Benzofuran derivatives and their use

Abstract

The application discloses a benzofuran derivative, which has a structure shown in formula (I), and discloses a pharmaceutical composition and application in preparation of anti-inflammatory drugs. The application can effectively inhibit LPS-induced inflammation in vivo and in vitro, and can play an anti-inflammatory role by inhibiting the release of inflammatory factors TNF-alpha and IL-6 through inhibition of NF-kappa B and MAPK signal pathways.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine. More particularly, the present application relates to a benzofuran derivative and its use. BACKGROUND

[0002] Inflammation is a self-defensive behavior of the body after being stimulated by external inflammatory factors. In most cases, inflammation is beneficial, but when the inflammatory response is excessive, it will cause excessive reaction and irreversible damage to the local tissues of the body, seriously affecting the health of the patient.

[0003] Anti-inflammatory drugs mainly act on the metabolic process of inflammatory mediators or their release, thereby playing an anti-inflammatory role. Anti-inflammatory drugs are divided into two categories according to their structure: one is steroidal anti-inflammatory drugs; the other is non-steroidal anti-inflammatory drugs. In the early 20th century, steroidal drugs such as adrenal cortex hormones were widely used in the treatment of inflammation and had good short-term efficacy, but long-term use of such drugs can produce strong drug dependence and serious side effects. In the mid-20th century, the research and development of anti-inflammatory drugs shifted to non-steroidal anti-inflammatory drugs. Non-steroidal anti-inflammatory drugs are the third largest class of drugs after vitamins and antibiotics due to their fast onset of action, clear efficacy, and good patient tolerance. However, non-steroidal anti-inflammatory drugs also have more adverse reactions in the gastrointestinal tract and have greater side effects, so there is an urgent need to find new anti-inflammatory drugs with obvious anti-inflammatory effects and fewer side effects. SUMMARY

[0004] The present application provides a benzofuran derivative and its use, which can effectively inhibit LPS-induced inflammation in vivo and in vitro, and may inhibit the release of inflammatory factors TNF-α and IL-6 by inhibiting the NF-κB and MAPK signaling pathways to play an anti-inflammatory role.

[0005] To achieve these objects and other advantages in accordance with the present application, a benzofuran derivative is provided, having the structure shown in formula (I):

[0006]

[0007] A pharmaceutical composition comprising a therapeutically effective amount of the benzofuran derivative and its salt, and a pharmaceutically acceptable carrier.

[0008] Preferably, the pharmaceutically acceptable carrier includes diluents, solubilizers, latent solvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, humectants, buffers, cross-linking agents.

[0009] Preferably, the pharmaceutical composition is prepared into a pharmaceutically acceptable dosage form.

[0010] Preferably, the dosage form comprises pills, tablets, powders, capsules, granules, powders, drop pills, drops, sprays, injections, suspensions, gels, suppositories.

[0011] Use of the benzofuran derivative or the pharmaceutical composition in the preparation of an anti-inflammatory drug.

[0012] The present application at least includes the following beneficial effects:

[0013] The present application takes C4-BM as the research object, and in vivo and in vitro explores the inhibitory effect of C4-BM on a LPS-induced RAW 264.7 macrophage inflammation model and a LPS-induced inflammation reaction of BALB / c mice in vivo, clarifies the anti-inflammatory effect of C4-BM and the possible mechanism, and provides a theoretical basis for treating inflammatory diseases by using the benzofuran derivative C4-BM.

[0014] In vitro experiment: a LPS-induced RAW 264.7 macrophage inflammation model is established, the influence of C4-BM on the activity of RAW 264.7 cells is determined by using an MTT method, the expression level of inflammatory factors in the supernatant of LPS-induced RAW 264.7 cells is detected by using a Griess method and an ELISA method, the levels of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) are detected by using a fluorescence microscope, the expressions of Ca 2+ , ROS and NO in cells are detected by using a flow cytometer, the nuclear translocation of NF-κB / p65 protein in cells is detected by using an immunofluorescence method, and the expressions of iNOS and COX-2 proteins and NF-κB and MAPK signaling pathway-related proteins in cells are determined by using a Western blot method. In vivo experiment: a BALB / c mouse inflammation model is established by intraperitoneal injection of LPS, the content of white blood cells (WBC), neutrophils (Neu) and lymphocytes (Lym) in blood is determined by using an automatic biochemical analyzer, the expressions of inflammatory factors in serum, lung and kidney tissues are detected by using an ELISA method, and the protective effect of C4-BM on lung and kidney tissues of LPS-induced inflammation mice is observed by using an H&E staining method. The anti-inflammatory activity of C4-BM and the possible mechanism are evaluated by combining in vivo and in vitro experiments.

[0015] MTT results in vitro showed that C4-BM had no cytotoxicity to RAW 264.7 cells at the concentration range of 20-80 μM (P>0.05), so 20, 40, 80 μM were used for the study of the anti-inflammatory activity of C4-BM; Griess results showed that compared with the normal control group, LPS significantly increased the content of Nitride in RAW 264.7 cells (P<0.001), and compared with the LPS model group, C4-BM could significantly inhibit the release of Nitride (P<0.001); ELISA results showed that compared with the normal control group, LPS significantly increased the levels of TNF-α and IL-6 in RAW 264.7 cells (P<0.001), and compared with the LPS model group, C4-BM could significantly inhibit the levels of TNF-α and IL-6 (P<0.001); JC-1 staining results showed that under the stimulation of LPS, the mitochondrial membrane potential of RAW 264.7 cells was significantly reduced, and a large amount of ROS was produced, while C4-BM treatment could reverse the loss of mitochondrial membrane potential in RAW 264.7 cells induced by LPS, and inhibit the production of ROS; flow cytometry results showed that under the stimulation of LPS, the contents of Ca 2+ , NO and ROS in RAW 264.7 cells were significantly increased (P<0.001), and compared with the LPS model group, C4-BM treatment could significantly inhibit the contents of Ca 2 +, NO, ROS release (P<0.001); immunofluorescence results showed that LPS stimulation made NF-κB / p65 from cytoplasm into the nucleus; and the pretreatment with C4-BM could effectively inhibit the transfer of NF-κB / p65 from cytoplasm to nucleus; Western blot results showed that after LPS stimulation of RAW 264.7 cells, the expression levels of iNOS, COX-2, p-IKKα / β, p-p65, p-IκBα, p-ERK and p-p38 proteins in cells were significantly increased (P<0.001), compared with the LPS model group, the low and high dose groups of C4-BM could reduce the expression levels of iNOS, COX-2, p-IKKα / β, p-p65, p-IκBα, p-ERK and p-p38 proteins to different degrees, especially the selective inhibition of the expression of COX-2 protein. The results of LPS-induced BALB / c mouse inflammation model in vivo showed that compared with the normal control group, under the induction of LPS, the contents of WBC, Neu and Lym in the blood of mice were significantly increased, the levels of BUN, CRE, TNF-α and IL-6 in serum were significantly increased, and the contents of TNF-α and IL-6 in lung tissue and kidney tissue were significantly increased (P<0.001), compared with the LPS model group, C4-BM treatment could significantly reduce the levels of WBC, Neu, Lym, TNF-α, IL-6, BUN and CRE; H&E staining results showed that C4-BM could reduce the infiltration of inflammatory cells in the lung and kidney tissues of LPS-induced inflammatory mice.

[0016] Other advantages, objects, and features of the application will be apparent from the following specification and appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 HRMS spectrum of C4-BM;

[0018] Figure 2 C4-BM 1 H-NMR spectrum of C4-BM;

[0019] Figure 3 C4-BM 13 C-NMR spectrum of C4-BM;

[0020] Figure 4 Effect of C4-BM on RAW 264.7 cell viability;

[0021] Figure 5 Effect of C4-BM on the release of Nitrite and NO of LPS-induced RAW 264.7 cells;

[0022] Figure 6Effect of C4-BM on LPS-induced TNF-α, IL-6 secretion in RAW 264.7 cells;

[0023] Figure 7 Effect of C4-BM on LPS-induced ROS production in RAW 264.7 cells;

[0024] Figure 8 Effect of C4-BM on LPS-induced mitochondrial membrane potential MMP and Ca 2+ level in RAW 264.7 cells;

[0025] Figure 9 Effect of C4-BM on LPS-induced iNOS, COX-2 protein expression in RAW 264.7 cells;

[0026] Figure 10 Effect of C4-BM on LPS-induced NF-κB / p65 nuclear translocation in RAW 264.7 cells;

[0027] Figure 11 Effect of C4-BM on LPS-induced key proteins of NF-κB pathway in RAW 264.7 cells;

[0028] Figure 12 Effect of C4-BM on LPS-induced key proteins of MAPK pathway in RAW 264.7 cells;

[0029] Figure 13 Effect of C4-BM on blood routine of LPS-induced inflammatory mice;

[0030] Figure 14 Effect of C4-BM on TNF-α, IL-6 content in serum of LPS-induced inflammatory mice;

[0031] Figure 15 Effect of C4-BM on TNF-α, IL-6 content in lung tissue of LPS-induced inflammatory mice;

[0032] Figure 16 Effect of C4-BM on TNF-α, IL-6 content in kidney tissue of LPS-induced inflammatory mice;

[0033] Figure 17 Effect of C4-BM on CRE, BUN content in serum of LPS-induced inflammatory mice;

[0034] Figure 18 Effect of C4-BM on lung tissue pathological morphology of LPS-induced inflammatory mice;

[0035] Figure 19 The effect of C4-BM on the pathological morphology of LPS-induced inflammatory mouse kidney tissue. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0037] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0038] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0039] Benzofuran derivatives, with the chemical formula C 32 H 32 N₂O₄, with a molecular weight of 508.2362, has the structure shown in formula (I):

[0040]

[0041] A pharmaceutical composition comprising a therapeutically effective amount of a benzofuran derivative of the structural formula shown in formula (I), a salt thereof, and a pharmaceutically acceptable carrier.

[0042] Pharmaceutically acceptable carriers include diluents, solubilizers, cosolvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, humectants, buffers, and crosslinking agents.

[0043] The pharmaceutical composition is formulated into a pharmaceutically permissible dosage form.

[0044] The dosage forms include pills, tablets, powders, capsules, granules, powders, drop pills, drops, sprays, injections, suspensions, gels, and suppositories.

[0045] The pharmaceutical preparations of the present invention are used in the form of a dose per unit body weight. The present invention is preferably administered to patients requiring treatment via oral or injectable administration. For oral administration, it can be formulated as tablets, sustained-release tablets, controlled-release tablets, capsules, pellets, microcapsules, suspensions, emulsions, powders or granules (nanoforms), oral liquids, etc.; for injectable administration, it can be formulated as sterile aqueous or oily solutions, sterile powder for injection, liposomes, or emulsions, etc.

[0046] Synthesis and Identification of Compound C4-BM

[0047] 1.1 Synthesis of C4-BM:

[0048]

[0049] wherein n = 6, R1= Ph.

[0050] 1.2 Identification of C4-BM:

[0051] The HRMS spectrum of C4-BM is shown in Figure 1 , 1 The H-NMR spectrum is shown in Figure 2 , 13 The C-NMR spectrum is shown in Figure 3 .

[0052] C4-BM: orange-yellow solid, yield 49%, m.p. 58.1-61.9 °C; HRMS (ESI) m / z: calcd for C 30 H 32 N2O4[M+H]+: 509.2362, found: 509.2437.1H-NMR (600 MHz, CDCI3) δ 8.00 - 7.95 (m, 2H), 7.78 (d, J = 7.8 Hz, 2H), 7.62 (s, 1H), 7.54 (s, 1H), 7.48 (d, J = 5.0 Hz, 3H), 7.42 - 7.35 (m, 3H), 7.25 - 7.20 (m, 2H), 6.94 (dd, J = 8.8, 1.3 Hz, 1H), 4.39 (q, J = 7.0 Hz, 2H), 4.02 (dt, J = 14.1, 6.6 Hz, 4H), 1.91 - 1.87 (m, 2H), 1.85 - 1.82 (m, 2H), 1.57 (dt, J = 15.3, 7.8 Hz, 2H), 1.44 (dt, J = 15.0, 7.7 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H).13C-NMR (151 MHz, CDCI3) δ 164.07, 161.35, 156.16, 148.81, 137.01, 130.18, 129.50, 128.66, 128.01, 126.99, 124.80, 114.62, 111.67, 108.94, 105.55, 68.32, 60.57, 47.43, 30.95, 29.12, 26.34, 25.71, 14.23.

[0053] 2C4-BM in vitro anti-inflammatory mechanism research

[0054] 2.1 Drug and cell lines

[0055] The experimental drug C4-BM was prepared by the aforementioned method (Section 1, synthesis and identification of compound C4-BM), and had an appearance of orange yellow powder and a content of more than 98% (high performance liquid chromatography (HPLC) pure); the experimental cell RAW 264.7 was a mouse monocyte macrophage cell line from the Cell Resource Center of Shanghai Branch of the Chinese Academy of Sciences.

[0056] 2.2 Experimental methods and steps

[0057] 2.2.1 MTT method for detecting the effect of the drug on cell viability

[0058] RAW 264.7 cells were inoculated in a 96-well plate at a concentration of 2×10 5 cells / well, incubated at 37°C in a 5% CO2 incubator for 12 h, and then treated with C4-BM (20, 40, and 80 μM) for 24 h. The supernatant in the wells was discarded, 100 μL of MTT working solution with a final concentration of 0.5 mg / mL was added to each well, and the OD value was detected at 490 nm. The relative viability of the cells was calculated as follows: cell relative viability (%) = OD (drug group) / OD (control group) × 100%.

[0059] 2.2.2 Griess reagent for determining the nitrite (Nitride) level in the cell supernatant

[0060] RAW 264.7 cells were inoculated in a 24-well plate at a concentration of 4×10 5 cells / mL, incubated at 37°C in a 5% CO2 incubator for 12 h, and then pretreated with C4-BM (20, 40, and 80 μM) for 1 h. A final concentration of 1 μg / mL of LPS inducer was added for co-incubation for 18 h. The cell supernatant (50 μL) of each group was collected in a 96-well detection plate, 50 μL of Griess reagent was added, and the plate was incubated at 37°C in the dark for 30 min. The OD value was determined at 540 nm.

[0061] 2.2.3 ELISA for determining the TNF-α and IL-6 content in the cells

[0062] RAW 264.7 cells were inoculated in a 24-well plate at a concentration of 4×10 5 cells / mL, incubated at 37°C in a 5% CO2 incubator for 12 h, and then pretreated with C4-BM (20, 40, and 80 μM) for 1 h. A final concentration of 1 μg / mL of LPS inducer was added for co-incubation for 18 h. The cell supernatant of each group was collected, and the TNF-α and IL-6 levels in the cells were detected according to the instructions of the ELISA kit.

[0063] 2.2.4 Fluorescent intracellular mitochondrial membrane potential MMP and reactive oxygen species ROS levels

[0064] RAW 264.7 cells were inoculated in a 24-well plate at a concentration of 2×105 RAW 264.7 cells were seeded in 96-well plates at 2 x 10

[0065] 2.2.5 Flow cytometry (FCM) detection of intracellular Ca 2+ , ROS, NO content

[0066] RAW 264.7 cells were seeded in 96-well plates at 2 x 10 5 2+ , ROS, NO content 2+

[0067] 2.2.6 Immunofluorescence (IF) detection of NF-κB / p65 protein nuclear translocation in cells

[0068] RAW 264.7 cells were seeded in confocal culture dishes at 1.5 x 10 5

[0069] 2.2.7 Western Blot analysis of intracellular protein expression

[0070] RAW 264.7 cells were seeded in 6-well plates at 3 x 10 5 ​​​The 6-well plates were inoculated and incubated at 37°C in a 5% CO2 incubator for 12 h. After 1 h of C4-BM (20, 40, and 80 μM) pretreatment, 1 μg / mL of LPS was added for co-incubation for 6 h. Western Blot was used to detect the expression levels of COX-2, iNOS, IKKα, IKKβ, p-IKKα / β, p65, p-p65, IκBα, p-IκBα, ERK, p-ERK, p38, p-p38, and GAPDH proteins.

[0071] 2.2.8 Statistical analysis of data

[0072] The experimental data were statistically analyzed using GraphPad Prism 8.0 software, and all results were presented in the form of mean ± standard deviation. The differences between groups were evaluated by one-way ANOVA, and P<0.05 was considered to be statistically significant.

[0073] 2.3 Experimental results

[0074] 2.3.1 Effect of C4-BM on the viability of RAW 264.7 cells

[0075] RAW 264.7 mononuclear macrophages were incubated with different concentrations of C4-BM drugs (final concentration of 20, 40, and 80 μM) for 24 h, and MTT was used to detect the cell viability. As shown in Figure 4 , the relative survival rates of cells in each drug concentration group were greater than 90%, and there was no significant difference compared with the normal control group (P>0.05), indicating that C4-BM had no toxic effect on RAW264.7 cells at concentrations of 20, 40, and 80 μM.

[0076] 2.3.2 Effect of C4-BM on the release of Nitride and NO in LPS-induced RAW 264.7 cells

[0077] The content of Nitride in the cell supernatant was detected by Griess reagent, and the results are shown in Figure 5 A. After LPS acted on RAW264.7 cells for 18 h, the content of Nitride in the supernatant of the model group cells significantly increased (P<0.001), and pretreatment with C4-BM significantly reduced the level of Nitride (P<0.001).

[0078] The flow results are shown in Figure 5B, 5C showed that after 8h of LPS induction, the NO content in the cells increased significantly (P<0.001), and the release of NO was significantly reduced (P<0.001) after C4-BM pretreatment. This indicated that C4-BM could inhibit the production and release of Nitrite and NO in LPS-induced RAW264.7 cells.

[0079] 2.3.3 Effect of C4-BM on TNF-α, IL-6 levels in LPS-induced RAW 264.7 cells

[0080] The content of TNF-α, IL-6 in the cell supernatant was determined by ELISA, and the results are shown in Figure 6 LPS significantly increased the release of TNF-α, IL-6 in RAW 264.7 cells (P<0.001); C4-BM pretreatment significantly reduced (P<0.001) the release of TNF-α, IL-6 in LPS-induced RAW 264.7 cells.

[0081] 2.3.4 Effect of C4-BM on ROS levels in LPS-induced RAW 264.7 cells

[0082] Fluorescence and flow detection of ROS levels in LPS-induced RAW 264.7 cells showed that Figure 7 LPS induction increased the level of ROS in the cells; C4-BM significantly inhibited the increase in ROS levels in LPS-induced cells.

[0083] 2.3.5 Effect of C4-BM on mitochondrial membrane potential MMP and Ca 2+ levels in LPS-induced RAW 264.7 cells

[0084] JC-1 staining results showed that Figure 8 LPS induction for 6h significantly reduced the mitochondrial membrane potential (red-green fluorescence ratio decreased significantly); compared with the model group, C4-BM (80μM) pretreatment could reverse the damage to the mitochondrial membrane potential in LPS-induced RAW264.7 cells (red-green fluorescence increased significantly). In addition, flow detection showed that LPS induced changes in intracellular Ca 2+ levels after 8h, and LPS (1μg / mL) induction significantly increased the intracellular Ca 2+ level (P<0.001), and C4-BM (20, 40, 80μM) significantly reduced the intracellular Ca 2+ level.

[0085] 2.3.6 Effect of C4-BM on iNOS, COX-2 protein expression in LPS-induced RAW 264.7 cells

[0086] Western blot results are shown in Figure 9 Figure 6. The protein levels of iNOS and COX-2 were low in normal RAW 264.7 cells. After 1 μg / mL LPS treatment for 18 h, the protein expression of iNOS and COX-2 was significantly increased (P < 0.001). C4-BM pretreatment significantly reduced the protein levels of iNOS and COX-2 (P < 0.001).

[0087] 2.3.7 Effect of C4-BM on LPS-induced NF-κB / p65 nuclear translocation in RAW 264.7 cells

[0088] NF-κB / p65 nuclear translocation can regulate the release of many inflammatory mediators, such as TNF-α, IL-6, IL-1β, NO, iNOS, etc. Immunofluorescence was used to detect the nuclear translocation of NF-κB / p65 protein in cells. As shown in Figure 10 Figure 7, under normal circumstances, NF-κB / p65 was in the cytoplasm of the cells. After LPS induction, NF-κB / p65 entered the nucleus from the cytoplasm (the fluorescence of NF-κB / p65 in the nucleus was significantly increased). C4-BM pretreatment significantly inhibited LPS-induced NF-κB / p65 nuclear translocation (the fluorescence of NF-κB / p65 in the nucleus was significantly reduced).

[0089] 2.3.8 Effect of C4-BM on LPS-induced key proteins of NF-κB pathway in RAW 264.7 cells

[0090] Western blot results are shown in Figure 11 Figure 8. After 1 μg / mL LPS treatment for 6 h, the protein expression of p-IKKα / β, p-p65 and p-IκBα was significantly increased (P < 0.001). C4-BM pretreatment reduced the protein levels of p-IKKα / β, p-p65 and p-IκBα in LPS-induced RAW 264.7 cells to different degrees. This indicates that C4-BM can effectively inhibit the phosphorylation of key proteins of the NF-κB pathway in LPS-induced RAW 264.7 cells, thereby exerting its anti-inflammatory activity.

[0091] 2.3.9 Effect of C4-BM on LPS-induced key proteins of MAPK pathway in RAW 264.7 cells

[0092] Western blot was used to detect the expression of key proteins of the MAPK signaling pathway in C4-BM, as shown in Figure 12As shown, compared with the normal control group, the expression of p-ERK and p-p38 in the cells was significantly increased after 1 pg / mL LPS for 6 h (P < 0.001), and compared with the model group, C4-BM pretreatment significantly reduced the level of p-ERK and p-p38 protein in LPS-induced RAW 264.7 cells (P < 0.001), indicating that C4-BM can effectively inhibit the phosphorylation of key proteins in the MAPK pathway in LPS-induced inflammatory RAW264.7 cells to exert its anti-inflammatory activity.

[0093] 3.2.2 Anti-inflammatory activity of C4-BM in LPS-induced inflammatory mice in vivo

[0094] 3.1 Drugs and animals

[0095] The experimental drug C4-BM was prepared by the aforementioned method (Section 1, Synthesis and Identification of Compound C4-BM), and its appearance was orange-yellow powder with a content of more than 98% (high-performance liquid chromatography (HPLC) pure).

[0096] The experimental animals were SPF grade BALB / c healthy mice (male, 6-8 weeks, 18-22 g) from Hunan Sylex Jingda Experimental Animal Co., Ltd. (Production License No.: SCXK (Hunan) 2019-0004).

[0097] After all the animals were purchased, they were housed in the Guangxi Center for Scientific Experiments of Traditional Chinese Medicine standard laboratory mouse room, with an environmental temperature of 18-24°C, a relative humidity of 45%-70%, and a strict control of 12h / 12h day / night alternating rhythm. During this period, they were fed with regular feed and pure water, and they were free to eat and drink.

[0098] 3.2 Experimental methods and steps

[0099] 3.2.1 Experimental dose design and preparation of drug solution

[0100] Test drug: C4-BM

[0101] Test drug dose design: Based on the previous laboratory team's research on lead compounds and the experimental results of the same mother nucleus compound, it was determined that the mouse dose should not be more than 40 mg / kg, while following the principles of safety, effectiveness, economy, and appropriateness of clinical application of traditional Chinese medicine, combined with the grouping of animal experiments, two dose groups were set up, with low dose group 5 mg / kg and high dose group 10 mg / kg.

[0102] Drug solution preparation: Accurately weigh an appropriate amount of C4-BM sample powder and dissolve it in pharmaceutical-grade soybean oil (the sample is insoluble in aqueous solvents and DMSO is highly toxic to animals; soybean oil can dissolve it without the need to add other co-solvents). Seal the container, wrap the outer wall with tin foil to protect it from light, and sonicate thoroughly by inverting the container to ensure uniform drug concentration. Prepare low- and high-dose drug solutions for mice with concentrations of 5 mg / kg and 10 mg / kg, respectively. Prepare the solution immediately before use.

[0103] Modeling agent: Lipopolysaccharide (LPS)

[0104] Based on the previous experimental dosage screening results of the research group, a concentration of 4 mg / kg was selected for LPS modeling. An appropriate amount of LPS powder was accurately weighed, dissolved in sterile saline for injection, and prepared into a 4 mg / kg LPS solution. The solution was prepared fresh for each use and protected from light throughout the process.

[0105] Positive test result: Dexamethasone (Dex) injection

[0106] The dosage design was based on careful reading of the drug instructions. The clinical injection dose for adults (60kg) was determined to be 30mg / day, which is equivalent to a daily dose of approximately 5mg / kg for mice. An appropriate amount of the purchased dexamethasone sodium phosphate injection (1mL: 5mg) was precisely pipetted and diluted with sterile saline for injection to the above concentration to obtain the experimental drug solution. The solution was prepared fresh for each use.

[0107] 3.2.2 Animal grouping, model establishment, and drug administration

[0108] After purchase, the mice were housed normally in the mouse house for 3 days to allow them to acclimatize to the experimental environment. Then, 40 mice were weighed and randomly divided into 5 groups of 8 mice each (with uniform weight distribution). Each mouse was labeled and assigned to one of the following groups: normal control group, model control group (LPS 4 mg / kg), positive control group (LPS + Dex 5 mg / kg), low-dose group (LPS + C4-BM 5 mg / kg), and high-dose group (LPS + C4-BM 10 mg / kg). Figures 13-17 The terms Control, LPS (4 mg / kg), Dex (5 mg / kg), C4-BM (5 mg / kg), and C4-BM (10 mg / kg) are used respectively. Figures 18-19 The terms are Control, LPS (4 mg / kg), LPS (4 mg / kg) + Dex (5 mg / kg), LPS (4 mg / kg) + C4-BM (5 mg / kg), and LPS (4 mg / kg) + C4-BM (10 mg / kg), respectively.

[0109] All mice were deprived of food and water for 12 h before modeling. At the beginning of the experiment, all mice except the normal control group were injected intraperitoneally with LPS (4 mg / kg) to induce inflammation. The normal control group was injected with an equal volume of sterile saline instead of LPS. After injection, the mice were allowed to eat and drink freely.

[0110] The low-dose group (LPS+C4-BM 5 mg / kg), high-dose group (LPS+C4-BM 10 mg / kg), and positive control group (LPS+Dex 5 mg / kg) were injected intraperitoneally with the corresponding drugs at 2 h before modeling and at 4 h and 10 h after modeling, respectively. The normal control group and model control group (LPS 4 mg / kg) were injected intraperitoneally with an equal volume of sterile saline at the same time points.

[0111] 3.2.3 Sample collection and processing

[0112] Blood samples:

[0113] After 12 h of LPS intraperitoneal injection modeling, the mice were euthanized by removing the eyeball to collect blood. Immediately, 45 μL of whole blood was taken for routine blood analysis.

[0114] Next, the mouse blood was collected in a 1.5 mL EP tube, and the serum was allowed to separate at room temperature for 2 h. Then, the serum was centrifuged at 4°C and 3000 r / min for 15 min. The upper serum was transferred to another 1.5 mL EP tube and stored in a -80°C freezer for subsequent determination of related inflammatory factors in the serum.

[0115] Tissue samples:

[0116] After blood collection, the mice were euthanized by dislocation and placed on ice. The lung and kidney tissues were then separated and removed, and the attached fat and fascia were removed. The tissues were washed with pre-cooled physiological saline and the surface moisture was absorbed with filter paper. The right lung and right kidney were placed in pre-prepared 4% paraformaldehyde fixing solution and stored at room temperature for subsequent H&E staining and pathological observation.

[0117] The remaining lung and kidney tissues were precisely weighed and packed in 1.5 mL EP tubes. Pre-cooled normal saline was added at a ratio of 1:9 (tissue weight: solution volume (mg:mL)), and the same number of magnetic beads were added to the EP tube. The mixture was ground thoroughly using a tissue grinder (operated on ice) to obtain a 10% tissue homogenate. The mixture was centrifuged at 4°C and 10,000 r / min for 15 min, and the supernatant was transferred to another 1.5 mL EP tube and stored in a -80°C refrigerator. The supernatant was used for subsequent determination of related inflammatory factors in the tissue.

[0118] 3.2.4 Detection of inflammatory factors in serum, lung and kidney tissues

[0119] The levels of inflammatory factors in serum, lung and kidney tissues were detected by ELISA according to the instructions.

[0120] 3.2.5 HE staining and pathological morphological observation of lung and kidney tissues

[0121] The lung and kidney tissues previously soaked in 4% paraformaldehyde for 24 h were transferred to a tube containing fresh 4% paraformaldehyde fixative to denature and coagulate the proteins in the tissues and cells for HE staining.

[0122] 3.2.6 Statistical analysis of data

[0123] The experimental data were statistically analyzed using GraphPad Prism 8.0 software, and all results were presented in the form of mean ± standard deviation. The differences between groups were evaluated by one-way ANOVA, and P<0.05 was considered to be statistically significant.

[0124] 3.3 Experimental results

[0125] 3.3.1 Effect of C4-BM on blood routine of LPS-induced inflammatory mice

[0126] The results of blood routine determination are as follows Figure 13As shown, compared with the normal control group, the number of white blood cells (WBC), neutrophils (Neu), and lymphocytes (Lym) in the blood of mice in the model control group (LPS 4 mg / kg) was significantly increased, and the difference was statistically significant (P<0.001), indicating that the LPS-induced inflammation mouse model has been successfully established. Compared with the model control group (LPS 4 mg / kg), the levels of WBC, Neu, and Lymd in the positive control group (LPS+Dex 5 mg / kg), the low-dose group (LPS+C4-BM 5 mg / kg), and the high-dose group (LPS+C4-BM 10 mg / kg) were all decreased to varying degrees, and the differences were statistically significant (P<0.05). This indicates that C4-BM can reduce the number of white blood cells, neutrophils, and lymphocytes in LPS-induced inflammation mice.

[0127] 3.3.2 Effects of C4-BM on inflammatory factors in serum, lung, and kidney tissues of LPS-induced inflammatory mice

[0128] 3.3.2.1 Effects of C4-BM on serum TNF-α and IL-6 levels in LPS-induced inflammatory mice

[0129] ELISA results as follows Figure 14 As shown, compared with the normal control group, the levels of inflammatory factors TNF-α and IL-6 in the model control group (LPS 4 mg / kg) were significantly increased (P<0.001), indicating that the inflammation model was successfully established. Compared with the model control group (LPS 4 mg / kg), the levels of TNF-α and IL-6 in the positive control group (LPS+Dex 5 mg / kg), the low-dose group (LPS+C4-BM 5 mg / kg), and the high-dose group (LPS+C4-BM 10 mg / kg) were significantly decreased, and the differences were statistically significant, indicating that C4-BM can reduce the expression of inflammatory factors in the serum of LPS-induced inflammatory mice.

[0130] 3.3.2.2 Effects of C4-BM on TNF-α and IL-6 levels in LPS-induced inflammatory mouse lung tissue

[0131] like Figure 15As shown, compared with the normal control group, the levels of inflammatory factors TNF-α and IL-6 in the model control group (LPS 4 mg / kg) were significantly increased (P<0.001); compared with the model control group (LPS 4 mg / kg), the levels of TNF-α and IL-6 in the positive control group (LPS+Dex 5 mg / kg), the low-dose group (LPS+C4-BM 5 mg / kg), and the high-dose group (LPS+C4-BM 10 mg / kg) were significantly decreased, and the differences were statistically significant (P<0.05), indicating that C4-BM can inhibit the release of TNF-α and IL-6 in the lung tissue of LPS-induced inflamed mice.

[0132] 3.3.2.3 Effects of C4-BM on TNF-α and IL-6 levels in the kidney tissue of LPS-induced inflamed mice

[0133] like Figure 16 As shown, compared with the normal control group, the levels of inflammatory factors TNF-α and IL-6 in the model control group (LPS 4 mg / kg) were significantly increased (P<0.001); compared with the model control group (LPS 4 mg / kg), the levels of TNF-α and IL-6 in the positive control group (LPS+Dex 5 mg / kg), the low-dose group (LPS+C4-BM 5 mg / kg), and the high-dose group (LPS+C4-BM 10 mg / kg) were significantly decreased, and the differences were all statistically significant (P<0.05). This indicates that C4-BM has an inhibitory effect on inflammatory factors in the kidney tissue of LPS-induced inflamed mice.

[0134] 3.3.2.4 Effects of C4-BM on serum CRE and BUN levels in LPS-induced inflammatory mice

[0135] like Figure 17 As shown, compared with the normal control group, the serum creatinine (CRE) and blood urea nitrogen (BUN) levels in the model control group (LPS 4 mg / kg) were significantly increased (P<0.001); compared with the model control group (LPS 4 mg / kg), the serum CRE and BUN levels in the positive control group (LPS+Dex 5 mg / kg), the low-dose group (LPS+C4-BM 5 mg / kg), and the high-dose group (LPS+C4-BM 10 mg / kg) were all significantly decreased (P<0.001). This indicates that C4-BM can reduce the expression levels of inflammatory mediators creatinine and blood urea nitrogen during LPS-induced nephritis.

[0136] 3.3.3 Effects of C4-BM on the histopathological morphology of LPS-induced inflammatory mice

[0137] 3.3.3.1 Effects of C4-BM on the pathological morphology of LPS-induced inflammatory mouse lung tissue

[0138] HE staining results are shown in Figure 18 The lung tissue structure of the normal control group mice was arranged in a clear grid, was evenly distributed, and had no obvious inflammatory cell infiltration. No inflammatory pathological changes were observed in the tissue, and the tissue morphology was normal. Compared with the normal control group, the lung tissue grid of the model control group (LPS 4 mg / kg) mice disappeared, the alveolar structure was destroyed, a large number of inflammatory cells infiltrated the lung interstitium, the lung wall was significantly thickened, and a large number of red blood cells exuded. The lung tissue structure was obviously destroyed, indicating that the LPS-induced mouse inflammation model was successfully constructed. Compared with the model control group (LPS 4 mg / kg), the number of inflammatory cells and red blood cells in the lung interstitium of the positive control group (LPS+Dex 5 mg / kg) and the low-dose group (LPS+C4-BM 5 mg / kg) and the high-dose group (LPS+C4-BM 10 mg / kg) mice was significantly reduced, and a clear grid was evenly distributed. The lung tissue morphology was normal. It can be seen from the pathological section results that C4-BM can reduce the damage of LPS-induced inflammatory mouse lung tissue, and its effect is equivalent to that of dexamethasone positive drug. It shows that C4-BM has an anti-pneumonia effect.

[0139] 3.3.3.2 Effect of C4-BM on the pathological morphology of LPS-induced inflammatory kidney tissue in mice

[0140] The pathological section results of the kidney tissue are shown in Figure 19 The kidney tissue structure of the normal group mice was clear and complete. The glomerular structure composed of capillaries was visible in the renal cortical area. A large number of epithelial cells were visible in the interstitial space of the glomerulus, and the proximal tubules with brush border and epithelial cells were cuboidal epithelial cells. The distal tubules without brush border structure, the tissue had no inflammatory, necrotic, and hemorrhagic pathological changes, and the tissue morphology was normal. In the model control group (LPS 4 mg / kg), partial renal tubular damage, renal tubular epithelial cell brush border shedding, partial glomerular atrophy, and a significant decrease in the number of intraglomerular capillaries were observed. Diffuse inflammatory cell infiltration was observed in the interstitial space of some renal tubules, and the tissue morphology was severely damaged, indicating that the LPS-induced inflammation model was successfully constructed. Compared with the model control group (LPS 4 mg / kg), the degree of kidney tissue damage in the positive control group (LPS+Dex 5 mg / kg) and the low-dose group (LPS+C4-BM 5 mg / kg) and the high-dose group (LPS+C4-BM 10 mg / kg) mice was significantly reduced. Partial renal tubular injury, renal tubular epithelial cell brush border shedding, and no inflammatory, necrotic, and hemorrhagic pathological changes were observed in the tissue. The tissue morphology was normal and relatively complete, and the degree of kidney tissue damage was significantly improved. It shows that C4-BM can alleviate LPS-induced kidney injury in mice.

[0141] 4Conclusion

[0142] In vitro LPS-induced RAW 264.7 cell inflammation model study found that C4-BM had no toxic effect on cells at 20, 40, 80 μM concentrations (P>0.05); C4-BM could significantly inhibit the release of LPS-induced RAW 264.7 cell inflammatory factors Nitride, NO, TNF-α, IL-6 (P<0.001), while inhibiting the expression of iNOS and COX-2 protein (P<0.001), especially for COX-2 protein may have a selective inhibitory effect; inhibit the nuclear translocation of NF-κB / p65 protein; inhibit the expression of p-IKKα / β, p-p65, p-IκBα, p-ERK and p-p38 protein (P<0.001); the results show that C4-BM has anti-inflammatory activity, and its mechanism of action may be through inhibiting the nuclear translocation of NF-κB, inhibiting the expression of key proteins in the NF-κB and MAPK signaling pathways to inhibit the release of inflammatory factors.

[0143] In vivo LPS-induced BALB / c mouse inflammation model study found that C4-BM could significantly reduce the levels of WBC, Neu, Lym and the contents of TNF-α, IL-6, CRE and BUN in LPS-induced BALB / c mice, and could also reduce the damage of LPS-induced BALB / c mouse lung tissue and kidney tissue, further confirming that C4-BM has anti-inflammatory effect.

[0144] The number of devices and the scale of processing described herein are intended to be illustrative of aspects of the present application. Changes in, or substitutions of, known equivalents for, each can be readily accommodated by those skilled in the art without departing from the application.

[0145] While the embodiments of the application have been disclosed as above, it is not limited to the application and implementation listed in the specification and embodiments, and can be fully applied to various fields suitable for the application, and additional modifications can be easily realized by those skilled in the art, therefore the application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and equivalent scope.

Claims

1. The use of a benzofuran derivative or a pharmaceutical composition comprising a therapeutically effective amount of said benzofuran derivative and its salts, and a pharmaceutically acceptable carrier, in the preparation of a medicament for treating pneumonia or alleviating LPS-induced inflammatory kidney injury in mice, characterized in that, The benzofuran derivative has the structure shown in formula (I): （I）。 2. The application as described in claim 1, characterized in that, Pharmaceutically acceptable carriers include diluents, solubilizers, cosolvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, humectants, buffers, and crosslinking agents.

3. The application as described in claim 1, characterized in that, The pharmaceutical composition is formulated into a pharmaceutically permissible dosage form.

4. The application as described in claim 3, characterized in that, The dosage forms include pills, tablets, powders, capsules, granules, powders, drops, sprays, injections, suspensions, gels, and suppositories.

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