Lactobacillus mediates the expression of NPC1L1, CYP7A1, and ABCG5 genes to regulate cholesterol

Abstract Hypercholesterolemia is the main cause of cardiovascular disease worldwide, and the regulation of cholesterol homeostasis is essential for human health. Lactobacillus is present in large quantities in the human intestine. As the normal flora in the gut, lactobacillus plays an important role in regulating metabolism in the human body. Lactobacillus can regulate the cholesterol content by regulating the expression of genes involved in cholesterol synthesis, metabolism, and absorption. This article reviews the biological effects and mechanisms of lactobacillus that mediate the expression of NPC1L1, CYP7A1, ABCG5, ABCG8, and other genes to inhibit cholesterol absorption, and discusses the mechanism of reducing cholesterol by lactobacillus in cells in vitro, to provide a theoretical basis for the development and utilization of lactobacillus resources.


| INTRODUC TION
With the development of the social economy and the improvement of living standards, coronary heart disease (CHD) has become the most important cause of death globally (Fabian et al., 2016); the most common form of coronary heart disease is coronary artery disease (CAD). There is a positive correlation between elevated serum total cholesterol levels (mainly low-density lipoprotein cholesterol (LDL-C)) and the risk of coronary heart disease. And there is a linear relationship between the increase in LDL-C concentration and the relative risk of CAD. Through a large number of articles, the effect of lactobacillus on the content of LDL-C in serum was analyzed. Studies have found that every 1 mmol increase in serum cholesterol levels increases the risk of CHD by about 35% and coronary heart diseaserelated mortality by 45%; every 1% decrease in serum cholesterol levels reduces the risk of CHD by 2%-3% (Liong & Shah, 2005).
Moreover, statins are effective in treating hypercholesterolemia; however, such drugs are expensive and have harmful side effects

| S TUDY ON CHOLE S TEROL REG UL ATI ON BY L AC TOBACILLUS
Several studies have shown that lactobacillus can reduce the cholesterol content. The initial determination of the cholesterollowering ability of lactobacillus only stayed in the ability to reduce cholesterol content in the culture medium, through coprecipitation (Klaver & van der Meer, 1993), assimilation, and absorption (Pereira & Gibson, 2002). Studies have found that lactobacillus can  (Young et al., 1988). Dao Dong Pan et al. showed that L. fermentum SM-7 can coprecipitate and absorb 38.5% of the cholesterol in the culture medium, and the coprecipitation of cholesterol and bile acid increased rapidly when pH <6 (Pan et al., 2011). Assimilation and absorption involve cholesterol absorption by lactobacillus through the cell wall, cell membrane, and cytoplasm in an anaerobic environment. Anila et al. found that in broth containing bile salts, the removal rate of total cholesterol (TC) by lactobacillus was significantly higher than that in the non-bile salt group (Anila et al., 2016).
Moreover, the assimilation effect of growing cells on cholesterol was significantly higher than that of resting cells and dead cells, whereas no significant difference between dormant cells and dead cells was observed for cholesterol removal and assimilation (p < .05); the ability of lactobacillus to absorb cholesterol during death and dormancy indicates that cholesterol may also be eliminated by binding to the cell surface. Lim et al. evaluated the cholesterol-lowering ability of Lactobacillus LAB4 and L. plantarum LAB12 in the growth medium (Lim et al., 2017); cholesterol reduction rate of both lactobacilli was greater than 98%; Nile red staining revealed that lactobacillus absorbed cholesterol directly.
Thereafter, lactobacillus with the ability to lower cholesterol began to be used in animal experiments, and the mechanism of regulating cholesterol content by lactobacillus was studied in vivo. Gilliland et al. dis-covered that under anaerobic conditions, L. acidophilus (isolated from pig feces) can reduce the cholesterol content in a cholesterol medium containing bile salts (Gilliland et al., 1985). The content of bile salts in the medium is different, and the amount of cholesterol reduction is also different. Moreover, serum cholesterol content in pigs fed on L. acidophilus did not increase significantly compared with pigs on a normal diet.
Yadav and other studies found that when rats on a high cholesterol diet were fed fermented milk containing L. fermentum for 90 days, serum TC, low-density lipoprotein cholesterol, triglycerides, very low-density lipoprotein cholesterol, atherosclerosis index, coronary artery risk index, liver lipid, and lipid peroxidation degree were reduced significantly (p < .001), and the mRNA expression of inflammatory cytokines, namely TNFα and IL-6, was found significantly (p < .001) higher in the cholesterol-enriched diet group compared to the group fed fermented milk containing L. fermentum in the liver. Research suggests that L. fermentum can be used as a potential probiotic for treating hypercholesterolemia (Yadav et al., 2018). Research on cholesterol-reducing lactobacillus has developed rapidly. The initial research focuses on the isolation and screening of lactobacillus with cholesterol-lowering ability. Recently, it has focused on the study of the cholesterol-lowering mechanism of lactobacillus, and research on its mechanism is warranted.

REG UL ATE CHOLE S TEROL
Cholesterol is an important component of the human body. It is not only an important component of biofilms, but also the precursor of substances, such as vitamin D and bile acid (Liong & Shah, 2005).
Cholesterol metabolism in the body is shown in Figure 1 Table 1. Therefore, the mechanism of cholesterol-lowering by lactobacillus will be demonstrated from three aspects.

| Lactobacillus-mediated AMPK phosphorylation affects the rate-limiting enzyme HMGCR for cholesterol synthesis
Human cholesterol is mainly biosynthesized in the liver by mevalonic acid. In addition, cholesterol is synthesized in the intestine and adrenal glands (Tahri et al., 1996). HMGCR reduces 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate, which is the precursor of cholesterol. HMGCR is the rate-limiting enzyme in cholesterol synthesis and is therefore the focus of the regulation of cholesterol synthesis (DeBose-Boyd & Russell, 2008).
HMGCR located in endoplasmic reticulum in mammals, composed of 888 amino acids, is divided into two domains. The C-terminal consists of 548 amino acids, and the C-terminal extends into the cytoplasm and possesses enzyme activity (Roitelman, 1992), shown in  (Chen et al., 2016). Although the NF-κB subunit is not directly phosphorylated by AMPK, the inhibition of NF-κB signaling is mediated by several downstream targets of AMPK. In addition, NF-κB is essential for lactobacillus mediated HMGCR gene regulation, and the NF-κB activation pathway may be a potential therapeutic target for HMGCR signaling.

| Lactobacillus mediates the effect of CYP7A1 on cholesterol catabolism
The synthesis of bile acids plays a vital role in maintaining cholesterol homeostasis in mammals. CYP7A1 expression is liver specific that  (Horton et al., 2002) encodes cholesterol 7α-hydroxylase, which catalyzes the first step of cholesterol catabolism and bile acid synthesis (Pullinger et al., 2002). In vivo, FXR is more efficient than LXRα in inhibiting CYP7A1 mRNA expression (Ando et al., 2005

| Lactobacillus mediates the effect of NPC1L1 and ABCG5/8 on the absorption and transport of intestinal cholesterol
Cholesterol absorption and excretion mainly occurs in the intestine. The intestine can absorb about 50% of dietary cholesterol every day, which is about 400 mg of dietary cholesterol (Grundy & Metzger, 1972;Wilson & Rudel, 1994); the rest is excreted through feces. The first step of intestinal cholesterol absorption is through NPC1L1, which is a unidirectional cholesterol transporter located in the brush border membrane of intestinal cells (Iyer et al., 2005) and is highly expressed in the jejunum (Wang et al., 2012). NPC1L1 regulates the transport of free cholesterol in the intestinal lumen. seems to be involves a complex crosstalk between PPARα and LXRα (Huang et al., 2014). The activation of PPAR reduces the intestinal expression of NPC1L1 and promotes reverse cholesterol transport.

| Lactobacillus mediates the effect of SREBPs on the absorption and transport of liver cholesterol
The liver is the main organ for the synthesis and secretion of endogenous cholesterol, secreting about 1 g of cholesterol daily. Sterol regulatory element binding proteins (SREBPs) are membrane-anchored transcription factors that are expressed in the liver. They can alter cholesterol synthesis and uptake by modulating genes encoding cholesterol biosynthetic enzymes, including HMGCR, and low-density lipoprotein (LDL) receptors (Horton et al., 2002). The uptake of low-density lipoprotein cholesterol (LDL-C) in the serum is achieved through LDL receptors in the liver (Brown & Goldstein, 1983). SREBPs not only stimulate the expression of LDL receptors, but also enhance lipid synthesis (Brown & Goldstein, 1997). The mammalian SREBP gene includes three subtypes, named SREBP-1a, SREBP-1c, and SREBP-2. SREBP-1a is an effective activator of all SREBP-responsive genes, including genes that mediate the synthesis of cholesterol, fatty acids, and triglycerides. SREBP-1c preferentially activates the transcription of genes required for fatty acid synthesis, but not cholesterol synthesis. Similarly, SREBP-2 has a transcription activation domain, but preferentially activates LDL receptor genes and genes required for cholesterol synthesis (Morgan et al., 2017). LDL receptor levels are regulated by the negative feedback of SREBP-2 (Shin & Osborne, 2003). Evidence suggests a log-linear relationship between LDL-C concentration and relative risk of CHD. Lactobacillus can reduce serum LDL-C (Costabile et al., 2017;Smet et al., 1995). Studies have found that lactobacillus can reduce cholesterol concentration by regulating SREBP-2 expression (Li et al., 2014). Segawa et al. found that gavage heat-killed L. brevis SBC8803 to C57BL/6N mice can inhibit the upregulation of SREBP-1 and SREBP-2 mRNA expression and reduce cholesterol accumulation (Segawa et al., 2008).
In summary, during the catabolism, absorption, and transporta-

| D ISCUSS I ON
The Therefore, intervention and regulation by lactobacillus is a promising treatment and prevention method for hypercholesterolemia.

ACK N OWLED G EM ENT
This work was supported by Regional project of National Natural  Writing-review & editing (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data of this study are openly available.