A new use of 25-hydroxycholesterol in treating rifampicin-induced liver lipid deposition

Abstract

The present invention provides a novel use of 25-hydroxycholesterol (25HC), a natural metabolite in vivo, for treating rifampicin (RFP)-induced hepatic lipid deposition. 25HC is a metabolite of cholesterol produced by cholesterol 25-hydroxylase (CH25H). Previously, it has been reported to exhibit antiviral, interferon-activating, anti-tumor, and lipid metabolism activities. The present invention finds that RFP can induce hepatic steatosis, lipid droplet deposition, and reduce CH25H expression. Therefore, 25HC deficiency may be one of the mechanisms by which RFP induces hepatic lipid deposition. The present invention is the first to discover that 25HC supplementation can alleviate RFP-induced lipid droplet deposition (reduction in both triglycerides and cholesterol). In particular, it is the first to discover that the mechanism by which 25HC reduces liver cholesterol content is related to promoting LXRα expression and entry into cells, and promoting the expression of cholesterol efflux genes ABCA1, 2, and 3. This suggests that 25HC may reduce liver lipid deposition and alleviate RFP-induced liver damage by promoting cholesterol efflux from hepatocytes, laying the foundation for the future clinical application of 25HC.

Description

Technical Field

[0001] The present invention relates to a new pharmaceutical use of 25-hydroxycholesterol (25HC). It is found that its chemical composition (oral administration) 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 a rare clinical manifestation of drug-induced liver injury. The main causes in China are steroids (glucocorticoids, tamoxifen), anti-tuberculosis drugs (such as rifampicin), antibiotics (tetracycline), anti-epileptic drugs (valproic acid), anti-arrhythmic drugs (amiodarone), and anti-tumor drugs (methotrexate). Rifampicin (RFP) is a common cause of DIHS. Rifampicin (RFP) is a first-line anti-tuberculosis drug recommended by the World Health Organization. Currently, traditional treatments for DIHS are ineffective. Traditional liver-protective drugs such as reduced glutathione, diammonium glycyrrhizinate, and glucuronolactone are merely symptomatic treatments, providing antioxidant and anti-inflammatory effects, but do not precisely target rifampicin-induced fatty liver. This is because the mechanism or pathogenic target remains unclear, hindering the implementation of precision medicine. Rifampicin-induced fatty liver is characterized by early or short-term hepatic lipid accumulation without elevated transaminases or inflammatory responses. Long-term exposure can lead to hepatocyte rupture, hepatic cholestasis, and elevated serum transaminases. However, there is currently no specific treatment for DIHS caused by RFP. Early intervention and protection against rifampicin-induced drug-induced fatty liver disease are urgent basic research priorities.

[0003] 25-Hydroxycholesterol (25HC) is a metabolite of cholesterol catalyzed by 25-hydroxylase (CH25H). Cholesterol-25-hydroxylase, a metabolic enzyme in the endoplasmic reticulum, uses oxygen and NADPH to add a hydroxyl group to the 25-carbon atom of cholesterol, forming the active 25HC. 25HC binds to various receptors (LXRα, SCAP / Insig-2, GPR155, and ACAT), contributing to biological effects such as antiviral activity, interferon activation, anti-tumor activity, and regulation of cholesterol synthesis. 25HC is readily soluble in DMSO and ethanol but poorly soluble in water. This study presents a novel solubilization method for 25HC, demonstrating its ability to protect against RFP-induced drug-induced fatty liver disease both in vitro and in vivo, and its underlying mechanisms, which will pave the way for future clinical applications. Summary of the Invention

[0004] In response to 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 triglycerides TG or cholesterol TC.

[0007] The 25-hydroxycholesterol is used as a drug to inhibit rifampicin from reaching liver fat droplets, thereby achieving the purpose of treating or improving rifampicin-induced liver lipid deposition.

[0008] The 25-hydroxycholesterol is used in the preparation of drugs for reducing or improving serum transaminases and bilirubin, wherein the serum transaminases include the level of ALT, and the bilirubin includes the levels of TBIL and DBIL.

[0009] 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.

[0010] 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 daily for 32 consecutive days) to establish a drug-induced fatty liver model in mice, inducing liver enlargement, increased liver weight, liver lipid deposition, and significant increases in hepatic triglycerides and cholesterol. The present invention, based on the discovery during treatment of RFP-induced fatty liver model with 25HC, demonstrated a strong protective effect, reducing liver lipid deposition, steatosis, and bilirubin, without causing mortality. The details are as follows:

[0011] The technical solution of the present invention has the following beneficial effects:

[0012] (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, with approximately a one-third mortality rate. Induction of fatty liver was difficult or required a prolonged period at 75 mg / kg, leading to the decision to use a moderate dose of 150 mg / kg. Results showed no mortality in this model, but significant hepatomegaly and hepatic steatosis were observed. Our model demonstrates that short-term RFP induces hepatic lipid deposition; see the relevant steps in Example 2.

[0013] (2) 25HC alleviates RFP-induced hepatic lipid droplet formation in a time-dependent and dose-dependent manner, as confirmed by both in vitro and in vivo experiments. In vitro experiments showed that RFP has a time-dependent and dose-dependent effect on lipid droplet formation, especially cholesterol accumulation in the liver, in normal mouse AML12 hepatocytes. 300mM RFP treatment of hepatocytes for 24 hours successfully induced lipid accumulation in hepatocytes, with significant increases in total cholesterol (TC) and total triglycerides (TG), but no significant increase in TC in the cell supernatant. The inconsistent trends in TC changes inside and outside the cells suggest that cholesterol efflux function may be impaired. At the same time, 25HC can significantly reduce intracellular TC to the normal range. See the relevant steps in Examples 2 and 3.

[0014] (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 entirely dependent on PXR, and other pathways should be involved in RFP-induced lipid droplet formation. This experiment further found that RFP can significantly inhibit the CH25H expression pathway, promote LXRα nuclear exocytosis, and inhibit ABCA1, 2, 3 gene expression, indicating that RFP inhibits the CH25H-LXRα-ABCA1, 2, 3 pathway, which leads to the obstruction of intracellular cholesterol efflux and the accumulation of large amounts 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.

[0015] (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 created a new method of "first dissolving with pure organic solvent and then diluting with saline solution". For animal experiments, 25HC is first dissolved in pure alcohol and then diluted with PBS. The final alcohol concentration in the gavage solution is only 0.3%, and the maximum gavage 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 in 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 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

[0016] Figure 1 : The figure shows the lipid droplet formation in normal AML12 mouse liver cell line induced by different concentrations of RFP (BODIPY staining, Oil Red O staining).

[0017] Figure 2 : Figures 2A and 2B show the enzymatic determination of intracellular cholesterol and triglyceride levels.

[0018] 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.

[0019] Figure 4 Figure A shows that RFP at different concentrations reduces the protein expression of CH25H in AML12 hepatocytes (Western blot). Figure B shows that RFP at different time points reduces the protein expression of CH25H in AML12 hepatocytes (Western blot).

[0020] Figure 5 : Liver morphology (including gross appearance and HE staining) of normal mice and mice with RFP-induced fatty liver after intraperitoneal injection of 25HC.

[0021] Figure 6 : Lipid droplet staining (Oil Red O staining) in the liver tissues of normal mice and mice with RFP-induced fatty liver after intraperitoneal injection of 25HC.

[0022] Figure 7 : Changes in body weight and food intake after intraperitoneal injection of 25HC in normal mice and mice with RFP-induced fatty liver (mouse metabolic cage method).

[0023] Figure 8 : Organ weights and liver tissue lipids (including total cholesterol TC and triglycerides TG) of normal mice and RFP-induced fatty liver mice injected intraperitoneally with 25HC.

[0024] Figure 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.

[0025] Figure 10 : The mRNA expressions of CH25H and ABCA family in liver tissues of normal mice and mice with RFP-induced fatty liver were evaluated by qRT-PCR after intraperitoneal injection of 25HC.

[0026] Figure 11 : Lipid droplet staining (Oil Red O staining) of Hep3B (human hepatocellular carcinoma cell line) treated with 25HC or (and) RFP.

[0027] Figure 12 : The intracellular cholesterol content of Hep3B cells treated with 25HC or (and) RFP was determined by enzymatic method.

[0028] Figure 13 :Effect of PXR knockdown (siRNA technology) on RFP-induced lipid droplet formation in hepatocytes.

[0029] Figure 14 Figure A: Immunofluorescence analysis to verify the effect of 25HC and / or RFP on LXRα nuclear translocation. Figure B: Western blotting analysis to verify the effect of 25HC and / or RFP on LXRα protein expression.

[0030] Figure 15 :The effects of 25HC and / or RFP on the mRNA expression of cholesterol efflux genes ABCA1, ABCA2 and ABCA3 in Hep3B in vitro. DETAILED DESCRIPTION

[0031] Example 1

[0032] Cell experiments

[0033] Preparation of 25HC solution

[0034] Dissolve 1 mg of 25HC (white solid powder, MCE, HY-113134) in 2.4835 mL of DMSO to make a 1 mM stock solution. Store the stock solution at -20°C for 1 month and at -80°C for 6 months.

[0035] Take 0.2 μL of the above stock solution and add 1.9998 mL of DMEM cell culture medium, mix well to obtain a 0.1 μM working solution (DMSO concentration is 0.01% when treating cells), and use it immediately.

[0036] Take 2 μL of the above stock solution and add 1.998 mL of DMEM cell culture medium, mix well to obtain a 1 μM working solution (DMSO concentration is 0.1% when treating cells), and use it immediately.

[0037] 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.

[0038] Animal experiments

[0039] Preparation of 25HC solution

[0040] Dissolve 50 mg of 25HC (white solid powder) in 30 μl of anhydrous ethanol, add 9.97 mL of PBS, and mix thoroughly to obtain a 5 mg / mL intragastric solution (the volume concentration of alcohol is 0.3%). Store at 4°C for one week.

[0041] Note: Phosphate Buffered Saline (PBS, Powder) is sterilized by steam sterilization at 121.3°C (103.4 kPa) under high pressure (30 minutes) and stored at room temperature or in a refrigerator at 4°C (use within 1 week).

[0042] PBS (pH 7.4, 10 mM) preparation:

[0043] 1. Preparation of mother liquor:

[0044] 0.2M Na2HPO4: Weigh 71.6g Na2HPO4·12H2O and dissolve in 1000mL water;

[0045] 0.2M NaH2PO4: Weigh 2.0g NaH2PO4·2H2O and dissolve in 1000mL water;

[0046] 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.

[0047] 2. Dilute the stock solution 20-fold to obtain a 10 mM working solution. 0.01 M PBS (pH 7.4): Take 50 mL of 0.2 M PBS and dilute to 1000 mL with water.

[0048] Example 2

[0049] In vitro experiments showed that RFP induced lipid droplet formation in hepatocytes.

[0050] 2.1 Figure 1 Normal mouse liver cell line AML12 was treated with different concentrations of RFP (0, 100, 200, 300 μM, treatment 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, pointed by red arrows); Figure 1 (Lower part) RFP acts on Hep3B, indicating that different concentrations of RFP induce an increase in lipid droplets in HepG2 (red dots, indicated by yellow arrows).

[0051] 2.2 Figure 2 : Figures 2A and 2B show the enzymatic determination of intracellular cholesterol TC and triglyceride TG levels. It was found that RFP (300μM, 24h) could increase the intracellular TC and TG levels by about 3 times.

[0052] 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. Calculation of the area under the curve (AUC). The inter-group comparison showed that the increase in TC in the cell supernatant of the RFP group was lower than that of the control group, while the increase in TC in the cells was higher than that of 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.

[0053] 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.

[0054] Example 3

[0055] In vivo study of 25HC in treating RFP-induced fatty liver.

[0056] C57Bl / 6J mice (8-week-old, male, SPF grade, free access to 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 administered orally once a day; the other group was the RFP group: 150 mg / kg·d was administered orally -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 The mice, weighing approximately 20-25g, were gavaged once daily (0.8-1.0mg, 5mg / mL, 0.16-0.2ml / d) for 32 consecutive days. A second group, the RFP+25HC group, received rifampicin (same dose) and 25HC (same dose) injections for 32 consecutive days. After isoflurane anesthesia, blood and fresh liver tissue were collected for genetic testing and morphological experiments. The results are as follows:

[0057] 3.1 Figure 5 Gross observation and HE staining demonstrate morphological changes in the liver. 25HC had no effect on the appearance of the liver in normal mice, with no significant changes observed in HE staining. RFP can induce fatty degeneration, cellular edema, paler cytoplasm, smaller lipid droplets within the liver, and geographic focal lesions in normal mice. Treatment of the livers of mice with RFP lesions with 25HC showed improvement in liver lesions, with a significant reduction in lipid droplets.

[0058] 3.2 Figure 6 The lipid droplets in the liver tissue stained with Oil Red O showed that 25HC did not increase the lipid droplets in the liver tissue of normal mice, and there was no difference between the livers of the normal group and those of the normal group; RFP could induce a large number of small lipid droplets in the livers of normal mice; 25HC could inhibit the lipid droplets in the livers of mice gavaged with RFP, which was close to that of normal mice.

[0059] 3.3 Figure 7 A represents the body weight change among the groups. No difference was found among the groups.

[0060] 3.4 Figure 7 B: Ordinary metabolic cages were used to observe the food intake of mice, and no difference was found between the groups.

[0061] 3.5 Figure 8 A represents the visceral weight / body weight ratio among the groups. 25HC slightly increased liver weight, while RFP significantly increased liver weight. Liver weight in the RFP+25HC group was significantly lower than that in the RFP group. However, the kidney weight / body weight ratio did not differ among the groups.

[0062] 3.6 Figure 8B shows the quantification of liver lipids. 25HC did not affect liver lipids (triglycerides (TG) and cholesterol (TC)) in normal mice. RFP significantly increased TC and TG in liver tissue; 25HC alleviated the RFP-induced increase in TC and TG, bringing them close to normal mouse levels.

[0063] 3.7 Figure 9 Serum transaminases (ALT, AST) and bilirubin (TBIL, DBIL, IBIL) in mice. RFP increased ALT and bilirubin (TBIL, DBIL, IBIL) but did not increase AST. 25HC decreased ALT (no statistical difference compared to normal mice) and bilirubin (TBIL, DBIL) but did not increase AST or IBIL.

[0064] 3.8 Figure 10 The expression of CH25H, ABCA1, and ABCA2 genes in mouse liver tissue was measured by qRT-PCR. RFP was found to reduce CH25H, ABCA1, and ABCA2 mRNA expression. 25HC had no effect on CH25H, ABCA1, and ABCA2 mRNA expression in normal mice. 25HC also had no effect on CH25H, ABCA1, and ABCA2 mRNA expression in mice with RFP-induced fatty liver disease.

[0065] Example 4

[0066] In vitro study of 25HC in treating RFP-induced fatty liver.

[0067] The human liver cancer cell line Hep3B was treated with RFP at different concentrations of 25HC (25HC concentrations were 0, 1, and 4 μM, RFP concentration was 300 μM, and treatment was for 24 h) to observe the changes in lipid droplets.

[0068] 4.1 Figure 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.

[0069] 4.2 Figure 12 The total cholesterol content in cells was detected by enzyme assay. 25HC at increasing concentrations (0.1, 1, and 4 μM) significantly reduced the intracellular cholesterol level induced by RFP.

[0070] 4.3 Figure 13Figure 3 shows the effect of PXR knockdown (siRNA) on RFP-induced lipid droplet formation in hepatocytes. Panel A demonstrates that siPXR can reduce PXR mRNA expression in Hep3B cells; Panel B demonstrates that siPXR can reduce PXR protein expression in Hep3B cells. Panels C (Oil Red O staining) and D (BODIPY staining) show the effects of siPXR on RFP-induced lipid droplet formation in Hep3B cells. Numerous lipid droplets were still visible after PXR knockdown. These findings suggest that PXR is not the sole receptor for RFP.

[0071] 4.4 Figure 14 Immunofluorescence analysis revealed that RFP can translocate LXRα from the nucleus to the cytoplasm. 25HC also promotes LXRα translocation from the cytoplasm to the nucleus. This suggests that 25HC can promote LXRα nuclear translocation, thereby enhancing its transcriptional regulatory activity and promoting the expression of the downstream gene ABCA.

[0072] 4.5 Figure 15 Gene expression in Hep3B cells treated with 25HC or / and RFP was assessed by qRT-PCR. RFP inhibited the mRNA expression of the cholesterol efflux genes ABCA1, ABCA2, and ABCA3 in Hep3B cells. 25HC increased the mRNA expression of ABCA1, ABCA2, and ABCA3 in normal hepatocytes. 25HC alleviated the RFP-induced downregulation of ABCA1, ABCA2, and ABCA3 mRNA expression in hepatocytes, with a concentration-dependent trend (0, 0.1, 1, and 4 μM).

[0073] Example 5

[0074] Effect of siRNA downregulation of PXR on RFP-induced lipid droplet formation in hepatocytes.

[0075] It has been reported that the pregnane X receptor (PXR), the primary receptor for rifampicin, promotes hepatic triglyceride synthesis by activating PXR. This study used in vitro siRNA knockdown of Hep3B hepatocytes to investigate whether RFP could still induce lipid droplet formation. The results showed that PXR inhibition did not completely block intracellular lipid droplet formation.

[0076] 5.1 Figure 13 A and 13B show the efficiency test 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.

[0077] 5.2 Figure 13Oil red O staining in C shows that siPXR cannot reduce lipid droplets in normal hepatocytes, but RFP can promote the increase of lipid droplets. siPXR can reduce RFP-induced lipid droplet formation, but it cannot completely block lipid droplet formation, and the lipid droplet formation is still higher than that of the normal group.

[0078] 5.3 Figure 13 BODIPY staining in D shows that siPXR can reduce RFP-induced lipid droplet formation, but it cannot completely block lipid droplet formation, and the level is still higher than that of the normal group.

Claims

1. Use of 25-hydroxycholesterol in the preparation of a medicament for treating or improving rifampicin-induced hepatic lipid deposition.

2. The use according to claim 1, characterized in that The liver lipids include TG or TC.

3. The use according to claim 1, characterized in that 25-Hydroxycholesterol inhibits hepatic lipid droplet formation.

4. The use according to claim 1, characterized in that 25-hydroxycholesterol reduces or improves serum transaminases and bilirubin. The serum transaminases include ALT, and the bilirubins include TBIL and DBIL.

5. The use according to claim 1, characterized in that 25-Hydroxycholesterol improves rifampicin-induced mRNA expression of ABCA1, ABCA2, and ABCA3 genes in hepatocytes.

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