CN116327749A

CN116327749A - Application of GW9662 in preparing medicament for treating nonalcoholic steatohepatitis

Info

Publication number: CN116327749A
Application number: CN202310434432.2A
Authority: CN
Inventors: 肖靖; 彭明利
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2023-04-21
Filing date: 2023-04-21
Publication date: 2023-06-27

Abstract

The invention relates to application of GW9662 in preparing a medicine for treating nonalcoholic steatohepatitis, wherein GW9662 can inhibit PPARgamma/CD 36 channel, down regulate the expression of SCD1, FABP1, FASN, PDGFR alpha, alpha-SMA and Col1a1, improve liver lipid metabolism, inhibit fatty acid transport and synthesis, promote oxidation of fatty acid to relieve choline deficiency, and induce disease progression of nonalcoholic steatohepatitis mice by amino acid-defined high fat diet (CDAA-HFD). Providing a new therapeutic strategy for NASH.

Description

Application of GW9662 in preparing medicament for treating nonalcoholic steatohepatitis

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of GW9662 in preparing a medicine for treating nonalcoholic steatohepatitis (NASH).

Background

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide, affecting at least 1/4 of the population worldwide. Nonalcoholic steatohepatitis (NASH) is an inflammatory subtype of NAFLD with steatosis, hepatocyte damage (balloon-like) and inflammation, with or without fibrosis. Although NASH is generally clinically asymptomatic, over time, about 20% of patients with NASH have NASH, of which about 20% develop cirrhosis, resulting in liver cancer and even requiring liver transplantation. NASH is closely associated with obesity, dyslipidemia, type 2 diabetes and metabolic syndrome, and is more prevalent in metabolic disease and obese patients. The main strategies for treatment focus on controlling dietary intake and increasing physical activity to reduce body weight, but generally patient compliance is poor, difficult to adhere to, and recurrence rate is high. Although research on NAFLD pathogenesis, therapeutic targets and drug development has been steadily advanced in recent years, development of NAFLD therapeutic drugs is basically in preclinical or clinical trial stages, and has not been approved for NAFLD/NASH treatment.

Current studies have found that GW9662 improves insulin resistance, reduces the expression of toll-like receptor 4 (tlir 4), bone marrow differentiation primary response 88 (Myd 88) and interleukin 1 beta (IL-1 beta) and nitrite (NO 2-) concentration in NAFLD mouse models constructed with fat, fructose and cholesterol rich diets (FFC).

In the research on the mechanism of improving NASH by GW9662, the applicant found that GW9662 can improve liver lipid metabolism, inhibit fatty acid transport and synthesis, promote fatty acid oxidation, alleviate liver fibrosis by inhibiting pparγ/CD36 pathway, down-regulating SCD1, FABP1, FASN, pdgfrα, α -SMA, col1a1 and the like, and relieve progression of NASH disease induced by CDAA-HFD diet. The result is not reported at home and abroad at present.

Disclosure of Invention

Based on this, the invention provides a new use of GW9662 in treating liver diseases. GW9662 can effectively improve liver lipid metabolism, inhibit fatty acid transport and synthesis, promote fatty acid oxidation, and improve non-alcoholic steatohepatitis disease progression. On the premise of lacking specific NASH therapeutic drugs at present, the application prospect of GW9662 is good. The invention is realized by the following technical scheme:

the invention discloses application of GW9662 in preparation of a medicament for treating nonalcoholic steatohepatitis.

The invention also discloses a medicine for treating nonalcoholic steatohepatitis, which comprises GW9662 and pharmaceutically acceptable carriers, additives or/and excipients.

Further, the GW9662 down regulates SCD1 expression in CDAA-HFD mouse liver tissue by inhibiting pparγ/CD36 pathway.

Further, the GW9662 down regulates FABP1 expression in CDAA-HFD mouse liver tissue by inhibiting pparγ/CD36 pathway.

Further, the GW9662 down regulates expression of FASN in CDAA-HFD mouse liver tissue by inhibiting pparγ/CD36 pathway.

Further, the GW9662 down regulates pdgfrα expression in CDAA-HFD mouse liver tissue by inhibiting pparγ/CD36 pathway.

Further, the GW9662 down regulates the expression of alpha-SMA in the liver tissue of CDAA-HFD mice by inhibiting PPARgamma/CD 36 pathway.

Further, the GW9662 down-regulates Col1a1 expression in CDAA-HFD mouse liver tissue by inhibiting PPARgamma/CD 36 pathway.

Further, the GW9662 inhibits fatty acid transport and synthesis.

Further, the GW9662 promotes oxidation of fatty acids.

The invention has the beneficial effects that:

the applicant shows that the medicine (compound GW 9662) can inhibit fatty acid transportation and synthesis and promote fatty acid oxidation through the treatment experiment of GW9662 on the liver of a non-alcoholic steatohepatitis mouse. Therefore, the medicament can improve liver lipid metabolism, inhibit fatty acid transport and synthesis, promote fatty acid oxidation and alleviate liver fibrosis by inhibiting PPARgamma/CD 36 pathway, downregulate SCD1, FABP1, FASN, PDGFR alpha, alpha-SMA, col1a1 and the like, and relieve CDAA-HFD diet-induced NASH disease progression. Providing a new therapeutic strategy for NASH.

Drawings

FIG. 1 is a technical roadmap of the invention;

FIG. 2 is a schematic diagram of the CDAA-HFD modeling and GW9662 process time axis according to the present invention;

fig. 3A is an effect of GW9662 on body weight;

fig. 3B is an effect of GW9662 on liver weight;

FIG. 3C is the effect of GW9662 on serum aminotransferase ALT and AST;

FIG. 4A is a liver pathology stain;

FIG. 4B is a liver pathology staining analysis;

FIG. 5A is a graph showing the effect of GW9662 on hepatic lipid synthesis and transport-related mRNA expression;

FIG. 5B is a graph showing the effect of GW9662 on hepatic lipid oxidation-related mRNA expression;

FIG. 5C is the effect of GW9662 on liver inflammation-associated mRNA expression;

FIG. 5D is the effect of GW9662 on liver fibrosis-associated mRNA expression.

Detailed Description

The following examples are further illustrative of the invention and are not intended to be limiting thereof.

Example 1

The experimental GW9662 of this embodiment was purchased from MCE corporation;

c57BL/6N mice were purchased from Beijing vitamin Torilhua;

ALT, AST, FFA, TG, HYP detection kit is purchased from Nanjing build company;

primer design and synthesis are completed by the Optimago company;

PCR experiment-related reagents were purchased from TAKARA company;

other reagents used in the experiments of this example are commercially available analytically pure reagents.

Technical roadmap (figure 1)

The invention constructs NASH models by feeding C57BL/6N mice with a choline deficient, amino acid defined high fat diet (CDAA-HFD) for 12 weeks and administers GW9662 treatment. After the modeling is finished, the materials are taken to measure the weight, liver weight, serum aminotransferase ALT, AST and the like, pathological section staining is carried out on the liver of the mouse, and the mRNA expression level in the liver tissue of the mouse is measured.

Animal treatment (figure 2)

30C 57BL/6N male mice aged 7 weeks in IVC room were acclimatized for one week and transferred to SPF room, after which the 30 mice were randomly divided into a normal diet group (Control group), a CDAA-HFD high fat diet group (CDAA-HFD group) and GW9662 group, 10 each. The normal diet group was fed with mice maintenance diet daily, the high fat diet group and the GW9662 group were fed with CDAA-HFD diet; the normal control group and the high fat diet group were intraperitoneally injected with a volume of 0.9% physiological saline based on body weight from week 5, and the GW9662 group was intraperitoneally injected with 1mg/kg body weight GW9662 daily from week 5. The administration time was continued for 8 weeks from week 5 to week 12.

Experimental results

(1) Liver function change of NASH mouse (figure 3)

After animal treatment, animals were sacrificed and livers were weighed and serum transaminase levels were determined using an ALT, AST detection kit.

The results are shown in figure 3A, with CDAA-HFD fed mice significantly reduced body weight compared to normal diet after 8 weeks of intervention, while GW9662 treatment had no significant effect on mouse body weight. Liver weight was significantly increased (p < 0.05) in high fat diet fed mice compared to normal diet mice, whereas liver weight was not significantly altered after GW9662 treatment (fig. 3B). ALT was increased approximately 4.8-fold and AST was increased approximately 3-fold in CDAA-HFD group mice, suggesting that feeding CDAA-HFD mice resulted in significant liver dysfunction. Liver function was significantly improved in NASH mice after 8 weeks of GW9662 intervention, with 50.46% and 34.73% decrease in ALT and AST, respectively, in GW9662 mice compared to CDAA-HFD mice (fig. 3C).

(2) NASH mice became fat and fibrotic (fig. 4)

After the experiment is finished, the mice are sacrificed to fix the livers, paraffin is embedded, sections are subjected to pathological staining, and the change of liver histology among groups is observed under a lens.

As shown in fig. 4, after HE and oil red O staining, the liver tissue of the control group mice showed a complete lobular structure, the cytoplasm was uniform, and no hepatocyte degeneration was present; compared with normal diet mice, the liver of CDAA-HFD fed mice can be densely distributed with a large number of steatosis liver cells, and the mice are in moderate steatosis (steatosis degree classification: 0-33% is mild, 33% -66% is moderate, and more than 66% is severe), the liver cells contain a large number of round vacuoles with different sizes, and the nuclei are extruded to one side by liver cell swelling. Compared with the high-fat diet group, the cavitation bubbles in the liver of GW9662 group are obviously reduced, the liver is slightly fat-changed, and only a small amount of small cavitation bubbles of fat drops exist. SR staining section shows that the Control group observes clear liver lobule structure and normal liver cell morphology, and the portal area hardly sees connective tissue; liver cable structural disorder of CDAA-HFD group, dou Zhouda amount of fibrous tissue hyperplasia, collagen deposition, occasional visible bridging fibrosis, fibrosis graded on the F2/3 scale (fibrosis graded: F0, no obvious fibrosis; F1, liver Dou Huomen perivenous fibrosis; F2 hepatic sinus and portal vein or manifold area fibrosis; F3 bridging fibrosis; F4, cirrhosis); liver fibrosis after GW9662 treatment was significantly reduced, collagen deposition was reduced, and fibrosis grading was dominated by F1. The oil red O staining area of GW9662 group was reduced by 68.81% compared to CDAA-HFD group.

The TG, FFA and HYP contents in the liver of mice are determined by using a TG, FFA and HYP detection kit.

FFA and TG levels were significantly increased in the CDAA-HFD group compared to the normal diet group (P < 0.01), and both FFA and TG levels were significantly decreased in the liver of mice after GW9662 treatment (P < 0.05, P < 0.01), liver lipid accumulation was significantly inhibited, and the GW9662 treatment significantly reduced steatosis in NASH mice (fig. 3B). Collagen Proportional Area (CPA) analysis was performed on pathological sections of mouse liver, and after CDAA-HFD diet fed mice, the liver collagen proportional area was significantly increased by 12.2-fold, while after GW9662 treatment, the liver collagen proportional area was significantly decreased by 49.95%. The change of Hydroxyproline (HYP) content in the liver of mice is consistent with the pathological section of the liver, the CDAA-HFD diet causes the increase of liver fibrosis degree, the HYP content is obviously increased by 2.38 times, and the HYP content is reduced by 20.22 percent after GW9662 treatment. We assessed the extent of lesions of steatohepatitis in mice by NAFLD Activity Score (NAS). As shown in FIG. 4, the NAS was increased to 5.50.+ -. 0.88 in the CDAA-HFD group of mice and decreased by 24.91% after GW9662 treatment compared to the Control group of mice.

(3) Potential mechanism of NASH mice (fig. 5)

The regulation of NASH mouse lipid metabolism and fibrosis related genes by GW9662 was detected by extracting mRNA from mouse liver tissue, and performing quantitative polymerase chain reaction (qPCR) experiments.

qPCR results are shown in fig. 5, and compared with Control group, mRNA expression levels of CDAA-HFD group liver pparγ, CD36, FABP1, LPL, FASN are significantly increased, indicating that high-fat diet promotes liver lipid transport and increase of synthetic gene expression, and pparγ, CD36, LPL, FASN is significantly decreased relative to CDAA-HFD group after GW9662 dry state (fig. 5A); whereas the level of the fatty acid oxidation-related gene pparα was reduced in the CDAA-HFD group and the liver pparα expression level was restored to normal level after GW9662 treatment, high-fat diet feeding down-regulated the expression of ACOX1, and GW9662 treatment had no effect on it (fig. 5B). Secondly, the expression levels of TNF-alpha, NF-kappa B and F4/80 in the CDAA-HFD group are obviously up-regulated compared with the normal group; the change in GW9662 group had no statistical significance compared to the high fat diet group, and the expression levels of IL-1 and IL-6 were not significantly changed in all three groups (FIG. 5C). At the gene level, the genes pdgfrα, α -SMA and Col1a1 suggesting the degree of liver fibrosis were significantly up-regulated in the high-fat diet group, with GW9662 treatment significantly down-regulating the mouse liver fibrosis gene (fig. 5D).

It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and the equivalents or alternatives made on the basis of the above description are all included in the scope of the present invention.

Claims

Use of gw9662 in the manufacture of a medicament for the treatment of non-alcoholic steatohepatitis.
2. A medicament for treating non-alcoholic steatohepatitis, wherein:

the medicament comprises GW9662 and pharmaceutically acceptable carriers, additives or/and excipients.
3. The use according to claim 1, wherein:

the GW9662 down-regulates the expression of SCD1, FABP1, FASN, PDGFR alpha, alpha-SMA and Col1a1 in liver tissue of CDAA-HFD mice by inhibiting PPARgamma/CD 36 pathway.
4. The use according to claim 1, wherein:

the GW9662 inhibits fatty acid transport and synthesis.
5. The use according to claim 1, wherein:

the GW9662 promotes oxidation of fatty acids.