Application of sesquiterpene lactone CJ in preparation of medicine for preventing and treating hepatic fibrosis

Abstract

The invention discloses an application of sesquiterpene lactone CJ shown as a formula I in preparation of a medicine for preventing and treating hepatic fibrosis, and relates to the technical field of biological medicines, and the sesquiterpene lactone CJ can obviously protect the liver from CCl4-induced injury and effectively improve hepatic fibrosis symptoms. The action mechanism is that HSP90AA1 is regulated in a targeted manner to inhibit an NLRP3 / caspase-1 signal channel, so that pyroptotic death of hepatocytes is generated. In addition, the sesquiterpene lactone CJ also shows a remarkable improvement effect in an LX-2 cell inflammation model induced by TGF-beta1. The callistephus chinensis extract sesquiterpene lactone CJ provided by the invention has a remarkable effect in the aspect of preventing and treating CCl4-induced hepatic fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of a rare 6 / 7 fused sesquiterpene skeleton compound CJ isolated from aster in the preparation of drugs or health foods for treating liver fibrosis. Background Technology

[0002] As a core metabolic organ in the human body, the liver's chronic inflammatory response is often induced by various factors such as toxic substances, pathogen infection, and metabolic abnormalities. Under the continuous stimulation of these pathogenic factors, resting hepatic stellate cells (HSCs) are activated and transformed into highly secretory myofibroblasts, leading to excessive deposition of collagen and other components in the extracellular matrix. As the pathological process progresses, collagen fibers accumulate in the liver tissue, gradually destroying the normal liver lobule structure, ultimately causing liver fibrosis, cirrhosis, and even liver failure, seriously threatening the patient's life and health. However, current clinical treatments mostly focus on symptomatic treatment and have not yet provided precise intervention targeting the core pathogenesis of liver fibrosis. Therefore, developing treatments that can directly regulate the fibrosis process and the accompanying chronic inflammatory response has become an urgent task. Pyroptosis is a form of programmed cell death mediated by GSDM family proteins. Studies have shown that HSP90AA1 can inhibit hepatocyte pyroptosis by regulating the classical NLRP3 / caspase-1 pyroptosis pathway.

[0003] Plants in the Asteraceae family possess both medicinal and edible value, serving both as tea and medicinal ingredients, and are often referred to as "longevity aids." Asters, the only species in the genus *Aster* within the Asteraceae family, have been proven to possess various pharmacological activities, including anti-inflammatory, hepatoprotective, and antihypertensive effects. Its monomeric component, compound CJ, is a newly discovered sesquiterpene compound with a 6 / 7 fused structure. Its pharmacological mechanism of action is currently unclear; therefore, in-depth research into whether compound CJ possesses highly effective and low-toxicity anti-hepatic fibrosis prevention and treatment potential, and clarifying its target and molecular mechanism, is of significant research value. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide an application of sesquiterpene lactone CJ in the preparation of drugs or health foods for the prevention and treatment of liver fibrosis.

[0005] The sesquiterpene lactone CJ described in this invention has the structure shown in Formula I:

[0006] This invention, through the establishment of a carbon tetrachloride (CCl4)-induced liver fibrosis mouse model, explores for the first time the protective effect and mechanism of sesquiterpene lactone CJ on CCl4-induced liver fibrosis. The study found that the mechanism by which sesquiterpene lactone CJ treats liver fibrosis may be through regulating HSP90AA1 to inhibit the NLRP3 / caspase-1 signaling pathway, thereby inhibiting hepatocyte pyroptosis.

[0007] This invention also investigated, for the first time, the protective effect and mechanism of sesquiterpene lactone CJ on TGF-β1-induced HSC activation model by establishing a transforming growth factor-β1 (TGF-β1)-induced LX-2 hepatic stellate cell (HSC) activation model. The study found that sesquiterpene lactone CJ may exert its anti-hepatic fibrosis effect by regulating HSP90AA1 to inhibit the NLRP3 / caspase-1 signaling pathway and suppressing hepatocyte pyroptosis.

[0008] The objective of this invention is achieved by at least one of the following technical solutions: The disease described is liver fibrosis. Liver fibrosis is an inevitable stage in the progression of many chronic liver diseases (such as viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, etc.) to cirrhosis.

[0009] The application of the sesquiterpene lactone CJ in the preparation of drugs for the prevention and treatment of liver fibrosis.

[0010] The drug mentioned is one that can improve liver tissue damage.

[0011] The drug described is capable of improving liver tissue structural disorder and the infiltration of a large number of inflammatory cells.

[0012] The drug described is one that can regulate the expression of genes related to inflammatory signaling pathways.

[0013] Preferably, the drug treats liver fibrosis by regulating the levels of inflammatory factors such as IL-1β, IL-6, and TNF-α.

[0014] The drug described is capable of regulating the expression of genes related to the HSP90AA1 / NLRP3 signaling pathway.

[0015] Preferably, the drug treats liver fibrosis by regulating genes related to the HSP90AA1 / NLRP3 signaling pathway.

[0016] The drug mentioned is one that can regulate serum AST and ALT.

[0017] The drug described is capable of regulating the expression of α-SMA and collagen I proteins.

[0018] The drug can be used alone as an active ingredient or in combination with other drugs for the prevention and treatment of liver fibrosis.

[0019] A pharmaceutical composition for the prevention and treatment of liver fibrosis includes a sesquiterpene lactone CJ as shown in Formula I and a pharmaceutically acceptable excipient or carrier.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Sesquiterpene lactone CJ is a stable and safe monomeric substance extracted from chrysanthemum. It has greatly ensured the reliability of the experiment by establishing a mature TGF-β1-induced LX-2 hepatic stellate cell (HSC) activation model and a CCl4-induced animal liver fibrosis model.

[0021] (2) Further experimental studies have shown that treatment with sesquiterpene lactone CJ can significantly improve ALT and AST levels in mouse serum and significantly reduce TNF-α and IL-1β levels, thus having a good therapeutic effect on liver fibrosis. Sesquiterpene lactone CJ can be used in the preparation of drugs or health foods that protect against liver fibrosis.

[0022] (3) Since aster has good anti-inflammatory effects and the inhibition of NLRP3 inflammasome is a key step in intervening in the progression of liver fibrosis, its monomeric component, sesquiterpene lactone CJ, can effectively inhibit hepatocyte pyroptosis and improve the pathological process of liver fibrosis by targeting and inhibiting the HSP90AA1 / NLRP3 signaling pathway. Therefore, sesquiterpene lactone CJ shows broad application prospects in the treatment of CCl4-induced liver fibrosis. Attached Figure Description

[0023] Figure 1 This is a comparison of the macroscopic pathological features of the livers of mice in each group in this invention; where CJ-L represents the low-dose group of sesquiterpene lactone CJ (25 mg / kg), CJ-H represents the high-dose group of sesquiterpene lactone CJ (50 mg / kg) and Silibinin represents the group of silybinin (10 mg / kg).

[0024] Figure 2 The results of pathological staining of mouse liver tissue in each group of this invention are shown below; HE staining: scale bar 50μm, magnification 20×; Sirius red staining: scale bar 100μm, magnification 10×; Masson staining: scale bar 100μm, magnification 10×; immunohistochemical staining α-SMA, Col-I: scale bar 200μm, magnification 5×.

[0025] Figure 3 The graph shows the results of detecting the levels of biochemical indicators AST and ALT in the serum of mice in each group in this invention; where A: comparison of ALT levels in mouse serum; B: comparison of AST levels in mouse serum.

[0026] Figure 4 The figure shows the effect of each group of mice on the expression of inflammatory factors in this invention. A: TNF-α gene expression result; B: IL-1β gene expression result; C: IL-6 gene expression result.

[0027] Figure 5 The relative mRNA expression levels of HSP90AA1 and NLRP3 in the liver of mice in each group were detected by qRT-PCR in this invention; where A: relative mRNA expression level of HSP90AA1; B: relative mRNA expression level of NLRP3.

[0028] Figure 6 The following is a Western blot analysis of HSP90AA1 / NLRP3 pathway-related protein expression in mice in this invention; where A: Western blot bands of HSP90AA1 / NLRP3 pathway-related proteins in each group; B: Western blot quantitative analysis of HSP90AA1 / NLRP3 pathway-related proteins in each group.

[0029] Figure 7 The figure shows the cell survival rate of TGF-β1-induced LX-2 cells by sesquiterpene lactone CJ in this invention.

[0030] Figure 8 The expression of HSP90AA1 / NLRP3 pathway-related proteins in each group of LX-2 cells in this invention was detected by Western blot; where A: Western blot band; B: Western blot quantitative analysis. Detailed Implementation

[0031] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0032] This invention explores for the first time the mechanism of action of sesquiterpene lactone CJ from aster extract in preventing and treating liver fibrosis and its application value in drugs or health foods for the prevention and treatment of liver fibrosis.

[0033] The sesquiterpene lactone CJ described in this invention was obtained by the preparation method described in the literature: Zhang XS, Cao JQ, Liu ZT, et al. Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower[J]. Phytochemistry Letters, 2015, 11: 5-8. https: / / doi.org / 10.1016 / j.phytol. 2014.10.030.

[0034] Example 1

[0035] Using sesquiterpene lactone CJ as the main research sample, the in vivo anti-hepatic fibrosis activity of sesquiterpene lactone CJ was evaluated through a carbon tetrachloride (CCl4)-induced liver fibrosis mouse model, and the molecular mechanism by which it exerts its anti-hepatic fibrosis effect by regulating the HSP90AA1 / NLRP3 signaling pathway was further explored.

[0036] First, a mouse model of liver fibrosis was established. Six- to seven-week-old male C57BL / 6 mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) weighing 22-24g were fed a standard pelleted diet with free access to water, at a temperature of 21-25℃ and humidity of 50-70%, with a 12-hour light-dark cycle. Mice were randomly divided into a blank control group, a model group, a low-dose CJ sesquiterpene lactone group (CJ-L, 25mg / kg), a high-dose CJ sesquiterpene lactone group (CJ-H, 50mg / kg), and a silibinin group (10mg / kg).

[0037] Except for the blank control group, all mice were intraperitoneally injected with 15% CCl4 (2 mL / kg, 3 times a week for 6 weeks) to induce liver fibrosis. The blank control group and model group were given the same dose of water, and the CJ-L group, CJ-H group, and Silibinin group were administered the drug by gavage for 3 weeks. Mice were weighed daily, and clinical symptoms of liver fibrosis were monitored. After the drug administration was completed, blood was collected from anesthetized mice, serum was obtained by centrifugation, and liver tissue was processed and photographed. Liver tissue was either embedded in paraffin or frozen at -80°C for subsequent analysis.

[0038] The levels of various indicators in serum were detected using commercially available kits to determine the changes in liver fibrosis caused by sesquiterpene lactone CJ in the CCl4 model.

[0039] Example 2

[0040] Using sesquiterpene lactone CJ as the main research sample, we verified its ability to inhibit HSC activation in a TGF-β1-induced LX-2 hepatic stellate cell (HSC) activation model and further explored its potential molecular mechanism.

[0041] First, a TGF-β1-induced activation model of LX-2 hepatic stellate cells (HSCs) was established. The human hepatic stellate cell line LX-2 was cultured in a sterile cell culture incubator at 37°C and 5% CO2 using DMEM high-glucose medium containing 10% FBS and 1% penicillin and streptomycin.

[0042] The effect of sesquiterpene lactone CJ on the viability of LX-2 cells was detected using the CCK8 assay. After cells reached the logarithmic growth phase, LX-2 cell suspensions were seeded into 96-well plates and cultured for 24 h before the experiment, with 6 replicates per group. Cells were treated with different concentrations (2.5, 5, 10, 20, 40, 60 μM) of sesquiterpene lactone CJ for 24 h, and cell viability was detected by CCK8 assay. The absorbance of each well at 450 nm was then measured using a microplate reader. Cell viability was calculated using the formula: Cell viability = (OD-treated group / OD-blank group) × 100%.

[0043] LX-2 cells were seeded in 6-well plates and cultured for 24 h. When the cells were in good condition and in the logarithmic growth phase, all groups except the blank control group were treated with TGF-β1 (5 ng / mL) inducer for 24 h. The groups were: blank control group, TGF-β1 model group, low-dose sesquiterpene lactone CJ group (CJ-L, 30 μM), high-dose sesquiterpene lactone CJ group (CJ-H, 60 μM), and silibinin group (2.5 μM).

[0044] Commercial kits were used to detect various parameters in cell supernatant and cells to determine the effects of sesquiterpene lactone CJ on a TGF-β1-induced LX-2 cell model.

[0045] Experimental results: (1) such as Figure 1 As shown, the liver tissue of mice in the Model group exhibited decreased luster and numerous granular protrusions, suggesting that this modeling method can effectively construct a mouse liver fibrosis model. Compared with the Model group, the appearance of the liver of mice in the sesquiterpene lactone CJ administration group was significantly improved.

[0046] (2) For example Figure 2As shown, histological analysis of liver tissue using H&E, Sirius Red, Masson staining, and immunohistochemical staining for α-SMA and collagen I revealed that treatment with the sesquiterpene lactone CJ significantly alleviated CCl4-induced pathological changes. The control group consistently exhibited normal liver structure in all staining assays. In contrast, the model group showed significant liver injury characteristics, including disorganized tissue structure, increased collagen fiber and fibrotic tissue deposition, and exacerbated inflammatory cell infiltration. The treated groups showed significant improvement after CJ treatment, including partial restoration of normal liver structure, reduced inflammation, and decreased collagen deposition. Immunohistochemical results further supported these findings, showing decreased α-SMA and collagen I immunoreactivity in the CJ-treated group compared to the model group. These findings strongly suggest that CJ, by inhibiting inflammation and collagen deposition, is a potentially effective therapeutic agent against CCl4-induced liver injury. In addition, quantitative detection based on various staining results showed that the collagen deposition (Sirius red, Masson), HSC activation (α-SMA), and type I collagen expression (collagen I) in the CJ-H group were closer to normal levels than those in the Silibinin group. The improvement effect of high-dose sesquiterpene lactone CJ was better than that of silybinin.

[0047] (3) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important indicators for assessing liver function, such as Figure 3 As shown, compared with the Control group, the serum AST and ALT levels in the Model group mice were significantly increased (P < 0.001). Compared with the Model group, the serum AST and ALT levels in the CJ-L, CJ-H, and silymarin groups were significantly decreased (P < 0.01), and the decrease in serum AST and ALT levels was more significant in the CJ-H group than in the silymarin group. This indicates that the sesquiterpene lactone CJ has a protective effect against CCl4-induced liver injury.

[0048] (4) The changes in the protein levels of inflammatory factors IL-6, IL-1β, and TNF-α in the serum of CCl4-induced mouse liver fibrosis were detected by enzyme-linked immunosorbent assay (ELISA). Figure 4As shown, compared with the Control group, the serum levels of IL-6, IL-1β, and TNF-α in the Model group mice were significantly increased. Compared with the Model group, the serum and liver tissue levels of IL-6, IL-1β, and TNF-α in the CJ-L, CJ-H, and Silibin groups were significantly decreased, and the decrease in IL-6, IL-1β, and TNF-α levels was more significant in the CJ-H group than in the Silibinin group. These results indicate that the sesquiterpene lactone CJ effectively inhibits the CCl4-induced inflammatory response, highlighting its potential application in the treatment of liver injury.

[0049] (5) such as Figure 5 As shown, compared with the control group, the levels of HSP90AA1 and NLRP3 mRNA were significantly increased in the model group; compared with the model group, the levels of HSP90AA1 and NLRP3 mRNA were significantly decreased in the sesquiterpene lactone CJ treatment group, and the decrease in HSP90AA1 and NLRP3 mRNA was more significant in the CJ-H group than in the silibinin group. This indicates that the mechanism of action of sesquiterpene lactone CJ in treating liver fibrosis may be related to the HSP90AA1 / NLRP3 pathway.

[0050] (6) For example Figure 6 As shown, compared with the Control group, the Model group significantly increased the relative expression levels of all components of the HSP90AA1 / NLRP3 signaling pathway, including HSP90AA1, NLRP3, Caspase-1, Cleaved Caspase-1, IL-1β, IL-18, and GSDMD-N, with statistically significant differences (P < 0.001). However, these elevated expression levels were reversed after treatment with the sesquiterpene lactone CJ, showing a corrective effect compared with the Model group, with statistically significant differences. These results confirm that treatment with the sesquiterpene lactone CJ can prevent the CCl4-induced increase in the expression of all components of the HSP90AA1 / NLRP3 signaling pathway, indicating that the sesquiterpene lactone CJ has an inhibitory effect on the HSP90AA1 / NLRP3 signaling pathway.

[0051] (7) A liver fibrosis model was established by inducing LX-2 cells with 5 ng / mL TGF-β1. The effect of different concentrations (2.5 μM-60 μM) of sesquiterpene lactone CJ on LX-2 cell viability was detected by the CCK-8 assay. Compared with the model group, cell viability decreased after treatment with different doses of sesquiterpene lactone CJ (concentrations of 2.5, 5, 10, 20, 40, and 60 μM), showing a dose-dependent effect. Figure 7 The results showed that sesquiterpene lactone CJ could inhibit HSC activation.

[0052] (8) Figure 8 This report presents the results of in vitro Western blot analysis of HSP90AA1 / NLRP3 pathway-related protein expression. Compared to the Control group, the expression levels of HSP90AA1, NLRP3, Caspase-1, Cleaved Caspase-1, and GSDMD-N were significantly increased in the Model group cells. However, these increases were reversed after administration of the sesquiterpene lactone CJ. These results indicate that the sesquiterpene lactone CJ also inhibits the HSP90AA1 / NLRP3 signaling pathway in vitro.

Claims

1. The application of a sesquiterpene lactone CJ in the preparation of drugs or health foods for the prevention and treatment of liver fibrosis, characterized in that, The structural formula of the sesquiterpene lactone CJ is: 。 2. The application according to claim 1, characterized in that, The drug in question is one that can improve liver tissue damage.

3. The application according to claim 1, characterized in that, The drug is capable of improving liver tissue structural disorders and inflammatory cell infiltration.

4. The application according to claim 1, characterized in that, The drug is capable of regulating TNF-α, IL-6 and IL-1β-related inflammatory factors.

5. The application according to claim 1, characterized in that, The drug is capable of targeting and regulating the HSP90AA1 / NLRP3 / caspase-1 signaling pathway.

6. The application according to claim 5, characterized in that, The drug is capable of inhibiting the NLRP3 / caspase-1 signaling pathway by targeting HSP90AA1.

7. The application according to claim 1, characterized in that, The drug is one that can regulate serum AST and ALT levels.

8. The application according to claim 1, characterized in that, The drug is capable of regulating the expression of α-SMA and collagen I proteins.

9. A pharmaceutical composition for preventing and treating liver fibrosis, characterized in that, Includes sesquiterpene lactones CJ as shown in Formula I and pharmaceutically acceptable excipients or carriers; 。

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