Elucidating the mechanism by which regulating spleen and lipid method improves the state of hyperlipidaemic rats

Backgound: Hyperlipidemia has been highlighted as global chronic that the overall metabolic mechanism is closely related to the target protein LDLR. Conclusions: Metabolomics combined with network pharmacology suggested that RSLD can improve hyperlipidemia by enhancing LDLR protein expression to regulate disordered biomarkers and their metabolic pathways. Therefore, we believe that RSLD represents an interesting candidate for further characterization as a treatment for hyperlipidemia.


Background
Hyperlipidemia is mainly characterized by dyslipidemia, which is defined as an increase in serum levels of triglyceride (TG), total cholesterol (TC), lowdensity lipoprotein cholesterol (LDL-C) and decrease in level of high-density lipoprotein cholesterol (HDL-C) in the serum [1]. Strikingly, in 2017, hyperlipidemia contributed to half of global mortality [2]. The danger of hyperlipidemia arises out of its causal role in the induction of coronary heart disease, atherosclerosis, pancreatitis, hepatic steatosis and diabetes [3,4]. Statins have been widely used in treating hyperlipidemia, but are also associated with occasional hepatotoxicity, myalgia, and rhabdomyolysis [5,6]. Therefore, further exploration of the pathogenesis of hyperlipidemia and potential additional effective therapeutics are urgently needed.
Traditional Chinese medicine (TCM) theory and related research suggests that the spleen has an important function in metabolising and processing nutritive elements (including lipids) following the injections of food [7]. Further, the importance of spleen in lipid metabolism has been elucidated in a number of preclinical studies [8,9]. Therefore, if the spleen's core functions are dysregulated, systemic metabolism and excretion of lipids is impacted, leading to hyperlipidemia.
As a result, the spleen plays a fundamental role in the occurrence of hyperlipidemia. The regulating spleen and lipid decoction (RSLD) is a TCM preparation that has been used to treat hyperlipidemia for decades, created by professors of this research group. RSLD  Next, another 111 ml of water was added for another 40 minutes of reflux after filtering, and the solution was concentrated using a rotary evaporator at 50°C. Finally, RSLD was prepared to a final total concentration of 3 g drug product/ml and stored at 4°C.

Reagents and equipment
Liquid chromatography was performed using the

Establishment of the hyperlipidemia model
Hyperlipidemia rat model was established based on the method of Ji et al 2018 [12]. Briefly, rats were housed in separate cages with a normal diet and free access to water at the temperature of 23°C, 50% relative humidity, and a 12 h light-dark cycle. After one week, the rats were randomly divided into control group (n=8) fed with the general diet, and hyperlipidemia model group fed with HFD every day. After 5 weeks, the rats were fasted for 12 h, and then blood samples were taken from the tail for measurement of blood lipid levels in order to verify a successful model of hyperlipidemia. Finally, the animals were randomly divided into model group (n=8), XZK group (n=8) and RSLD group (n=8).

Dosing measurement design
The control group was given saline (0.5 ml/100 g), model, XZK and RSLD groups were given HFD (0.5 ml/100 g) every morning. In the afternoon, the control group was given saline (0.5 ml/100 g, according to their body weight), the XZK group was given XZK (0.5 ml/100 g) and the rats in the RSLD group was administered RSLD (0.5 ml/100 g, according to the transforming formula= (daily dose of RSLD in human (g))/human weight (kg)) ×9.1, the usual human weight is 60kg in China and 40g/d is the daily dose in humans, so the rat dose is 6g/d ) from the 6 th week to 9 th week.

Sample preparation
After 4 weeks treatment, each rat was placed alone in a metabolic cage to collect urine samples for 24 hours.
Next, 40 μl of urine was added in a microcentrifuge tube, followed by the addition of 120 μl of cold methanol, and centrifugation at 3,500rmp for 10 min. Next, 25 μl of supernatant was placed into a new eppendorf tube and diluted with 225 μl of 50% methanol. Finally, 25 μl of each sample was mixed into a quality control (QC) sample for liquid chromatography and mass spectrometry analysis. Blood was collected from the carotid artery, and the serum was separated by centrifugation at 3,500rpm for 10 min. Serum was stored at −80 ℃ for further biochemistry examinations.

Western blotting
First, the hepatic tissue was extracted and the total protein was obtained by using RIPA buffer containing PMSF, and then centrifuging at 12000 rpm for 10 min.
Second, the nuclear protein was extracted and the concentration was determined by the BCA protein assay.
Third, the total protein was subjected to SDS-PAGE gel electrophoresis and electroblotted onto a PVDF membrane, and the membrane was blocked in 5% BSA for 1 h and then incubated with the following primary antibody overnight at 4 °C. Antibodies: ACTIN (mouse monoclonal antibody 1:1000), low density lipoprotein receptor (LDLR, rabbit monoclonal antibody 1:1000 ), and then incubated the membrane with 1:3000 secondary antibody and visualized the conjugate using the ECL system. Finally, the protein expression was analyzed using the odyssey imaging system.

Statistical analysis
The total ion current (TIC) data was collected by UPLC-Q-TOF-MS and the data matrix of mass detection, retention time and peak intensity was processed by peak detection, matching, alignment and control processing. scipps.edu) and HMDB (http://www.hmdb.ca) to explain the mass spectra and identify the structure of the compounds.

The network pharmacology analysis
We set the criterias oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18 to screen the active ingredients in RSLD. Finally, the component-targetdisease network indicated that 43 target proteins were closely associated with 22 active components in RSLD ( As shown in Figure 1 and Table S1 ). In addition, the LDLR target protein was closely related to RSLD in improving hyperlipidemia.

Body weight and blood lipid level
We found that, compared with the model group, the body weight of rats in the XZK and RSLD group decreased significantly after treatment (Figure 2, P<0.01). As shown in Figure 3, after treatment, the HDL-C level was significantly increased in the XZK and RSLD groups (P<0.01), and TG, TC and LDL-C levels in XZK and RSLD groups were obviously lower than that of model group (P<0.01).

Figure 2 Body weight in each group(x±s(
Note: Compared with the model group at the same time period, * P < 0.01.

Figure 3 Blood lipid levels after treatment(x±s(
Note: Compared with the model group at the same time period, * P < 0.01.

Western blotting
LDLR plays an important role in improveing hyperlipidemia [13], so the protein expression of LDLR was analyzed in this study. As shown in the result ( Figure   4), the protein expression of LDLR in the model group was obviously decreased compared with the control group. While in XZK and RSLD group, the LDLR protein expression was obviously increased compared with the model group.

Metabolomic analysis
The overlaid chromatograms in the POS and NEG ion modes demonstrated good reproducibility ( Figure 5). The orthogonal partial least-squares discriminant analysis (OPLS-DA) score plot suggested that the inter-groups were well separated and the sample modelling was acceptable ( Figure 6). In addition, RSLD group was closer to control than XZK and model groups, which indicated that RSLD has a more powerful therapeutic effect than  (Table 1). We found that after treatment with RSLD, twenty-two potential biomarkers were upregulated, and four potential biomarkers were downregulated in the urine. The potential metabolic pathways disturbed in hyperglycaemic rats were presented in Figure 8. As shown in Figure 9, the following pathways had strong impacts: linoleic acid metabolism; taurine and hypotaurine metabolism; valine, leucine and isoleucine biosynthesis; glyoxylate and dicarboxylate metabolism and tricarboxylic acid (TCA) cycle.

XZK (Chinese National Food and Drug Administration
Standard YBZ01592004), whose main component is red yeast, is an effective antilipidemic drug developed by Chinese biopharmaceutical companies [14,15] Thus, we chose XZK to compare the lipid-lowering effect of RSLD.
We found that the body weight and blood lipid level improved significantly and some metabolites related to lipid metabolism showed the same trend after treatment with XZK and RSLD. Therefore, like XZK, RSLD appears to have a beneficial effect in regulating blood lipid levels.

The correlation between metabolomic results and hyperlipidemia
Abnormal lipid metabolism is the most obvious manifestation of hyperlipidemia. Linoleic acid is a polyunsaturated fatty acid and precursor of prostaglandins, which is helpful in lowering blood lipid [16]. In addition, linoleic acid has been identified via metabolomic profiling as being differentially regulated in hyperlipidemia progression [17]. In this study, compared with the model group, the level of linoleic acid in RSLD group increased obviously, and the impact on the linoleic acid metabolic pathway was significant (Figure 9), suggesting that RSLD may improve hyperlipidemia via a mechanism involving a change in linoleic acid metabolism.
Leucine and valine are essential amino that act as signalling molecules to control energy homeostasis involving lipid metabolism [18,19]. Additionally, leucine can regulate fat metabolism, reduce fat synthesis, promote fat decomposition and increase energy consumption in mammals [20,21]. It has been shown that supplementation with leucine can inhibit the action and synthesis of fatty acids, assisting in clinical weight loss [22,23]. In this work, compared with the model group, RSLD group led to increased concentrations of leucine and valine, indicating that RSLD may affect amino acid metabolism and regulate fat metabolism.
TCA cycle is responsible for acetyl coenzyme A (CoA) oxidation, releasing energy from fat, protein and sugar [24]. Citric acid is an important intermediate product in TCA cycle linking oxidative metabolism of carbohydrate, protein and fat [25]. Taurine in the liver combines with bile acids to form taurocholic acid, which can increase the solubility of lipids that play an important part in the absorption of lipids in the digestive tract [26,27], and a study found that taurine supplementation led to a reduction of TG, TC and LDL-C in alloxan-induced rats [28]. Uric acid is an important antioxidant involved in the body's acid-base balance in physiology and is closely related to the occurrence of hyperglycaemia [29,30]. In this work, we also found that the levels of citric acid, taurine and uric acid in hyperlipidemic rats were changed obviously after treatment with RSLD, providing a beneficial effect contributing to the regulation of dyslipidemia.

The correlation between network pharmacology and metabolomics
Prior work has shown that deficiency in LDLR proteins induces hyperlipidemia [13], and the LDLR (-/-) rats model have been widely used to mimic cardiovascular and cerebrovascular diseases [31,32]. The western blotting suggested that treatment with RSLD appears to enhance LDLR protein expression in the liver.
The network pharmacology study showed that the LDLR target protein is associated with RSLD. Previous metabolomic studies have shown that LDLR protein deficiency causes a significant metabolic perturbation in TCA cycle, amino acid metabolism and fatty acid metabolism, and influences levels of taurine, linoleic acid, citric acid, leucine, and valine [33][34][35]. As shown in Figure   10, we observed similar changes in concentration of taurine, linoleic acid, citric acid, leucine, and valine in these related metabolic pathways. Indeed, we believe that RSLD enhances protein expression of LDLR to restore function of amino acid metabolism, fatty acid metabolism and TCA cycle as a means of attenuating hyperlipidemia.

Ethics approval
This study was approved by the Animal Ethics Committee of Shandong University of Traditional Chinese Medicine.

Data availability
The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of interest
The authors declare that have no competing interests.