Hederagenin Exerts Potential Antilipemic Effect via p38MAPK Pathway in Oleic Acid-induced HepG2 cells and in Hyperlipidemic Rats

: Hederagenin, a natural compound distributed in many medicinal plants, has a variety of pharmacological properties including anti-bacteria, anti-infl ammation, anti-oxidation, and anti- apoptosis.. The aim of this study was to evaluate the effects of hederagenin on decreasing blood lipid and anti-oxidative stress in oleic acid-induced HepG2 cells and hyperlipidemic rats, and explore underlying mechanisms. In vitro , TG was used as the index to verify the lipid-lowering effect of hederagenin in oleic acid-induced HepG2 cells. In vivo , TC, TG, LDL-C, and HDL-C were used as direct indicators to study the antilipemic effect of hederagenin in hyperlipidemic rats. MDA, SOD, and GSH-PX were measured to analyze the anti-oxidative effect of hederagenin. The signaling pathways of anti-oxidation were evaluated using Western blot. Our results showed that hederagenin (250μmol/L) increased signifi cantly TG clearance rate. In addition, treatment with hederagenin, XZK and simvastatin reduced effectively TC, TG, LDL-C and MDA content, and increased HDL-C, SOD and GSH-PX in HFD rats. Moreover, the phosphorylation level of p38 MAPK was inhibited after administration of hederagenin, XZK and simvastatin. Our results revealed that hederagenin possessed benefi cial potentials for hypolipidemic effects, especially in TG clearance. The mechanism might be associated with inhibition of lipid absorption, reduction of lipid oxidation, and down-regulation of phosphorylation.


INTRODUCTION
H yperlipoidemia (H LP), a disorder of lipid metabolism, refers to the pathological state that the level of t otal cholesterol (TC) and/or t riglyceride (TG) in serum or plasma increases independently or simultaneously in fasting condition (Liu et al. 2011).
According to the results of the 2012 Chinese lipid census, the morbidity of various types of abnormal lipid metabolism was 11.43% (Moran et al. 2010). Among patients with lipid metabolism disorder, the average TC and TG in adult serum were 4.50 mmol/L and 1.38 mmol/L, with prevalence rates of 4.9% and 13.1% respectively. Abnormal lipid metabolism greatly increased the occurrence of cardiovascular and cerebrovascular diseases (coronary heart disease, ischemic stroke, etc.) (Wu et al. 2006, Sarwar et al. 2007). As scientifi c investigation showed that about 4.4 million people died every year from ischemic stroke and heart disease induced by elevated cholesterol in serum (Conroy et al. 2003, Guilbert 2003.
Clinical treatment of high blood fat disease was broadly divided into cholesterollowering drugs (statins, cholic acid chelators etc. (H et al. 2001)) and triglyceride-lowering drugs (bate, niacin, etc.) (Rubins et al. 1999, Keech et al. 2005, Das et al. 2010, Group et al. 2010. However, some hypolipidemic agents could lower both cholesterol and triglycerides, especially for severe hyperlipidemia. Although statins are effective in lowering lipids, they have serious side effects, such as liver damage and rhabdomyolysis. Hederagenin, a pentacyclic triterpene are widely distributed in many medicinal plants, such as dipsacus asperoids, clematis chinensis, radix pulsatillae, lonicerae japonicae, etc. Studies have found that hederagenin has a variety of properties such as anti-tumor, antidepression, anti-bacteria and anti-inflammation and anti-diabetes (Kim et al. 2017a, b). However, a function of hederagenin in lowering blood lipid or anti-hyperlipidemia is not clear.
Hyperlipidemia is often accompanied by lipid oxidation caused by oxygen free radicals (Omari-Siaw et al. 2016). In the process of producing oxidized lipids, malondialdehyde (MDA) is the product of reaction of polyunsaturated fatty acids and an active oxygen, whose changes in concentration reflects the level of lipid oxidation (Wang et al. 2015, Lykkesfeldt 2007. Superoxide dismutase (SOD) is an antioxidant enzyme with a level that reflects the ability of free radicals to remove oxygen in the body (Wang et al. 2015). As an important peroxidase decomposing enzyme, glutathione-peroxidase (GSH-PX) can transform the lipid peroxides into more stable hydroxyl compounds in time and block the free radical chain reaction in lipid peroxidation (Bednarek-Tupikowska et al. 2001).
It is well known that activated molecules can indirectly trigger a variety of stresssensitive serine/threonine kinase signaling pathways including the p38 isoform mitogenactivated protein kinase (p38MAPK) (Klaunig et al. 2010). The current research had shown that hederagenin was documented to inhibit MAPK activation (Tian et al. 2020). Moreover, a high fat-diet (HFD) could lead to an increase of TC and TG in serum (Fu et al. 2019). Therefore, in this study, we investigated the lipid-lowering effects of using oleic acid-induced HepG2 cells and HFD-induced hyperlipidemia animal model. Furthermore, the p38MAPK signaling pathway related to lipid metabolism and antioxidant was evaluated and validated. Together, we provided evidence that hederagenin could lower the level of lipid, which might be associated with the inhibition of p38MAPK signaling pathway.

Cell Culture and Treatment
HepG2 cells with good growth status were cultured in Dulbecco's modified Eagle's medium (DMEM) high sugar medium containing 1% fetal bovine serum (FBS) and 2% Bovine serum protein (BSA). Cells were grown in 37°C with a humidified atmosphere containing 5% CO 2 . HepG2 cells was seeded in in 24-well plates when the concentration was adjusted to about 10 5 / mL. After incubation for 24 h, HepG2 cells were set as blank group, model group, XZK group, Simvastatin group, and different concentration hederagenin group, each with 3 parallel wells. The blank group was added to 1% FBS high glucose DMEM medium containing 2% BSA, and the model group was added 1% FBS high glucose DMEM medium containing a final concentration of 1 mmol/L oleic acid to each well. In the XZK group, 1% FBS high-sugar DMEM medium containing a final concentration of 1 mmol/L oleic acid and 200 μg/mL drug was added to each well. In the simvastatin group, 1% FBS high-sugar DMEM medium containing a final concentration of 1 mmol/L oleic acid and 20 μg/mL drug was added to each well. The hederagenin group was supplemented with 1% FBS high glucose DMEM medium containing a final concentration of 1 mmol/L oleic acid and different concentrations of drugs. Then place the 24-well plate in a constant temperature incubator and incubate for 24 hours, then remove the culture medium, wash it twice with PBS, and then use Western and IP cell lysate for ice bath for 30 min, collect the lysate, and centrifuge at 4 12000 r/min for 10 minutes, take the supernatant, and determine the triglyceride and protein content according to the operating steps on the kit, each group has 6 parallel holes, and the results are expressed in mmol/gprot, and the intracellular TG clearance rate of each administration group is calculated at the same time.

Animals and Treatment
Male Sprague-Dawley (SD) rats (180-220 g) were purchased from Comparative Medical Center in Yangzhou University, Jiangsu Province, China (Certificate No: SCXK2019-0117), and maintained on a 12-hour light-dark cycle, 22 ± 1°C,55 ± 3% humidity, with ad libitum feeding. All the animal experiments were carried out according to the Guidelines of Chinese Experimental Animal Administration Legislation, and were approved by the Animal Ethics Committee of China (Document No: 55, 2019).

Blood Samples and Liver Tissue Preparation
After the last intragastric administration, blood samples were drawn from the abdominal aorta when rats were anesthetized deeply with 20% urethane, then the liver was excised from rats and was stored in ice-cold normal saline.
Determination of TC, TG, Low-Density Lipoprotein-Cholesterol (LDL-C) and High-Density Lipoprotein-Cholesterol (HDL-C) The serum obtained from blood samples was operated according to the instructions of the TC, TG, LDL-C and HDL-C kits respectively, and the absorbance was measured with a microplate reader.

Determinatio n of SOD, MDA, and GSH-PX
The liver ti ssue was homogenized by cold normal saline (1 mg liver tissue in 9 ml of normal saline). The supernatants were separated in condition of 3,000 rpm, 20 min. The protein concentrati ons were detected by Coomassie blue protein binding using bovine serum album as a standard. Test kits were used to detect the levels of SOD, MDA and GSH-PX in supernatants, whose procedures followed strictly the manufacturer's specifi cation (Wei et al. 1997, Katiyar et al. 2011).

Western blots
The liver samples were lysed with cold lysis buffer (Solarbio, Beijing, China), and the samples homogenates were centrifuged at 13,000 rpm for 15 min at 4 °C. The protein concentrations of supernatant were determined with a BCA protein assay kit (Beyotime, Jiangsu, China). The protein samples were mixed with loading buffer in a volume ratio of 4:1 and then boiled for 10 min. Subsequently, the protein samples were separated on 5% SDS-PAGE and transferred to a polyvinylidene fl uoride (PVDF) membrane . The PVDF membranes were blocked for 1.5 h in 5% skim milk and subjected to immunoblotting using primary anti-MAPK antibody overnight at 4 °C. Then, the PVDF membranes were washed three time of 10 min each with TBST and incubated with secondary horseradish peroxidase (HRP)-conjugated antibody for 2 h at room temperature. The blotting of membranes was detected by chemiluminescence (ECL) kits (Millipore, Billerica, MA, USA) and Bio-Rad imaging systems. Relative intensities of protein bands were analyzed by Image J software.

Data analysis
Data are represented as means ±standard deviation ( ). Statistical analysis of differences between groups was performed by one-way ANOVA using SPSS 19 statistical software. Differences were considered signifi cant at P < 0.05.

Effect of Hederag enin on TG in HepG2 Cells Induced by Oleic Acid
Compared with the blank group, the TG in the model g roup increased significantly, indicating that the oleic acid-induced HepG2 cell fat accumulation model was successfully established. As shown in Table I, compared with the model group, XZK, simvastatin, and hederagenin (100 μmol/L, 250 μmol/L) signifi cantly reduced the level of TG (P <0.01); TG clearance rates were: 30.84%, 41.94%, 24.76%, and 35.81%, respectively. Meanwhile, the result showed that hederagenin administration led to decreased TG in a dose-dependent manner.

Effect of Hederagenin on the Level of Lipid
Next, we tested whether the decrease of weight in HFD-induced models is related to hederagenin effect on the level of lipid. The levels of TC, TG and LDL-C were increased remarkably in the model group compared with the blank group, and the level of HDL-C decreased significantly, indicating that hyperlipidemia model was established successfully. The serum lipid profile markedly improved, especially in the hederagenin highdose group (H-H) after drug administration for 8 weeks. The levels of TC, TG, and LDL-C in the H-H group were 24.2%, 27.3% and 23.1% lower than those in the model group, respectively (P<0.05), and the HDL-C level in the H-H group was 6.4% higher than that in the model group (Fig. 1ad). The simvastatin treatment also significantly improved the serum TC, TG, LDL-C and HDL-C levels compared to the model group (P < 0.05).

Effect of Hederagenin on Response to Oxidative Stress
Furthermore, this study investigated whether hederagenin decreased the level of lipid in HFDinduced rats is associated with the effect of that on anti-oxidation. As shown as Fig. 2a

Effect of Hederagenin on the p38MAPK Pathway
How hederagenin improved the response to oxidative stress in rats induced by HFD is not clear. Therefore, the proteins related to p38MAPK pathway were tested with Western blot. Fig. 3 displayed the effects of hederagenin on the levels of total-p38 and p-p38. There was no striking difference in total-p38 expression between blank group and other groups. However, HFD induced p38MAPK phosphorylation, leading to p38MAPK signaling activation. Compared to the blank group, phosphorylation of p38MAPK in the model group significantly overexpressed. In treatment groups (simvastatin, XZK, H-L, H-M, and H-H), however, the expression of phosphorylation of p38MAPK significantly decreased. Interestingly, the hederagenin treatment groups were observed in dose-dependent manner in decrease of phosphorylation of p38MAPK.

DISCUSSION
Hyperlipidemia with TC, TG, LDL-C and HDL-C as main reference indicators is a leading cause of death from cardiovascular diseases throughout the globe (Le 2008). It is well known that hyperlipidemia is key risk factor of heart diseases, including atherosclerosis and coronary heart disease (Rosenson 2006, Le & Walter 2007, Das et al. 2010. Statins are the mainstay of lipidlowering therapy in clinic which primarily act on LDL, also slightly lower the level of TG, and induce a moderate increase in HDL-C. However, statins have additional risk factors of increasing side effects and adverse drug interactions. In the meantime, previous studies had shown that hederagenin could effectively treat tumors through anti-oxidation (Kim et al. 2017a) and antiinflammation (Lee et al. 2015). Lowering blood lipids is often closely related to properties of antioxidation (Stein et al. 2020, Gimeno-Mallench . However, there are no studies have reported that hederagenin can decrease blood lipid. Therefore, we hypothesized that hederagenin had the effect of reducing blood lipids and conducted a preliminary study on its mechanism. In vitro studies, preliminary results showed that hederagenin (100 mol/L, 250 mol/L) had beneficial effect on oleic acid induced-HepG2 cells. This proved that hederagenin could protect HepG2 cells from oleic acid by reducing intracellular TG level.
This study adopted a HFD to establish an animal model of hyperlipidemia similar to human disease, with highly reproducible results and easy operation. Previous studies had shown that rats who were fed a high cholesterol diet for 6 weeks showed a significant increase levels of TC and LDL-C in serum (Fki et al. 2005), which was consistent with the results of our study. Our study demonstrates that hederagenin could effectively slow the trend of weight gain in rats fed a HFD. In addition, this study also showed that hederagenin had a dose-dependent effect, the reasons for this were unclear, and we suspected that it might be related to the suppression of lipid absorption.
Our data demonstrated hederagenin notably improved lipid profile, thereby exerting hypolipidemic effect. Among them, hederagenin was effective in reducing TG which is a key risk factor of atherosclerosis, heart disease and fatty liver. If there is overabundance of TG, hoarding in the subcutaneous would lead to body obesity; hoarding in the blood vessel wall would cause atherosclerosis; hoarding in the heart would lead to heart hypertrophy; and hoarding in the liver would cause fatty liver. These were the leading causes of mortality throughout the globe (Le 2008). As we know, liver is one of the main organs of TG synthesis and metabolism. Liver cells are involved in many stages of lipid metabolism; therefore, it is possible that hyperlipidemia drives the development of fatty liver (Lee et al. 2014, Tessari et al. 2009). It is reported that most cholesterol is synthesized by the body itself, and a little is obtained from the diet (Kapourchali et al. 2016). On the contrary, TG are mostly obtained from the diet and a small part is made by the body itself (Alves-Bezerra & Cohen 2017). Therefore, the best way to reduce TG is to inhibit TG absorption. Our results show that Hederagenin has a good TG clearance rate and is dose-dependent, which may be the reason why hederagenin has an effective anti-lipid effect.
Oxidative stress is the unifying mechanism for causing many cardiovascular diseases, especially in atherogenesis (Singh & Jialal 2006, Marchio et al. 2019, Peluso et al. 2012). In addition, we learned that the main characteristics of antioxidation was to reduce TG, increase HDL and adiponectin, and prevent LDL from being oxidized. Therefore, we selected SOD, MDA, and GSH-PX as anti-oxidative indicators to verify the antioxidant capacity of hederagenin. The results showed HFD induced oxidative stress reaction in rats, which were consistent with previous studies (Meng et al. 2011). Our results also showed that the blood lipid levels and anti-oxidative index of hederagenin showed a precise correlation ( Fig. 1 and Fig. 2). Therefore, we believed that anti-oxidative stress may be one of the lipid-lowering mechanisms of hederagenin, which was consistent with other studies (Wang et al. 2020, Pengrattanachot et al. 2020) on lipid lowering and anti-oxidation results. Thus, hederagenin might be used as an antioxidant to treat hyperlipidemia.
Oxidative stress is well known to stimulate the MAPK pathway activation, especially the phosphorylation of p38MAPK. Furthermore, in order to further confirm hederagenin antioxidation activity, we conducted experiments from a molecular perspective and offered a plausible explanation for the molecular mechanism of hederagenin on decreasing blood lipid. The results of this study showed that HFD promoted phosphorylation of p38MAPK. The affections of oxidative stress on cellular signaling pathways and activation of p38MAPK pathways were inhibited by treatment with hederagenin, simvastatin and XZK.

CONCLUSION
In conclusion, the present study identified the scientific basis for the therapeutic effects of hederagenin in the treatment of hyperlipidemia. Our results found that hederagenin possessed the anti-oxidative potential for lipids-lowering in the rats received HFD, which might be related to suppression of p38MAPK phosphorylation. Hederagenin could inhibit MAPK pathway activation, thus exert anti-oxidation and lipidslowering effect in rats induced by HFD.