Application of phellodendrine in preparation of medicine for treating or improving acute lung injury

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of phellodendrine in preparation of medicine for treating or improving acute lung injury. Through systematic animal experiment verification, the research finds that the phellodendrine (PDE) has a remarkable and comprehensive technical effect in improving the acute lung injury (ALI) induced by lipopolysaccharide (LPS), the effect of the phellodendrine (PDE) covers multiple dimensions such as overall state improvement, tissue injury repair, inflammation regulation and control, signal channel regulation and the like, and the phellodendrine (PDE) shows definite dose dependence and has the optimal effect under the dose of 12 mg / kg. Through the multi-dimensional synergistic effect of improving the overall state, repairing tissue damage, protecting the barrier function, inhibiting inflammatory response and regulating and controlling a signal channel, the phellodendrine effectively improves ALI induced by LPS, the safety is good, and a new effective medicine and an action target are provided for treatment of acute lung injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of berberine in the preparation of drugs for treating or improving acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is an acute inflammatory response syndrome of the lungs caused by a variety of factors such as infection, trauma, and sepsis. Its core pathological features include damage to the alveolar epithelium and capillary endothelial barrier, cytokine storm, excessive oxidative stress, and abnormal polarization of macrophages. In severe cases, it can progress to acute respiratory distress syndrome (ARDS), which has a high clinical mortality rate.

[0003] Lipopolysaccharide (LPS), a key component of the cell wall of Gram-negative bacteria, is a common pathogenic factor inducing ALI. It triggers downstream inflammatory cascade reactions by activating signaling pathways such as TLR4 / NF-κB, leading to inflammatory cell infiltration, pulmonary edema, and impaired gas exchange in lung tissue, making it a challenging problem for clinical treatment.

[0004] Currently, clinical treatment of ALI mainly focuses on symptomatic support, lacking specific therapeutic drugs. Current treatment interventions primarily concentrate on anti-inflammatory, antioxidant, and barrier protection approaches, but all have significant technical limitations. For example, prior art CN202010293140.8A discloses the synergistic therapeutic effect of a composition of dexamethasone and WX-UK1 or WX-671 in the treatment of acute lung injury. However, clinical practice shows that long-term use of dexamethasone easily causes serious side effects such as gastrointestinal bleeding, immunosuppression, and secondary infections, and its efficacy for advanced ALI patients is limited, failing to fundamentally repair the damaged lung tissue barrier, thus limiting its clinical application. Existing patent CN202410176863.8A discloses the application of N-acetylcysteine ​​(NAC) in the preparation of drugs for treating lung injury. Its research confirms that diabetic myocardial infarction / reperfusion can cause acute lung injury, manifested as increased circulating inflammatory factors, aggravated lung tissue pathological damage, increased pulmonary oxidative stress, aggravated pulmonary edema, and increased lung permeability index. N-acetylcysteine ​​(NAC) is a commonly used antioxidant that reduces oxidative damage to lung tissue by replenishing intracellular glutathione reserves and scavenging reactive oxygen species (ROS). However, NAC has poor water solubility and low bioavailability, and targets only a single pathological link of oxidative stress, without significant regulatory effects on cytokine storms and abnormal macrophage polarization, resulting in poor clinical efficacy and difficulty in achieving multi-target synergistic therapy. Existing technology CN202211694554.7A discloses a stem cell exosome preparation, its preparation method, and its application in the preparation of drugs for the prevention and treatment of lung injury. The stem cell exosome preparation consists of water, stem cell exosomes, and PBS containing berberine. Experiments show that this stem cell exosome preparation has good anti-inflammatory effects, significantly reduces the level of inflammatory factors, and has a significant repair effect on lung tissue damage caused by acute lung injury. Mesenchymal stem cell (MSC) therapy has been a hot topic in ALI research in recent years, promoting lung tissue repair through paracrine anti-inflammatory and repair factors. However, MSC therapy carries risks such as tumorigenesis, allogeneic transplant rejection, and difficulties in storage and transportation. Furthermore, its mechanism of action is not fully understood, and its clinical translation faces ethical controversies and technological bottlenecks, making widespread application unlikely in the short term. Natural products, due to their multi-target and low-toxicity advantages, have become an important direction for ALI treatment drug development.

[0005] Phellodendron alkaloid, an alkaloid extracted from the traditional Chinese medicine Phellodendron bark, has been shown to have various pharmacological activities such as anti-inflammatory, antioxidant, and immunomodulatory effects. However, there are currently no reports on its use in treating or improving ALI, and the specific molecular mechanism by which it regulates the pathological process of ALI is not clear.

[0006] To address the shortcomings of existing technologies, we are developing ALI treatment drugs based on berberine. These drugs can improve inflammatory storms, repair lung barrier damage, and regulate macrophage polarization through multi-target synergistic effects, which has significant clinical value and application prospects. Summary of the Invention

[0007] To address the aforementioned technical problems, the purpose of this invention is to provide the application of berberine in the preparation of drugs for treating or improving acute lung injury. Through systematic animal experiments, it has been confirmed that berberine (PDE) has significant and comprehensive technical effects in improving lipopolysaccharide (LPS)-induced acute lung injury (ALI). Its effects encompass multiple dimensions, including overall condition improvement, tissue damage repair, inflammation regulation, and signaling pathway modulation, exhibiting a clear dose-dependent effect, with optimal efficacy at a dose of 12 mg / kg. This active ingredient of berberine effectively improves LPS-induced ALI through a multi-dimensional synergistic effect of improving overall condition, repairing tissue damage, protecting barrier function, inhibiting inflammatory responses, and regulating signaling pathways, with good safety profile. This provides a new effective drug and target for the treatment of acute lung injury.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: Application of berberine in the preparation of drugs for treating or improving acute lung injury.

[0009] Preferably, the drug is applied by any one or more of the following methods: It improves the degree of weight loss in mice, improves pulmonary edema in mice, improves pathological damage in mouse lung tissue, improves the integrity of the air-blood barrier, reduces the level of pro-inflammatory factors in serum, downregulates the expression level of pro-inflammatory factor mRNA in lung tissue, inhibits M1 polarization of mouse lung tissue macrophages, and regulates the activity of cAMP / PKA / Akt / CREB signaling pathway.

[0010] Preferably, the improvement of pathological damage to mouse lung tissue includes: reducing leukocyte infiltration in lung tissue, reducing alveolar wall thickening, alleviating lung hemorrhage, and reducing the number of immune cells in bronchoalveolar lavage fluid.

[0011] Preferably, the improvement of air-blood barrier integrity includes: reducing BALF protein concentration in mice, alleviating pulmonary inflammatory exudation, and reducing alveolar-capillary permeability.

[0012] Preferably, reducing the level of pro-inflammatory factors in serum includes reducing the levels of TNF-α, IL-1β, and IL-6 in serum.

[0013] Preferably, the downregulation of pro-inflammatory factor mRNA expression levels in lung tissue includes reducing the mRNA expression levels of TNF-α, IL-1β, and IL-6.

[0014] Preferably, the inhibition of M1 polarization of mouse lung macrophages includes: inhibiting the expression of iNOS protein and CD86 protein in lung macrophages.

[0015] Preferably, the regulation of the cAMP / PKA / Akt / CREB signaling pathway activity includes: reducing the level of cAMP and inhibiting the expression of PKA, Akt and CREB proteins.

[0016] A drug for treating or improving acute lung injury, wherein the main active ingredient of the drug is berberine.

[0017] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0018] Compared with the prior art, the present invention has at least the following technical effects: This invention provides the application of berberine in the preparation of drugs for treating or improving acute lung injury. This study, through systematic animal experiments, has clarified that berberine (PDE) has significant and comprehensive technical effects in improving lipopolysaccharide (LPS)-induced acute lung injury (ALI). Its effects cover multiple dimensions, including overall condition improvement, tissue damage repair, inflammation regulation, and signaling pathway regulation, and show a clear dose-dependent effect, with the best effect at a dose of 12 mg / kg.

[0019] 1) Regarding the improvement of overall condition, berberine can effectively reverse the weight loss in ALI mice. The weight of mice decreased significantly after LPS induction, while the weight loss in the LPS+PDE 12mg / kg group was significantly improved (P<0.01), suggesting that it can alleviate the systemic wasting symptoms caused by ALI and improve the overall health of mice.

[0020] 2) In terms of lung tissue repair, berberine exhibited a potent effect in improving pulmonary edema. The lung coefficient and wet-to-dry weight ratio (W / D) were significantly increased in the LPS group, while the PDE 12mg / kg group significantly reduced these indicators (P<0.05), effectively alleviating pulmonary fluid retention. Simultaneously, H&E staining of lung tissue showed that berberine significantly improved pathological damage caused by LPS, such as leukocyte infiltration, alveolar wall thickening, and pulmonary hemorrhage, and significantly reduced the number of immune cells in bronchoalveolar lavage fluid (BALF) (P<0.01), alleviating the degree of inflammatory infiltration in lung tissue and repairing the normal structure of lung tissue.

[0021] 3) In terms of protecting the air-blood barrier, berberine can effectively reduce LPS-induced increase in alveolar-capillary permeability, significantly reduce BALF supernatant protein concentration (P<0.05), alleviate pulmonary inflammatory exudation, thereby improving the damaged state of the air-blood barrier and maintaining the structural basis of pulmonary gas exchange function.

[0022] 4) In terms of inflammation regulation, berberine can inhibit inflammatory responses at multiple levels. Serological tests showed that it could significantly reduce the levels of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 in the serum of ALI mice (P<0.05 or P<0.01); qRT-PCR results confirmed that berberine could downregulate the mRNA expression levels of the above-mentioned pro-inflammatory factors in lung tissue (P<0.05 or P<0.01), blocking the cytokine storm at the transcriptional and translational levels. Meanwhile, Western blotting showed that berberine could significantly inhibit the protein expression of M1 macrophage markers iNOS and CD86 (P<0.01), and the inhibitory effect of a 12 mg / kg dose was better than that of a 6 mg / kg dose, suggesting that it exerts its anti-inflammatory effect by regulating macrophage polarization balance.

[0023] 5) At the molecular level, berberine can precisely regulate the activity of the cAMP / PKA / Akt / CREB signaling pathway. The expression of cAMP, PKA, p-PKA, Akt, p-Akt, CREB, and p-CREB proteins in the lung tissue of LPS group mice was significantly downregulated (P<0.01), while berberine administration could upregulate the expression of the above proteins in a concentration-dependent manner (P<0.01). By activating this signaling pathway, it blocks the pathological process of ALI, providing clear molecular mechanism support for its therapeutic effect.

[0024] Phellodendron chinense effectively improves LPS-induced ALI through a multi-dimensional synergistic effect of "improving overall condition, repairing tissue damage, protecting barrier function, inhibiting inflammatory response, and regulating signaling pathways," and has good safety profile, providing a new effective drug and target for the treatment of acute lung injury. Attached Figure Description

[0025] Figure 1 A schematic diagram illustrating the effect of berberine on LPS-induced changes in body weight in ALI mice. Figure 2 A schematic diagram showing the effect of berberine on the lung coefficient of LPS-induced ALI mice. Figure 3 A schematic diagram showing the effect of berberine on the wet / dry weight ratio (W / D) of the lungs in LPS-induced ALI mice; Figure 4 Schematic diagram of H&E staining results of lung tissue from mice in each group, magnified ×100; Figure 5 A schematic diagram showing the effect of berberine on immune cells in BALF of LPS-induced ALI mice. Figure 6 A schematic diagram showing the effect of berberine on the number of immune cells in the BALF of LPS-induced ALI mice. Figure 7Schematic diagram of the Wright-Giemsa staining morphology of BALF cells in each group of mice, magnification ×400; Figure 8 Schematic diagram of the effect of palmatine on the protein concentration in the BALF supernatant of LPS-induced ALI mice; Figure 9 Schematic diagram of the effect of palmatine on the content of serum inflammatory factors in LPS-induced ALI mice; Figure 10 Schematic diagram of the effect of palmatine on the relative expression level of iNOS protein in the lung tissue of LPS-induced ALI mice; Figure 11 Schematic diagram of the effect of palmatine on the relative expression level of CD86 protein in the lung tissue of LPS-induced ALI mice; Figure 12 Protein electrophoresis diagram of Western blotting of lung tissue in each group of mice; Figure 13 Diagram of the effect of palmatine on the protein content of cAMP in the lung tissue of LPS-induced ALI mice; Figure 14 Schematic diagram of the effect of palmatine on the relative expression levels of various proteins in the lung tissue of LPS-induced ALI mice. Specific implementation manners

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the present invention in combination with the accompanying drawings and the description of the embodiments or the prior art. Obviously, the following description of the structures of the accompanying drawings is only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings. It should be noted here that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation to the present invention.

[0027] The technical solution of a specific implementation manner of the present invention is as follows: 1. Experimental materials 1.1 Experimental animals: C57BL / 6J mice, male, 72, 7 weeks old, SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SCXK (Beijing) 2021-0006. Raised in the animal center of Tianjin University of Traditional Chinese Medicine, SYXK (Tianjin) 2020-0005, 12 h day / 12 h night cycle, temperature 23-25°C.

[0028] 1.2 Drugs and Reagents: Lipopolysaccharide (LPS): Sigma-Aldrich (L2630); Phellodendron chinense (PDE): Chengdu Lemeitian Pharmaceutical Technology Co., Ltd. (DH0031-0050); Dexamethasone: Beijing Solarbio Science & Technology Co., Ltd. (D8040); Anti-iNOS antibody: Abcam (ab283655); Anti-CD86 antibody: Selleck Biotechnology Co., Ltd. (A5611); β-actin antibody: Wuhan Sanying Biotechnology Co., Ltd. (81115-1-RR).

[0029] 2. Methods 2.1 Animal grouping, model establishment, drug administration and tissue collection C57BL / 6J mice were acclimatized for one week, weighing 23±2 g, and then randomly divided into 6 groups: Control, Control+PDE 12 mg / kg, LPS, LPS+PDE 6 mg / kg, LPS+PDE 12 mg / kg, and LPS+Dexamethasone (DXM). The mice were induced to develop the model and were simultaneously injected intraperitoneally with the drugs. The other groups were given an equal dose of physiological saline. The drugs were administered once a day for 3 consecutive days before the mice were harvested.

[0030] 2.2 Difference in mouse body weight between modeling and pre-sampling levels The body weight of mice after modeling and before sampling were measured, and the difference was calculated to observe the effect of berberine on improving the body weight of mice.

[0031] 2.3 Evaluation and detection of pulmonary edema Freshly harvested right middle lobe of the lung was weighed using an electronic balance to obtain wet weight (W), and then dried in an oven at 80°C for 48 h until constant weight was obtained to obtain dry weight (D). W / D = wet weight of lung / dry weight is the lung edema index, which is used to assess the degree of lung edema in mice.

[0032] 2.4 H&E staining of lung tissue H&E staining of lung tissue was used to observe the inflammatory cell infiltration and edema in different groups.

[0033] 2.5 BALF cell count The number of immune cells in BALF is counted to reflect the extent of inflammatory cell infiltration and the degree of damage to the air-blood barrier.

[0034] 2.6 Wright-Gymsa staining The resuspended BALF cell suspension was dropped onto a slide (10 μL), dried, and then stained with Wright-Giemsa to observe the amount of different immune cell types.

[0035] 2.7 Detection of protein concentration in BALF supernatant Protein concentration was detected by thawing BALF supernatant, which reflects the degree of damage to the air-blood barrier.

[0036] 2.8 ELISA method for detecting inflammatory factors and cAMP protein levels Mouse serum was collected, and the levels of TNF-α, IL-1β, and IL-6 were measured according to the ELISA kit instructions. The levels of TNF-α, IL-1β, and MCP-1 were also measured according to the ELISA kit instructions. Mouse lung tissue proteins were extracted, and the cAMP protein level was measured according to the ELISA kit instructions.

[0037] 2.9 Detection of inflammatory factor mRNA expression levels by qRT-PCR Lung tissues from mice in each group were sonicated, and the supernatant was collected. Trizol reagent was added to extract RNA, which was then reverse transcribed into cDNA. Amplification and qRT-PCR analysis were performed according to the amplification kit instructions. Two [samples were used]. -ΔΔCt The relative expression levels of each gene can be calculated.

[0038] 2.10 Western blotting detection of related protein expression Lung tissues from mice in each group were sonicated, and the supernatant was collected. Proteins were extracted using lysis buffer, and protein concentration was determined using the BCA method. Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a membrane, and blocked. Then, p-PKA, p-Akt, p-CREB, PKA, Akt, CREB, iNOS, CD86, and β-actin antibodies were added, and the membranes were incubated overnight at 4°C. The membranes were washed with TBST. Secondary antibody was added and incubated for 1 hour, followed by washing and imaging. The gray values ​​of the target bands were calculated using SWE Image Gray Analysis Software. β-actin was used as an internal control band to calculate the relative expression level of the target protein.

[0039] 3. Results Interpretation of the attached chart: In the attached chart, "-" indicates that the experimental group did not add the substance on that vertical axis; "+" indicates that the experimental group added the substance on that vertical axis; the horizontal axis of the attached chart is based on the blank control group (Control). The attached chart is analyzed using vertical columns.

[0040] by Figure 1 For example, the attached figure contains 6 columns of information with "-" and "+". The first column indicates that no substance was added, belonging to the Control group; the second column indicates Control + PDE 12 mg / kg; the third column indicates LPS; the fourth column indicates LPS + PDE 6 mg / kg; the fifth column indicates LPS + PDE 12 mg / kg; and the sixth column indicates LPS + dexamethasone (DXM).

[0041] 3.1 Phellodendron chinense improves the degree of weight loss in mice after modeling. like Figure 1 The diagram shows the effect of berberine on LPS-induced changes in body weight in ALI mice.

[0042] The difference between the body weight before modeling and before sampling is calculated, such as... Figure 1 The results showed that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group showed the greatest difference in mouse weight, with a significant decrease in mouse weight (P<0.01). Compared with the LPS group, there was no significant difference in the LPS+PDE 6 mg / kg group, while both the LPS+PDE 12 mg / kg group and the LPS+DXM group significantly improved the degree of weight loss in mice (P<0.01), indicating that both PDE 12 mg / kg and DXM could improve the degree of weight loss in LPS-induced ALI mice to varying degrees.

[0043] 3.2 Phellodendron chinense improves pulmonary edema in ALI mice like Figure 2 The diagram shown illustrates the effect of berberine on the lung coefficient of LPS-induced ALI mice. like Figure 3 The diagram shows the effect of berberine on the wet / dry weight ratio (W / D) of the lungs in LPS-induced ALI mice.

[0044] The lung coefficient is an important indicator for evaluating pulmonary edema. The lung coefficient statistics for each group are as follows: Figure 2 As shown, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group showed a significantly increased lung coefficient (P<0.01). Compared with the LPS group, there was no significant difference in the LPS+PDE 6 mg / kg group, while both the LPS+PDE 12 mg / kg group and the LPS+DXM group significantly reduced the lung coefficient (P<0.05). This indicates that both PDE 12 mg / kg and DXM can reduce the lung coefficient of LPS-induced ALI mice to varying degrees, with PDE 12 mg / kg and DXM showing the most significant effects.

[0045] Lung W / D weight is another important indicator for evaluating pulmonary edema. The lung W / D weight statistics for each group are as follows: Figure 3The results showed that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, and the W / D weight of mice in the LPS group was higher than that in the Control group; compared with the LPS group, there was no significant difference in the LPS+PDE 6 mg / kg group, and both the LPS+PDE 12 mg / kg group and the LPS+DXM group significantly improved pulmonary edema in mice, which was statistically significant (P<0.05).

[0046] 3.3 Phellodendron chinense improves pathological damage in lung tissue of ALI mice Each group of lung tissue was prepared into paraffin sections, stained with H&E, and tissue near the trachea was selected for direct observation of the degree of pathological damage to the lungs.

[0047] like Figure 4 The image shows a schematic diagram of the H&E staining results of mouse lung tissue in each group, with magnifications of 4×, 20×, and 40×.

[0048] Preliminary observations and statistics, such as Figure 4 As shown, the LPS group exhibited significant pathological damage, including severe leukocyte infiltration, alveolar wall thickening, pulmonary edema, and hemorrhage. The LPS+PDE 6 mg / kg group, the LPS+PDE 12 mg / kg group, and the LPS+DXM group all showed varying degrees of improvement in leukocyte infiltration, alveolar wall thickening, pulmonary edema, and hemorrhage, with the PDE 12 mg / kg group showing the best treatment effect.

[0049] 3.4 Phellodendron chinense reduces the degree of inflammatory infiltration in the lung tissue of ALI mice. like Figure 5 The diagram shows the effect of berberine on the number of immune cells in the BALF of LPS-induced ALI mice. like Figure 6 The diagram shown illustrates the statistical significance of berberine's effect on the number of immune cells in the BALF of LPS-induced ALI mice. like Figure 7 The image shows a schematic diagram of the Wright-Gymsa staining morphology of BALF cells in each group of mice, magnified 20×.

[0050] The results are as follows Figure 5 , Figure 6 It was found that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group significantly increased the number of alveolar immune cells (P<0.01). Compared with the LPS group, the LPS+PDE 6 mg / kg group slightly reduced the number of alveolar immune cells, while the LPS+PDE 12 mg / kg group and DXM significantly reduced the number of LPS-induced alveolar immune cells (P<0.01).

[0051] The above data results indicate that LPS at a concentration of 2 mg / kg can successfully establish a mouse ALI model, significantly increasing the number of immune cells in alveolar BALF, and PDE at 12 mg / kg can significantly reduce the number of immune cells in alveolar BALF and alleviate the degree of lung tissue damage.

[0052] The results are as follows Figure 7 As can be seen, the Wright-Gymsa staining results also showed that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group had a significantly increased number of cells in BALF; compared with the LPS group, the LPS+PDE 6 mg / kg group slightly reduced the number of cells in BALF, while the LPS+PDE 12 mg / kg group and the LPS+DXM group significantly reduced the number of cells in BALF.

[0053] In summary, LPS at a concentration of 2 mg / kg can successfully establish a mouse ALI model, significantly increasing the number of immune cells in alveolar BALF, and PDE at 12 mg / kg can significantly reduce the number of immune cells in alveolar BALF and alleviate the degree of lung tissue damage.

[0054] 3.5 Phellodendron chinense improves the increase in alveolar-capillary permeability in ALI mice. BALF supernatant protein concentration reflects the degree of inflammatory exudation in the lungs and the extent of damage to the air-blood barrier.

[0055] like Figure 8 The diagram shows the effect of berberine on the concentration of BALF supernatant protein in LPS-induced ALI mice.

[0056] The results are as follows Figure 8 It was found that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group significantly increased the protein concentration in the BALF supernatant (P < 0.01). Compared with the LPS group, there was no significant difference in the LPS+PDE 12 mg / kg group, while the LPS+PDE 12 mg / kg group and the DXM group significantly reduced the protein concentration in the BALF supernatant (P < 0.05). These results suggest that LPS+PDE 12 mg / kg can reduce the protein concentration in the BALF of LPS-induced ALI mice, alleviate pulmonary inflammatory exudation, improve the degree of air-blood barrier damage, and reduce alveolar-capillary permeability, with a clear therapeutic effect.

[0057] 3.6 Phellodendron chinense downregulates serum inflammatory factor levels and lung tissue inflammatory factor mRNA expression. like Figure 9The diagram shows the effect of berberine on the serum inflammatory factor levels in LPS-induced ALI mice, where A represents TNF-α level; B represents IL-1β level; C represents IL-6 level; D represents TNF-α mRNA expression level; E represents IL-1β mRNA expression level; and F represents IL-6 mRNA expression level.

[0058] Results combined Figure 9 As shown in A, B, and C, PDE significantly reduced the serum TNF-α level in LPS-induced ALI mice. Compared with the Control group, there was no significant difference in the Control + PDE 12 mg / kg group, while the LPS group significantly increased the serum TNF-α level in mice (P<0.01). Compared with the LPS group, both the LPS + PDE 6 mg / kg group and the LPS + PDE 12 mg / kg group significantly reduced the serum TNF-α concentration in mice (P<0.05; P<0.01).

[0059] PDE significantly reduced serum IL-1β levels in LPS-induced ALI mice. Compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group significantly increased serum IL-1β levels in mice (P<0.01). Compared with the LPS group, the LPS+PDE 12 mg / kg group significantly reduced serum IL-1β concentration in mice (P<0.05).

[0060] PDE significantly reduced serum IL-6 levels in LPS-induced ALI mice. Compared with the Control group, there was no significant difference in the Control + PDE 12 mg / kg group, while the LPS group significantly increased serum IL-6 levels in mice (P<0.05). Compared with the LPS group, the LPS + PDE 12 mg / kg group significantly reduced serum IL-6 concentration in mice (P<0.05).

[0061] Results combined Figure 9 As shown in D, E, and F, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group. The LPS group significantly upregulated the mRNA expression level of TNF-α in lung tissue, which was statistically significant (P<0.01). Compared with the LPS group, the LPS+PDE 12 mg / kg group significantly reduced the mRNA expression level of TNF-α, which was statistically significant (P<0.01).

[0062] Compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group. The LPS group significantly upregulated the mRNA expression level of IL-1β in lung tissue, which was statistically significant (P<0.01). Compared with the LPS group, the LPS+PDE 12 mg / kg group significantly decreased the mRNA expression level of IL-1β, which was statistically significant (P<0.05).

[0063] Compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group. The LPS group significantly upregulated the mRNA expression level of IL-6 in lung tissue, which was statistically significant (P<0.01). Compared with the LPS group, the LPS+PDE 6 mg / kg and LPS+PDE 12 mg / kg groups significantly reduced the mRNA expression level of IL-6, which was statistically significant (P<0.01).

[0064] 3.7 Phellodendron chinense inhibits the expression of M1 polarization marker-related proteins in macrophages of ALI mice. like Figure 10 The diagram shows the effect of berberine on the relative expression level of iNOS protein in the lung tissue of LPS-induced ALI mice.

[0065] The results are as follows Figure 10 It can be seen that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group was able to upregulate the expression of iNOS protein, which was statistically significant (P<0.01). Compared with the LPS group, the LPS+PDE 6 mg / kg and LPS+PDE 12 mg / kg groups were able to significantly inhibit the expression of iNOS protein, which was statistically significant (P<0.01). Compared with the LPS+PDE 6 mg / kg group, the LPS+PDE 12 mg / kg group was able to significantly inhibit the expression of iNOS protein.

[0066] like Figure 11 The diagram shows the effect of berberine on the relative expression level of CD86 protein in the lung tissue of LPS-induced ALI mice.

[0067] The results are as follows Figure 11It can be seen that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group was able to upregulate the expression of CD86 protein, which was statistically significant (P<0.01). Compared with the LPS group, the LPS+PDE 6 mg / kg and LPS+PDE 12 mg / kg groups were able to significantly inhibit the expression of CD86 protein, which was statistically significant (P<0.01). Compared with the LPS+PDE 6 mg / kg group, the LPS+PDE 12 mg / kg group was able to significantly inhibit the expression of CD86 protein.

[0068] 3.8 Phellodendron chinense modulates the cAMP / PKA / Akt / CREB signaling pathway to improve LPS-induced ALI like Figure 12 The image shown is a Western blotting electrophoresis image of proteins in the lung tissues of mice in each group. like Figure 13 The figure shown is a graph illustrating the effect of berberine on the cAMP protein content in the lung tissue of LPS-induced ALI mice. like Figure 14 The diagram shows the effect of berberine on the relative expression levels of various proteins in the lung tissue of LPS-induced ALI mice. In the diagram, A represents the p-PKA protein content; B represents the PKA protein content; C represents the p-Akt protein content; D represents the p-CREB protein content; and E represents the CREB protein content.

[0069] The results are as follows Figure 12 The swim lanes, from left to right, represent the Control group, Control+PDE 12mg / kg group, LPS group, LPS+PDE 6mg / kg group, and LPS+PDE 12mg / kg group. The figure shows the grayscale values ​​of the bands for proteins with molecular weights of 40kDa (p-PKA, PKA), 60kDa (p-Akt, Akt), 43kDa (p-CREB, CREB), 42kDa (β-actin), and the target protein (iNOS, CD86), reflecting the differences in protein expression levels among the groups.

[0070] The results are as follows Figure 13 The results showed that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group could downregulate cAMP protein expression, which was statistically significant (P<0.01). Compared with the LPS group, LPS+PDE 6 mg / kg could upregulate cAMP protein expression, and significantly upregulated cAMP protein expression in the LPS+PDE 12 mg / kg group (P<0.01).

[0071] The results are as follows Figure 14Figures A-E show that, compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group, while the LPS group downregulated p-PKA protein expression. Compared with the Control+PDE 12 mg / kg group, the LPS group downregulated p-PKA protein expression, which was statistically significant (P<0.01). Compared with the LPS group, LPS+PDE 6 mg / kg upregulated p-PKA protein expression, and significantly upregulated p-PKA protein expression in the LPS+PDE 12 mg / kg group (P<0.01).

[0072] Compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group. The LPS group was able to downregulate the expression of PKA protein, which was statistically significant (P<0.01). Compared with the LPS group, LPS+PDE 6 mg / kg was able to upregulate the expression of PKA protein and significantly upregulate the expression of cAMP protein in the LPS+PDE 12 mg / kg group (P<0.01), indicating that berberine can activate the cAMP / PKA / Akt / CREB signaling pathway.

[0073] Compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group. The LPS group downregulated p-Akt protein expression, which was statistically significant (P<0.01). Compared with the LPS group, the LPS+PDE 12 mg / kg group upregulated p-Akt protein expression, which was statistically significant (P<0.01). Compared with the LPS+PDE 6 mg / kg group, the LPS+PDE 12 mg / kg group significantly upregulated p-Akt protein expression.

[0074] Compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group. The LPS group was able to downregulate the expression of p-CREB protein, which was statistically significant (P<0.01). Compared with the LPS group, LPS+PDE 6 mg / kg was able to upregulate the expression of p-PKA protein, and significantly upregulated the expression of p-PKA protein in the LPS+PDE 12 mg / kg group (P<0.01).

[0075] Compared with the Control group, there was no significant difference in the Control+PDE 12 mg / kg group. The LPS group was able to downregulate the expression of CREB protein, which was statistically significant (P<0.01). Compared with the LPS group, the LPS+PDE 6 mg / kg group and the LPS+PDE 12 mg / kg group were able to upregulate the expression of CREB protein.

[0076] In summary, berberine can promote the phosphorylation and activation of PKA, regulate the total protein expression level of PKA, promote the phosphorylation and activation of Akt, promote the phosphorylation and activation of CREB, and regulate the total protein expression level of CREB.

[0077] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of phellodendrine in the preparation of a drug for treating or ameliorating acute lung injury.

2. Use of phellodendrine according to claim 1 for the preparation of a medicament for the treatment or amelioration of acute lung injury, characterized in that, The drug is applied by any one or more of the following ways: ameliorating the weight loss of mice, ameliorating the lung edema of mice, ameliorating the pathological injury of lung tissue of mice, ameliorating the integrity of gas-blood barrier, reducing the level of pro-inflammatory factors in serum, down-regulating the mRNA expression level of pro-inflammatory factors in lung tissue, inhibiting the M1 polarization of macrophages in lung tissue of mice, regulating the activity of cAMP / PKA / Akt / CREB signaling pathway.

3. Use of phellodendrine according to claim 2 for the preparation of a medicament for the treatment or amelioration of acute lung injury, characterized in that, The ameliorating the pathological injury of lung tissue of mice includes: reducing the leukocyte infiltration of lung tissue, reducing the thickening degree of alveolar wall, reducing the hemorrhage of lung tissue and reducing the number of immune cells in bronchoalveolar lavage fluid.

4. Use of phellodendrine according to claim 2 for the preparation of a medicament for the treatment or amelioration of acute lung injury, characterized in that, The ameliorating the integrity of gas-blood barrier includes: reducing the protein concentration of BALF of mice, reducing the inflammatory exudation of lung and reducing the alveolar-capillary permeability.

5. Use of phellodendrine according to claim 2 for the preparation of a medicament for the treatment or amelioration of acute lung injury, characterized in that, The reducing the level of pro-inflammatory factors in serum includes: reducing the content of TNF-α, IL-1β and IL-6 in serum.

6. Use of phellodendrine according to claim 2 for the preparation of a medicament for the treatment or amelioration of acute lung injury, characterized in that, The down-regulating the mRNA expression level of pro-inflammatory factors in lung tissue includes: reducing the mRNA expression amount of TNF-α, IL-1β and IL-6.

7. Use of phellodendrine according to claim 2 for the preparation of a medicament for the treatment or amelioration of acute lung injury, characterized in that, The inhibiting the M1 polarization of macrophages in lung tissue of mice includes: inhibiting the expression of iNOS protein and CD86 protein of macrophages in lung tissue.

8. Use of phellodendrine according to claim 2 for the preparation of a medicament for the treatment or amelioration of acute lung injury, characterized in that, The regulating the activity of cAMP / PKA / Akt / CREB signaling pathway includes: reducing the level of cAMP, inhibiting the expression of PKA, Akt and CREB protein.

9. A medicament for treating or ameliorating acute lung injury, comprising a compound of claim 1. The main active ingredient of the drug is phellodendrine as claimed in claim 1.

10. The medicament for treating or ameliorating acute lung injury according to claim 9, wherein The drug further comprises pharmaceutically acceptable excipients.

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