Application of iridoid compounds in the preparation of drugs for the treatment of acute lung injury or pulmonary fibrosis

Abstract

The invention discloses the use of iridoid compounds in preparing a drug for treating acute lung injury or pulmonary fibrosis. By using RLE-6TN and Raw264.7 to construct an acute lung injury cell model, an LPS-induced mouse acute lung injury model and a bleomycin-induced mouse pulmonary fibrosis model, the protective effect of iridoid compounds, especially morroniside and loganin, on acute lung injury and their mechanism of action are discussed from the aspects of the expression of proinflammatory cytokines, pathological manifestations of lung tissue, anti-oxidation, and expression of proteins related to the NF-κB / Stat3 signaling pathway. By replicating a model of pulmonary fibrosis, the protective effect of loganin and morroniside on pulmonary fibrosis is preliminarily confirmed; and it is preliminarily verified that the protective effect of loganin and morroniside on acute lung injury may be achieved through the NF-κB / Stat3 signaling pathway.

Description

Technical Field

[0001] The present invention relates to a new use of iridoid compounds, and in particular to the use of iridoid compounds in preparing drugs for treating acute lung injury or pulmonary fibrosis, belonging to the field of medical technology. Background Art

[0002] Acute lung injury (ALI) and its severe stages of acute respiratory distress syndrome (ARDS) and pulmonary fibrosis are difficult to cure diseases of the respiratory system, which seriously affect respiratory function and can lead to respiratory failure.

[0003] A large proportion of patients with acute lung injury develop a severe fibroproliferative process characterized by mesenchymal cell proliferation, neovascularization, and deposition of alveolar granulation tissue composed of type I and type III collagen and fibronectin-rich matrix within 10 to 14 days of clinical manifestation. Although IL-1β is generally not considered an effective fibrogenic factor in lung injury, studies have shown that transient overexpression of IL-1β in rat lungs using adenoviral vectors induces tissue injury, followed by local production of platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), and leads to pulmonary fibrosis.

[0004] Acute respiratory distress syndrome (ARDS) refers to a respiratory disease characterized by alveolar epithelial cell barrier dysfunction and alveolar capillary endothelial cell damage caused by various pathogenic factors inside or outside the lungs, resulting in diffuse alveolar and interstitial edema, hypoxemia, respiratory distress, and non-cardiogenic pulmonary edema. It is a common critical illness in the intensive care unit.

[0005] Pulmonary fibrosis (PF) refers to a chronic interstitial lung disease caused by drug toxicity, connective tissue disease, radiotherapy and other reasons, which can develop into respiratory failure. Idiopathic pulmonary fibrosis (IPF) is the most common and deadly type of idiopathic interstitial pneumonia, with a 5-year survival rate of about 20%.

[0006] There are many factors that cause ALI / ARDS, which can be divided into direct factors (intrapulmonary factors) and indirect factors (extrapulmonary factors). Intrapulmonary factors include: lung contusion, pneumonia caused by various bacteria and viruses (such as pneumonia caused by SARS, pneumonia caused by the new coronavirus), inhalation lung injury, etc. Extrapulmonary factors include: severe trauma, severe burns, drowning, drug overdose, non-cardiogenic shock, etc.

[0007] Clinically, the treatment of ALI / ARDS mainly includes comprehensive therapies such as mechanical ventilation, fluid management and drug therapy, but the mortality rate is still very high. There is also a lack of ideal drugs for the treatment of pulmonary fibrosis. Therefore, seeking highly effective and low-toxic drugs for the treatment of acute lung injury and pulmonary fibrosis in order to improve the quality of life of patients and save their lives has become an urgent issue to be addressed.

[0008] Among the current therapeutic drugs, pirfenidone and nintedanib can effectively relieve mild to moderate functional impairment in patients with idiopathic pulmonary fibrosis and slow down the deterioration of lung function, highlighting the importance of drugs in early detection, early diagnosis, and early treatment of the disease. However, in clinical applications, pirfenidone and nintedanib can cause severe gastrointestinal reactions (such as nausea, vomiting, abdominal discomfort, indigestion, and diarrhea) and adverse events such as elevated liver enzymes and bleeding that require special attention. In addition, the two drugs are relatively expensive, which will lead to poor patient compliance. It is difficult for ordinary patients to adhere to long-term medication. Therefore, it is of far-reaching significance to develop drugs with few side effects, economy, and high efficiency.

[0009] Iridoids are effective ingredients in traditional Chinese medicine, with anti-inflammatory, anti-tumor, diabetes prevention and treatment properties. As a member of the terpenoid family, iridoids are a class of monoterpene compounds with a cyclopentadienylpyran ring structure. They are acetal derivatives of ant dialdehyde, first obtained from the secretions of ants, have defensive effects, and are the first antibiotics obtained from animals. Iridoids are widely present in a variety of dicotyledonous plants, with Rubiaceae, Scrophulariaceae, and Gentianaceae being the most common. The basic core structure of iridoids is iridoid alcohol, which can be divided into three types according to the variation of its basic core structure: iridoid acetal lipids, cleaved iridoid glycosides, and iridoid glycosides. Because the hemiacetal alcohol hydroxyl group of iridoids is unstable and easily combined with sugar, most of the iridoids isolated and extracted from plants are iridoid glycosides. At present, there are more than 800 kinds of iridoid compounds obtained from plant species, among which there are only more than 30 kinds of cleocyclic iridoid compounds, more than 60 kinds of non-glycoside iridoid compounds, and the rest are iridoid glycoside compounds. Commonly used Chinese medicines such as Cornus officinalis, Honeysuckle leaves, Strychnos nux vomica, Gardenia jasminoides, Picrorhizon, Plantago, etc. all contain iridoid glycoside compounds.

[0010] Currently, there is no report on the use of iridoid glycosides for the preparation of drugs for the treatment of acute lung injury or pulmonary fibrosis. Summary of the invention

[0011] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide an application of an iridoid ether terpenoid compound in the preparation of a drug for treating acute lung injury or pulmonary fibrosis.

[0012] To achieve the above object, the present invention adopts the following technical solutions:

[0013] 1. Application of cyclopentadiene compounds in the preparation of drugs for the treatment of acute lung injury or pulmonary fibrosis.

[0014] Preferably, the iridoid compound is loganin or morroniside.

[0015] Preferably, the acute lung injury comprises a severe stage of acute respiratory distress syndrome.

[0016] 2. A drug for treating acute lung injury or pulmonary fibrosis, wherein the active ingredient is a terpene compound.

[0017] Preferably, the iridoid compound is loganin or morroniside.

[0018] Preferably, the acute lung injury comprises a severe stage of acute respiratory distress syndrome.

[0019] Beneficial effects of the present invention:

[0020] The present invention constructs an acute lung injury cell model with RLE-6TN and Raw264.7, an LPS-induced mouse acute lung injury model, and a bleomycin-induced mouse pulmonary fibrosis model, and discusses the protective effect and mechanism of iridoid ether terpenoid compounds, especially morroniside and loganin, on acute lung injury from the aspects of the expression of proinflammatory cytokines, pathological manifestations of lung tissue, anti-oxidation, and expression of proteins related to the NF-κB / Stat3 signaling pathway. By replicating a model of pulmonary fibrosis, the protective effect of loganin and morroniside on pulmonary fibrosis is preliminarily confirmed; and it is preliminarily verified that the protective effect of loganin and morroniside on acute lung injury may be achieved through the NF-κB / Stat3 signaling pathway.

[0021] The results showed that the cyclopentane compounds loganin and morroniside can reduce the expression of inflammatory factors IL-6mRNA, IL-1βmRNA, TNF-αmRNA in the acute lung injury cell model, and down-regulate the expression of p-p65 and stat3 proteins. In animal experiments, it was found that pretreatment with loganin and morroniside can improve the general condition of mice with LPS-induced acute lung injury and pathological changes such as inflammatory cell infiltration in lung tissue and alveolar wall thickening; at the same time, it can reduce the expression of IL-6mRNA, IL-1βmRNA, TNF-αmRNA in lung tissue and down-regulate the expression of p-p65 and p-stat3; the detection of antioxidant function indicators found that the SOD content of mice increased and the MDA content decreased after pretreatment with loganin and morroniside, indicating that loganin and morroniside can improve the antioxidant capacity of mice. In terms of preventing pulmonary fibrosis, loganin and morroniside can increase the weight growth rate of mice and the number of CD4 in peripheral blood. + / CD8 + The ratio of T lymphocytes and down-regulated the content of collagen fibers and HYP and the expression of TGF-β1mRNA, α-SMA mRNA, CollagenⅠmRNA and α-SMA protein in the lung tissue of mice with pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure shows the effects of morroniside and loganin on the proliferation of RLE-6TN cells, where A is morroniside and B is loganin.

[0023] Figure 2 The differences in the expression of inflammatory factors produced by RLE-6TN stimulated by different concentrations of LPS, including: A. IL-6 mRNA expression; B. IL-1β mRNA expression; C. TNF-α mRNA expression.

[0024] Figure 3 The expressions of IL-6 mRNA, IL-1β mRNA and TNF-α mRNA in RLE-6TN treated with LPS at different time periods: A. IL-6 mRNA was upregulated significantly at 1 hour compared with other times; B. IL-1β mRNA was upregulated more significantly at 0.5 hour; C. TNF-α mRNA was most significantly upregulated at 2 hours (compared with the Control group, **P<0.01, ***P<0.005, ****P<0.0001, respectively).

[0025] Figure 4The results showed that morroniside and loganin could inhibit the up-regulation of inflammatory factors induced by LPS stimulation on RLE-6TN. After the addition of morroniside, A. IL-6 mRNA was down-regulated (compared with the LPS group, ****P < 0.0001), B. IL-1β mRNA was down-regulated (compared with the LPS group, ****P < 0.0001), C. TNF-α mRNA was down-regulated (compared with the LPS group, **P < 0.01); after the addition of loganin, D. IL-6 mRNA was down-regulated (compared with the LPS group, ****P < 0.0001), E. IL-1β mRNA was down-regulated (compared with the LPS group, ***P < 0.005), F. TNF-α mRNA was down-regulated (compared with the LPS group, *P < 0.05).

[0026] Figure 5 The effect of morroniside and loganin on the expression of p-p65 and stat3 proteins in RLE-6TN cells.

[0027] Figure 6 The effects of morroniside and loganin on the proliferation of RAW 264.7, including A. The effect of morroniside concentration gradient treatment on RAW264.7 for different time periods (12h, 24h, 48h). B. The effect of loganin concentration gradient treatment on RAW264.7 for different time periods (12h, 24h, 48h). ns means no statistical difference, *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001, n=5.

[0028] Figure 7 Figure 3 The differences in the expression of inflammatory factors IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA in RAW 264.7 cells stimulated by different concentrations of LPS. A. The expression of IL-6 mRNA was upregulated under the stimulation of different concentrations of LPS (compared with the Control group, ***P<0.005, ****P<0.0001), and the concentration after 100 ng / mL was concentration-dependent; B. The expression of IL-1β mRNA was upregulated under the stimulation of different concentrations of LPS (compared with the Control group, **P<0.01, ****P<0.0001), C. The expression of TNF-α mRNA was upregulated under the stimulation of LPS (compared with the Control group, ****P<0.0001), and there was no significant difference in the increase multiple within the concentration range of 1 μg / mL.

[0029] Figure 8Morroniside and loganin can inhibit the up-regulation of cellular inflammatory factors in RAW 264.7 cells induced by LPS stimulation, among which, ABC: the expression of proinflammatory factors IL-6mRNA, IL-1βmRNA, and TNF-αmRNA were down-regulated after pretreatment with morroniside; DEF: the expression of proinflammatory factors IL-6mRNA, IL-1βmRNA, and TNF-αmRNA were down-regulated after treatment with loganin.

[0030] Fig. 9 This is the effect of morroniside and loganin on the changes in p-p65 and stat3 protein expression in RAW264.7 cells.

[0031] Fig.10 The effects of morroniside and loganin on body weight changes in an acute lung injury mouse model, where A. morroniside treatment had no effect on alleviating LPS-induced weight loss in mice; B. loganin treatment had no effect on alleviating LPS-induced weight loss in mice.

[0032] Fig.11 HE staining of mouse lung injury tissue sections (×400), where A is morroniside and B is loganin.

[0033] Fig.12 The effects of Morroniside and Loganin on the SOD activity of mouse lung tissue, A. The average SOD activity of the Control group was 26.83±0.7363 (U / mg lung tissue wet weight), n=8; the average SOD activity of the LPS group was 17.07±0.5678 (U / mg lung tissue wet weight), compared with the Control group, the SOD activity of the LPS group decreased, ***P<0.005, n=8; the average SOD activity of the LPS+Morroniside group was 29.66±1.317 (U / mg lung tissue wet weight), compared with the LPS group, the SOD activity of the LPS+Morroniside group increased, ###P<0.005, n=8. B. The data of the Loganin preprotection group had the same trend as the Morroniside preprotection (LPS vs Control, **P<0.01; LPS+Loganin vs LPS, ##P<0.01, n=8).

[0034] Fig.13The effects of morroniside and loganin on the content of MDA in the lung tissue of mice, among which, A. The content of MDA in the lung tissue of mice in the LPS group was higher than that in the Control group, **P<0.01, n=8. After oral administration of morroniside before modeling, the content of MDA in the lung tissue of mice decreased, ##P<0.01, n=8. B. The content of MDA in the lung tissue of mice in the LPS group was higher than that in the Control group (**P<0.01). Compared with the LPS group, the content of MDA in the lung tissue of mice in the LPS+Loganin group was reduced (#P<0.05).

[0035] Fig.14 The results are as follows: Effects of Morroniside and Loganin on the expression levels of IL-6mRNA, IL-1βmRNA and TNF-αmRNA in lung tissues. ABC: In the mice in the Morroniside pre-protection experimental group, the expression of proinflammatory factors IL-6mRNA, IL-1βmRNA and TNF-αmRNA in the LPS group was higher than that in the Control group (****P<0.0001, n=8), and the expression of proinflammatory factors in the LPS+Morroniside group was lower than that in the LPS group (####P<0.0001, n=8); DEF: In the mice in the Loganin pre-protection experimental group, compared with the Control group, the expression of IL-6mRNA in the LPS group was higher (**P<0.01, n=8), the expression of IL-1βmRNA was higher (****P<0.0001, n=8), and the expression of TNF-αmRNA was higher (**P<0.01, n=8). Compared with the LPS group, the expressions of IL-6 mRNA and IL-1β mRNA in the LPS+Loganin group were reduced (##P<0.01, n=8), and the expression of TNF-α mRNA also showed a downward trend (#P<0.05, n=8).

[0036] Fig.15 Morroniside and loganin can downregulate the expression of p-p65 and p-stat3 proteins in the NF-κB / STAT3 signaling pathway in mouse lung tissue. A. The increased expression of p-p65 and p-stat3 caused by LPS was downregulated by morroniside; B. The increased expression of p-p65 and p-stat3 caused by LPS was downregulated by loganin.

[0037] Fig.16 The results show that loganin and morroniside can improve the weight growth rate of mice with pulmonary fibrosis. A. is the weekly growth trend of mice weight, B. is the statistical chart of weight growth rate, compared with the BLM group, the control group and the treatment group were **P < 0.01, ***P < 0.005, ****P < 0.0001.

[0038] Fig.17Staining of lung tissue sections of mice with pulmonary fibrosis, including A. HE staining; B. Masson staining.

[0039] Fig.18 The changes of HYP content in lung tissue of mice after loganin and morroniside treatment.

[0040] Fig.19 CD4 in peripheral blood of mice in different treatment groups + / CD8 + Changes in T cell ratios, including A. Control group and BLM group mice peripheral blood CD4 + 、CD8 + T cell expression, B. Peripheral blood CD4 + 、CD8 + Expression of CD4 in peripheral blood of mice in different Morroniside concentration groups + 、CD8 + D. Expression of peripheral blood CD4 in mice in different treatment groups + / CD8 + Statistical graph of changes in T cell ratio (compared with the BLM group, *P<0.05, **P<0.01, ***P<0.005, n=8).

[0041] Fig. 20 The expression of TGF-β1 mRNA, α-SMA mRNA, and CollagenⅠ mRNA in the lung tissues of mice in different treatment groups, among which, A. The expression of TGF-β1 mRNA in the blank control group and the loganin and morroniside treatment groups was lower than that in the BLM group, and ****P<0.0001 (not marked in the figure). BC. The expression of α-SMA mRNA and CollagenⅠ mRNA in the blank control group and the loganin and morroniside treatment groups was lower than that in the BLM group, **P<0.01, ***P<0.005, ****P<0.0001, n=8.

[0042] Fig.21 Figure 2 shows the expression of α-SMA protein in the lung tissue of mice in different treatment groups. A. The expression of α-SMA protein in the lung tissue of mice in the bleomycin model group was upregulated compared with that in the control group. B is the relative expression of α-SMA. CE shows a downward trend in the expression of α-SMA from low to high concentrations of loganin treatment group and morroniside treatment group. DF shows the relative expression of α-SMA from low to high concentrations of loganin treatment group and morroniside treatment group. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0044] Experimental procedures

[0045] 1. 10% APS: Weigh 2 g of ammonium persulfate (APS), dissolve it in 20 mL of ultrapure water, mix well by vortexing, dispense into 1.5 mL EP tubes, and store at -20°C.

[0046] 2. 10% SDS: Weigh 5 g of sodium dodecyl sulfate (SDS), dissolve it in 40 mL of ultrapure water, make up to 50 mL after complete dissolution, and store at room temperature.

[0047] 3. pH 6.8 Tris-HCl (1M): Weigh 60.57 g of tris (hydroxymethyl)aminomethane (Tris) and dissolve it in 400 mL of ultrapure water. Adjust the pH to 6.8 with concentrated hydrochloric acid, make up to 500 mL with ultrapure water, and store at 4°C.

[0048] 4. pH 8.8 Tris-HCl (1.5 M): Weigh 90.9 g of Tris and dissolve it in 400 mL of ultrapure water. Adjust the pH to 8.8 with concentrated HCl, make up to 500 mL with ultrapure water, and store at 4°C.

[0049] 5. Loading buffer (5×): Take 2g sodium dodecyl sulfate (SDS), 10g bromophenol blue, 5mL 1mol / L Tris-HCl with pH 6.8, 10mL glycerol, and add ddH2O to make the volume to 20mL. Take 900ul and add 100ul of mercaptoethanol, divide into 1.5mL EP tubes, and store at room temperature.

[0050] 6. Running Buffer (10×): Use an electronic balance to weigh 144g of glycine, 30g of Tris, and 10g of SDS, and dissolve them in 1000mL of double distilled water to make 10× electrophoresis buffer. Store at room temperature. Take 100mL of 10× Running buffer and dilute it to 1L with ultrapure water before use.

[0051] 7. Transfer Buffer (10×): Weigh 720 g of glycine and 152 g of Tris, dissolve in 4 L of ultrapure water, stir evenly with a magnetic stirrer, add ultrapure water to make up to 5 L, and store at room temperature. When using, take 100 mL of transfer buffer and 200 mL of methanol, and dilute in 700 mL of ultrapure water.

[0052] 8. PBS (10×): weigh 80g NaCl, 36.3g Na2HPO4.12H2O, 2g KCl, 2.4g KH2PO4 and dissolve in 800mL ultrapure water. Stir with a magnetic stirrer, dilute to 1L, and adjust the pH to 7.2-7.4. Dilute to 1× with ultrapure water before use. If used for cell culture, autoclave.

[0053] 9. TBS buffer (10×): weigh 88g NaCl and 12.11g Tris and dissolve them in 800mL ultrapure water, adjust the pH to 7.5, dilute to 1L with ultrapure water, and store at room temperature. When needed, take 500mL TBS (10×) and 5mL Tween-20 and dissolve them in 4000mL ultrapure water, stir evenly with a magnetic stirrer, dilute to 5L, and store at room temperature.

[0054] 10. RIPA cell lysis buffer (1×): Add 0.0292 g EDTA, 0.038 g EGTA, 0.121 g Tris, 0.87 g NaCl, and 1 mL Triton-100 to 90 mL ultrapure water, adjust the pH to 7.4, make up to 100 mL, and store at 4°C.

[0055] 11. PMSF (100 mM, 100×): Dissolve 1.74 g of PMSF in 100 mL of isopropanol, stir well, and store at 4°C.

[0056] 12. β-Glycerol phosphate (500mM, 100×): Weigh 1.08g of β-glycerol phosphate, dissolve in 10mL of ultrapure water, divide into 1.5mL EP tubes, and store at -20℃.

[0057] 13. 8% SDS-PAGE separation gel (15 mL): Use a pipette to draw 6.9 mL of ddH2O, 4 mL of 30% Acr-Bis, 3.8 mL of 1.5 M Tris-HCl (pH 8.8), 150 μL of 10% SDS, 150 μL of 10% ammonium persulfate (APS), and 10 μL of TEMED into a 50 mL EP tube and use it immediately.

[0058] 14. 5% SDS-PAGE stacking gel (6 mL): 4.1 mL ddH2O, 1 mL 30% Acr-Bis, 750 μL 1.0 M Tris-HCl (pH 6.8), 80 μL 10% SDS, 80 μL 10% ammonium persulfate (APS), and 6 μL TEMED in a 10 mL EP tube, prepare and use immediately.

[0059] 15. Blocking solution (5% skim milk): Before use, weigh 2 g of milk powder and dissolve it in 40 mL of TBST, then shake to mix.

[0060] 16. 5 mg / mL MTT solution: Weigh 0.05 g of MTT powder and dissolve it in 10 mL of 1× PBS solution. Filter and sterilize it through a 0.22 μm filter membrane in a clean bench. Dispense it into 1.5 mL EP tubes wrapped in tin foil and store it at -20°C.

[0061] 17. 4% paraformaldehyde: Weigh 40g paraformaldehyde and dissolve it in 900mL 1xPBS solution. Since paraformaldehyde is not easy to dissolve, slowly add a small amount of sodium hydroxide to promote dissolution. After the paraformaldehyde is dissolved, adjust the pH value to 7.4, make the volume into 1L, and store it in a cold storage at 4℃.

[0062] 18. Preparation of LPS solution: Dissolve 10 mg of LPS lyophilized powder in 2 mL of normal saline to make 5 mg / mL LPS solution, dispense into 1.5 mL EP tubes, and store in a refrigerator at -20°C. Dilute to 1 mg / mL before use.

[0063] 19. Preparation of morroniside solution: Weigh 90 mg of morroniside lyophilized powder (Chengdu Ruifensi) and dissolve it in 5 mL of normal saline to prepare 18 mg / mL morroniside solution, and store it in a -20°C refrigerator.

[0064] 20. Preparation of loganin solution: Accurately weigh 120 mg of loganin lyophilized powder (Chengdu Ruifensi) and dissolve it in 6 mL of normal saline to prepare 20 mg / mL loganin solution, store in a -20°C refrigerator, and dilute as needed when used.

[0065] 1.1 Experimental methods

[0066] 1.1.1 Cell experiments

[0067] 1.1.1.1 Cell culture and passaging

[0068] 1. Inoculate RLE-6TN in complete culture medium (volume percentage 89% DMEM, 10% FBS, 1% penicillin / streptomycin double antibody).

[0069] 2. The cells were cultured in a constant temperature incubator at 37°C, 5% CO2 volume content, and 90% relative humidity.

[0070] 3. Observe the cell density and morphology under a microscope. When the cells grow to cover 80% of the culture dish area, they can be subcultured.

[0071] 4. Discard the old culture medium and add PBS to rinse along the edge of the culture dish. Add 1 mL of trypsin to the culture dish, shake gently to cover the entire bottom of the dish, place it in the incubator for 3 minutes, and observe under a microscope. If the cells shrink and become round, discard the trypsin and add 1 mL of culture solution to stop digestion. Gently blow the cells to make them fall off, transfer the cell suspension to a 1.5 mL centrifuge tube, and centrifuge at 1000 rpm for 3 minutes.

[0072] 5. Take out the centrifuge tube, discard the supernatant, and perform cell passage at a ratio of 1:2-1:3. Observe under a microscope and transfer the cells to a cell culture incubator after the cells are evenly spread to the bottom of the dish. Observe the cell status every day and replace the culture medium if necessary.

[0073] 1.1.1.2 Cell cryopreservation

[0074] 1. Take out the culture medium, trypsin, PBS, cell freezing solution, etc. from the 4℃ refrigerator and rewarm them.

[0075] 2. When the cells grow to an appropriate density and are in good condition, observe under the microscope and collect the cells and freeze them.

[0076] 3. Discard the old culture medium in the culture dish and rinse it with 1mL of PBS to remove the residual culture medium and cell metabolic waste.

[0077] 4. Discard PBS, add 1 mL of trypsin for digestion, blow down the cells and transfer them to a 1.5 mL EP tube, and centrifuge at 1000 rpm for 3 min.

[0078] 5. Discard the supernatant, add 1 mL of freezing solution to resuspend, and transfer to a cell cryopreservation tube. Store at -80℃ or in liquid nitrogen.

[0079] 6. Arrange the clean bench and wipe the surface with 75% alcohol. Turn off the clean bench, centrifuge, microscope, etc.

[0080] 1.1.1.3 Cell recovery

[0081] 1. Heat the water bath to 37°C. Take out the cells from -80°C and put them into a 37°C water bath, shaking constantly to thaw the cells quickly.

[0082] 2. Centrifuge the thawed cells at 1000 rpm for 3 minutes.

[0083] 3. Discard the supernatant in the cryopreservation tube, resuspend the cells in 1 mL of complete culture medium and evenly spread them into a culture dish of appropriate specifications. Shake well and place them in the incubator for culture.

[0084] 1.1.1.4 Protein extraction

[0085] 1. Prepare an appropriate amount of cell lysis buffer, the mass ratio of each component is RIPA lysis buffer: PMSF (phenylmethylsulfonyl fluoride): β-phosphoglycerate: protein phosphatase inhibitor: Na3VO4 = 100: 1: 1: 1: 1

[0086] 2. Discard the culture medium in the culture dish and wash twice with PBS. Add 120μL of lysis solution to each well of the six-well plate and scrape the cells with a scraper. Note: After using the scraper, wash it with 75% alcohol and ultrapure water and wipe it dry with paper to avoid cross-contamination of cells and affect the experimental results. Operate on ice throughout the process.

[0087] 3. Collect the cells into a 1.5 mL EP tube, turn on the ultrasonic cell disruptor and adjust its frequency to 25%.

[0088] 4. Wash the probe with ultrapure water, wipe it dry with paper, and then perform ultrasonic crushing, 2 seconds each time, 3 seconds interval, a total of 10 times. Be careful not to insert the probe too deep or too shallow or beyond the foam, and do it on ice throughout the process.

[0089] 5. Precool the centrifuge to 4°C, 13200rpm, 4°C, 15-30min.

[0090] 6. Carefully aspirate the supernatant and transfer it to a labeled new EP tube. Mix thoroughly and measure the protein concentration.

[0091] 7. When measuring protein concentration, make a standard curve. Take a 96-well plate, select the first well and add 10μL NaCl; add 9μL NaCl and 1μL BSA (bovine serum albumin) to the second well; add 8μL NaCl and 2μL BSA to the third well; add 6μL NaCl and 4μL BSA to the fourth well; add 2μL NaCl and 8μL BSA to the fifth well; add 10μL BSA to the sixth well. Add 8μL NaCl and 2μL of the protein sample to be tested to the subsequent measurement wells in turn. To ensure the accuracy of protein concentration, duplicate wells can be set as needed, with a mass concentration of NaCl of 9% and a concentration of BSA of 2mg / mL.

[0092] 8. Take the A solution and B solution from the BCA protein concentration assay kit, and prepare the working solution according to the instructions, based on the number of standard curve wells and measurement wells, 200 μL / well, A solution: B solution = 50:1, mix well and store in a dark place, use immediately after preparation, and do not store for too long. Due to the error of the pipette, you can prepare 1-2 more test solutions.

[0093] 9. Add the mixed working solution of solution A and solution B to the 96-well plate and incubate in a 37°C biochemical incubator for 20-25 minutes;

[0094] 10. After the incubation is over, turn on the microplate reader, call the protein concentration measurement program of this laboratory, and measure the absorbance values ​​of the standard curve wells and protein sample wells;

[0095] 11. Copy the absorbance value to an Excel table, with the absorbance value as the horizontal axis and the BSA protein concentration as the vertical axis, insert a scatter plot, add a trend line, display the formula, and display R 2 value, draw the standard curve, R 2 Must be greater than 0.99.

[0096] 12. According to the formula obtained from the standard curve, substitute the absorbance value of the protein sample to obtain the concentration of the protein sample. According to the protein concentration, add 5× or 2× Loading buffer and boil for 15-20 minutes to denature the protein, and use it directly for western blot loading or store it at -20℃. Calculate the actual concentration of the protein to be tested after adding the Loading buffer.

[0097] 1.1.1.5 Western Blot detection of protein expression

[0098] 1. Preparation of separation gel and concentration gel

[0099] (1) Clean and dry the thin and thick plates required for glue making, place the dried thin and thick plates in the glue making clamp, align the edges and fasten them;

[0100] (2) Prepare the separation gel of corresponding concentration according to the molecular weight of the protein to be detected by referring to the SDS-PAGE separation gel formula table;

[0101] (3) After vortexing, carefully pour the prepared separation gel into the interlayer between the thin plate and the thick plate. The liquid layer should be 1 cm below the comb. Seal with 75% ethanol.

[0102] (4) Let the separation gel stand for about 30 minutes. A clear dividing line can be seen between the separation gel and the upper alcohol layer, indicating that the lower separation gel has solidified. Discard the upper 75% ethanol layer and invert to dry.

[0103] (5) Prepare the required stacking gel according to the SDS-PAGE stacking gel recipe table, vortex and oscillate, and inject between the thin and thick gel plates until it reaches the upper edge of the thin plate;

[0104] (6) Insert the sample comb into the concentrated gel. Hold the comb with both hands and insert from one side.

[0105] (7) Let the concentrated gel stand for 20-30 minutes to solidify, remove the comb and use it directly for sample loading or store it at 4°C for later use.

[0106] 2. Loading of protein to be tested, electrophoresis and electrotransfer

[0107] (1) Take out the protein sample to be tested from the refrigerator at -20℃, place it in a boiling water bath for 5-10 minutes, and centrifuge it in a palm-type centrifuge for 20-30 seconds to allow the vapor liquid hanging on the tube wall to completely sink to the bottom of the tube. Freshly prepared protein samples can be directly centrifuged and loaded;

[0108] (2) Place the prepared gel plate into the electrophoresis gel plate holder and place it into the matching electrophoresis tank filled with 1× Running Buffer;

[0109] (3) Calculate the loading volume based on the actual concentration of the sample to be tested and the loading mass of the sample to be tested (30-50 μg). After the protein sample to be tested is loaded, add 2-3 μL of protein marker close to the sample well to mark the protein molecular weight;

[0110] (4) Turn on the electrophoresis instrument and adjust the voltage to 90 V. Run for 30 min until the markers are separated. At this time, adjust the voltage to 120-150 V. When the marker bands are separated to the required interval, end the electrophoresis.

[0111] (5) Prepare electrotransfer solution, transfer clips, sponges, filter paper, PVDF membrane, ice cubes and other items;

[0112] (6) Open the rubber plate, cut a corner to mark the loading order, and also cut a small corner of the PVDF membrane and soak it in methanol;

[0113] (7) Pour the electrotransfer solution into a special container for electrotransfer, open the transfer clamp in the electrotransfer solution, with the black side of the transfer clamp at the bottom and the white side at the top. Place the sponge, thick filter paper, glue, PVDF membrane, thick filter paper, and sponge between the black and white sides of the transfer clamp in sequence, and make sure there are no bubbles between the layers.

[0114] (8) Place the inner electroporation tank into the electroporation tank, place the transfer clip containing gel and ice cubes into the electroporation tank, pour the electroporation solution into the electroporation tank, cover the lid, turn on the power supply, adjust the voltage to 100 V, and adjust the time to 60 min.

[0115] (9) After the electroporation device stops working, quickly remove the PVDF membrane from the transfer clip. Clearly visible red and blue bands (Protein Maker) can be seen on the membrane. Place it in the blocking solution prepared in advance.

[0116] (10) Place the blocking tank containing the blocking solution on a shaker, slow down the speed, and incubate at room temperature for 60 min;

[0117] (11) After blocking, discard the blocking solution and wash off the remaining blocking solution on the PVDF membrane with TBST;

[0118] (12) Cut the protein band, put it into plastic film, add primary antibody incubation solution, put it into a 4°C cold storage shaker, slow down the speed, and incubate overnight;

[0119] (13) Recover the primary antibody, place the protein band incubated with the primary antibody into the membrane washing tank, and wash three times with TBST, each time for 10 minutes;

[0120] (14) After washing, incubate with the secondary antibody using the same incubation method as the antibody at room temperature with slow shaking on a shaker for 1 hour;

[0121] (15) The secondary antibody is recovered or directly discarded, and the protein bands incubated with the secondary antibody are washed three times with BST, also for 10 minutes each time;

[0122] (16) Prepare the ECL luminescent liquid, turn on the developing machine, precool the machine, call the program, evenly spread the ECL luminescent liquid on the PVDF membrane containing the protein strips, put it into the dark box of the developing machine, wait for exposure, and save the developed strips.

[0123] 1.1.1.6 RNA extraction and qPCR

[0124] 1. Extraction of total RNA

[0125] (1) Prepare RNA free pipette tips and total RNA extraction kit.

[0126] (2) Processing cell samples: After digestion with trypsin, collect the cells into a 1.5 mL centrifuge tube, add 100 μL of lysis buffer R1, mix well by pipetting, and let stand at room temperature for 1 min.

[0127] (3) Add 600 μL of lysis buffer R2 to the pretreated cell sample, invert to mix, and let stand at room temperature for 5 min.

[0128] (4) Transfer the supernatant to a purification column connected to a pipette and centrifuge at 12,000 rpm for 1 min.

[0129] (5) Discard the waste liquid in the liquid collecting tube, add 600 μL of washing solution to the purification column, and centrifuge at 12000 rpm for 1 min.

[0130] (6) Repeat step (5).

[0131] (7) Discard the liquid in the receiving tube, centrifuge the empty tube and column for 1 min, and transfer the purification column to a new 1.5 mL centrifuge tube.

[0132] (8) Add 20 μL of eluent to the purification column, let stand at room temperature for 1 min, and centrifuge at 12,000 rpm for 1 min. The liquid in the 1.5 mL centrifuge tube is the total RNA.

[0133] 2. Reverse transcription to synthesize cDNA

[0134] (1) Preparation. Prepare a simple ice box, place the measured RNA and the reagent kit required for RNA reverse transcription on ice, centrifuge the gDNA digester and HifairⅡSuperMix plus briefly, and mix them evenly. Prepare PCR reaction tubes, pipettes, etc.

[0135] (2) Take 1 μg of RNA for inversion. Calculate the required RNA volume based on the RNA concentration;

[0136] (3) Remove residual genomic DNA. Take the prepared RNase free PCR reaction tube, add RNA, 2 μL of 5×gDNAdigester Buffer, 1 μL of gDNA digester, and then add RNase free ddH2O to the total volume of the PCR reaction tube to 10 μL;

[0137] (4) Use a pipette to gently pipette the premixed solution in the PCR tube to mix well and incubate in a PCR machine at 42°C for 2 minutes;

[0138] (5) Take out 10 μL of the premixed solution incubated in the PCR instrument, add 10 μL of 2×HifairⅡSuperMix plus to prepare a 20 μL reverse transcription reaction system, mix well by pipetting, and set the reverse transcription reaction program of the PCR instrument: 25℃5min, 42℃30min, 85℃5min. After the program is completed, the cDNA is stored at -20℃.

[0139] 3. Real-time quantitative PCR

[0140] (1) Preparation: Make a simple ice box, place the qPCR kit and cDNA on ice, and prepare a pipette, eight-tube strips, etc.;

[0141] (2) Calculate the MonAmp required for each gene according to the qPCR reaction system recommended by Monad TM ChemoHSqPCR Mix, forward primer, reverse primer, and Nucleasse-Free water were premixed in a 1.5EP tube. The reaction system is shown in Table 1.

[0142] Table 1. Reaction system

[0143]

[0144] The sequences and sizes of primers for fluorescence quantitative PCR are shown in Table 2.

[0145] Table 2. PCR primers

[0146]

[0147] (3) The premix was added to 8 tube strips, 8 μL per well, and then the cDNA template was added to the 8 tube strips according to the reaction system. The mixture was vortexed to mix well, centrifuged to the bottom of the tube, and placed in a Roche PCR instrument for qPCR. The three-step reaction procedure is shown in Table 3.

[0148] Table 3. Reaction procedures

[0149]

[0150] Denaturation, annealing, and extension require 40 cycles.

[0151] Note: The annealing temperature is set according to the Tm value of the primer. Generally, when designing primers, the Tm value of the primer is generally selected between 55-65°C.

[0152] (4) Copy the results and analyze the data using the computer LC96 software.

[0153] 1.1.1.7 MTT assay for cell viability

[0154] 1. Resuspend the cells in 1 mL of culture medium on the clean bench between cells.

[0155] 2. Count the cells using a cell counter and inoculate into a 96-well plate with 5,000 to 10,000 cells per well.

[0156] 3. After the cells have attached to the wall and grown to a suitable density, add drugs and set up negative control wells.

[0157] 4. After culturing to the preset time point, add 20 μL of 5 mg / mL MTT and continue culturing.

[0158] 5. After 4 hours, discard the supernatant in the 96-well plate, add 150 μL of DMSO to each well, wrap it in a dark place and place it on a shaker for 15 minutes, turn on the microplate reader, and use the MTT program to measure the absorbance value of each well.

[0159] 1.1.2 Animal experiments

[0160] 1.1.2.1 Animal models

[0161] BALB / c mice were purchased from the Experimental Animal Center of Xiamen University and were fed adaptively in our laboratory for one week before being weighed.

[0162] 1. Acute lung injury model

[0163] (1) BALB / c mice (24 mice, 6 weeks old, weighing 18-20 g) were randomly divided into three groups, with 8 mice in each group: normal saline group (Control group), lipopolysaccharide (LPS) 5 mg / kg injury group (LPS group), 5 mg / kg LPS + 180 mg / kg Morroniside group (Morroniside group), or 5 mg / kg LPS + 150 mg / kg Loganin group (Loganin group).

[0164] (2) Animal modeling method: LPS was intraperitoneally injected into the LPS group, Morroniside group, and Loganin group, while an equal volume of saline was intraperitoneally injected into the Control group. Morroniside group and Loganin group were intragastrically administered with Morroniside solution and Loganin solution, respectively, one day before LPS intraperitoneal injection, twice a day. The Control group and LPS group were intragastrically administered with an equal volume of saline.

[0165] (3) Observe general condition and separate lung tissue

[0166] After intraperitoneal injection of LPS, the three groups of mice were observed for general conditions such as breathing, eating, oral, nasal and ocular secretions, and defecation. Twelve hours later, the mice were killed by cervical dislocation, and the chest was opened to obtain lung tissue.

[0167] 2. Pulmonary fibrosis model

[0168] (1) Animal grouping: BALB / c mice were randomly divided into 8 groups: normal saline group (Control group), bleomycin + normal saline group (BLM group), bleomycin + low concentration loganin group (BLM + loganin 80 mg / kg), bleomycin + medium concentration loganin group (BLM + loganin 160 mg / kg), bleomycin + high concentration loganin group (BLM + loganin 200 mg / kg), bleomycin + low concentration morroniside group (BLM + morroniside 50 mg / kg), bleomycin + medium concentration morroniside group (BLM + morroniside 100 mg / kg), bleomycin + high concentration morroniside group (BLM + morroniside 200 mg / kg), 8 mice in each group.

[0169] (2) Animal modeling method: BLM group, BLM+loganin group and BLM+morroniside group were intraperitoneally injected with BLM 35 mg / kg, 3 times / week for 8 weeks. At the same time, morroniside and loganin were intragastrically administered, 2 times / week, starting from the first week.

[0170] (3) During the modeling period, the mice were weighed periodically. After 8 weeks, the mice were killed by cervical dislocation and the lung tissue was obtained by thoracotomy.

[0171] 3. Divide the whole lung into 5 parts and place them in EP tubes. Perform lung tissue pathology test, mouse lung dry / wet ratio, protein extraction, RNA extraction, and enzymatic test respectively. If not tested in time, the lung tissue should be stored at -80℃.

[0172] 1.1.2.2 Extraction of total RNA from mouse lung tissue

[0173] 1. Mark Trizol (Takara), anhydrous ethanol, isopropanol, chloroform, DEPC water, RNase-free gun tips, RNase-free EP tubes, high-pressure grinding rods and other items needed for the experiment, place them on the clean bench, and irradiate them with ultraviolet light for 10 minutes in advance;

[0174] 2. Cut the lung tissue in the 1.5mL EP tube into small pieces with scissors, add 500μL Trizol, grind until the lung tissue is finely minced, then add 300μL Trizol to each tube, pipette and mix evenly, cover the EP tube tightly, shake vigorously for 10 seconds, and leave at room temperature for 10 minutes;

[0175] 3. After 10 minutes, add 160 μL of chloroform (chloroform: Trizol volume ratio is 1:5) to the EP tube, cover tightly, shake vigorously until the color changes to pink turbid liquid, and leave at room temperature for 10 minutes. Turn on the centrifuge and precool to 4°C;

[0176] 4. Transfer the EP tube to a centrifuge and centrifuge at 13200 rpm at 4°C for 15 minutes;

[0177] 5. Take out the EP tube. You can see that the liquid has been divided into three layers. Carefully transfer the upper layer of the clear liquid in the EP tube to the 1.5mL RNase free EP tube. Be careful not to absorb the middle and lower layers of liquid.

[0178] 6. Add 1 mL of isopropanol to the RNase free EP tube containing the supernatant, invert to mix, let stand at room temperature for 10 minutes, and transfer to a centrifuge.

[0179] 7. Centrifuge at 13200rpm and 4℃ for 15 minutes. Take out the centrifuge tube and you can see the white precipitate at the bottom of the EP tube, which is the extracted total RNA.

[0180] 8. Discard the supernatant and use a small-range pipette to carefully suck the remaining liquid at the bottom of the tube into a waste bottle. Add 500 μL of 70% ethanol (prepared from anhydrous ethanol and DEPC water) to each tube, and use a pipette to gently blow up the protein precipitate (be careful not to blow it apart), and transfer it to a pre-cooled centrifuge.

[0181] 9. Centrifuge at 13200 rpm and 4°C for 5 minutes, discard the washing solution in the EP tube, and repeat the previous step.

[0182] 10. After centrifugation at 13200rpm and 4℃, absorb all the residual liquid in the EP tube, open all the EP tube caps, adjust the ultra-clean hood wind speed to the maximum, and dry for 10-20 minutes.

[0183] 11. Add an appropriate amount of DEPC water to dissolve the precipitated RNA at the bottom of the EP tube.

[0184] 12. Take 2 μL of dissolved RNA and measure its concentration and purity. If the concentration is too high, dilute it to the required concentration as needed. After measuring the concentration, reverse transcribe it in time and store it at -80℃.

[0185] 13. The reverse transcription synthesis of cDNA and real-time PCR methods are as described in the previous cell experiment and will not be repeated here.

[0186] 1.1.2.3 Protein extraction from mouse lung tissue

[0187] 1. Cut the mouse lung tissue in 1.5mL EP tubes with scissors, add appropriate amount of lysis buffer according to the size of the lung tissue (the lysis buffer is prepared and used immediately, and the composition is: RIPA lysis buffer, PMSF, protein phosphatase inhibitor, β-phosphoglycerate, Na3VO4), grind with a grinding rod, and let it stand for 5 minutes. The whole process should be operated on ice.

[0188] 2. The remaining steps are the same as the corresponding parts in the cell experiment.

[0189] 1.1.2.4 Western Blot detection of changes in tissue protein expression

[0190] The Western Blot part in the same cell experiment is omitted here.

[0191] 1.1.2.5 Pathological examination of lung tissue

[0192] 1. Place the right upper lobe of the mouse lung in a 4% paraformaldehyde aqueous solution and fix it at room temperature for more than 48 hours. The amount of fixative is 10-15 times the volume of the fixed tissue. After the tissue is fixed, rinse the tissue with running water for 2-3 hours and then put it into the embedding frame for subsequent experiments.

[0193] 2. Tissue dehydration and transparent wax immersion. The purpose is to replace the water in the tissue with ethanol, then replace the ethanol with xylene, and then replace the xylene with paraffin. This process is carried out in the central laboratory of Xiamen University Medical College. The instrument used is the fully automatic tissue dehydration machine ASP200S. The setting program is as follows:

[0194] (1) 50% ethanol for 6 hours; (2) 70% ethanol for 7 hours; (3) 80% ethanol for 1 hour; (4) 90% ethanol for 1 hour; (5) 95% ethanol for 1 hour; (6) anhydrous ethanol ① for 1 hour; (7) anhydrous ethanol ② for 1 hour; (8) xylene ① for 1 hour; (9) xylene ② for 1 hour; (10) soft wax for 30 minutes; (11) hard wax ① for 1 hour; (12) hard wax ② for 1 hour;

[0195] 3. Embedding is carried out in the central laboratory of the Medical College, and the instrument used is the embedding machine EG1150H. The instrument is turned on 30 minutes in advance to heat and melt the wax, and the tissue angle is adjusted according to needs for embedding;

[0196] 4. The sections were made in the central laboratory of the Medical College. The instrument used was the paraffin slicer RM2245. The slide spreader and slide baking machine were the supporting instruments of the paraffin slicer. The section thickness was 4 μm.

[0197] 5. Mark the slide with a pencil, adjust the temperature of the slide spreader to 37℃, spread the slide until the wrinkles are fully unfolded and there is no loose separation, pick up the tissue wax slide with a slide, place it in a slide baking machine, bake it at 42℃ overnight or 60℃ for 1h, and after drying for a year, place it in a section box for storage or directly use it for wax dewaxing;

[0198] 6. The purpose of dewaxing and rehydrating is to replace the paraffin in the paraffin tissue section with the organic solvent xylene (paraffin and xylene are mutually soluble), then replace the organic solvent with ethanol (ethanol and xylene are mutually soluble), and finally use gradient ethanol to replace the ethanol in the tissue with water, so that it can be stained with water-soluble dyes. The specific steps of dewaxing and rehydrating are as follows:

[0199] Dewaxing: (1) xylene ① 15 min; (2) xylene ② 15 min;

[0200] Rehydration: (1) Anhydrous ethanol ① 5 min; (2) Anhydrous ethanol ② 5 min; (3) 95% ethanol 3 min; (4) 80% ethanol 2 min; (5) 70% ethanol 2 min; (6) Ultrapure water

[0201] 7. HE staining: Hematoxylin combines with intracellular acidic substances to present a blue-purple color, and eosin combines with cytoplasm to present a red color. The specific steps are as follows:

[0202] (1) After 5 minutes of hematoxylin staining, wash off the excess dye with tap water and observe under a microscope;

[0203] (2) Stain with eosin for 3 minutes. The staining time can be appropriately increased or shortened depending on whether the tissue is easy to stain. Excess dye is washed off with tap water. The staining effect is confirmed by microscopic examination before sealing.

[0204] (3) After the tissue is stained with hematoxylin and eosin, it is dehydrated and mounted. The purpose of dehydration is to replace the water in the slice with ethanol, and then replace the ethanol with xylene, so that it is convenient for the non-water-soluble mounting medium neutral gum used in this laboratory to mount the slice. The steps are as follows:

[0205] 1) 70% ethanol by volume for 5 seconds;

[0206] 2) 80% ethanol by volume for 10 seconds;

[0207] 3) 95% ethanol for 1 minute;

[0208] 4) Anhydrous ethanol ① 2 minutes;

[0209] 5) Anhydrous ethanol②2 minutes;

[0210] 6) Xylene ① 5 minutes;

[0211] 7) Xylene②5 minutes;

[0212] 8) Seal the slide with neutral gum;

[0213] (4) After sealing, examine the slide again under a microscope. If the staining is not satisfactory, immerse the slide in xylene, remove the coverslip, and stain again.

[0214] 8. Masson staining

[0215] (1) Check whether there is enough R1 nuclear stain, R2 pulp stain, R3 color separation solution, R4 counterstain, and R5 rinse solution in the kit.

[0216] (2) Sample processing: Dewax the paraffin sections and rehydrate them with a gradient of ethanol (volume): 95%, 70%, 30%, and ultrapure water, 2 min each. After rehydration, wash twice with warm water at 30-40°C.

[0217] (3) Use a 200 μL pipette to draw up R1 nuclear stain solution and drop it on the location of the tissue section on the slide. After staining for 60 seconds, rinse with tap water.

[0218] Use a pipette to draw up R2 slurry stain solution to stain tissue sections for 30-60 seconds, and rinse with tap water for 30 seconds.

[0219] (5) Separate with R3 colorimetric solution for 6-8 minutes. Under microscope, the collagen fibers in the tissue sample should be light pink. Discard the colorimetric solution.

[0220] (6) Stain with R4 counterstain for 5 minutes, discard the counterstain, dehydrate with graded ethanol, and examine under a microscope.

[0221] 1.1.2.6 Lung tissue wet / dry (W / D) ratio

[0222] Take out the right middle lobe of the lung from the 1.5mL EP tube, use filter paper to absorb the liquid on the surface of the tissue, weigh and record its wet weight (W) using an electronic analytical balance, put it in a 65℃ oven to dry for 48 hours until constant weight, weigh and record the dry weight (D). The wet / dry (W / D) value of the lung tissue can reflect the degree of edema of the lung tissue.

[0223] 1.1.2.7 Determination of superoxide dismutase (SOD) content in mouse lung tissue

[0224] 1. Prepare WST-8 / enzyme working solution according to the number of samples and standards to be tested. Each sample to be tested requires 160μL of working solution, including 151μL of SOD detection buffer, 8μL of WST-8, and 1μL of enzyme solution. The working solution should be prepared and used immediately, and should not be stored at 4℃ for more than 24 hours.

[0225] 2. Prepare the reaction start working solution according to the number of samples and standards to be tested. Dilute the 40× reaction start solution with SOD detection buffer, the ratio is 1μL of 40× reaction start solution plus 39μL of SOD detection buffer. Prepare and use immediately or store at 4℃ and use on the same day.

[0226] 3. Weigh part of the left lung tissue placed in the EP tube. Add 100 μL of sample preparation solution to 10 mg of tissue, homogenize in an ice bath, centrifuge at 12,000 g, 4°C for 5 min, take the supernatant, and determine its concentration. Each sample to be tested needs to prepare 20-100 μg of protein for subsequent testing.

[0227] 4. Since the inhibition percentage in subsequent tests must be between 30% and 70%, when the inhibition percentage is high, dilute the sample; when the inhibition percentage is low, re-prepare a higher concentration of the sample to be tested.

[0228] 5. Add buffer, working solution and sample to be tested in order according to Table 4. Operate on ice and mix thoroughly by pipetting. Because lung tissue is pink, blank control 3 is required to eliminate interference.

[0229] Table 4. Preparation of samples to be tested

[0230] Sample hole Blank control well 1 Blank control well 2 Blank control group 3 Samples to be tested 20μL － － 20μL SOD Assay Buffer － 20μL 40μL 20μL WST-8 / Enzyme working solution 160μl 160μl 160μl 160μl Reaction start-up solution 20μL 20μL － －

[0231] 6. Incubate at 37°C for 30 min.

[0232] 7. Use the full-wavelength microplate reader in the central laboratory of the Medical College to measure the absorbance, with 650nm as the reference wavelength and 450nm as the measurement wavelength. The actual reading is the absorbance value at 450nm minus the absorbance value at 650nm.

[0233] 8. Calculate the total SOD activity of the sample to be tested

[0234] The calculation formula is as follows:

[0235]

[0236] (Note: A is absorbance)

[0237] In the sample to be tested

[0238] The protein mass was calculated based on the protein concentration and dilution multiple, and the SOD activity unit was converted into U / g or U / mg.

[0239] 1.1.2.8 Determination of malondialdehyde (MDA) content in mouse lung tissue

[0240] 1. Prepare TBA stock solution: 25 mg of TBA is added to 6.76 mL of TBA stock solution to obtain 0.37% TBA stock solution. The TBA stock solution needs to be heated to 70°C to promote dissolution. The prepared 0.37% TBA stock solution can be stored at room temperature for three months.

[0241] 2. Prepare the MDA working solution. The MDA working solution consists of TBA storage solution, TBA diluent and antioxidant, and is used on the same day. The proportions are shown in Table 5.

[0242] Table 5. Preparation of MDA detection working solution

[0243] Number of samples tested 10 20 50 TBA diluent 1500μL 3000μL 7500μL TBA storage solution 500μL 1000μL 2500μL Antioxidants 30μL 60μL 150μL

[0244] 3. Dilute the standard. Dilute the 1mM standard with ultrapure water to 1, 2, 5, 10, 20, 50, 100μM, with a total volume of 100μL. The specific steps are shown in Table 6.

[0245] Table 6. Dilution of Standards

[0246]

[0247]

[0248] In order to ensure the accuracy of the standard curve, the 1mM standard can be diluted to 10μM first, and then 10μM is diluted to 5μM, 2μM, and 1μM.

[0249] 4. Prepare tissue samples: Lyse the tissue with Western cell lysis buffer. After lysis, centrifuge at 12000g for 10 min at 4°C, take the supernatant and measure the protein concentration with BCA protein content assay kit to calculate the MDA content in the tissue per unit protein weight.

[0250] 5. Mix the detection reaction system in a centrifuge tube. The reaction system is shown in Table 7.

[0251] Table 7. Reaction system

[0252] Blank control tube Standard quality control Sample tube Lysis buffer 0.1mL － － Standards － 0.1mL － Samples to be tested － － 0.1mL MDA detection working solution 0.2mL 0.2mL 0.2mL

[0253] After mixing the liquid in each tube, boil it in a 100°C water bath for 15°C. After cooling to room temperature, centrifuge at 1000 g for 10 minutes.

[0254] 6. After centrifugation, take 200 μL of the upper clear liquid into a 96-well plate, turn on the full-wavelength microplate reader, use λ=450nm as the reference wavelength and λ=532nm as the measurement wavelength, and measure the absorbance value of each sample.

[0255] 7. Draw a standard curve based on the absorbance value and concentration of the standard and calculate the MDA content of the sample to be tested. Calculate the protein mass based on the protein concentration and volume, and finally calculate the MDA content per unit mass of protein or tissue based on the MDA content and protein mass, expressed in μmoL / mg protein or μmoL / mg tissue.

[0256] 1.1.2.9 Determination of hydroxyproline content in mouse lung tissue

[0257] 1. Prepare Reagent 1: Dissolve the powder of Reagent 1 in the Hydroxyproline Test Kit in 10 mL of Solution A. After mixing thoroughly, add 20 mL of Solution B. It is best to prepare it before use and store it at 4°C for 3 months.

[0258] 2. Prepare Reagent 3: Add the Reagent 3 powder in the kit into 30 mL of solvent to dissolve. Prepare and use immediately. Store at 4°C away from light for 30 days.

[0259] 3. Prepare standard stock solution: Dissolve the hydroxyproline standard in 30 mL of ultrapure water, dilute to 50 mL, and prepare a 100 μg / mL standard stock solution. Store at 4°C for 2 weeks. Prepare a 5 μg / mL standard application solution before use.

[0260] 4. Accurately weigh 15-50 mg of wet weight of lung tissue and place it in a 1.5 mL EP tube. Add 500 μL of hydrolyzate and mix well by pipetting. Then place in a boiling water bath for 20 minutes. Remember to mix every 10 minutes.

[0261] 5. Adjust the pH value to around 6.0-6.8: rinse and cool each EP tube with running water, then add 5 μL of indicator and mix well. Add 500 μL of pH adjustment solution A and mix well. Use a 200 μL pipette to add pH adjustment solution B (about 50-250 μL). Add one drop at a time and mix well until the red color of the indicator disappears and turns to yellow-green.

[0262] 6. Add ultrapure water to 5 mL and mix well. Take 2 mL of the hydrolyzate and add an appropriate amount of activated carbon (about 15 mg). Centrifuge at 3500g for 10 min and take 500 μL of the supernatant for detection.

[0263] 7. Follow the instructions in Table 8.

[0264] Table 8. Operating conditions

[0265]

[0266]

[0267] 8. Cool, centrifuge at 3500g / min for 10min, take the supernatant and add it to a 96-well plate, adjust the wavelength to 550nm, and measure the absorbance of each well.

[0268] 9. Calculate tissue hydroxyproline content using the following formula:

[0269]

[0270] 1.1.2.10 Flow cytometry

[0271] 1. Sample processing: Add 200μL anticoagulant and 500μL red blood cell lysis solution to the EP tube, take 2-3 drops of blood from the mouse tail vein and add to the above EP tube, lyse at room temperature for 30 minutes, centrifuge at 1500rpm for 3min. Discard the supernatant and add 500μL PBS to resuspend, centrifuge at 1500rpm for 3min, repeat twice. Discard the supernatant and resuspend in 100μL PBS for later use.

[0272] 2. Prepare antibody premix: Add 20 μL of antibody premix to each sample. The antibody premix contains CD8 (0.1 μL), CD4 (0.02 μL), and PBS (19.88 μL).

[0273] 3. Add the antibody premix to each sample, stain on ice for 30 minutes, centrifuge at 1500 rpm for 3 minutes, wash twice with PBS, resuspend in 300 μL PBS and transfer to a flow tube for later use.

[0274] 4. Open the flow cytometer Fortessa, adjust various parameters, load samples, copy data, and analyze the results.

[0275] 1.2 Statistical analysis

[0276] Prism 7.0 was used for data analysis. All data were from at least three independent experiments. The measured data were expressed as mean±SD. T-test was used for pairwise comparison between groups. P<0.05 was considered statistically significant.

[0277] Experimental Results

[0278] 1.3 Cell experiments

[0279] Acute lung injury causes varying degrees of damage to lung cell function. Alveolar cells and immune cells are target cells for inflammatory responses during lung injury. When the inflammatory response gets out of control, it can lead to damage to alveolar cell function and a decrease in surfactant, which in turn leads to small airway closure, alveolar collapse and atelectasis, which in turn causes respiratory distress, aggravates pulmonary edema and lung injury. Therefore, the applicant selected rat alveolar type II cells RLE-6TN and mouse macrophages RAW 264.7 as cell models for acute lung injury.

[0280] 1.3.1 RLE-6TN cell experiments

[0281] 1.3.1.1 Effects of morroniside and loganin on the proliferation activity of rat alveolar type II RLE-6TN cells

[0282] Current studies have shown that cyclopentadiene compounds have anti-tumor and nervous system protection effects, but there is no report on whether morroniside and loganin have a killing effect on normal alveolar cells when used alone. We consulted relevant information and selected appropriate concentrations and action times to conduct MTT experiments to verify the effects of morroniside and loganin on the proliferation activity of normal rat alveolar type II cells RLE-6TN. The experimental results showed that ( Figure 1 ): When the doses of morroniside and loganin were 10μM, 50μM, and 100μM, there was no effect on cell proliferation within 48 hours.

[0283] exist Figure 1 A. Effects of different doses (10 μM, 50 μM, 100 μM) of morroniside on RLE-6TN cells for different time periods (12h, 24h and 48h) on cell proliferation (P>0.05, n=5). B. Effects of different doses (10 μM, 50 μM, 100 μM) of loganin on RLE-6TN cells for different time periods (12h, 24h and 48h) on cell proliferation (P>0.05, n=5). There was no significant difference in the effects of different doses of morroniside and loganin on RLE-6TN cells for the same time period, and the results are not shown.

[0284] 1.3.1.2 Differences in the relative expression of inflammatory factors IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA in RLE-6TN cells stimulated by different concentrations of LPS

[0285] Although alveolar type II cells only account for 14%-16% of alveolar epithelial cells, they are considered to be stem cells of the alveolar epithelium. They can differentiate into alveolar type I epithelial cells, proliferate into new alveolar type II epithelial cells, and secrete surfactant. Therefore, rat alveolar type II epithelial cells RLE-6TN were selected as a cell model of lung injury. The LPS working solution was diluted with DMEN culture medium to prepare DMEM culture medium containing different concentrations of LPS. The cells were divided into 4 groups, culture medium group (Control), LPS 2μg / mL treatment group, LPS 5μg / mL treatment group, and LPS 10μg / mL treatment group. The cells were cultured in 6-well plates. When the cells grew to an appropriate concentration, DMEM culture medium containing the corresponding concentration of LPS was added and cultured for another hour. RT-PCR was used to detect changes in the expression levels of IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA. Figure 2 (A. IL-6 mRNA expression; B. IL-1β mRNA expression; C. TNF-α mRNA expression) shows that RLE-6TN can actively respond to LPS stimulation. At the three preset concentrations, the inflammatory factors IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA were all upregulated (compared with the Control group, ***P < 0.005, ****P < 0.0001, respectively). IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA were most significantly upregulated when the LPS concentration was 2μg / mL. Subsequent experiments used LPS 2μg / mL to model lung injury cell models.

[0286] 1.3.1.3 Effects of LPS treatment at different times on the relative expression of inflammatory factors IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA in RLE-6TN cells

[0287] We have previously demonstrated that LPS treatment of RLE-6TN cells can upregulate the expression of inflammatory factors. In order to explore the optimal LPS action time for cell modeling, the LPS working solution was diluted to 2 μg / mL with DMEM complete medium. When the cells grew to an appropriate density, the complete culture medium was replaced with cell culture medium supplemented with LPS (2 μg / mL) 6 h, 3 h, 2 h, 1 h, and 0.5 h before cell collection and continued to be cultured. RT-PCR detected changes in the expression levels of IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA. The results are shown in Figure 3As shown: Within 6 hours of LPS treatment, the expression of inflammatory factors was upregulated, and the subsequent experiments selected LPS for 1 hour as the modeling time of the cell model. Among them, A. IL-6 mRNA was upregulated more significantly at 1 hour than at other times; B. IL-1β mRNA was upregulated more significantly at 0.5 hours; C. TNF-α mRNA was most significantly upregulated at 2 hours (compared with the Control group, **P<0.01, ***P<0.005, ****P<0.0001, respectively).

[0288] 1.3.1.4 The iridoid compounds morroniside and loganin inhibit the upregulation of inflammatory cytokines IL-6mRNA, IL-1βmRNA, and TNF-αmRNA produced by RLE-6TN stimulated by LPS

[0289] The applicant has obtained the time and concentration of LPS for stimulating RLE-6TN cell modeling through experiments, the time is 1 hour, and the concentration is 2μg / mL. It is not known whether Morroniside and Loganin have a protective effect on acute lung injury. Therefore, we used Morroniside and Loganin to pre-treat the lung injury RLE-6TN cell model to preliminarily verify the anti-inflammatory effect of Morroniside and Loganin. Dilute the prepared Morroniside and Loganin working solution to 10μM. Divide the cells into three groups: Control group, LPS group, Morroniside+LPS group or Loganin+LPS group. After the cells are cultured to the appropriate density, 10μM Loganin or Morroniside is added, and the culture is continued for 24 hours. 2μg / mL LPS is added to the corresponding wells and the cells are collected for 1 hour. q-PCR is used to detect the expression levels of IL-6mRNA, IL-1βmRNA, and TNF-αmRNA. The results are as follows Figure 4 As shown, morroniside and loganin can reduce the upregulation of inflammatory factors IL-6mRNA, IL-1βmRNA, and TNF-αmRNA caused by LPS stimulation of RLE-6TN. A. After adding morroniside, IL-6mRNA was downregulated (compared with the LPS group, ****P < 0.0001), IL-1βmRNA was downregulated (compared with the LPS group, ****P < 0.0001), and TNF-αmRNA (compared with the LPS group, **P < 0.01 were all downregulated); B. After adding loganin, IL-6mRNA was downregulated (compared with the LPS group, ****P < 0.0001), IL-1βmRNA was downregulated (compared with the LPS group, ***P < 0.005), and TNF-αmRNA was downregulated (compared with the LPS group, *P < 0.05).

[0290] 1.3.1.5 Morroniside and loganin alleviate the inflammatory response of RLE-6TN cells stimulated by LPS and are related to the NF-κB / Stat3 signaling pathway

[0291] The pathogenesis of acute lung injury involves a complex inflammatory cascade reaction. LPS is an activator of the NF-κB signaling pathway. After activation, the NF-κB signaling pathway can stimulate the transcription of inflammatory cytokines. IL-6 can regulate the Stat3 signaling pathway. Therefore, we considered exploring the mechanism of action of Morroniside and Loganin from the NF-κB / Stat3 signaling pathway. The cells were divided into four groups: Control group, LPS group, Morroniside+LPS group and Loganin+LPS group. The action time and concentration of Morroniside, Loganin and LPS were consistent with those in 3.1.1.4. The results are shown in Figure 5 : Under LPS stimulation, the expression of p-p65 and stat3 was upregulated, while when morroniside and loganin were added in advance, the protein expression of p-p65 and Stat3 was downregulated.

[0292] 1.3.2 RAW 264.7 cell experiments

[0293] 1.3.2.1 Effects of morroniside and loganin treatment on the proliferation activity of mouse macrophage cell line RAW 264.7

[0294] Mouse macrophage cell line RAW 264.7 can actively respond to external stimuli and is often used to establish a cell inflammation model. Similarly, although the anti-inflammatory effects of cyclopentadiene ether terpenoids have been reported, there are no reports on whether the single application of morroniside and loganin will affect the proliferation of RAW 264.7. MTT was used to detect the effect of morroniside and loganin treatment on the proliferation of RAW 264.7. The experimental results are as follows Figure 6 , A. Effect of morroniside concentration gradient treatment on cell proliferation of RAW 264.7 for different time periods (12h, 24h, 48h). B. Effect of loganin concentration gradient treatment on cell proliferation of RAW 264.7 for different time periods (12h, 24h, 48h). Morroniside and loganin 10μM, 100μM, 200μM acted on RAW 264.7 cells for 12 hours and 24 hours, and did not show a killing effect on the cells, but instead promoted cell proliferation; at 48 hours, each concentration of morroniside had no significant effect on cell proliferation, while loganin at 10μM could promote the proliferation of RAW 264.7 cells.

[0295] 1.3.2.2 Differences in the relative expression of inflammatory factors IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA in RAW 264.7 cells stimulated by different concentrations of LPS

[0296] RAW 264.7, as an immune cell, is different from normal alveolar type II cells. Low concentrations of LPS stimulation can promote dramatic changes in the expression of proinflammatory factors. According to literature data, a concentration gradient of LPS below 1μg / mL was made to determine the optimal concentration for modeling. The cells were divided into 6 groups: Control group, LPS 50ng / mL group, LPS 100ng / mL group, LPS200ng / mL group, LPS 500ng / mL group, and LPS 1μg / mL group. When the cells grew to an appropriate concentration, complete culture medium containing the corresponding LPS concentration was added to each well according to the marked groups and cultured for 12 hours. The cells were collected and q-PCR was used to detect the expression of proinflammatory factors IL-6mRNA, IL-1βmRNA, and TNF-αmRNA. The results are as follows. Figure 7 As shown in the figure, under the stimulation of relatively low LPS concentration, the expression of pro-inflammatory factor mRNA in RAW 264.7 was significantly upregulated. Among them, A. The expression of IL-6 mRNA was upregulated under the stimulation of different concentrations of LPS (compared with the Control group, ***P < 0.005, ****P < 0.0001), and the concentration after 100ng / mL was concentration-dependent; B. The expression of IL-1β mRNA was upregulated under the stimulation of different concentrations of LPS (compared with the Control group, **P < 0.01, ****P < 0.0001), C. The expression of TNF-α mRNA was upregulated under the stimulation of LPS (compared with the Control group, ****P < 0.0001), and there was no significant difference in the upregulation multiple within the concentration range of 1μg / mL.

[0297] 1.3.2.3 The iridoid compounds morroniside and loganin can inhibit the expression of IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA in RAW 264.7 cells stimulated by LPS

[0298] Before LPS treatment, terpenoid glycosides morroniside and loganin were added to RAW 264.7 to verify that morroniside and loganin can protect against LPS-induced cellular inflammatory response. Unlike rat alveolar type II cells, the LPS concentration acting on RAW 264.7 was 500 ng / mL and the treatment time was 12 hours; the treatment concentration and time of loganin were 25 μM and 24 hours; the treatment concentration and time of morroniside were the same as those of RLE-6TN, which were 10 μM and 24 hours. The results are shown in Figure 8 As shown: Morroniside and loganin alone can inhibit the increase in the expression of IL-6mRNA, IL-1βmRNA, and TNF-αmRNA in RAW 264.7 cells stimulated by LPS. Figure 8A. The expression of proinflammatory factors IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA was downregulated after pretreatment with morroniside; B. The expression of proinflammatory factors IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA was downregulated after treatment with loganin.

[0299] 1.3.2.4 Effects of morroniside and loganin pretreatment on the expression of proteins related to the NF-κB / Stat3 signaling pathway in RAW264.7 cells

[0300] The previous experiment detected the changes of signaling pathway proteins in the rat alveolar type II cell lung injury model induced by morroniside and loganin. Similarly, we performed similar grouping and treatment on RAW 264.7 cells. Fig. 9 As shown, the addition of the iridoid compounds morroniside and loganin can reduce the upregulation of p-p65 and stat3 protein expression caused by LPS stimulation.

[0301] In summary, by using LPS-induced RLE-6TN and RAW 264.7 acute lung injury cell models, it can be preliminarily determined that the iridoid ether terpenoid compounds morroniside and loganin can alleviate further damage to lung cells and tissues by reducing the production of inflammatory factors; changes in the expression of p-p65 and Stat3 proteins indicate that the inhibitory effects of morroniside and loganin on inflammatory responses may be regulated through the NF-κB / Stat3 signaling pathway.

[0302] 1.4 Animal experiments

[0303] The animal experiment of acute lung injury was conducted in two batches. The first batch used morroniside (180 mg / kg) to pre-protect the mice with acute lung injury, and the second batch used loganin (150 mg / kg) to prevent and protect the mice with acute lung injury. The experimental data were also processed in two batches, so if there is no special note in the following experimental results of acute lung injury animal model, Group A represents the first batch of mice prevented and protected by morroniside, and Group B represents the second batch of mice prevented and protected by loganin.

[0304] 1.4.1 General Condition of Mice

[0305] The mice in the Control group had normal hair, moved freely, ate normally, and had normal mouth, nose, eyes, and anus, with no abnormal secretions. The mice in the LPS group and the Morroniside+LPS group or the Loganin+LPS group had dull hair, no appetite, and were lazy. A large amount of secretions could be seen blocking their eyes and anus. However, the mice in the Morroniside+LPS group or the Loganin+LPS group recovered and had increased activity before being killed. Weight changes can reflect the basic conditions of mice, such as food intake and activity, to a certain extent. Since the samples were collected 12 hours after modeling, the weight changes were not obvious. The weight changes and weight change rates of the mice were statistically analyzed. The results are as follows: Fig.10 As shown: Compared with the Control group, the weight loss rate (weight loss in grams / weight before modeling in grams) of the LPS group was significantly increased (****P<0.0001, n=8), and there was no significant difference in weight loss rate between the LPS group and the Morroniside+LPS group or the Loganin+LPS group. The weight change of mice in the Control group was the smallest, and its weight change rate was less than 5%; the weight loss rate of the LPS group and the weight loss rate of the Morroniside+LPS group or the Loganin+LPS group was about 15%.

[0306] 1.4.2 HE staining of mouse lung tissue

[0307] like Fig.11 (A is morroniside, B is loganin) As shown: In the mice of the Control group, the alveolar cell structure was clear, and there was no infiltration of inflammatory cells in the alveoli and alveolar interstitium. In the mice of the LPS group, a large number of inflammatory cells infiltrated the alveoli and alveolar interstitium, and the alveolar septa thickened. Compared with the LPS group, the infiltration of inflammatory cells in the alveoli and interstitium in the morroniside group and loganin group was significantly reduced, and the thickening of the alveolar septa was also alleviated. It can be concluded that cyclopentadiene ether terpenoid compounds can reduce the infiltration of inflammatory cells and the destruction of cell structure in acute lung injury in mice caused by LPS.

[0308] 1.4.3 Wet / dry ratio (W / D) of mouse lung tissue

[0309] Acute lung injury will cause pulmonary edema due to imbalance in the generation and reflux of tissue fluid in the lungs, which will accumulate in the alveoli and interstitial lungs, leading to ventilation and gas exchange dysfunction and dyspnea. We used the ratio of wet weight to dry weight of lung tissue to represent the degree of pulmonary edema, and the results are shown in Tables 9 and 10: Morroniside and loganin can alleviate pulmonary edema caused by acute lung injury in mice induced by intraperitoneal injection of LPS.

[0310] Table 9. Ratio of wet / dry mass of mouse lung tissue

[0311]

[0312] Table 10. Ratio of wet / dry mass of lung tissue

[0313]

[0314] As shown in Tables 9 and 10, compared with the Control group, the W / D value of the lung tissue in the LPS group increased, indicating that effusion occurred in the lung tissue of mice after LPS treatment; compared with the LPS group, the W / D ratio of morroniside or loganin treatment decreased. **P<0.01vs Control group, *P<0.05vs LPS group, Mean±SD, n=8.

[0315] 1.4.4 Detection of SOD and MDA, antioxidant function indicators in mouse lung tissue

[0316] 1.4.4.1 Detection of superoxide dismutase (SOD) activity in mouse lung tissue

[0317] Superoxide dismutase (SOD) is a metal enzyme widely found in the biological world. It can catalyze the dismutation reaction of superoxide free radicals in the body, remove the excess superoxide anion free radicals produced by cell metabolism or damage, protect cells from damage, and has antioxidant, immunity-enhancing, anti-inflammatory, and anti-aging effects. The level of SOD activity can indirectly reflect the body's ability to remove oxygen free radicals. Acute lung injury is a complex pathophysiological process, and the activity of superoxide dismutase in the body also plays a very important role in the occurrence of the disease. Our experimental results show that ( Fig.12 ): When mice have acute lung injury, the activity of SOD decreases; when mice with acute lung injury are given the iridoid compounds morroniside and loganin for preventive care, the activity of SOD in the mice can be increased, improving the mice's ability to scavenge oxygen free radicals. Fig.12 A. The average SOD activity of the Control group was 26.83±0.7363 (U / mg lung tissue wet weight), n=8; the average SOD activity of the LPS group was 17.07±0.5678 (U / mg lung tissue wet weight), compared with the Control group, the SOD activity of the LPS group decreased, ***P<0.005, n=8; the average SOD activity of the LPS+Morroniside group was 29.66±1.317 (U / mg lung tissue wet weight), compared with the LPS group, the SOD activity of the LPS+Morroniside group increased, ###P<0.005, n=8. B. The data of the loganin preprotection group had the same trend as the morroniside preprotection (LPS vs Control, **P<0.01; LPS+Loganin vs LPS, ##P<0.01, n=8).

[0318] 1.4.4.2 Malondialdehyde (MDA) in mouse lung tissue

[0319] When the body's ability to remove oxygen free radicals decreases, excessive oxygen free radicals act on biological membranes, causing lipid peroxidation and producing lipid peroxidation products, leading to cell damage and metabolic dysfunction in the body. Malondialdehyde (MDA) is an important product of lipid peroxidation. The content of MDA can indirectly reflect the severity of the body's attack by free radicals. We tested the MDA content using an MDA kit, and the results are as follows: Fig.13 As shown in the figure: Morroniside and loganin weakened the toxic effect of oxygen free radicals on lung tissue. Fig.13 A. The MDA content in the lung tissue of mice in the LPS group was higher than that in the Control group, **P<0.01, n=8. After oral administration of morroniside before modeling, the MDA content in the lung tissue of mice decreased, ##P<0.01, n=8. B. The MDA content in the lung tissue of mice in the LPS group was higher than that in the Control group (**P<0.01). Compared with the LPS group, the MDA content in the lung tissue of mice in the LPS+Loganin group was decreased (#P<0.05).

[0320] 1.4.5 Changes in the expression of pro-inflammatory factors IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA in mouse lung tissue

[0321] In the mouse acute lung injury cell model, it was detected that the expression of proinflammatory factors IL-6mRNA, IL-1βmRNA, and TNF-αmRNA was upregulated after LPS stimulation of RLE-6TN cells and RAW 264.7. However, lung tissue is composed of alveolar type I cells, alveolar type II cells, alveolar macrophages, alveolar capillary endothelial cells and other cells, which is much more complicated than the inflammatory response of a single type of cell. Therefore, we extracted total RNA from the lung tissue and detected the expression of proinflammatory factors IL-6mRNA, IL-1βmRNA, and TNF-αmRNA in mouse lung tissue. The results are as follows: Fig.14 As shown: The iridoid compounds morroniside and loganin can reduce the expression of proinflammatory factors IL-6mRNA, IL-1βmRNA, and TNF-αmRNA in lung tissue of mice with acute lung injury. Fig.14In the experiment, A. In the mice given Morroniside pre-protection experimental group, the expression of pro-inflammatory factors IL-6mRNA, IL-1βmRNA, and TNF-αmRNA in the LPS group was higher than that in the Control group (****P<0.0001, n=8), and the expression of pro-inflammatory factors in the LPS+Morroniside group was lower than that in the LPS group (####P<0.0001, n=8). B. In the mice given Loganin pre-protection experimental group, compared with the Control group, the expression of IL-6mRNA and TNF-αmRNA in the LPS group was higher (**P<0.01, n=8), and the expression of IL-1βmRNA was also higher (****P<0.0001, n=8). Compared with the LPS group, the expression of IL-6mRNA and IL-1βmRNA in the LPS+Loganin group was lower (##P<0.01, n=8), and the expression of TNF-αmRNA also showed a downward trend (#P<0.05, n=8).

[0322] 1.4.6 Expression of proteins related to the NF-κB / Stat3 signaling pathway in mouse lung tissue

[0323] Acute lung injury is a complex pathophysiological process, which is the result of an imbalance between systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome. We detected the expression of related proteins in the more classic inflammatory signaling pathways and tried to understand the mechanism by which the iridoid compounds morroniside and loganin have a preventive and protective effect on acute lung injury. The results are as follows Fig.15 Shown: Morroniside and loganin can downregulate the expression of p-p65 and p-stat3 proteins in the NF-κB / Stat3 signaling pathway.

[0324] From the cell model and mouse acute lung injury model, we can draw a preliminary conclusion: iridoid compounds can have a certain preventive and therapeutic effect on acute lung injury by reducing the release of inflammatory mediators and anti-oxidation. However, whether it has a protective effect on pulmonary fibrosis or even idiopathic pulmonary fibrosis caused by chronic inflammation, drug toxicity, radiotherapy and chemotherapy, we conducted a series of studies using the bleomycin-induced mouse pulmonary fibrosis model and conducted a preliminary exploration of the effects of loganin and morroniside on mouse pulmonary fibrosis.

[0325] 1.4.7 Body weight changes in mouse pulmonary fibrosis model

[0326] The modeling period was 8 weeks, and the mice were weighed and recorded every week. The weight of the mice is shown in the figure below: the weight of the mice in the control group (Control group) showed a steady growth trend, the weight of the mice in the intraperitoneal injection of bleomycin group (BLM group) increased slowly, and the growth rate of the groups treated with different concentrations of iridoid compounds loganin and morroniside was between the control group and the BLM group. At the same time, we counted the weight growth rate of mice in each group at the end of the eighth week, and the results are as follows Fig.16 As shown: The weight growth rate of mice in the BLM group was the lowest. Fig.16 A. is the weekly growth trend of mouse body weight, B. is the statistical chart of body weight growth rate, compared with the BLM group, the control group and the treatment group, **P < 0.01, ***P < 0.005, ****P < 0.0001.

[0327] 1.4.8 HE staining and Masson staining of lung tissues of mice with pulmonary fibrosis

[0328] Masson staining is one of the staining methods used to show fibers in tissues. It is an authoritative and classic technical method for collagen fiber staining. We performed HE staining and Masson staining on lung tissue sections of mice in different experimental groups. The results are as follows: Fig.17 As shown, A. HE staining; B. Masson staining. The alveolar structure of mice in the BLM group was damaged, the lung interstitium was thickened, the inflammatory cell infiltration in the alveoli and interstitium was obvious, and the content of blue collagen fibers was also increased. However, the alveolar structure and inflammatory infiltration of the lung interstitium of mice pre-intervention with loganin or morroniside were significantly improved, and the collagen fiber content was also lower than that of the BLM group.

[0329] 1.4.9 Determination of Hydroxyproline Content in Lung Tissue of Mice with Pulmonary Fibrosis

[0330] Hydroxyproline (HYP) is the main component of collagen tissue. By measuring the content of HYP in tissue, the degree of fibrosis can be determined. We measured the content of HYP in lung tissue of different groups of mice. The results are as follows: Fig.18 As shown: Compared with the Control group, the HYP content of mice in the BLM group increased (****P<0.0001, n=8), and the HYP content of the drug treatment group showed a downward trend compared with the BLM group. The results showed that loganin and morroniside could reduce the increase in HYP content caused by bleomycin-induced pulmonary fibrosis in mice, and alleviated the process of pulmonary fibrosis in mice to a certain extent.

[0331] Loganin and morroniside can increase the number of peripheral blood CD4 in mice with pulmonary fibrosis + / CD8 + T cell ratio

[0332] In the normal immune system of the body, T lymphocyte subsets are a very important cell subset. + After recognizing antigenic peptides, T cells differentiate into helper T cells (Th), which secrete cytokines to regulate cellular immunity and humoral immunity. + After activation, T cells differentiate into cytotoxic T cells (cytotoxic T lymphocytes), which can specifically recognize endogenous antigen peptides-MHC class I molecules and kill target cells. + / CD8 + Under normal circumstances, T cells maintain an appropriate ratio, CD4 + / CD8 + The reduction in the proportion of T cells leads to damage to the immune system and corresponding pathological changes. Fig.19 ) showed that compared with the Control group, the CD4 + / CD8 + The ratio of T cells decreased, while the peripheral blood CD4 + / CD8 + The T cell ratio was increased. Fig.19 In the peripheral blood of mice in different treatment groups, CD4 + / CD8 + Changes in T cell ratios, including A. Control group and BLM group mice peripheral blood CD4 + 、CD8 + T cell expression, B. Peripheral blood CD4 + 、CD8 + Expression of CD4 in peripheral blood of mice in different Morroniside concentration groups + 、CD8 + D. Expression of peripheral blood CD4 in mice in different treatment groups + / CD8 + Statistical graph of changes in T cell ratio (compared with the BLM group, *P<0.05, **P<0.01, ***P<0.005, n=8).

[0333] 1.4.10 Loganin and morroniside can reduce the expression of TGF-β1 mRNA, α-SMA mRNA, and Collagen I mRNA in lung tissue of fibrotic mice

[0334] In the process of pulmonary fibrosis, transforming growth factor-β1 (TGF-β1) plays a very important role

[42] , which can lead to abnormal activation of myofibroblasts, excessive production of extracellular matrix components such as α-smooth muscle actin (α-SMA) and collagen, elastin, and proteoglycans, resulting in lung tissue structural remodeling and impaired lung function. From the experimental results of HE and Masson, it can be known that the cyclopentadiene ether terpenoid compounds loganin and morroniside can alleviate bleomycin-induced pulmonary fibrosis in mice, but the specific mechanism and pathway of action are not yet known. Therefore, the expression of fibrosis-related genes TGF-β1mRNA, α-SMA mRNA, and CollagenⅠmRNA was detected. The results are as follows Fig. 20 As shown in the figure: In the mice in the bleomycin group, the expression levels of TGF-β1mRNA, α-SMA mRNA and CollagenⅠmRNA increased, while the expression levels of the three mRNAs in the drug treatment group showed a downward trend and had a certain concentration dependence. Fig. 20 A. The expression of TGF-β1 mRNA in the blank control group and the loganin and morroniside treatment groups was lower than that in the BLM group, and ****P<0.0001 (not marked in the figure). BC. The expression of α-SMA mRNA and CollagenⅠ mRNA in the blank control group and the loganin and morroniside treatment groups was lower than that in the BLM group, **P<0.01, ***P<0.005, ****P<0.0001, n=8.

[0335] 1.4.11 Loganin and morroniside can reduce the expression of α-SMA protein in lung tissue of mice with pulmonary fibrosis

[0336] Similarly, in this study, the expression of α-SMA protein in mice of different treatment groups was also detected ( Fig.21 .AC left), and grayscale analysis was performed ( Fig.21 .AC right). First, we detected the expression of α-SMA protein in the control group and BLM group mice. Fig.21 As shown in A: The expression of α-SMA protein in the lung tissue of mice in the BLM group showed an up-regulated trend, which proved that we established an effective mouse pulmonary fibrosis model. Secondly, we detected the expression of α-SMA protein in the Loganin group and Morroniside group at different concentrations. The results are shown in Fig.21As shown in BC: from low concentration to high concentration of loganin group, the expression of α-SMA showed a downward trend, that is, loganin showed a concentration-dependent trend in regulating the expression of α-SMA protein in the lung tissue of mice with pulmonary fibrosis; Morroniside also showed a certain concentration-dependent trend in the expression of α-SMA protein in the lung tissue of mice with pulmonary fibrosis. The expression of α-SMA protein in the low concentration group (Morroniside 100mg / kg) was higher than that in the medium concentration group (Morroniside 100mg / kg) and the high concentration group (Morroniside 200mg / kg), and there was no difference in the expression of α-SMA protein between the medium concentration group and the high concentration group. The above results suggest that loganin and morroniside may have a certain protective effect on mouse pulmonary fibrosis by downregulating the expression of proteins (such as α-SMA) in the TGF-β1-related signaling pathway.

[0337] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. Use of an iridoid terpenoid compound in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The iridoid compound is loganin or morroniside.

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