Lactobacillus plantarum LRCC 5273 isolated from Kimchi ameliorates diet-induced hypercholesterolemia in C57BL/6 mice

ABSTRACT

This study was designed to select potent cholesterol-lowering probiotic strains on HepG2 cell and investigate the effect of selected strain, Lactobacillus plantarum LRCC 5273 and LRCC 5279 in hypercholesterolemic mice. In the results, LP5273 group showed significantly reduced total and LDL cholesterol compared to HCD group. In addition to significantly up-regulated hepatic mRNA expression of LXR-α and CYP7A1, intestinal LXR-α and ABCG5 were significantly up-regulated in LP5273 group. With activation of hepatic and intestinal LXR-α and its target genes, fecal cholesterol and bile acid excretion were increased in LP5273 fed mice. These results suggest that LP5273 ameliorates hypercholesterolemia in mice through the activation of hepatic and intestinal LXR-α, resulting in enhancement of fecal cholesterol and bile acids excretion in the small intestine. The results of present study suggest mechanistic evidences for hypocholesterolemic effects of L. plantarum spp., and may contribute to future researches for prevention of hypercholesterolemia and cardiovascular disease.

Graphical Abstract

LP5273 ameliorates hypercholesterolemia through the activation of hepatic and intestinal LXR-alpha, resulting in enhancement of fecal cholesterol and bile acids excretion.

KEYWORDS:

• Hypercholesterolemia
• LXR-α
• Lactobacillus plantarum LRCC 5273
• high-cholesterol diet
• kimchi

Cardiovascular disease (CVD), the most common cause of death in modern society, results from hypercholesterolemia. Hypercholesterolemia is defined as the presence of high levels of cholesterol in the blood, which are caused due to hereditarily impaired cholesterol metabolism [1] or increased dietary cholesterol intake [2]. Elevated cholesterol levels in plasma leads to an increase in low density lipoprotein, forming lipid-laden macrophages known as foam cells. Foam cells form plaques on the blood vessel walls thereby impeding blood flow. The primary onset of atherosclerosis is signaled by plaque formation accompanied by inflammation. Hence, regulation of plasma cholesterol levels is essential to prevent CVD and hypercholesterolemia. Commonly, hypercholesterolemia is treated or controlled with drugs such as statins and ezetimibe, regulation of diet, or exercise [3–5]. However, the undesirable side effects of drugs have caused concerns about their therapeutic use [6]. Therefore, recently there has been great interest in using biomaterials for their cholesterol-regulating effects, because of their low toxicity and fewer side effects. Especially, lactic acid bacteria (LAB) as probiotics have become the focus of intensive international research for their health-promoting effects. The numerous health benefits of LAB have turned them into promising probiotic candidates that are being studied for their desirable properties. Thus, investigators are paying close attention to the cholesterol-lowering effects of LAB, among their many functional effects.

Probiotics is defined as “live micro-organism which provides beneficial effects to human health when it is consumed.” It has several health-promoting effects, such as improvement of immune responses, anti-inflammatory, anti-diabetic, and anti-hypercholesterolemic effects [7]. For decades, many researchers have tried to evaluate and elucidate the hypocholesterolemic effects of Lactobacillus strains. A number of in vitro and in vivo evidences have demonstrated that Lactobacillus strains reduce serum cholesterol levels through incorporation of cholesterol into cell wall, oxidation of cholesterol, promotion of bile acid excretion, and regulation of gene expression involved in cholesterol metabolism [8–12]. Lactobacillus strains have been shown to regulate key genes involved in cholesterol metabolism and transport [8,13,14]. In addition, Lactobacillus strains play an important role in balancing cholesterol in enterohepatic circulation. Cholesterol, being a precursor of bile acids, converts its molecules to bile acids replacing those lost during excretion, leading to a reduction in serum cholesterol. This mechanism could be utilized in the control of serum cholesterol levels by conversion of deconjugated bile acids into secondary bile acids by intestinal microbiota. Bile acids in human body are secreted from liver into the intestinal lumen in the conjugated form with glycine and taurine. Since conjugated form of bile acids is amphipathic, it acts as an emulsifier and helps in the absorption of dietary lipids. It is re-absorbed into the body, emulsifying lipophilic materials in the intestinal lumen [15]. It has been reported that Lactobacillus plantarum ssp. have bile salt hydrolase (BSH) enzyme that deconjugates the bond between glycosides and bile acids. As the deconjugated form of bile acids is much less soluble than conjugated form, it may not be dissolved in intestinal fluid and re-absorbed into enterohepatic circulation. Consequently, BSH-activity could inhibit the re-absorption, while promoting the excretion of bile acids resulting in promotion of cholesterol consumption in the body [16].

In the present study, we investigated the effects of the administration of L. plantarum LRCC 5273 and LRCC 5279 having bile salt hydrolyzing activity on serum cholesterol metabolism in diet-induced hypercholesterolemic C57BL/6 mice.

Materials and methods

Maintenance and preparation of bacterial cultures

Thirty-eight probiotic strains isolated from kimchi (Lotte Foods Co. Seoul, Korea) were screened. Bacterial cultures were maintained by inoculation (2%, v/v) in MRS broth (BD Korea, Inc., Seoul, Korea) and incubation for 24 h at 37°C. The cultures were sub-cultured twice before use.

Cell culture

The human hepatocellular carcinoma cell line, HepG2 cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). HepG2 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution in an incubator with 5% CO₂ at 37°C.

Stimulation of HepG2 cells with probiotics

HepG2 cells (1 × 10⁶ cells/mL) were treated with probiotic crude cell extracts (10 ± 0.2 mg/1 × 10⁸ cfu), followed by incubation at 37°C for 20 hours. The cells were harvested and assayed for cholesterol content and mRNA expression.

Determination of cholesterol regulation in cell

HepG2 cells were treated with chloroform: isopropanol (2:1) for 30 min at room temperature, and then the lipid-extracted solvent was transferred to test tubes. The organic solvent was removed using a vacuum centrifuge, and the lipids were re-suspended in 95% ethanol. The intracellular cholesterol level was quantified with a cholesterol/cholesteryl ester kit (Cellbiolabs, San Diego, CA, USA) according to the manufacturer’s instructions. After lipid extraction, cells were lysed with RIPA buffer and centrifuged at 13,000 x g for 10 min to collect the supernatant for the measurement of cellular protein concentrations. Lipid levels were normalized to the total cellular protein concentration determined using the Bradford protein assay.

Use of animals and experimental design

Seven-week-old C57BL/6 female mice were purchased from Samtako Co. (O-san, Gyeong-gi, Korea). All mice were fed a commercial chow diet (modified TD 02028, Harlan-Teklad) for a week and randomly divided into four groups (8 mice in each group); CON, HCD, LP5273, and LP5279. Mice in CON group and HCD group were fed with commercial normal chow diet or high cholesterol diet (TD 02028, Harlan-Teklad, supplementary Table 1) and orally administered 100 µL of saline (0.9% NaCl, w/v). LP5273 and LP5279 groups were fed with high cholesterol diet and orally administered 10⁸ CFU of live L. plantarum LRCC 5273 or LRCC 5279 in 100 µL saline daily for 6 weeks. The mice were housed at 24 ± 2°C under the 12 h–12 h light dark cycles. Body weight and feed intake were measured every week. At the end of 6 weeks, mice were euthanized by Avertin (2,2,2-Tribromoethanol, Sigma-Aldrich, Yong-in, Gyeong-gi, Korea). Serum, liver, and intestine samples were immediately removed, weighed, and then stored in liquid nitrogen for measurement of lipids and assays of gene expression. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Korea University (KUIACUC-2015–146).

Table 1. Effects of L. plantarum LRCC 5273 on body weight, food intake, and organ weights.

		High-cholesterol diet
Groups	Saline	Saline	LP5273	LP5279
Initial body weight (g)	20.40 ± 0.30	20.40 ± 0.60	20.40 ± 0.40	20.40 ± 0.40
Final body weight (g)	22.60 ± 0.40	22.30 ± 0.40	22.20 ± 0.60	22.50 ± 0.40
Liver weight (g)	0.87 ± 0.01^a	1.31 ± 0.05^b	1.30 ± 0.03^b	1.35 ± 0.03^b
Visceral fat depot (g)	0.37 ± 0.03	0.39 ± 0.05	0.38 ± 0.03	0.42 ± 0.05
Food intake (g day^−1)	3.90 ± 0.10	3.30 ± 0.10	3.20 ± 0.10	3.50 ± 0.20

^a,bDifferent letters indicate significant differences (p < 0.05)

Analysis of fecal microbial quantity

Fecal samples of all groups were collected daily for three days before the end of the 6-week period. Bacterial genomic DNA was extracted from the fecal samples using a QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Quantitative real-time PCRs were performed in duplicate using ABI 7500 system with a SensiFAST Probe Lo-ROX mastermix according to the method described by Han et al. [17].

Analysis of lipid contents in serum and liver

Blood samples were collected by cardiac puncture and centrifuged at 2,000 × g for 20 min at 4°C. Total cholesterol level in serum was measured using total cholesterol assay kit (Cellbiolabs), and HDL & LDL contents were measured using HDL and LDL/VLDL cholesterol assay kit (Cellbiolabs). For serum triglyceride (TG) measurement, Serum TG determination kit (Sigma-Aldrich) was used.

For measurement of hepatic total cholesterol, hepatic tissues were minced and homogenized with micro-homogenizer in the extraction solvent, a mixture of chloroform:isopropanol:NP-40 (7:11:0.1), and further analysis was performed according to manufacturer’s instruction. For determination of hepatic TG content, tissue saponification in ethanolic KOH and neutralization with MgCl₂ was performed as previously described.

Determination of fecal cholesterol and bile acids

Fecal cholesterol was determined by same procedure employed for hepatic cholesterol determination, using total cholesterol assay kit (Cellbiolabs). Fecal bile acids were extracted using the slightly modified method of de Wael et al. [18]. Dried fecal specimen was weighed, powdered and 100 mg transferred to a glass tube. After adding 1 mL of 4% KOH in ethylene glycol, the samples were heated at 200°C for 15 min with an air reflux condenser on the test tube. Meanwhile, the contents were mixed three or four times. After cooling, 1 mL of 20% NaCl solution was added. After mixing, 0.2 mL of concentrated HCl was added and the contents mixed. Following this, 6 mL of diethyl ether was added to the acidified solution. The tube was shaken for 1 min, followed by centrifugation at 2,000 × g for 3 min. The upper layer was collected in a new glass tube. This procedure was repeated three times. From the combined extracts, the diethyl ether was completely evaporated at 40°C and the residue dissolved in 1 mL of methanol. This was used for enzymatic determination of bile acids using total bile acids assay kit (Crystal Chem, Downers Grove, IL, USA).

Gene expressions

HepG2 cells were homogenized in 3000 µL of easy-BLUETM reagent (iNtRON Biotechnology, Seong-nam, Gyeong-gi, Korea). Liver and small intestinal tissues were homogenized in 1000 µL of easy-BLUETM reagent (iNtRON). Following this, RNA was isolated according to the manufacturer’s instruction. Total RNA was quantified by spectroscopy. From each sample, 1 µg of total RNA was reverse transcribed to cDNA with high capacity cDNA reverse transcription kit (Thermo Scientific) according to manufacturer’s instruction in a total volume of 20 µL. cDNAs were used in qPCR with primers for each of the target genes. The amplification was performed in a total volume of 20 µL, which included 2 µL of cDNA (50 ng/µL), 1 µL each target primer (10 pmol/µL), 1 µL reference primer, 10 µL SensiFAST Probe Lo-ROX mastermix (Bioline), and 6 µL diethyl pyrocarbonate treated water (iNtRON). Reaction were carried out in and ABI7500 system (Life Technologies) using the following thermal cycling parameters: 95°C for 3 min, and then 40 cycles of 95°C for 30 sec and the appropriate annealing temperature for 30 sec. All samples were also examined in parallel for ACTB, and relative quantities of each gene were presented in terms of 2^−ΔΔCt, calculated using the ΔCt (Ct value of target gene – Ct value of ACTB) and ΔΔCt values (ΔCt value of tested sample – ΔCt value of control sample).

Statistical analysis

All statistical analyses were performed by Student’s t-test and one-way ANOVA using SAS software (SAS Institute Inc., Cary, NC, USA). Multiple comparisons were performed with a Tukey–Kramer adjustment. Results are presented as the mean ± standard error (SE). A value of p < 0.05 was considered significant.

Results

Isolation and identification of cholesterol-lowering lactobacillus strains

Thirty-eight probiotic strains were isolated from kimchi samples. Effect of all strains on cholesterol levels of HepG2 cells was analyzed. We selected only 7 strains among the 38 total strains (Figure 1a & b). Examination of cholesterol-lowering effects of the 7 selected Lactobacillus strains showed down-regulation of mRNA expression of cholesterol-related genes (Figure 1c). Based on this result, we selected two strains (LRCC 5273 and LRCC 5279) for further experimentation.

Figure 1. Effect of LP5273 and LP5279 on cellular cholesterol level and mRNA expression in HepG-2 cells.

Data expresses Mean± SE. Cellular cholesterol was measured at 24 hours after treatment of LAB lysate on HepG-2 cells, n = 1 (a). The measurement was repeated with the reduced number of LAB strains, n = 3 (b). Real-time PCR is performed to measure mRNA expression of SREBP-2, HMGCR, and LDLR (c). *p < 0.05, **P < 0.01, and ****P < 0.0001 vs CON. Slashed bars, commercial strains; Empty bars, isolated strains.

Effects of L. plantarum LRCC 5273 on body weight, food intake, and organ weights

As shown in Table 1, although food intake in mice fed with normal chow diet was higher compared with that of the other groups, there were no differences in body weight gain and visceral fat depot between the groups. However, liver weight in mice fed with high-cholesterol diet significantly increased.

Effects of L. plantarum LRCC 5273 on population of fecal bacteria

There is some evidence that LAB could modulate bile acid excretion and this could lower plasma cholesterol levels [19,20]. The cholesterol-decreasing activity of some Lactobacillus spp. resulted from their co-precipitation with deconjugated bile salts [16]. There are some reports on presence of bile-salt hydrolases in different species of Lactobacillus, Enterococcus, Peptostreptococcus, Bifidobacterium, Clostridium, and Bacteroides [21]. Bile-salt hydrolase is the enzyme that transforms deconjugated bile acids into free bile salts. Fecal loss of bile salts may cause an increased requirement for cholesterol as a precursor for new bile-salt synthesis, thereby decreasing the serum level of cholesterol [22]. This suggested that a high bile-salt hydrolase activity of fecal bacteria may lower serum cholesterol levels and bile-salt hydrolases may thus play an important role in the overall decrease in cholesterol. Therefore, we confirmed the fecal bacterial count. The results showed a significant increase in the total count of bacteria in the HCD-fed group. The number of Lactobacillus spp. was also reduced in HCD group but administration of LP5273 and LP5279 restored the amount of Lactobacillus spp. significantly. The number of Bifidobacterium spp. was also increased in mice fed with LP5273 and LP5279 compared to CON group. In addition, Escherichia ssp. was decreased in LP5273 group but increased population of Enterococcus spp. was observed (Table 2). This result suggests that LP5273 and LP5279 may have a cholesterol-lowering effect.

Table 2. Effects of L. plantarum LRCC 5273 on population of fecal bacteria.

		High-cholesterol diet
Groups(log CFU · g feces^−1)	Saline	Saline	LP5273	LP5279
Total bacteria	12.30 ± 0.28^b	15.22 ± 0.06^a	14.85 ± 0.01^a	14.98 ± 0.04^a
Lactobacillus spp.	7.81 ± 0.20^a	6.13 ± 0.14^c	6.84 ± 0.07^b	6.85 ± 0.15^b
Bifidobacterium spp.	5.10 ± 0.31^b	5.93 ± 0.11^ab	6.51 ± 0.09^a	6.51 ± 0.15^a
Enterococcus spp.	7.81 ± 0.27^c	9.76 ± 0.13^b	10.83 ± 0.06^a	9.12 ± 0.14^b
Escherichia coli ssp.	5.89 ± 0.19^c	11.05 ± 0.14^a	10.65 ± 0.07^b	11.11 ± 0.13^a

^a,bDifferent letters indicate significant differences (p < 0.05)

L. plantarum LRCC 5273 lowered the levels of TC and LDL in serum

To confirm the cholesterol-lowering effect on LP5273 and LP5279, we investigated the lipid profiles of blood. With significant decrease of serum LDL-cholesterol level, total cholesterol level in mice fed with LP5273 was significantly reduced compared with HCD group (Figure 2a and c, p < 0.05). However, there were no significant differences in HDL-cholesterol and triglyceride levels upon administration of LP5273 (Figure 2b and d).

Figure 2. Effect of LP5273 on serum cholesterol and triglyceride level.

Data expresses Mean± SE. Serum total cholesterol (a), HDL cholesterol (b), LDL cholesterol (c), and triglyceride (d) were measured by enzymatic assay. Mice in CON group were fed with normal chow diet and 0.9% saline; Mice in HCD with group were fed with high-cholesterol diet and 0.9% saline; Mice in LP5273 and LP5279 group were fed with high-cholesterol diet and 10⁸ CFU of LP5273 or LP5279 per mouse. *P < 0.05 vs CON.

Effects of L. plantarum LRCC 5273 on hepatic cholesterol and triglyceride levels

The effects of L. plantarum LRCC 5273 on hepatic cholesterol and triglyceride levels were investigated. As shown in Figure 3a, hepatic cholesterol levels were increased in the HCD group. However, there were no significant differences among the HCD, LP5273, and LP5279 groups. The hepatic triacylglycerol levels were also not significantly different (Figure 3b).

Figure 3. Effect of LP5273 on hepatic cholesterol, triglyceride level, fecal cholesterol and bile acid excretion.

Data expresses Mean ± SE. Hepatic total cholesterol (a), triglyceride (b), fecal total cholesterol (c) and bile acids (d) were measured by enzymatic assay. Mice in CON group were fed with normal chow diet and 0.9% saline; Mice in HCD group were fed with high-cholesterol diet and 0.9% saline; Mice in LP5273 and LP5279 group were fed with high-cholesterol diet and 10⁸ CFU of LP5273 or LP5279 per mouse^a,bDifferent letters indicate significant differences (P < 0.05).

Effects of L. plantarum LRCC 5273 on fecal total cholesterol and bile acids

Bile acids, metabolites synthesized from cholesterol in the liver, are excreted into the intestinal lumen and reabsorbed into the enterohepatic circulation. Some of them are not absorbed again and disposed along with feces. Since cholesterol is a precursor of bile acids, loss of bile acids results in cholesterol consumption in the body. Therefore, we examined the effect of L. plantarum LRCC 5273 feeding on fecal cholesterol and bile acid excretion. As shown in Figure 3c, fecal total cholesterol level was significantly increased in LP5273 group (p < 0.05). Fecal bile acids in LP5273 group tended to increase but not significantly (Figure 3d).

Effect of L. plantarum LRCC 5273 feeding on transcription of key genes in hepatic and intestinal cholesterol metabolism

We examined the influence of L. plantarum LRCC 5273 feeding on the transcription of the HMG-CoA reductase, LDL receptor, SREBP-2, CYP7A1, LXR-α, ABCG5, ABCG8 and ABCB11 in mouse liver using RT-PCR. Hepatic CYP7A1, a rate limiting enzyme in bile acid synthesis from cholesterol, was significantly increased with induction of hepatic LXR-α expression in LP5273 group. And also, ABCG 8, limiting intestinal absorption and facilitating biliary secretion of cholesterol, was significantly increased in LP5273 group. However, there were no effects on ABCG5 and ABCB11 expression. Also, HMGCR and LDLR levels as well as SREBP-2 expression were not altered by LP5273 and LP5279 (Figure 4a). On the other hand, intestinal ABCG5 and ABCG8 were remarkably up-regulated while NPC1L1 was significantly down-regulated with significant LXR-α induction in LP5273 group, while ABCA1 levels remained unchanged (Figure 4b).

Figure 4. Effect of LP5273 on mRNA expression of cholesterol transport- and synthesis- related genes in the liver (a) and jejunum (b).

Data expresses Mean ± SE. mRNA level is measured by real-time qPCR and calculated by the ∆∆Ct method. Mice in CON group were fed with normal chow diet and 0.9% saline; Mice in HCD group were fed with high-cholesterol diet and 0.9% saline; Mice in LP5273 and LP5279 group were fed with high-cholesterol diet and 10⁸ CFU of LP5273 or LP5279 per mouse. *P < 0.05 vs HCD.

Discussion

LAB in food products have been researched as candidates for probiotics and have shown several beneficial effects on human health [23]. Many researchers have demonstrated that LAB could improve metabolic syndromes such as obesity, diabetes, hypertension, diarrhea, and hyperlipidemia [24–28]. Meanwhile, L. plantarum spp. was revealed to have cholesterol-lowering effects through in vitro and in vivo studies [9,20,29–31]. In the present study, we observed that L. plantarum LRCC 5273 lowered cholesterol level accompanied with significant reduction of LDL-cholesterol in serum. Although HDL-cholesterol was also reduced, the decrease was not significant. Administration of L. plantarum LRCC 5273 did not change cholesterol and triglyceride levels in the liver. The expression of the hepatic LDL receptor and HMG-CoA reductase were not affected upon administration of L. plantarum LRCC 5273, however, interestingly, transcription rates of the CYP7A1 and LXR-α gene in the LP5273 group were significantly elevated compared with HCD-group. These gene expression changes, together with the increase tendency in fecal bile acid excretion, may contribute to the decrease of plasma cholesterol levels upon administration of L. plantarum LRCC 5273.

Cholesterol is excreted by several mechanisms including excretion through biliary tract from the liver, and direct excretion through enterocyte. Reverse cholesterol transport is a pathway that describes the transport of excess cholesterol from peripheral cells to the liver for excretion into the bile, either directly or indirectly after conversion into bile acids [32]. This pathway is mediated by macrophages having ApoA-I proteins [33]. In this manner, cholesterol is catabolized in the liver or excreted through the bile duct. However, it has been reported that direct excretion of cholesterol through enterocytes from the plasma into the intestinal lumen comprises the largest amount of total cholesterol excretion [34]. The pathway called trans-intestinal cholesterol excretion (TICE) is non-biliary and involves direct excretion of cholesterol through enterocytes [35]. Cholesterol in the plasma is absorbed into enterocytes by unknown transporters and excreted by ABCG5/G8. Since LXR-α is the transcription factor of ABCG5/G8, activation of LXR-α is essential for intestinal cholesterol excretion [36]. ABCG5/G8 also play a key role in the intestinal absorption of dietary cholesterol. These transporters are localized on the apical membrane of enterocytes and their primary function is to transport absorbed cholesterol back into the lumen of the intestines [37]. Expression of ABCG5/G8 is significantly increased upon LXR activation in both, murine intestine and in human enterocyte Caco-2 cell line [38,39]. Consequently, administration of LXR agonists markedly decreases intestinal net cholesterol absorption in mice. Initially this effect was associated with increased ABCA1 expression in enterocytes [40]. However, subsequent experiments on mice lacking either ABCA1 or ABCG5 and ABCG8 revealed that only the latter two transporters are involved in the LXR-induced inhibition of dietary cholesterol absorption [41]. In addition, activation of LXR-α down-regulates expression of NPC1L1, a key factor of cholesterol absorption in the intestinal lumen [38]. LXR-α, a nuclear receptor controlling intercellular transport and efflux of cholesterol, is one of the genes affected by Lactobacillus strains. LXR-α expression predominates in metabolically active tissues such as the liver, small intestine, kidney, macrophages, and adipose tissue, while LXR-β is expressed ubiquitously [42]. Activation of LXR-α induces cholesterol conversion and excretion, up-regulation of several genes such as cholesterol 7-alpha hydrolase, and ATP binding cassette transporter families [42]. Studies in Caco-2 cell demonstrated that Lactobacillus strains up-regulated ABCG5/G8 expression through induction of LXR expression and may assist in the lowering of the blood cholesterol level [43]. Studies in THP-1 cells revealed that L. plantarum NR74 activated LXR and increased cholesterol efflux of THP-1 cells by promoting ABCA1 and ABCG1 expressions [44]. Studies in Caco-2 cells demonstrated that L. plantarum NR74 promoted cholesterol efflux by up-regulation of ABCG5/8 with increased LXR expression, and furthermore, cholesterol absorption was decreased with down-regulated expression of NPC1L1 by L. plantarum NR74 [43,45]. However, mechanism of action of L. plantarum spp. in animal intestine has still not been elucidated. In our study, mice administered with LP5273 showed significantly decreased LDL-cholesterol level and increased fecal cholesterol. In addition, bile acid excretion tended to increase but not significantly. Since bile acids are converted from cholesterol in the liver by CYP7A1, and it induced fecal bile acid excretion may affect serum cholesterol levels. In agreement to this result, the gene expression of hepatic CYP7A1, cholesterol-7α-hydrolase synthesizing cholic acid from cholesterol, was found to be up-regulated in LP5273 group. We believed that administration of LP5273 can help increase fecal bile acids excretion. In conclusion, our results show that L. plantarum LRCC 5273 has hypocholesterolemic effects. L. plantarum LRCC 5273 was more effective than L. plantarum LRCC 5279 in improving various plasma lipid levels. In particular, cholesterol excretion by ABCG5/G8 in intestine, were significantly higher than in the HCD-group. However, exact mechanism of effects on humans should be confirmed in the future.

Author contributions

Y.J.K., W.H., E.S.L., and H.T.C. carried out the animal study and analyzed data. S.M.Y., H.T.K., and S.Y. performed bacteria identification and in vitro experiment contributed. J.H.K., and J.H.L. contributed to the discussions of data and reviewed the manuscript. Y.J.K., W.H., and E.S.L. prepared the article.

Acknowledgments

This work was supported by a Korea University Grant fund.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplemental data

Supplementary data for this article can be accessed here.

Additional information

Funding

This work was supported by the Korea University [KU20170301].