Application of siCD36 delivered by lipid nanoparticles in relieving liver injury induced by ligation of bile duct

Abstract

The invention provides a novel cholestatic liver disease treatment method based on circadian rhythm. Researches find that in a biliary duct ligation (BDL)-induced liver injury model, the rhythmic expression of CD36 is enhanced and reaches a peak value at ZT16. SiCD36 is delivered by using lipid nanoparticles (LNP) at a ZT10 time point, and expression increase of CD36 can be effectively inhibited at ZT16, so that liver injury and inflammatory response caused by cholestasis are remarkably relieved. Serum and liver biochemical analysis shows that the strategy reduces the levels of alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), cholesterol and total bile acid, and improves liver lipid metabolism. The invention provides a new treatment thought and target for chronic cholestatic liver diseases such as PBC and PSC, and shows the potential application value of circadian rhythm in precision medicine.

Description

Technical Field

[0001] The present invention relates to the treatment of cholestatic liver disease, and in particular to a therapeutic method for alleviating cholestatic liver injury induced by bile duct ligation by delivering siCD36 at the circadian rhythm ZT10 time point using lipid nanoparticles. Background Art

[0002] Cholestatic liver diseases are characterized by the accumulation of toxic bile acids, which triggers a series of inflammatory responses and metabolic remodeling. Impairment of liver function and often progression to fibrosis, ultimately leading to liver failure. Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are the most common chronic cholestatic liver diseases, and their incidence and prevalence have been increasing in recent years. The pathogenesis of PSC and PBC is complex, and current treatments mainly intervene by altering the bile acid pool. However, two drugs, ursodeoxycholic acid (UDCA) and obeticholic acid (OCA), have limited efficacy in treating many patients and are associated with poor outcomes. Therefore, it remains necessary to explore the underlying mechanisms and develop innovative therapies to improve the prognosis of PBC and PSC.

[0003] Fatty acid transporter protein (CD36) is a multifunctional class B scavenger receptor that plays a key role in lipid homeostasis and immune response. Elevated levels of CD36 in the liver may lead to increased activities of key enzymes for bile acid synthesis such as cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1). In addition, CD36 modulates gallstone susceptibility in mice by altering bile lipid content and gallbladder contractility, thereby affecting bile acid metabolism and secretion. These studies suggest that CD36 may have multiple regulatory effects on bile acids. Our group previously found that CD36 is an important regulator of the mammalian circadian clock and is essential for the circadian coordination of the liver clock and glucose homeostasis. In subsequent screening, we observed that CD36 was associated with the circadian rhythm of bile acid metabolism. However, there is little research on the circadian regulation of CD36 in bile acid metabolism and its role in PBC and PSC. Summary of the invention

[0004] The present invention aims to provide a novel circadian rhythm-based treatment strategy for cholestatic liver disease to address the deficiencies in the prior art.

[0005] In order to achieve the purpose of the present invention, the present invention first provides the application of CD36 as a target for chronotherapy of cholestatic liver disease.

[0006] The inventors found that compared with the livers of mice treated with sham surgery, the rhythmic expression of CD36 in the mouse model of cholestatic liver injury induced by bile duct ligation was enhanced and reached a peak at ZT16.

[0007] This invention provides a new therapeutic strategy using lipid nanoparticles to deliver drugs at the circadian ZT10 time point siCD36 targeted to the liver of bile duct ligated mice inhibited the BDL-induced increase in CD36 levels at ZT16 and suppressed the pathological manifestations of cholestatic liver injury.

[0008] The beneficial effects of the present invention are:

[0009] The present invention found that CD36 reshapes the bile acid metabolism rhythm, and found that inhibiting CD36 during the peak period of the circadian rhythm can significantly restore the size of the bile acid pool, thereby alleviating cholestatic liver damage and inflammation. The present invention provides a new strategy and target for studying the treatment of cholestatic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The expression of CD36 at RNA and protein levels in the liver of mice in the sham operation (Sham) group and bile duct ligation (BDL) group at different time points; N = 4 (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0011] Figure 2 Flow chart of the mouse model injected with Lip-CTRL-siRNA and Lip-CD36-siRNA, gross images of the livers of the two groups of mice, as well as liver weight and liver weight / body weight ratio; N=7 (*, p<0.05; **, p<0.01; ***, p<0.001).

[0012] Figure 3 The expression of lipid content in the liver of mice in the Lip-CTRL-siRNA and Lip-CD36-siRNA groups; N=7 (*, p<0.05; **, p<0.01; ***, p<0.001).

[0013] Figure 4 The expression of serum related indexes in mice in Lip-CTRL-siRNA and Lip-CD36-siRNA groups; N=7 (*, p<0.05; **, p<0.01; ***, p<0.001).

[0014] Figure 5The expression of bile acid synthesis, regulation and secretion genes in the liver of mice in the Lip-CTRL-siRNA and Lip-CD36-siRNA groups; N=7 (*, p<0.05; **, p<0.01; ***, p<0.001).

[0015] Figure 6 Pathological sections of mice in the Lip-CTRL-siRNA and Lip-CD36-siRNA groups. Specific implementation methods

[0016] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0017] Example 1

[0018] 1. Materials and Methods

[0019] 1. Animal model and treatment: 10-week-old C57 / BL6J male mice were selected and treated with sham surgery (Sham) and bile duct ligation (BDL) for 1 week to construct a cholestatic liver disease mouse model and its control. Mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0020] 2. Real-time fluorescence quantitative PCR: Tissue RNA was extracted with Trizol (TAKARA, Japan) and then reverse transcribed and subjected to real-time fluorescence quantitative PCR.

[0021] 3. Western blot analysis: Liver tissue protein extracts were lysed in RIPA lysis buffer, separated by SDS-PAGE electrophoresis, and transferred to PVDF membranes. The membranes were incubated with primary antibodies overnight at 4°C. After three TBST washes (10 minutes each), the blots were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour, followed by three TBST washes (15 minutes each). Protein expression was detected using an ECL kit (Amersham Bioscience, USA).

[0022] 2. Results

[0023] The inventors selected 10-week-old C57 / BL6J male mice and performed sham surgery (Sham) and bile duct ligation. (BDL) was treated for 1 week to construct a cholestatic liver disease mouse model and its control. Real-time fluorescence quantitative PCR and protein immunoblotting results showed that the rhythmic expression of CD36 in the liver of BDL mice was stronger and reached a peak at ZT16. (Figure 1).

[0024] Example 2

[0025] 1. Materials and Methods

[0026] 1. Animal model and treatment: Ten-week-old male C57 / BL6J mice were treated with bile duct ligation (BDL) for 1 week. At ZT10 on day 7, lipid nanoparticles encapsulating CTRL-siRNA and CD36-siRNA were injected into the tail vein. The material was taken at ZT16.

[0027] 2. Liver and serum biochemical index detection: Alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bile acid (TBA) levels in serum were measured by using enzymatic kits (Nanjing Jiancheng, China). The concentration of 7α-hydroxy-4-cholesten-3-one (C4) was determined using an ELISA kit (Enzyme Science). In liver samples, total cholesterol (TC) (Nanjing Jiancheng), free cholesterol (FC) (Addison), non-esterified fatty acids (NEFA) (Nanjing Jiancheng), phospholipids (PL) (Addison) and TBA; cholesterol ester (CE) was quantitatively analyzed by ELISA kit (Enzyme Science).

[0028] 3. Real-time fluorescence quantitative PCR: Tissue RNA was extracted with Trizol (TAKARA, Japan) and then reverse transcribed and subjected to real-time fluorescence quantitative PCR.

[0029] 4. HE staining: After paraffin embedding, the tissue was sliced, dewaxed and hydrated, dripped with hematoxylin for 5 minutes, rinsed with running water, then dripped with eosin for 2 minutes, rinsed with running water, then dehydrated and sealed, and imaged with an automatic slice scanner for observation and analysis.

[0030] 5. Sirius red staining: After paraffin embedding, the tissue is sliced, dewaxed and hydrated, and iron hematoxylin staining solution is added for 5-10 minutes. The excess staining solution is washed with distilled water for 10-20 seconds, and then washed with tap water for 5 minutes. The Sirius red staining solution is then added for 15-30 minutes. The surface of the slice is rinsed with running water to remove the staining solution, and then the slice is dehydrated and sealed. The slice is imaged with an automatic slice scanner for observation and analysis.

[0031] 6. CK19 and F4 / 80 immunohistochemical detection: The tissue was embedded in paraffin and sliced, fixed with 4% paraformaldehyde, incubated with CK19 (cholangiocyte marker) and F4 / 80 (macrophage marker) antibodies at 4°C overnight, then combined with secondary antibodies, observed and photographed under a microscope, and the inflammatory cell infiltration and bile duct cell proliferation in the liver were analyzed.

[0032] 2. Results

[0033] Liver macroscopic images and liver weight / body weight ratio showed that the liver damage in the Lip-CD36-siRNA group mice was alleviated compared with that in the Lip-CTRL-siRNA group mice ( Figure 2 ). The results of liver biochemical index detection showed that compared with the mice in the Lip-CTRL-siRNA group, the total cholesterol (TC) and bile acid (BA) levels in the liver of the mice in the Lip-CD36-siRNA group were decreased, and the phospholipid (PL) and free fatty acid (FFA) levels were increased ( Figure 3 The results of serum biochemical indexes showed that compared with the mice in the Lip-CTRL-siRNA group, the serum alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), 7α-hydroxy-4-cholesten-3-one (C4) and total bile acid (TBA) levels of the mice in the Lip-CD36-siRNA group were significantly higher than those in the mice in the Lip-CTRL-siRNA group. The levels are reduced ( Figure 4 ). The results of real-time fluorescence quantitative PCR showed that compared with the mice in the Lip-CTRL-siRNA group, In the Lip-CD36-siRNA group, the mRNA level of Cyp7a1 in the liver was decreased, and the mRNA levels of SHP and FXR, the negative regulatory genes of Cyp7a1, were increased ( Figure 5 HE staining, picrosirius red staining, F4 / 80 and CK19 immunohistochemical staining results showed that compared with the mice in the Lip-CTRL-siRNA group, the liver injury, collagen deposition, inflammatory cell infiltration and bile duct cell proliferation in the mice in the Lip-CD36-siRNA group were alleviated ( Figure 6 ).

[0034] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of CD36 as a therapeutic target for cholestatic liver disease.

2. An application of a lipid nanoparticle delivery carrier, characterized in that: This vector is used to precisely deliver siCD36 to the liver and regulate the circadian expression of CD36.

3. The application according to claim 2, characterized in that: The use can restore the size of the bile acid pool, thereby alleviating cholestatic liver damage.

4. The application according to claim 2, characterized in that: The application can reduce collagen deposition, inflammatory response and bile duct cell proliferation caused by cholestatic liver disease.

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