Application of curcumin analogue C1 in preparation of medicine for treating endotoxemia

Abstract

The invention relates to application of a curcumin analogue C1 in preparation of a medicine for treating endotoxemia, and relates to the technical field of biological medicines. According to the application, after an endotoxin-induced mouse model is intervened by adopting the curcumin analogue C1, TNF-alpha, IL-6, MCP-1, BUN and BNP in serum are all found to be remarkably reduced, and the pathological injury condition of lung tissues and the survival rate / clinical state of mice are all remarkably improved, the pharmacological value of the curcumin analogue C1 in endotoxemia is disclosed, and the curcumin analogue C1 can be used for treating endotoxemia. And a new thought is provided for treatment of inflammatory diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a curcumin analog C1 in the preparation of a drug for treating endotoxemia. Background Technology

[0002] Endotoxins, also known as lipopolysaccharides (LPS), are a major component of the cell walls of Gram-negative bacteria and possess strong pro-inflammatory properties. When the body is injured or infected, Gram-negative bacteria invade the bloodstream and multiply, producing endotoxins that lead to endotoxemia. Clinical manifestations include fever, tachycardia, hypotension, tachypnea, and leukocytosis. Endotoxins activate inflammatory cells to release inflammatory mediators, leading to systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MOPS), and even multiple organ failure (MOF), ultimately endangering life. Currently, the pathophysiological process and pathogenesis of endotoxemia remain unclear, and effective treatments are lacking. Increasing research has found that autophagy plays a crucial role in the pathophysiological process of endotoxemia.

[0003] Curcumin is a natural phenolic compound with the following structural formula: Figure 1 As shown in Figure A, its derivative, curcumin analog C1, is a potent and orally active transcription factor EB (TFEB) activator, and also a TFEB-dependent and mTOR-independent autophagy enhancer. Its structural formula is shown below. Figure 1 As shown in B, it can be used for research on neurodegenerative diseases. Compared with curcumin, it significantly enhances anti-inflammatory and anti-tumor activity. However, whether curcumin analog C1 can be used for endotoxemia and organ damage caused by endotoxins still needs further investigation. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by providing an application of curcumin analog C1 in the preparation of drugs for treating endotoxemia.

[0005] A first aspect of the present invention provides the use of curcumin analog C1 in the preparation of a medicament for treating endotoxemia, said curcumin analog C1 having the following structural formula:

[0006]

[0007] Formula I.

[0008] A second aspect of the invention provides the use of curcumin analog C1 in the preparation of a medicament for treating endotoxin-induced lung injury.

[0009] Preferably, in the above application technical solution, the active ingredient of the drug is curcumin analog C1.

[0010] Preferably, in the above application technical solution, the curcumin analog C1 has at least one of the following functions:

[0011] (1) Reduce or alleviate clinical symptoms;

[0012] (2) Reduce the expression levels of pro-inflammatory factors such as TNF-α, IL-6, and MCP-1 in serum;

[0013] (3) Promotes the autophagic degradation of PKM2;

[0014] (4) Improves tissue pathological damage.

[0015] Preferably, in the above application technical solution, the drug is a pharmaceutical composition.

[0016] Preferably, in the above application technical solution, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0017] Preferably, in the above application technical solution, the dosage form of the drug includes at least one of injection, oral liquid, pill, tablet, capsule, and granule.

[0018] Preferably, in the above application technical solution, the route of administration of the drug includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

[0019] In summary, this application includes at least one of the following beneficial technical effects:

[0020] This application, through an endotoxin-induced mouse model, found that after intervention with curcumin analog C1, serum TNF-α, IL-6, MCP-1, BUN, and BNP levels were significantly reduced, and lung tissue pathological damage and mouse survival / clinical status were significantly improved. This reveals the pharmacological value of curcumin analog C1 in endotoxemia and provides a new approach for the treatment of inflammatory diseases. Attached Figure Description

[0021] Figure 1 This is a comparison diagram of the structural formulas of curcumin and its analogue C1. Figure 1 A is the structural formula of curcumin. Figure 1 B is the structural formula of curcumin analog C1;

[0022] Figure 2 These are the clinical evaluation results of mice in the model group (LPS) and the drug treatment group (LPS+Curcumin analog C1) of this application. Figure 2 A represents the clinical score of the mouse. Figure 2B represents the result of mouse rectal temperature measurement;

[0023] Figure 3 These are the serum test results of four groups of mice in this application, among which... Figure 3 A represents the detection result of TNF-α in mouse serum. Figure 3 Figure B shows the results of IL-6 detection in mouse serum. Figure 3 C shows the detection results of MCP-1 in mouse serum. Figure 3 Figure D shows the results of dsDNA content detection in mouse serum. Figure 3 E shows the results of BUN content detection in mouse serum. Figure 3 F shows the results of BNP detection in mouse serum;

[0024] Figure 4 These are images showing the pathological results of lung tissue from four groups of mice in this application. Figure 4 Image A shows the results of HE staining. Figure 4 B represents the histological score;

[0025] Figure 5 These are the results of four mouse protein imprinting experiments (WB experiments) in this application, among which... Figure 5 A represents the expression bands of autophagy-related proteins. Figure 5 B is a quantitative analysis diagram of LC3B-II expression. Figure 5 C represents the quantitative analysis chromatogram of p62 expression. Figure 5 D is the PKM2 protein expression band diagram. Figure 5 E is a quantitative analysis graph of PKM2 expression. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should all be covered within the protection scope of this invention.

[0027] Unless otherwise specified, the reagents, instruments and equipment used in the following examples are all commercially available products. Other specific conditions not specified shall be performed according to standard conditions or the manufacturer's recommendations.

[0028] The Chinese definitions of chemical abbreviations in this application are based on common industry understanding. Unless otherwise specified, the experimental methods in the following examples are all conventional methods in the industry.

[0029] Example 1

[0030] Methods of this study: Thirty-two male mice were selected and randomly divided into four groups: normal control group (control), drug control group (Curcumin analog C1), model group (LPS), and drug treatment group (LPS+Curcumin analog C1). Clinical status was evaluated within 8 hours of drug injection. Mice were sacrificed 8 hours later, and serum and lung tissue samples were collected. HE staining was used to detect the pathological state of lung tissue. The expression levels of TNF-α, IL-6, and MCP-1 in serum and the content of dsDNA in serum were detected. The levels of disease markers BUN and BNP in serum were also detected.

[0031] The specific process is as follows:

[0032] 1. Materials

[0033] 1.1 Laboratory Animals

[0034] Animal source: 32 male C57BL / 6J mice aged 6-8 weeks were obtained from the Animal Experiment Center of Chongqing Medical University.

[0035] Husbandry conditions: Ensure mice have sufficient water and food and observe their weight, food intake, and water intake daily; keep them in an environment with a temperature of 22 ± 2℃ and a relative humidity of 50%~60%, and avoid prolonged exposure to strong light (suitable light avoidance time: 12 hours of light and 12 hours of darkness); for small mice, feed them peanuts, sunflower seeds, etc.; ensure the bedding is clean and comfortable, and change it every five days.

[0036] 1.2 Main Reagents

[0037] Lipopolysaccharide (LPS) (CAS: 01473370) is sourced from Sigma-Aldrich, USA.

[0038] Curcumin analogue C1 (CAS: 39777-61-2) is derived from Aladdin Pharmaceuticals, USA. Its structural formula is 1,5-bis-(2-methoxyphenyl)-pentadien-3-one, and its English name is 1,5-bis-(2-methoxyphenyl)-1,4-pentadien-3-one. Its structural formula is shown below (other aliases are not mentioned; please refer to the aliases published by Aladdin Pharmaceuticals):

[0039] .

[0040] 2. Methods

[0041] 2.1 Animal Model Construction

[0042] Thirty-two male mice were randomly and equally divided into four groups: a normal control group (control), a drug control group (Curcumin analog C1), a model group (LPS), and a drug treatment group (LPS + Curcumin analog C1). The mice were fed normally for seven days. On the eighth day, the animal model was constructed. The normal control group was fed normally, while the drug control and drug treatment groups were administered curcumin analog C1 via gavage (80 mg / kg, dissolved in DMSO) according to mouse weight. Half an hour later, the model and drug treatment groups were injected intraperitoneally with LPS (10 mg / kg, dissolved in physiological saline) according to mouse weight. The mice were placed in a constant-temperature operating room. Eight hours later, all experimental animals were sacrificed, and serum and lung tissue samples were collected for further experiments. The experimental procedure was approved by the Animal Protection and Utilization Committee of Chongqing Medical University and complied with animal experimental ethics standards.

[0043] 2.2 Clinical status assessment

[0044] At 0h, 2h, 4h, 6h and 8h after LPS injection, the rectal temperature of the mice was measured using a rectal thermometer (China Zhongqiao Co., Ltd.). The lower the body temperature of the mice, the worse their clinical condition and the more severe their inflammatory response. At the same time points, three blind evaluators scored the mice according to Table 1. The higher the score, the worse the clinical condition and the more severe the inflammatory response.

[0045] Table 1 Clinical Science Rating Scale

[0046] Score Hair Activity Respiration Posture Stool 1 Smooth Normal Normal Move or Stimulated Movement Slightly upright or wrinkled 2 Slow movement without stimulation Labored respiration Crouched mass Slightly loose, soft stool Not smooth, upright 3 Move only with stimulation Irregular respiration Severe diarrhea, loose stool - Do not move with stimulation 4 - Figure 2 - - -

[0047] 2.3 Enzyme-linked immunosorbent assay (ELISA)

[0048] After LPS injection into 8 mice, ocular blood was centrifuged at 12000g for 15 minutes at 4℃, and the supernatant serum was collected to detect the expression levels of serum IL-6, MCP-1, and TNF-α. Unused kits were stored at -80℃ for later use. The kits were from the QuantiCyto® ELISA series (MCP-1#EMC113.96, TNF-α#EMC102a, IL-6#EMC004.96) from China.

[0049] Preparation before testing: Remove the kit from the refrigerator 20 minutes in advance to allow it to equilibrate to room temperature; dilute the 20× concentrated wash buffer with double-distilled water to prepare a 1× working solution; Standards: Centrifuge at 1000 rpm for 1 min before opening, add 1.0 ml of universal standard and sample diluent to the lyophilized standard, let stand for 15 minutes until fully dissolved, then gently mix (concentration 1000 pg / ml), and then dilute as needed; Biotinylated antibody working solution: Dilute the 30× concentrated biotinylated antibody with biotinylated antibody diluent to prepare a 1× working solution 20 minutes before use, according to the amount required for the current test, and use on the same day; Enzyme conjugate working solution: Dilute the 30× concentrated enzyme conjugate with enzyme conjugate diluent to prepare a 1× working solution 20 minutes before use, according to the amount required for the current test, and use on the same day.

[0050] Operating Procedure: Remove the required strips from the sealed bag that has been equilibrated to room temperature. Return unused strips and desiccant to the aluminum foil bag, press firmly the self-sealing strip, seal the bag, and return to 4°C. Add standard and specimen general diluent to the blank wells, and specimen or different concentrations of standards (100 μL / well) to the corresponding wells. Seal the reaction wells with sealing tape and incubate at 37°C in the dark for 90 minutes. Prepare biotinylated antibody working solution 20 minutes in advance. Wash the plate 5 times. Add biotinylated antibody diluent to the blank wells, and biotinylated antibody working solution (100 μL / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate at 37°C in the dark for 60 minutes. Prepare enzyme conjugate working solution 20 minutes in advance. Incubate at room temperature (22-25°C) in the dark. Wash the plate 5 times. Add enzyme conjugate diluent to the blank wells, and enzyme conjugate working solution (100 μL / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate at 37 ℃ in the dark for 30 minutes; turn on the microplate reader, preheat the instrument, and set the detection program; wash the plate 5 times; add 100 μL of chromogenic substrate (TMB) per well and incubate at 37 ℃ in the dark for 15 minutes; add 100 μL of reaction stop solution per well, mix well, and immediately measure the OD450 value (within 3 minutes).

[0051] 2.4 Double-stranded DNA detection

[0052] The kit was sourced from Thermo Fisher Scientific (#Q33263). The main steps were as follows: Remove serum samples from the -80°C freezer and thaw on ice; remove the dsDNA kit from the 4°C freezer 20 minutes in advance and allow it to reach room temperature; prepare 1× TE working solution with pure water according to the instructions; then prepare the required reagent A working solution using 1× TE working solution according to the sample quantity, ensuring this step is performed in the dark; first, dilute the standards sequentially to the appropriate concentrations according to the instructions, adding 100 μL of the graded concentration of standard and the sample to be tested to each well. First, add the first sample to a 96-well plate; then add 100 μL of the prepared working solution A to each well, avoiding light and bubble formation, and gently shake to mix; preheat the microplate reader; seal the plate with a sealing strip, and incubate the plate in a 37°C water bath for 5 minutes; use the preheated microplate reader to detect the absorbance (OD) value of each sample at excitation light of 480 nm and emission light of 520 nm; set a standard curve based on the OD value and concentration of the standard, and substitute the OD value of the sample to be tested into the standard curve to calculate the concentration, thereby statistically analyzing the dsDNA content in the serum of each sample.

[0053] 2.5 Detection of Blood Urea Nitrogen (BUN) Content

[0054] The kit was obtained from Nanjing Jiancheng Biotechnology Co., Ltd. (#C013-2-1). The specific operating steps are as follows: Take mouse serum from -80℃ and thaw it on ice; prepare 10 mmol / L standard working solution and buffer enzyme solution according to the kit instructions, and use immediately after preparation; add 20 μL of double-distilled water to the blank well, add 20 μL of 10 mmol / L BUN standard working solution to the standard well, add 20 μL of the sample to be tested to the assay well, and then add 250 μL of buffer enzyme solution to each well in sequence. Add the sample first and then add the buffer enzyme solution in the order that cannot be reversed. Mix thoroughly, centrifuge, and place in a 37℃ water bath for 10 min. All samples were loaded into 2 mL EP tubes; the microplate reader was preheated in advance to prepare for the test; 500 μL of phenol chromogenic reagent was added to each well, followed by 500 μL of alkaline sodium hypochlorite to the EP tubes, and the mixture was thoroughly mixed and placed in a 37°C water bath for 10 min; 300 μL of the reaction solution was then added sequentially to an 8-tube array and immediately tested on the microplate, with an absorbance value of 630 nm.

[0055] The content is calculated using the following formula: Blood Urea Nitrogen (BUN) (mmol / L) = [(OD value of test well - OD value of blank well) / (OD value of standard well - OD value of blank well)] × standard concentration (10 mmol / L) × sample dilution factor.

[0056] 2.5 Detection of Brain Natriuretic Peptide (BNP) Content

[0057] The kit is from Wuhan Youersheng Technology Co., Ltd. (#SEA541Mu). The specific operating steps are as follows: Set up standard wells, sample wells, and blank wells. Set up 7 standard wells and add 100 μL of different concentrations of standard to each well. Add 100 μL of standard diluent to the blank wells and 100 μL of sample to the remaining wells. Cover the plate with a membrane and incubate at 37°C for 1 hour. Discard the liquid, shake off the excess water, and do not wash. Add 100 μL of detection solution A working solution (prepare immediately before use) to each well. Cover the plate with a membrane and incubate at 37°C for 1 hour. Discard the liquid in the wells. Wash each well with 350 μL of washing buffer and soak for 1-2 minutes. Gently tap the plate on absorbent paper to remove all liquid from the wells. Repeat the washing process 3 times. After the last wash, aspirate or pour out the remaining washing buffer, invert the plate on absorbent paper, and absorb all the remaining liquid from the wells. This process can also be completed using spray bottles, multi-channel pipettes, or automated plate washers. Add 100 μL of detection solution B working solution (prepared immediately before use) to each well, cover the plate with a membrane, and incubate at 37°C for 30 minutes. Discard the liquid in the wells, shake off excess water, and wash the plate 5 times. Add 90 μL of TMB substrate solution to each well, cover the plate with a membrane, and incubate at 37°C in the dark (control the reaction time to 10-20 minutes, do not exceed 30 minutes; stop the reaction when the first 3-4 wells show a clear gradient of blue, while the gradient is not obvious in the last 3-4 wells). Add 50 μL of stop solution to each well to stop the reaction; the blue color will immediately turn yellow. The order of adding the stop solution should be as similar as possible to the order of adding the substrate solution. If uneven color occurs, gently shake the plate to mix the solution evenly. After ensuring that there are no water droplets on the bottom of the plate and no air bubbles in the wells, immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using a microplate reader.

[0058] 2.6 Pathological examination of lung tissue

[0059] 2.6.1 Preparation of tissue sections

[0060] Left mouse lung tissue was fixed in 4% paraformaldehyde for at least 24 hours. The tissue was then removed, rinsed with running water, and dehydrated sequentially with 70%, 85%, 90%, and 100% ethanol concentrations, for 1 hour at each concentration. The dehydrated tissue was then placed in xylene I and xylene II staining tanks for 30 minutes each. Cleared samples were then placed in paraffin cups I, II, and III, immersing for 1 hour at each stage, for a total time of approximately 3 hours. Melted paraffin was poured into a paraffin frame, and the paraffin-immersed sample was quickly placed in the center and gently pressed flat until a clear paraffin film formed. The sample was then cooled in cold water. A 5μm thick paraffin section was cut using a paraffin microtome, gently placed in warm water to float, spread with a brush, and then slowly fixed onto a glass slide. The slide was then baked in a 50°C oven for 30 minutes to obtain a clear slide, which was then used as a sample for subsequent HE staining.

[0061] 2.6.2 Hematoxylin and eosin staining (HE staining)

[0062] Dewaxing the paraffin sections sequentially in xylene I and xylene II. After dewaxing, rehydrate the samples sequentially with gradient alcohol concentrations of 100%, 90%, 80%, and 70%, maintaining each step for 3 minutes in the correct order. Finally, rinse with tap water. Place the cleaned paraffin sections in a hematoxylin staining jar and let stand for 10 minutes until the cell nuclei are stained. Then, rinse the sections with tap water. Separate the pre-stained sections in 1% hydrochloric acid ethanol for approximately 3 seconds, then rinse with tap water. Wash with water; place the washed sections into a 0.5% eosin staining jar for about 3 to 5 minutes, and observe the cytoplasmic staining under a microscope. If the cytoplasm is stained, stop this step; immerse the stained sections in a gradient of alcohols (90%, 95%, and 100%) for dehydration, and finally clear the dehydrated sections with xylene I and II for 5-8 minutes; wipe the xylene around the tissue with filter paper, and place a drop of resin at the tissue edge, quickly cover with a coverslip, being careful to avoid air bubbles. Then place the sections at room temperature to air dry naturally. The HE staining experiment is complete.

[0063] Finally, the HE staining was observed under an optical microscope, and photographs were taken at different magnifications.

[0064] 2.7 Verification Experiment of Anti-inflammatory Properties of Curcumin Analog C1

[0065] The effects of curcumin analog C1 on the expression of autophagy-related and metabolism-related proteins in LPS-induced mouse lung tissue were investigated using Western blotting experiments.

[0066] The specific steps are as follows:

[0067] Lysing tissue proteins: 30 mg of lung tissue was weighed from each of the four groups of samples, and 300 μL of protein lysis buffer (6 μL protease inhibitor, 294 μL RIPA lysis buffer) was added (protease inhibitor was from Beyotime P1045, RIPA lysis buffer was from Beyotime P0013B). The lung tissue was minced and sonicated, AMPL 80%, on for 10 s, off for 10 s; centrifuged at 14000 RPM at 4℃ for 10 minutes, and the supernatant was collected. The supernatant was diluted 40 times and subjected to BCA assay (BCA kit from Thermo Scientific, 23225). The loading volume of 30 μg was calculated based on the OD value. The remaining protein was added to 5X protein loading buffer (Yaxin, LT103), and incubated in a metal bath at 99℃ for 10 minutes for electrophoresis.

[0068] Electrophoresis: Prepare a 12.5% ​​gel according to the instructions for Yamei (PG112). Lower gel: Mix 2 mL of lower gel solution + 2 mL of lower gel buffer + 40 μL of modified coagulant thoroughly and add it to the groove between the glass plates. Add anhydrous ethanol to flatten the liquid. Let it stand for 15 minutes, then discard the anhydrous ethanol. Add the upper gel solution (500 μL upper gel solution + 500 μL upper gel buffer + 10 μL modified coagulant), insert 10 combs, and let it stand for 15 minutes before electrophoresis.

[0069] Prepare 1.5 L of electrophoresis buffer, 4.53 g Tris (BioFroxx, 1115 KG001), 28.2 g Gly (BioFroxx, 1275 KG2P5), 1.5 g SDS (BioFroxx, 3250 GR500), and 1500 mL of pure water, and mix thoroughly. Remove the denatured protein sample and thaw slowly at room temperature. Centrifuge at high speed for 1 min before loading. Add different volumes of marker (Yamei, WJ102) to the outermost wells on both sides. Add 30 μg of sample to the remaining wells in sequence. After loading, cover the electrophoresis tank, connect the power supply, and set the parameters to: constant voltage 75 V. After the bromophenol blue indicator band enters the lower separating gel, change to constant voltage 100 V and continue electrophoresis. Stop electrophoresis when the marker bands are fully separated and the bromophenol blue indicator band is approximately 1 cm from the bottom edge of the glass plate.

[0070] Electrophoresis buffer preparation: 3.02g Tris, 14.4g Gly, 200ml methanol (Chuandong Chemical) were thoroughly mixed and then subjected to electrophoresis. Following the marking instructions, a PVDF membrane (Merck, IPVH00010) of appropriate size was cut out to the desired molecular weight. A "sandwich" electrophoresis clamp was constructed, starting from the black side and moving to the transparent side, following the order of sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad. During the process, air bubbles between the gel and the PVDF membrane were carefully removed. The transfer apparatus was connected to a power source, ensuring the connection was correct. Ice was added, and the power switch was turned on. The electrophoresis conditions were: constant current 250 mA, and transfer time set at a rate of 1 kDa per minute.

[0071] Preparation of TBST: 1.21g Tris, 8.77g NaCl, 1ml Tween, 1000ml pure water. After thorough mixing, take an appropriate volume of TBST according to the number of bands, add 5X protein-free rapid blocking buffer (Yamei Pharmaceuticals PS108) and block for 15 minutes. After blocking, immerse the bands in TBST.

[0072] Antibody incubation: Primary antibodies: The p62 (#18420-1-AP) antibody was purchased from Proteintech (Wuhan, China), the LC3B (#AF5225) antibody was provided by Beyotime Biotech (Shanghai, China), the PKM2 (#4053) antibody was purchased from Cell Signaling Technology (Danvers, MA, United States), and the β-actin (#4ab000001) antibody was provided by 4A Biotech (Beijing, China); prepared according to the number of bands and antibody instructions, and incubated the primary antibodies overnight.

[0073] Secondary antibodies: Biosharp goat anti-mouse IgG-HRP BL001A and Biosharp goat anti-rabbit IgG-HRP BL003A. Antibody working solutions were prepared according to the band count and antibody instructions, and incubated for two hours.

[0074] Luminescence: Development and detection were performed using ECL luminescent solution (biosharp, BL520A).

[0075] 3. Data Statistical Analysis

[0076] All experimental data were statistically analyzed using GraphPad Prism 9.0.0 and IBM SPSS Statistics 26 software. Since 32 is a small sample size, the Shapiro-Wilk test was used to test whether the data followed a normal distribution. If they did, one-way ANOVA and two-way ANOVA were used to compare the differences among the four groups. P < 0.05 was considered statistically significant.

[0077] 4. Results

[0078] 4.1 Clinical Results

[0079] Clinical evaluation results of mice in the model group (LPS) and the drug treatment group (LPS+Curcumin analog C1) are as follows: Figure 2 As shown, where Figure 2 A represents the clinical score of the mouse. Figure 3B shows the results of rectal temperature measurement in mice. As can be seen from the figure, after LPS modeling, the clinical score of mice increased with time, while the body temperature decreased with time. After intervention with curcumin analog C1, the initial clinical score and body temperature of mice were basically the same as those of LPS mice. However, as time increased, the overall clinical score of mice was lower than that of LPS mice. After 6 hours, the score showed a downward trend. The decrease in body temperature of mice was less than that of LPS mice, and the overall body temperature of mice was higher than that of LPS mice. This proves that curcumin analog C1 can indeed alleviate the clinical symptoms of LPS mice.

[0080] 4.2 Serum test results

[0081] The serum test results of the four groups of mice are as follows: Figure 3 As shown, where, Figure 3 A represents the detection result of TNF-α in mouse serum. Figure 3 Figure B shows the results of IL-6 detection in mouse serum. Figure 3 C shows the detection results of MCP-1 in mouse serum. Figure 3 Figure D shows the results of dsDNA content detection in mouse serum. Figure 3 E shows the results of BUN content detection in mouse serum. Figure 4 F shows the results of BNP detection in mouse serum.

[0082] As shown in the figure, the TNF-α level was significantly increased in the LPS group, while the TNF-α level in the LPS + curcumin analog C1 group was significantly decreased compared to the LPS group (P<0.05), indicating that curcumin analog C1 can reduce the LPS-induced increase in TNF-α levels and has an inhibitory effect on inflammation. The IL-6 level was significantly increased in the LPS group, while the IL-6 level in the LPS + curcumin analog C1 group was significantly lower than that in the LPS group (P<0.01), indicating that it can effectively inhibit the LPS-induced increase in IL-6 and reduce the inflammatory response. The MCP-1 level was significantly increased in the LPS group, while the MCP-1 level in the LPS + curcumin analog C1 group was significantly lower than that in the LPS group (P<0.01), further demonstrating that curcumin analog C1 has an inhibitory effect on LPS-induced TNF-α levels. The LPS group showed an inhibitory effect on the increase of inflammatory factors induced by LPS. The extracellular dsDNA content was increased in the LPS group, while the dsDNA content in the LPS + curcumin analog C1 group was significantly lower than that in the LPS group (P<0.01), suggesting that it may have an inhibitory effect on the release of dsDNA related to LPS-induced cell damage. The BUN level was increased in the LPS group, while the BUN level in the LPS + curcumin analog C1 group was significantly lower than that in the LPS group (P<0.01), indicating that it has an ameliorative effect on changes in renal function-related indicators induced by LPS. The BNP level was increased in the LPS group, while the BNP level in the LPS + curcumin analog C1 group was significantly lower than that in the LPS group (P<0.01), indicating that it has a certain ameliorative effect on changes in cardiac-related indicators induced by LPS.

[0083] This indicates that curcumin analog C1 has an inhibitory effect on LPS-induced inflammatory response (reducing TNF-α, IL-6, and MCP-1 levels), and improves LPS-induced cell damage (reducing dsDNA content), changes in renal function (reducing BUN levels), and changes in cardiac function (reducing BNP levels).

[0084] 4.3 Histopathological Results

[0085] The pathological results of lung tissue in the four groups of mice are as follows: Figure 4 As shown, where, Figure 4 Image A shows the results of HE staining. Figure 4 B represents the histological score.

[0086] Depend on Figure 4 As shown in A, the lung tissue pathology of the LPS group exhibited obvious damage characteristics, possibly including inflammatory cell infiltration and tissue structure destruction. In contrast, the lung tissue pathology of the LPS + curcumin analogue C1 group showed improvement compared to the LPS group, with a relatively more intact tissue structure and reduced inflammatory cell infiltration. Figure 5 As shown in B, the histological score of the LPS group was significantly higher (P<0.01), indicating that LPS caused severe pathological changes in the lung tissue. In contrast, the histological score of the LPS + curcumin analog C1 group was significantly lower than that of the LPS group (P<0.01), further indicating that curcumin analog C1 can alleviate the pathological damage to the lung tissue caused by LPS.

[0087] Based on the combined HE staining results and histological scoring results, curcumin analog C1 has an ameliorative effect on LPS-induced pathological damage to mouse lung tissue, reducing pathological changes such as inflammatory cell infiltration and tissue structure destruction in lung tissue, and lowering the histological score.

[0088] 4.4 Results of protein imprinting experiment

[0089] The results of the Western blot (WB) assay are as follows: Figure 5 As shown, where, Figure 5 A represents the expression bands of autophagy-related proteins. Figure 5 B is a quantitative analysis diagram of LC3B-II expression. Figure 5 C represents the quantitative analysis chromatogram of p62 expression. Figure 5 D is the PKM2 protein expression band diagram. Figure 5 E is a quantitative analysis graph of PKM2 expression.

[0090] Autophagy dysfunction is an important mechanism of inflammatory response, and transcription factor EB (TFEB) is a major regulator of autophagy. Therefore, activating autophagy to promote the degradation of key components of the inflammatory response and thus achieve anti-inflammatory effects is a novel anti-inflammatory intervention strategy. LC3B-I and LC3B-II are autophagy marker proteins; LC3B-II is a direct indicator of autophagosome formation. p62 is an autophagy substrate protein, and its accumulation reflects impaired autophagic flux. β-actin is an internal control protein used to correct loading volume. PKM2 plays a crucial role in the development and progression of endotoxemia and is related to cellular metabolism, inflammation, and autophagy regulation.

[0091] Depend on Figure 5 As shown in A, B, and C, the expression level of LC3B-II was low and the expression level of p62 was high in the LPS group, indicating that autophagic flux was inhibited. After intervention with curcumin analog C1, the LC3B-II expression level in the LPS group was significantly increased (*P<0.05, **P<0.01*), indicating that the formation of autophagosomes was increased. The expression level of p62 was significantly decreased, confirming that autophagic flux was activated.

[0092] Numerous studies have demonstrated that autophagy-driven PKM2 degradation is emerging as a novel pathway for controlling inflammatory responses, and this application also found ( ​ D and ​ In the E group, PKM2 expression was elevated in the LPS group, while treatment with curcumin analog C1 resulted in a decrease in PKM2. This suggests that curcumin analog C1 may inhibit endotoxemia by promoting the autophagic degradation of PKM2.

[0093] In summary, this application, through an endotoxin-induced mouse model, found that serum TNF-α, IL-6, MCP-1, BUN, and BNP levels were significantly reduced after intervention with curcumin analog C1. Furthermore, lung tissue pathological damage and mouse survival / clinical status were significantly improved, revealing the pharmacological value of curcumin analog C1 in endotoxemia and providing a new approach for the treatment of inflammatory diseases.

Claims

1. Use of a curcumin analogue C1 in the preparation of a medicament for the treatment of endotoxemia, characterized in that: The curcumin analog C1 has the following structural formula: Formula I.

2. Use of the curcumin analog C1 in the preparation of a drug for treating endotoxin-induced lung injury.

3. Use according to any one of claims 1-2, characterized in that: The active ingredient of the drug is the curcumin analog C1.

4. Use according to claim 3, characterized in that: The curcumin analog C1 has at least one of the following effects: (1) alleviating or relieving clinical symptoms; (2) reducing the expression levels of TNF-α, IL-6, MCP-1 and other pro-inflammatory factors in serum; (3) promoting autophagy degradation of PKM2; (4) improving histopathological injury.

5. Use according to claim 3, characterized in that: The drug is a pharmaceutical composition.

6. Use according to claim 5, characterized in that: The drug further comprises a pharmaceutically acceptable carrier or excipient.

7. Use according to claim 5, characterized in that: The dosage form of the drug comprises at least one of injection, oral liquid, pill, tablet, capsule, granule.

8. Use according to claim 5, characterized in that: The administration route of the drug comprises intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

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