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Journal of Traditional Chinese Medicine logoLink to Journal of Traditional Chinese Medicine
. 2022 Feb 25;42(3):364–371. doi: 10.19852/j.cnki.jtcm.20220225.002

Efficacy of Kushen decoction (苦参汤) on high-fat-diet-induced hyperlipidemia in rats

Jiri Mutu ZHANG 1,2, Shilong LIANG 1, Peng NIE 1, Yong’an LIAO 1, Qinying AI 1, Xiaojun YAN 1, Hongning LIU 1, Yanhua JI 1,✉, Zhijun ZENG 1,✉
PMCID: PMC9924673  PMID: 35610005

Abstract

OBJECTIVE:

To investigate the efficacy and underlying mechanisms of action of Kushen decoction on high-fat-diet-induced hyperlipidemia in rats using RNA-seq technology.

METHODS:

The efficacy of a Kushen decoction (苦参汤), at a concentration of 1 mL/g of crude medicine prepared according to the method commonly used in clinical practice, was investigated on 24 specific pathogen-free male Sprague-Dawley rats. Liver tissues were compared using RNA-Seq technology. The differentially expressed genes were further investigated by real-time fluorescent quantitative polymerase chain reaction (qPCR and Western blot (WB).

RESULTS:

Serum triglycerides (TG), liver low-density lipoprotein cholesterol (LDL-C), body weight, body length, and Lee’s index were significantly increased in the untreated hyperlipidemia-induced group (model) compared with the control group, whereas liver high-density lipoprotein cholesterol (HDL-C) was significantly decreased. Serum TG, liver LDL-C, bodyweight, and Lee’s index were decreased in the high-dose Kushen decoction group (HDKS) compared with the model group, whereas liver HDL-C was significantly increased. Similarly, liver TG tended to decline in the HDKS group. Comparison of the gene expression profiles in the livers from different groups indicated that the Kushen decoction significantly affected metabolic pathways, PPAR signalling pathway, and circadian rhythm (Q ≤ 0.05), with the genes ARNTL, PER3, and CLOCK being differentially expressed. qPCR and WB analysis confirmed the differential expression of the genes discovered by transcriptomics analysis.

CONCLUSION:

The Kushen decoction may achieve a lipid-lowering effect on hyperlipidemic rats by regulating metabolic pathways and the circadian rhythm pathway and in particular, their related genes ARNTL, PER3, and CLOCK.

Keywords: liver, hyperlipidemias, RNA-seq, transcriptome, Kushen decoction

1. INTRODUCTION

Lipids are transported in the blood as part of lipoprotein complexes. Furthermore, hyperlipidemia is a condition in which serum lipid level is elevated. The prevalence of hyperlipidemia in China has gradually increased with economic development, improved living standards, and changing lifestyles.1 In the USA, individuals with hyperlipidemia may be twice as likely as individuals with normal total cholesterol (TC) level to develop cardiovascular disease.2 The level of some lipids and lipoproteins, such as low-density lipoprotein cholesterol (LDL-C), TC, and apolipoprotein A, are key risk factors for cardiovascular disease and stroke.3,4 Increased risk of hyperlipidemia is related to several factors. Diets rich in saturated fat or trans-fat, lack of exercise, smoking, obesity, hypertension, chronic kidney disease, type 2 diabetes, and hypothyroidism are variable risk factors.2 In addition, some drugs can contribute to a high LDL-C level.5

Changes in diet and lifestyle have a big impact on dyslipidemia. Lipid-lowering drugs produce better results when combined with diet control and lifestyle improvements. In terms of pharmacotherapy, common LDL-C-lowering drugs used as a primary intervention in clinics are niacin, resins, statins, fibrates, intestinal cholesterol absorption inhibitors, and probucol.6 However, these lipid-lowering drugs can cause different adverse reactions, mainly headache, respiratory infections, musculoskeletal pain, and gastrointestinal events.7-11 Elevated blood glucose and glycosylated haemoglobin (GHb) levels are also side effects of statins.12-14 In addition, 20% of the patients are resistant or intolerant to statins, while these drugs reduce the risk of atherosclerotic cardiovascular disease. Therefore, it is necessary to explore other therapies to achieve an ideal LDL-C level.15

Kushen is a widely used traditional herbal medicine in China (China Pharmacopoeia, 2020 Edition). It had been mentioned in the first Chinese medical classic, “Shen-Nung’s Pen-Ts’ao”, in 200 A.D. In the past, Kushen would be combined with other medicines to form a compound decoction and used orally. The various biological activities of Kushen have since been proven by modern pharmacological research. In a mouse model of contact dermatitis induced by 1-fluoro-2,4-dinitrofluorobenzene (DNFB), the levels of TNF-α and IFN-γ are reduced by a Kushen treatment (10 mg/mL).16 Airway responsiveness in mice with allergic asthma is significantly reduced by Kushen water extraction.17 An ethanol extract of Kushen relaxes phenylephrine-precontracted aortic rings of isolated rat aorta in a dose-dependent manner.18 The capacity of peritoneal macrophages to ingest H22 tumour cells and produce nitric oxide via upregulation of the inducible nitric oxide synthase activity is significantly enhanced by Kushen polysaccharides.19 Ethyl acetate extract of Kushen lowers blood lipid level. Finally, Kushen extract sig-nificantly decreases triglycerides (TG), LDL-C, and TC levels in rat serum, while increasing high-density lipoprotein cholesterol (HDL-C) levels. However, this specific antihyperlipidemic mechanism was not elucidated.20

Kushen contains many chemical components, of which more than 200 have been isolated. These constituents can be divided into four categories, i.e., flavonoids, alkaloids, terpenes, and other compounds. The main active ingredients are considered to be alkaloids and flavonoids.21

Next-generation mRNA sequencing offers detailed, quantitative, and rapid measurement for comparing transcriptomic variations between different cells, tissues, breeds, or species under different conditions.22 In this study, we investigated the lipid-lowering effect of Kushen by sequencing the transcriptomes of hyperlipidemic rat livers.

2. MATERIALS AND METHODS

2.1. Reagents and drugs

Kushen (lot No. 150826) was purchased from Jiangxi Jiangzhong Chinese Medicine decoction Pieces Co. Ltd. and confirmed by Professor Liu Yong, Department of Chinese Medicinal Resources, School of Pharmacy, Jiangxi University of Chinese Medicine, China. The detailed preparation method of the Kushen decoction was as follows: first, 0.5 kg of Kushen was placed into a boiling pot. Second, the herb was soaked for 1 h in 10 times its weight of water. Third, the herb was boiled for 50 min, and the resulting decoction was filtered through a filter of less than 0.5-mm diameter pores. Fourth, the decoction was concentrated to 1 g/mL crude medicine. This concentrated Kushen decoction was then stored at -80 ℃ until use.

Notably, 60% kcal high-fat food (catalogue No. D12492) was obtained from Research Diets, Inc (USA). TRIzol reagent (lot No. 149105) was purchased from Thermo Scientific. Bicinchoninic acid (BCA) Protein Quantitative Kit (lot No. 40238) was purchased from Kangwei Century Biotechnology Co. Ltd. RIPA tissue cell lysate (lot No. 20170302) was purchased from Solarbio Science& Technology Co. Ltd. The additional reagents used in this study were all commercially available and of analytical grade.

2.2. Animal model and drug administration

Twenty-four male Sprague-Dawley rats were acquired from Hunan SJA Laboratory Animal Co. Ltd. The rats were kept in a room at (22 ± 2) °C, under controlled light conditions, had access to standard rodent chow diet and water, and their litter was changed every day. The rats were randomly assigned to the control group (control; n = 6) or the model group (n = 18). The control group was fed normal food containing 10% of lipids. The model group was fed a high-fat diet containing 60% of lipids. After five weeks of high fat regimen, termed “modelling” thereafter, the model group was randomly assigned to the untreated model group (model, n = 6), low-dose Kushen decoction group (LDKS; n = 6; 0.09 g/100 g), or high-dose Kushen decoction (苦参汤) group (HDKS; n = 6; 0.54 g/100 g). The Kushen decoction was administrated once a day orally for five weeks. During this treatment period, the model, LDKS, and HDKS groups were fed a high-fat diet, while the control group was fed a normal diet. The animal body weight and body lengths were measured at 4, 9, and 10 weeks. Then, Lee’s index was calculated according to the following formula: 23 Lee’s index = [Body mass (g) × 1000]1/3 / body length (cm). Sera were collected at 10 and 11 weeks. Five weeks after administration, the rats were fasted for 12 h and injected with 3% sodium pentobarbital (50 mg/kg) according to their body weight for terminal anaesthesia. After withdrawal, the abdominal aortic blood was left to coagulate for 2 h and centrifuged at 3000 rpm for 10 min. The upper serum was collected and stored at -80 ℃. The liver homogenates were prepared as follows: 100 mg liver accurately weighed was placed in 0.9 mL of normal saline, mashed in a tissue homogenizer, centrifuged for 10 min at 1500 rpm. Finally, the supernatant was removed and stored at -80 ℃.

2.3. Histopathological analysis of the liver

The rat liver tissues were fixed in 4% paraformaldehyde and cut into 3-mm thick slices, embedded, and packed. Then, the slices were washed under running water for 6 h. The sections were further dehydrated, embedded, dewaxed, stained, sealed, and examined under a microscope.

2.4. Total RNA extraction

Fifty milligrams of liver tissue was mashed in a mortar. A small amount of liquid nitrogen was poured over the tissue to grind to a powder. Next, 1 mL of trizol reagent was added into the mortar, and the tissue powder was left to completely dissolve until being transferred into a microtube. Then, 200 μL of chloroform was added, and the tube was shaken vigorously by hand for 15 s, incubated for 3 min at room temperature, and centrifuged (4 ℃, 12 000 rpm/min, 15 min). The supernatant was removed and precipitated with 500 μL of isopropanol at room temperature for 10 min, mixed, and centrifuged (4 ℃, 12 000 rpm/min, 10 min). The pellet was washed with 800 μL of 75% ethanol and centrifuged (4 ℃, 8000 rpm/min, 5 min) twice. The supernatant was discarded, and the pellet was left to dry on a clean bench for 5 min. The RNA concentration was measured using a Nanodrop ND-2000 instrument, and the quality of the RNA was checked by 1% agarose gel electrophoresis. The samples passing the quality control were stored at -80 ℃.

2.5. RNA sequencing and assembling

First, the RNA and DNA probes were mixed. The DNA/RNA hybrids were digested and the DNA probe was removed by the DNase I digestion. The target RNA was obtained after purification. The target RNA fragments and the N6 random primers were reversely transcribed to double-stranded cDNA (dscDNA). The dscDNA was repaired by introducing a phosphate residue in 5’-Ter and a sticky ‘A’ end in 3’-Ter, and then was sealed and connected to a sticky ‘T’ in 3’-Ter. The amplification of the ligation product was performed with two specific primers. The PCR product was denatured by heating, and the single-stranded DNA was circularized with a splint oligonucleotide and the DNA ligase. The resulting library was sequenced. The quality control (QC) of the primary sequencing data indicated whether resequencing was necessary. Upon satisfactory QC, the raw reads were filtered to clean the reads. Again, a QC comparison was performed to determine whether resequencing was required. The read distribution and normalization with the reference genes were calculated by comparing the data.

2.6. Analysis of the differentially expressed genes (DEGs)

RNA-Seq by Expectation-Maximization (RSEM)24 is a thread used to estimate gene expression levels from RNA-Seq data and is used for expectation-maximisation (EM) algorithms, single-ended and double-ended read data, quality scores, variable length reads, and parallel computing threads for read start position distribution estimation. The fragments per kilobase of transcript per million mapped reads method can eliminate the influence of different gene lengths and sequencing differences on gene expression calculations. NOI seq25 method was used to screen the DEGs between the two groups. The relevant thresholds were fold change ≥ 2 and divergence probability ≥ 0.8.

2.7. Enrichment analysis in GO and KEGG pathways

GO analysis is a bioinformatics tool that can determine the main biological functions carried out by the DEGs. It helps to understand the biological functions of the genes. The calculated P value goes through Bonferroni correction,26 taking corrected P value ≤ 0.05 as the cut-off standard. The pathway enrichment analysis with KEGG determines which metabolic pathways or signal transduction pathways are significantly enriched in the DEGs.27 Pathways with Q value ≤ 0.05 were defined as significantly enriched in the DEGs.

2.8. Real-time fluorescent quantitative polymerase chain reaction (qPCR)

To confirm the results obtained by RNA sequencing, the expression of the most relevant DEGs was quantified by qPCR. We assessed the differential expression of ARNTL, PER3, and CLOCK in the liver tissues from the different experimental groups. Gene expression levels were calculated using the 2-ΔΔCt method.

2.9. Western blot analysis of the protein expression of the DEGs

One hundred milligrams of liver tissue were ground, lysed on ice using lysis solution, centrifuged, and the protein concentration was determined by BCA. An aliquot of 40 μg/well of each sample was analysed by 12% SDS-PAGE. After electrophoresis, the separated proteins were transferred onto membranes, which were then blocked with a 5% skimmed milk powder solution for 1 h. After being washed three times, the membranes were incubated with primary antibodies (rabbit anti-ARNTL, PER3, CLOCK and β-actin, all used at a 1∶1000 dilution) overnight at 4 ℃ on a shaker. The day after, the membranes were washed three times, incubated for 1 h with secondary antibodies and washed three times again. A developing solution was added onto the membranes and the signal was detected by chemiluminescent gel imaging. Image Lab was used to calculate the ratio of the grayscale values of the target band normalized against the internal reference band to obtain the relative protein expression. Each quan-tification was repeated three times.

2.10. Statistical analysis

The results of the experimental data are expressed as mean ± standard error of mean. The differences between the two groups were analysed by t-test; the differences between multiple groups were compared by one-way analysis of variance (GraphPad Prism software version 8.0). P < 0.05 was considered statistically significant.

3. RESULTS

3.1. Kushen decoction reduces lipids in high-fat diet-induced hyperlipidemia rats

We found that rats fed a high-fat diet for four weeks had significantly increased body weight, body length, and Lee’s index (Figure 1A-1C) (P < 0.01). Compared with the model group, after administration of Kushen decoction for four weeks (with a total high-fat diet regimen, or “modelling” of nine weeks), or five weeks (10-week modelling) at a low dose (LDKS group), body weight, body length, Lee’s index tended to decrease (P > 0.05). The comparison between these same two groups at five (10-week modelling) and six weeks (11 week-modelling) indicated that the Kushen decoction had decreased the level of serum TG in the treated group (Figure 1F) (P < 0.01).

Figure 1. Effect of high-fat diet and treatment with low- or high-dose Kushen.

Figure 1

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(A-F) Different parameters were compared between the control group, the untreated model group (high-fat diet), the model group treated with low-dose Kushen decoction (LDKS), and the model group treated with high-dose decoction (HDKS). (A) body weight; (B) body length; (C) Lee’s index; (D) serum high-density lipoprotein cholesterol (HDL-C); (E) serum low-density lipoprotein cholesterol (LDL-C); and (F) serum triglycerides (TG). Compared with the control group, a high-fat diet significantly increased body weight, body length, Lee’s index, and serum TG (aP < 0.01). Compared with the model group, low-dose Kushen decoction (LDKS group) tended to decrease body weight, body length, and Lee’s index, but the differences were not significant (P > 0.05), while the levels of serum HDL-C (cP < 0.05) and serum LDL-C (bP < 0.01) increased significantly, and the level of serum TG decreases significantly (bP < 0.01). Compared with the model group, high-dose Kushen decoction (HDKS group) decreased significantly body weight, Lee’s index, and serum TG (bP < 0.01), while serum HDL-C and serum LDL-C increased significantly (bP < 0.01).

When the model group and HDKS group were compared, we found that after a 4-week (9-week modelling) and 5-week administration (10-week modelling), the bodyweight of the HDKS rats was reduced significantly (P < 0.01); body length tended to decrease (P > 0.05), and Lee’s index decreased significantly (P < 0.05). Compared with the model group, after 5-week administration (10-week modelling), the level of serum HDL-C in the rats from the LDKS group was increased significantly (Figure 1D; P < 0.05). Similarly, after 5-week administration (10-week modelling), the level of serum HDL-C in the HDKS rats was increased significantly (Figure 1D) (P < 0.01).

Furthermore, after 5-week administration (10-week modelling), the rats from both the LDKS and the HDKS groups had a significant increase in serum LDL-C level compared with those from the model group (Figure 1E) (P < 0.01). After 6-week administration (11-week modelling), the levels of serum HDL-C and serum LDL-C in the HDKS rats were increased significantly (Figure 1D-1E) (P < 0.01). In contrast, after 5-week (10-week modelling) or 6-week administration (11-week modelling), the level of serum TG in the HDKS rats was decreased significantly (Figure 1F) (P < 0.01).

Altogether, these results implied that Kushen could reduce body weight, Lee’s index, and serum TG, and increase HDL-C. In addition, we performed histo-pathological analysis of liver biopsies (Supplementary Figure 1 in Supplementary Information). Compared with the model group, the liver cells from the control group and the groups treated with different doses of Kushen decoction displayed a normal shape and were regularly organized. In contrast, in the livers from the model group, missing cells or cell disruption were visible, resulting in cavities, and the typical cell lineages were not neatly distinguishable, with increased intercellular spaces. Subsequently, to examine the effect of a high-fat diet on liver tissue and the regulatory effect of different doses of Kushen decoction, we assessed different liver function parameters (Supplementary Figure 2a-2k in Supple-mentary Information). This analysis showed that there were no significant alterations in the assessed liver functions in the model group compared with the control group, except for high HDL-C and LDL-C levels (P < 0.01). In addition, the TG in the liver tended to be higher in the model group. When compared with the model group, there were no significant differences in the liver functions of the LDKS (P > 0.05) or the HDKS groups, except for HDL-C and LDL-C levels (P < 0.01). Interestingly, there was a downward trend in liver TG in the HDKS group.

Figure 2. Analysis of ARNTL, CLOCK, and PER3 mRNAs by real-time fluorescent quantitative polymerase chain reaction.

Figure 2

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Bar graphs showing ARNTL (A), CLOCK (B), and PER3 (C) mRNA levels in the different groups. Model group compare with the control group, aP < 0.05; LDKS group and HDKS group compare with the model group, bP < 0.01 or cP < 0.05.

3.2. RNA Sequencing and gene prediction

Total RNA was extracted from the liver tissue and analysed on 1% agarose gel electrophoresis (Supplementary Figure 3 in Supplementary Information). The 28S and 18S rRNAs were visible, indicating that the total RNA was not degraded and could be used for subsequent loading and sequencing. About 20 000 000 raw readings were detected from each sample. After filtering, about 91% of the clean reads were mapped, and a total of 17 407 predicted protein-coding genes were obtained.

Figure 3. Quantification of the proteins encoded by the DEGs in livers from the different experimental groups.

Figure 3

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Representative immunoblots assessing the target proteins are shown. Molecular weight markers are indicated in kilodaltons (kDa). Protein levels were normalized to internal controls. β-actin was used as a loading control. Bar charts showing ARNTL (C), CLOCK (D), and PER3 (E) protein expression in the different groups. DEGs: differentially expressed genes; LDKS: low-dose Kushen decoction; HDKS: high-dose decoction. Compared with the control group, the expression of ARNTL, PER3, and CLOCK in the model group was decreased significantly (aP < 0.01 versus control group). It can be noted that the expression of ARNTL, PER3, and CLOCK was increased significantly in the LDKS or HDKS group (bP < 0.05 and cP < 0.01 versus model group).

3.3. DEG Screening

After comparing the model group with the control group, there were a total of 139 DEGs, including 59 up-regulated and 80 down-regulated genes (Supplementary Figure 4a in Supplementary Information). Compared with the model group, 45 DEGs were found in the LDKS group, including 32 up-regulated and 13 down-regulated genes (Supplementary Figure 4b in Supplementary Information).

The comparison between the HDKS and the model group yielded a total of 114 DEGs, including 82 up-regulated and 32 down-regulated genes (Supplementary Figure 4c in Supplementary Information).

3.4. Effect of Kushen decoction on DEGs involved in metabolism

Between the model group and the control group, 38 DEGs involved in metabolism were detected by RNA-seq analysis, including 30 upregulated and 8 downregulated genes. Between the LDKS group and the model group, 22 DEGs involved in metabolism were detected, including 16 upregulated and 6 downregulated genes. Finally, between the HDKS and the model group, 42 DEGs involved in metabolism were detected, including 35 upregulated and 7 downregulated genes.

3.5. Effect of Kushen decoction on DEGs involved in the circadian rhythm

In the detailed analysis of the effects of Kushen decoction on biological clock genes, 4 DEGs related to the circadian clocks were detected between the model and the control group, including 2 up-regulated and 2 down-regulated genes. Between the LDKS and the model group, only one upregulated DEGs involved in circadian clocks was detected. Similarly, between the HDKS and the model group, only one upregulated DEGs involved in circadian clocks was detected.

3.6. Enrichment analysis for GO and KEGG pathways

GO function annotation of the DEGs between the model and the control group revealed 43 significant GO terms (Supplementary Figure 5a in Supplementary Information), of which the total enrichment in biological processes retrieved 23 entries. The molecular functions were enriched for up to 9 terms, and cellular components were enriched for up to 11 terms. The DEGs between the model and the LDKS group were enriched for 32 significant GO terms (Supplementary Figure 5b in Supplementary Information), of which 17 terms were related to biological processes, five terms were related to molecular functions, and 10 terms were related to cellular components. The GO function annotation for the DEGs between the model and HDKS group (Supplementary Figure 5c in Supplementary Information) retrieved a total of 41 significant GO terms, of which 23 terms related to biological processes, 9 terms related to molecular functions, and 9 terms related to cellular components. The GO analysis between the model, the control, the LDKS, and the HDKS group demonstrated that changes in biological processes mainly occurred in the single-organism process, cellular process, metabolic process, biological regulation, and response to a stimulus. Changes in molecular functions were significantly enriched in binding, and catalytic activity. Changes in cellular components were enriched in the cell, cell part, organelle, membrane, macromolecular complex.

Comparing the control group with the model groups, the model group with the LDKS group, and the model group with the HDKS group, yielded respectively, 134, 68, and 134 entries of significantly enriched pathways. The co-enriched pathways were screened and 47 entries were retained. An annotated classification (Supplementary Figure 6a-6c in Supplementary Information) indicated that these significant pathways were mainly associated with metabolic pathways, PPAR signalling pathway, and circadian rhythm.

3.7. Confirmation of the DEGs by qPCR

As shown in Figure 2A-2C, compared with the control group, the expression level of ARNTL, PER3, and CLOCK mRNA in the model group was decreased significantly (aP < 0.05). Compared with the model group, the level of ARNTL mRNA in the LDKS group was increased, but this difference was not statistically significant, whereas it was significant for the HDKS group (bP < 0.01; Figure 2A). Compared with the model group, the expression of CLOCK mRNA in the LDKS and the HDKS groups was increased, but this difference was not statistically significant (Figure 2B). Compared with the model group, the LDKS group had increased PER3 mRNA expression, but this difference was not significant, whereas it was significant for the HDKS group (cP < 0.05; Figure 2C).

3.8. Confirmation of the DEGs by Western blot

The results are shown in Figure 3. Compared with the control group, the expression of the ARNTL, PER3, and CLOCK proteins in the model group was decreased significantly (aP < 0.01; Figure 3A-3E).

Compared to the model group, the LDKS group and HDKS group had a significant increase in ARNTL protein expression (bP < 0.05 and cP < 0.01, respectively; Figure 3C). Interestingly, CLOCK and PER3 protein expression followed the same trend as ARNTL expression, in a dose-dependent manner (Figure 3D-3E).

4. DISCUSSION

In this study, we used a high-fat diet to induce obesity in SD rats, who typically developed high weight, Lee’s index, and lipids levels. We found a significant decrease in serum and liver lipid levels in rats fed treated orally with a Kushen decoction, suggesting a clear effect of the treatment on obesity hypertriglyceridemia.

Metabolic abnormalities occur in diseases such as cancer, diabetes, obesity, dementia, and cardiovascular disease.28-30 Adipogenesis is the process of cell differentiation into adipocytes, which involves the transformation of pre-adipocytes into mature adipocytes and the accumulation of intracellular lipids. Among the genes involved in this process, PPARγ is the main transcription factor that regulates early adipocyte differentiation, mid-term expression, and target gene expression in adipogenesis. It promotes fat production by upregulating the expression of fat cell fatty acid-binding proteins, and adiponectin.31-34 The results obtained by RNA-seq on liver tissue showed that the Kushen decoction mainly induced genes related to 47 pathways, including metabolic pathways, circadian rhythms, and PPAR signalling.

Circadian rhythms regulate important bodily activities at the transcriptional and translation levels by sharing a set of molecular mechanisms, including interconnected feedback loops.35,36 The positive loop involves CLOCK and ARNTL, which are E-box transcription factors that conjointly induce the transcription of the negative feedback regulatory loci PER1-3 and cryptochrome (CRY) 1-2. The proteins encoded by the PER and CRY loci form a complex that can translocate to the nucleus, inhibit the transcription of CLOCK and BMAL1, and exert a complete transcription-translation negative feedback pathway.37,38 CLOCK and BMAL1 play an important role in protecting the body from diseases caused by metabolic abnormalities. Mutations in the CLOCK gene lead to the development of hyperlipidemic and hyperinsulinemic hyperglycemia.39,40 The knock out BMAL1 in mouse increases sleep time, alters blood pressure regulation, glucose homeostasis, lipid metabolism, and fat production.41,42 Importantly, a high-fat diet suppresses the circadian rhythm regulated by the CLOCK gene because it increases the proportion of energy consumed in unconventional phases. This results in an increase in calorie consumption during abnormal circadian rhythms.43-45 A link between CLOCK gene polymorphism and susceptibility to obesity have been found in a small sample population study.46,47 In the present study, the level of the ARNTL, PER3, and CLOCK mRNAs in liver tissue was increased in the LDKS and the HDKS groups compared with the model group. Among these differences in mRNA expression, the increase of ARNTL and PER3 mRNA was statistically significant in the HDKS group. A possible reason for this result was that the Kushen decoction reduces the level of serum TG, which controls body weight.48, 49 Alternatively, fat consumption increases under the control of upregulated PER3 mRNA.50, 51

In summary, we used RNA-seq technology to analyse the changes in gene expression provoked by a Kushen decoction during treatment for hyperlipidemia in rats. In total, 17 407 predicted protein-coding genes were identified. After comparing the model group with the control group, 59 upregulated genes and 80 downregulated genes totalizing 139 DEGs were found. After comparing the LDKS group with the model group, a total of 45 DEGs were identified, including 32 upregulated and 13 downregulated genes. Finally, by comparing the HDKS group with the model group, a total of 114 DEGs were identified, including 82 upregulated and 32 downregulated genes.

In conclusion, our findings revealed DEGs enriched in metabolic pathways, PPAR signalling pathways, and circadian rhythm pathways. Further, the results obtained by qPCR and WB analyses were consistent with those obtained by RNAseq. Overall, this study may provide new insights into the mechanism of action of Kushen decoction to treat hyperlipidemia.

Contributor Information

Yanhua JI, Email: 20162018@jxutcm.edu.cn.

Zhijun ZENG, Email: zhijunzeng@aliyun.com.

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