Application of phellodendrine in preparation of medicine for preventing and / or treating acute pancreatitis

Abstract

The invention discloses application of phellodendrine in preparation of a medicine for preventing and / or treating acute pancreatitis, and belongs to the technical field of biological medicine. The invention discloses a key link in acute pancreatitis pathogenesis that phellodendrine directly acts on neutrophil-mediated excessive inflammatory response and upstream regulation of extracellular trap formation (NETs) for the first time, and reduces the generation of active oxygen by inhibiting the generation of neutrophil extracellular traps of pancreatic tissues, thereby reducing the damage degree of acinus cells. As a small molecule compound, phellodendrine has better tissue permeability and metabolic stability. Compared with a synthetic NETs inhibitor, the natural product has more advantages in the aspect of reducing immunotoxicity. Therefore, the invention proves that the phellodendrine (PHE) realizes the effects of relieving the inflammatory response of the acute pancreatitis and reducing the severity of the acute pancreatitis by targeting immune cells (inhibiting the formation of extracellular traps of neutrophils).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of berberine in the preparation of drugs for the prevention and / or treatment of acute pancreatitis. Background Technology

[0002] Acute pancreatitis (AP) is a leading cause of hospitalization for digestive system diseases, characterized by high morbidity and mortality. Various triggers, such as alcohol consumption, biliary tract disease, hyperlipidemia, and medications, can induce the self-activation of trypsinogen, ultimately leading to acinar cell death and local and systemic inflammation. Although most AP cases are mild and self-limiting, approximately 20% progress to moderate to severe AP, causing serious local complications and systemic organ failure. Therefore, early intervention is crucial for acute pancreatitis.

[0003] Following the onset of acute pancreatitis (AP), neutrophils are the first white blood cells to infiltrate pancreatic tissue. Infiltrating neutrophils can release reactive oxygen species, enzymes, and inflammatory factors, exacerbating pancreatic damage. Activated neutrophils can also release a network of decondensed DNA, histones, and granule proteins—neutrophil extracellular traps (NETs). Increasing evidence suggests that NET levels are significantly elevated in AP patients, inducing trypsinogen activation and promoting pancreatic tissue damage. In AP mice, injection of DNase I to eliminate NET formation or inhibit key components of NET formation significantly reduces inflammation and tissue damage. This evidence suggests that NET formation is an important and effective target in AP treatment.

[0004] Phellodendron bark ( Phellodendron amurense The bark of the Phellodendron amurense tree is an important basic medicinal herb, possessing antiarrhythmic, antibacterial, anti-ulcer, and antihypertensive effects. Phellodendrine (PHE) is an active alkaloid derived from the bark of the Phellodendron amurense tree. Recent studies have shown that Phellodendrine has various pharmacological activities, including inhibiting the production of inflammatory factors, improving intestinal inflammation, anti-allergy, and anti-arthritis. However, the function of Phellodendrine in acute pancreatitis (AP) remains unknown, and there are no reports on its preventive or therapeutic effects in acute pancreatitis. The effects of Phellodendrine on neutrophils and acute pancreatitis still need further investigation. Summary of the Invention

[0005] The purpose of this invention is to provide the use of berberine in the preparation of medicaments for the prevention and / or treatment of acute pancreatitis.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses the use of berberine in the preparation of drugs for the prevention and / or treatment of acute pancreatitis.

[0007] Phellodendron alkaloid refers to a benzyl isoquinoline alkaloid extracted from the bark of the Phellodendron amurense plant (Rutaceae family). Specifically, it can be purified by ethanol extraction combined with column chromatography. The quaternary ammonium group in its chemical structure can interact with the cell membrane and freely enter and exit the cell.

[0008] Specifically, one method of this invention involves preparing berberine into an orally administered dosage form. Through oral administration and absorption, the hydrophobic groups in its molecular structure can bind to the lipid layer of neutrophil membranes, freely penetrating into the cell and affecting the assembly of NADPH oxidase 2 (NOX2), thereby inhibiting NOX2 activity and blocking the explosive production of ROS. When ROS levels decrease, neutrophils are unable to initiate the NETosis process, thus reducing NET deposition in pancreatic tissue. This mechanism effectively interrupts the vicious cycle of abnormal trypsinogen activation, preventing the expansion of pancreatic autodigestion. Compared to existing technologies, traditional treatments primarily target the inhibition of inflammatory factors, while this invention directly acts on the upstream regulatory links of NET formation. Compared to DNase I enzyme preparations that require repeated injections, berberine, as a small molecule compound, has better tissue permeability and metabolic stability. Compared to synthetic NET inhibitors, this natural product has a greater advantage in reducing immunotoxicity.

[0009] Furthermore, the drug is a drug that inhibits the formation of neutrophil extracellular traps (NETs) in pancreatic tissue during acute pancreatitis.

[0010] Neutrophil extracellular traps refer to the network structure composed of decondensed DNA, histones, and granular proteins released by activated neutrophils. This can be detected by measuring the DNA-histone complex content in pancreatic tissue or by immunofluorescence labeling. This structure exacerbates pancreatic damage by activating trypsinogen. Therefore, inhibiting the formation of neutrophil extracellular traps means blocking the biological process of neutrophils releasing this network structure, which controls the inflammatory response by reducing abnormal trypsinogen activation.

[0011] Specifically, the berberine selected in this invention inhibits reactive oxygen species-mediated deoxyribonucleic acid release and histone citrullination modification by regulating the neutrophil activation pathway, thereby blocking the formation of neutrophil extracellular traps. The reduction in the level of neutrophil extracellular traps in pancreatic tissue can decrease the abnormal activation of trypsinogen, thereby inhibiting acinar cell autodigestion and the release of inflammatory factors, ultimately alleviating pathological damage to pancreatic tissue.

[0012] Furthermore, the drug is a drug that reduces ROS levels in acute pancreatitis.

[0013] ROS level refers to the concentration of reactive oxygen species in a living organism. It can be specifically measured by detecting the content of free radicals such as hydrogen peroxide and superoxide anion in pancreatic tissue. Excessive accumulation of reactive oxygen species can lead to oxidative stress and cell damage.

[0014] Specifically, berberine reduces superoxide anion production by regulating intracellular signaling pathways in neutrophils and inhibiting the assembly and activation of the NADPH oxidase complex. During the pathogenesis of acute pancreatitis, activated neutrophils produce large amounts of reactive oxygen species (ROS) through a respiratory burst, leading to mitochondrial dysfunction in acinar cells and abnormal activation of lysosomal enzymes. After berberine intervention, the decrease in ROS levels alleviates oxidative stress damage to pancreatic tissue and simultaneously blocks the excessive activation of the ROS-mediated NF-κB signaling pathway, further inhibiting the release of inflammatory factors.

[0015] Furthermore, the drug is a drug that reduces serum amylase and lipase levels.

[0016] Serum amylase is a digestive enzyme secreted by the pancreas. During acute pancreatitis, it is released into the bloodstream due to damage to acinar cells. The degree of pancreatic damage can be assessed by detecting the level of amylase activity in the blood. Serum lipase is a lipase secreted by the pancreas. In acute pancreatitis, its levels are abnormally elevated due to pancreatic duct obstruction or acinar cell rupture. The functional status of the pancreas can be reflected by detecting the level of lipase activity in the blood.

[0017] Specifically, the berberine selected in this invention reduces the production of reactive oxygen species by inhibiting the formation of extracellular traps in pancreatic neutrophils, thereby decreasing the degree of damage to acinar cells. Reduced acinar cell damage decreases the abnormal release of amylase and lipase into the bloodstream, thus lowering the activity levels of these two enzymes in serum. This mechanism of action, by regulating the neutrophil-mediated inflammatory response, blocks the vicious cycle of pancreatic tissue damage, ultimately achieving effective control of serum amylase and lipase levels.

[0018] Furthermore, the drug is a drug that improves the pathological damage of pancreatic tissue.

[0019] Improving the pathological damage of pancreatic tissue refers to restoring the normal structure of the tissue by reducing edema, necrosis, and inflammatory cell infiltration.

[0020] Specifically, the berberine selected in this invention inhibits the release of reactive oxygen species and the formation of extracellular traps by neutrophils, thereby blocking the self-activation process of trypsinogen, reducing acinar cell death and local inflammatory response, and ultimately improving the pathological damage state of pancreatic tissue. By regulating neutrophil activity, berberine can reduce the infiltration of inflammatory cells in pancreatic tissue and alleviate the spread of tissue edema and necrosis.

[0021] This application further proposes the use of berberine in the preparation of drugs that reduce serum inflammation levels in acute pancreatitis.

[0022] Serum inflammation level refers to the degree of systemic inflammatory response reflected by detecting changes in the concentration of inflammatory factors in serum. Specifically, it can be achieved by using enzyme-linked immunosorbent assay (ELISA) or flow cytometry to detect the levels of pro-inflammatory factors such as interleukin-6 and tumor necrosis factor-α.

[0023] Specifically, berberine inhibits the release of pro-inflammatory cytokines and the formation of extracellular traps by regulating the functional state of neutrophils. This compound can block reactive oxygen species-mediated inflammatory signaling pathways, thereby reducing the amount of inflammatory mediators entering the bloodstream after pancreatic tissue injury. Under the pathological conditions of acute pancreatitis, berberine intervention significantly reduced the concentration of pro-inflammatory factors in serum, effectively controlling the systemic inflammatory response.

[0024] This application further proposes that berberine inhibits the formation of extracellular traps and the production of reactive oxygen species in pancreatic tissue during acute pancreatitis.

[0025] The present invention also provides a medicament for the prevention and / or treatment of acute pancreatitis, said medicament being made from the active ingredient berberine with or without pharmaceutically acceptable excipients.

[0026] Furthermore, the excipients include one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, and lubricants.

[0027] Furthermore, the drug can be formulated into tablets, granules, injections, suspensions, or powders; The drug can be introduced into body tissues via oral administration, injection, spray, nasal drops, eye drops, penetration, absorption, and physical or chemical mediated methods; or it can be introduced into body tissues after being mixed with or encapsulated by other substances.

[0028] More preferably, the body tissues include muscle, intradermal, subcutaneous, venous, or mucosal tissues.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of berberine (PHE) in the preparation of drugs for inhibiting acute pancreatitis (AP). An AP model was simulated in C57BL / 6 mice by intraperitoneal injection of berberine (100 μg / kg, 1 injection per hour for 10 consecutive hours). Experimental results demonstrated that gavage treatment with berberine (PHE) in mice significantly improved the development and progression of AP, and can be used for clinical prevention / treatment of AP. Furthermore, this invention experimentally verified that the therapeutic effect of berberine (PHE) on AP is achieved by targeting immune cells (inhibiting the formation of extracellular traps on neutrophils), thereby reducing the inflammatory response and severity of AP.

[0030] The drug for inhibiting acute pancreatitis provided by this invention has high safety, strong pharmacological effects, and clear efficacy. This invention provides a new drug source for the prevention, diagnosis, detection, protection, treatment, and research of pancreatitis, and is easily applicable in clinical practice, with significant clinical application prospects and social benefits in a short period. Attached Figure Description

[0031] Figure 1 The results of gavage treatment with berberine (PHE) in Example 1 significantly improved pancreatic damage (edema, inflammatory exudation, necrosis) in mice with acute pancreatitis induced by baicalin. Among them, (a) is a pathological image of mouse pancreatic tissue stained with HE; (b) is a statistical graph of pancreatic damage pathological score.

[0032] Figure 2 The figure shows the results of treatment with berberine (PHE) via gavage in Example 1, which reduced serum amylase and serum lipase levels in mice with baicalin-induced acute pancreatitis; where (a) represents serum lipase level and (b) represents serum amylase level.

[0033] Figure 3 In Example 2, oral administration of berberine (PHE) reduced the production of NETs in pancreatic tissue of mice with acute pancreatitis induced by baicalin; where (a) is MPO; (b) is PAD4; and (c) is NE.

[0034] Figure 4 In Example 2, oral administration of berberine (PHE) reduced the level of total NETs production in peripheral blood of mice with acute pancreatitis induced by baicalin.

[0035] Figure 5 In Example 2, oral administration of berberine (PHE) reduced the levels of inflammatory factors IL-6, TNF-α, and IL-18 in the peripheral serum of mice with acute pancreatitis induced by baicalin; where (a) is the serum IL-6 level; (b) is the serum TNF-α level; and (c) is the serum IL-18 level.

[0036] Figure 6 This is a diagram showing the results of PHE (perberlein acetate) inhibiting PMA-induced ROS generation in neutrophils in a dose-dependent manner in Example 3.

[0037] Figure 7 The image shows the results of PHA-induced neutrophil NETs generation inhibited by berberine (PHE) in a dose-dependent manner in Example 2; where (a) is PAD4; (b) is NE; and (c) is MPO-CitH3 immunofluorescence. Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0039] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0040] The present invention will now be described in further detail with reference to the accompanying drawings: Unless otherwise specified, the experimental methods used in this embodiment are conventional methods. All reagents and materials used in the embodiments are readily available. All quantitative experiments involved in the embodiments were performed in at least three replicates, and the results were averaged.

[0041] C57BL / 6 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0042] The caerulein (Cae) used was purchased from Selleck Chemicals, catalog number S9690. The berberine (PHE) used was purchased from MCE, catalog number HY-N0427. The molecular formula of berberine is C2. 20 H24 NO4, CAS No.: 6873-13-8, structural formula as follows:

[0043] Example 1: The role of berberine (PHE) in the prevention / treatment of acute pancreatitis 1. Acute pancreatitis model induced by taurine in mice: Experimental animals: 6-8 week old male C57BL / 6 mice (weighing 20-25g).

[0044] Experimental grouping: Experimental animals were divided into a normal control group, a model group, and low-dose (25 mg / kg), medium-dose (50 mg / kg), and high-dose (100 mg / kg) berberine groups. The model group and all berberine groups were intraperitoneally injected with berberine (100 μg / kg, 1 hour apart, for 10 consecutive injections) to establish an acute pancreatitis model. Control group mice received a strict PBS control via intraperitoneal injection.

[0045] 2. Phellodendron chinense (PHE) gavage therapy PHE was dissolved in PBS. Mice in the PHE drug group were administered PHE at doses of 25 mg / kg, 50 mg / kg, and 100 mg / kg by gavage 0 h before the start of the peritoneal modeling with basil. Mice in the model group received a statistically significant amount of the solvent control (PBS). All mice were anesthetized 12 h after the first intraperitoneal injection of basil, and serum was collected for serum enzyme and inflammatory factor levels. Subsequently, the mice underwent in vivo cardiac perfusion to remove circulating blood, and pancreatic tissue was rapidly extracted, fixed, dehydrated, and prepared for HE-stained paraffin sections.

[0046] The results are as follows Figure 1 As shown, the 50 mg / kg PHE treatment group improved pancreatic tissue damage (edema, inflammatory exudation, and acinar cell necrosis). Therefore, it is suggested that 50 mg / kg PHE can improve the severity of acute pancreatitis induced by hygroscopic ... Figure 2 As shown, 50 mg / kg PHE significantly reduced serum lipase in mice. Figure 2 (a) and amylase levels Figure 2 (b) Example 2: Phellodendron chinense inhibits NET production in AP mice 1. Acute pancreatitis model induced by taurine in mice: Experimental animals: 6-8 week old male C57BL / 6 mice (weighing 20-25g).

[0047] Experimental grouping: The experimental animals were divided into a normal control group, a model group, and a berberine treatment group. The model group and all berberine treatment groups were intraperitoneally injected with berberine (100 μg / kg, 1 hour apart, for 10 consecutive injections) to establish an acute pancreatitis model. The control group mice received a strict PBS intraperitoneal injection.

[0048] 2. Phellodendron chinense (PHE) gavage therapy Mice in the PHE drug group were administered PHE 50 mg / kg by gavage 0 h before the start of the intraperitoneal modeling with baicalein, while mice in the model group received a statistically significant amount of solvent control (PBS). All mice were anesthetized 12 h after the first intraperitoneal injection of baicalein, and plasma was collected for MPO-DNA detection. Subsequently, the mice underwent in vivo cardiac perfusion to remove circulating blood, and the pancreas was rapidly extracted. A portion of the pancreatic tissue was fixed and dehydrated, and paraffin sections were prepared. The remaining pancreatic tissue was digested with collagen into a single-cell suspension for flow cytometry analysis.

[0049] The results are as follows Figure 3 As shown in (a), PHE treatment significantly inhibited neutrophil infiltration in the pancreatic tissue of AP mice. Figure 3 (b)-(c) and Figure 4 As shown, PHE treatment significantly reduced the expression of PAD4 and NE in AP mice, as well as the level of MPO-DNA in plasma. This suggests that PHE treatment can significantly inhibit NETs production in AP mice. Furthermore, as... Figure 5 As shown, PHE can significantly reduce the level of inflammatory factors in mouse serum.

[0050] Example 3: In vitro inhibition of ROS generation by berberine Primary neutrophils: Mouse bone marrow neutrophils were extracted, resuspended in RPMI 1640 medium containing 5% FBS, and seeded into 12-well plates. The cells were then cultured stably at 37°C in a 5% CO2 incubator. Neutrophils were incubated with PMA (100 nm) for 4 hours to induce ROS production.

[0051] The specific groups are as follows: Control group: cultured in 1640 containing 5% FBS for 4 hours.

[0052] PMA group: Add 100 nm of phorbol ester (PMA) to each well and incubate for 4 hours.

[0053] PMA+PHE group: 100 nm phorbol ester (PMA) and different concentration gradients of Rha (2.5, 5, 10, 20, 40, 80 μM) were added to each well and cultured for 4 hours.

[0054] Cells from each group were collected and reactive oxygen species (ROS) generation was detected.

[0055] The results are as follows Figure 6 As shown, different concentrations of PHE significantly reduced PMA-induced ROS production in neutrophils, with 20 μM showing the best inhibitory effect.

[0056] Example 4: In vitro inhibition of NETs production by berberine Primary neutrophils: Mouse bone marrow neutrophils were extracted, resuspended in RPMI 1640 medium containing 5% FBS, and seeded into 12-well plates. The cells were then cultured stably at 37°C in a 5% CO2 incubator. Neutrophils were incubated with PMA (100 nm) for 4 hours to induce NET production.

[0057] The specific groups are as follows: Control group: cultured in 1640 containing 5% FBS for 4 hours; PMA group: Add 100 nm of phorbol ester (PMA) to each well and incubate for 4 hours.

[0058] PMA+PHE group: Add 100 nm phorbol ester (PMA) and 20 μM PHE to each well and incubate for 4 hours.

[0059] Cells from each group were collected and analyzed by flow cytometry and immunofluorescence. Results are as follows: Figure 7 As shown, 20 μMPHE significantly inhibited the expression of PAD4 and NE in PMA-activated neutrophils, and immunofluorescence further confirmed that PHE inhibited the production of extracellular traps (NETs) in neutrophils.

[0060] In summary, existing drugs for treating acute pancreatitis mostly focus on inhibiting pancreatic enzyme activity or controlling inflammatory factors, failing to target the crucial pathological link of neutrophil extracellular trap formation. This invention is the first to incorporate berberine into a pharmaceutical application for acute pancreatitis, achieving intervention at the molecular level at the root cause of the disease through a synergistic mechanism of dual blocking of reactive oxygen species bursts and DNA network release. Through this technical solution, this application can effectively reduce abnormally elevated serum amylase and lipase levels, alleviate pancreatic tissue edema and hemorrhagic necrosis, and simultaneously inhibit the spread of systemic inflammatory response by regulating neutrophil function, providing a targeted drug option for the treatment of acute pancreatitis.

[0061] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. The use of berberine in the preparation of drugs for the prevention and / or treatment of acute pancreatitis.

2. The application as described in claim 1, characterized in that, The drug described is a drug that inhibits the formation of extracellular traps in neutrophils in pancreatic tissue during acute pancreatitis.

3. The application as described in claim 1, characterized in that, The drug mentioned is one that reduces ROS levels in acute pancreatitis.

4. The application as described in claim 1, characterized in that, The drug mentioned is one that lowers serum amylase and lipase levels.

5. The application as described in claim 1, characterized in that, The drug described is a drug that improves the pathological damage of pancreatic tissue.

6. The application as described in claim 1, characterized in that, The drug described is used to reduce serum inflammatory levels in patients with acute pancreatitis.

7. A medicament for the prevention and / or treatment of acute pancreatitis, characterized in that, The drug is made from the active ingredient berberine with or without pharmaceutically acceptable excipients.

8. The medicament for the prevention and / or treatment of acute pancreatitis according to claim 7, characterized in that, The excipients include one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, and lubricants.

9. The medicament for the prevention and / or treatment of acute pancreatitis according to claim 7, characterized in that, The drug can be formulated into tablets, granules, injections, suspensions, or powders; The drug can be introduced into body tissues via oral administration, injection, spray, nasal drops, eye drops, penetration, absorption, and physical or chemical mediated methods; or it can be introduced into body tissues after being mixed with or encapsulated by other substances.

10. The medicament for the prevention and / or treatment of acute pancreatitis according to claim 9, characterized in that, The body tissues include muscle, intradermal, subcutaneous, venous, or mucosal tissues.

Images