Application of alkaloid bicuculline in liver fibrosis resistance

Abstract

The invention provides an application of alkaloid bicuculline in preparation of a medicine for preventing and / or treating hepatic fibrosis diseases and hepatic inflammation, and also provides an application of bicuculline in preparation of an inflammatory factor expression inhibitor and an application of bicuculline in preparation of a Toll-like receptor 4 signal channel inhibitor. 7.5 mu g / mL of bicuculline can be used for relieving the activation of hepatic stellate cells induced by TGF-beta1, remarkably reducing the expression of alpha-SMA and COL1A1, and reducing the expression of MyD88 and NF-kB as well as downstream inflammatory factors IL-6, IL-1beta and TNF-alpha in a TLR4 / MyD88 / NF-kB signal channel. By intraperitoneal injection treatment of 0.6 mg / kg of bicuculline, the liver morphology in a fibrosis model can be remarkably improved, the levels of ALT and AST in serum can be remarkably reduced, and collagen deposition and expression of alpha-SMA and COL1A1 in the liver can be remarkably reduced.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of a natural alkaloid, bicuculline, in the preparation of a drug for the prevention and / or treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathological response secondary to various chronic liver injuries (such as chronic viral hepatitis, alcoholic liver disease, metabolic dysfunction-related fatty liver disease, autoimmune liver disease, and drug-induced liver injury). Its core pathological feature is the excessive deposition and abnormal distribution of extracellular matrix (especially collagen) in the liver, which is essentially an abnormal repair response of the liver to persistent damage.

[0003] If this process is not effectively contained, the disease may gradually progress along the trajectory of "chronic liver damage—liver fibrosis—cirrhosis—liver failure / hepatocellular carcinoma," seriously threatening human health. It is worth noting that the liver fibrosis stage is considered the last potentially reversible link in the progression of liver disease; once decompensated cirrhosis is reached, the pathological changes are essentially irreversible.

[0004] The core driving force behind liver fibrosis is the imbalance between hepatic stellate cells (HSCs) activation and extracellular matrix (ECM) metabolism. Under normal physiological conditions, HSCs are in a quiescent state, primarily functioning to store vitamin A. However, under chronic injury stimulation, HSCs are activated and transform into myofibroblasts (MFBs). Activated HSCs synthesize large amounts of ECM components such as collagen, leading to liver structural remodeling and fibrous scar formation.

[0005] The activation and fibrosis process of hepatocellular carcinoma (HSCs) are precisely regulated by multiple signaling pathways, including the Hedgehog, Hippo, Notch, Wnt / β-catenin, transforming growth factor-β (TGF-β), and platelet-derived growth factor (PDGF) pathways. TGF-β1 is widely recognized as the most potent pro-fibrotic cytokine, strongly driving HSC activation and proliferation through the activation of the TGF-β / Smad signaling pathway. Therefore, targeting and inhibiting the TGF-β signaling pathway has become a highly promising therapeutic direction and a current focus of drug development.

[0006] Further elucidating the pathogenesis of liver fibrosis and developing strategies to effectively block, delay, or even reverse its progression has become a core goal and major challenge in the field of liver disease research. Achieving this breakthrough is crucial for fundamentally curbing the progression of chronic liver disease and reducing the incidence of cirrhosis and hepatocellular carcinoma. For example, Fu et al. (The protective effect of forsythiaside A on 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholestatic liver injury in mice: Based on targeted metabolics and molecular biology technology). Biochimica et Biophysica Acta- Molecular Basis of Disease , 2023, 1869(8): 166822.) Studies have shown that the natural compound forsythoside A can reduce mitochondrial damage and ferroptosis through multiple mechanisms, such as regulating NLRP3 inflammasome-mediated pyroptosis and activating the Nrf2 signaling pathway, demonstrating its potential value in combating liver fibrosis.

[0007] Despite significant efforts, there is still a lack of specific drugs that can definitively reverse liver fibrosis. Current clinical interventions mainly focus on controlling the underlying cause, such as antiviral therapy, alcohol abstinence, and weight management. While these measures can alleviate persistent liver damage and create favorable conditions for the natural reversal of liver fibrosis, their effectiveness in reversing already significant fibrosis is very limited.

[0008] Therefore, there are significant unmet clinical needs in the field of liver fibrosis, highlighting the urgency of related basic and translational research. In the complex cellular network of liver fibrosis, the activation and proliferation of hepatic fibrotic cells (HSCs) and the large amount of extracellular matrix (ECM) they generate are core components driving disease progression. Inhibiting these pathological behaviors of HSCs is considered a key strategy for anti-fibrotic therapy.

[0009] Toll-like receptor 4 (TLR4), a key pattern recognition receptor in the innate immune system, can induce inflammatory immune responses after recognizing exogenous pathogen-associated molecular patterns (PAMPs), endogenous damage-associated molecular patterns (DAMPs), and xenogeneic substance-associated molecular patterns (XAMPs). A moderate TLR4 response is crucial for the body's resistance to infection and tissue repair, but persistent or excessive activation of its signaling pathway can drive chronic inflammation, leading to various tissue damages.

[0010] ZHU et al. (Progress in lipopolysaccharide / Toll-like receptor 4 signaling transduction in hepatic fibrosis. Chinese Journal of Pharmacology and Toxicology (2013, 27(1): 106-109.) Research indicates that TLR4 plays an important role in the development of liver fibrosis. It promotes inflammatory response and HSC activation through the MyD88-dependent pathway, ultimately leading to excessive ECM deposition. During the process of liver fibrosis, the activation of the TLR4 / MyD88 / NF-κB signaling pathway, on the one hand, prompts hepatic macrophages (KCs) to release a large number of pro-inflammatory cytokines and chemokines. On the other hand, these inflammatory mediators, together with TLR4 signaling, directly act on HSCs, inducing their activation, proliferation, and transformation into MFBs, leading to excessive ECM deposition, thereby driving the fibrosis process.

[0011] Seki, E. et al. (TLR4 enhances TGF-β signaling and hepatic fibrosis. Nature Medicine Further research (2007, 13(11): 1324-1332.) shows that activation of the TLR4 / MyD88 / NF-κB signaling pathway can not only directly promote the release of inflammatory factors, but also directly activate HSCs by regulating factors such as TGF-β. Crucially, activated HSCs highly express TLR4 and produce a large amount of endogenous DAMPs. These DAMPs, in turn, continuously activate the TLR4 signaling pathway and the expression of its downstream inflammatory and fibrotic factors, thus forming a vicious cycle of "TLR4-HSC activation" that drives the continuous progression of liver fibrosis.

[0012] Given the central role of TLR4 in liver fibrosis, several TLR4 signaling pathway inhibitors have shown anti-fibrotic potential in preclinical studies. Inhibiting the activation of the TLR4 / MyD88 / NF-κB signaling pathway can curb the vicious cycle at its source, which is an effective anti-liver fibrosis treatment strategy.

[0013] Bicuculline, also known as bicuculline alkaloid or pericarp alkaloid, is an isoquinoline alkaloid extracted from plants in the Papaveraceae and Dipterocarpaceae families. It has the structural formula shown below and plays a crucial role in neuroscience research and potential drug applications.

[0014] Bicuculline, an important natural product, is best known for its role as a competitive antagonist of the type A receptor for γ-aminobutyric acid (GABA).

[0015] GABA is the main inhibitory neurotransmitter in the mammalian central nervous system, which reduces neuronal excitability. By binding to GABA type A receptors, bicuculline can specifically block the inhibitory effect of GABA, thereby increasing neuronal excitability and even inducing seizures.

[0016] Based on this mechanism, bicuculline is used as a standard research tool in basic research on neurological diseases, for verifying the function of GABAergic signaling pathways in in vitro experiments; for inducing seizures in animal models for screening and research of antiepileptic drugs; and for studying the excitation / inhibition balance of neurons.

[0017] Alkaloids have become an important source of drug discovery due to their structural diversity and wide range of biological activities. However, precisely because of bicuculline's strong convulsive effect, it has not long been considered as a therapeutic drug for the treatment of human diseases.

[0018] However, Malaguarnera M et al. (Bicuculline Reduces Neuroinflammation in Hippocampus and Improves Spatial Learning and Anxiety in Hyperammonemic Rats. Role of Glutamate Receptors. Frontiers in Pharmacology In a study on reducing hippocampal neuroinflammation in rats with hyperammonemia using bicuculline, it was noted that no signs of seizures were observed in any rats injected with it because its intraperitoneal injection dose of 0.3 mg / kg was lower than the prescribed seizure induction dose of >1 mg / kg.

[0019] To date, no published literature has shown any association between bicuculline and the prevention or treatment of liver fibrosis. Summary of the Invention

[0020] The purpose of this invention is to provide a novel TLR4 signaling pathway inhibitor for the prevention and / or treatment of liver fibrosis and related liver diseases.

[0021] Based on the core role of the TLR4 / MyD88 / NF-κB signaling pathway in the development of liver fibrosis and its vicious cycle with hepatic stellate cell activation, this invention screened and obtained a novel natural alkaloid compound that can effectively inhibit the activity of this signaling pathway.

[0022] This invention has shown through in vitro experiments that the natural alkaloid bicuculline can inhibit the activation of the TLR4 / MyD88 / NF-κB signaling pathway and downregulate the expression of downstream inflammatory factors, thereby inhibiting the activation of hepatic stellate cells and alleviating the formation of liver fibrosis.

[0023] Therefore, the present invention primarily provides the use of the natural alkaloid bicuculline in the preparation of medicaments for the prevention and / or treatment of liver fibrosis, and in the preparation of medicaments for the prevention and / or treatment of liver inflammation.

[0024] Furthermore, the applications described in this invention also include the use of the alkaloid bicuculline as a lead compound in the preparation of medicaments for the prevention and / or treatment of liver fibrosis and liver inflammation.

[0025] Specifically, the liver fibrosis disease described in this invention is caused by alcoholic liver disease and / or drug-induced liver injury; the liver inflammation includes alcoholic, drug-induced, fatty, and autoimmune liver inflammation.

[0026] The drug described in this invention exerts its effect by inhibiting the activity of hepatic stellate cells, thereby inhibiting the production of fibrosis markers.

[0027] This invention has shown through experiments that the alkaloid bicuculline can be used to inhibit the activation of the Toll-like receptor 4 signaling pathway, thereby suppressing the expression of inflammatory factors.

[0028] Therefore, the application described in this invention also includes the use of the alkaloid bicuculline in the preparation of Toll-like receptor 4 signaling pathway inhibitors, and more specifically, the factors in the signaling pathway include MyD88 and / or NF-κB.

[0029] Furthermore, the application described in this invention also includes the use of the alkaloid bicuculline in the preparation of inhibitors of inflammatory factor expression.

[0030] Preferably, the inflammatory factors include one or more of IL-6, IL-1β, and TNF-α.

[0031] Furthermore, this invention also verified the therapeutic effect of the alkaloid bicuculline in vivo using an animal model of liver fibrosis, ultimately achieving the goal of preparing an anti-liver fibrosis drug from this alkaloid.

[0032] Therefore, the present invention ultimately provides a medicament for the prevention and / or treatment of liver fibrosis and liver inflammation, wherein the medicament contains the active pharmaceutical ingredient bicuculline and pharmaceutically acceptable excipients.

[0033] Preferably, the dosage form of the drug of the present invention may include, but is not limited to, any one of injectable formulations, oral formulations, or spray formulations.

[0034] Compared with the prior art, the present invention provides the application of the alkaloid bicuculline in the preparation of anti-liver fibrosis and liver inflammation drugs and their lead compounds, as well as the application of bicuculline in the preparation of TLR4 signaling pathway inhibitors and the application of bicuculline in the preparation of inflammatory factor expression inhibitors.

[0035] In vitro experiments of this invention confirmed that bicuculline has a better inhibitory effect on HSCs than previously reported forsythosides and forsythoside A, and can alleviate TGF-β1-induced HSC activation, significantly reducing the expression of α-SMA and COL1A1. Simultaneously, bicuculline can also reduce the expression of MyD88 and NF-κB in the TLR4 / MyD88 / NF-κB signaling pathway, as well as their downstream inflammatory factors IL-6, IL-1β, and TNF-α.

[0036] This invention demonstrates through in vivo experiments that intraperitoneal injection of bicuculline can significantly improve liver morphology in fibrosis models, significantly reduce serum ALT and AST levels, and significantly reduce collagen deposition and α-SMA and COL1A1 expression in the liver. Attached Figure Description

[0037] Figure 1 This refers to the inhibitory effect of bicuculline on the activation and proliferation of hepatic stellate cells.

[0038] Figure 2 This refers to the inhibitory effect of bicuculline on the expression of α-SMA and COL1A1 in hepatic stellate cells.

[0039] Figure 3 This refers to the inhibitory effect of bicuculline on the activation of the Toll-like receptor 4 signaling pathway in hepatic stellate cells.

[0040] Figure 4 This describes the inhibitory effect of bicuculline on the expression of inflammatory factors IL-6, IL-1β, and TNF-α in hepatic stellate cells.

[0041] Figure 5This refers to the alleviating effect of bicuculline on liver fibrosis induced by carbon tetrachloride and bile duct ligation.

[0042] Figure 6 This describes the inhibitory effect of bicuculline on the expression of α-SMA and COL1A1 induced by carbon tetrachloride and bile duct ligation.

[0043] Figure 7 This describes the inhibitory effect of bicuculline on the activation of the Toll-like receptor 4 signaling pathway in carbon tetrachloride- and bile duct ligation-induced fibrotic liver.

[0044] Figure 8 This describes the inhibitory effect of bicuculline on the expression of inflammatory factors IL-6, IL-1β, and TNF-α in carbon tetrachloride- and bile duct ligation-induced fibrotic liver. Implementation

[0045] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. It should be noted that the embodiments are merely illustrative and are intended to provide a thorough understanding of the technical solutions of the present invention and to provide guidance for those skilled in the art to implement and apply the present invention. It should be understood that these descriptions do not constitute any limitation on the scope of protection of the present invention.

[0046] Unless otherwise expressly stated, the production processes, experiments, tests or analysis methods involved in the embodiments of the present invention are all considered to be conventional methods known to those skilled in the art, and only need to be implemented in accordance with conventional conditions or relevant product instructions. The steps and names involved are also generally clear and unambiguous in the art.

[0047] The instruments, equipment, raw materials, reagents, or samples used in the embodiments are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels or prepared by known methods, and their source does not have a substantial impact on the implementation results of the present invention.

[0048] Unless otherwise expressly defined, the scientific and technical terms used in this invention have the meanings commonly understood by one of ordinary skill in the art. In case of any conflict, the definitions in this specification shall prevail.

[0049] The terms “comprising,” “including,” “having,” etc., used in this invention should be understood as open-ended, meaning “including but not limited to.” The term “and / or” includes any and all combinations of one or more of the associated listed items. Quantitative terms such as “a,” “one,” etc., do not exclude multiples; “multiple” or “a variety” refers to quantities greater than or equal to two.

[0050] The terms "preferred", "better", and "exemplary" used in this invention are only used to describe specific solutions or effects and are not intended to limit the necessary scope of the solution or the scope of protection.

[0051] This invention relates to the description of numerical parameters (such as quantity, concentration, temperature, time, etc.), and it should be understood that reasonable deviations naturally exist due to measuring instruments, operational errors, statistical fluctuations, etc. The range of such deviations should be within limits acceptable to those skilled in the art based on common sense.

[0052] This invention provides the application of the alkaloid bicuculline in the treatment of liver fibrosis and its use as an inhibitor of the TLR4 signaling pathway. According to this invention, the alkaloid bicuculline is preferably used to inhibit liver fibrosis induced by TGF-β1, carbon tetrachloride (CCl4), and bile duct ligation (BDL).

[0053] This invention does not limit the source of the alkaloid bicuculline; it can be commercially available or prepared using conventional methods.

[0054] This invention provides the use of the alkaloid bicuculline as a lead compound for anti-liver fibrosis.

[0055] Existing technologies report that after liver damage, hepatic stellate cells are activated and secrete a large amount of fibrous material to form scar tissue. Among them, α-SMA and COL1A1 have received widespread attention as markers of hepatic stellate cell activation.

[0056] This invention experimentally demonstrates that bicuculline plays an important role in inhibiting hepatic stellate cell activation and resisting liver fibrosis, and is an effective lead compound for anti-liver fibrosis.

[0057] This invention verifies through in vitro experiments that bicuculline has a significant inhibitory effect on TGF-β1-induced activation and proliferation of hepatic stellate cells, and has a better inhibitory effect on hepatic stellate cell activation than previously reported forsythoside and forsythoside A.

[0058] This invention verifies through in vivo experiments that bicuculline has a significant alleviating effect on liver fibrosis induced by carbon tetrachloride and bile duct ligation.

[0059] This invention provides the application of bicuculline in the preparation of TLR4 signaling pathway inhibitors.

[0060] Toll-like receptor 4 (TLR4), a typical innate immune receptor, can recognize exogenous pathogen-associated molecular patterns (PAMPs), endogenous damage-associated molecular patterns (DAMPs), and foreign substance-associated molecular patterns (XAMPs) to induce inflammatory immune responses. While immune responses can protect the body, persistent inflammation can cause damage.

[0061] Existing technologies disclose that TLR4 is highly expressed in activated hepatic stellate cells and Kuff cells, producing a large number of inflammatory factors, thereby enhancing the formation of liver fibrosis.

[0062] This invention experimentally demonstrates that bicuculline plays an important role in the immune response mediated by the innate immune receptor TLR4 and is a major inhibitor of the TLR4 signaling pathway.

[0063] This invention, through in vivo and in vitro detection of the expression of key genes and proteins in the TLR4 / MyD88 / NF-κB signaling pathway, found that bicuculline has a significant inhibitory effect on the TLR4-mediated signaling pathway of the innate immune receptor.

[0064] This invention provides the application of bicuculline in the preparation of inhibitors of inflammatory cytokine expression, wherein the inflammatory cytokines preferably include one or more of IL-6, IL-1β and TNF-α.

[0065] According to the present invention, the inhibitor is preferably used to inhibit the expression of inflammatory factors induced by TGF-β1, carbon tetrachloride, and bile duct ligation.

[0066] This invention demonstrates through in vivo and in vitro experiments that the levels of downstream inflammatory factors are significantly increased in TGF-β1-activated hepatic stellate cells and in the liver induced by carbon tetrachloride and bile duct ligation, while administration of bicuculline can significantly inhibit the expression of downstream inflammatory factors.

[0067] In summary, this invention provides the use of bicuculline in the preparation of medicaments for the prevention and / or treatment of liver fibrosis and liver inflammation.

[0068] This invention provides a medicament for the prevention and / or treatment of liver fibrosis and liver inflammation, wherein the medicament contains the active pharmaceutical ingredient bicuculline and pharmaceutically acceptable excipients.

[0069] The dosage form of the drug described in this invention may include, but is not limited to, any one of injectable formulations, oral formulations, or spray formulations.

[0070] This invention does not limit the pharmaceutically acceptable excipients, which can be any excipient well known to those skilled in the art, such as solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, and release inhibitors, etc., any one or more of these.

[0071] Furthermore, the present invention also provides the application of bicuculline in the preparation of health products for improving liver diseases.

[0072] This invention provides the application of the alkaloid bicuculline in the preparation of anti-liver fibrosis and liver inflammation drugs and their lead compounds, as well as the application of bicuculline in the preparation of TLR4 signaling pathway inhibitors and the application of bicuculline in the preparation of inflammatory factor expression inhibitors.

[0073] In vitro experiments of this invention confirmed that bicuculline has a better inhibitory effect on hepatic stellate cells than previously reported forsythosides and forsythoside A; 7.5 μg / mL of bicuculline can alleviate TGF-β1-induced hepatic stellate cell activation and significantly reduce the expression of α-SMA and COL1A1. P <0.05); 7.5 μg / mL bicuculline can also reduce the expression of MyD88 and NF-κB in the TLR4 / MyD88 / NF-κB signaling pathway and their downstream inflammatory factors IL-6, IL-1β and TNF-α. P <0.05).

[0074] In vivo experiments of this invention have demonstrated that intraperitoneal injection of 0.6 mg / kg bicuculline significantly improves liver morphology and significantly reduces serum ALT and AST levels in a fibrosis model. P <0.05), and can also significantly reduce collagen deposition and α-SMA and COL1A1 expression in the liver ( P <0.05); RT-qPCR and WB results showed that intraperitoneal injection of 0.6 mg / kg bicuculline could inhibit the expression of MyD88 and NF-κB in the TLR4 / MyD88 / NF-κB signaling pathway, accompanied by a decrease in the expression of inflammatory factors such as IL-6, IL-1β and TNF-α. P <0.05).

[0075] To further illustrate the present invention, the following describes in detail the application of bicuculline provided by the present invention with reference to embodiments. Example

[0076] Example 1

[0077] Phlygenol and phyryrin are two main active components extracted from the traditional Chinese medicine Forsythia suspensa. Numerous studies have shown that both have a clear anti-liver fibrosis effect, one of the core mechanisms being the inhibition of hepatic stellate cells (HSCs) activation and proliferation, and the induction of their apoptosis. Therefore, this embodiment uses phylygenol and phyryrin as positive controls to examine and compare the inhibitory effect of bicuculline on hepatic stellate cells.

[0078] Hepatic stellate cells cultured according to conventional methods were seeded into 96-well plates and set up blank groups, solvent groups, TGF-β1 induction groups, and various small molecule inhibitor treatment groups with different concentration gradients. After culturing in starvation medium for 24 h, 2 ng / mL of TGF-β1 was added to each group except the blank group.

[0079] Once the cells reached 60-70% cell growth, each small molecule inhibitor treatment group was given a corresponding concentration of small molecule inhibitor, while the solvent group received an equal volume of DMSO solvent. Treatments were performed for 6, 12, and 24 hours, respectively. The culture medium in each well was aspirated, and then 10 μL of CCK-8 solution was added, taking care to avoid generating air bubbles. The wells were incubated for 1-4 hours, and the absorbance of each well was measured at 450 nm using a microplate reader.

[0080] The inhibition rate of different concentrations of small molecule inhibitors on cells was calculated using the formula: Inhibition rate = (Absorbance of control wells - Absorbance of experimental wells) / (Absorbance of control wells - Absorbance of blank wells) × 100%. Specific results are shown below. Figure 1 As shown.

[0081] Figure 1 In the study, within a 24-hour treatment period, bicuculline at a concentration of 7.5 μg / mL showed significantly better inhibitory effects on hepatic stellate cells (A) than forsythoside (B) and forsythoside (C). Although forsythoside and forsythoside showed good inhibitory effects on hepatic stellate cells at concentrations of 100 μg / mL and 20 μg / mL, respectively, within 24 hours, the cell survival rate had already been greatly reduced by this time.

[0082] Example 2

[0083] Hepatic stellate cells cultured according to standard methods were seeded into T-25 cell culture flasks, and blank, TGF-β1-induced, and bicuculline-treated groups were set up. After culturing in starvation medium for 24 h, 2 ng / mL of TGF-β1 was added to the TGF-β1-induced and bicuculline-treated groups, respectively.

[0084] When the cells grew to 60-70%, the bicuculline-treated group was treated with 7.5 μg / mL bicuculline for 24 h. The cell state was observed under a microscope. After 24 h of TGF-β1 stimulation and starvation culture, the cells changed from the original round shape to a spindle shape resembling fibroblasts.

[0085] Cells were washed three times with PBS, digested with trypsin, and centrifuged to collect the cell pellet. Total RNA was extracted with Trizol reagent, and the RNA concentration was measured before storage at -80°C.

[0086] RNA was reverse transcribed using a reverse transcription kit to obtain cDNA, which was then analyzed by qPCR to detect the mRNA expression levels of α-smooth muscle actin (α-SMA) and type I collagen α1 chain (COL1A1).

[0087] Figure 2 The inhibitory effects of bicuculline on the expression of fibrosis markers α-SMA and COL1A1 in hepatic stellate cells are presented. The figure shows that hepatic stellate cells are activated upon TGF-β1 stimulation, leading to increased gene expression of the related fibrosis markers α-SMA (A) and COL1A1 (B). However, this upregulation of gene expression was significantly reversed after bicuculline treatment.

[0088] Example 3

[0089] In this embodiment, Western blotting was used to detect the protein expression of MyD88, NF-κB and P-NF-κB in the TLR4 / MyD88 / NF-κB signaling pathway in the blank group, TGF-β1 group and bicuculline treatment group; RNA extracted in Example 2 was used to detect the expression levels of inflammatory factors IL-6, IL-1β and TNF-α in hepatic stellate cells.

[0090] Figure 3 The inhibitory effect of bicuculline on the activation of the Toll-like receptor 4 signaling pathway in hepatic stellate cells was presented. It was found that bicuculline treatment could reduce the protein expression of MyD88 and phosphorylated NF-κB in the TLR4 signaling pathway in activated hepatic stellate cells.

[0091] Figure 4The results showed that after TGF-β1 stimulation, the gene expression levels of inflammatory factors IL-6, IL-1β and TNF-α in hepatic stellate cells were significantly increased, but the increase in gene expression of these inflammatory factors was reduced after treatment with bicuculline.

[0092] Example 4

[0093] A liver fibrosis model was established using C57BL / 6N mice to evaluate the in vivo anti-liver fibrosis pharmacodynamics of bicuculline.

[0094] A CCl4 liver fibrosis model was established by intraperitoneal injection of 20% CCl4, 5 mL / kg, twice a week for 8 weeks; a bile duct ligation surgery was performed to establish a bile duct ligation liver fibrosis model.

[0095] The CCl4 liver fibrosis model was divided into an olive oil control group, a CCl4 model group, a low-dose bicuculline group (0.3 mg / kg), and a high-dose bicuculline group (0.6 mg / kg), with 5 mice in each group. Starting from week 6, mice were treated with intraperitoneal injection of bicuculline for 2 weeks. After 8 weeks, the mice were anesthetized and sacrificed, and liver tissue was obtained.

[0096] The bile duct ligation liver fibrosis model was divided into a sham operation group, a BDL model group, a low-dose bicuculline group (0.3 mg / kg), and a high-dose bicuculline group (0.6 mg / kg), with 5 mice in each group. After bile duct ligation surgery, the mice were fed for 4 weeks. In the 3rd week, they were treated with intraperitoneal injection of bicuculline for 2 weeks. Subsequently, the mice were anesthetized and sacrificed to obtain liver tissue.

[0097] A portion of liver tissue was embedded, sectioned, and stained with Masson's stain. The remaining liver tissue was flash-frozen at -80°C in liquid nitrogen.

[0098] Figure 5 In the study, CCl4 and bile duct ligation induced a significant amount of blue collagen deposition in the liver tissue sections of mice. After treatment with 0.3 mg / kg bicuculline, the blue collagen deposition in the liver tissue sections of these two liver fibrosis models was reduced to some extent. After treatment with 0.6 mg / kg bicuculline, the collagen deposition in the liver tissue sections of both liver fibrosis models was significantly reduced, and the liver fibrosis was significantly alleviated.

[0099] We took frozen liver tissue, ground it to extract RNA, and performed qPCR to test the expression levels of α-SMA and COL1A1 in the liver tissue.

[0100] Figure 6In the study, the expression levels of α-SMA and COL1A1 genes in both liver fibrosis models were significantly higher than those in the olive oil control group and the sham surgery group. However, after treatment with low and high doses of bicuculline, the gene expression of these two fibrosis indicators was significantly downregulated.

[0101] Example 5

[0102] The liver tissue was lysed using RIPA to extract proteins. Protein concentration was determined by BCA method. Western blotting was used to detect the protein expression of MyD88, NF-κB and P-NF-κB in the TLR4 / MyD88 / NF-κB signaling pathway in the olive oil or sham operation group, control group, CCl4 and bile duct ligation group, and bicuculline low and high treatment group. The expression levels of inflammatory factors IL-6, IL-1β and TNF-α in liver tissue were detected using RNA.

[0103] Figure 7 In terms of the inhibitory effect of bicuculline on the activation of the Toll-like receptor 4 signaling pathway in CCl4- and bile duct ligation-induced fibrotic liver tissue, bicuculline treatment can reduce the protein expression of MyD88 and phosphorylated NF-κB in the TLR4 signaling pathway in CCl4- and bile duct ligation-induced fibrotic liver tissue. Figure 8 The inhibitory effects of bicuculline on inflammatory factors IL-6, IL-1β, and TNF-α in CCl4- and bile duct ligation-induced fibrotic liver tissue were observed. In CCl4- and bile duct ligation-induced fibrotic liver tissue, the gene expression of inflammatory factors IL-6, IL-1β, and TNF-α was significantly increased. However, after intraperitoneal injection of bicuculline, the level of increased gene expression of these inflammatory factors was significantly reduced.

[0104] The present invention provides the application of bicuculline through preferred embodiments. It should be noted that the above embodiments do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Those skilled in the art can make appropriate improvements based on the above content. All similar substitutions and modifications are obvious to those skilled in the art. Modifications or appropriate changes and combinations made to the methods and applications of the present invention without departing from the content, spirit and scope of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of the alkaloid bicuculline in the preparation of drugs for the prevention and / or treatment of liver fibrosis and liver inflammation.

2. The use of alkaloid bicuculline as a lead compound in the preparation of drugs for the prevention and / or treatment of liver fibrosis and liver inflammation.

3. The application according to claim 1, wherein the liver fibrosis is caused by alcoholic liver disease and / or drug-induced liver injury.

4. The application according to claim 1, wherein the liver inflammation includes alcoholic, drug-induced, fatty, and autoimmune liver inflammation.

5. The application according to claim 1 or 2, wherein the drug exerts its effect by inhibiting hepatic stellate cell activity.

6. Application of the alkaloid bicuculline in the preparation of inhibitors of the Toll-like receptor 4 signaling pathway, wherein the signaling pathway includes MyD88 and / or NF-κB.

7. Application of the alkaloid bicuculline in the preparation of inhibitors of inflammatory factor expression.

8. The application according to claim 7, wherein the inflammatory factor is one or more of IL-6, IL-1β and TNF-α.

9. A medicament for the prevention and / or treatment of liver fibrosis and liver inflammation, wherein the medicament contains the pharmaceutically active ingredient bicuculline and pharmaceutically acceptable excipients.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug is any one of an injectable formulation, an oral formulation, or a spray formulation.

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