The Anti-hyperlipidemia Effects of Raw Polygonum multiflorum Extract in Vivo

Abstract

Polygonum multiflorum is widely used in the prevention and treatment of hyperlipidemia in traditional Chinese Medicine. In this study, the effects and relevant mechanisms of lipid-regulation by raw Polygonum multiflorum (RPM) were investigated. The results indicated that the basal plasma lipids, such as low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG), were significantly decreased in RPM treatment groups compared with the model group, especially in the RPM high dose group. The key enzymes involved in lipid metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) in plasma were generally reduced after oral administration, which was consistent with the transcription levels of their target genes. In addition, the hepatotoxicity of RPM was investigated, and RPM showed slightly less liver injury than that induced by simvastatin. Histological analysis indicated that the fat vacuoles and steatosis in hepatocytes were relieved after oral administration of RPM extract at a high dose of 16.2 g/kg, which was more obvious than that induced by simvastatin. These results revealed that RPM exerted its lipid-lowering effect by regulating the expression of related genes, and performed better than simvastatin in the treatment of hyperlipidemia.

Hyperlipidemia is a common metabolic syndrome characterized by increased total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) and decreased high-density lipoprotein cholesterol (HDL-C) levels,¹⁾ which facilitate endothelial dysfunction and atherogenesis, the major risk factor for cardiovascular disease.²⁾ Statins are commonly used in clinical treatment as first-line lipid-lowering drugs, especially in the treatment of hyperlipidemia. They act to suppress the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and thus affect the formation of mevalonic acid, the rate-limiting step in the biosynthesis of cholesterol,³⁾ but this treatment is often accompanied by adverse reactions such as gastrointestinal tract issues, myopathy and hepatotoxicity.^4,5) Traditional Chinese Medicine (TCM) is a healthcare-focused medical system based on more than 3000 years of continuous practice experience, and therefore has its own advantages and characteristics in early intervention and personalized treatment. Some Chinese medicinal herbs have shown lipid-lowering effects by reducing the absorption of exogenous lipids and the synthesis of endogenous lipids, and also by facilitating the transportation, metabolism and excretion of lipids.⁶⁾

TCM performs multi-target network regulation based on the function of the whole body; between its characteristic good efficiency and low toxicity, TCM has received extensive attention all over the world. There are various Chinese medicinal herbs clinically prescribed to cure hyperlipidemia. Among these herbs, Polygonum multiflorum ranked as the fifth most frequently used crude drug.⁶⁾ Recent studies have demonstrated that the extract of Polygonum multiflorum THUNB. (PM) exhibited prominent effects, particularly in cardiovascular disease.^6,7) PM possesses such pharmacological effects as anti-hyperlipidaemia,⁸⁾ anti-aging,⁹⁾ antioxidant,¹⁰⁾ hepatoprotective¹¹⁾ and anti-inflammatory.¹²⁾ Although both raw Polygonum multiflorum (RPM) and processed Polygonum multiflorum (PMP, which was originated from RPM steamed with black beans) feature a history of treatment in the cure of hyperlipidemia, RPM exerted the most beneficial effects.¹³⁾ It was found that processing decreases the contents of some ingredients correlated with its hypolipidemic effect.¹⁴⁾ Therefore, in our work, the anti-hyperlipidemia activities of RPM were chosen for deeper study. Most research has shown that RPM and its preparations significantly decrease TC, TG, and LDL-C levels regardless of the monomer component¹⁵⁾ or extract.¹⁶⁾ Mechanism studies were mainly focused on the molecular level,¹⁷⁾ with a lack of systematic assessment based on the animal level. In addition, an adverse hepatic reaction of RPM and its preparations have been reported.^18,19) However, its toxicity and efficacy were studied separately, and again lack systematic research. Therefore, it is necessary to now investigate both the lipid-lowering effect and hepatotoxicity of RPM extract simultaneously.

In this study, we have evaluated the lipid-lowering effect and revealed the relevant mechanism of RPM extract in rats fed a high fat diet (HFD). At the same time, its hepatotoxicity was investigated during our experiments.

MATERIALS AND METHODS

Chemicals

Simvastatin (Hangzhou MSD Pharmaceutical Co., Ltd., China) was used as a positive control. Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Bioengineering Institute. SYBR Green PCR kit, RNAprep Pure Tissue kit and FastQuant reverse transcription kit came from Tiangen Biotech Co., Ltd. (Beijing). The primer was provided by Sangon Biotech Co., Ltd. (Shanghai).

Extraction of RPM

RPM was purchased from Beijing San He Co., Ltd. (Beijing, China). Air-dried, powdered RPM (43 kg) was extracted with 70% ethanol (430 L×1.5 h×3). The extraction was combined, condensed and lyophilized to yield 5.4 kg RPM powder. The final RPM powder was used as a therapeutic drug for hyperlipidemia rats.

Chromatographic Analysis of RPM Extract

RPM extract was analyzed by HPLC using an Agilent 1260 Infinity (America) gradient liquid chromatograph. A poroshell 120 EC-C18 column (50 mm×4.6 mm×5 µm) was used. Column temperature was kept at 35°C, and a 5 µL sample was injected into the column and eluted with a constant flow rate of 1.0 mL/min. The UV detection wavelength was set at 254 nm and the main components of RPM extract were determined. The contents of 2,3,5,4′-tetrahydroxy-stilbene-2-Ο-β-D-glucoside, emodin-8-O-β-D-glucoside, physcion-8-O-β-D-glucoside, emodin, chrysophanol and physcion were 2.93, 0.16, 0.01, 0.06, 0.006 and 0.04%, respectively, as determined by HPLC analysis. The chromatograms of standards and RPM are listed in Supplementary Figure S1.

Animals and Experimental Design

Thirty six male Sprague-Dawley (SD) rats were purchased from Beijing HFK Bioscience Co., Ltd., China. After one week of acclimation, rats (weighing 230–240 g) were randomly divided into 6 groups (Table S1) and fed a standard diet (C), a high-fat diet (M), a simvastatin-supplemented high-fat diet (S, 1.2 mg/kg), or a RPM extract-supplemented high-fat diet (M+PML, 2.7 g/kg; M+PMM, 8.1 g/kg; M+PMH, 16.2 g/kg) for 4 months. Groups C, M and S served as the blank control, model control and positive control group, respectively. Group M+PML, M+PMM and M+PMH served as RPM treatment groups. Standard diets and HFD²⁰⁾ which contained 2% cholesterol, 10% lard, 10% powdered egg yolk, 0.2% bile salt, and 78.8% standard diet were provided by Beijing HFK Bioscience Co., Ltd., China. The animals were maintained at a steady temperature and humidity (20–23°C, 50–60%) under a 12 h light/dark cycle). All animal procedures were conducted in accordance with the guidelines for Animal Experimentation of the China Academy of Chinese Medical Sciences.

Body weights were measured once a week to adjust the dosage. Rats were sacrificed at the end of the experiment. Liver tissue was rapidly excised, weighed, and stored at −80°C until use. Samples of the resected liver were later used for analysis of the histology, and in RT-PCR.

Biochemical Parameters

Blood samples were collected from the retro-orbital venous plexus, in the amount of about 1.5 mL once a month. Plasma was then obtained by centrifugation (3500 rpm, 15 min, 4°C). Levels of LDL-C, HDL-C, TC, TG, alkaline phosphatase (ALP), total bile acid (TBA), alanine aminotransferase (ALT) and glutamate transaminase (AST) were quantitatively analyzed by an automatic biochemical analyzer (Toshiba 40-FR, Japan).

Lipid Regulation Mechanisms Investigation

Plasma 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) were tested by ELISA assay kits with a microplate reader (Tecan-Infinite 200 Pro NanoQuant, Switzerland). The protein expression of phosphorylated acetyl CoA carboxylase (p-ACC) and ACC in the liver of the rats were also tested by ELISA assay kits. Gene expression levels in liver tissues obtained from different groups were detected by quantitative real-time PCR. Then, 1 µg of total RNA was extracted for reverse transcription. The primer sequences of the target genes are listed in Table S2. In addition, 20 µL reaction volumes were used for data analysis using Rotor-gene Q Software (Qiagen). PCR reactions for each gene were repeated 3 times. The relative levels of each gene expression were determined by the 2^−ΔΔCt method.²¹⁾

Determination of Inflammatory Factor in Plasma

Tumor necrosis factor alpha (TNF-α) and glutathione-S-transferase alpha (GST-α) were determined in plasma using commercial ELISA assay kits with a microplate reader (Tecan-Infinite 200 Pro NanoQuant, Switzerland).

Histological Analysis

Each liver was fixed in 10% neutral-buffered formalin and processed routinely for embedding in paraffin. Tissue sections (3 µm) were stained with hematoxylin and eosin (HE) and examined under a light microscope.

Statistical Analysis

All data in this study are expressed in the form of mean±standard deviation (S.D.) The data were evaluated by one-way ANOVA, and the differences between means assessed using Duncan’s test with a significance level of p<0.05 and p<0.01.

RESULTS

Effects of RPM Extract on the Physical Condition of Rats

Body weight can directly reflect a lipid-lowering effect. We found that the body weights in the model group were generally higher than in the control group, especially in the last month (Table 1); however, they were remarkably decreased in RPM treatment groups. The high dose of RPM extract exhibited a significant weight loss effect, and the weight was returned to normal over time; moreover, liver weight gave similarly consistent results. Simvastatin did not have the ability to reduce body and liver weight. There was no significant difference in food intake or feces between the RPM treatment groups and the model group (Table S3).

Table 1. The Effects of RPM Extract on Body Weight

Groups	0 Month (g)	1 Month (g)	2 Months (g)	3 Months (g)	4 Months (g)
C	239.00±8.87	378.33±9.95	431.67±21.75	490.77±22.67	526.00±29.02
M	248.95±3.51	431.50±24.32**	494.73±20.77**	539.48±25.70*	603.00±31.26**
S	245.78±5.50	422.65±17.81**	490.75±25.06**	572.67±35.43**	620.18±45.04**
M+PML	243.62±5.44	410.17±14.71	477.37±8.05	546.55±15.86	582.96±15.01
M+PMM	244.77±5.92	410.12±17.78	480.16±28.58	542.12±42.11	589.84±43.56
M+PMH	245.14±9.14	375.71±9.49#	444.46±14.85#	497.94±18.46#	539.07±25.38##

Values are given as mean±S.D. (n=6). * p<0.05, ** p<0.01 compared with the control group. ^# p<0.05, ^## p<0.01 compared with the model group.

Effects of RPM Extract on Lipid Regulation in Plasma

The biochemical index clearly reflected the effects of RPM extract on lipid regulation. Plasma levels of LDL-C, TC, TG were consistently increased in the model group compared with the blank control group. LDL-C levels in the model group were increased to the highest level in the last month, to 4 times that in blank control group, whereas it was reduced in RPM treatment groups (Table 2). In addition, we found that RPM extract exerted a stable and fast lipid-lowering effect in the initial treatment at a high dose. A similar regulating effect of RPM extract was found in TC-regulation (Table 3). The TG content was decreased in RPM treatment groups, but the regulation of TG (Table 4) was not as significant as LDL-C and TC. The results also indicated that the high concentration of RPM extract showed an advantage in its LDL-C, TC and TG-lowering effect compared with that by simvastatin. However, the HDL-C level was not changed in the RPM treatment groups relative to the model group (Table S4).

Table 2. Effects of RPM Extract on the Levels of LDL-C in Plasma of Rats

LDL-C (mmol/L)	0 Month	1 Month	2 Months	3 Months	4 Months
C	0.36±0.13	0.43±0.08	0.37±0.05	0.35±0.06	0.32±0.04
M	0.40±0.05	0.56±0.07*	0.61±0.09**	0.89±0.12**	1.20±0.15**
S	0.37±0.08	0.58±0.17	0.47±0.11#	0.65±0.12##	0.63±0.15##
M+PML	0.41±0.08	0.51±0.04	0.49±0.07#	0.71±0.10#	0.66±0.19##
M+PMM	0.39±0.04	0.62±0.09	0.46±0.04##	0.65±0.11#	0.70±0.14##
M+PMH	0.40±0.06	0.53±0.09	0.50±0.15	0.52±0.14##	0.50±0.17##

Values are given as mean±S.D. (n=6). * p<0.05, ** p<0.01 compared with the control group. ^# p<0.05, ^## p<0.01 compared with the model group. The plasma LDL-C levels were quantitatively analyzed by an automatic biochemical analyzer (Toshiba 40-FR, Japan) once a month.

Table 3. Effects of RPM Extract on the Levels of TC in the Plasma of Rats

TC (mmol/L)	0 Month	1 Month	2 Months	3 Months	4 Months
C	1.33±0.06	1.40±0.21	1.37±0.13	1.22±0.09	1.34±0.14
M	1.39±0.51	1.65±0.20*	2.04±0.33**	2.41±0.46**	3.55±0.36**
S	1.43±0.43	1.49±0.25	1.57±0.17#	1.67±0.14##	1.84±0.38##
M+PML	1.39±0.07	1.63±0.11	1.61±0.07#	1.71±0.16#	1.87±0.41##
M+PMM	1.40±0.18	1.61±0.35	1.63±0.19#	1.66±0.04#	1.92±0.17##
M+PMH	1.38±0.10	1.56±0.12	1.50±0.18##	1.47±0.26##	1.69±0.30##

Values are given as mean±S.D. (n=6). * p<0.05, ** p<0.01 compared with the control group. ^# p<0.05, ^## p<0.01 compared with the model group. The plasma TC levels were quantitatively analyzed by an automatic biochemical analyzer (Toshiba 40-FR, Japan) once a month.

Table 4. Effects of RPM Extract on the Levels of TG in the Plasma of Rats

TG (mmol/L)	0 Month	1 Month	2 Months	3 Months	4 Months
C	0.67±0.06	0.69±0.04	0.70±0.22	0.82±0.15	0.96±0.19
M	0.69±0.09	0.81±0.09*	0.95±0.17*	1.26±0.33**	1.53±0.13**
S	0.61±0.09	0.70±0.19	0.53±0.10#	0.84±0.09##	1.26±0.33
M+PML	0.56±0.15	0.70±0.05#	0.70±0.13#	1.33±0.19	1.41±0.09
M+PMM	0.67±0.22	0.78±0.19	0.67±0.10##	1.20±0.19	1.30±0.09#
M+PMH	0.59±0.06	0.71±0.11	0.59±0.13##	0.98±0.20#	1.02±0.28##

Values are given as mean±S.D. (n=6). * p<0.05, ** p<0.01 compared with the control group. ^# p<0.05, ^## p<0.01 compared with the model group. The plasma TG levels were quantitatively analyzed by an automatic biochemical analyzer (Toshiba 40-FR, Japan) once a month.

Lipid-Regulating Mechanisms of RPM Extract

To investigate the potential mechanism of RPM in the treatment of hyperlipidemia, the activities of HMGR, ACC and FAS in plasma, as well as the relative expression levels of its target gene in liver, were tested by ELISA kits and RT-PCR, respectively. The results indicated that HFD enhanced the levels of HMGR, ACC and FAS by 52, 57, and 60%, respectively (Fig. 1). Low, medium and high doses of RPM extract all showed down-regulation effects on HMGR, ACC and FAS. Additionally, the high concentration of RPM treatment group resulted in a significant down-regulation of the expression levels of HMGR, ACC, FAS and SREBP-1c mRNA in the liver by 37, 54, 62, and 70%, respectively, which was correlated with enzyme activity results; thus its regulating effect was better than simvastatin at a high dose (Fig. 2). Also, the ratio of p-ACC to ACC in liver was significantly reduced in the model group compared with the blank control group, whereas it was increased in RPM treatment groups and the positive control group. The higher the dose of the RPM extract, the greater the proportion of p-ACC to ACC (Fig. 3).

Fig. 1. Effects of RPM Extract on the Expression of the Key Enzymes Involved in Lipid Metabolism, Including HMGR (A), FAS (B) and ACC (C) in Plasma of Hyperlipidemic Rats

Values are given as mean±S.D. (n=6). ** p<0.01 compared with the control group. ^# p<0.05, ^## p<0.01 compared with the model group.

Fig. 2. Effects of RPM Extract on Gene Expression Involved in Lipid Metabolism, Including HMGR mRNA (A), FAS mRNA (B), ACC mRNA (C) and SREBP-1c mRNA (D) in the Liver of Hyperlipidemic Rats

Values are given as mean±S.D. (n=6). ** p<0.01 compared with the control group. ^# p<0.05, ^## p<0.01: compared with the model group.

Fig. 3. Effects of RPM Extract on the Protein Expression of ACC in the Liver of Hyperlipidemic Rats

The ratio of ACC and its phosphorylated form (p-ACC) were evaluated by ELISA kits. Values are given as mean±S.D. (n=6). ^# p<0.05, ^## p<0.01 compared with the model group.

Histological Analysis

Fatty degeneration, inflammation, necrosis and cytoplasmic vacuoles in hepatocytes were observed in the model group, while no histological abnormalities were found in the blank control group (Fig. 4). Simvastatin could control the progression of fatty degeneration, while it did not alleviate the inflammatory reaction. Additionally, a medium dose of RPM extract exhibited a therapeutic effect by reducing hepatic steatosis, whereas the high dose of RPM extract not only markedly reduced the hepatic steatosis but also alleviated inflammation. There was no significant difference between the model group and low dose RPM treatment group. Histopathological signs of inflammation and fatty degeneration in the liver were scored semi-quantitatively and referred to Brunt’s criteria²²⁾ and other references.²³⁾ Three typical fields of vision were randomly chosen under ×100 magnification. Steatosis was graded 0–3 (0, the percentage of steatosis is less than 5%; 1, the percentage of steatosis cells is between 5 and 33%; 2, the percentage of steatosis is between 34 and 66%; 3, the percentage of steatosis is more than 66%), as shown in Table 5. The portal area inflammation was graded 0–3 (0 is none, 1 is mild; 2 is moderate; 3 is severe), also as shown in Table 5. The cellular components of inflammation (lymphocytes) were noted (Figure S2).

Fig. 4. Liver Histological Analysis Cytoplasmic Vacuoles, Inflammatory Infiltration and Fatty Degeneration Were Observed in the Model Group

(A) blank control group, (B) model group, (C) positive control group (simvastatin), (D) low-dose RPM treatment group, (E) medium-dose RPM treatment group, (F) high-dose RPM treatment group. The magnification is 200×.

Table 5. The Effects of RPM Extract on Histopathological Changes

Parameters	C	M	S	M+PML	M+PMM	M+PMH
Fatty generation (grade)	0	2.83±0.41**	2.33±0.52#	2.00±0.71#	1.80±0.84#	1.00±0.00##
Inflammation (grade)	0	3.00±0.00**	2.00±1.10#	2.20±1.10#	2.60±0.89	1.33±0.82##

** p<0.01 compared with the control group. ^# p<0.05, ^## p<0.01 compared with the model group.

Study on Liver Toxicity

The results above indicate that RPM extract has a remarkable lipid-lowering effect, especially at the high dose. However, the adverse hepatic reaction of RPM and its preparations have increasingly been reported.^18,19) Thus, we investigated the hepatotoxicity of RPM extract comprehensively in this study.

Effects of RPM Extract on Plasma Biochemical Indexes

ALP, TBA, ALT and AST were commonly used to evaluate liver function. The contents of ALP and TBA increased by 3- and 2.6-fold, respectively, in the model group in the last month (Table S5, Table S6), which was markedly higher compared with the blank control group, whereas levels in the RPM treatment groups were markedly decreased. Simvastatin could reduce the ALP and TBA levels, but the contents of ALP remained at a higher level compared with that of the high concentration of RPM treatment group, which implied that simvastatin should not be taken for a long time. At the same time, ALT and AST showed no significant change (data not shown).

Effects of RPM Extract on the Inflammatory Factor and Detoxifying Enzyme in Plasma

To corroborate whether the liver was damaged, levels of TNF-α and GST-α were evaluated. The levels of TNF-α and GST-α increased 54% and 91%, respectively, in the model group compared with the blank control group (p<0.01, Fig. 5). RPM-treatment resulted in a remarkable reduction in TNF-α and GST-α content, especially at the high concentration, which was similar to the results in the regulation of ALP and TBA. Simvastatin could reduce the levels of TNF-α and GST-α, but these remained at a higher level compared with the high concentration of RPM treatment group, which implied that simvastatin was not suitable for long-term use.

Fig. 5. Effects of RPM Extract on the Expression of Inflammatory Factor and Detoxifying Enzyme, Including TNF-α(A) and GST-α(B) in the Plasma of Rats

Values are given as mean±S.D. (n=6). ** p<0.01 compared with the control group. ^# p<0.05, ^## p<0.01 compared with the model group.

DISCUSSION

Hyperlipidemia is a common metabolic syndrome characterized by increased TC, TG, and LDL-C, and decreased HDL-C levels which, in combination, facilitate the occurrence of lipid metabolism disorders. High lipid levels present a risk factor for hypertension, coronary heart disease and cerebrovascular damage. Polygonum multiflorum is widely used in the prevention and treatment of hyperlipidemia in TCM and reduces the occurrence of these complications. Although the lipid-lowering effect of Polygonum multiflorum has previously been studied, those studies have focused mainly on PMP; the research of RPM was not sufficient. Moreover, mechanism studies were mainly focused on the molecular level, with a lack of systematic assessment based on the animal level. This study has focused on the lipid regulatory effect of RMP, and reveals the relevant mechanisms of RPM extract. A high-fat diet was used to induce a hyperlipidemia model in rats. The rats in this model group had higher body weight, plasma TC, TG and LDL-C levels compared to rats in the blank control group. These results demonstrated that hyperlipidemia rats with TC and TG accumulation was successfully induced by HFD. In order to explore further the mechanism regulating lipid metabolism in vivo, the key enzymes and genes involved in lipid metabolism were chosen for study.

In this study, we found a remarkable improvement of dyslipidemia in HFD-fed rats after oral administration of RPM. RPM extract may decrease the levels of cholesterol by down-regulating the expression of HMGR, the rate-controlling enzyme in the biosynthesis of TC,²⁴⁾ and they suppressed the synthesis of fatty acids by down-regulating ACC. By contrast, simvastatin down-regulated the expression of HMGR, thereby affecting the formation of mevalonic acid, the rate-limiting step in the biosynthesis of cholesterol.³⁾ Thus, RPM extract appeared to exhibit advantages in the treatment of hyperlipidemia.

Plasma TG must first be resolved into fatty acids and glycerol, after which it is ingested by tissue in the form of FFA.^3,25) Therefore, TG levels are associated closely with fatty acids. A decrease in the content of fatty acids is accompanied by a reduction in the synthesis of TG.^26,27) ACC and FAS, on the other hand, play a major role in the biosynthesis of fatty acids. ACC is the rate-limiting enzyme in the synthesis of fatty acids, which facilitates acetyl-CoA to generate malonyl-CoA, and fatty acids are formed by malonyl-CoA after a series of reactions.²⁸⁾ Thus, ACC plays an important role in the synthesis of lipids and regulated by phosphorylation and dephosphorylation. Phosphorylation caused ACC to lose its effect. The greater the levels of p-ACC, the lower the activities of ACC. RPM extract increased the ratio of p-ACC to ACC in liver in our study, and the activity of ACC was reduced correspondingly, indicating that RPM could decrease TG levels and thus display favorable lipid reducing effects. FAS directly catalyzed the biosynthesis of fatty acids.²⁹⁾ Moreover, sterol regulatory element-binding protein 1c (SREBP-1c), a membrane-bound transcription factor, is important for the development of hyperlipidemia: it modulates the transcription levels of genes involved in the synthesis of TC and TG,^28,30) such as FAS. Thus, HMGR, ACC, FAS and SREBP-1c were closely related to lipid biosynthesis and are worth studying. The transcription levels of HMGR mRNA, ACC mRNA, FAS mRNA and SREBP-1c mRNA were promoted after rats were fed a HFD, but decreased after treatment with RPM. Thus, we investigated the expression of HMGR, ACC and FAS, and found that they were all down-regulated in RPM treatment groups. Therefore, the mechanism of RPM extract’s lipid lowering effect in vivo might be the suppression of the expression of HMGR and ACC, thus affecting the biosynthesis of TC and TG.

Aside from the disorder of lipid metabolism in hyperlipidemia rats, an inflammatory reaction in the liver was observed. Simvastatin was able to decrease the contents of ALP, TBA, TNF-α and GST-α, but the high concentration of RPM extract exhibited even greater effects. Thus, the hepatotoxicity caused by RPM extract was less than that caused by simvastatin. Histopathological findings in the liver showed that there was inflammatory infiltration around portal areas (Figure S2). Lymphocytes were noted. The inflammation score in rats fed a high fat diet alone was significantly higher than in rats in the control group. However, the inflammation score was significantly decreased after treatment with RPM extract. These results indicate that RPM extract can improve hepatocyte steatosis and alleviate the inflammation caused by a high fat diet. Additionally, there were no significant changes in ALT and AST plasma levels in rats during the experiment, although liver injury did occur according to histological analysis. Thus, we need to find new indicators. GST-α was mainly found in centrolobular cells in a high concentration, thus this metabolic zone of the liver was more sensitive to injury.³¹⁾ TNF-α is a typical feature in animal models of hepatic injury and inflammation. The proinflammatory cytokine TNF-α is the central mediator of apoptosis and necrotic liver damage.³²⁾ Therefore, we can obtain more useful information by examining changes in TNF-α and GST-α. Long-term feeding of a high-fat diet to rats may increase TNF-α and GST-α levels, resulting in lipid metabolism disorder and inducing inflammation. However, RPM was shown to reduce TNF-α and GST-α levels and demonstrated an anti-inflammatory effect that helped to regulate lipid levels.

In summary, the results of this study indicate that RPM extract may play a significant role in the treatment of hyperlipidemia by inhibiting the expression of HMGR, ACC and FAS, and by down-regulating the transcription levels of its target genes. In addition, RPM extract could alleviate the inflammation caused by a high fat diet.

Acknowledgments

This work was supported by the National Science and Technology Major Project (2014ZX09304307-001), the surface project of the National Natural Science Foundation (81573839) and by the China Postdoctoral Science Foundation (2016M591362).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Dembowski E, Davidson MH. A review of lipid management in primary and secondary prevention. J. Cardiopulm. Rehabil. Prev., 29, 2–12 (2009).
• 2) Toth PP. Drug treatment of hyperlipidaemia: a guide to the rational use of lipid-lowering drugs. Drugs, 70, 1363–1379 (2010).
• 3) Davidson MH. Safety profiles for the HMG-CoA reductase inhibitors. Drugs, 61, 197–206 (2001).
• 4) Garnett WR. Interactions with hydroxymethylglutaryl-coenzyme A reductase inhibitors. Am. J. Health Syst. Pharm., 52, 1639–1645 (1995).
• 5) Matsuyama K, Nakagawa K, Nakai A, Konishi Y, Nishikata M, Tanaka H, Uchida T. Evaluation of myopathy risk for hmg-coa reductase inhibitors by urethane infusion method. Biol. Pharm. Bull., 25, 346–350 (2002).
• 6) Zhang TJ, Chen CQ. Modern research and application of traditional chinese medicine that have blood lipid-regulating activity. People’s medical Publishing House, Beijing, pp. 3–4 (2007).
• 7) Yang PY, Almofti MR, Lu L, Kang H, Zhang J, Li TJ, Rui YC, Sun LN, Chen WS. Reduction of atherosclerosis in cholesterol-fed rabbits and decrease of expressions of intracellular adhesion molecule-1 and vascular endothelial growth factor in foam cells by a water-soluble fraction of Polygonum multiflorum. J. Pharmacol. Sci., 99, 294–300 (2005).
• 8) Gao X, Hu YJ, Fu LC. Blood lipid-regulation of stilbene glycoside from Polygonum multiflorum. Chung Kuo Chung Yao Tsa Chih, 32, 323–326 (2007).
• 9) Liu QF, Lee JH, Kim YM, Lee S, Hong YK, Hwang S, Oh Y, Lee K, Yun HS, Lee IS, Jeon S, Chin YW, Koo BS, Cho KS. In vivo screening of traditional medicinal plants for neuroprotective activity against aβ42 cytotoxicity by using drosophila models of Alzheimer’s disease. Biol. Pharm. Bull., 38, 1891–1901 (2015).
• 10) Zhou XX, Yang Q, Xie YH, Sun JY, Qiu PC, Cao W, Wang SW. Protective effect of tetrahydroxystilbene glucoside against D-galactose induced aging process in mice. Phytochem. Lett., 6, 372–378 (2013).
• 11) Bhadauria M. Dose-dependent hepatoprotective effect of emodin against acetaminophen-induced acute damage in rats. Exp. Toxicol. Pathol., 62, 627–635 (2010).
• 12) Wang X, Zhao L, Han T, Chen S, Wang J. Protective effects of 2, 3,5,4′-tetrahydroxystilbene-2-O-beta-D-glucoside, an active component of Polygonum multiflorum Thunb, on experimental colitis in mice. Eur. J. Pharmacol., 578, 339–348 (2008).
• 13) Wang M, Zhao R, Wang W, Mao X, Yu J. Lipid regulation effects of Polygoni Multiflori Radix, its processed products and its major substances on steatosis human liver cell line L02. J. Ethnopharmacol., 139, 287–293 (2012).
• 14) Yu J, Xie J, Mao X, Wei H, Zhao SL, Ma YG, Li N, Zhao R. Comparison of laxative and antioxidant activities of raw, processed and fermented Polygoni Multiflori Radix. Chin. J. Nat. Med., 10, 63–67 (2012).
• 15) Wang CY, Gu JM, Liu WN, Yang W, Zhang LT. Studies on pharmacokinetics and tissue distribution of stilbene glycoside in the hyperlipidemia model rats. Chinese Journal of Pharmaceutical Analysis, 29, 1073–1078 (2009).
• 16) Li N, Chen Z, Mao X, Yu J, Zhao R. Effects of lipid regulation using raw and processed radix polygoni multiflori in rats fed a high-fat diet. Evid. Based Complement. Alternat. Med., 2012, 329171 (2012).
• 17) Wang W, He Y, Lin P, Li Y, Sun R, Gu W, Yu J, Zhao R. In vitro effects of active components of Polygonum multiflorum Radix on enzymes involved in the lipid metabolism. J. Ethnopharmacol., 153, 763–770 (2014).
• 18) Dong H, Slain D, Cheng J, Ma W, Liang W. Eighteen cases of liver injury following ingestion of Polygonum multiflorum. Complement. Ther. Med., 22, 70–74 (2014).
• 19) Park GJH, Mann SP, Ngu MC. Acute hepatitis induced by Shou-Wu-Pian, a herbal product derived from Polygonum multiflorum. J. Gastroenterol. Hepatol., 16, 115–117 (2001).
• 20) Gao H, Liu Z, Wan W, Qu X, Chen M. Aqueous extract of yerba mate tea lowers atherosclerotic risk factors in a rat hyperlipidemia model. Phytother. Res., 27, 1225–1231 (2013).
• 21) Ballester M, Castelló A, Ibáñez E, Sánchez A, Folch JM. Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals. Biotechniques, 37, 610–613 (2004).
• 22) Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am. J. Gastroenterol., 94, 2467–2474 (1999).
• 23) Kirsch R, Clarkson V, Shephard EG, Marais DA, Jaffer MA, Woodburne VE, Kirsch RE, Hall PdeL. Rodent nutritional model of non-alcoholic steatohepatitis: species, strain and sex difference studies. J. Gastroenterol. Hepatol., 18, 1272–1282 (2003).
• 24) Istvan ES. Structural mechanism for statin inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Am. Heart J., 144 (Suppl.), S27–S32 (2002).
• 25) Takeuchi K, Reue K. Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis. Am. J. Physiol. Endocrinol. Metab., 296, E1195–E1209 (2009).
• 26) McGarry JD, Foster DW. Regulation of hepatic fatty acid oxidation and ketone body production. Annu. Rev. Biochem., 49, 395–420 (1980).
• 27) Sham TT, Chan CO, Wang YH, Yang JM, Mok DKW, Chan SW. A review on the traditional Chinese medicinal herbs and formulae with hypolipidemic effect. BioMed Research International, 2014, 925302 (2014).
• 28) Raghow R, Yellaturu C, Deng X, Park EA, Elam MB. SREBPs: the crossroads of physiological and pathological lipid homeostasis. Trends Endocrinol. Metab., 19, 65–73 (2008).
• 29) Semenkovich CF. Regulation of fatty acid synthase (FAS). Prog. Lipid Res., 36, 43–53 (1997).
• 30) Begriche K, Igoudjil A, Pessayre D, Fromenty B. Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it. Mitochondrion, 6, 1–28 (2006).
• 31) Ozer J, Ratner M, Shaw M, Bailey W, Schomaker S. The current state of serum biomarkers of hepatotoxicity. Toxicology, 245, 194–205 (2008).
• 32) Tilg H. Cytokines and liver diseases. Can. J. Gastroenterol., 15, 661–668 (2001).