New application of 25-hydroxycholesterol in treatment of rifampicin-induced liver lipid deposition

Abstract

The invention provides a new application of a natural metabolite 25-hydroxycholesterol (25-hydroxycholesterol (25 HC for short) in a body to treatment of liver lipid deposition induced by rifampicin (RFP for short), and particularly provides a new application of the natural metabolite 25-hydroxycholesterol (25 HC for short) to treatment of liver lipid deposition induced by rifampicin (RFP for short). The < 25 > HC is a metabolite generated by catalyzing cholesterol by using in-vivo cholesterol 25-hydroxylase (CH25H), and the < 25 > HC is a metabolite generated by catalyzing cholesterol by using the in-vivo cholesterol 25-hydroxylase (CH25H). The compound is reported to have the biological activities of resisting viruses, activating interferon, resisting tumors, participating in lipid metabolism and the like in the past. The invention finds that RFP can induce liver fatty degeneration and lipid droplet deposition and reduce CH25H expression, so that the lack of 25HC may be one of the mechanisms of RFP-induced liver lipid deposition. The invention firstly discovers that the supplement of 25HC can relieve RFP-induced lipid droplet deposition (both triglyceride and cholesterol are reduced), and particularly firstly discovers that the mechanism of reducing the content of cholesterol in the liver by 25HC is related to promotion of expression and entry of LXR alpha into cells and promotion of expression of cholesterol efflux genes ABCA1, 2 and 3. Therefore, the 25HC possibly reduces lipid deposition in the liver by promoting cholesterol excretion of liver cells and relieves the RFP-induced liver injury, and a foundation is laid for future clinical application of the 25HC.

Description

Technical Field

[0001] The present invention relates to a new pharmaceutical use of 25-hydroxycholesterol (25HC for short). It is found that the chemical composition (oral administration) of 25HC has a strong protective effect against rifampicin-induced hepatic lipid deposition, which helps to expand its indications and provide a new option for liver protection treatment of anti-tuberculosis drugs. Background Art

[0002] Drug-induced hepatic steatosis (DIHS) is one of the characteristics of a few clinical drug-induced liver injuries. The main causes in China are hormone drugs (glucocorticoids, tamoxifen), anti-tuberculosis drugs (such as rifampicin), antibiotics (tetracycline), anti-epileptic drugs (valproic acid), anti-arrhythmic drugs (amiodarone), and anti-tumor drugs (methotrexate). Among them, rifampicin (RFP) is one of the common causes of DIHS. Rifampicin (RFP) is a first-line anti-tuberculosis drug recommended by the World Health Organization. At present, the traditional treatment of DIHS is not ideal. Traditional liver protection drugs such as reduced glutathione, diammonium glycyrrhizinate, and glucuronolactone are only symptomatic treatments, which play an antioxidant and anti-inflammatory role, and are not precise targets for rifampicin-induced fatty liver. The reason is that the mechanism or pathogenic target is not yet clear, and precision medicine cannot be achieved. Fatty liver caused by rifampicin has significant characteristics, with early or short-term liver lipid deposition, no elevated transaminase and inflammatory response. Long-term exposure can lead to hepatocyte rupture, hepatic cholestasis, and elevated serum transaminases. However, there is currently no specific drug for the treatment of DIHS caused by RFP. How to intervene early and protect against rifampicin-induced drug-induced fatty liver is an urgent task in basic research.

[0003] 25-hydroxycholesterol (25HC) is a metabolite of cholesterol produced by 25-hydroxylase (Cholesterol-25-Hydroxylase, CH25H). As a metabolic enzyme in the endoplasmic reticulum of cells, the latter uses oxygen and NADPH to add a hydroxyl group to the 25-carbon atom of cholesterol to form active 25HC. 25HC participates in biological effects such as antiviral, interferon activation, anti-tumor, and cholesterol synthesis regulation by binding to various receptors (LXRα, SCAP / Insig-2, GPR155, ACAT). 25HC is easily soluble in DMSO and ethanol, but difficult to dissolve in water. The present invention proposes a new dissolution method for 25HC for the first time, and can protect RFP-induced drug-induced fatty liver and internal mechanisms both in vivo and in vitro, which will lay the foundation for future clinical applications. Summary of the invention

[0004] In view of the above technical problems, the present invention provides the use of 25-hydroxycholesterol in preparing a drug for treating or improving liver lipid deposition.

[0005] A new use of 25-hydroxycholesterol in preparing a method for treating or improving rifampicin-induced hepatic lipid deposition.

[0006] The liver lipids include triglyceride TG or cholesterol TC. The 25-hydroxycholesterol is used as a drug for inhibiting rifampicin from reaching liver fat droplets, thereby achieving the purpose of treating or improving rifampicin-induced liver lipid deposition. The 25-hydroxycholesterol is used in the preparation of drugs for reducing or improving serum transaminase and bilirubin, wherein the serum transaminase includes the level of ALT, and the bilirubin includes the levels of TBIL and DBIL.

[0007] The 25-hydroxycholesterol is used in preparing a drug for treating or improving the mRNA expression of ABCA1, ABCA2 and ABCA3 genes in hepatocytes induced by rifampicin.

[0008] The present invention discloses that 25HC can alleviate liver lipid deposition induced by RFP. This model adopts a medium dose of RFP (150 mg / kg·d -1 , once a day for 32 consecutive days) to create a drug-induced fatty liver model, inducing liver enlargement, increased liver weight, liver lipid deposition, and significant increase in intrahepatic triglycerides and cholesterol in mice. The present invention is based on the discovery in the process of 25HC treating RFP-induced fatty liver model that 25HC has a very strong protective effect, reducing liver lipid deposition, fatty degeneration, and bilirubin, and no death occurs. The specific contents are as follows: The technical solution of the present invention has the following beneficial effects: (1) RFP-induced fatty liver is characterized by increased liver weight, fatty degeneration of hepatocytes, and the formation of a large number of small lipid droplets in hepatocytes. This study used a medium dose of RFP (150 mg / kg·d -1 , once a day) was administered orally to C57BL / 6J mice (about 20 g, male, 8 weeks old) for 32 days. -1 According to the conversion ratio (9.1 times), 91 mg / kg is needed for oral administration. We consulted the literature and conducted preliminary experiments. Our preliminary experiments found that RFP 300 mg / kg·d -1, about 1 / 3 mortality, 75mg / kg is difficult to induce or requires a long time to induce fatty liver, and finally decided to use a medium dose of 150mg / kg to induce fatty liver. The results showed that there were no deaths in this model, and there was significant liver enlargement and liver fatty degeneration. From our model, it can be found that the short-term RFP induces liver lipid deposition; see the relevant steps in Example 2.

[0009] (2) 25HC alleviates RFP-induced liver lipid droplet formation in a time-dependent and dose-dependent manner, which can be confirmed by both in vitro and in vivo experiments. Through in vitro experiments, RFP has a time-dependent and dose-dependent effect on lipid droplet formation in normal mouse AML12 hepatocytes, especially cholesterol accumulation in the liver. 300mM RFP treatment of hepatocytes for 24h can successfully induce lipid accumulation in hepatocytes, with significant increases in total cholesterol (TC) and total triglycerides (TG), but the increase in TC in the cell supernatant is not obvious; the inconsistent trend of TC changes inside and outside the cells suggests that there may be impaired cholesterol efflux function; at the same time, 25HC can significantly reduce intracellular TC to the normal range. See the relevant steps of Examples 2 and 3.

[0010] (3) The intrinsic mechanism of 25HC alleviating RFP-induced cholesterol accumulation in the liver. PXR is currently recognized as the receptor of RFP. RFP causes triglyceride deposition in the liver by activating PXR. The present invention found that lipid deposition in the liver is not completely dependent on PXR, and other pathways should be involved in the formation of lipid droplets induced by RFP. This experiment further found that RFP can significantly inhibit the CH25H expression pathway, promote LXRα nuclear exocytosis, and inhibit the expression of ABCA1, 2, 3 genes, indicating that RFP inhibits the CH25H-LXRα-ABCA1, 2, 3 pathway, which leads to the obstruction of cholesterol efflux in cells and leads to a large accumulation of cholesterol; while 25HC can increase the expression of CH25H, LXRα, ABCA1, 2, 3, promote LXRα to enter the cell nucleus, thereby promoting the activation of the CH25H-LXRα-ABCA1, 2, 3 pathway, thereby promoting cholesterol efflux, alleviating cholesterol accumulation, and reducing liver lipid deposition. See the relevant steps of Examples 4 and 5.

[0011] (4) Solvent formula of 25HC: 25HC is a fat-soluble substance that is difficult to dissolve in water, but easily soluble in DMSO and alcohol. The present invention takes into account that DMSO and alcohol may have toxic effects on cells and animals. Therefore, the present invention has newly created a method of "first dissolving with pure organic solvent and then diluting with saline solution". For animal experiments, 25HC is first dissolved with pure alcohol and then diluted with PBS. The final alcohol concentration in the intragastric solution is only 0.3%, and the maximum intragastric volume for each mouse is only 0.2 ml / d, which reduces the impact of the solvent on subsequent experiments. For cell experiments, 25HC is first dissolved with a certain small volume of pure DMSO and then frozen as a storage solution. In cell experiments, it is added to DMEM solution. The final DMSO concentration in the cell culture solution during this process is 0.01-0.4% (lower than the toxic limit value of DMSO 1.0%). See the relevant steps in Example 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : The figure shows the lipid droplet formation (BODIPY staining, Oil Red O staining) induced by different concentrations of RFP in normal AML12 mouse liver cell line.

[0013] Figure 2 : Figures 2A and 2B show the enzymatic determination of intracellular cholesterol and triglyceride levels.

[0014] Figure 3 :Figure 3A shows the determination of intracellular cholesterol levels by different concentrations of RFP. Figure 3 The figure shows the determination of cholesterol content in cell supernatant at different time points (all enzymatic methods). Figure 3 C is correct Figure 3 B Calculate the area under the curve and make comparisons between groups.

[0015] Figure 4 :Figure A shows that RFP at different concentrations reduces the protein expression of CH25H in AML12 hepatocytes (Western blotting). Figure B shows that RFP at different times reduces the protein expression of CH25H in AML12 hepatocytes (Western blotting).

[0016] Figure 5 :Liver morphology (including gross observation and HE staining) of normal mice and mice with RFP-induced fatty liver after intraperitoneal injection of 25HC.

[0017] Figure 6 : Lipid droplet staining (Oil Red O staining) of liver tissues of normal mice and mice with RFP-induced fatty liver after intraperitoneal injection of 25HC. Figure 7 :The changes of body weight and food intake in normal mice and mice with RFP-induced fatty liver after intraperitoneal injection of 25HC (mouse metabolic cage method).

[0018] Figure 8 : Organ weight and liver tissue lipids (including total cholesterol TC and triglyceride TG) of normal mice and mice with RFP-induced fatty liver were injected intraperitoneally with 25HC.

[0019] Fig. 9 : The levels of serum ALT (alanine aminotransferase), AST (aspartate aminotransferase), and bilirubin (total bilirubin, direct bilirubin, and indirect bilirubin) in normal mice and mice with RFP-induced fatty liver after intraperitoneal injection of 25HC.

[0020] Fig.10 : The mRNA expression of CH25H and ABCA family in liver tissues of normal mice and mice with RFP-induced fatty liver was evaluated by qRT-PCR after intraperitoneal injection of 25HC.

[0021] Fig.11 : Lipid droplet staining (Oil Red O staining) of Hep3B (human hepatoma cell line) treated with 25HC or (and) RFP.

[0022] Fig.12 : Enzymatic method was used to determine the intracellular cholesterol content of Hep3B cells after treatment with 25HC or (and) RFP.

[0023] Fig.13 :Effect of PXR knockdown (siRNA technology) on RFP-induced lipid droplet formation in hepatocytes. Fig.14 :Figure A is the effect of 25HC or (and) RFP on LXRα nuclear translocation verified by cell immunofluorescence. Figure B is the effect of 25HC or (and) RFP on LXRα protein expression verified by immunoblotting.

[0024] Fig.15 :In vitro experiments showed that 25HC or (and) RFP affected the mRNA expression of cholesterol efflux genes ABCA1, ABCA2, and ABCA3 in Hep3B. DETAILED DESCRIPTION

[0025] Example 1 Cell experiments 25HC solution preparation Take 1 mg of 25HC (white solid powder, MCE, HY-113134) and 2.4835 mL of DMSO, dissolve into a 1 mM storage solution, store the storage solution at -20°C for 1 month and at -80°C for 6 months; Take 0.2 μL of the above storage solution and add 1.9998 mL of DMEM cell culture medium, mix well to get a working solution with a concentration of 0.1 μM (DMSO concentration is 0.01% when treating cells), and use it immediately after preparation; Take 2 μL of the above storage solution and add 1.998 mL of DMEM cell culture medium, mix well to get a working solution with a concentration of 1 μM (DMSO concentration is 0.1% when treating cells), and use it immediately after preparation; Take 8 μL of the above storage solution and add 1.992 mL of DMEM cell culture medium, mix well to obtain a working solution with a concentration of 4 μM (DMSO concentration is 0.4% when treating cells), and use it immediately after preparation.

[0026] Animal experiments 25HC solution preparation Take 25HC 50 mg (white solid powder) + anhydrous ethanol 30 μl, dissolve it, add PBS 9.97 mL, mix well to obtain an intragastric solution with a concentration of 5 mg / mL (the volume concentration ratio of alcohol is 0.3%), and store it at 4°C for one week.

[0027] Note: Phosphate Buffered Saline (PBS, Powder) is sterilized by steam sterilization at 121.3℃ (103.4KPa) under high pressure (30 minutes), and stored at room temperature or in a refrigerator at 4℃ (use up within 1 week).

[0028] PBS (pH 7.4, 10 mM) preparation: 1. Preparation of mother solution: 0.2M Na2HPO4: weigh 71.6g Na2HPO4·12H2O and dissolve in 1000mL water; 0.2M NaH2PO4: weigh 2.0g NaH2PO4·2H2O and dissolve in 1000mL water; Preparation of PBS (pH=7.4): first prepare 0.2M PBS (pH=7.4, 100mL): take 19mL 0.2mol / L Na2HPO4, 81ml 0.2mol / L Na2HPO4.

[0029] 2. Dilute the stock solution 20 times to obtain a 10mM working solution. 0.01M PBS (pH=7.4): Take 50mL of 0.2M PBS and dilute to 1000mL with water.

[0030] Example 2 In vitro experiments showed that RFP induced lipid droplet formation in hepatocyte cell lines.

[0031] 2.1 Figure 1 Normal mouse liver cell line AML12 was treated with different concentrations of RFP (0, 100, 200, 300 μM, treated for 24 h), then fixed with formaldehyde fixative, and stained for cell lipid droplets using BODIPY staining kit and Oil Red O staining. Figure 1 (Upper part) shows that as the concentration increases, RFP can induce the formation of a large number of lipid droplets in Hep3B cells (green dots, indicated by red arrows); Figure 1 (Lower part) The effect of RFP on Hep3B showed that different concentrations of RFP induced an increase in lipid droplets in HepG2 (red dots, indicated by yellow arrows).

[0032] 2.2 Figure 2 : Figures 2A and 2B show the enzymatic determination of intracellular cholesterol TC and triglyceride TG content. It was found that RFP (300μM, 24h) can increase the intracellular TC and TG levels by about 3 times.

[0033] 2.3 Figure 3 :Figure 3A shows the determination of intracellular cholesterol levels by different concentrations of RFP. Figure 3 The figure shows the determination of cholesterol content in cell supernatant at different time points (all enzymatic methods). Figure 3 C is correct Figure 3 B. Area under the curve (AUC) calculation. The inter-group comparison showed that the increase in TC in the cell supernatant of the RFP group was lower than that in the control group, while the increase in TC in the cells was higher than that in the control group. This indicates that RFP promotes the increase in intracellular cholesterol, while the increase in extracellular cholesterol is not obvious, indicating that RFP has a cholesterol excretion disorder.

[0034] 2.4 The total protein of the above cells was extracted and the expression of CH25H in AML12 cells treated with RFP at different concentration gradients and different time gradients was detected by western blot (see Figure 2 A, 2B), it was found that with the increase of RFP concentration, the mRNA expression of CH25H decreased significantly; with the extension of time, the mRNA expression of CH25H decreased significantly.

[0035] Example 3 In vivo study on the effect of 25HC on RFP-induced fatty liver.

[0036] C57Bl / 6J mice (8 weeks old, male, SPF grade, free drinking water and diet) were divided into 4 groups (6 mice in each group): one group was the negative control group (Veh group): 0.2 ml of 25HC solvent was gavaged once a day; the other group was the RFP group: 150 mg / kg·d was gavaged -1 , once a day for 32 consecutive days; one group was the 25HC group: normal mice were intraperitoneally injected with 25HC (40 μg / g·d -1, once a day, each mouse is about 20-25g, 0.8~1.0mg is administered orally every day, the concentration of 25HC is 5mg / mL, and the volume of each mouse is about 0.16-0.2ml / d), for 32 consecutive days; one group is the RFP+25HC group, that is, while administering rifampicin (the same dose as before), 25HC (the same dose as before) is injected for 32 consecutive days, and then isoflurane anesthesia is taken, blood and fresh liver tissue are taken for gene detection and morphological experiments. The results are as follows: 3.1 Figure 5 The gross appearance and HE staining show the morphological changes of the liver. 25HC had no effect on the appearance of the liver of normal mice, and no significant changes were observed in HE staining. RFP can induce fatty degeneration of the liver in normal mice, edema of cells, lighter cytoplasm color, smaller lipid droplets in the liver, and map-like focal lesions in the liver. After using 25HC to treat the liver of mice with RFP lesions, the liver lesions improved and the lipid droplets were significantly reduced.

[0037] 3.2 Figure 6 The lipid droplets in liver tissue stained with Oil Red O show that 25HC does not increase the lipid droplets in liver tissue of normal mice, and there is no difference with the liver of normal group; RFP can induce a large number of small lipid droplets in the liver of normal mice; 25HC can inhibit the lipid droplets in the liver of mice gavaged with RFP, which is close to that of normal mice.

[0038] 3.3 Figure 7 A is the weight change between groups. No difference was found between groups.

[0039] 3.4 Figure 7 B: The mice were kept in ordinary metabolic cages and their food intake was observed, and no difference was found between the groups.

[0040] 3.5 Figure 8 A is the ratio of visceral weight to body weight among the groups. 25HC slightly increased liver weight, while RFP significantly increased liver weight; the liver weight of the RFP+25HC group was significantly lower than that of the RFP group. However, there was no difference in the ratio of kidney weight to body weight among the groups.

[0041] 3.6 Figure 8 B is the quantification of lipids in the liver. 25HC does not affect the liver lipids (triglyceride TG, cholesterol TC) of normal mice. RFP significantly increases TC and TG in liver tissue; 25HC can alleviate the increase of TC and TG induced by RFP, close to the level of normal mice.

[0042] 3.7 Fig. 9The levels of serum transaminases (ALT, AST) and bilirubin (TBIL, DBIL, IBIL) in mice. RFP can increase ALT, bilirubin (TBIL, DBIL, IBIL), but not AST. 25HC can reduce ALT (no statistical difference with normal mice) and bilirubin (TBIL, DBIL), but not AST and IBIL.

[0043] 3.8 Fig.10 The CH25H, ABCA1, 2 gene expression in mouse liver tissue (qRT-PCR method). It was found that RFP can reduce the mRNA expression of CH25H, ABCA1, and ABCA2. 25HC did not affect the mRNA expression of CH25H, ABCA1, and ABCA2 in normal mice. 25HC did not affect the mRNA expression of CH25H, ABCA1, and ABCA2 in mice with RFP-induced fatty liver.

[0044] Example 4 In vitro experiment of 25HC treating RFP-induced fatty liver.

[0045] The human liver cancer cell line Hep3B was treated with RFP using different concentrations of 25HC (25HC concentrations were 0, 1, and 4 μM, RFP concentration was 300 μM, and treatment was performed for 24 h) to observe the changes in lipid droplets.

[0046] 4.1 Fig.11 Oil red O staining is shown. The RFP group had significantly more lipid droplets than the normal control group. The number of lipid droplets in the 25HC (1 μM) + RFP group was significantly lower than that in the RFP group; the number of lipid droplets in the 25HC (4 μM) + RFP group was also significantly lower than that in the 25HC (1 μM) + RFP group and the RFP group; 4.2 Fig.12 The total cholesterol content in cells was detected by enzymatic kit. 25HC with increasing concentration (0.1, 1, 4 μM) could significantly reduce the intracellular cholesterol level induced by RFP.

[0047] 4.3 Fig.13 Figure 2 shows the effect of PXR knockdown (siRNA technology) on RFP-induced hepatocyte lipid droplet formation. Figure A verifies that siPXR can reduce the mRNA expression of the PXR gene in Hep3B cells; Figure B verifies that siPXR can reduce the protein expression of the PXR gene in Hep3B cells. Figures C (Oil Red O staining) and D (BODIPY staining) show the changes of siPXR on RFP-induced Hep3B cell lipid droplets. It was found that a large number of lipid droplets were still visible after knockdown. These suggest that PXR is not the only receptor for RFP.

[0048] 4.4 Fig.14Cell immunofluorescence showed that RFP could move LXRα from the nucleus to the cytoplasm. 25HC could promote the transfer of LXRα from the cytoplasm to the nucleus. This suggests that 25HC can promote the transcriptional regulation activity of LXRα through nuclear translocation and promote the expression of the downstream gene ABCA gene.

[0049] 4.5 Fig.15 The gene expression of Hep3B cells treated with 25HC or (and) RFP was detected by qRT-PCR. It was found that RFP inhibited the mRNA expression of Hep3B cholesterol efflux genes ABCA1, ABCA2, and ACBA3. 25HC increased the mRNA expression of ABCA1, ABCA2, and ABCA3 genes in normal hepatocytes. 25HC can alleviate the RFP-induced downregulation of mRNA expression of ABCA1, ABCA2, and ABCA3 genes in hepatocytes, and there is a concentration trend (0, 0.1, 1, and 4 μM).

[0050] Example 5 Effect of downregulation of PXR by siRNA on RFP-induced lipid droplet formation in hepatocytes.

[0051] It has been reported that the pregnane X receptor (PXR) is the main receptor of rifampicin, which can promote the synthesis of triglycerides in the liver by activating liver PXR. In this study, Hep3B hepatocytes were knocked down by siRNA in vitro, and then RFP was observed to induce the appearance of lipid droplets. The results showed that inhibition of PXR could not completely block the formation of intracellular lipid droplets.

[0052] 5.1 Fig.13 A and 13B show the efficiency detection of knocking down PXR by siRNA technology, and it was found that both the mRNA and protein expressions of PXR were significantly downregulated by siPXR.

[0053] 5.2 Fig.13 Oil red O staining in C showed that siPXR could not reduce lipid droplets in normal hepatocytes, but RFP could promote the increase of lipid droplets. siPXR could reduce the formation of lipid droplets induced by RFP, but it could not completely block the formation of lipid droplets, which was still higher than the normal group.

[0054] 5.3 Fig.13 BODIPY staining in D showed that siPXR can reduce RFP-induced lipid droplet formation, but it cannot completely block lipid droplet formation, which is still higher than the normal group.

Claims

1. Use of 25-hydroxycholesterol in preparing a drug for treating or improving liver lipid deposition.

2. The use according to claim 1, characterized in that A new use of 25-hydroxycholesterol in preparing a method for treating or improving rifampicin-induced hepatic lipid deposition.

3. The use according to claim 2, characterized in that: The liver lipids include triglyceride TG or cholesterol TC.

4. The use according to claim 2, characterized in that: The invention discloses a drug for inhibiting rifampicin from reaching liver lipid droplets, thereby achieving the purpose of treating or improving rifampicin-induced liver lipid deposition.

5. The use according to claim 2, characterized in that: The invention discloses an application of 25-hydroxycholesterol in the preparation of a drug for reducing or improving serum transaminase and bilirubin, wherein the serum transaminase includes the level of ALT, and the bilirubin includes the levels of TBIL and DBIL.

6. The use according to claim 2, characterized in that: Use of 25-hydroxycholesterol in preparing a drug for treating or improving the mRNA expression of ABCA1, ABCA2 and ABCA3 genes in hepatocytes induced by rifampicin.

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