Octacosanol Modifies Obesity, Expression Profile and Inflammation Response of Hepatic Tissues in High-Fat Diet Mice

The incidence of obesity has increased significantly on account of the alterations of living habits, especially changes in eating habits. In this study, we investigated the effect of octacosanol on lipid lowering and its molecular mechanism. High-fat diet (HFD)-induced obesity mouse model was used in the study. Thirty C57BL/6J mice were divided into control, HFD, and HFD+Oct groups randomly, and every group included ten mice. The mice of HFD+Oct group were intragastrically administrated 100 mg/kg/day of octacosanol. After 10 weeks for treatment, our results indicated that octacosanol supplementation decreased the body, liver, and adipose tissues weight of HFD mice; levels of TC, TG, and LDL-c were reduced in the plasma of HFD mice; and level of HDL-c were increased. H&E staining indicated that octacosanol supplementation reduces the size of fat droplets of hepatic tissues and adipose cells comparing with the HFD group. Gene chip analysis found that octacosanol regulated 72 genes involved in lipid metabolism in the tissues of liver comparing to the HFD group. IPA pathway network analysis indicated that PPAR and AMPK may play a pivotal role in the lipid-lowering function of octacosanol. Real-time quantitative PCR and Western blot showed that the octacosanol supplementation caused change of expression levels of AMPK, PPARs, FASN, ACC, SREBP-1c, and SIRT1, which were closely related to lipid metabolism. Taken together, our results suggest that octacosanol supplementation exerts a lipid-decreasing effect in the HFD-fed mice through modulating the lipid metabolism-related signal pathway.


Introduction
The incidence of obesity has increased significantly on account of the alterations of living habits, especially changes in eating habits. Hyperlipidemia is vital factor of obesity, characterized by the abnormal increase of one or more lipid components, mainly referring
All animal experiments involved in this study were reviewed and approved by the Guidelines for the Care and Use of Experimental Animals, the Hunan Normal University, College of Medicine (SYXK-Xiang, 2015-0007). All mice were obtained from air-conditioned animal rooms under specific pathogen-free and 12/12 h reverse light/dark cycles conditions, and free access to water and food. Wild-type C57BL/6J male mice of 7 to 8 weeks old were individually housed and maintained at an environment of 23 ± 2 • C and adapted to the experimental condition based on stable food and water intake.

Octacosanol Treatment
All mice were randomly divided into three groups (10 per group) at random after an adaptation period of one week: the control (Con), HFD, and HFD+Octacosanol (HFD+Oct) group. The Con group was given basic diet and water ad libitum, while the HFD or HFD+Oct group were given a high-fat diet. Octacosanol was additionally administrated to mice of HFD+Oct group by gavage (200 mg/Kg) 3 days before HFD feeding until the end of the experiment about 11 weeks later. As a parallel measurement, the other two groups were intragastric administrated the same volume of water simultaneously every day.

Glucose Tolerance Testing
After fasting for 6 h, the glucose tolerance of mice was performed by intraperitoneal (i.p.) injection of 2 g of 25% glucose (Sigma-Aldrich, St. Louis, MO, USA). The blood glucose concentrations in the tail of mice were measured by a glucometer (Hemocue, Angelholm, Sweden) at the time points of 0, 15, 30, 60, and 120 min. Data are described as mean concentration of blood glucose per group using area under the curve (AUC; glucose challenge).

Analysis of Blood Biochemical Indicators
The whole blood samples of mice were obtained from anterior cubital veins by venipuncture, adding heparin and ethylenediaminetetraacetic acid (EDTA) immediately, and swiftly centrifuging to separate plasma. The plasma parameters of TC, TG, highdensity lipoprotein (HDL), and low-density lipoprotein (LDL) were measured. The assay method refers to our previous publication [12].

Histological Analysis
The whole liver and retroperitoneal adipose tissues were carefully removed after sacrifice of mice. About 100 mg liver and retroperitoneal adipose tissues from the same site were intercepted for histological analysis, fixed in 10% (100 g/L) formalin solution for over 24 h, and dehydrated in absolute ethanol. Then the tissues were transplanted with xylene before paraffin imbedding, cut into 5 µm sections with microtome, mounted on clean glass slides, and finally dried overnight at 37 • C. After dewaxing with xylene and dehydrating with absolute ethanol, prepared tissue sections were subjected to hematoxylin and eosin (H&E) staining, followed by observing under microscope [12].

RNA Isolation and Array Processing
Liver tissues were lysed using Transzol Up (TransGen, Beijing, China) under liquid nitrogen cooling to extract the total RNA. The obtained RNA samples were stored in −80 • C refrigerator. NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific Company, Waltham, MA, USA) and standard denaturing agarose gel electrophoresis were used to evaluate RNA concentration quality and integrity, respectively. Using Invitrogen SuperScript ds-cDNA synthesis kit, 5 µg of total RNA was synthesized into double-stranded cDNA (ds-cDNA), which next were washed and labeled according to the NimbleGen Gene Expression Analysis Protocol (NimbleGen Systems, Inc., Madison, WI, USA): 4 µg of RNase was incubated with ds-cDNA for 10 min at 37 • C and cleaned with chloroform: isopropyl alcohol, then precipitated with absolute ethanol at 4 • C. The purified cDNA was quantitatively analyzed using NanoDrop ND-2000. According to the manufacturer's guidelines in the Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA), the cDNA was labeled with Cy3 using NimbleGen One-Color DNA Labeling Kit. One OD Cy3 primer was incubated with 1 µg of ds-cDNA for 10 min at 98 • C, followed by addition of 100 pmol of deoxynucleoside triphosphates and 100 U of the Klenow enzyme (New England Biolabs, Ipswich, MA, USA), and incubation at 37 • C for 2 h. Thereafter, the reaction was terminated by adding 0.1 vol of 0.5 M EDTA, and the purified ds-cDNA was precipitated with isopropanol/ethanol. In a hybridization chamber (Hybridization System-NimbleGen Systems, Madison, WI, USA), the microarray was subjected to hybridization with 4 µg of Cy3-labeled ds-cDNA in the hybridization buffer/hybridization fraction A at 42 • C for 16 h. After carefully washing with Wash Buffer (NimbleGen Systems, Madison, WI, USA), the gene chips were scanned (Axon GenePix 4000B microarray scanner) after washing in an ozone-free environment. Differentially expressed genes between the two groups were identified by fold-change filtration or by t-test filtration. Hierarchical clustering was performed using MeV software (4.4v). Protein molecular network obtained from octacosanol-modified genes of upregulated and downregulated expressions were analyzed by IPA software.

RT-PCR Analyzing mRNA Expressions
The CFX96 Real-Time PCR system (Applied Biosystems Co., Ltd., Foster City, CA, USA) was used for real time quantitative RT-qPCR analysis performed by SYBR ® Select Master Mix Kit (Applied Biosystems Co., Ltd.). According to the manufacturer's protocol (Applied Biosystems Co., Ltd., Foster City, CA, USA), expression levels of genes involved in proinflammatory cytokines (IL-6 and TNF-α), lipid metabolism (SIRT1, PPAR, ACC, and CD36), and β-actin (control) were analyzed. The relative expression level of mRNA was measured by PCR system (Applied Biosystems, USA) software represented by the ratio of expression level of target gene to endogenous gene showed in bar graph. The PCR primers used in the experiments was described in our previous publication [12].

Statistical Analysis
Each experiment was repeated three times. Statistical analyses were performed using SPSS Statistics software (Version 2.50, SPSS Inc., Chicago, IL, USA). The differences among groups were analyzed using one-way analysis of variance test. Data shown as the mean ± standard deviation (SD) were considered statistically significant at p-value less than 0.05. Correlations between two variables were determined by Pearson's correlation coefficient.

Octacosanol Enhances Glucose Tolerance of HFD Mice
Glucose tolerance refers to the ability of the body to regulate blood glucose concentration. After eating high-starch foods, such as rice and flour staple foods, or taking glucose, the glucose is absorbed by the intestine, makes the body's blood sugar rise, and stimulates the insulin secretion, and the liver glycogen synthesis increases, decomposition is suppressed, the liver glycogen output reduces, and after, the body of blood sugar is maintained in a relatively stable range. After 11 weeks' supplement of octacosanol in mouse experiments, the results showed that the area under the glucose tolerance curve (AUC) increased by 23.74%, and the AUC decreased by 17.44% in mice fed with octacosanol compared with normal control mice; the results showed that octacosanol could enhance the glucose tolerance of obese mice, as shown in Figure 1. standard deviation (SD) were considered statistically significant at p-value less than 0.05. Correlations between two variables were determined by Pearson's correlation coefficient.

Octacosanol Enhances Glucose Tolerance of HFD Mice
Glucose tolerance refers to the ability of the body to regulate blood glucose concentration. After eating high-starch foods, such as rice and flour staple foods, or taking glucose, the glucose is absorbed by the intestine, makes the body's blood sugar rise, and stimulates the insulin secretion, and the liver glycogen synthesis increases, decomposition is suppressed, the liver glycogen output reduces, and after, the body of blood sugar is maintained in a relatively stable range. After 11 weeks' supplement of octacosanol in mouse experiments, the results showed that the area under the glucose tolerance curve (AUC) increased by 23.74%, and the AUC decreased by 17.44% in mice fed with octacosanol compared with normal control mice; the results showed that octacosanol could enhance the glucose tolerance of obese mice, as shown in Figure 1.

Octacosanol Ameliorate the Obesity Phenotype of HFD-Induced Mice
The mice fed with octacosanol for 11 weeks had a slight decrease in food intake, but there was no statistically significant difference. The weight gain of mice in the Oct+HFD group was significantly lower than that in HFD group but still higher than that in the Con group. The average body weight of HFD mice raised from 23.50 ± 0.91 to 26.72 ± 2.86 g after 11 weeks of HFD (p < 0.01). The average weight of mice treated with octacosanol was 25.35 ± 1.93 g (p < 0.05). As shown in Figure 2A, the weight gain of the mice in HFD group was significantly higher than that in Con group, while octacosanol reduced the weight gain These results suggest that octacosanol partially inhibited the weight gain in the HFD mice.

Octacosanol Ameliorate the Obesity Phenotype of HFD-Induced Mice
The mice fed with octacosanol for 11 weeks had a slight decrease in food intake, but there was no statistically significant difference. The weight gain of mice in the Oct+HFD group was significantly lower than that in HFD group but still higher than that in the Con group. The average body weight of HFD mice raised from 23.50 ± 0.91 to 26.72 ± 2.86 g after 11 weeks of HFD (p < 0.01). The average weight of mice treated with octacosanol was 25.35 ± 1.93 g (p < 0.05). As shown in Figure 2A, the weight gain of the mice in HFD group was significantly higher than that in Con group, while octacosanol reduced the weight gain. These results suggest that octacosanol partially inhibited the weight gain in the HFD mice.
The liver of mice in HFD group was larger, whiter, smoother, and greasier than those in the normal group, and octacosanol significantly inhibited the liver enlargement and whitening induced by HFD, as shown in Figure 2B. Compared with Con group, the liver weight in the HFD group raised from 1.49 ± 0.15 g to 1.65 ± 0.27 g (p < 0.01). Octacosanol supplementation significantly improved liver damage induced by HFD, with liver weight reduced to 1.52 ± 0.13 g, as shown in Figure 2B. These data show that octacosanol can alleviate liver weight gain in hyperlipidemic mice and liver fat accumulation induced by HFD.
At the end of the experiment, the weight of the retroperitoneal fat pad was measured. HFD diet resulted in more significant abdominal fat deposits than the Con group, while octacosanol observably reduced the weight and size of the retroperitoneal fat tissue, as shown in Figure 2. The weight of adipose tissue in Con group, HFD group, and HFD+Oct group were 1.46 ± 0.09, 1.84 ± 0.17, and 1.56 ± 0.07 g, respectively, as shown in Figure 2. The above evidence shows that octacosanol can effectively inhibit the HFD-induced abdominal fat deposition in mice. The liver of mice in HFD group was larger, whiter, smoother, and greasier than those in the normal group, and octacosanol significantly inhibited the liver enlargement and whitening induced by HFD, as shown in Figure 2B. Compared with Con group, the liver weight in the HFD group raised from 1.49 ± 0.15 g to 1.65 ± 0.27 g (p < 0.01). Octacosanol supplementation significantly improved liver damage induced by HFD, with liver weight reduced to 1.52 ± 0.13 g, as shown in Figure 2B. These data show that octacosanol can alleviate liver weight gain in hyperlipidemic mice and liver fat accumulation induced by HFD.
At the end of the experiment, the weight of the retroperitoneal fat pad was measured. HFD diet resulted in more significant abdominal fat deposits than the Con group, while octacosanol observably reduced the weight and size of the retroperitoneal fat tissue, as shown in Figure 2. The weight of adipose tissue in Con group, HFD group, and HFD+Oct group were 1.46 ± 0.09, 1.84 ± 0.17, and 1.56 ± 0.07 g, respectively, as shown in Figure 2. The above evidence shows that octacosanol can effectively inhibit the HFD-induced abdominal fat deposition in mice.

Histological Alterations of Liver Tissues and Epididymal Fat
To estimate the effect of octacosanol on the histology and morphology of mice, livers and epididymal fat were cut into slices and stained by H&E. The image of liver pathological sections of mice in each group ( Figure 3A) showed that the hepatocyte in Con group were normal in morphology and distribution, and the cytoplasm was homogeneous without fatty degeneration, while hepatocyte of mice in HFD group were swollen and arranged as loosely scattered with the white vacuole formed by different lipid droplets. The hepatocyte of mice in HFD+Oct group was near the normal hepatocyte in Con group, and the white vacuole was less than HFD group. Compared with HFD group, octacosanol

Histological Alterations of Liver Tissues and Epididymal Fat
To estimate the effect of octacosanol on the histology and morphology of mice, livers and epididymal fat were cut into slices and stained by H&E. The image of liver pathological sections of mice in each group ( Figure 3A) showed that the hepatocyte in Con group were normal in morphology and distribution, and the cytoplasm was homogeneous without fatty degeneration, while hepatocyte of mice in HFD group were swollen and arranged as loosely scattered with the white vacuole formed by different lipid droplets. The hepatocyte of mice in HFD+Oct group was near the normal hepatocyte in Con group, and the white vacuole was less than HFD group. Compared with HFD group, octacosanol treatment can reduce the epididymal fat cell hypertrophy induced by high-fat diet ( Figure 3B). All these data indicate that octacosanol may lower the lipids of liver by reducing the lipid accumulation and prevent the formation of fatty liver to a certain extent.
Foods 2022, 11, x FOR PEER REVIEW 7 of 18 treatment can reduce the epididymal fat cell hypertrophy induced by high-fat diet ( Figure  3B). All these data indicate that octacosanol may lower the lipids of liver by reducing the lipid accumulation and prevent the formation of fatty liver to a certain extent.

Octacosanol Improves the Plasma Lipid Level in High-Fat Diet Mice
The blood samples of mice were collected to acquire the lipid profiles of plasma. Compared with Con group, TG level was obviously increased in the HFD group mice; the TG level in HFD+Oct group was 45.96% (p < 0.05, Figure 4A) lower than that in HFD group. The results indicate that octacosanol could obviously reduce TG level of plasma in HFD mice. Likewise, compared with Con group, TC level was increased in the HFD group mice; TC level in HFD+Oct group was significantly reduced by 26.93% compared with HFD group (p < 0.05, Figure 4B), indicating that intervention with octacosanol can significantly inhibit the increase of TC level in HFD mice. Compared with HFD group, LDL-c level in HFD+Oct group was decreased by 47.93% (p < 0.05, Figure 4C). Compared with HFD group, HDL-c level in HFD+Oct group decreased by 11.22% (p = 0.06, Figure 4D), and the difference was not significant (p ˃ 0.05, Figure 4D).

Octacosanol Improves the Plasma Lipid Level in High-Fat Diet Mice
The blood samples of mice were collected to acquire the lipid profiles of plasma. Compared with Con group, TG level was obviously increased in the HFD group mice; the TG level in HFD+Oct group was 45.96% (p < 0.05, Figure 4A) lower than that in HFD group. The results indicate that octacosanol could obviously reduce TG level of plasma in HFD mice. Likewise, compared with Con group, TC level was increased in the HFD group mice; TC level in HFD+Oct group was significantly reduced by 26.93% compared with HFD group (p < 0.05, Figure 4B), indicating that intervention with octacosanol can significantly inhibit the increase of TC level in HFD mice. Compared with HFD group, LDL-c level in HFD+Oct group was decreased by 47.93% (p < 0.05, Figure 4C). Compared with HFD group, HDL-c level in HFD+Oct group decreased by 11.22% (p = 0.06, Figure 4D), and the difference was not significant (p > 0.05, Figure 4D).

Octacosanol Modulated the Gene Expression Profile of Liver Tissues
cDNA microarray was applied to analyze the gene expression in mouse liver tissues. There were 72 differentially expressed genes in related to lipid, which was initially confirmed by PubMed database retrieval among more than 15,000 genes, including 44 downregulated genes (Table 1) and 28 upregulated genes ( Table 2). By combining with hierarchical clustering analysis, it can be seen from the heatmap that there are 72 obviously different expression profiles between the two groups ( Figure 5). Comparing with Con group, 44 genes were upregulated expressions in the liver tissues and octacosanol supplementation inhibited the 44 gene expressions. Comparing with Con group, 28 genes were downregulated expressions in the liver tissues, and octacosanol supplementation upregulated

Octacosanol Modulated the Gene Expression Profile of Liver Tissues
cDNA microarray was applied to analyze the gene expression in mouse liver tissues. There were 72 differentially expressed genes in related to lipid, which was initially confirmed by PubMed database retrieval among more than 15,000 genes, including 44 downregulated genes (Table 1) and 28 upregulated genes ( Table 2). By combining with hierarchical clustering analysis, it can be seen from the heatmap that there are 72 obviously different expression profiles between the two groups ( Figure 5). Comparing with Con group, 44 genes were upregulated expressions in the liver tissues and octacosanol supplementation inhibited the 44 gene expressions. Comparing with Con group, 28 genes were downregulated expressions in the liver tissues, and octacosanol supplementation upregulated the 44 gene expressions (Figure 4).  . Clustering analysis showed the changed genes of HFD and HFD+ with control group. Seventy-two differentially expressed genes were screene in both HFD and HFD+Oct groups. Color represents the log intensities. Red r expression genes; green represents the decreased expression genes. The den clustering according to gene classification. The heatmap was plotted using octacosanol; HFD, high-fat diet.

GO and KEGG Analyzed the 72 Different Expression Genes
The genes were divided into 18 categories according to their biological functions, including fatty acid metabolic process, response to muscle stretch, metabolism of lipids, lipid homeostasis, regulation of lipid metabolic process, insulin resistance, and so on. About half of the alternated genes are closely associated with lipid metabolism (see Figure 6). KEGG analysis indicated that octacosanol supplementation modulated PPAR pathway, sphingolipid pathway, fatty acid degradation pathway, insulin resistance pathway, and lectin receptor pathway, and all of those pathways are closely related with lipid metabolism. Meanwhile, octacosanol supplementation also regulated the Toll-like receptor pathway, which is a key pathway of inflammation, suggesting octacosanol may inhibit HFD-induced inflammation through the Toll-like receptor pathway (see Figure 7).

GO and KEGG Analyzed the 72 Different Expression Genes
The genes were divided into 18 categories according to their biological functions, including fatty acid metabolic process, response to muscle stretch, metabolism of lipids, lipid homeostasis, regulation of lipid metabolic process, insulin resistance, and so on. About half of the alternated genes are closely associated with lipid metabolism (see Figure  6). KEGG analysis indicated that octacosanol supplementation modulated PPAR pathway, sphingolipid pathway, fatty acid degradation pathway, insulin resistance pathway, and lectin receptor pathway, and all of those pathways are closely related with lipid metabolism. Meanwhile, octacosanol supplementation also regulated the Toll-like receptor pathway, which is a key pathway of inflammation, suggesting octacosanol may inhibit HFD-induced inflammation through the Toll-like receptor pathway (see Figure 7).

IPA Analysis Indicated That Octacosanol Modulated AMPK a
To explore the lipid-lowering underlying mechanism of octa expressed genes were further analyzed by signal pathway ana protein network via IPA software. After octacosanol supplemen pressed genes affecting the lipid-lowering function were signif pathway and AMPK signaling pathway (Figure 8). These data ERK and AMPK might play an important role in the octacosan

IPA Analysis Indicated That Octacosanol Modulated AMPK and ERK Pathways
To explore the lipid-lowering underlying mechanism of octacosanol, the 72 differentially expressed genes were further analyzed by signal pathway analysis and the construction of protein network via IPA software. After octacosanol supplementation, the differentially expressed genes affecting the lipid-lowering function were significantly enriched in the ERK pathway and AMPK signaling pathway (Figure 8). These data showed that the two kinases ERK and AMPK might play an important role in the octacosanol lipid-lowering function. expressed genes were further analyzed by signal pathway analysis and protein network via IPA software. After octacosanol supplementation, th pressed genes affecting the lipid-lowering function were significantly en pathway and AMPK signaling pathway (Figure 8). These data showed t ERK and AMPK might play an important role in the octacosanol lipid-l Figure 8. Gene network of octacosanol regulating the lipid metabolism. The n data were uploaded into IPA software to produce the gene network through an ison between our data with the IPA database. Red, increased expression gen expression genes; and the darkness of the color stands for the ratio of fold chan octacosanol regulating the lipid metabolism. Figure 8. Gene network of octacosanol regulating the lipid metabolism. The normalized gene chip data were uploaded into IPA software to produce the gene network through an automatic comparison between our data with the IPA database. Red, increased expression genes; green, decreased expression genes; and the darkness of the color stands for the ratio of fold change. Gene network of octacosanol regulating the lipid metabolism.

Octacosanol Affected Lipid Metabolism-Related Gene Expressions
To further reveal the possible mechanism of the lipid-lowering function of octacosanol, the mRNA levels and protein levels of lipid metabolism-related genes were examined by RT-qPCR analysis and WB test, respectively. The data of RT-qPCR showed that the mRNA expressions of those genes, such as PPAR, SIRT-1, ACC, and SREBP-1c, were evidently increased in HFD mice compared with the Con group ( Figure 9A). WB data indicated that the protein expressions of those genes, such as PPAR, SIRT-1, and ACC, were also significantly increased in HFD mice compared with the Con group (p < 0.05) ( Figure 9B). However, the protein levels of SREBP-1c did not change between HFD group and HFD+Oct group. It suggests that octacosanol may exert lipid-lowering function through regulating the expression of genes related to lipid metabolism.
by RT-qPCR analysis and WB test, respectively. The data of RT-qPCR showed that the mRNA expressions of those genes, such as PPAR, SIRT-1, ACC, and SREBP-1c, were evidently increased in HFD mice compared with the Con group ( Figure 9A). WB data indicated that the protein expressions of those genes, such as PPAR, SIRT-1, and ACC, were also significantly increased in HFD mice compared with the Con group (p < 0.05) ( Figure  9B). However, the protein levels of SREBP-1c did not change between HFD group and HFD+Oct group. It suggests that octacosanol may exert lipid-lowering function through regulating the expression of genes related to lipid metabolism.

Octacosanol Affected PPAR and AMPK Signal Pathways
To further reveal the possible signal pathways of the lipid-lowering effect of octacosanol, protein samples of liver tissues were analyzed by WB. The test results showed that HDF promoted phosphorylations of PPAR-α (p-PPAR-α) and p-PPAR-γ but did not affect p-PPAR-δ, suggesting that HDF can activate PPAR signal pathway. Octacosanal supplementation could decrease HDF-induced phosphorylations of PPAR-α and PPAR-γ, which means octacosanol can inhibit PPAR signal pathway ( Figure 10A). Compared with the HFD group, octacosanol promoted phosphorylation of AMPK (pAMPK-T172) in the liver tissue of mice and significantly increased the protein level of pAMPK ( Figure 10B). These showed that octacosanol may inhibit lipid metabolism-related gene expressions by modulating the AMPK signal pathway and PPAR signal pathway.
HDF promoted phosphorylations of PPAR-α (p-PPAR-α) and p-PPAR-γ but did not affect p-PPAR-δ, suggesting that HDF can activate PPAR signal pathway. Octacosanal supplementation could decrease HDF-induced phosphorylations of PPAR-α and PPAR-γ, which means octacosanol can inhibit PPAR signal pathway ( Figure 10A). Compared with the HFD group, octacosanol promoted phosphorylation of AMPK (pAMPK-T172) in the liver tissue of mice and significantly increased the protein level of pAMPK ( Figure 10B). These showed that octacosanol may inhibit lipid metabolism-related gene expressions by modulating the AMPK signal pathway and PPAR signal pathway.

Octacosanol Decreases Inflammatory Factor Expressions
Considering that the inflammatory reaction is related to the increase of blood lipids [13], the expression levels of inflammatory cytokines were analyzed by RT-qPCR and WB. The experiment results showed that HFD can increase the expressions of TNF-α, IL-6, and iNOS and octacosanol can remarkably reduce the expression levels of these factors by RT-qPCR, p < 0.01, and p < 0.05, respectively ( Figure 11). WB analysis confirmed that octacosanol could inhibit the protein expression of these inflammatory cytokines ( Figure 11). It suggests that octacosanol can restrain the expression levels of inflammatory cytokines in RNA and protein layers, thus reducing the inflammation accompanied with obesity caused by a high-fat diet.

Octacosanol Decreases Inflammatory Factor Expressions
Considering that the inflammatory reaction is related to the increase of blood lipids [13], the expression levels of inflammatory cytokines were analyzed by RT-qPCR and WB. The experiment results showed that HFD can increase the expressions of TNF-α, IL-6, and iNOS and octacosanol can remarkably reduce the expression levels of these factors by RT-qPCR, p < 0.01, and p < 0.05, respectively ( Figure 11). WB analysis confirmed that octacosanol could inhibit the protein expression of these inflammatory cytokines ( Figure 11). It suggests that octacosanol can restrain the expression levels of inflammatory cytokines in RNA and protein layers, thus reducing the inflammation accompanied with obesity caused by a high-fat diet.

Discussions
Unhealthy diets and lacking exercise result in a growing population of people obesity. Obesity patients are usually characterized by a significant increase of triglyce and total cholesterol. Now, most of lipid-lowering drugs used in clinical treatments different side effect in human bodies. It is necessary to explore novel natural compo

Discussions
Unhealthy diets and lacking exercise result in a growing population of people with obesity. Obesity patients are usually characterized by a significant increase of triglycerides and total cholesterol. Now, most of lipid-lowering drugs used in clinical treatments have different side effect in human bodies. It is necessary to explore novel natural compounds to exert the lipid-lowering effect and decrease the side effect of lipid-lowering drugs. Previous investigations showed that some foods or natural compounds, such as polysaccharides, flavonoids, and glycosides, could reduce plasma lipid levels without obvious side effects [14][15][16]. In this study, our research found that octacosanol supplementation in diets significantly reduced HFD-induced obesity in mice. Octacosanol reduced the weight gain in mouse bodies, lipid accumulation in the liver, and epididymal tissues in the HFD-induced mice. Plasma lipid profiles of TC, TG, and LDL-c was decreased, and that of HDL-c was upregulated, meaning octacosanol could significantly ameliorate blood lipid index of HFD mice. The result is consistent with recent reports [10].
To explain lowering-lipid mechanisms of octacosanol, gene chips analysis was used to analyze the gene expression profiles of hepatic tissues. Our study is the first investigation to report expression profile alternated by octacosanol supplementation in HFD mice, which benefit us to comprehensively understand the lowering-lipid effect of octacosanol. The alternated expression genes are related with lipid metabolism, including the changed expressions of lipid metabolism-related genes and multiple signal pathways of lipid metabolism. Further IPA analysis also confirms the discovery and constructed the protein network modulating by octacosanol supplementation.
In this study, our data showed that the classic lipid metabolism-related genes, such as SREBP1, ACC, PPARα, and FASN, are modulated by octacosanol, and it also regulated AMPK pathway in the liver tissues of HFD mice. The kinase AMPK is the vital regulator of energy metabolism, which could reduce ATP-consuming processes and upregulate ATP regeneration [12,17]. AMPK takes part in many biological functions: the pathological processes of obesity, hyperlipidemia, and diabetes. The AMPK has different types of heterotrimeric complexes, which include an alpha-catalytic, a beta-regulatory, and a gamma-regulatory subunit. Threonine phosphorylation of AMPK is necessary to activate the kinase, and activated AMPK promotes the oxidation of fatty acids and also reduces the synthesis of cholesterol, fat, and TG, which causes the decrease of lipid accumulation. On the contrary, AMPK inactivation could reduce the oxidation of fatty acids and cause hyperlipidemia [16][17][18]. In the study, we found that octacosanol supplementation promoted AMPK phosphorylation and activation, which means the lipid-lowering effect of octacosanol may be mediated by AMPK pathway.
SIRT1 is an important metabolism and energy sensor and belongs to NAD+-dependent deacetylase. SIRT1 usually takes part in homeostatic responses to nutrient availability, and SIRT1 is the key regulator of lipid metabolism-related gene expressions [19,20]. In the study, our results indicated that octacosanol supplementation promoted SIRT1 expression in the liver tissues of HFD mice. In fact, SIRT1 can activate AMPK and decrease Fas expression, which can reduce lipid accumulation in liver tissues. Interestingly, AMPK can also activate SIRT1 and trigger deacetylation by upregulating NAD+ levels, which modulates lipid metabolism-related gene expressions and exerts a lipid-lowering effect.
Many investigations showed that AMPK activation, SREBP-1, and ACC inhibition could ameliorate hyperlipemia therapy. AMPK can phosphorylate ACC and promote ACC inactivation. ACC can catalyze malonyl-CoA production from acetyl-CoA, and it is an important rate-limiting enzyme of fat synthesis. ACC plays a vital role in the fatty acids metabolism, and ACC activation could modulate fatty acid synthesis [21,22]. SREBP is an important lipogenic transcription factor and controls lipogenesis, which includes the synthesis of fatty acids and triglycerides. AMPK can prevent SREBP-1 activation, thereby decreasing the transcriptional expressions of fatty acid and TG synthesis-related enzymes [23]. In the study, HFD decreased AMPK activation and promoted SREBP1-mediated lipogenesis. Meanwhile, FAS, an SREBP1 target protein, upregulated expression. The FAS gene is fatty acid synthase and directly participates in fatty acid synthesis, which causes an accumulation of TC and TG [24]. AMPK can prevent ACC activation and FASN expression, which decreases lipid accumulation, suggesting that octacosanol supplementation exerts a lipid-lowering effect that may be mediated by the AMPK/SREBP1/ACC pathway.
Plasma cholesterol content can be modulated by transcription factors SREBPs and LXRs. LXRs are the most abundantly expressed in the liver and control multiple important processes of cholesterol metabolism [25,26]. For example, LXRs can prevent cholesterol absorption in the gut and increase its reverse transport, which causes the convert of bile acid and then promotes bile acid excretion [25]. In the study, LXR expression was obviously reduced in the liver tissues of HFD mice, which causes the imbalance of cholesterol metabolism and leads to hyperlipidemia. On the contrary, octacosanol increased LXR expression in the liver tissues of HFD mice, suggesting that the octacosanol may modulate the LXR pathway and regulate cholesterol metabolism.
PPARs belong to the ligand-activated nuclear receptor superfamily, and they have three subtypes (PPARα, PPARγ, and PPARδ). PPARs have many different biological functions, including carcinogenesis, inflammation, and lipids metabolism [27][28][29]. PPARs especially modulate many gene expressions of lipids metabolism. PPARα is highly expressed in liver tissues and has high mitochondrial and â-oxidation activity, which participates in lipid metabolism; PPARδ regulates gene expressions of lipid oxidation and energy dissipation; PPARγ is a transcription factor and takes part in adipocyte differentiation and fat formation. Activated PPARα activation can promote fatty acid oxidation and reduces the level of circulating triglyceride; meanwhile, it also can reduce lipid storage. AMPK could modulate expressions of PPARα/PPARγ and their target genes [27,30]. In this study, our results indicated that octacosanol can activate AMPK and partly reduce the expressions of PPARγ and PPARδ; meanwhile, octacosanol increased the PPARα expression in the HFD mouse model, suggesting that PPARs are also important target and participates in the anti-hyperlipidemic function of octacosanol.
Previous investigations indicate that obesity and high-fat symptoms can cause chronic inflammation in the body. Excessive fat accumulation can promote MAPK-NF-κB activation, which promotes the expressions and secretions of pro-inflammatory factors [11]. In the study, our results indicated that HFD also increased the expressions of pro-inflammatory factors in mouse liver tissues, and octacosanol supplementation decreased the expressions of pro-inflammatory factors. MAPK is the important signal pathway to regulate the expressions of inflammatory factor [31,32]. MAPK can activate NF-κB, and it is the vital transcript factor and controls the transcript expressions of many inflammatory factors, and the promoters of most of inflammatory factors include one or more binding sites of NF-κB [33][34][35]. Octacosanol supplementation could inhibit NF-κB activation, suggesting that octacosanol supplementation prevents inflammation and may be mediated by regulating NF-κB in the HFD mice. This result matches our previous investigation that octacosanol inhibits inflammation through the MAPK/NF-κB pathway.
In conclusion, our investigation showed that octacosanol could ameliorate hyperlipidemia in the HFD-induced mouse model, and the lipid-lowering function was mediated by multiple pathways, including AMPK/SREBP-1c/ACC, AMPK/SIRT1, PPARs, and LXR pathways. Octacosanol could reduce ameliorate inflammatory response in the liver tissues of HFD-induced mice, and the anti-inflammation effect was mediated by MAPK/NF-κB pathway. Taken together, dietary interventions using natural compounds such as octacosanol might be a candidate for an adjuvant therapy for hyperlipidemia.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Animal Ethics Committee of Hunan Agricultural University (protocol code 43321575 and 1 December 2014).

Informed Consent Statement:
The study did not involve humans.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.