were significantly decreased (P < 0.01). Figure 3 A~C; after knocking down the expression of Chrm3 receptor by 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). Figure 3 D~F; on the contrary, after overexpression of Chrm3 receptor, the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α increased significantly (P<0.001, P<0.0001, P<0.01), such as Figure 3 G~I. It can be concluded that downregulating the expression of Chrm3 receptor can effectively reduce Lbp - / - The expression of inflammatory factors in primary mouse hepatocytes, and up-regulating Chrm3 expression can aggravate the expression of inflammatory factors in primary hepatocytes.

[0067] In Lbp - / - Chrm3 receptor was also observed in mouse peritoneal macrophages to regulate LPS-induced Lbp - / - Expression of inflammatory factors in mice. Figure 4 As shown in A to C, the inhibitor 4-damp can reduce the changes of inflammatory factors in peritoneal macrophages induced by LPS (P < 0.0001, P < 0.001, P < 0.01); Figure 4As shown in D to F, the elimination of Chrm3 by interfering RNA can also significantly reduce the expression of IL-1β, IL-6, and TNF-α (P < 0.0001, P < 0.001, P < 0.0001); and after overexpression of lentivirus treatment, the high expression of Chrm3 caused 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 receptor mediates the expression of Lbp under LPS stimulation. - / - Expression of inflammatory factors in mouse cells.

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

[0070] CCK-8 was used to verify whether changes in Chrm3 receptors would affect cell survival under LPS stimulation.

[0071] The results showed that compared with the control group, LPS treatment of Lbp - / - The survival rate of primary mouse hepatocytes was significantly decreased (P < 0.0001), while after the use of 4-damp to inhibit the expression of Chrm3, the survival rate of cells increased slightly (P < 0.05). Figure 5 A; using si-Chrm3 to downregulate Chrm3 expression, it can be seen that the cell survival rate was significantly increased compared with the LPS group (P < 0.01). Figure 5 B; then the changes in cell survival rate were observed by overexpressing Chrm3. The cell survival rate of the overexpression group was significantly decreased compared with the LPS group (P < 0.05). Figure 5 C.

[0072] For Lbp - / - The cell survival rate of mouse peritoneal macrophages was significantly decreased after LPS stimulation (P < 0.01). After 4-damp was used to inhibit the expression of Chrm3, the cell survival rate was slightly increased (P < 0.05). Figure 6 A; after downregulating Chrm3 expression by si-Chrm3, the cell survival rate was also significantly increased (P < 0.05). Figure 6 B; As the expression of Chrm3 receptor increased, the cell survival rate of the overexpression group decreased significantly (P < 0.05). Figure 6 C.

[0073] Therefore, the changes of Chrm3 receptor regulate the Lbp - / - Downregulation of Chrm3 increased cell survival rate, while upregulation of Chrm3 decreased cell survival rate.

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

[0075] The MAPK signaling pathway plays a vital role in the normal growth of cells. ERK protein mainly regulates the expression of inflammatory factors (such as IL-1β, IL-6, TNF-α), p38 protein participates in regulating the proliferation and apoptosis of cells, and JNK protein is related to the oxidative stress response of cells. Studies have shown that Chrm3 receptor can activate the MAPK signaling pathway and affect the downstream reactions of the pathway. Therefore, the present invention is in Lbp - / - Western Blot was used to study the phosphorylation of ERK, p38, and JNK proteins in the MAPK pathway by inhibiting, interfering, and overexpressing Chrm3 in primary mouse hepatocytes and peritoneal macrophages.

[0076] The results showed that reducing Chrm3 by inhibitors and interfering RNA could effectively reduce the protein phosphorylation of ERK, p38, and JNK caused by LPS stimulation (P < 0.01). Figure 7 A to F. After Chrm3 was upregulated by overexpression lentivirus transfection, the protein phosphorylation levels of ERK, p38, and JNK in the MAPK pathway were further increased (P < 0.001, P < 0.01, P < 0.01). Figure 7 G~I.

[0077] In Lbp - / - Chrm3 also plays a similar role in mouse peritoneal macrophages. Figure 8 As shown in A to C, the protein levels of p-ERK, p-p38, and p-JNK in the MAPK pathway decreased significantly after Chrm3 was inhibited by the inhibitor 4-damp (P < 0.01, P < 0.01, P < 0.001); Figure 8 As shown in D to F, the protein levels of p-ERK, p-p38, and p-JNK were significantly reduced after interfering with the Chrm3 receptor (P < 0.01, P < 0.001, P < 0.01); while overexpression of Chrm3 significantly increased the protein expressions 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 receptor is involved in the LPS-stimulated Lbp - / - The MAPK signaling pathway in mouse cells mediates the occurrence and development of inflammatory responses.

[0079] 10. Co-immunoprecipitation

[0080] In order to determine how LPS achieves signal transduction through Chrm3, the present invention verifies the relationship between LPS and Chrm3 by immunoprecipitation.

[0081] Use cell lysis buffer to extract protein. Wash A / G magnetic beads 5 times with 1×TBST, add 5μg antibody, and rotate and mix at 4℃ for 3-4 hours. After taking out, wash with 1×TBST 5 times, discard the supernatant, and harvest the magnetic beads (antibody-magnetic bead coupling complex). Wash A / G magnetic beads 5 times with 1×TBST, add protein solution, and rotate and mix at 4℃ for 3-4 hours. Collect the supernatant. Combine the previously collected magnetic beads with the supernatant, and rotate and mix at 4℃ overnight. Wash with 1×TBST 5 times, discard the supernatant, and harvest the magnetic beads (protein-antibody-magnetic bead coupling complex). Then rotate and mix the protein-antibody-magnetic bead coupling complex with 1×loading buffer at room temperature for 20 minutes. Collect the supernatant and denature at 100℃ for 15 minutes. Perform SDS-PAGE electrophoresis on the denatured protein to detect the binding of Chrm3 protein and LPS. The results are as follows Fig. 9 As shown, it can be seen that when LPS is pulled down by the magnetic beads, the Chrm3 bound to it is also pulled down, indicating that LPS and Chrm3 bind to each other to transmit signals.

[0082] Example 2 In vivo experiment

[0083] 1. Preparation of Inflammation Mouse Model

[0084] In order to further study the role of Chrm3 receptor in mice, the mouse inflammation model was induced by intraperitoneal injection of LPS (10 mg / kg). The most appropriate stimulation time was screened by time gradient. - / - Mice were injected with the same dose of LPS at the same time and the expression of inflammatory factors in their livers was detected. The results showed that although the expression of inflammatory factors gradually decreased over time, the expression of inflammatory factors in WT mice and Lbp - / - The gap between mice is also narrowing. Therefore, we chose to construct a validation mouse model at the 6-hour time point of LPS stimulation for the next step of research.

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

[0086] The inhibitor 4-damp was injected into mice in advance at a concentration gradient of 1mg / kg, 5mg / kg, and 10mg / kg, and then LPS was injected for stimulation. The expression of inflammatory factors was used as an indicator to screen the best concentration of the inhibitor 4-damp. The results showed that as the concentration of the inhibitor increased, the expression of inflammatory factors decreased, so the concentration of the inhibitor in vivo was selected to be 10mg / kg.

[0087] 3. Infection of mice with overexpression lentivirus

[0088] The previously synthesized overexpression lentivirus was diluted to 1×10 7 TU concentration was injected into mice, and the mice were divided into control group (blank control), empty vector group (empty vector lentivirus injection), overexpression-24h group (overexpression lentivirus injection 24h), overexpression-48h group (overexpression lentivirus injection 48h), overexpression-72h group (overexpression lentivirus injection 72h), and then the mRNA expression level of Chrm3 was detected by RT-PCR to determine the appropriate overexpression time. The results showed that the expression of Chrm3 receptor had reached a very high level 72h after injection, so the overexpression time point of 72h was selected as the basis for subsequent experiments.

[0089] 4. Animal Grouping

[0090] (1) WT mice and Lbp - / - LPS stimulation time gradient experiment in mice: 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), and LPS-24h group (LPS stimulation for 24h).

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

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

[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 10 mg / kg was injected 1 hour after LPS stimulation), combined inhibition group (Ulinastatin 5×10 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 test showed that when Lbp - / - After LPS injection, the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the liver of mice was significantly increased (P < 0.0001). When pretreated with the inhibitor 4-damp, the expression of inflammatory factors was significantly decreased (P < 0.0001, P < 0.001, P < 0.0001). Fig.10 A~C; after the expression of Chrm3 was increased by overexpression lentivirus, the inflammatory factors in the mouse liver also increased significantly (P<0.0001, P<0.001, P<0.001). Fig.10 D~F.

[0096] LbP - / - The same results were obtained in mouse peritoneal macrophages. Fig.11 As shown in A-C, blocking Chrm3 by injecting inhibitors in advance can reduce the increase of inflammatory factors caused by LPS stimulation (P < 0.001, P < 0.01, P < 0.01); Fig.11 As shown in D to F, overexpression of Chrm3 receptor had the opposite result, that is, the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α were significantly increased (P < 0.001, P < 0.0001, P < 0.0001).

[0097] Therefore, it can be determined that Chrm3 receptor plays an important role 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 MAPK signaling pathway in vivo

[0099] In order to further verify whether Chrm3 regulates Lbp at the protein level - / - The expression of MAPK pathway-related factors in mice was first detected by detecting the protein phosphorylation levels of ERK, p38, and JNK in the liver. Fig.12 As shown in the figure, 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). - / - Chrm3 regulates LPS-induced cell necrosis via MAPK pathway in mouse liver injury.

[0100] Afterwards, in Lbp - / - The protein phosphorylation levels of ERK, p38, and JNK were also detected in mouse peritoneal macrophages. The same results were obtained. After inhibiting the expression of Chrm3, the protein phosphorylation levels induced by LPS were significantly reduced (P < 0.01, P < 0.01, P < 0.05). Fig.13 A~C; however, after overexpression of Chrm3, the protein phosphorylation level of MAPK pathway was further increased (P<0.05, P<0.01, P<0.01). Fig.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. When liver cells are damaged, these enzymes are released into the blood, so they are often used as indicators of liver damage. Myeloperoxidase (MPO), as an indicator of neutrophil surface activity, plays a key role in inducing oxidative stress and tissue damage in the inflammatory process. - / - Effects of Chrm3 on the activities of the above enzymes in mouse liver injury Fig.14 As shown by Fig.14 As can be seen from A to C, compared with the control group, the activities of ALT, AST, and MPO in the LPS group increased significantly (P < 0.0001, P < 0.001, P < 0.001), and after inhibiting Chrm3, their enzyme activities decreased significantly (P < 0.01, P < 0.01, P < 0.05). Fig.14 As shown in Figures D to 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, the most direct evidence is to evaluate the histological changes in the liver after LPS stimulation. Fig.15 As shown in the results, inflammatory infiltration, hepatocyte degeneration, hemorrhage and hepatic cord dissociation appeared in the liver of mice treated with LPS. The addition of the inhibitor effectively alleviated liver hemorrhage and inflammatory infiltration and restored the structure of the liver lobule. In addition, compared with the LPS group, the overexpression group further aggravated the histopathological damage of the liver, and increased the bleeding and tissue structure changes.

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

[0105] Ulinastatin (UTI) is a serine protease inhibitor that plays a protective role in multiple organs. UTI has strong anti-inflammatory and cytoprotective effects in various cell and animal models, and can regulate innate immunity and pro-inflammatory responses. The present invention used UTI, 4-damp, and UTI and 4-damp combined to intervene in LPS-treated WT mice, and found that the inflammatory factors in the combined treatment group were significantly reduced compared with other groups, such as Fig.16 At the same time, the present invention also detected liver injury indicators and found that the combined treatment group had a significantly better therapeutic effect on liver injury indicators than other groups. Fig.16 As shown in D~F.

[0106] The present invention is through Lbp - / - When mice were stimulated with LPS, it was found that the expression of Chrm3 protein increased significantly, and then the phosphorylation level of proteins related to the MAPK signaling pathway also increased significantly. After adding the inhibitor 4-damp to specifically inhibit the expression of Chrm3 protein, the phosphorylation level of proteins related to the MAPK pathway was significantly reduced, and the expression of inflammatory factors downstream of the pathway also decreased, and the pathological damage of liver tissue was improved and the biochemical indicators were reduced. On the contrary, after the injection of Chrm3 overexpression lentivirus, the damage to the liver was aggravated. Then we confirmed that LPS can bind to the Chrm3 receptor. Therefore, we speculate that LPS activates the conduction of the MAPK signaling pathway through the Chrm3 protein, thereby causing the production of inflammatory factors and leading to liver damage, indicating that Chrm3 may play a compensatory role for LBP protein.

[0107] In summary, the present invention discovered a new pathway for LPS signal transduction, which can bind to Chrm3 receptor and aggravate Lbp through MAPK pathway. - / - Sepsis-induced liver dysfunction and injury in mice. The present findings suggest that Chrm3 may be a promising therapeutic target for sepsis-induced liver injury.

[0108] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of an agent that inhibits Chrm3 receptor expression in the preparation of a drug for treating lipopolysaccharide-induced diseases.

2. The use according to claim 1, characterized in that: The related diseases include inflammation related diseases.

3. The use according to claim 2, characterized in that: The inflammation-related diseases include sepsis, hepatitis, liver dysfunction and liver injury.

4. The use according to claim 1, characterized in that: The reagents for inhibiting the expression of Chrm3 receptor include Chrm3 receptor antagonists and interfering RNA for inhibiting the expression of Chrm3 receptor.

5. The use according to claim 4, characterized in that: The Chrm3 receptor antagonist includes 4-damp.

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

7. The use according to claim 6, characterized in that: The active ingredient includes ulinastatin.

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