Uses of celastrol in preventing and/or treating cholestatic liver disease and liver fibrosis
11607417 · 2023-03-21
Assignee
- ZHEJIANG CHONGLING TECHNOLOGY CO., LTD. (Zhejiang, CN)
- KUNMING INSTITUTE OF BOTANY, THE CHINESE ACADEMY OF SCIENCES (Yunnan, CN)
Inventors
Cpc classification
International classification
Abstract
The present disclosure relates to a celastrol or a pharmaceutically acceptable salt thereof, and a use thereof in the manufacture of a medicament for preventing and/or treating a cholestatic liver disease or a liver fibrosis.
Claims
1. A method for treatment of a cholestatic liver disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of celastrol or a pharmaceutically acceptable salt thereof, or the method comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises celastrol or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant or excipient.
2. The method according to claim 1, wherein the cholestatic liver disease is a chemical substance-induced cholestatic liver disease, a drug-induced cholestatic liver disease, a primary cholestatic liver disease or a cholestatic liver disease caused by excessive estrogen in vivo.
3. The method of claim 1, wherein the celastrol or a pharmaceutically acceptable salt thereof decreases bile acid components in a patient's plasma, or reduces liver injury, liver necrosis caused by cholestasis, or inhibits or alleviates inflammation caused by cholestasis.
4. The method of claim 3, wherein the bile acid component includes at least one component selected from the group consisting of: ω-MCA, DCA, TβMCA, TαMCA, TCA, THDCA, TUDCA, TCDCA, and TDCA.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1) The drawings described herein are provided for further understanding of the present disclosure, which is a part of the present disclosure, and the exemplary examples of the present disclosure and the description thereof are for illustrating the present disclosure and do not constitute an undue limitation of the disclosure. In the drawings:
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SPECIFIC MODELS FOR CARRYING OUT THE INVENTION
(9) The substantive content of the present disclosure will be illustrated in detail by reference to the following specific examples of the present disclosure. It is understood that the following examples are only intended to illustrate the disclosure, but are not intended to limit the scope of the disclosure. When the specific conditions are not indicated in the Examples, the Examples are carried out under conventional conditions or the conditions recommended by the manufacturers. The drugs or reagents used herein, the manufacturers of which are not indicated, are the all conventional products that are commercially available.
(10) Although many materials and operational methods used in the following examples are well known in the art, the present disclosure still describes them in detail as far as possible. It would be apparent to those skilled in the art that the materials and operation methods used in the following examples are well known in the art unless otherwise stated.
Example 1: Protection and/or Prevention Effects of Celastrol on ANIT-Induced Mouse Cholestatic Liver Injury
1.1 Experimental Animals
(11) C57BL/6 Mice, SPF Grade, Body Weight 20˜23 g, Male; Certificate Number: SCXK (Xiang) 2013-0004, purchased from Hunan SJA Laboratory Animal Co., Ltd.
1.2 Drugs and Reagents
(12) Celastrol, red amorphous crystalline powder, purchased from Chengdu Ruifensi Biotechnology Co., Ltd., Lot number: L-003-150420.
(13) Aspartate aminotransferase (AST) kit (Cat. No. C010-3, Lot No. 20161031), alanine aminotransferase (ALT) kit (Cat. No. C009-3, Lot No. 20161031), and alkaline phosphatase (ALP) kit (Cat. No. A059-3, Lot No. 20161031), produced by Nanjing Jiancheng Bioengineering Institute.
(14) α-Naphthyl isothiocyanate (ANIT, Cat. No. N4525-10G, Lot No. 101734146), DMSO (Cat. No. V900090-500m1, Lot No. 101669350), Tween 80 (Cat. No. P1754-500m1, Lot No. 101761909), Formic Acid (Cat. No. 94318-250m1-F, Lot No. 101721592), chlorpropamide (Cat. No. C1290-25G, Lot No. 1002213299), cholic acid (CA, Cat. No. C1129-25G), deoxycholic acid (DCA, Cat. No. D2510-10G), taurocholic acid (TCA, Cat. No. T4009-1G), glycocholic acid (GCA, Cat. No. G2878-500MG), taurohyodeoxycholic acid (THDCA, Cat. No. T0682-250MG) and taurolithocholic acid (TLCA, Cat. No. T7515-100MG), purchased from Sigma Co.
(15) Corn oil (Cat. No. C116025-500G, Lot No. 11524089), purchased from Aladdin Co.
(16) Chromatographic grade acetonitrile (Cat. No. 1.00030.4008), purchased from Merck Co.
(17) ω-muricholic acid (ω-MCA, Cat. No. sc-396738) and tauro-β-muricholic acid (TβMCA, Cat. No. SC-361829), purchased from Santa Cruz Co.
(18) Tauroursodeoxycholic acid (TUDCA, Cat. No. HY-19696A), purchased from MCE Co.
(19) β-muricholic acid (β-MCA, Cat. No. C008852) and tauro-α-muricholic acid (TαMCA, Cat. No. C1893-000), purchased from Steraloids Co.
1.3 Main Instruments
(20) Precision electronic balance, purchased from Sartorius Co.
(21) Automatic microplate reader, purchased from BioTek Instruments Co.
(22) SHA-C water bath constant temperature shaker, purchased from Changzhou Zhongcheng Instrument Manufacturing Co., Ltd.;
(23) Refrigerated centrifuge, purchased from Eppendorf Co.
(24) Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry, including 1290 sample injector, 1290 pump, column oven, XDB-C18 column (2.1×100 mm, 1.8 μM), and 6530 quadrupole time-of-flight mass spectrometry, purchased from Agilent Co.
(25) Precellys 24 tissue homogenizer, purchased from Bertin Technologies; upright microscope, purchased from Nikon Co.
(26) Tissue microtome, purchased from Leica Co.
1.4 Experimental Methods
1.4.1 Experimental Grouping
(27) A total of 18 male C57BL/6 mice were divided into three groups: blank group (n=6); model group (n=6); treatment group (n=6).
1.4.2 Preparation and Dosage of Drug Solution
(28) Celastrol was dissolved in a solvent to obtain a celastrol solution at a concentration of 2 mg/mL, wherein the solvent consisted of 1% DMSO, 2% Tween 80, and 97% water (v:v:v). For intragastrical administration, the dosage of celastrol was 10 mg/kg.Math.d. ANIT was dissolved in corn oil to obtain a corn oil solution of ANIT at a concentration of 15 mg/mL, and the dosage of ANIT for intragastrical administration was 75 mg/kg.
1.4.3 Experimental Procedure
(29) The treatment group was intragastrically administered with the celastrol solution at a dosage of 10 mg/kg.Math.d for five consecutive days. At the same time, the blank group and the model group were intragastrically administered with the same dosage of a solvent, wherein the solvent consisted of 1% DMSO, 2% Tween 80 and 97% water (v:v:v), and the solvent was administrated for five consecutive days.
(30) On the third day of administration, ANIT model was established. 4 Hours after administration with the solvent or celastrol solution, the model group and the treatment group were respectively intragastrically administered with the ANIT corn oil solution to induce cholestatic liver injury at a dosage of 75 mg/kg for single administration, and the blank group was fed with an equal dosage of corn oil. After the model was established, the treatment group was continuously administrated with the celastrol solution, and the blank group and the model group was continuously administrated with the same dosage of solvent.
(31) 48 Hours after the model was established, the mice were sacrificed by CO.sub.2 anesthesia, and mouse livers and plasma samples were taken. The eyeballs of the mice were taken for blood collection. The plasma samples were placed on ice for 1-2 hours, centrifuged at 4000 g for 5 minutes at 4° C., and the supernatant was taken to determine the plasma biochemical indicators, the levels of ALT, AST and ALP.
(32) At the same time, plasma samples were prepared: 10 μL of plasma sample was taken, mixed with 190 μL of 67% (v/v) acetonitrile (containing 5 μM internal standard chlorpropamide), centrifuged at 18000 g for 20 minutes at 4° C., and the supernatant was taken for sample injection.
(33) Liver samples were prepared: 100 mg of liver samples were mixed well with 1 mL of 50% (v/v) acetonitrile (containing 5 μM internal standard chlorpropamide), shaken for 20 minutes at room temperature, and then centrifuged at 18000 g for 20 minutes at 4° C. Thereafter, 200 μL of the supernatant was taken and added with 200 μL of pure acetonitrile, vortexed for 1 minute, centrifuged at 18000 g for 20 minutes at 4° C., and the supernatant was taken for sample injection.
(34) The liquid chromatography conditions were as follows: the injection volume was 5 μL; the column temperature was 45° C.; the flow rate of mobile phase was 0.3 mL/min; the gradient elution was that acetonitrile concentration changed from 2% to 98% during 16 minutes of operation, wherein the mobile phase (water/acetonitrile solution) contained 0.01% formic acid. The elution procedure was as follows: during 0-12.sup.th minute, the acetonitrile concentration changed from 2% to 98% at a constant rate; during 12-14.sup.th minute, the acetonitrile concentration was maintained at 98%; during 14-14.1.sup.th minute, the acetonitrile concentration changed from 98% to 2%; during 14.1-16.sup.th minute, the acetonitrile concentration was maintained at 2%. Mass spectrometry conditions were as follows: the temperature of dry gas was 350° C., the pressure of nebulizer was 35 psi, and the voltage of capillary was 3.5 kV.
(35) Part of the collected mouse liver tissue was fixed overnight in formalin and tissue sections were stained with hematoxylin and eosin (H&E).
1.5 Experimental Results
(36) The H&E staining results of mouse liver tissues of each group were shown in
(37) The changes in body weight and liver weight of mouse in each group were shown in Table 1. The results showed that the mice in the blank group, the model group and the treatment group had uniform body weight, and there was no significant difference. However, when the mice were sacrificed, the liver weight (1.22 g) of the model group was significantly higher than that (1.00 g) of the blank group, and the liver weight/body weight ratio (5.78%) of the model group was significantly higher than that (4.56%) of the blank group, indicating the ANIT model group had severe liver injury. When the mice were sacrificed, the liver weight (1.01 g) of the treatment group was significantly lower than that (1.22 g) of the model group, and there was no significant difference in comparison with the mice of the blank group; and the liver weight/body weight ratio (4.66%) of the treated group was significantly lower than that (5.78%) of the model group, and there was no significant difference in comparison with the blank group, indicating that celastrol could significantly inhibit the increases in mouse liver weight and liver weight/body weight ratio induced by ANIT.
(38) TABLE-US-00001 TABLE 1 Changes in body weight and liver weight of mouse in each group Body weight of Liver weight of Liver weight/ mouse when mouse when body Group sacrificed (g) sacrificed (g) weight (%) Blank group 21.02 ± 0.94 1.00 ± 0.03 4.56 ± 0.10 ANIT model group 21.03 ± 0.94 1.22 ± 0.04** 5.78 ± 0.12** Treatment group 21.63 ± 1.15 1.01 ± 0.05.sup.## 4.66 ± 0.23.sup.## Note: as comparing the model group with the blank group, **P < 0.01; as comparing the treatment group with the model group, .sup.##P < 0.01.
(39) The changes of AST, ALT and ALP in mouse plasma of each group were shown in Table 2. The results showed that the levels of AST, ALT and ALP in plasma of the model group were significantly higher than those of the blank group (P<0.01), indicating that the liver tissue of the model group was severely damaged. The levels of AST, ALT and ALP of the treatment group were significantly lower than those of the model group (P<0.01), and there was no significant difference in comparison with the blank group, indicating that celastrol could significantly inhibit the increases of AST, ALT and ALP levels in plasma induced by ANIT.
(40) TABLE-US-00002 TABLE 2 Changes of AST, ALT and ALP in mouse plasma of each group Group AST (U/l) ALT (U/l) ALP (U/l) Blank group 43.0 ± 2.1 33.2 ± 4.6 20.0 ± 3.5 ANIT model 1869.2 ± 582.5** 1366.7 ± 548.3** 333.6 ± 63.2** group Treatment 72.0 ± 34.4.sup.## 54.0 ± 36.4.sup.## 27.3 ± 3.7.sup.## group Note: as comparing the model group with the blank group, **P < 0.01; as comparing the treatment group with the model group, .sup.##P < 0.01.
(41) The relative content changes of various bile acid components in mouse plasma of each group were shown in
(42) The relative content changes of various bile acid components in mouse liver of each group were shown in
(43) The above experimental results showed that celastrol had prevention and/or protection effects on ANIT-induced cholestatic liver injury, was capable of inhibiting the severe inflammatory infiltration and periportal hemorrhage induced by ANIT, inhibiting the increases of the levels of AST, ALT and ALP in mouse plasma induced by ANIT, and inhibiting the increases of the levels of bile acids in plasma and liver induced by ANIT.
Example 2: Prevention and/or Protection Effects of Celastrol on TAA-Induced Mouse Cholestasis Liver Disease
2.1 Experimental Animals
(44) C57BL/6 mice, SPF grade, body weight 20-23 g, male; certificate number: SCXK (Xiang) 2013-0004, purchased from Hunan SJA Laboratory Animal Co., Ltd.
2.2 Drugs and Reagents
(45) Celastrol, red amorphous crystalline powder, Chengdu Ruifensi Biotechnology Co., Ltd., Lot No. L-003-150420.
(46) Aspartate aminotransferase (AST) kit (Cat. No. C010-3, Lot No. 20161031), alanine aminotransferase (ALT) kit (Cat. No. C009-3, Lot No. 20161031) and alkaline phosphatase (ALP) kit (Cat. No. A059-3, Lot No. 20161031), produced by Nanjing Jiancheng Bioengineering Institute.
(47) Thioacetamide (TAA, Cat. No. C1290-25G, Lot No. 1002213299), DMSO (Cat. No. V900090-500m1, Lot No. 101669350), Tween 80 (Cat. No. P1754-500m1, Lot No. 101761909), formic acid (Cat. No. 94318-250m1-F, Lot No. 101721592), chlorpropamide (Cat. No. C1290-25G, Lot No. 1002213299), cholic acid (CA, Cat. No. C1129-25G), deoxycholic acid (DCA, Cat. No. D2510-10G), taurocholic acid (TCA, Cat. No. T4009-1G), glycocholic acid (GCA, Cat. No. G2878-500MG), taurohyodeoxycholic acid (THDCA, Cat. No. T0682-250MG), taurochenodeoxycholic acid (TCDCA, Cat. No. T6260-25MG), taurodeoxycholate acid (TDCA, Cat. No. T0875-1G) and taurolithocholic acid (TLCA, Cat. No. T7515-100MG), purchased from Sigma Co.
(48) Chromatographic grade acetonitrile (Cat. No. 1.00030.4008), purchased from Merck Co.
(49) NaCl injection (Lot No. B16080E1), purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.
(50) ω-muricholic acid (ω-MCA, Cat. No. sc-396738) and tauro-β-muricholic acid (TβMCA, Cat. No. SC-361829), purchased from Santa Cruz Co.
(51) Tauroursodeoxycholic acid (TUDCA, Cat. No. HY-19696A), purchased from MCE Co.
(52) β-muricholic acid (β-MCA, Cat. No. C008852) and tauro-α-muricholic acid (TαMCA, Cat. No. C1893-000), purchased from Steraloids Co.
2.3 Main Instruments
(53) Precision electronic balance, purchased from Sartorius Co.
(54) Automatic microplate reader, purchased from BioTek Instruments Co.
(55) SHA-C water bath constant temperature shaker, purchased from Changzhou Zhongcheng Instrument Manufacturing Co., Ltd.
(56) Refrigerated centrifuge, purchased from Eppendorf Co.
(57) Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry, including 1290 sample injector, 1290 pump, column oven, XDB-C18 column (2.1×100 mm, 1.8 μM), and 6530 quadrupole time-of-flight mass spectrometry, purchased from Agilent Co.
(58) Precellys 24 tissue homogenizer, purchased from Bertin Technologies Co.
(59) Upright microscope, purchased from Nikon Co.
(60) Tissue microtome, purchased from Leica Co.
2.4 Experimental Methods
2.4.1 Experimental Grouping
(61) A total of 18 male C57BL/6 mice were divided into three groups: blank group (n=6); model group (n=6); treatment group (n=6).
2.4.2 Preparation and Dosage of Drug Solution
(62) Celastrol was dissolved in a solvent to obtain a solution of celastrol at a concentration of 2 mg/mL, wherein the solvent consisted of 1% DMSO, 2% Tween, and 97% water (v:v:v). For intragastrical administration, the dosage of celastrol was 10 mg/kg.Math.d. TAA was dissolved in 0.9% NaCl solution to prepare a NaCl solution of TAA at a concentration of 60 mg/kg, and the dosage of TAA for intraperitoneal administration was 300 mg/kg.
2.4.3 Experimental Procedure
(63) The treatment group was administered intragastrically with the celastrol solution at a dosage of 10 mg/kg.Math.d for 4 consecutive days. At the same time, the blank group and the model group were intragastrically administered with the same dosage of a solvent, wherein the solvent consisted of 1% DMSO, 2% Tween 80 and 97% water (v:v:v), and the solvent was administrated for 4 consecutive days.
(64) On the 4.sup.th day, TAA model was established. 1 Hour after administration with the solvent or celastrol solution, the mice of the treatment group and the model group were respectively intraperitoneally injected with the NaCl solution of TAA at a dosage of 300 mg/kg to induce cholestatic liver injury. 24 Hours after treatment with TAA, the mice were sacrificed by CO.sub.2 anesthesia, and plasma samples and liver samples were collected. The eyeballs of the mice were taken for blood collection. The plasma samples were placed on ice for 1-2 hours, centrifuged at 4000 g for 5 minutes at 4° C., and the supernatant plasma samples were taken to determine the plasma biochemical indicators, the levels of ALT, AST and ALP.
(65) At the same time, plasma samples were prepared: 10 μL of plasma sample was taken, mixed with 190 μL of 67% acetonitrile (containing 5 μM internal standard chlorpropamide), centrifuged at 18000 g for 20 minutes at 4° C., and the supernatant was taken for sample injection.
(66) Liver samples were prepared: 100 mg liver samples were mixed well with 1 mL of 50% acetonitrile (containing 5 μM internal standard chlorpropamide), shaken for 20 minutes at room temperature, and then centrifuged at 18000 g for 20 minutes at 4° C. Thereafter, 200 μL of the supernatant was taken and added with 200 μl of pure acetonitrile, vortexed for 1 minute, centrifuged at 18000 g for 20 minutes at 4° C., and the supernatant was taken for sample injection. The liquid chromatography conditions were as follows: the injection volume was 5 μL; the temperature of column was 45° C.; the flow rate of mobile phase was 0.3 mL/min; the gradient elution was that acetonitrile concentration changed from 2% to 98% during 16 minutes of operation, wherein the mobile phase (water/acetonitrile solution) contained 0.01% formic acid. The elution procedure was as follows: during 0-12.sup.th minute, the acetonitrile concentration changed from 2% to 98% at a constant rate; 12-14.sup.th minute, the acetonitrile concentration was maintained at 98%; 14-14.1.sup.th minute, the acetonitrile concentration changed from 98% to 2%; 14.1-16.sup.th minute, the acetonitrile concentration was maintained at 2%. Mass spectrometry conditions were as follows: the temperature of dry gas was 350° C., the pressure of nebulizer was 35 psi, and the voltage of capillary was 3.5 kV.
(67) Part of the collected mouse liver tissue was fixed overnight in formalin and tissue sections were stained with hematoxylin and eosin (H&E).
2.5 Experimental Results
(68) The H&E staining results of mouse liver tissues of each group were shown in
(69) The changes in body weight and liver weight of mouse in each group were shown in Table 3. The results showed that the mice in the blank group, the model group and the treatment group had uniform body weight, and there was no significant difference. However, the liver weight (1.54 g) of the model group was significantly higher than that (1.18 g) of the blank group, and the liver weight/body weight ratio (6.37%) of the model group was significantly higher than that (4.92%) of the blank group, indicating the mice of the TAA model had severe liver injury. When the mice were sacrificed, there was no significant difference in liver weight (1.55 g) of the treatment group in comparison with the model group; and there was no significant difference in the liver weight/body weight ratio of (6.48%) the treatment group in comparison with the model group, indicating that the prevention and/or protection effects of celastrol had no significant influence on liver weight in the mice of the TAA model.
(70) TABLE-US-00003 TABLE 3 Changes in body weight and liver weight of mouse in each group Body weight of Liver weight of Liver mouse when mouse when weight/body Group sacrificed (g) sacrificed (g) weight (%) Blank group 24.02 ± 1.09 1.18 ± 0.09 4.92 ± 0.38 TAA model group 24.10 ± 0.75 1.54 ± 0.18** 6.37 ± 0.62** Treatment group 23.92 ± 0.63 1.55 ± 0.21 6.48 ± 0.93 Note: as comparing the model group with the blank group, **P < 0.01.
(71) The changes of AST, ALT and ALP in mouse plasma of each group were shown in Table 4. The results showed that the levels of AST, ALT and ALP in plasma of the TAA model group were significantly higher than those of the blank group (P<0.05), indicating that a severe liver injury occurred in the TAA model group. The levels of AST, ALT and ALP of the treatment group were lower than those of the model group (P<0.01), indicating that celastrol could significantly inhibit the increases of the levels of AST, ALT and ALP in plasma induced by TAA.
(72) TABLE-US-00004 TABLE 4 Changes of AST, ALT and ALP in mouse plasma of each group Group AST (U/l) ALT(U/l) ALP(U/l) Blank group 81.7 ± 45.0 8.0 ± 4.2 101.6 ± 13.0 TAA model group 2204.4 ± 172.0** 1407.5 ± 350.1** 121.9 ± 11.1* Treatment group 920.3 ± 433.4.sup.## 333.3 ± 211.3.sup.## 50.1 ± 7.8.sup.## Note: as comparing the model group with the blank group, *P < 0.05, **P < 0.01; as comparing the treatment group with the model group, .sup.##P < 0.01.
(73) The relative content changes of various bile acid components in mouse plasma of each group were shown in
(74) The relative content changes of various bile acid components in mouse liver of each group were shown in
(75) The above experimental results showed that celastrol had prevention and/or protection effects on TAA-induced cholestatic liver injury, was capable of inhibiting the severe inflammatory infiltration and periportal hemorrhage induced by TAA, inhibiting the increases of the levels of AST, ALT and ALP in mouse plasma induced by TAA, and inhibiting the increased bile acids levels in plasma and liver induced by TAA.
Example 3: Effect of Sirtuin 1 (SIRT1) on Celastrol Activity in ANIT Model
3.1 Experimental Animals
(76) C57BL/6 mice, SPF grade, body weight 20-23 g, male; certificate number: SCXK (Xiang) 2013-0004, purchased from Hunan SJA Laboratory Animal Co., Ltd.
3.2 Drugs and Reagents
(77) Celastrol, red amorphous crystalline powder, purchased from Chengdu Ruifensi Biotechnology Co., Ltd., Lot No. L-003-150420.
(78) EX527 (Selisistat, purchased from MedChem Express Co., Cat. No. HY-15453).
(79) Aspartate aminotransferase (AST) kit (Cat. No. C010-3, Lot No. 20161031), alanine aminotransferase (ALT) kit (Cat. No. C009-3, Lot No. 20161031) and alkaline phosphatase (ALP) kit (Cat. No. A059-3, Lot No. 20161031), produced by Nanjing Jiancheng Bioengineering Institute.
(80) α-Naphthyl isothiocyanate (ANIT, Cat. No. N4525-10G, Lot No. 101734146), DMSO (Cat. No. V900090-500m1, Lot No. 101669350), Tween 80 (Cat. No. P1754-500m1, Lot No. 101761909), formic acid (Cat. No. 94318-250m1-F, Lot No. 101721592), chlorpropamide (Cat. No. C1290-25G, Lot No. 1002213299), cholic acid (CA, Cat. No. C1129-25G), deoxycholic acid (DCA, Cat. No. D2510-10G), taurocholic acid (TCA, Cat. No. T4009-1G) and taurohyodeoxycholic acid (THDCA, Cat. No. T0682-250MG), purchased from Sigma Co.
(81) Corn oil (Cat. No. C116025-500G, Lot No. 11524089), purchased from Aladdin Co.
(82) Chromatographic grade acetonitrile (Cat. No. 1.00030.4008), purchased from Merck Co.
(83) Tauro-β-muricholic acid (TβMCA, Cat. No. SC-361829), purchased from Santa Cruz Co.
(84) Tauroursodeoxycholic acid (TUDCA, Cat. No. HY-19696A), purchased from MCE Co.
(85) Tauro-α-muricholic acid (TαMCA, Cat. No. C1893-000), purchased from Steraloids Co.
3.3 Main Instruments
(86) Precision electronic balance, purchased from Sartorius Co.
(87) Automatic microplate reader, purchased from BioTek Instruments Co.
(88) SHA-C water bath constant temperature shaker, purchased from Changzhou Zhongcheng Instrument Manufacturing Co., Ltd.
(89) Refrigerated centrifuge, purchased from Eppendorf Co.
(90) Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry, including 1290 sample injector, 1290 pump, column oven, XDB-C18 column (2.1×100 mm, 1.8 μM), and 6530 quadrupole time-of-flight mass spectrometry, purchased from Agilent Co.
(91) Upright microscope, purchased from Nikon Co.
(92) Tissue microtome, purchased from Leica Co.
3.4 Experimental Methods
3.4.1 Experimental Grouping
(93) A total of 24 male C57BL/6 mice were divided into four groups: blank group (n=6); model group (n=6); treatment group (n=6); and EX527 group (n=6).
3.4.2 Preparation and Dosage of Drug Solution
(94) Celastrol was dissolved in a solvent to obtain a celastrol solution at a concentration of 2 mg/mL, wherein the solvent consisted of 1% DMSO, 2% Tween 80, and 97% water (v:v:v). For intragastrical administration, the dosage of celastrol was 10 mg/kg.Math.d. ANIT was dissolved in corn oil to obtain a corn oil solution of ANIT at a concentration of 15 mg/mL, and the dosage of ANIT for intragastrical administration was 75 mg/kg. EX527 was dissolved in corn oil to obtain a corn oil solution of EX527 at a concentration of 2 mg/mL, and the dosage of EX527 for intraperitoneal injection was 10 mg/kg.Math.d.
3.4.3 Experimental Procedure
(95) The EX527 group was simultaneously administrated with the celastrol solution and the EX527 corn oil solution for 5 consecutive days. The treatment group was administrated with the celastrol solution and an equal dosage of corn oil for 5 consecutive days. At the same time, the blank group and the model group were administrated with equal dosages of solvent (1% DMSO, 2% Tween 80 and 97% water (v:v:v)) and corn oil, and the solvent was administrated for 5 consecutive days.
(96) On the 3.sup.rd day of administration, ANIT model was established. 4 Hours after administration, the mice in the model group, the treatment group and the EX527 group were respectively intraperitoneally injected with the ANIT corn oil solution at a dosage of 75 mg/kg for single administration to induce cholestatic liver injury, and the blank group was fed with an equal dosage of corn oil. After the model was established on the 3.sup.rd day of administration, the treatment group was continuously administrated with celastrol, the EX527 group was continuously administrated with celastrol and EX527, and the blank group and the model group were continuously administrated with the same dosage of solvent. 48 Hours after administration with ANIT, the mice were sacrificed by CO.sub.2 anesthesia, and mouse liver samples and plasma samples were taken. The eyeballs of the mice were taken for blood collection. The plasma samples were placed on ice for 1-2 hours, centrifuged at 4000 g for 5 minutes at 4° C., and the supernatants of the plasma samples were taken to determine plasma biochemical indicators, the levels of ALT, AST and ALP.
(97) At the same time, plasma samples were prepared: 10 μL of plasma sample was taken, mixed well with 190 μL of 67% acetonitrile (containing 5 μM internal standard chlorpropamide), centrifuged at 18000 g for 20 minutes at 4° C., and the supernatant was taken for sample injection. The liquid chromatography conditions were as follows: the injection volume was 5 μL; the temperature of column was 45° C.; the flow rate of mobile phase was 0.3 mL/min; the gradient elution was that acetonitrile concentration changed from 2% to 98% during 16 minutes of operation, wherein the mobile phase (water/acetonitrile solution) contained 0.01% formic acid. The elution procedure was as follows: during 0-12.sup.th minute, the acetonitrile concentration changed from 2% to 98% at a constant rate; during 12-14.sup.th minute, the acetonitrile concentration was maintained at 98%; during 14-14.1.sup.th minute, the acetonitrile concentration changed from 98% to 2%; during 14.1-16.sup.th minute, the acetonitrile concentration was maintained at 2%. Mass spectrometry conditions were as follows: the temperature of dry gas was 350° C., the pressure of nebulizer was 35 psi, and the voltage of capillary was 3.5 kV.
(98) Part of the collected mouse liver tissue was fixed overnight in formalin and tissue sections were stained with hematoxylin and eosin (H&E).
3.5 Experimental Results
(99) The H&E staining results of mouse liver were shown in
(100) The changes of AST, ALT and ALP in mouse plasma of each group were shown in
(101) The relative content changes of various bile acid components in mouse plasma of each group were shown in
Example 4: Effect of SIRT1 on Celastrol Activity in TAA Model
4.1 Experimental Animals
(102) C57BL/6 mice, SPF grade, body weight 20-23 g, male; certificate number: SCXK (Xiang) 2013-0004, purchased from Hunan SJA Laboratory Animal Co., Ltd.
4.2 Drugs and Reagents
(103) Celastrol, red amorphous crystalline powder, Chengdu Ruifensi Biotechnology Co., Ltd., Lot No. L-003-150420.
(104) EX527 (Selisistat, purchased from MedChem Express Co., Cat. No. HY-15453).
(105) Aspartate aminotransferase (AST) kit (Cat. No. C010-3, Lot No. 20161031), alanine aminotransferase (ALT) kit (Cat. No. C009-3, Lot No. 20161031) and alkaline phosphatase (ALP) kit (Cat. No. A059-3, Lot No. 20161031), produced by Nanjing Jiancheng Bioengineering Institute.
(106) Thioacetamide (TAA, Cat. No. C1290-25G, Lot No. 1002213299), DMSO (Cat. No. V900090-500m1, Lot No. 101669350), Tween 80 (Cat. No. P1754-500m1, Lot No. 101761909), formic acid (Cat. No. 94318-250m1-F, Lot No. 101721592), chlorpropamide (Cat. No. C1290-25G, Lot No. 1002213299), deoxycholic acid (DCA, Cat. No. D2510-10G), taurocholic acid (TCA, Cat. No. T4009-1G), taurohyodeoxycholic acid (THDCA, Cat. No. T0682-250MG), taurochenodeoxycholic acid (TCDCA, Cat. No. T6260-25MG) and taurodeoxycholic acid (TDCA, Cat. No. T0875-1G), purchased from Sigma Co.
(107) Chromatographic grade acetonitrile (Cat. No. 1.00030.4008), purchased from Merck Co.
(108) NaCl injection (Lot No. B16080E1), purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.
(109) ω-muricholic acid (ω-MCA, Cat. No. sc-396738) and tauro-β-muricholic acid (TβMCA, Cat. No. SC-361829), purchased from Santa Cruz Co.
(110) Tauroursodeoxycholic acid (TUDCA, Cat. No. HY-19696A), purchased from MCE Co.
(111) Tauro-α-muricholic acid (TαMCA, Cat. No. C1893-000), purchased from Steraloids Co.
4.3 Main Instruments
(112) Precision electronic balance, purchased from Sartorius Co.
(113) Automatic microplate reader, purchased from BioTek Instruments Co.
(114) SHA-C water bath constant temperature shaker, purchased from Changzhou Zhongcheng Instrument Manufacturing Co., Ltd.
(115) Refrigerated centrifuge, purchased from Eppendorf Co.
(116) Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry, including 1290 sample injector, 1290 pump, column oven, XDB-C18 column (2.1×100 mm, 1.8 μM), and 6530 quadrupole time-of-flight mass spectrometry, purchased from Agilent Co.
(117) Upright microscope, purchased from Nikon Co.
(118) Tissue microtome, purchased from Leica Co.
4.4 Experimental Methods
4.4.1 Experimental Grouping
(119) A total of 24 male C57BL/6 mice were divided into four groups: blank group (n=6);
(120) model group (n=6); treatment group (n=6); EX527 group (n=6).
4.4.2 Preparation and Dosage of Drug Solution
(121) Celastrol was dissolved in a solvent to obtain a solution of celastrol at a concentration of 2 mg/mL, wherein the solvent consisted of 1% DMSO, 2% Tween, and 97% water (v:v:v). For intragastrical administration, the dosage of celastrol was 10 mg/kg.Math.d. TAA was dissolved in 0.9% NaCl solution to prepare a NaCl solution of TAA at a concentration of 60 mg/mL, and the dosage of TAA for intraperitoneal administration was 300 mg/kg. EX527 was dissolved in corn oil to prepare a corn oil solution of EX527 at a concentration of 2 mg/mL, and the dosage of EX527 for intraperitoneal administration was 10 mg/kg. d.
4.4.3 Experimental Procedure
(122) The EX527 group was simultaneously administered with the celastrol solution and the EX527 corn oil solution for 4 consecutive days. The treatment group was administrated with the celastrol solution and an equal dosage of corn oil for 4 consecutive days. At the same time, the blank group and the model group were administrated with equal dosages of solvent (1% DMSO, 2% Tween 80 and 97% water (v:v:v)) and corn oil, and the solvent was administrated for 4 consecutive days.
(123) On the 4.sup.th day, the establishment of TAA model was performed. 1 Hour after administration, the mice in the model group, the treatment group and the EX527 group were intraperitoneally injected with TAA at a dosage of 300 mg/kg to induce cholestatic liver injury. 24 Hours after administration with TAA, the mice were sacrificed by CO.sub.2 anesthesia, and plasma samples and liver samples were collected. The eyeballs of the mice were taken for blood collection. The plasma samples were placed on ice for 1-2 hours, centrifuged at 4000 g for 5 minutes at 4° C., and the supernatants of plasma samples were taken to determine the plasma biochemical indicators, the levels of ALT, AST, and ALP.
(124) At the same time, plasma samples were prepared: 10 μL of plasma sample was mixed with 190 μL of 67% acetonitrile (containing 5 μM internal standard chlorpropamide), centrifuged at 18000 g for 20 minutes at 4° C., and the supernatant was taken for sample injection. The liquid chromatography conditions were as follows: the volume of injection was 5 μL; the temperature of column was 45° C.; the flow rate of mobile phase was 0.3 mL/min; the gradient elution was that acetonitrile concentration changed from 2% to 98% during 16 minutes of operation, wherein the mobile phase (water/acetonitrile solution) contained 0.01% formic acid. The elution procedure was as follows: during 0-12.sup.th minute, the acetonitrile concentration changed from 2% to 98% at a constant rate; during 12-14.sup.th minute, the acetonitrile concentration was maintained at 98%; during 14-14.1.sup.th minute, the acetonitrile concentration changed from 98% to 2%; during 14.1-16.sup.th minute, the acetonitrile concentration was maintained at 2%. Mass spectrometry conditions were as follows: the temperature of dry gas was 350° C., the pressure of nebulizer was 35 psi, and the voltage of capillary was 3.5 kV.
(125) Part of the collected mouse liver tissue was fixed overnight in formalin, and tissue sections were stained with hematoxylin and eosin (H&E).
4.5 Experimental Results
(126) The H&E staining results of mouse liver tissues of each group were shown in
(127) The changes of AST, ALT, and ALP in mouse plasma of each group were shown in
(128) The relative content changes of various bile acid components in mouse plasma of each group were shown in
Example 5: Effect of Farnesoid X Receptor (FXR) on Celastrol Activity in ANIT Model
(129) The same experimental method of Example 1 was used, wherein the experimental animals were FXR knockout mice (C57BL/6J as background), and the FXR knockout mice were presented by Frank J. Gonzalez, a researcher of the Cancer Institute of National Institutes of Health, USA.
(130) The changes of AST, ALT, and ALP in plasma of FXR knockout mice of each group were shown in
(131) The relative content changes of various bile acid components in plasma of FXR knockout mice of each group were shown in
Example 6: Effect of FXR on Celastrol Activity in TAA Model
(132) The same experimental method of Example 2 was used, wherein the experimental animals were FXR knockout mice (C57BL/6J as background), and the FXR knockout mice were presented by Frank J. Gonzalez, a researcher of the Cancer Institute of National Institutes of Health, USA.
(133) The H&E staining results of liver tissue of FXR knockout mice of each group were shown in
(134) The changes of AST, ALT, and ALP in plasma of FXR knockout mice of each group were shown in
(135) The relative content changes of various bile acid components in plasma of FXR knockout mice of each group were shown in
Example 7: Therapeutic Effect of Celastrol on TAA-Induced Liver Fibrosis in Mice
(136) 7.1 Experimental Animals: C57BL/6 Mice, SPF Grade, Body Weight 20-23 g, Male; Certificate Number: SCXK (Xiang) 2013-0004, Purchased from Hunan SJA Laboratory Animal Co., Ltd
(137) 7.2 Drugs and Reagents
(138) Celastrol, red amorphous crystalline powder, purchased from Chengdu Ruifensi Biotechnology Co., Ltd., Lot No. L-003-150420.
(139) Aspartate aminotransferase (AST) kit (Cat. No. C010-3, Lot No. 20161031), alanine aminotransferase (ALT) kit (Cat. No. C009-3, Lot No. 20161031), and alkaline phosphatase (ALP) kit (Cat. No. A059-3, Lot No. 20161031), produced by Nanjing Jiancheng Bioengineering Institute.
(140) Thioacetamide (TAA, Cat. No. C1290-25G, Lot No. 1002213299), DMSO (Cat. No. V900090-500m1, Lot No. 101669350) and Tween 80 (Cat. No. P1754-500m1, Lot No. 101761909), purchased from Sigma Co.
(141) NaCl injection (Lot No. B16080E1), purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.
(142) 7.3 Main Instruments: Precision Electronic Balance, Purchased from Sartorius Co.; Automatic Microplate Reader, Purchased from BioTek Instruments Co.; SHA-C Water Bath Constant Temperature Shaker, Purchased from Changzhou Zhongcheng Instrument Manufacturing Co., Ltd.; Refrigerated Centrifuge, Purchased from Eppendorf Co.; Upright Microscope, Purchased from Nikon Co.; Tissue Microtome, Purchased from Leica Co
(143) 7.4 Experimental Method
(144) 7.4.1 Experimental Grouping
(145) A total of 30 male C57BL/6 mice were divided into three groups: blank group (n=10); TAA liver fibrosis group (n=10); treatment group (n=10).
(146) 7.4.2 Preparation and Dosage of Drug Solution
(147) Celastrol was dissolved in a solvent to obtain a celastrol solution at a concentration of 2 mg/mL, wherein the solvent consisted of 1% DMSO, 2% Tween 80, and 97% water (v:v:v); for intragastrical administration, the dosage of celastrol was 10 mg/kg/time, three times per week, and the administration was absent in the first week. TAA was dissolved in 0.9% NaCl solution to prepare a NaCl solution of TAA at a concentration of 20 mg/kg, and the dosage for intraperitoneal administration was 100 mg/kg/time in the first week and 160 mg/kg/time in the second to sixth week, three times per week.
(148) 7.4.3 Experimental Procedure
(149) The model group and the treatment group were administrated with TAA three times per week, and administered every other day. The dosage of TAA was 100 mg/kg each time during the first week. From the second week onwards, the dosage of TAA administered to the model group and the treatment group was changed to 160 mg/kg each time. The blank group was given an equal dosage of NaCl solution.
(150) The treatment group was administrated with celastrol from the second week, in which celastrol at a dosage of 10 mg/kg was administered 1 hour after administration with TAA. The blank group and the model group were administrated with an equal dosage of solvent (1% DMSO, 2% Tween 80, and 97% water (v: v: v)).
(151) Six weeks later, mice were sacrificed by CO.sub.2 anesthesia, and mouse plasma samples and liver samples were collected. The eyeballs of the mice were taken for blood collection. The plasma samples were placed on ice for 1-2 hours, centrifuged at 4000 g for 5 minutes at 4° C., and the supernatants of plasma samples were taken to determine the plasma biochemical indicators, the levels of ALT, AST, and ALP.
(152) Part of the collected mouse liver tissue was fixed overnight in formalin, and tissue sections were stained with hematoxylin and eosin (H&E).
(153) 7.5 Experimental Results
(154) The H&E staining results of mouse liver tissue of each group were shown in
(155) The changes of AST, ALT, and ALP in mouse plasma of each group were shown in
(156) It should be noted that the above examples are only used to illustrate the technical solutions of the present disclosure and are not to be construed as limitation thereof; although the present disclosure will be described in detail with reference to the preferred examples, those skilled in the art should understand that the specific embodiments as disclosed can be amended and partial technical features of the present disclosure are can be replaced without departing from the spirit of the technical solution of the present disclosure, all these changes fall into the protection scope sought to be protected by the present application.