Effects of sorghum rice and black rice on genes associated with cholesterol metabolism in hypercholesterolemic mice liver and intestine

Abstract The effects of different proportions of dietary sorghum rice and black rice on the expression of genes related to cholesterol metabolism in mice liver, intestine, and the characteristics of the small intestinal microbiota were investigated. Six types of diets were used to feed C57BL/6 mice: AIN‐93M standard diet, high‐cholesterol model diet, high‐cholesterol and low‐dose sorghum grain or black rice diet, and high‐cholesterol and high‐dose sorghum grain or black rice diet. The results showed that black rice or sorghum grain diets had no effect on the serum TC, LDL‐C levels in the hypercholesterolemic mice, whereas these diets decreased serum TG level, and black rice diets increased serum HDL‐C level. The diets containing black rice and sorghum grain had no effect on liver TC, TG, HDL‐C levels. However, these diets decreased LDL‐C levels significantly except high dose of black rice. The black rice or sorghum grain diets reduced the expression of the genes encoding liver 3‐hydroxyl‐3‐methyl‐glutarate monoacyl coenzyme A reductase (HMG‐CoA‐R) and increased the expression of SREBP‐2, thereby partially inhibiting the synthesis of cholesterol in liver. The diets containing different proportions of black rice and a low proportion of sorghum grain reduced the expression level of Niemann–Pick type C 1 like 1 (NPC1L1) mRNA and increased the mRNA level of the ATP‐binding cassette transporters, ABCG5/ABCG8, in the small intestine, thereby reducing cholesterol absorption. A diet containing a low proportion of black rice promoted the expression of ABCA1 mRNA and increased the expression of high‐density lipoprotein (HDL) mRNA, thereby promoting reverse cholesterol transport. Black rice diets significantly increased the relative abundances of microbiota in the small intestine and maintained biodiversity, while sorghum grain had no positive effect on the abundance of microbiota.


| INTRODUC TI ON
Cardiovascular disease is the leading cause of death in China.
Abnormal cholesterol metabolism in the body leads directly to atherosclerosis, which is the main cause of cardiovascular disease (Kianoush et al., 2016). Ezetimibe is a common drug that acts on the RCT pathway in the intestines to achieve cholesterol reduction, but it has no effect on cholesterol synthesis. Statin drugs are the most common and effective drugs for the treatment and prevention of hypercholesterolemia, but they have certain toxicity, such as increased gastrointestinal discomfort, elevated blood sugar, increased risk of cataract, liver damage, and myopathy (Hu et al., 2012). Therefore, choosing long-term food therapy with low side effects is one of the most effective methods for lowering cholesterol.
The cholesterol required by the body is mainly synthesized in the liver, and 3-hydroxyl-3-methyl-glutarate monoacyl coenzyme A reductase (HMG-CoA-R) is the main rate-limiting enzyme in cholesterol synthesis (Reihner et al., 1990). Sterol regulatory element-binding proteins (SREBPs) are major transcription factors in lipid metabolism, and SREBP-2 mainly regulates the genes involved in intracellular cholesterol metabolism via the INSIG-SREBP-SCAP pathway (Joseph et al., 2002). The excretion of cholesterol mainly depends on bile acids. Liver X receptor (LXR-ɑ) regulates DNA transcription factors and regulates cholesterol and lipid metabolism in the body via SREBP-2 and ATP-binding cassette subfamily A1 (ABCA1) (Wang et al., 2001), respectively. LXR-ɑ regulates the expression of the cholesterol 7-alpha hydroxylase gene (Cyp7a1) (Mitchell et al., 1991;Pullinger et al., 2002), thus inhibiting bile acid absorption and increasing cholesterol excretion. They are involved in the hepatic cholesterol metabolism and stimulate the excretion of cholesterol into the bile. Therefore, studying biochemical indices and genetic changes of hepatocyte cholesterol metabolism is important in the prevention and treatment of hypercholesterolemia.
Most organs, except for the liver, lack the enzymes that break down steroid hormones. Therefore, cholesterol cannot be excreted by forming bile acids in these organs. High-density lipoprotein (HDL) is a cholesterol carrier, which transports cholesterol back to the liver for degradation. This process is called reverse cholesterol transport (RCT). The RCT pathway is regulated by a variety of genes, such as the ATP-binding cassette transporter A and G family (ABCA1, ABCG5/8) (Jia et al., 2011), which are responsible for the binding of free cholesterol and apolipoprotein to form HDL. When the cholesterol level in the body is high, cholesterol is decomposed to form bile acids, which enter the intestine with bile. After bile acids enter the intestine, some are absorbed by the mucosa and return to the liver, the unabsorbed bile acids are degraded to fecal sterols by intestinal microorganisms and excreted (Anderson et al., 2019). The undecomposed cholesterol passes through the surface of the small intestine where it is reabsorbed by the Niemann-Pick C1 Like 1 (NPC1L1) and returns to the liver. This process is the classic enterohepatic circulation (Ahmed & Byrne, 2010). Therefore, the intestine is another important organ for cholesterol metabolism.
In addition, the intestinal microbiota is an important part of the human ecosystem and also associated with cholesterol absorption.
A high-fat diet not only raises cholesterol levels but also severely and rapidly changes the structure of intestinal microbiota. Cani et al. (2007) found that a high-fat diet causes damage to intestinal permeability and eventually allows harmful bacteria to enter in large quantities. Diets can affect the distribution of intestine microbiota, which in turn seriously affect human health (Possemiers et al., 2009).
More and more researchers have begun to focus on the effects of diet on the structure of intestinal microbiota.
In recent years, more attentions have been paid to the functional researches of grains due to their large daily consumptions. Sorghum rice, obtained after the decortication of sorghum seeds, is used as both a medicine and a food in China. However, sorghum rice has drawbacks, such as difficult digestion, astringent taste, which limits its edible value. In addition, the digestibility of sorghum protein is the lowest in cereal crops. Nonetheless, because of the high tannin content in sorghum, it has a certain physiological function. Kim et al. (2015) found that sorghum extract significantly reduced serum triglyceride and total cholesterol in a hypercholesterolemic mouse model and also significantly promoted the expression of CYP7A1 protein in mice. Park et al. (2012) found that sorghum extract significantly increased adiponectin expression in mice, reduced blood glucose levels, and reduced the expression of antitumor necrosis factor in serum.
Black rice refers to coarse black rice with husks removed. It is mainly distributed in Yunnan, Guizhou, and Guangxi, China. Black rice is rich in various trace elements which are at significantly higher levels than that in polished rice, and its nutrient use efficiency is also high. A variety of unique biologically active substances, such as anthocyanin flavonoids, cardiac glycosides, and alkaloids, which are mainly concentrated in the seed coat, confer black rice with strong free radical scavenging effects, cardiovascular protection, and sedation. Hou et al. (2013) found that black rice bran extracts significantly protected liver cells from damage and experiments in vitro showed that anthocyanin was the main active component in liver protection. Watanabe (2016) found that anthocyanin extract promoted mouse bile acid metabolism, improved lipid metabolism, and inhibited oxidative stress in the body.
According to modern Chinese dietary patterns, sorghum and black rice containing foods are only occasionally consumed and are generally not staple foods. As the consumption is low, it is associated with limited health benefits. In order to promote the applications of sorghum and black rice, it is necessary to study different proportions of sorghum and black rice in the diet and their consumption.
We hypothesized that different doses of sorghum and black rice may produce different effects on cholesterol metabolism and intestinal microbiota. Therefore, in this study, the effects of black rice and sorghum rice on hepatic cholesterol metabolism, intestinal microbiota, and cholesterol absorption and transport in the intestinal tract of hypercholesterolemic mice were then investigated to provide a basis for dietary treatment of hypercholesterolemia. The molecular mechanisms of these effects were analyzed and the suitable proportions of black rice or sorghum rice in the diet were investigated, to provide useful information on the application of black rice and sorghum rice.

| Design of animal experiments
Eight-week-old male C57BL/6 mice (specific-pathogen-free grade, 20 ± 2 g, n = 60) were purchased from the Zhaoyan New Drug The animals were maintained on a 12-hr light/12-hr dark cycle in an environmentally controlled room (temperature 23 ± 2°C and humidity 60 ± 10%). After 1 week of acclimatization, they were randomly divided into six groups (n = 10): group N (normal group, fed an AIN-93M diet), group H (high-cholesterol group, given a high-cholesterol diet), group B (low-dose black rice group, black rice replaced half the maltodextrin and corn starch in the high-cholesterol diet), group C (high-dose black rice group, black rice replaced all the maltodextrin and corn starch in the high-cholesterol diet), S (lowdose sorghum group, sorghum replaced half the maltodextrin and corn starch in the high-cholesterol diet), and T (high-dose sorghum group, sorghum replaced all the maltodextrin and corn starch in the high-cholesterol diet). The grain compositions were determined in advance, while the change in protein content was regulated by casein and the change in the fat in the feed was regulated by soybean oil.
All feeds were prepared by Trophic Animal Feed High-Tech Co., Ltd, (Jiangsu China). The mice were fed for 12 weeks. The dietary compositions in each group are listed in Table 1. The black rice and sorghum used in the experiment were purchased from Shandong Helaixiang Food Co., LTD. Black rice was from Jingzhou City, Hubei Province, China, and sorghum was from Jinan City, Shandong Province, China.

| Collection of samples
After 12 weeks of treatment, the mice were fasted for 12 hr overnight. They were anesthetized with ethyl ether, and blood was collected from their eyes. Their livers were removed, washed with phosphate-buffered saline (PBS), wiped dry, and weighed. The intact mouse small intestines were taken out. The contents of the small intestine were completely removed with sanitized tweezers in clean bench. Because the intestinal content of each mouse was not enough after fasting, the intestinal contents of three mice were mixed into one sample and placed in cryotubes. The tubes were immediately placed in liquid nitrogen and then stored in a freezer at − 80°C for analysis of microbiota diversity in the mouse intestinal feces. A section of the small intestine near the cecum was taken and fixed in formalin solution for H&E staining and observation. In addition, a section of small intestine was placed in 1 ml of Trizol reagent to determine the expression levels of genes related to cholesterol metabolism in the small intestine.

| Biochemical factors in sera and liver homogenates
The blood samples were allowed to stand and were then centri-

| RNA extraction and gene expression analysis
The total RNA in the livers and intestines was extracted with an RNA extraction kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's protocol, and

| Statistical analysis
The data are presented as means ± standard deviations (SD) and were analyzed with GraphPad Prism 6.0 (GraphPad Software, Inc.).
Student's t test was used to detect differences between group H and group N, B, C, S, T, respectively. Statistical significance was set at p < .05 and p < .01.

| Effects of black rice and sorghum rice intake on bodyweight and liver weight
In this study, there were no significant differences in body weight and food intake of the high-cholesterol diet between the groups (Figure 1

| Effects of black rice and sorghum rice diets on blood lipids in mice
The results of the blood lipid analysis showed that the serum TC levels in the H were significantly higher than those in N (Figure 1), indicating that the mouse model of hypercholesterolemia was successfully established (Matsuno et al., 2001). Diets containing different proportions of sorghum and black rice did not lower the serum TC levels in the hypercholesterolemic mice after the mice were fed the diets for 12 weeks. The trend of serum LDL-C levels in different groups is similar to that of serum TC levels. There was no significant difference in the serum TG levels between the group H and N, which differed from the serum TC results. However, the diets containing different proportions of sorghum and black rice significantly reduced the serum TG levels in the mice. The serum HDL-C levels in the group H were significantly lower than those in the group N, but the diets containing Sorghum rice did not change the serum HDL-C levels in the hypercholesterolemic mice, whereas the serum HDL-C levels in the group B and C were significantly increased. Black rice and sorghum rice stimulated the body to produce HDL-C. TC entered the bloodstream; it was then absorbed by HDL-C and transported back to the liver. This should be the reason for the higher serum TC and HDL-C levels in each grain group. Kim et al. (2006) demonstrated that high-purity anthocyanins had strong antioxidant effects, and the high-purity anthocyanins extracted from black rice reduced the TC, LDL-C, and TG levels in the sera of hypercholesterolemic mice (Ho et al., 2012). In the present study, whole black rice was added directly to the mouse diets, so the amounts of anthocyanins in the mouse diets were limited. However, in addition to anthocyanins, black rice also contains other flavonoids, terpenoids, and some beneficial proteins and polysaccharides (Dias et al., 2017), which may have some beneficial effects on the body. Shen et al. (2017) reported that adding 51% of sorghum flour to the diet had significant beneficial effects on TG, TC, HDL-C, and LDL-C. However, Moraes et al. (2018) found that the addition of 19% whole grain sorghum flour or decorticated sorghum flour or 12% sorghum bran to the diet did not significantly affect the TG, TC, HDL-C, or LDL-C levels in obese mice. The results of the present study showed that sorghum rice had an effect similar to that of black rice. The antioxidants in sorghum rice probably played a role in relieving oxidative stress in the hypercholesterolemic mice, but they did not reach the effect shown by Shen et al. This may be related to the use of fully decorticated sorghum rice, rather than whole sorghum rice, in the present study, or to the type of animal model used.

| Effects of black rice and sorghum rice diets on the morphology of mice liver
Hepatic steatosis is a typical nonalcoholic fatty liver disease associated with hyperlipidemia and obesity (Zheng et al., 2008). High cholesterol is also associated with reduced anti-inflammatory ability, which may contribute to hepatocyte death (Yeap et al., 2015).

| Effects of black rice and sorghum rice diets on liver lipids in mice
The results of the liver lipid analysis showed that the liver TC levels in H were significantly higher than those in N (Figure 2). Diets con-

| Effects of black rice and sorghum rice diets on the expression of genes involved in cholesterol metabolism in the mice liver
The expression of liver SREBP-2 mRNA was significantly lower in the group H than in the group N ( Figure 2). The expression of liver SREBP-2 mRNA was significantly higher in all the black rice and sorghum rice groups than in the group H, and the increases in the highdose groups (C,T) were greater than those in the low-dose groups.
The expression of liver HMG-CoA reductase mRNA was significantly higher in the group H than in the group N. The expression of liver HMG-CoA reductase mRNA was significantly lower in all the black rice and sorghum rice groups than in the group H, and the reductions in the high-dose groups were greater than those in the lowdose groups.  (Mitchell et al., 1991).
Our experimental results showed that the synthesis of cholesterol in the liver was inhibited through HMG-CoA reductase and SREBP-2 pathway, and cholesterol excretion increased through LDL-R pathway by eating black rice and sorghum rice for a long time.

| Effects of black rice and sorghum rice diets on intestinal villus structure
Villous morphology reflects the function of intestinal absorption.

High and dense intestinal villi increase the intestinal absorption area
and can fully absorb nutrients from food. On the contrary, damaged intestinal villi and villous loss will weaken digestion and absorption.
It can be observed in Figure

| Effects of black rice and sorghum rice diets on the expression of genes related to mouse intestinal cholesterol metabolism
The expression of NPC1L1 mRNA in the intestinal tract of group H was significantly increased relative to group N ( The expression of SR-B1 mRNA was significantly lower in H than in N (Figure 3). The expression of liver SR-B1 mRNA was significantly higher in all the black rice and sorghum rice groups than in H. The mechanism of hypercholesterolemia may be related to the low expression of LDL-R (LDL receptor) and SR-B1. Scavenger receptor type B type I (SR-BI) is a receptor protein which is currently recognized the only HDL receptor (Kozarsky et al., 1997). After entering the intestine, free cholesterol will bind to HDL under the mediation of SR-BI and form HDL-C, which will be transported back to the liver for further decomposition.
Black rice and sorghum rice diets both increased the expression of SR-BI mRNA, thus improving the reverse cholesterol transport.
The expression of ABCA1 mRNA was significantly decreased in

| Effects of black rice and sorghum rice diets on microbiota diversity in the intestinal feces of hypercholesterolemic mice
The alpha diversity indices were used for comparison. Common alpha diversity indices include ACE, Chao1, Shannon. ACE and  not significantly changed. However, compared with group H, the microbial species of sorghum groups showed a decreasing trend.
The results indicate that high proportion of black rice diet can effectively repair the intestinal microbiota ecosystem damaged by high-cholesterol diet, but there were no similar benefits from eating sorghum.
Beta diversity was calculated, and principal component analysis (PCA) plots were drawn to compare microbial communities from different intestinal feces samples, respectively. It can be observed in Figure 4; intestinal microbiota from N and H demonstrated higher divergence and were separated clearly by PCA.
In addition, intestinal microbiota from H and C were separated clearly. In contrast, samples from H, S, and T displayed closer relative distances and were difficult to distinguish (Figure 4). These results indicated that the intestinal microbial communities in H became obviously different from those in N after a high-cholesterol diet. The intestinal microbiota damaged from high-cholesterol diet could be improved significantly by high-dose black rice diet, while sorghum diets had little effect on the damage. The reason might be that sorghum contains plenty of tannins, which reduce intestinal digestibility to sorghum and might inhibit the growth of some intestinal flora (Sarwar et al., 2012).  Proteobacteria, and Verrucomicrobia. The total relative abundance of these four phyla was over 95% in all groups. The relative abundance of Bacteroidetes in N was the highest, reaching 50%. There was a big difference in intestinal microbiota between H and N. Firmicutes was dominant in the intestinal microbiota in H, and the relative abundance of Bacteroidetes was significantly reduced (p < .05) to about 25%. The composition of intestinal microbiota in S and T were similar to those in H. In addition, the relative abundances of Verrucomicrobia increased obviously in S (30%) and T (47%) compared to H, whereas those of Bacteroidetes decreased significantly (17% in S and 12% in T) accompanied by the increase of sorghum rice proportion in diet.
However, compared to H, the relative abundances of Bacteroidetes in two black rice groups were increased (30% in B and 50% in C) substantially and were higher than that of Firmicutes.
At taxonomic levels, more differences in relative abundances were observed. Figure 4b shows that Desulfovibrionaceae, Bacteroides, Akkermansia, and Alistipes were the main dominant flora in N, with a total relative abundance of 53%. Compared with N, the relative abundances of Bacteroides and Alistipes in H decreased significantly, whereas that of Akkermansia increased obviously. Compared with H, the relative abundances of Desulfovibrionaceae, Bacteroides and Alistipes increased and those of Akkermansia decreased in the intestinal microbiota in B and C. On the contrary, the relative abundances of Akkermansia increased, but that of Bacteroides decreased in the intestinal microbiota in S and T. Ley et al. (2006) believed that the relative proportion of Bacteroidetes was decreased in obese people by comparison with lean people. Turnbaugh et al. (2006) thought that obesity was associated with changes in the relative abundance of the two intestinal dominant bacterial divisions, the Bacteroidetes and the Firmicutes. It could be observed in Figure 4 that a lower ratio of B/F (Bacteroidetes/ Firmicutes) was found in group H. It indicates that a high-cholesterol diet can induce a lower ratio of B/F, just like a high-fat diet (Huang et al., 2019). As reported by Turnbaugh et al. (2006), low ratio of B/F had a negative impact on health because it may lead to obesity.
High dose of black rice diet promoted the health of the intestinal microbiota for its higher ratio of B/F, but high dose of sorghum diet continued to reduce the ratio of B/F after a high-cholesterol diet.

| CON CLUS ION
Our hypothesis was proved in this study that different doses of sorghum rice and black rice produced different effects on cholesterol metabolism and intestinal microbiota. The effects of black rice and sorghum rice on the lipid metabolism in hypercholesterolemic mice were investigated in this study. Our results showed that at the genetic level, diets containing different proportions of black rice or sorghum rice had certain beneficial effects on cholesterol synthesis in the liver after the mice were fed the diets for 12 weeks. However, at the biochemical level, the diets containing different proportions of black rice or sorghum rice had no significant effect on serum and liver TC in these mice, but they significantly reduced serum TG and liver LDL-C levels after the mice were fed the diets for 12 weeks.
Diets containing different proportions of black rice can reduced the absorption of cholesterol and promoting the reverse transportation of cholesterol in the intestine by regulating the expression levels of relative genes. In addition, black rice diets can also repair intestinal tissues damaged from a high-cholesterol diet and increase the proportions of beneficial intestinal bacteria. Although sorghum diets can also reduce the absorption of cholesterol in intestine, they have no beneficial effects on the intestinal mucosal structure and intestinal bacteria. Therefore, eating black rice seems more beneficial to cholesterol metabolism and intestinal flora stability than eating sorghum through overall consideration.

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest to declare.

AUTH O R CO NTR I B UTI O N
Haiying Liu involved in conceptualization, methodology, project administration, resources, writing-review and editing, and funding acquisition. Lu Huang involved in software and writing-original draft.
Xinli Pei involved in data curation, software, and formal analysis.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in [repository name, e.g. "figshare"] at xxxx, reference number [ref- erence number].