Application of genipin β-methyl derivative Gen-17 in the preparation of drugs for preventing or treating acute severe pneumonia

Abstract

The present invention relates to the field of medical technology, and specifically to the use of a β-methyl derivative of genipin, Gen‑17, in the preparation of a drug for preventing or treating acute severe pneumonia. The present invention simulates human acute severe pneumonia for the first time with an acute lung injury mouse model, and systematically evaluates the preventive and therapeutic effects of a β-methyl derivative of genipin, Gen‑17, on mice with acute lung injury. The results show that: the β-methyl derivative of genipin, Gen‑17, can significantly reduce the pathological morphological damage of the lung tissue of ALI mice induced by LPS, and inhibit the inflammatory response and oxidative stress of the lung tissue; the protective mechanism of Gen‑17 on the lungs of ALI mice is: it exerts an anti-inflammatory effect by inhibiting the NF‑κB and MAPK signaling pathways, and exerts an antioxidant effect by activating the Keap1 / Nrf2 / HO‑1 signaling pathway. The present application can provide a certain experimental basis for the use of Gen‑17 in the prevention and treatment of acute severe pneumonia.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of a genipin β-methyl derivative Gen-17 in the preparation of a drug for preventing or treating acute severe pneumonia. Background Art

[0002] Acute severe pneumonia is a common clinical critical illness caused by a variety of reasons (bacterial or viral infection, trauma, etc.), with high morbidity and mortality, seriously threatening the life safety of patients. A variety of pathogenic factors directly or indirectly cause acute lung injury (ALI), systemic inflammatory response syndrome or multiple organ dysfunction syndrome, which is mainly manifested clinically as refractory hypoxemia, severe edema in the alveoli and lung parenchyma, and may lead to acute respiratory distress syndrome (ARDS) in severe cases.

[0003] Acute lung injury (ALI) is a common clinical acute severe pneumonia with complex etiology. The occurrence of ALI is related to environmental factors, severe lung infection, lung trauma, acute pancreatitis, drowning, sepsis, genetics, drugs and other factors. Although ALI / ARDS has been studied in depth at home and abroad in recent years, due to its complex etiology and unclear pathogenesis, there is no clear clinical cure, which makes the overall quality of life and survival rate of ALI / ARDS patients low. Therefore, it is necessary to actively explore and develop new and efficient drugs to reverse ALI.

[0004] Although a variety of comprehensive treatments are used for acute severe pneumonia in clinical practice, it is usually difficult for patients' lung function to return to normal levels after treatment. At present, the main anti-inflammatory drugs widely used in clinical treatment of acute severe pneumonia are dexamethasone (DEX), prednisolone, prednisone and ulinastatin. However, the quality of life and mortality of patients have not been substantially improved, and these drugs can also cause various adverse side effects, including decreased immune function, coagulation disorders, gastric ulcers and osteoporosis. Therefore, it is urgent to find new drugs with high efficiency and low toxicity for the prevention and treatment of acute severe pneumonia. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a use of a β-methyl derivative of genipin, Gen-17, in the preparation of a drug for preventing or treating acute severe pneumonia.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0007] Provided is a use of a genipin β-methyl derivative Gen-17 in the preparation of a drug for preventing or treating acute severe pneumonia, wherein the chemical structure of Gen-17 is:

[0008]

[0009] Furthermore, the medicine is a tablet, capsule, drop, pill, sustained-release or controlled-release preparation, injection, dry powder inhaler, metered-dose inhaler, or aerosol.

[0010] The beneficial effects of the present invention are:

[0011] For the first time, an acute lung injury mouse model was used to simulate human acute severe pneumonia, and the preventive and therapeutic effects of genipin β-methyl derivative Gen-17 on acute lung injury mice were systematically evaluated. The results showed that genipin β-methyl derivative Gen-17 could significantly reduce the pathological morphological damage of lung tissue in ALI mice induced by LPS, and inhibit the inflammatory response and oxidative stress of lung tissue; the protective mechanism of Gen-17 on the lungs of ALI mice was: it exerted anti-inflammatory effects by inhibiting the NF-κB and MAPK signaling pathways, and exerted antioxidant effects by activating the Keap1 / Nrf2 / HO-1 signaling pathways. This application can provide certain experimental basis for the application of Gen-17 in the prevention and treatment of acute severe pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a gross specimen of mouse lung tissue;

[0013] Figure 2 is the mouse lung coefficient ( n=6);

[0014] Figure 3 This is the pathological picture of mouse lung tissue;

[0015] Figure 4 The results of pathological scoring of mouse lung tissue;

[0016] Figure 5 is the total protein concentration of BALF ( n=6);

[0017] Figure 6 is the total cell count in BALF ( n=6);

[0018] Figure 7 The levels of TNF-α, IL-1β and IL-6 in BALF ( n=6);

[0019] Figure 8 Effect of Gen-17 on the expression of inflammatory mediator genes in lung tissue of ALI mice ( n=6);

[0020] Fig. 9 The expression of NF-кBp65 protein in lung tissue ( n=6);

[0021] Fig.10 The expression of p38, ERK, JNK and their phosphorylated proteins in lung tissue ( n=3);

[0022] Fig.11 The content of SOD and MDA in lung tissue ( n=6);

[0023] Fig.12 Effect of Gen-17 on the gene expression of oxidative stress factors in lung tissue of ALI mice ( n=6);

[0024] Fig.13 Keap1, HO-1, and Nrf2 protein expressions in lung tissues ( n=3);

[0025] in, Figure 2 , Figure 4-13 Compared with the NS group, * P<0.05, ** P<0.01, *** P<0.001; compared with the LPS group, # P<0.05, ## P<0.01, ### P<0.001; compared with the DEX group, ★ P<0.05, ★★ P<0.01, ★★★ P<0.001; compared with the low-dose group, ▲ P<0.05, ▲▲ P<0.01, ▲▲▲ P<0.001. DETAILED DESCRIPTION

[0026] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0027] Example 1

[0028] Synthesis of Genipin β-methyl derivative Gen-17:

[0029]

[0030] Structural characterization of the synthesized Gen-17:

[0031] Yellowish-brown oily liquid, 75% yield. 1H NMR (500 MHz, CDCl3) δ 8.17 (dd, J = 2.1, 0.9 Hz, 1H), 8.00–7.96 (m, 1H), 7.69–7.64 (m, 1H), 7.52 (s, 1H), 7.31 (t, J = 7.9 Hz, 1H), 5.93 (s, 1H), 4.99 (d, J = 13.8 Hz, 1H), 4.94–4.86 (m, 1H), 4.50 (d, J = 8.0 Hz, 1H), 3.71 (s, 3H), 3.56 (s, 3H), 3.27–3.18 (m, 1H), 2.91 (dd, J = 16.8, 8.5 Hz, 1H), 2.68 (t, J = 7.9 Hz, 1H), 2.16–2.05 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 167.7, 164.9, 152.2, 137.9, 136.0, 132.6, 132.1, 131.0, 130.0, 128.2, 122.5, 110.8, 102.7, 63.4, 57.2, 51.3, 46.3, 39.0, 35.5. HRMS (ESI) calcd. for C19H19BrNaO6 [M+Na]+: 445.0263, found 445.0266.

[0032] Example 2

[0033] Application of genipin β-methyl derivative Gen-17 in acute severe pneumonia:

[0034] 1 Experimental materials

[0035] 1.1 Experimental animals

[0036] 144 SPF-grade male Kunming mice, 5–7 weeks old, weighing 18–22 g, provided by the Animal Experiment Center of Kunming Medical University, animal production license number: SCXK(Dian)K 2020-0004), housed in the experimental animal room of the School of Pharmacy, Kunming Medical University. Experimental environment: constant temperature (22 ± 2) °C, humidity 60%–70%, mice had free access to water and food, followed the day-night rhythm, and the mice were monitored daily. Animal experiments were started after the mice had adapted to the environment for about 1 week. Animal welfare complied with relevant animal ethics regulations and was approved by the Animal Ethics Committee of Kunming Medical University (batch number KMMU2020111).

[0037] 1.2 Main Reagents

[0038]

[0039]

[0040]

[0041] 2 Experimental methods

[0042] 2.1 Experimental Grouping

[0043] A total of 144 SPF healthy Kunming mice aged 5 to 7 weeks and weighing 18 to 22 g were weighed and randomly divided into 6 groups: normal control group (NS), model group (LPS), dexamethasone group (DEX, 0.39 mg / kg), low-dose group of β-methyl derivative of genipin (12.5 mg / kg), medium-dose group of β-methyl derivative of genipin (25 mg / kg), and high-dose group of β-methyl derivative of genipin (50 mg / kg), with 24 mice in each group, of which 12 mice had lung tissues taken for detection of other indicators, and the other 12 mice had BALF taken.

[0044] 2.2 Drug administration and establishment of acute lung injury model

[0045] All mice were gavaged with the same volume of 0.9% saline once a day in the NS group and LPS group, DEX solution (0.39 mg / kg) was gavaged once a day in the DEX group, low-dose Gen-17 solution (12.5 mg / kg) was gavaged once a day in the Gen-17 medium-dose group, medium-dose Gen-17 solution (25 mg / kg) was gavaged once a day in the Gen-17 high-dose group, and high-dose Gen-17 solution (50 mg / kg) was gavaged once a day in the Gen-17 high-dose group; the dosage was converted according to the weight of the mice, and the gavage volume was 20 mL / kg, and the gavage was continued for 3 days. One hour after the gavage on the third day, the acute lung injury model was established by tracheal instillation of LPS solution. The specific experimental operation method is as follows:

[0046] ① Before the operation, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30 mg / kg) according to their body weight.

[0047] ② Use rubber bands to fix the limbs of the anesthetized mouse, put a thin wire around its teeth, and disinfect the neck skin with 75% alcohol.

[0048] ③ Make a longitudinal incision along the middle of the mouse's neck and separate the subcutaneous tissue until the trachea is exposed and cartilage rings appear.

[0049] ④ The microinjector was inserted into the trachea about 1 cm parallel to the proximal end. Sterile NS was slowly dripped into the trachea of ​​the control group, and LPS was dripped into the trachea of ​​the other groups.

[0050] ⑤ After pulling out the injector, immediately stand the mouse upright, rotate and shake it evenly for 2 minutes, then suture the neck wound, observe the condition of the mouse, and feed it as usual after it wakes up.

[0051] 2.3 Experimental animal treatment and lung tissue specimen collection

[0052] Samples were collected 24 hours after the animal model was established. The mice were weighed and 12 mice in each group were killed. Lung tissues were collected and the whole lung wet weight was measured. The lung tissues were packaged for HE staining, MDA and SOD content determination, gene and protein detection. The specific experimental operations are as follows:

[0053] ① After weighing the 12 mice in each group, they were killed by cervical dislocation;

[0054] ②Fix the mouse, wipe the abdomen with 75% alcohol, cut the chest cavity, remove the lung tissue, organize and take photos, remove the trachea and other excess tissues, weigh the wet weight of the whole lung, and calculate the lung coefficient;

[0055] ③ The left upper lobe of the lung was fixed in 4% neutral paraformaldehyde, and after fixation for 24 hours, it was routinely paraffin-embedded and sectioned for HE staining;

[0056] ④ The remaining lung tissues were placed in centrifuge tubes and quickly frozen in liquid nitrogen. After the experiment, they were transferred to -80℃ for storage and used for detection of indicators such as MDA, SOD, WB and RT-PCR.

[0057] 2.4 BALF collection and processing

[0058] Bronchoalveolar lavage fluid was collected 24 hours after the animal model was established. First, the remaining 12 mice in each group were weighed, and then BALF was extracted. The extracted BALF was used for total cell count, total protein content determination, and ELISA determination of IL-1β, IL-6, and TNF-α in BALF. The specific experimental operations were as follows:

[0059] ① After weighing, the mice were moderately anesthetized by intraperitoneal injection of sodium pentobarbital according to their body weight;

[0060] ② When the tail root, hind limbs, eyelash reflex and other activities of the mouse disappear, and the mouse breathes slowly and evenly, fix the mouse's limbs and teeth, disinfect the mouse's neck skin with 75% alcohol, make a longitudinal opening, separate the neck tissue, expose the tracheal cartilage ring, and use a 19G cannula catheter to puncture the trachea. Insert the needle about 1 cm, pull out the needle core, connect a 1mL syringe, and slowly inject pre-cooled saline (0.8mL / time, repeat 2 times), collect the lavage fluid in a 1.5mL centrifuge tube, and store it on ice (BALF recovery rate 80% to 95%);

[0061] ③ The BALF lavage fluid was centrifuged at 3000 g for 10 min at 4°C, the supernatant was aliquoted and stored at -80°C, and the total protein concentration was determined by BCA method and ELISA kit;

[0062] ④ Add a certain amount of PBS to the cell pellet for resuspending, mix well, take 20 μL and add it to a 1.5 mL centrifuge tube with 180 μL PBS in advance, mix well and use it for total cell counting.

[0063] 2.5 Calculation of mouse lung coefficient

[0064] The mice in each group were killed by cervical dislocation, and the whole lungs of the mice were removed by dissection. The lungs were weighed after removing excess tissues such as the trachea to obtain the wet weight of the whole lungs of the mice. The lung coefficient was calculated according to the formula: lung coefficient = lung wet weight / body weight × 100%.

[0065] 2.6 Analysis of lung tissue pathomorphology

[0066] The left upper lobe of the lung was fixed with 4% paraformaldehyde solution, embedded in paraffin and sectioned, and stained with HE. The pathological changes of the lung tissues of mice in each group were observed under a microscope. The specific procedures were as follows:

[0067] 2.6.1 Preparation of paraffin sections

[0068] ① Take the left upper lobe of the lung and fix it in a sufficient amount of 4% paraformaldehyde solution for 24 hours;

[0069] ② Dehydration: Remove the lung tissue fixed in 4% neutral paraformaldehyde, soak the tissue in 70%, 80%, 90%, and 95% ethanol for 25 minutes each, and soak it in 100%Ⅰ, 100%Ⅱ, and 100%Ⅲ anhydrous ethanol for 30 minutes each to remove all the water in the tissue;

[0070] ③ Xylene transparent: After absorbing the excess ethanol with absorbent paper, transparentize in xylene I, II, and III for 30 minutes each. If the transparent effect is not good, the xylene transparent time can be adjusted according to the reagent conditions;

[0071] ④ Embedding: Place the transparent tissue in melted paraffin I and II for 30 minutes each, and in paraffin III for 60 minutes. After the wax immersion is completed, embed it and store it at room temperature for later use;

[0072] ⑤ Sectioning: Before sectioning, place the wax block stored at room temperature in a 4℃ refrigerator for 30 minutes, then cut thin slices of 4-6μm thickness on a microtome, smooth the surface of the paraffin block, slice continuously, select complete fragments, spread the slices in a 45℃ warm water bath, and adhere them to the slides, bake them on a 37℃ baking machine for 2 hours, then place the slices in a 60℃ oven overnight, and store them in a 37℃ constant temperature box the next day for use.

[0073] 2.6.2 Hematoxylin-eosin staining (HE staining)

[0074] ① Baking: Place the tissue slide in a constant temperature box and bake at 60-65℃ for 25 minutes;

[0075] ② Dewaxing: Place tissue slides in xylene I, II, and III for 10 minutes each (the specific time is adjusted according to the room temperature and the freshness of the xylene. The higher the room temperature and the fresher the xylene, the shorter the immersion time, and the time is based on the absence of paraffin residue on the tissue);

[0076] ③ Hydration: After absorbing the excess xylene with absorbent paper, hydrate the sample and place it in 100% ethanol I and 100% ethanol II.

[0077] 5 min each, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol each for 1 min and then dried in the sun;

[0078] ④ After staining with hematoxylin for 3.5 minutes, rinse with running water for 5 minutes;

[0079] ⑤After differentiation with 1% hydrochloric acid alcohol for 6 seconds, immediately rinse with running water for 15 minutes;

[0080] ⑥ 90% ethanol for 10 seconds, eosin staining for 1 minute, rinse with tap water until the basin water is clear;

[0081] ⑦ Gently lift and lower the ethanol gradient to 70% × 10 times, 80% × 10 times, 90% × 10 times;

[0082] ⑧95% ethanol for 2 min, 100% ethanol I and II for 2 min each;

[0083] ⑨ Transparency: After absorbing excess ethanol, place in xylene I and II for 5 minutes each, and in xylene III for 15 minutes;

[0084] ⑩ Sealing: Air-dry and seal with neutral gum.

[0085] 2.6.3 Observation of lung tissue pathology

[0086] After sealing, a Nikon DS-Ri2 automatic microscope was used to observe and take pictures for analysis.

[0087] 2.6.4 Alveolitis score of lung histopathology

[0088] Observe the pathological features of HE and Masson-stained sections under a microscope, and score alveolitis and fibrosis. The scoring method for graded data is: (-) is 1 point, (+) is 2 points, (++) is 3 points, and (+++) is 4 points, as follows:

[0089] Alveolitis scoring criteria: refer to the method reported by Szapiel

[48] , lung tissue sections were scored for alveolitis.

[0090] Grade 0: No alveolitis (-), normal alveolar structure, no thickening of alveolar walls, and no widening of alveolar spacing.

[0091] Grade 1: Mild (+), the alveolar structure is still intact, the alveolar wall is thickened, the alveolar septum is widened, and the lesion area accounts for less than 20% of the whole lung.

[0092] Grade 2: Moderate (++), with significant thickening of the alveolar wall and widening of the alveolar septa, and the lesion area accounting for 20% to 50% of the entire lung.

[0093] Grade 3: Severe (+++), diffuse alveolitis, massive inflammatory cell infiltration, lung parenchymal changes caused by alveolar hemorrhage, and the lesion area exceeds 50% of the whole lung.

[0094] 2.7 Total cell count

[0095] Use a pipette to take 20 μL of the mixed cell pellet in the cell counting plate, and use the Count Star automatic cell counter to count the total cell count of each group of ALI mice.

[0096] 2.8 BCA method to detect total protein concentration in BALF

[0097] ① According to the instructions of the BCA kit, dilute the protein standard with double distilled water in a concentration gradient, and prepare the BCA working solution with A solution: B solution = 50:1 for use;

[0098] ② In a 96-well plate, add 20 μL / well of different concentrations of standard to prepare a standard curve (3 duplicate wells), add 20 μL / well of the sample to be tested (3 duplicate wells), and then add 200 μL / well of BCA working solution, mix well and incubate in a 37°C incubator for 30 min;

[0099] ③Measure the OD value at a wavelength of 562nm and calculate the total protein concentration of BALF according to the standard curve.

[0100] 2.9ELISA detection of IL-1β, IL-6, and TNF-α in BALF

[0101] Take out the packaged samples and place them on ice to melt. Use ELISA kits to detect the levels of IL-1β, IL-6, and TNF-α in the cell supernatant. The specific operation is the same as Part 1 2.4.

[0102] 2.10 qRT-PCR detection of related gene expression in lung tissue

[0103] Remove the lung tissue from -80℃ and weigh it accurately. Place the sheared lung tissue in a glass homogenizer that has been rinsed with Trizol and pre-cooled. Add Trizol to the glass homogenizer. Add 1mL of Trizol to every 50-100mg sample for grinding. Transfer the tissue homogenate to a 1.5mL centrifuge tube and place on ice for 15min. Invert to mix, then centrifuge at 4℃, 12000rpm for 15min. Subsequent steps are consistent with Part I 2.5.

[0104] 2.11 Detection of MDA activity in lung tissue

[0105] Remove the lung tissue from -80℃, weigh it accurately, add 9 times the volume of pre-cooled physiological saline according to the ratio of lung weight (g): volume (mL) = 1:9, use a tissue grinder to grind the lung tissue into a 10% tissue homogenate on ice, centrifuge at 4℃, 3000rpm for 10min, and take the supernatant for testing. Use the BCA method to determine the protein concentration of the 10% tissue homogenate. Subsequent operations follow the first part 2.7.

[0106] 2.12 Detection of SOD activity in lung tissue

[0107] Lung tissue was removed from -80℃, accurately weighed, and 9 times the volume of pre-cooled NS was added at a ratio of lung weight (g): volume (mL) = 1:9. The lung tissue was ground into 10% tissue homogenate on ice using a tissue grinder, and centrifuged at 4℃, 3000rpm for 10min, and the supernatant was taken for testing; a certain amount of supernatant after centrifugation was taken, and the protein concentration was determined by the BCA method; before the formal experiment began, 2 samples were randomly selected from normal tissues and diluted to different concentrations for preliminary testing of the SOD inhibition rate of the samples, and the concentration of the well with an inhibition rate of 40%-60% was selected for the formal experimental concentration; according to the preliminary experimental results, the 10% tissue homogenate supernatant of the mouse was diluted 30 times; the subsequent experimental operations were consistent with Part I 2.8.

[0108] 2.13 Western blot analysis of related protein expression

[0109] 2.13.1 Preparation of tissue samples

[0110] The lung tissue was taken out from -80℃ and accurately weighed. The sheared lung tissue was placed in a glass homogenizer that had been rinsed with protein lysis buffer and precooled. Lysis buffer was added to the glass homogenizer. 100L of protein lysis buffer was added for every 100mg of sample. The tissue was fully ground on ice. After grinding, the tissue homogenate was transferred to a 1.5mL centrifuge tube and crushed with an ultrasonic crusher for 10s, 10s interval, repeated 3 times, and then placed on ice.

[0111] Lyse for 30 minutes to fully lyse the lung tissue; then centrifuge at 12000rpm for 15 minutes, transfer the supernatant to a new 1.5mL centrifuge tube and mark it. Detect protein concentration by BCA method. According to the total protein concentration, add 5× Loading Buffer and protein lysis buffer to adjust all proteins to a uniform concentration of 6μg / μL, mix well, boil in a boiling water bath for 10 minutes to denature the protein, cool and store in aliquots at -80℃. Subsequent experimental operations are consistent with Part 1 2.10.

[0112] 3 Statistical methods

[0113] The experimental data were statistically analyzed using SPSS23.0 statistical analysis software. The measurement data were expressed as mean ± standard deviation (x ± s). When the measurement data obeyed the normal distribution and the variance was equal, the differences among multiple groups were tested by one-way analysis of variance (One-Way ANOVA). SNK test or q test was used for pairwise comparison between groups, and P < 0.05 was considered statistically significant. GraphPad Prism 8 software was used to draw the relevant results.

[0114] 4 Experimental results

[0115] 4.1 Mouse lung tissue specimens and lung coefficients

[0116] like Figure 1 As shown in the figure, the lung tissue surface of mice in NS group was shiny, without edema, and the tissue was pink and uniform in color; compared with NS group, the lung tissue of mice in LPS group was obviously edematous, with obvious spots on the surface and dark tissue color, while the lung tissues of mice in each drug group had different degrees of edema, but compared with LPS group, the degree of edema and the glossiness of tissue surface were improved to varying degrees.

[0117] The lung coefficient is an important indicator to characterize the degree of lung tissue edema. Figure 2 As shown in the figure, compared with the NS group, the changes in the lung system of mice in the LPS group and Gen-17 group were statistically significant (P < 0.05), indicating the occurrence of acute pulmonary edema; after drug treatment, the lung coefficient of mice decreased to varying degrees. Compared with the LPS group, the lung coefficient of mice in the Gen-17 medium and high dose groups and the DEX group changed statistically significantly (P < 0.01); compared with the DEX group, the most obvious increase in the lung coefficient of mice was in the Gen-17 low dose group, and the difference in changes was statistically significant (P < 0.05). The results show that Gen-17 can reduce the degree of pulmonary edema in ALI mice to varying degrees.

[0118] 4.2 Effect of β-methyl genipin derivatives on histopathology of acute lung injury in mice induced by LPS Histopathological morphology is a good way to evaluate tissue damage. HE staining was used to evaluate the effect of Gen-17 on LPS-induced mouse lung tissue. The results of mouse lung tissue pathology and pathological scoring are shown in Figure 2. Figure 3-4 As shown in the figure, the lung tissue structure and alveolar septum of the NS group were normal, the alveolar wall was thin, and a small number of inflammatory cells were visible; compared with the NS group, the lung tissue of the LPS group showed obvious inflammatory cell infiltration and hemorrhage, thickening of the lung interstitium, loss of alveolar structure, and partial congestion of the lung interstitium. The pathological score was significantly increased, which was statistically significant (P<0.001); compared with the LPS treatment group, the Gen-17 group and the DEX group could significantly alleviate the pathological damage of the lung tissue caused by LPS, and the high-dose Gen-17 group and the DEX group could significantly improve the pathological lesions of the lung tissue, and the pathological score was significantly reduced, which was statistically significant (P<0.01); compared with the DEX group, the alveolitis score of the mice in the low-dose and medium-dose Gen-17 groups increased, and the difference in changes was statistically significant (P<0.05). The results showed that Gen-17 could reduce the inflammatory response of ALI mice induced by LPS, and improved the pathological structure of the lung tissue and the alveolitis score of the mice to varying degrees.

[0119] 4.3 Total protein concentration in BALF

[0120] When inflammation occurs in lung tissue, the permeability of lung capillaries decreases, causing a large amount of protein to enter the interstitial space and increase the protein concentration. Therefore, the total protein concentration in BALF can reflect the degree of inflammation. Figure 5 As shown in the figure, compared with the NS group, the total protein concentration in BALF of the LPS group was significantly increased, and the difference was statistically significant (P<0.01); compared with the LPS group, after drug intervention, the total protein concentration in BALF of each drug dose group decreased, among which the Gen-17 medium dose group, high and low dose groups, and DEX group could significantly reduce the total protein content in BALF, and the difference was statistically significant (P<0.05). The results showed that Gen-17 reduced the permeability of pulmonary capillaries and reduced the exudation of proteins.

[0121] 4.4 Total cell count in BALF

[0122] The experimental results are as follows Figure 6As shown in the figure, compared with the NS group, the total number of cells in BALF of the LPS group, the low-dose Gen-17 group, and the medium-dose group increased significantly, and the difference was statistically significant (P<0.001); compared with the LPS group, the total number of cells in BALF of the Gen-17 group and the DEX group decreased significantly after drug treatment, and the difference was statistically significant (P<0.001); in addition, compared with the DEX group, the total number of cells in BALF of the low-dose Gen-17 group and the medium-dose group increased significantly, and the difference was statistically significant (P<0.01). The results showed that Gen-17 could reduce the total cell count in BAFL of ALI mice, reduce cell infiltration, and alleviate lung tissue inflammation.

[0123] Effect of Gen-17 on the expression of inflammatory mediators TNF-α, IL-1β, and IL-6 in BALF of ALI mice

[0124] ELISA results Figure 7 The results showed that the levels of inflammatory factors TNF-α, IL-1β and IL-6 in mice were significantly increased after LPS induction. Compared with the NS group, the levels of inflammatory factors TNF-α, IL-1β and IL-6 in BALF of mice in the LPS group were significantly increased, and the difference was statistically significant (P<0.05); compared with the LPS group, the levels of inflammatory factors TNF-α, IL-1β and IL-6 in BALF of mice in the Gen-17 group and the DEX group were reduced to varying degrees after drug treatment. Except for TNF-α and IL-6 in the low-dose Gen-17 group, the differences in other cytokines were statistically significant (P<0.05); compared with the DEX group, Gen-17 had no statistically significant difference in reducing the level of TNF-α (P>0.05), but the low-dose Gen-17 group had statistically significant difference in reducing the levels of IL-1β and IL-6 (P<0.01). The results showed that after treatment with Gen-17, the levels of TNF-α, IL-1β, and IL-6 were significantly inhibited, indicating that Gen-17 can affect the progression of ALI through anti-inflammatory effects.

[0125] Effect of Gen-17 on the expression of inflammatory mediator genes in lung tissue of ALI mice

[0126] In order to explore the inhibitory effect of Gen-17 on pro-inflammatory factors, the expression of pro-inflammatory cytokine mRNA in mouse lung tissue was detected. Figure 8The results showed that compared with the NS group, the expression of proinflammatory cytokines TNF-α, IL-1β, IL-6, IKKα / β, IkB-α, and iNOS in the lung tissue of mice was significantly upregulated after LPS induction (P<0.001); compared with the LPS group, Gen-17 could significantly reduce the expression of proinflammatory cytokines TNF-α, IL-1β, IL-6, IKKα / β, IkB-α, and iNOS in the lung tissue, and the difference was statistically significant (P<0.001); compared with the DEX group, only the low-dose Gen-17 group had statistical significance in inhibiting the expression of TNF-α and IL-6 (P<0.001). The results showed that Gen-17 had an inhibitory effect on the expression of inflammatory mediator genes in the lung tissue of ALI mice.

[0127] 4.7 Effect of Gen-17 on the expression of NF-кBp65 protein in lung tissue of ALI mice

[0128] like Fig. 9 As shown in the results, after LPS induction, the expression of NF-кBp65 protein in the lung tissue of mice was significantly upregulated. Compared with the NS group, the expression of NF-кBp65 protein in the lung tissue of the LPS group and Gen-17 group was significantly increased (P<0.001, P<0.05); compared with the LPS group, after drug treatment, the expression of NF-кBp65 protein in the lung tissue of the drug-treated group decreased to varying degrees. Except for the low-dose Gen-17 group, the differences in the other groups were statistically significant (P<0.05). The results showed that Gen-17 could downregulate the expression of NF-кBp65 protein in the lung tissue of ALI mice and inhibit the activation of the NF-кB signaling pathway.

[0129] Effect of 4.8Gen-17 on the expression of MAPK signaling pathway proteins in lung tissue of ALI mice

[0130] like Fig.10 As shown in the results, the expression of p38, ERK, and JNK phosphorylated proteins in the lung tissue of mice was significantly upregulated after LPS induction. Compared with the NS group, the expression of p38, ERK, and JNK phosphorylated proteins in the lung tissue of the LPS group increased significantly, and the difference was statistically significant (P<0.05). Compared with the LPS group, the expression of p38, ERK, and JNK phosphorylated proteins in the lung tissue of mice decreased to varying degrees after drug treatment. Except for the Gen-17 low-dose group, the changes in the other groups were statistically significant (P<0.05). Compared with the DEX group, the expression of p38 phosphorylated protein in the lung tissue of the Gen-17 group had no statistically significant change (P>0.05), while the expression of ERK and JNK phosphorylated proteins in the lung tissue of the Gen-17 low-dose group had statistically significant changes (P<0.05). The results showed that Gen-17 could downregulate the expression of p38, ERK, and JNK proteins by regulating the MAPK signaling pathway.

[0131] Effects of Gen-17 on oxidative stress in lung tissue of ALI mice

[0132] like Fig.11 As shown in the figure, after LPS induction, the SOD activity in the lung tissue of mice decreased significantly, and the MDA content increased significantly. Among them, in terms of SOD activity, compared with the NS group, the SOD activity in the LPS group decreased (P<0.05), and compared with the LPS group, after drug treatment, the SOD activity increased to varying degrees, but only the Gen-17 high-dose group and the DEX group were statistically significant (P<0.05); in terms of MDA content, compared with the NS group, the MAD content in the LPS group increased significantly (P<0.001), and compared with the LPS group, after drug treatment, the MAD in the lung tissue homogenate decreased significantly (P<0.001). Therefore, Gen-17 can significantly increase the SOD activity and MDA content of lung tissue, thereby reducing the degree of damage to lung tissue.

[0133] Effect of 4.10Gen-17 on the gene expression of oxidative stress factors in lung tissue of ALI mice

[0134] like Fig.12 As shown in the figure, after LPS induction, the expression of Keap1 gene in lung tissue of mice was significantly upregulated, and the expression of Nrf2 and HO-1 genes was significantly downregulated. Among them, in the expression of Keap1 gene, compared with the NS group, the expression of Keap1 in the LPS group and Gen-17 group was significantly increased (P<0.001), compared with the LPS group, after drug intervention, the expression of Keap1 was significantly decreased (P<0.001), compared with the DEX group, the expression of Keap1 in the Gen-17 group was significantly increased, and the change was statistically significant (P<0.001). In the expression of Nrf2 gene, compared with the NS group, the expression of Nrf2 in the LPS group was significantly downregulated (P<0.001), and after drug treatment, the drug group was able to increase the content of Nrf2. In HO-1 gene expression, compared with the NS group, HO-1 expression was significantly downregulated in the LPS group and Gen-17 group (P<0.001). Compared with the LPS group, HO-1 expression was upregulated after drug treatment, and except for the low-dose Gen-17 group, the other groups were statistically significant (P<0.001). Compared with the DEX group, Gen-17 was statistically significant in upregulating the expression of HO-1 (P<0.001).

[0135] Effect of 4.11Gen-17 on the expression of Keap1 / Nrf2 / HO-1 signaling pathway proteins in lung tissue of ALI mice

[0136] like Fig.13As shown in the figure, after LPS induction, the expression of Keap1 protein in the lung tissue of mice was significantly upregulated, and the expression of Nrf2 and HO-1 proteins was significantly downregulated. Compared with the NS group, the expression of Keap1 protein in the LPS group was significantly increased, and the expression of Nrf2 and HO-1 proteins was significantly decreased, and the differences were statistically significant (P<0.05). Compared with the LPS group, after drug treatment, the expression of Keap1 protein decreased to varying degrees, and only the high-dose group of Gen-17 and the DEX group had statistically significant differences (P<0.001), and the changes in protein expression in the other groups were not statistically significant (P>0.05). While the expression of Nrf2 protein increased to varying degrees, and only the medium-dose group of Gen-17 and the DEX group had statistically significant differences (P<0.05), and the changes in protein expression in the other groups were not statistically significant (P>0.05). The expression of HO-1 protein also increased to varying degrees, and except for the low-dose group of Gen-17, the changes in the other groups were statistically significant (P<0.05). The results showed that Gen-17 could downregulate the expression of Keap1 protein and upregulate the expression of Nrf2 and HO-1 proteins by regulating the Keap1 / Nrf2 / HO-1 signaling pathway.

[0137] Acute lung injury caused by LPS is a classic modeling method in current ALI animal experiments. Since mice have similar structures to humans, inducing mice with LPS can well simulate human inflammatory responses. LPS is the main component of the cell wall of Gram-negative bacteria. As the preferred inducer of the inflammatory model, it is the most common in the establishment of animal models of ALI. It is a single instillation of LPS into the animal trachea. This model has high cost performance, good reproducibility, many excellent cases, and can induce strong lung injury reactions. When ALI occurs, there will be phenomena such as inflammatory exudation, cell swelling, and capillary congestion, which will cause an increase in lung wet weight. However, due to the short ALI modeling time, the weight of mice does not change significantly. Therefore, the lung coefficient (ratio of lung wet weight to body weight) is one of the most important indicators that can reflect lung tissue edema. In the experiment of this application, the lung coefficient increased significantly after LPS induced ALI in mice, and decreased to varying degrees after intervention with Gen-17, indicating that it can improve pulmonary edema.

[0138] The occurrence of ALI is accompanied by the infiltration of inflammatory cells such as macrophages, neutrophils, and lymphocytes, and the release of inflammatory factors, leading to an inflammatory cascade reaction, which destroys the integrity of vascular endothelial and alveolar epithelial cells and damages lung tissue. In this experiment, after LPS induction, the total number of cells and total protein concentration in BALF increased significantly, and HE staining of lung tissue showed a large number of inflammatory cell infiltration, alveolar wall thickening, and obvious alveolar inflammation; after the administration of Gen-17, the infiltration of inflammatory cells and proteins decreased, and the inflammation of lung tissue was alleviated to varying degrees, indicating that Gen-17 can improve the alveolitis and lung injury pathological process of ALI mice.

[0139] Inflammation is an important cause of ALI. Many reports indicate that the molecular mechanism of ALI is related to the excessive release of inflammatory cytokines such as IL-1β, TNF-α, and IL-6. NF-κB / MAPK is the most typical pro-inflammatory signal transduction pathway, which can cause the occurrence and development of ALI by promoting the release of inflammatory genes. Therefore, timely control of disease progression before the onset of ALI or elimination of inflammation in the early stage of ALI can effectively improve the condition, increase the cure rate of patients, and reduce the damage suffered before the disease is resolved. In this study, according to the results of RT-PCR and ELISA, the mRNA transcription level and secretion of IL-1β, TNF-α, and IL-6 increased after LPS treatment, and decreased to varying degrees after Gen-17 intervention. When inflammatory cells are released in large quantities, the NF-κB signaling pathway is activated, which activates the downstream signaling molecule p65 in the NF-κB pathway and regulates the occurrence of ALI. In addition. The MAPK signaling pathway plays a vital role in the development of inflammation and is a classic inflammatory pathway. The phosphorylation and activation of downstream proteins p38, ERK, and JNK in this signaling pathway abnormally increase the levels of inflammatory factors such as IL-1β, TNF-α, and IL-6. The increased concentrations of various inflammatory factors further promote the cascade reaction of MAPK family proteins, leading to the occurrence of inflammatory storms. Zhang et al. found that in the ALI animal model, by specifically inhibiting the NF-κB and MAPK signaling pathways to transmit cascade reactions to reduce the release of inflammatory cells, it exerts an anti-ALI effect, which is consistent with the results reported in the above literature. In addition, iNOS is a NO synthase, whose expression directly determines the secretion of NO and is an important indicator for detecting inflammatory responses. The expression of iNOS is considered an important tool for treating inflammatory diseases. In this experiment, after LPS induction, the mRNA content of iNOS in lung tissue increased, indicating that the model was successful and inflammation occurred; the mRNA content of IκB-α and IKKα / β in lung tissue increased, and the expression of p65 protein was significantly upregulated, indicating the activation of NF-κB signaling pathway; the expression of p38, ERK1 / 2, and JNK phosphorylated proteins in lung tissue increased significantly, indicating the activation of MAPK signaling pathway; after Gen-17 intervention, the mRNA content of IκB-α and IKKα / β decreased, and the expression of p65, p38, ERK1 / 2, and JNK proteins decreased, indicating that Gen-17 can reduce the inflammatory response by inhibiting NF-κB and MAPK signaling pathways, thereby inhibiting the occurrence of ALI. The above experimental results are consistent with the in vitro experiments.

[0140] Oxidative stress is caused by excessive production of reactive oxygen species or dysfunction of reactive oxygen scavenging function in the body, which eventually leads to abnormal increase of reactive oxygen species in the body's tissues or cells, thus breaking the balance between oxidation and anti-oxidation in the body, making the balance tend to the oxidative state, leading to a large number of inflammatory cell infiltration and a large number of protease secretions in the tissues, thereby producing accumulated oxidative intermediates. Existing literature shows that oxidative stress can serve as the pathological basis for the onset of various types of ALI and plays an important role in different stages of ALI. MDA is a sensitive marker for lipid peroxidation that is broken by the oxidative stress balance state, while CAT and SOD are antioxidant enzymes that scavenge free radicals in the body. Both are important indicators for evaluating the body's antioxidant capacity. In this experiment, LPS induced ALI in mice, resulting in a significant increase in the detection results of MDA. On the contrary, the activity of SOD decreased significantly, proving that the balance of oxidative stress in the body was broken after LPS stimulation. After intervention with Gen-17, the MDA content in lung tissue decreased, while the activity of SOD increased, which was consistent with the in vitro results. In addition, the Nrf2-ARE pathway is an intrinsic mechanism of antioxidant defense, and Nrf2 participates in the anti-inflammatory process by participating in the recruitment of inflammatory cells and regulating the expression of antioxidant response element (ARE) genes. Keap1 is a Cullin3 (Cul3)-based E3 ligase bridge protein that strictly regulates the activity of Nrf2. Under normal physiological conditions, Keap1 selectively targets Nrf2, leading to ubiquitin-dependent proteasomal degradation. When oxidative stress occurs, Keap1 inactivation can inhibit Nrf2 ubiquitination, ultimately leading to the accumulation of newly synthesized Nrf2 and accompanied by Nrf2 activation. Studies have shown that Nrf2 plays an important role in the adaptive response of cells to oxidative stress and other types of stress, and may be a prospective target for controlling oxidative stress in ALI. Therefore, the present application detects the mRNA transcription level and protein expression of Keap1, Nrf2, and HO-1 by PT-PCR and Western Blot. The results show that after LPS-induced ALI in mice, the Keap1 mRNA content and protein expression in the lung tissue increased, while the Nrf2 and HO-1 mRNA content and protein expression levels decreased. After intervention with Gen-17, the Keap1 mRNA content and protein expression can be significantly reduced, and the Nrf2 and HO-1 mRNA content and protein expression can be increased. This shows that Gen-17 exerts an antioxidant effect through Keap / Nrf2 / HO-1 signals, thereby inhibiting the occurrence and development of ALI. The present application can provide a certain experimental basis for the application of Gen-17 in the prevention and treatment of acute severe pneumonia.

[0141] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. Use of genipin β-methyl derivative Gen-17 in the preparation of a drug for preventing or treating acute severe pneumonia, characterized in that: The chemical structure of Gen-17 is:

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