Application of TLR4 / TRAF6 / NF-kappa B pathway inhibitor in preparation of anti-hepatic fibrosis drugs

Abstract

The invention discloses an application of a TLR4 / TRAF6 / NF-kappa B pathway inhibitor in preparation of anti-hepatic fibrosis drugs, the development of the anti-hepatic fibrosis drugs is realized based on negative regulation of the TLR4 / TRAF6 / NF-kappa B pathway for the first time, in a hepatic fibrosis animal model, the expression of key molecules of the TLR4-TRAF6-NF-kappa B signaling pathway is down-regulated, the level of proinflammatory factors (TNF-alpha, IL-6 and TGF-beta 1) is reduced, and the anti-hepatic fibrosis effect is realized. The traditional Chinese medicine composition has the effects of relieving inflammatory response, inhibiting activation of hepatic stellate cells and reducing collagen deposition, thereby playing a role in resisting hepatic fibrosis. Therefore, the dihydromyricetin (DHM) is applied to the development of the anti-hepatic fiber medicine on the basis of negative regulation of a TLR4 / TRAF6 / NF-kappa B pathway, and a new thought and basis are provided for the development of the anti-hepatic fiber medicine.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and in particular to the application of TLR4 / TRAF6 / NF-κB pathway inhibitors in the preparation of anti-hepatic fibrosis drugs. Background Art

[0002] Hepatic fibrosis is an irreversible intermediate link in the development of various chronic liver diseases (such as viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, etc.). Its basic pathological feature is the abnormal activation of hepatic stellate cells (HSCs) and the excessive deposition of extracellular matrix such as collagen fibers. If hepatic fibrosis cannot be effectively intervened, it will further develop into liver cirrhosis and even liver cancer. Currently, there is no internationally recognized specific anti-hepatic fibrosis drug, and the development of safe and effective treatment strategies has become a clinical problem to be solved urgently. Inflammatory response is closely related to the occurrence of hepatic fibrosis. The Toll-like receptor 4 (TLR4) signaling pathway is considered to play a key role in the initiation and progression of hepatic fibrosis. After TLR4 is activated, it can activate the downstream NF-κB pathway by recruiting TRAF6 (TNF receptor-associated factor 6), inducing the release of inflammatory cytokines (such as TNF-α, IL-6, TGF-β1), thereby promoting the activation of HSCs and the fibrosis process. Therefore, developing an intervention in the TLR4 / TRAF6 / NF-κB signaling pathway to achieve anti-fibrosis is a promising direction, but there are few or no publicly disclosed related technical solutions at present. Summary of the Invention

[0003] Therefore, based on the above background, the present invention provides the application of TLR4 / TRAF6 / NF-κB pathway inhibitors in the preparation of anti-hepatic fibrosis drugs, providing new ideas and basis for the development of anti-hepatic fibrosis drugs.

[0004] The technical solution provided by the present invention is as follows:

[0005] The application of TLR4 / TRAF6 / NF-κB pathway inhibitors in the preparation of anti-hepatic fibrosis drugs.

[0006] Further, the TLR4 / TRAF6 / NF-κB pathway inhibitor contains an effective amount of the active ingredient dihydromyricetin.

[0007] Further, the active ingredient dihydromyricetin is in monomer form or a plant extract containing dihydromyricetin.

[0008] Based on the same inventive concept, the present invention also provides:

[0009] Application of dihydromyricetin in preparing anti-hepatic fibrosis drug by negatively regulating TLR4 / TRAF6 / NF-κB pathway.

[0010] Furthermore, the drug contains an effective amount of the active ingredient dihydromyricetin.

[0011] Furthermore, the active ingredient dihydromyricetin is in monomer form or is a plant extract containing dihydromyricetin component.

[0012] The plant extract is from at least one of Ampelopsis grossedentata extract and Hovenia dulcis extract.

[0013] Furthermore, the drug is in oral dosage form.

[0014] Furthermore, the drug is any one of powder, tablet, pill, emulsion, dripping pill, capsule, suspension or tincture.

[0015] The beneficial effects achieved by the present invention are as follows:

[0016] The present invention realizes the development of anti-hepatic fibrosis drug for the first time based on negatively regulating TLR4 / TRAF6 / NF-κB pathway. In the hepatic fibrosis animal model, it can be seen that by down-regulating the expression of key molecules in TLR4-TRAF6-NF-κB signaling pathway, reducing the levels of pro-inflammatory factors (TNF-α, IL-6, TGF-β), alleviating the inflammatory reaction, inhibiting the activation of hepatic stellate cells and reducing collagen deposition, so as to play an anti-hepatic fibrosis role. In this regard, the present invention first applies dihydromyricetin (DHM) to the development of anti-hepatic fibrosis drug based on negatively regulating TLR4 / TRAF6 / NF-κB pathway, providing new ideas and basis for the development of anti-hepatic fibrosis drug.

[0017] Description of the drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments or the description of the prior art. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0019] Appendix Figure 1 Pathological morphology HE staining of liver tissue (100×): A: normal group; B: model group; C: dihydromyricetin 100 mg·kg -1 group (low dose group); D: dihydromyricetin 200 mg·kg -1 group (medium dose group); E: dihydromyricetin 400 mg·kg -1 group (high dose group).

[0020] Appendix Figure 2 Liver tissue pathological Masson staining (200×). A: Normal group; B: Model group; C: Dihydromyricetin 100 mg·kg -1 group; D: Dihydromyricetin 200 mg·kg -1 group; E: Dihydromyricetin 400 mg·kg -1 group.

[0021] Appendix Figure 3 Effect of dihydromyricetin on the contents of HA, Ⅳ-C, LN and PCⅢ in the serum of liver fibrosis rats (*P<0.05, **P<0.01 vs normal group; #P<0.05, ## P<0.01 vs model group).

[0022] Appendix Figure 4 Effect of dihydromyricetin on the gene expression of TNF-α, IL-6, TGF-β1 and NF-κB in the liver tissue of liver fibrosis rats (*P<0.05, **P<0.01 vs normal group; #P<0.05, ## P<0.01 vs model group).

[0023] Appendix Figure 5 Effect of dihydromyricetin on the protein expression of TLR-4, TRAF6 and NF-κB in the liver tissue of liver fibrosis rats (*P<0.05, **P<0.01 vs normal group; # P<0.05, ## P<0.01 vs model group). Specific embodiments

[0024] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0025] Example 1: In this example, a carbon tetrachloride-induced rat liver fibrosis model was constructed to investigate the protective effect of DHM on liver fibrosis.

[0026] The CAS number of dihydromyricetin used in this example is 27200-12-0, which was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0027] Construction of the model

[0028] Fifty SPF - level SD rats, 6 - 8 weeks old, weighing 200 ± 20 g, were provided by Hunan Slack Jingda Experimental Animal Co., Ltd. The experimental animal production license number is: SCXK(Xiang)2021 - 0002.

[0029] Experimental grouping: Fifty rats were randomly and evenly divided into 5 groups: blank group, model group, DHM 100 mg / kg group, DHM 200 mg / kg group, and DHM 400 mg / kg group.

[0030] Construction of the model: Except for the blank control group, other groups were intraperitoneally injected with 0.5% DMZ solution (1.6 ml / kg) three times a week. The modeling was completed at the end of 8 weeks. The control group was gavaged with an equal volume of normal saline. DHM was dissolved in normal saline (8 ml) and administered by gavage once a day for 6 consecutive weeks.

[0031] The serum and tissues of mice in each group were stained to observe the pathology.

[0032] The results are as follows:

[0033] (1) Through HE staining ( Figure 1 ) and Masson staining ( Figure 2 ), it was observed that the hepatocytes of normal rats were arranged evenly, with abundant cytoplasm, the hepatic lobule structure was complete, and there was no inflammatory infiltration in the portal area. The liver tissue of the model group was severely damaged. Under the microscope, the hepatic lobule structure was destroyed, the number of hepatocytes decreased, the hepatocytes in the lobule showed varying degrees of edema, obvious vacuoles were visible, and obvious fibrous deposition occurred. In each DHM treatment group, the degree of hepatocyte necrosis was significantly improved compared with the model group. The hepatic lobule structure was basically normal, the cell cords were arranged more neatly, the number of vacuoles and the area of fibrous deposition were significantly reduced, focal necrosis was occasionally seen, and the number of hepatocytes also gradually increased. Histopathological examination by HE staining and Masson staining indicated that DHM could effectively improve liver fibrosis in rats. By observing the hepatic tissue structure through HE and Masson staining, the hepatocytes in the DHM treatment group were arranged more regularly, the lobule structure was basically retained, and the fibrous deposition and necrotic foci were significantly reduced, showing significant improvement compared with the model group.

[0034] (2) ALT, AST, MDA, and SOD in the serum of liver fibrosis rats were detected, and the results are shown in Table 1.

[0035] Table 1: Effects of dihydromyricetin on serum ALT, AST, MDA, and SOD in liver fibrosis rats ( n = 10)

[0036]

[0037] *P < 0.05, **P < 0.01 vs normal group; # P < 0.05,## P < 0.01 vs the model group.

[0038] As can be seen from the data in Table 1, dihydromyricetin can reduce the contents of ALT, AST and MDA in the serum of rats with liver fibrosis, and increase the SOD activity, suggesting that DHM has a certain protective effect on the liver of rats with liver fibrosis, and has the effects of reducing transaminase and antioxidation. Compared with the model group, DHM (100, 200, 400 mg / kg) can significantly reduce the levels of ALT, AST and MDA in the serum of rats, suggesting its role in alleviating liver injury and oxidative stress; at the same time, the SOD activity is significantly increased, indicating its antioxidant potential and dose-dependence.

[0039] (3) Detect the contents of HA, Ⅳ-C, LN and PCⅢ in the serum of rats with liver fibrosis, and the results are shown in the appendix Figure 3 .

[0040] From Figure 3 it can be seen that the levels of HA, Ⅳ-C, LN and PCⅢ in the serum of rats in the liver fibrosis model group are significantly higher than those in the normal group, with significant differences, indicating that the liver fibrosis model is successfully established; the levels of HA, Ⅳ-C, LN and PCⅢ in the serum of rats in the DHM administration group are significantly lower than those in the model group, with significant differences, indicating that DHM has an obvious inhibitory effect on liver fibrosis and is positively correlated with the dose.

[0041] (4) Detect the gene expressions of TNF-α, IL-6, TGF-β1 and NF-κB in the liver tissues of rats with liver fibrosis, and the results are shown in the appendix Figure 4 .

[0042] From Figure 4 it can be seen that compared with the normal group, the expressions of TNF-α, IL-6, TGF-β1 and NF-κB in the model group are significantly increased (P < 0.01); compared with the model group, dihydromyricetin can significantly inhibit the expressions of TNF-α, IL-6, TGF-β1 and NF-κB in each administration group (P < 0.05, P < 0.01). Real-time PCR detection shows that DHM can dose-dependently inhibit the mRNA expressions of TNF-α, IL-6, TGF-β1 and NF-κB in liver tissues, with significant differences compared with the model group.

[0043] (5) Detect the protein expressions of TLR-4, TRAF6 and NF-κB in the liver tissues of rats with liver fibrosis, and the results are shown in Figure 5 . From Figure 5It can be seen that compared with the normal group, the protein expressions of TLR-4, TRAF6 and NF-κB in the model group were all significantly increased (P<0.01); compared with the model group, DHM could significantly inhibit the protein expressions of TLR4, TRAF6 and NF-κB in each administration group (P<0.05, P<0.01), indicating that the anti-hepatic fibrosis effect of DHM was related to the regulation of the expressions of these three proteins. The results of Western blot experiment showed that DHM could significantly inhibit the expressions of TLR4, TRAF6 and NF-κB proteins in liver tissues, and showed a downward trend with the increase of dose (P<0.05 or P<0.01).

[0044] The present invention first found that dihydromyricetin can achieve anti-hepatic fibrosis by inhibiting the TLR4 / TRAF6 / NF-κB pathway. Based on this, an anti-hepatic fibrosis drug that can negatively regulate the TLR4 / TRAF6 / NF-κB pathway can be developed.

[0045] The above description of the present invention and its implementation manners is not restrictive. What is shown in the embodiments is only one of the implementation manners of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural manners and embodiments without creative efforts without departing from the purpose of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. Application of TLR4 / TRAF6 / NF-κB pathway inhibitor in preparing anti-hepatic fibrosis drug.

2. The application according to claim 1, wherein The TLR4 / TRAF6 / NF-κB pathway inhibitor contains an effective amount of active ingredient dihydromyricetin.

3. The application according to claim 2, wherein The active ingredient dihydromyricetin is in monomer form or a plant extract containing dihydromyricetin component.

4. Application of dihydromyricetin in preparing anti-hepatic fibrosis drug by negatively regulating TLR4 / TRAF6 / NF-κB pathway.

5. The application according to claim 4, wherein The drug contains an effective amount of active ingredient dihydromyricetin.

6. The application according to claim 5, characterized in that The active ingredient dihydromyricetin is in monomer form or a plant extract containing dihydromyricetin component.

7. The application according to claim 5, characterized in that, The drug is in oral dosage form.

8. The application according to claim 7, wherein The drug is any one of powder, tablet, pill, emulsion, dripping pill, capsule, suspension or tincture.

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