A traditional Chinese medicine compound for treating fatty liver

Abstract

The present application relates to the field of traditional Chinese medicine compound, disclose a kind of traditional Chinese medicine compound for treating fatty liver, its raw material composition by mass ratio is: Gynostemma pentaphyllum 28~32, including 13~17, Cassia obtusifolia 11~14, turmeric 6~12, red yeast 7~11.The traditional Chinese medicine compound of the present application-Jianyu Xiaozhi recipe, with the effect of transporting spleen and soothing heart, resolving turbidity and removing blood stasis, the curative effect is remarkable, not only significantly reduce the body mass index and blood lipid level of NASH patient, also can improve liver damage and fibrosis degree.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine compound prescriptions, specifically to a traditional Chinese medicine compound prescription for treating fatty liver. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has become the most common chronic liver disease. Its spectrum includes metabolic-associated fatty liver, metabolic-associated steatohepatitis (MASH), and multiple metabolic-associated steatohepatitis. Studies show that approximately 25% of patients with metabolic-associated fatty liver will progress to MASH (formerly known as NASH), an inflammatory disease state of MAFLD associated with persistent hepatocellular damage. When MASH progresses to the stage of liver fibrosis, the condition often becomes irreversible, and the risk of cirrhosis and liver cancer increases significantly. Given the large number of patients with MASH, its serious health risks, and the strong clinical need for intervention, researching interventional drugs and therapeutic targets is a crucial issue urgently needing to be addressed in the life sciences. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a traditional Chinese medicine compound for treating fatty liver, aiming to solve the problem that existing prescriptions are difficult to cope with the large number of patients with MASH and difficult to intervene in and treat fatty liver.

[0004] The present invention solves the above-mentioned technical problems through the following technical means:

[0005] A traditional Chinese medicine compound for treating fatty liver, wherein the raw materials of the traditional Chinese medicine compound are composed of: Gynostemma pentaphyllum 28-32, Artemisia capillaris 13-17, Cassia tora 11-14, Curcuma longa 6-12, and red yeast rice 7-11 by mass ratio.

[0006] Furthermore, the raw materials of the traditional Chinese medicine compound are as follows by mass ratio: Gynostemma pentaphyllum 30, Artemisia capillaris 15, Cassia tora 12, Curcuma longa 10, and red yeast rice 12.

[0007] Furthermore, formulations suitable for oral administration are adopted.

[0008] Beneficial effects:

[0009] 1. Guided by the basic theories of Traditional Chinese Medicine and combined with the theory of "the liver governs qi", this invention summarizes the key pathogenesis of fatty liver treatment through long-term clinical practice, which is to grasp the spleen qi stagnation, phlegm disturbance of the heart, and qi stagnation and meridian obstruction. The invention is a compound Chinese medicine formula for treating fatty liver - Juanyu Xiaozhi Formula, which has the effects of invigorating the spleen and calming the heart, resolving turbidity and removing blood stasis. It has significant curative effect, and animal experiments have fully verified its effectiveness.

[0010] 2. The present invention has significant therapeutic effects, not only significantly reducing the body mass index and blood lipid levels of NASH patients, but also improving the degree of liver damage and fibrosis.

[0011] 3. This invention uses metabolomics to discover that UDP-N-acetylglucosamine (UDP-GlcNAc), a key metabolite in the hexosamine biosynthetic pathway (HBP), is significantly downregulated after intervention with Juanyu Xiaozhi Formula. Combined with preliminary experiments, Juanyu Xiaozhi Formula intervention can significantly downregulate the glycosylation modification level of O-linked β-N-acetylglucosamine (O-GlcNAc) mediated by the HBP pathway and the expression of the key enzyme OGT. Further glycosylation modification profiling revealed that O-GlcNAc modification of TGF-β-activated kinase 1 / MAP3K7-binding protein 2 (TAB2) in the MAPK pathway is significantly downregulated. Therefore, it can be understood that the specific molecular mechanism by which the present invention inhibits the O-GlcNAc modification of TAB2 protein mediated by the HBP pathway, thereby leading to the inactivation of the MAPK signaling pathway and thus inhibiting the progression of NASH, not only helps the clinical application and promotion of the formula and promotes the transformation of research results, but also provides a new pharmacological target with Chinese medicine characteristics for the treatment of NASH and provides new ideas for the design of innovative drugs.

[0012] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0013] Figure 1 The formula significantly inhibited liver damage in NASH mice.

[0014] Figure 2 The formula, called "Juanyu Xiaozhi Fang," was used to treat and inhibit lipid deposition and fibrosis in NASH mice.

[0015] Figure 3 A diagram showing the positive and negative ions in the original herbal extract of Juanyu Xiaozhi Fang.

[0016] Figure 4 To detect differential metabolites after intervention with Juanyu Xiaozhi Formula using non-targeted metabolomics;

[0017] Figure 5.1KEGG analysis of the effective active ingredients in Juanyu Xiaozhi Formula;

[0018] Figure 5.2 This is a compound-target-signaling pathway network diagram;

[0019] Figure 5.3 This is a schematic diagram of molecular docking between the active compounds quercetin (left, binding energy -8.5) or curcumin (right, binding energy -8.45) and OGT.

[0020] Figure 6 The intervention of the Juanyu Xiaozhi formula significantly downregulated O-GlcNAc modification in the liver tissue of NASH mice;

[0021] Figure 7 Serum intervention with the drug-containing formula significantly inhibited lipid deposition in adipocytes;

[0022] Figure 8 Serum treatment with the drug-containing formula significantly inhibited lipid deposition in adipocytes and downregulated O-GlcNAc modification.

[0023] Figure 9 The results of O-GlcNAc modification analysis after intervention with Juanyu Xiaozhi Fang;

[0024] Figure 10 Enrichment of differentially modified peptides and proteins after intervention with Juanyu Xiaozhi Fang;

[0025] Figure 11 After intervention with the Juanyu Xiaozhi formula, the O-GlcNAc modification of TAB2 and the MAPK pathway were significantly downregulated. Detailed Implementation

[0026] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can understand the advantages and effects of the present invention from the content disclosed in this specification. It should be noted that the illustrations provided in the following embodiments are for illustrative purposes only and represent schematic diagrams, not actual pictures, and should not be construed as limiting the present invention. In order to better illustrate the embodiments of the present invention, some components in the figures may be omitted, enlarged, or reduced, and do not represent the actual product size; it is understandable for those skilled in the art that some well-known structures and their descriptions may be omitted in the figures.

[0027] In the figures of this invention, the same or similar reference numerals correspond to the same or similar components. In the description of this invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the figure, they are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the figures are only for illustrative purposes and should not be construed as limiting this invention. For those skilled in the art, the specific meaning of the above-mentioned terms can be understood according to the specific circumstances.

[0028] This embodiment discloses a traditional Chinese medicine compound for treating fatty liver, the raw materials of which are: 30g of Gynostemma pentaphyllum, 15g of Artemisia capillaris, 12g of Cassia tora, 10g of Curcuma longa, and 12g of red yeast rice.

[0029] Experimental Analysis

[0030] (1) The formula for reducing lipid deposition and fibrosis in NASH mice can significantly reduce lipid deposition and fibrosis and improve liver function.

[0031] Preparation of decoction: The dosage of Juanyu Xiaozhi Fang was calculated according to the equivalent dose ratio of human and rat based on body surface area. Soak in 20 times the amount of water for 30 minutes, boil, filter to obtain filtrate, add 15 times the amount of water and boil for 30 minutes. Repeat the above steps, filter and combine the three filtrates.

[0032] Low-dose Qianyu Xiaozhi Formula: The drug content in the liquid is 5g / kg;

[0033] Medium-dose Qianyu Xiaozhi Formula: The drug content in the liquid is 10g / kg;

[0034] High-dose Qianyu Xiaozhi Formula: The drug content in the liquid is 20g / kg;

[0035] In this in vivo study, NASH mice fed a high-fat, high-sugar diet were administered low, medium, and high doses of the purpura-zofylline-reducing formula (JYXZF) by gavage. The negative control group was administered physiological saline by gavage, and the positive control group was administered atorvastatin by gavage. Figure 1 As shown, after 12 weeks of treatment, tests revealed that compared with the negative control group, the medium- and high-dose lipid-lowering formula gavage groups showed significant weight reduction, decreased liver specific gravity, and significantly lower serum levels of ALT, AST, FFA, TG, and TC, indicating reduced liver damage and improved lipid metabolism disorders (see Table 1). Furthermore, HE staining, Oil Red O staining, F4 / 80 staining, and Sirius Red staining experiments confirmed that the lipid-lowering formula significantly inhibited lipid accumulation, inflammatory response, and fibrosis in the liver of NASH mice. Figure 2 As shown, the scale bar is 100 μm.

[0036] Figure 1 In the table, A is a schematic diagram of the NASH diet-fed mouse model; B is the mouse body weight curve; C is the mouse food intake; DF is the mouse body weight, liver weight, and liver weight-to-body weight ratio; GH is the serum ALT and AST levels; IJ is the blood glucose level measured in the ITT and GTT experiments (NCD normal control group, HFD high-fat group, JYXZF high-fat + Juanyuxiaozhifang intervention group, ATO high-fat + atorvastatin intervention group, n=6, *P<0.05, **P<0.01, ***P<0.001).

[0037] Table 1. Treatment of NASH mice with the "Juanyu Xiaozhi Formula" to inhibit lipid deposition.

[0038]

[0039] Serum TG / TC / FFA / Blood Glucose levels (NCD normal control group, HFD high-fat group, JYXZF high-fat + Juanyu Xiaozhi Fang intervention group, ATO high-fat + atorvastatin intervention group, n=6, *P<0.05, **P<0.01, ***P<0.001, ####P<0.001).

[0040] (2) Identification of the components of Juanyu Xiaozhi Formula.

[0041] Seven days after rats were administered the fat-reducing formula via gavage, blood was collected to obtain serum containing the formula. Chromatographic analysis revealed 2831 components from the original herbal extract and 591 components from the serum containing the formula. The positive and negative ion diagrams are shown below. Figure 3 As shown.

[0042] (3) Metabolomics combined with network pharmacology screening and prediction of the HBP pathway is a key target of Juanyu Xiaozhi Formula.

[0043] Non-targeted metabolomics analysis was performed on serum from NASH patients who took and did not take Juanyu Xiaozhi Fang, such as... Figure 4 As shown, the results indicated that compared to the untreated group, the levels of HBP pathway metabolites F6P, GlcNAc-1-P, and UDP-GlcNAc (the final product of HBP, a substrate modified with O-GlcNAc) were significantly reduced in the treated group. Figure 4In the diagram, NASH represents the group that did not take the fat-reducing formula, XZF represents the group that took the fat-reducing formula, and Fructose 6-phosphate (F6P), Acetyglucosamine 1-phosphate (GlcNAc-1-P), and UDP-N-acetylglucosamine (UDP-GlcNAc). Simultaneously, this embodiment uses network pharmacology to predict the target of the Juanyu fat-reducing formula. KEGG analysis showed significant enrichment of the glucose metabolism pathway, and the compound-target-signal pathway network diagram also showed that the main components of the Juanyu fat-reducing formula may target the key enzyme OGT (O-GlcNAc modification) downstream of the HBP pathway. Figures 5.1–5.3 Molecular docking also showed that the active ingredients quercetin and curcumin in the compound had high binding energies to OGT. These results suggest that HBP pathway-mediated O-GlcNAc modification and its key enzyme OGT may be key targets of Juanyu Xiaozhi Formula.

[0044] (4) The Juanyu Xiaozhi formula significantly downregulated O-GlcNAc modification and key enzyme expression in NASH mice.

[0045] To clarify the changes in O-GlcNAc modification in NASH mice after treatment with the fat-reducing formula, this example examined the liver tissue of mice after initial gavage. Immunohistochemical staining and Western blot results showed that, compared with the control group, the O-GlcNAc modification level in the liver tissue of NASH mice decreased in a dose-dependent manner with increasing concentration of the fat-reducing formula administered via gavage. Figure 6 AB). Furthermore, target metabolomics analysis revealed a significant decrease in the level of the key metabolite UDP-GlcNAc, which is modified by O-GlcNAc. Figure 6 C). Further examination of key enzyme levels revealed that after intervention with the fat-reducing formula, the levels of O-GlcNAc modification key enzyme OGT protein and mRNA were downregulated. It is speculated that the fat-reducing formula may regulate HBP pathway-mediated protein O-GlcNAc modification by downregulating the expression of the metabolite UDP-GlcNAc and the key enzyme OGT. Figure 6 DE).

[0046] Figure 6 In the table, A represents immunohistochemical staining of O-GlcNAc modification, scale bar = 100 μm; B represents the O-GlcNAc modification level in liver tissue of NASH mice; C represents the level of metabolite UDP-GlcNAc; D represents the protein levels of key enzymes OGA, OGT, and GFPT1; E represents the mRNA levels of key enzymes OGA, OGT, and GFPT1 (NCD normal control group, HFD high-fat group, JYXZF high-fat + Juanyuxiaozhifang intervention group, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001).

[0047] (5) In vitro treatment of serum containing the drug Juanyu Xiaozhi Formula significantly inhibited lipid deposition in adipocytes and downregulated O-GlcNAc modification.

[0048] To verify the efficacy of Juanyu Xiaozhi Formula in vitro, this embodiment used serum containing Juanyu Xiaozhi Formula to treat FFA-induced normal human hepatocytes (MIHA), with DON, a key enzyme in the HBP pathway GPFT1, as a positive control. First, serum treatment with different concentrations of the drug was performed to screen for the optimal concentration of 10% (…). Figure 7 A). Oil Red O staining then revealed that treatment with drug-containing serum significantly reduced FFA-induced lipid accumulation in MIHA cells, and the levels of TG and TC in the supernatant also significantly decreased. Figure 7 (BC) suggests that the serum containing the drug Juanyu Xiaozhifang can significantly inhibit lipid deposition in adipocytes.

[0049] Figure 7 In the study, A represents FFA-induced MIHA cells treated with serum containing different concentrations of Juanyu Xiaozhi Fang, and cell viability was detected by CCK8 assay; BC represents cells treated with serum containing the optimal concentration of Juanyu Xiaozhi Fang, and the lipid accumulation was detected by Oil Red O staining (B), and the TG and TC levels in the cell supernatant were detected by ELISA (C). Among them, BSA is the normal control group, FFA is the adipogenic group, JYXZF adipogenic group + Juanyu Xiaozhi Fang serum intervention group, and DON adipogenic group + DON intervention group, n=3, *P<0.05, **P<0.01, ***P<0.001.

[0050] Next, Western blot results showed that, compared with the control group, the O-GlcNAc modification level in cells of the JYXZF group and the DON group was significantly decreased. Figure 8 A), target metabolism assays also revealed a significant decrease in the level of UDP-GlcNAc, a key metabolite modified by O-GlcNAc. Figure 8 B). Furthermore, this embodiment examined changes in key enzyme levels and found results consistent with in vivo animal experiments: after intervention with the fat-reducing formula, the expression level of the key enzyme OGT, which modifies O-GlcNAc, was downregulated, further confirming our hypothesis: the fat-reducing formula regulates HBP pathway-mediated protein O-GlcNAc modification by regulating and downregulating the expression of the metabolite UDP-GlcNAc and the key enzyme OGT. Figure 8 CD).

[0051] Figure 8In the table, A represents the total O-GlcNAc modification level in cells; B represents the UDP-GlcNAc metabolite level; C represents the protein levels of key enzymes OGA, OGT, GFPT1, and p-GFPT1; and D represents the mRNA levels of key enzymes OGA, OGT, and GFPT1 (BSA normal control group, FFAs adipogenic group, JYXZF adipogenic group + serum intervention with Juanyu Xiaozhi formula, DON adipogenic group + DON intervention group, n=3, *P<0.05, **P<0.01, ***P<0.001).

[0052] (6) Glycosylation modification proteomics depicts the differential modification profile of NASH mice after intervention with the gluten-reducing fat-reducing formula.

[0053] To further characterize the differential modification profile after intervention with the fat-reducing formula, this embodiment performed label-free O-GlcNAc modification proteomics analysis on NASH mouse liver tissue after fat-reducing formula intervention. Figure 9 A). In 6 samples, we detected a total of 488 unique peptides in 416 proteins, including 1236 O-GlcNAc modified protein sites, of which 299 modified proteins had 569 quantifiable O-GlcNAc peptides. Figure 9 B). Next, the Venn diagram showed 342 overlapping modified peptides between the two groups, indicating good overlap. Figure 9 C). Figure 9 In the diagram, A is a flowchart of proteomics enrichment and identification of O-GlcNAc modified peptides; B is a bar chart of the identification and quantification results of all modified peptides; C is a Venn diagram of O-GlcNAc peptides identified between groups (A: saline gavage group, B: Juanyu Xiaozhi formula gavage group).

[0054] To analyze the differentially expressed O-GlcNAc peptides among different groups, we quantified the experimental results. The results showed that, compared with the control group, the lipid-lowering intervention group had 51 peptides with downregulated modification levels. Figure 10 AB). Furthermore, we performed KEGG analysis on the proteins containing the downregulated differentially modified peptides. The results showed that the MAPK, NF-κB, and HIF-1 pathways were significantly enriched. These pathways are all related to lipid regulation, suggesting that the jujube fat-reducing formula does indeed inhibit NASH progression by affecting protein O-GlcNAc modification. Figure 10 C). Figure 10In the table, A is a bar chart showing the quantitative differences in O-GlcNAc peptides between groups; B is a volcano plot of differentially modified O-GlcNAc peptides between groups, with significantly downregulated modified peptides marked in blue, significantly upregulated modified peptides marked in red, and no difference in modified peptides marked in gray, FC≤0.67 or FC≥1.5, P<0.05; C is a KEGG analysis of the proteins to which the differentially expressed peptides belong, P<0.05 (A: saline gavage group, B: Juanyu Xiaozhi formula gavage group).

[0055] (7) O-GlcNAc modification of TAB2 and its downstream MAPK pathway were significantly downregulated after intervention with the Trichomonas lipid-lowering formula.

[0056] This embodiment selected the differentially modified protein TAB2 of the MAPK pathway for further study. We first used a Co-IP experiment to confirm the interaction between TAB2 and the O-GlcNAc-modified OGT key enzyme. Then, an sWGA affinity experiment confirmed that TAB2 is modified with O-GlcNAc, and that its modification level significantly decreased after intervention with the suanyuxiaozhi formula. Figure 11 TAB2 is a binding protein of the MAPK pathway kinase TAK1. Therefore, we examined the MAPK pathway activity in the liver tissue of NASH mice treated with Juanyu Xiaozhi Fang. The results showed that compared with the saline gavage group, the high-fat group had significantly upregulated p-TAK1, p-p38, and p-65 (NF-κB), while these indicators were significantly decreased after Juanyu Xiaozhi Fang intervention. Figure 11 C), while inflammatory factors such as TNF-α and IL-6 were also significantly downregulated after treatment. Figure 11 D) suggests that the activity of TAK1-mediated MAPK and NF-κB decreased significantly after intervention with Juanyu Xiaozhi Fang.

[0057] Figure 11 In the table, A represents the Co-IP experiment of TAB2 and OGT; B represents the sWGA affinity experiment of TAB2, and anti-TAB2 detection of the O-GlcNAc modification level of TAB2 protein; C represents the protein levels of MAPK and NF-κB pathway related indicators; D represents the ELISA detection of TNF-α and IL-6 levels in the liver of NASH mice (NCD normal control group, HFD high-fat group, JYXZF high-fat + Juanyuxiaozhifang intervention group, n=6, *P<0.05, **P<0.01, ***P<0.001).

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A traditional Chinese medicine compound for treating metabolic-associated steatohepatitis, characterized in that, The raw materials of the traditional Chinese medicine compound are as follows by mass ratio: Gynostemma pentaphyllum 30, Artemisia capillaris 15, Cassia tora 12, Curcuma longa 10, and red yeast rice 12.

2. The traditional Chinese medicine compound for treating metabolic-associated steatohepatitis according to claim 1, characterized in that: The traditional Chinese medicine compound is an oral preparation.

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