To examine how Jiang-Zhi-Ning (JZN) regulates cholesterol metabolism and compare the role of its four main components. We established a beagle model of hyperlipidemia, fed with JZN extract and collected JZN-containing serum 0, 1, 2, 4, and 6 h later. Human liver cells Bel-7402 were stimulated with 10% JZN-containing serum as well as the four main components of JZN and Atorvastatin. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoAR), cytochrome P450 7A1 (CYP7A1), and acetyl-Coenzyme A acetyltransferase 2 (ACAT2) was measured by real-time PCR. LDL-R surface expression and LDL-binding and internalization were examined by flow cytometry. The results showed that JZN-containing serum significantly increased the mRNA expression of LDL-R, HMG-CoAR, and CYP7A1 in Bel-7402 cells. All the four components significantly increased the mRNA and protein expression of LDL-R and HMG-CoAR and decreased the mRNA and protein expression of ACAT2 in Bel-7402 cells. Hyperinand chrysophanol also markedly increased the mRNA expression of CYP7A1. Stimulation with stilbene glycosidesignificantly increased the surface expression of LDL-R and the binding and internalization of LDL. In conclusion, JZN and its four components have close relationship with the process of cholesterol metabolism, emphasizing their promising application as new drug candidates in the treatment of hyperlipidemia.
Numerous studies have shown that cholesterol plays a key role in the development of atherosclerosis, which is the main pathological basis of cardiovascular diseases [
Cholesterol metabolism is a complicated homeostasis involving multiple steps, including cholesterol absorption, synthesis, conversion, and modification. LDL receptor (LDL-R) plays a critical role in cholesterol absorption. In the serum, cholesterol mainly exists in the form of cholesterol ester and is carried and transported by lipoproteins, such as LDL, apolipoprotein B100 (Apo B100), and apolipoprotein E (Apo E). LDL-R binds to these lipoproteins and internalizes them into cells, providing lipids for cell proliferation and synthesis of steroid hormones and bile salt. These animals metabolized 7.1 pools of LDL-cholesterol (LDL-C) per day, and 79% of this degradation took place in the liver. Of this total turnover, the LDLR accounted for 88% while the remaining 12% was receptor independent. 91% of the receptor-dependent transport identified in these animals was located in the liver while only 38% of the receptor-independent uptake was found in this organ [
Cholesterol in the body can be obtained from food intake or from liver biosynthesis, the latter accounting for generating 70% to 80% of serum total cholesterol [
Another important step in cholesterol metabolism is the conversion of cholesterol into bile acid through cytochrome P450-meidiated oxidation. The rate-limiting enzyme for the dominant pathway of bile acid synthesis, the so-called classic pathway, is cytochrome P450 7A1 (CYP7A1). Human CYP7A1 gene defect can cause cholesterol accumulation in the liver, which has been associated with hypercholesterolemia [
Another interesting participant in cholesterol metabolism is acetyl-CoA acetyltransferase 2 (ACAT2), which esterifies cholesterol in the small intestine and liver. Cholesterol esters synthesized by ACAT2 are packed into very-low-density lipoprotein and secreted into blood. It is observed in an atherosclerosis model of Apo E-deficient mice that the formation of atherosclerosis can be prevented by simultaneous knockout of ACAT2, suggesting that ACAT2-mediated cholesterol esterification is important for atherosclerosis [
Among all current lipid-regulating drugs, Atorvastatin shows the most significant clinical effects. Atorvastatin decreases the cholesterol level by inhibiting HMG-CoAR and promotes the transcription of LDL-R by activating the transcription factor steroid response element binding protein 2 (SREBP2). The defined targets and marked lipid-lowering effects of Atorvastatin have made it a major breakthrough in lipid-lowering drugs. Atorvastatin has now become a preferred first-line lipid-regulating drug. However, statins may cause serious side reactions, such as liver toxicity and statin myopathy, with symptoms including increases in alanine aminotransferase and aspartate aminotransferase, dermatomyositis and polymorphic myositis, and so forth. In severe cases, statins can lead to rhabdomyolysis. Therefore, it is imperative to develop novel, safe, and effective lipid-regulating drugs.
Traditional Chinese Medicine (TCM) has been used to prevent and cure atherosclerosis and lower lipid for thousands of years. Jiang-Zhi-Ning (JZN), a widely used ready-made Chinese medicine, is composed of stilbene glycoside (from ShouWu, fleeceflower root), hyperin (from ShanZha, fructus crataegi), nuciferine (from HeYe, folium nelumbinis), and chrysophanol (from JueMingZi, semen cassiae). JZN has been in clinical application for more than 1300 years and has been shown to significantly lower serum cholesterol levels. The four herbs in JZN, Fleeceflower Root, Fructus Crataegi, Folium Nelumbinis, and Semen Cassiae have been used in clinic on obesity for centuries as in “QianJinFang” (Prescriptions Worth Thousands Gold). Though at that time the disease is not called as hyperlipidemia, but recent research has confirmed that those herbs have significant effect on lowing serum cholesterol levels [
This study aims to examine the underlying mechanisms of the lipid-lowering role of JZN and compare how its four main components contribute to this function. Cholesterol metabolism after drug treatment was examined by analyzing the expression of LDL-R, HMG-CoAR, CYP7A1, and ACAT2.
Adult laboratory beagles weighted between
Fleeceflower root is the processed product of the dried root of polygonum multiflorum thunb. Folium nelumbinis is dried leaf of nelumbo nucifera gaertn. Fructus crataegi and semen cassiae are dried mature fruits of
Fleeceflower root (25 g) and folium nelumbinis (75 g) were mixed and 25-fold of 50% ethanol was added. The mixture was heated and refluxed for 1.5 h. Then, ethanol extract was swilled and the residues were filtered and distilled twice. The three extracts were combined and concentrated into ointment, which was named as whole solid I. Fructus crataegi (500 g) and semen cassiae (25 g) were mixed and 7-fold of water was added. The mixture was heated and refluxed for 2 h. Then, the liquid extract was swilled and the residues were filtered and distilled. The two liquid extracts were combined and concentrated into ointment, which was named as whole solid II. The two whole solids were mixed and then dried using decompression drying method at 50°C.
Hyperin (from ShanZha) and stilbene glycoside (from ShouWu) were dissolved in sterilized water. Nuciferine (from HeYe) was dissolved in hydrochloric acid and then the pH was adjusted to 7.0 by sodium hydroxide. Chrysophanol and Atorvastatin were dissolved in DMSO.
To establish a beagle model of hyperlipidemia, beagles were adaptively fed for one week and then fed with high-fat feed for two months. The fat feed was composed of 88% of usual feed, 10% of lard, and 2% of cholesterol. Beagles with hyperlipidemia were then fasted for 12 h and fed with 0.4 g/kg of JZN extract. 10 mL of vein blood was withdrawn from the back limb of beagles after 0, 1, 2, 4, and 6 h. The blood was centrifuged at 3000 r/min for 10 min to obtain serums, which were heat-inactivated for 30 min at 56°C and filtered through 0.22
The human serum was collected and centrifuged with gradient solution in a density of 1.0, 1.1, and 1.3 at 4500 r/min for 10 min. The yellow lipid in the center of the solution was extracted as LDL. After a 48-h dialysis in the dialysate, which contains 0.02 M Tris, 0.01% EDTA, and 0.9% NaCl, pH 7.4, the isolated LDL was kept at 4°C. The separation of LDL was confirmed by gelose gel electrophoresis with oil red O staining. LDL concentration was determined by the bicinchoninic acid method.
Bel-7402 cells were cultured in 6-well plates at
Bel-7402 cells in logarithmic growth were cultured in 35 mm or 100 mm flasks at
Total RNA was extracted from drug-stimulated Bel-7402 cells using Trizol one-step method and then reverse-transcribed into cDNA using AMV Reverse Transcriptase. RT-PCR was conducted by the CYBR green method using following primers (Beijing Bioko Biotechnology Company, Beijing, China).
Equal amount of protein (50
Cells from the high-dosage group of stilbene glycoside treatment were washed twice with PBS and then once with serum-free medium. Cells were incubated for 1 h in serum-free medium containing 2% BSA and then washed with 0.5% BSA. For examining surface expression of LDL-R, cells were incubated with FITC-conjugated anti-LDL-R primary antibody and then analyzed by flow cytometry. For analyzing LDL binding and internalization, cells were incubated with 2
We first established a beagle model of hyperlipidemia, fed these beagles with JZN extract, and collected their serum after 0, 1, 2, 4, and 6 h. In Bel-7402 cells, 10% of JZN-containing serum from these beagles significantly increased the mRNA expression of LDL-R, HMG-CoAR, and CYP7A1 after X h stimulation (
The effects of JZN-containing serum on cholesterol metabolism in liver cells.
Time point | LDL-R | HMG-CoAR | CYP7A1 | ACAT2 |
---|---|---|---|---|
0 h | 1 | 1 | 1 | 1 |
1 h | ||||
2 h | ||||
4 h | ||||
6 h |
Note: comparison with 0-h time point,
As a positive control, we first tested the effects of Atorvastatin on cholesterol metabolism. Atorvastatin treatment increased the mRNA expression of LDL-R, HMG-CoAR, and CYP7A1 and decreased the mRNA expression of ACAT2 (
The effects of stilbene glycoside treatment on cholesterol metabolism in liver cells (mRNA analysis).
Treatment | LDL-R | HMG-CoAR | CYP7A1 | ACAT2 |
---|---|---|---|---|
No stilbene glycoside | ||||
Stilbene glycoside: low dose | ||||
Stilbene glycoside: medium dose | ||||
Stilbene glycoside: high dose | ||||
Atorvastatin |
Note: comparison with no-drug control group, *
The effects of nuciferine treatment on cholesterol metabolism in liver cells (mRNA analysis).
Treatment | LDL-R | HMG-CoAR | CYP7A1 | ACAT2 |
---|---|---|---|---|
No nuciferine | 1 | 1 | 1 | 1 |
Nuciferine: low dose | ||||
Nuciferine: medium dose | ||||
Nuciferine: high dose | ||||
Atorvastatin |
Note: comparison with no-drug control group, *
The effects of hyperin on cholesterol metabolism in liver cells (mRNA analysis).
Treatment | LDL-R | HMG-CoAR | CYP7A1 | ACAT2 |
---|---|---|---|---|
No hyperin | 1 | 1 | 1 | 1 |
Hyperin: low dose | ||||
Hyperin: medium dose | ||||
Hyperin: high dose | ||||
Atorvastatin |
Note: comparison with no-drug control group, *
The effects of chrysophanol treatment on cholesterol metabolism in liver cells (mRNA analysis).
Treatment | LDL-R | HMG-CoAR | CYP7A1 | ACAT2 |
---|---|---|---|---|
No chrysophanol | ||||
Chrysophanol: low dose | ||||
Chrysophanol: medium dose | ||||
Chrysophanol: high dose | ||||
Atorvastatin |
Note: comparison with no-drug control group, *
Stilbene glycoside and nuciferine showed a similar pattern of influence on cholesterol metabolism in liver cells. Compared with the no-drug control group, mRNA expression of LDL-R and HMG-CoAR in liver cells was significantly increased after stilbene glycoside or nuciferine stimulation (
Hyperin and chrysophanol showed similar influence on cholesterol metabolism in liver cells. Compared with the control group, stimulation with hyperin or chrysophanol also significantly increased the mRNA expression of LDL-R and HMG-CoAR (
Atorvastatin and the four components of JZN showed similar affects. Protein expression of LDL-R and HMG-CoAR was significantly increased under all treatments (
The effects of stilbene glycoside treatment on cholesterol metabolism in liver cells (protein analysis).
Treatment | LDL-R | HMG-CoAR | ACAT2 |
---|---|---|---|
No stilbene glycoside | |||
Stilbene glycoside: low dose | |||
Stilbene glycoside: medium dose | |||
Stilbene glycoside: high dose | |||
Atorvastatin |
Note: comparison with no-drug control group, *
The effects of nuciferine treatment on cholesterol metabolism in liver cells (protein analysis).
Treatment | LDL-R | HMG-CoAR | ACAT2 |
---|---|---|---|
No nuciferine | |||
Nuciferine: low dose | |||
Nuciferine: medium dose | |||
Nuciferine: high dose | |||
Atorvastatin |
Note: comparison with no-drug control group, *
The effects of hyperin on cholesterol metabolism in liver cells (protein analysis).
Treatment | LDL-R | HMG-CoAR | ACAT2 |
---|---|---|---|
No hyperin | |||
Hyperin: low dose | |||
Hyperin: medium dose | |||
Hyperin: high dose | |||
Atorvastatin |
Note: comparison with no-drug control group, *
The effects of chrysophanol treatment on cholesterol metabolism in liver cells (protein analysis).
Treatment | LDL-R | HMG-CoAR | ACAT2 |
---|---|---|---|
No chrysophanol | |||
Chrysophanol: low dose | |||
Chrysophanol: medium dose | |||
Chrysophanol: high dose | |||
Atorvastatin |
Note: comparison with no-drug control group, *
LDL-R, HMG-CoAR, and ACAT2 protein expression under stilbene glycoside treatment. Notes: Lane 1: no-drug control; 2: low dose of stilbene glycoside; 3: medium dose of stilbene glycoside; 4: high dose of stilbene glycoside; 5: Atorvastatin.
LDL-R, HMG-CoAR, and ACAT2 protein expression under nuciferine treatment. Notes: Lane 1: no-drug control; 2: low dose of nuciferine; 3: medium dose of nuciferine; 4: high dose of nuciferine; 5: Atorvastatin.
LDL-R, HMG-CoAR, and ACAT2 protein expression under hyperin treatment. Notes: Lane 1: no-drug control; 2: low dose of hyperin; 3: medium dose of hyperin; 4: high dose of hyperin; 5: Atorvastatin.
LDL-R, HMG-CoAR, and ACAT2 protein expression under chrysophanol treatment. Notes: Lane 1: No-drug control; 2: Low dose of chrysophanol; 3: Medium dose of chrysophanol; 4: High dose of chrysophanol; 5: Atorvastatin.
Our flow cytometry analysis showed that similar to Atorvastatin, stilbene glycoside significantly increased surface expression of LDL-R, as well as binding and internalization of LDL (
The effects of stilbene glycoside treatment on the surface expression of LDL-R.
Treatment | LDL-R Expression | LDL Binding | LDL Internalization |
---|---|---|---|
No-drug | |||
Stilbene glycoside | |||
Atorvastatin |
Note: comparison with no-drug control group, **
The effects of stilbene glycoside treatment on the surface expression of LDL-R. Treatment of stilbene glycoside or atorvastatin increased LDL-R surface expression as well as LDL binding and internalization.
Hyperlipidemia causes progressive atherosclerosis, which is the major cause of cardiovascular diseases. Clinically, 80% to 90% of acute coronary events are triggered by sudden collapse of atherosclerotic plaques [
Many different types of lipid-regulating drugs, including resins, statins, niacin, and unsaturated fatty acid lipid-regulating drugs, have been developed. However, most of these drugs only focus on one step of cholesterol metabolism. Few currently available drugs target cholesterol synthesis, absorption, modification, and conversion all together. In addition, most lipid-regulating drugs cause serious side effects, including gastrointestinal problems and liver and kidney dysfunction.
Numerous lipid-lowering formulae have been recorded in TCM. However, their application has been limited due to a lack of thorough understanding of both their effective ingredients and underlying mechanisms. There is also no standardized quality control in drug preparation. Therefore, it is imperative to examine active ingredients from known lipid-lowering TCM for their pharmacological effects. This has been proven to be an effective strategy to develop novel, more efficacious lipid-regulating drugs with fewer side effects. It has been reported that the activity of LDL-R can be enhanced by tea polyphenol as well as the water-soluble and ethanol-soluble extracts of turmeric [
JZN shows significant cholesterol-lowering effects. Polygonum multiflorum thunb, hawthorn, lotus leaf, and cassia seed are all frequently used as lipid-lowering TCM. Total glycoside from polygonum multiflorum thunb shows significant lipid-lowering and antioxidant effects [
However, the current understanding of JZN or its four major components is still far from satisfactory. Stilbene glycoside has been shown to exert neuroprotectivity, antioxidation, and lipid-lowering effects [
In the current study, we first examined the role of JZN in lipid metabolism in liver cells. It is worth noting that for a long time, a routine TCM pharmacological experiment is conducted as follows: ingredients of TCM are first extracted and separated and then added directly to an
Medicine-containing serum is collected from animals fed with TCM after a certain period of time. This TCM serum method mimics the genuine route of TCM to take effects
Cholesterol absorption and synthesis are negatively regulated by their end products. When cholesterol levels are low (e.g., when treated with Atorvastatin), the transcription factor SREBP is activated and triggers the transcription of LDL-R and HMG-CoAR [
In this study, all four major components of JZN, stilbene glycoside, nuciferine, hyperin, and chrysophanol, significantly increase the mRNA and protein expression of LDL-R and HMG-CoAR and significantly reduce the mRNA and protein expression of ACAT2. Hyperin and chrysophanol also significantly increase the mRNA expression of CYP7A1. In addition, stilbene glycoside increases the surface expression of LDL-R as well as the surface binding and internalization of LDL. Our results showed that the components of JZN may exert lipid-lowering effects by increasing the surface expression and activity of LDL-R in liver cells, inhibiting intracellular cholesterol synthesis, increasing the conversion of cholesterol into bile acid, and reducing ACAT2-mediated cholesterol esterification. Our study suggested that these four components of JZN are promising candidates for developing new medications to treat hyperlipidemia and related diseases.
Jianxin Chen, Huihui Zhao, and Xueling Ma contributed equally to this work.
This work was supported by the National Basic Research Program of China (973 Program) under Grant no. 2011CB505106, the Creation for Significant New Drugs under Grant no. 2009ZX09502-018, the International Science and Technology Cooperation of China under Grant no. 2008DFA30610, National Science Foundation of China under Grant no. 81173463 30902020 and 81102730, the New Century Excellent Talent Support Plan of the Ministryof Education under Grant no. NCET-11-0607, the Beijing Science and Technology Star under Grant no. 2011069, the Beijing Common special construction projects, and the Foundation of Beijing University of Chinese Medicine of Education Ministry of China under Grant no. 2011-CXTD-06 and 2011JYBZZ-JS090.