Protective Effect of Saponins-Enriched Fraction of Gynostemma pentaphyllum against High Choline-Induced Vascular Endothelial Dysfunction and Hepatic Damage in Mice

Abstract

Choline as a quaternary amine nutrient is metabolized to trimethylamine by gut microbiota and subsequently oxidized to circulating trimethylamine-N-oxide (TMAO), a gut-derived metabolite associated with liver toxicity and cardiovascular disease. The study was to probe the possible vasoprotective and hepatoprotective effects of total saponins of Gynostemma pentaphyllum (TSGP) in 3% high-choline water-feeding mice. The purified TSGP was obtained with content of 83.0% saponins, and its antioxidant activities were evaluated in vitro. Furthermore, the mice fed with high choline for 8 weeks significantly expressed vascular endothelial dysfunction and liver oxidative stress (p < 0.01 vs. Normal). Administration of TSGP at 400 and 800 mg/kg·body weight (b.w.) significantly lowered the serum total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), endothelin-1 (ET-1) and thromboxane A₂ (TXA₂) levels, as well as hepatic malondialdehyde (MDA) formation, but effectively elevated the serum nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and prostaglandin I₂ (PGI₂) levels, as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST), T-superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in high choline-fed mice. Hematoxylin–eosin (H&E) and oil red O staining also suggested that TSGP could exert the significant protection against endothelial dysfunction and liver injury in high choline-treated mice. These findings suggest that TSGP is of the saponins-enriched extract, and is a good candidate of dietary supplement and therapeutic application in vascular and hepatic oxidative injury.

INTRODUCTION

Choline has been considered as an essential nutrient for the human body, which may exert many biological activities.^1,2) However, recent reports have discovered that the gut microbes-dependent metabolite trimethylamine (TMA) of dietary choline and its precursor phosphatidylcholine can be further oxidized by the host liver flavin monooxygenases (FMOs) to circulating baleful trimethylamine-N-oxide (TMAO) with association with cardiovascular diseases (CVD) and hepatotoxicity.^3–7) Several reports have demonstrated that ingestion of some diets with high choline can effectively lead to endothelial dysfunction, and our previous study also showed that dietary ingestion of high choline or TMAO is highly linked to liver oxidative stress injury.^8–10) As a result, high choline intake in animals has been developed as an ideal experimental model for nutritional evaluation against vascular endothelial injury and liver toxicity.

Gynostemma pentaphyllum Makino (G. pentaphyllum) is a famous edible and medicinal plant in China and some other Asian countries, which is widely supplied as classical medicine or herbal tea.¹¹⁾ It has been clinically used to adjust blood pressure, strengthen the immune system, lower cholesterol levels, treat chronic bronchitis and gastritis, and reduce inflammation.^11–15) In recent years, the consumption of G. pentaphyllum as food or tea has increased steadily in China and some European countries. The principal active constituents of G. pentaphyllum are various saponins, which are also known as gypenosides, and more than 100 gypenosides have been segregated and identified from G. pentaphyllum.¹¹⁾ The saponins as the most important active components of G. pentaphyllum have been widely used in health care.¹⁶⁾ Studies have also indicated that G. pentaphyllum saponins exhibit the benefit effects in treatment of metabolic and vascular diseases.^17–19) Because the bioactive components of cheap G. pentaphyllum was similar to expensive ginseng root, it was named as “the second ginseng,” and hence, the cultures of G. pentaphyllum or their extracts for health care have been put into mass production.^13,20)

To our knowledge, the beneficial protection of G. pentaphyllum saponins on the dysfunction of the blood vessel and liver injury caused by high choline intake is still unreported. In this context, the current study was dedicated to isolate the total saponin of G. pentaphyllum (TSGP) from G. pentaphyllum herb, and its antioxidant activities were evaluated in vitro. Furthermore, we for the first time try to discover whether consumption of TSGP may antagonize the high choline diet-caused vascular functional disorder and liver oxidative stress injury in mice.

MATERIALS AND METHODS

Isolation of TSGP

G. pentaphyllum was purchased from Wanshou Road Medicine Market (Xi’an, China), and identified by Yonghui Sun from Xi’an Medical College. TSGP was prepared using the previous method with some adjustments.²¹⁾ In brief, the dried G. pentaphyllum herb material was shattered and screened, and then the stive (100 g) was extracted in 70% ethanol (1 : 8 (w/v)) for three times and refluxed 90 min each time at 80°C for three times. After leaching, the filtrate was combined and collected using rotary evaporators under vacuum to volatilize the solvents at 60°C, and the condensed solution was dissolved in deionized water, and then extracted with petroleum ether, ethyl acetate and n-butanol, sequentially. The n-butanol-extracted solution was collected and concentrated. The condensed solution was further purified through D101 resin column chromatography, and eluted with 70% ethanol to get the saponins-enriched fraction solution, followed by drying as TSGP through concentration and evaporation at 60°C.

Determination of Total Saponins in TSGP

The total saponins of TSGP were determined by our the previously described method with slight adjustments.^22,23) In brief, 100 µL definite TSGP (4.0 mg/mL) was added to 0.2 mL of 5% vanillin in acetic acid and 0.8 mL of 70% perchloric acid. The mixture was incubated at 60°C for 15 min. Ginsenosides Rb1 was viewed as the standard sample. The absorbance was read at 550 nm as the maximum absorbance wavelength after the sample was chilled down at room temperature. A standard curve was worked out using the standard ginsenosides Rb1. Namely, ginsenoside Rb1 was separately dissolved in ethanol for an ultimate concentration of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL, and the absorbance at 550 nm were determined, respectively.

Assay for in Vitro Antioxidant Activities of TSGP

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals-scavenging capacity of TSGP: The radicals-scavenging capacity of TSGP against DPPH was tested by previous procedure.²¹⁾ Hydroxyl radicals-scavenging activity of TSGP: The eliminating ability of TSGP against hydroxyl radical (HO^・) was evaluated by previous method.²¹⁾ Superoxide radicals-scavenging assay of TSGP: The Superoxide radicals-scavenging assay was tested by previous method.²⁴⁾ Ferric-reducing antioxidant ability assay of TSGP: The deoxidizing capacity of TSGP was evaluated by a previous method.²⁴⁾

Animals and Experimental Design

Healthy male Kunming mice (body weight 18 ± 2 g) were supplied by the Experimental Animal Center of the Fourth Military Medical University (Xi’an, China). Mice were housed under controlled laboratory conditions (temperature 22 ± 2°C, humidity 60 ± 5% with a 12/12 h light-dark cycle) and permitted free intake of a standard rodent chow and tap water. After acclimation for a week, they were stochastically separated into 5 groups (n = 10). Namely, normal control group, HC control group (3% dietary choline water alone), and three TSGP-treated groups (dose of 200, 400 and 800 mg/kg·b.w., supplemented with 3% dietary choline water, respectively). The mouse was permitted free intake of tap water or 3% dietary choline water. TSGP was dissolved in 0.5% CMC-Na solution and administered intragastrically (i.g.) at 200, 400 and 800 mg/kg·b.w. once daily (0.4 mL) for 8 consecutive weeks, where the dose was designed according the previous results of our pre-experiment in animals.^8,9) The normal and dietary choline groups of mice were also administered with the same volume of 0.5% CMC-Na solution (0.4 mL, i.g.). All the administrations were conducted between five and six o’clock in the afternoon. The mice were fed with 3% dietary choline water every two days and weighed once a week. Two hours after the last administration, all mice were fasted overnight and were only allowed to drink water freely for 12 h, and then all mice were given isoflurane for complete anesthesia and killed, and Blood, liver and vessels were collected. All samples were disposed and stored according to our previous experiment. All animal experiments were conducted in accordance with the protocols approved by the Committee on Care and Use of Laboratory Animals of the Fourth Military Medical University, China (XJYYLL-2015689).

Assay for Endothelial Nitric Oxide Synthase (eNOS), Nitric Oxide (NO), Endothelin 1 (ET-1), Thromboxane A₂ (TXA₂) and Prostaglandin I₂ (PGI₂)

Serum levels of NO, eNOS, ET-1, TXA₂ and PGI₂ were assessed to reflect the vasal injury of mice, respectively. Assay kits were products from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and the measurements of eNOS, NO, ET-1, TXA₂ and PGI₂ were performed on the basis of the instructions, and the consequences were expressed as U/L, µmol/L, pg/mL, pg/mL and pg/mL, respectively.

Analysis of Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) Activities for Monitoring Liver Function

The blood of mice was gathered and centrifuged for enzyme activity analysis of the serum ALT and AST as assay kits from Changchun Huili Biotechnology Co., Ltd. (Changchun, China). Serum AST and ALT activities were detected according to the instructions, and the consequences were expressed as U/L.

Determination of Hepatic T-Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-Px) and Malondialdehyde (MDA)

Detection kits were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The liver tissue homogenates were prepared in a ratio of 1 : 9 (w/v, liver: saline), and homogenates were centrifuged at 2000 × g for 10 min and gathered for the test of activities of T-SOD, GSH-Px and MDA concentration, and the consequences were expressed as U/mg prot., U/mg prot. and nmol/mg prot., respectively. Determination was performed using the kit instructions. The total protein content in the homogenate was tested by Coomassie bright blue method.²⁵⁾

Serum Lipid Measurement and Histopathological Examination

The mouse blood was gathered and centrifuged at 2000 × g for 20 min to collect the supernatant as the isolated serum. Assay kits were supplied by Changchun Huili Biotechnology Co., Ltd. (Changchun, China). Dyslipidaemia was evaluated by testing the TC, TG, high density lipoprotein (HDL)-C and low density lipoprotein (LDL)-C levels according to the instructions of the assay kits. The atherogenic index (AI), an important determinant of cardiovascular risk, was evaluated by the TC/HDL-C ratio.²⁶⁾ Besides, the histopathological examination of pectoral aortas and liver tissues was done according to our previous procedure.^25,27)

Statistical Analysis

All experiments were performed in triplicate and the values were expressed as mean ± standard deviation (S.D.). Data analysis was using one-way ANOVA and Duncan’s multiple range tests (DPS 7.05) for differences in data of biochemical parameters among the different groups. The p-value <0.05 was considered statistically significant.

RESULTS

Chemical Analysis of Total Saponins Content in TSGP

Total saponins of herbal G. pentaphyllum were extracted with 70% ethanol, and 3.9% (w/w) extraction yield of the herb powder was achieved. The TSGP as saponins-enriched fraction was further obtained from the rough extracts by separation on a D101 macroporous resin column. The total saponins content determined using the method of vanillin colorimetry was up to 83.0%. In this work, the regression equation for ginsenoside Rb1 was y = 7.8377x−0.0018 with the correlation coefficient (R2) of 0.9977, where y and x was absorbance value at 550 nm and concentration of Rb1, respectively, and R was linear correlation coefficient. This quantitation demonstrated that TSGP was of a saponins-enriched extract.

In Vitro Antioxidant Activity of TSGP

The assay for in vitro antioxidant activities of TSGP through the classical DPPH^・, HO^・, O₂^・− and ferric reducing ability was performed. The free radicals-scavenging ability of TSGP on DPPH radicals was 35.5, 65.9, 83.2, 89.5 and 92.0% at concentration of 0.1, 0.5, 1.0, 2.0 and 4.0 mg/mL, respectively (Fig. 1A). Similarly, the reducing ability of TSGP on HO^・ was 36.5, 47.0, 61.5, 77.1 and 82.0% at various concentrations (0.1–3.0 mg/mL, Fig. 1B). Meanwhile, TSGP also exhibited obvious scavenging activities (22.3, 32.1, 43.1, 69.7 and 72.2%) against O₂^・− at various concentrations (0.1–3.0 mg/mL, Fig. 1C). It was also found that ferric-reducing antioxidant power values of TSGP ranged from 0.08 to 0.57 in 0.1–3.0 mg/mL (Fig. 1D). These findings indicated that TSGP had a strong antioxidant capacity.

Fig. 1. In Vitro Antioxidant Effects and Free Radical-Scavenging Activities of TSGP

(A) DPPH^・-scavenging activity of various concentrations of TSGP and reference Vitamin C (Vit. C), (B) HO^・-scavenging effect of TSGP and Vit.C, (C) O₂^・−-scavenging capacity of TSGP and Vit. C, (D) Ferric-reducing antioxidant power of TSGP and Vit. C.

In Vivo Effects of TSGP Administration on Body Weight

Table 1 summarized the influences of high choline (HC, model) water with or without TSGP administration on the body weights of the tested mice. It could be seen that the mice significantly gained weight after intake of 3% dietary HC water for 8 weeks in comparison with the normal control group (p < 0.05). Interestingly, administration of TSGP at 400 and 800 mg/kg·b.w. effectually decreased the body weight gain caused by HC feeding (p < 0.05), particularly at the last two weeks. It was worth noting that there was no obvious discrepancy in average ingestion of food and water observed in the daily food intake (data not shown). All these results suggest that the mice with ingestion of HC water can gain the body weight and the protective administration of HC-fed mice with TSGP could effectively prevent this weight gain.

Table 1. Effects of TSGP on Body Weight (b.w.) of the Mice Fed with High-Choline (HC) Water for Consecutive 8 Weeks

Group	Body weight (g)
	0th week	2th weeks	4th weeks	6th weeks	8th weeks
Normal	28.6 ± 1.5	33.6 ± 2.7	35.9 ± 1.9	39.7 ± 2.1	44.7 ± 2.2
HC	29.0 ± 1.0	33.2 ± 2.6	37.4 ± 2.3	43.4 ± 1.9#	47.5 ± 2.1#
HC + TSGP (200 mg/kg·b.w.)	28.2 ± 1.7	33.3 ± 2.7	36.3 ± 1.7	41.9 ± 2.4	45.9 ± 1.6
HC + TSGP (400 mg/kg·b.w.)	27.5 ± 1.2	33.7 ± 1.7	36.1 ± 2.0	41.1 ± 2.6	44.6 ± 2.2*
HC + TSGP (800 mg/kg·b.w.)	27.3 ± 1.3	33.1 ± 2.6	36.2 ± 1.9	40.9 ± 1.2*	44.1 ± 2.8*

HC, 3% high-choline water. Values are expressed as mean standard deviation of 10 mice in each group. #p < 0.05, relative to normal group. * p < 0.05 and ** p < 0.01, relative to the HC group

Effects of TSGP on Serum eNOS, NO and ET-1 Levels

NO exerts a markedly protective effect on vassal integrity and endothelial function, which is synthesized by the eNOS.²⁸⁾ In this study, the levels of eNOS and NO were decreased by 25.9 and 59.0% after the normal mice were given with HC water, respectively (p < 0.01, Figs. 2A, B). But the decline of eNOS and NO levels could be effectually antagonized by consumption of TSGP at 400 and 800 mg/kg·b.w. (Figs. 2A, B). In contrast, it is well known that the elevated release of ET-1 is one of the endothelial dysfunctional characters.²⁹⁾ Interestingly, HC feeding led to a 32.0% increase in ET-1 levels of mice, relative to the normal feeding control (p < 0.01, Fig. 2C), but administration of TSGP at 400 and 800 mg/kg·b.w. could inhibited the HC-induced increase of the serum ET-1 release, respectively (p < 0.05, p < 0.01). Although a mild elevation in eNOS level and a slight reduction in ET-1 release were observed following the administration of TSGP at 200 mg/kg·b.w., there was not the statistical significance when compared to HC feeding control, respectively (Figs. 2A–C, p > 0.05). These findings suggested that feeding of 3% HC water in mice caused endothelial dysfunction clearly, and TSGP might significantly suppress the endothelial damage in a dose-dependent manner.

Fig. 2. Effects of TSGP on the Serum Levels of eNOS (A), NO (B), ET-1 (C), TXA₂ (D) and PGI₂ (E) in the Mice Fed 3% Dietary High-Choline (HC, Model) Water for Consecutive 8 Weeks

Values are presented as means ± standard deviation (S.D.) for 10 mice in each group. ^## p < 0.01, vs. the normal group. * p < 0.05, ** p < 0.01, compared to the HC model group.

Effects of TSGP on Serum TXA₂ and PGI₂ Levels

TXA₂ and PGI₂ are the most common prostanoids in cardiovascular system, which display extensive modification against endothelial damage and atherosclerosis.³⁰⁾ Herein, we further measured the serum levels of TXA₂ and PGI₂ of the tested mice with or without HC feeding. In particular, the serum TXA₂ level in HC-treated mice was markedly enhanced to 546.8 ± 47.8 pg/mL with an enhancement of above 4.0-fold (p < 0.01, Fig. 2D). Meanwhile, the serum PGI₂ level was reduced to 19.4 ± 3.4 pg/mL in HC-treated mice from 50.7 ± 5.2 pg/mL of the normal control group (p < 0.01, Fig. 2E). As expected, following administration of mice with TSGP at 200 mg/ kg·b.w., the TXA₂ level was significantly decreased to 211.5 ± 34.8 pg/mL (p < 0.01), and TSGP could dose-dependently inhibit the elevation of TXA₂ level with different degrees at 400 and 800 mg/kg·b.w., respectively (p < 0.01). Conversely, PGI₂ level was significantly enhanced to 24.3 ± 4.3 pg/mL in the mice following administration of HC-fed mice with 400 mg/kg·b.w. TSGP (p < 0.05), and a further increase could be seen at 800 mg/kg·b.w. TSGP (p < 0.01) (Fig. 3).

Effects of TSGP on Enzymatic Activities of Serum AST and ALT

AST and ALT were deemed to be valid biochemical indexes of early liver injury.²⁵⁾ Compared with the normal mice, the enzymatic activities of the serum AST and ALT were dramatically increased to 37.6 ± 3.2 U/L (p < 0.01) and 66.55 ± 5.8 U/L (p < 0.01) from 24.0 ± 3.4 and 43.2 ± 2.5 U/L of HC feeding mice, respectively (Figs. 4A, B). However, TSGP could dose-dependently inhibit the elevation of the AST and ALT activities with different degrees. The serum AST activities were markedly decreased to 30.7 and 27.4 U/L of the mice treated with TSGP at 400 and 800 mg/kg·b.w., respectively (p < 0.05, p < 0.01), and the ALT activities were also reduced to 48.0 and 44.7 U/L, following the treatments, respectively (p < 0.01). Our consequences demonstrated that HC consumption in mice caused the hepatic injury, and TSGP administration could effectively attenuate the adverse effect of HC-caused liver damage.

Effects of TSGP on Serum Lipid Profiles

As is well-known, lipid disorder is correlative to CVD, and the elevation in the circulating TC, TG and LDL-C/HDL-C values is to be targets of CVD treatment.³¹⁾ As illustrated in Table 2, the levels of the serum TC, TG, and LDL-C in HC-fed mice had a sharp elevation by 46.9% (p < 0.01), 75.0% (p < 0.01) and 50.0% (p < 0.01), and HDL-C had a significance decline by 45.5% (p < 0.01) when compared to the normal mice, respectively. Meanwhile, AI, an important determinant index of cardiovascular risk, had an obvious increase by 168.9% in HC-feeding mice in comparison with the normal group (p < 0.01). However, the elevation of TC, TG and LDL-C levels and the reduction of HDL-C concentration in HC-fed mice were effectively attenuated by treatment of the mice with 400 and 800 mg/kg·b.w. of TSGP, respectively (p < 0.05, p < 0.01). A slight change (p > 0.05) in TC, TG, LDL-C and HDL-C levels was also observed following administration of TSGP at 200 mg/kg·b.w. in comparison with HC control. As a result, TSGP might return to normal status for the dyslipidemia of HC-induced mice.

Table 2. Effects of High-Choline (HC) Ingestion and/or TSGP Administration on the Serum TC, TG, HDL-C and LDL-C Levels of the Tested Mice (n = 10)

Parameters	TC	TG	LDL-C	HDL-C	AI
Normal	3.2 ± 0.6	0.8 ± 0.1	1.2 ± 0.2	1.1 ± 0.2	2.9 ± 0.1
Choline	4.7 ± 0.3##	1.4 ± 0.1##	1.8 ± 0.1##	0.6 ± 0.1##	7.8±0.2##
HC + TSGP (200 mg/kg·b.w.)	4.2 ± 0.4	1.2 ± 0.2	1.6 ± 0.2	0.6 ± 0.1	7.0 ± 0.1
HC + TSGP (400 mg/kg·b.w.)	3.7 ± 0.7**	0.9 ± 0.1*	1.4 ± 0.2*	0.9 ± 0.1**	4.1 ± 0.1**
HC + TSGP (800 mg/kg·b.w.)	3.5 ± 0.3**	0.8 ± 0.1**	1.3 ± 0.2**	1.0 ± 0.1**	3.5 ± 0.1**

Data are expressed as mean ± S.D. of 10 mice in each group. Values of serum TC, TG, HDL-C and LDL-C levels are all expressed in mmol/L, respectively. Atherogenic index (AI) = TC /HDL-C. ^## p < 0.01, relative to normal group. * p < 0.05 and ** p < 0.01, relative to the HC group.

Histological Observation for Thoracic Aortas of TSGP-Treated Mice

To further provide the evidence of the biochemical assay, the mouse thoracic aorta was stained through hematoxylin–eosin (H&E). As shown in Fig. 3A, the histopathological alteration suggested the proliferation of the vascular wall or incrassation of media of the pectoral aorta of HC water-fed mice occurred. Nevertheless, the damage extent of vascular endothelium in HC-treated mice together with TSGP treatment had the significant improvement, where the tissue thickness of the thoracic aorta had the remarkable decrease in a dose-dependent manner. Furthermore, we analyzed the ratio of intima-media/lumen, suggesting that TSGP effectually steadied the vessels and reduced the wall thickness ratio against HC-caused damage. As shown in Fig. 3B, the intima-media/lumen ratio in HC feeding mice had an elevation by 91.1% as compared to the normal control group (p < 0.01), indicating that HC consumption severely caused the damage of the vascular structure. However, administration of HC-fed mice with 200, 400 and 800 mg/kg·b.w. of TSGP led to the decrease in intima-media/lumen ratio by 5.5% (p > 0.05), 14.3% (p < 0.05) and 30.0% (p < 0.01) when compared to HC feeding control, respectively, suggesting that TSGP could protect vessel injury or vascular dysfunction in HC-fed mice.

Fig. 3. Effects of TSGP on Thoracic Aortas Stained by H&E (Magnification 400×)

(A) Representative histological section of the aorta of the normal mice, high choline-induced mice, low-, middle- and high-doses of TSGP-treated mice. The medial thickness was typically showed by the arrows (values followed at the bottom) and analyzed with image pro-plus 6.0 software. (B) Representative intima-media/lumen ratio of the thoracic aorta of the tested mice in the normal group, high choline-induced group, low-, middle- and high-doses of TSGP-treated group. Data denoted were means ± S.D. (n = 10). ^## p < 0.01, ^# p < 0.05, vs. the normal group. * p < 0.05, ** p < 0.01, compared to high choline-treated mice.

Hepatic Protection of TSGP against Oxidative Stress or Histopathologic Injury

The enzyme activities of T-SOD and GSH-Px, and the formation of MDA were considered to be important indicators of liver tissue damage.²⁵⁾ Figures 4C–E exhibited the alteration of these biochemical parameters of hepatic damage in mice. It was found that TSGP remarkably alleviated the oxidative stress injury of HC feeding mice, where treatment of HC-fed mice with TSGP at 800 mg/kg·b.w. remarkably elevated T-SOD activity by 144.4% (p < 0.01, Fig. 4C) and GSH-Px activity by 11.2% (p < 0.01, Fig. 4D), but significantly decreased the MDA concentration by 35.9% (p < 0.01, Fig. 4E), respectively. Notably, administration of mice with the lower levels of TSGP at 200 and 400 mg/kg·b.w. could also effectually enhance the T-SOD and GSH-Px activities, and reduced MDA levels, respectively (p < 0.05, p < 0.01).

Fig. 4. Effects of TSGP on the Serum AST (A) and ALT (B) Activities, and Hepatic Levels of T-SOD (C), GSH-Px (D) and MDA (E) against High Choline-Induced Damage in Mice for Consecutive 8 Weeks

Values are presented as means ± S.D. for 10 mice in each group. ^## p < 0.01, vs. the normal group. * p < 0.05, and ** p < 0.01, compared to the high choline-treated group.

As shown in Figs. 5A–E, the histological change of the cellular structure in the liver tissue section of the normal mice was not observed. However, the severe parenchymal disarrangement with characteristics of degeneration of cells, cellular karyopyknosis, cytoplasmic vacuolation, massive steatosis, necrosis and the loss of cellular boundaries appeared following HC treatment in mice for 8 weeks (Fig. 5B). As expected, administration of TSGP obviously ameliorated the hepatic lesions (Figs. 5C–E), especially at 800 mg/kg·b.w., where the hepatic tissues of the tested mice exhibited nearly normal appearance with well-preserved cytoplasm, prominent nuclei and legible nucleoli. As depicted in Fig. 6 with photomicrographs of Oil Red O-stained liver tissues in the tested mice, feeding of 3% HC water led to the extensive lipid droplets in parenchymal cells, and the boundaries were ambiguity, relative to the normal mice. However, TSGP dose-dependently displayed effective protection against liver damage caused by HC consumption (Figs. 6C–E). TSGP at 400 and 800 mg/kg·b.w. had more valid protection, in comparison to 200 mg/kg·b.w.. Together with biochemical analysis, histopathologic examination confirmed that TSGP exerted the good hepatoprotection and remission of oxidative stress in HC-fed mice.

Fig. 5. Effects of TSGP on Histopathological Changes of the Liver Hepatocytes Stained with H&E in High Choline-Induced Mice (Original Magnification of 400×)

(A) Normal group, (B) high choline-induced group, (C) 200 mg/kg·b.w. TSGP (low-dose) + 3% choline, (D) 400 mg/kg·b.w. TSGP (medium-dose) + 3% choline, (E) 800 mg/kg·bw TSGP (high-dose) + 3% choline. The green arrows indicate normal cellular architecture with clear hepatic cell nucleus. The red arrows indicate the hepatic cell necrosis. The yellow arrows indicate the enlarged sinusoids between the plates of hepatocytes. (F) Representative hepatocytes stained area of the tested mice in the normal group, high choline-induced group, low-, middle- and high-doses of TSGP-treated group. Data denoted were means ± S.D. (n = 10). ^## p < 0.01, ^# p < 0.05, vs. the normal group. * p < 0.05, ** p < 0.01, compared to high choline-treated mice.

DISCUSSION

CVD is one of the major diseases threatening worldwide human health, and its intervention with naturally occurring bioactive ingredients is of a hotspot in current studies. Metabolic syndrome, such as obesity, liver dysfunction, dyslipidemia and vascular endothelial damage, is one of the key dangerous factors of CVD.¹²⁾ Interestingly, TMA-containing nutriments, such as choline, lecithin, L-carnitine, are rich in a Western diet with high consumption, and human gut microbial TMA lyases can use these nutrients as substrates to produce TMA, which is absorbed to the liver via the portal circulation, and is further converted into TMAO by host hepatic flavin monooxygenases (FMOs), where TMAO can lead to vascular and liver injury.^14,16,32) Herein, our result demonstrated that TSGP as saponins-enriched fraction exerted the protective effect against L-carnitine-induced vascular endothelial damage, which was in accordance with previous investigation.³³⁾ A similar report has shown that oolong tea extract or citrus peel polymethoxyflavones may decrease vascular inflammation in L-carnitine feeding mice by reducing TMAO formation and hepatic FMO3 mRNA levels.³⁴⁾ Therefore, we speculate that the protective effects of TSGP on endothelial damage may be related to gut microbiota-derived TMAO formation of dietary L-carnitine ingestion in mice. Recently, naturally occurring saponins from G. pentaphyllum herb have been found to be effective in the prevention and treatment of metabolic and vascular diseases.^13,17,19,22) The cheaper G. pentaphyllum is generally named as “the second ginseng” because its bioactive components are similar to chemical composition of ginseng roots which have potentially value in treating cardiovascular diseases.^13,20) The active compounds of G. pentaphyllum saponins for protecting CVD have been reported to be Gypenoside LVI, Gypenoside XVII, Gypenoside XLIX and so on.^35–37) Gypenoside LVI attenuated the ox-LDL-induced foam cell formation mainly by promoting cholesterol efflux and inhibiting inflammatory response, and the protective effect of Gypenoside XLIX against cardiovascular disease was involved in the inhibition of cytokine-induced vascular cell adhesion molecule-1 expression.^35,36) Gypenoside XVII prevented atherosclerosis by attenuating endothelial apoptosis and oxidative stress, and alleviated atherosclerosis via the ERα-mediated phosphatidylinositol 3-kinase (PI3K)/Akt pathway.³⁷⁾ However, there are no reports on the beneficial effects of G. pentaphyllum saponins with prophylaxis and treatment of vascular diseases and liver damage resulted from the intake of high dietary choline.

In this study, TSGP as saponins-enriched fraction was extracted from G. pentaphyllum and subsequently separated with D101 macroporous resin column chromatography. The total saponins content determined using the method of vanillin colorimetry was up to 83.0% in TSGP, indicating that the non-saponin compounds occupied 17.0%, which was coincident with previous studies regarding that G. pentaphyllum contained saponins, flavonoids, polysaccharide, sterol and other chemical compositions.^38–40) In this regard, the 17.0% non-saponin compounds derived from nonpolar D101 macroporous resin column might be sterol, flavonoids and other chemical ingredients,^38–40) but the significantly lower concentration of flavonoids and sterols in TSGP was possibly insufficient to show biological activities, relative the saponins-enriched fraction, although sterol and flavonoids was approved to exert the protective effects against atherosclerosis and other benefits.^40,41) Furthermore, antioxidant activities of TSGP were verified by DPPH^・, O₂^・−, HO^・ and ferric reducing ability of plasma, indicating that TSGP had a significant antioxidant function (Fig. 1). Moreover, the mice receiving HC water showed the obvious vascular and liver damage by oxidative injury and lipid peroxidation, which was in accordance with the recent researches.^16,18,42) Besides, it was of interesting that TSGP exhibited the high protective effect against hypercholine-induced the vascular and liver injury in mice.

It is widely recognized that endothelial cells exist on the internal surface of blood vessel to control the balance between vasodilation and vasoconstriction for holding vasal tension and structure equilibrium.^28,43) However, when suffered oxidative stress and inflammation, the endothelium undergoes structural and functional changes and finally loses its protective role, which is known as endothelial dysfunction.²⁸⁾ Endothelial dysfunction is characterized by decreasing the NO secretion from eNOS, which plays a key role in protecting the vascular injury, and releasing constrictor molecules such as ET-1 and TXA₂, resulting in an increase in vascular permeability.^28,29) In our hands, feeding of mice given 3% HC water remarkably resulted in the reduction in the NO and eNOS levels (p < 0.01), demonstrating that vascular damage occurred in HC-treated mice. However, following the mouse ingestion of TSGP for 8 weeks, the eNOS and NO levels were sharply enhanced in HC-fed mice, respectively (Fig. 2), indicating that TSGP might effectually protect endothelial damage by promoting the NO synthesis. On the other hand, ET-1 has been distinguished as the most potent endogenous vasoconstrictor and a mitogen in vascular smooth muscle cells.^44,45) In our hands, ET-1 levels were significantly elevated in the model mice, but the administration of TSGP remarkably antagonized the increase in ET-1 levels (Fig. 2C). NO, PGI₂ and ET-1 as vascular mediators can maintain the relaxation of vascular tension and a certain degree of oxidative injury.⁴⁶⁾ Herein, TSGP was demonstrated to effectively elevate the NO level and inhibit the ET-1 release, suggesting its protection against vascular dysfunction.

TXA₂ and PGI₂ are two main prostaglandins for coordinating cardiovascular system homeostasis, which are mainly produced by thrombocytes and vasal endothelial cells.⁴⁷⁾ TXA₂ can cause platelet activation and vascular smooth muscle contraction, which is blockaded in high cardiovascular risk patients,⁴⁸⁾ while PGI₂ is the most effective endogenous vascular protector and plays a crucial role in the prevention of atherosclerosis and other cardiovascular diseases.⁴⁹⁾ Our results clearly demonstrated that after ingestion of HC water for consecutive 8 weeks, circulating TXA₂ concentration was obviously enhanced, while PGI₂ level was dramatically decreased as compared to the normal mice (Fig. 2), indicating that the vascular system homeostasis injury occurred by exposure of 3% dietary choline to the tested mice. However, management with TSGP could restrain the elevation in the serum TXA₂ levels and the decline in the serum PGI₂ levels. In addition, histopathological examination indicated that the aortic vascular lesion was significantly remitted after TSGP treatment (Fig. 3A), and the intima-media/lumen ratio of mouse pectoral aorta and ply of the tunica media of the main artery were reduced (Fig. 3B) in comparison with HC intake in mice. All the alterations indicated that TSGP has a beneficial effect on the damaged endothelium.

Dyslipidaemia is one of the risk factors of CVD, accompanied by chronic liver disease,^50,51) and systemic lipid disorder involved in abnormal TG, TC and LDL-C/HDL-C alteration was used to estimate cardiovascular diseases and atherosclerosis process.^31,51) As showed in Table 2, the elevation of the serum TG, TC and AI (TC/HDL-C) was observed after HC feeding in mice, showing that high dietary choline consumption caused circulating lipid disorders. However, the blood lipid abnormality was ameliorated following TSGP management, indicating that TSGP obviously improved the lipid disorders to protect cardiovascular and hepatic function. The oil red O-stained liver tissues in mice further demonstrated that TSGP could preserve the liver from HC-induced histopathological change (Fig. 6).

Fig. 6. Lipid Staining of the Liver Section in Mice (Oil Red O Staining, 400×)

(A) Normal group, (B) high choline-induced group, (C) 200 mg/kg·bw TSGP (low-dose) + 3% choline, (D) 400 mg/kg·b.w. TSGP (medium-dose) + 3% choline, (E) 800 mg/kg·b.w. TSGP (high-dose) + 3% choline. The arrows indicates widespread deposition of lipid droplets inside the parenchyma cells. (F) Representative Lipid staining of the liver section area of the tested mice in the normal group, high choline-induced group, low-, middle- and high-doses of TSGP-treated group. Data denoted were means ± S.D. (n = 10). ^## p < 0.01, ^# p < 0.05, vs. the normal group. * p < 0.05, ** p < 0.01, compared to high choline-treated mice.

In spite of this, we also evaluated the oxidative stress injury of the liver in HC-fed mice. AST and ALT are viewed as significantly biochemical indexes for indicating inchoate liver damage since these enzymes may seep into the bloodstream from the liver tissue when hepatic cell structure is damaged.^25,52,53) Herein, the AST and ALT activities elevated through feeding of mice with 3% HC. However, administration of TSGP could obviously reduce the elevation in AST and ALT activities of HC feeding mice (Fig. 4), suggesting the protective effects of TSGP by conservation of the integrated structure of the hepatocellular membrane. Production of endogenous ROS as oxidative stress molecules is well known to usually cause endothelial dysfunction or/and liver damage through covalent binding and lipid peroxidation.^54,55) However, the free radicals-scavenging enzymes of SOD and GSH-Px can exert protective effects on oxidative stress and display the significant effect in ROS detoxification.^53,54) Meanwhile, MDA is a mediate target of lipid peroxidation.^52,56) In our work, tissue injures and failure of antioxidant defense system to prevent the formation of excessive ROS could be reflected in the decreased GSH-Px and T-SOD activities and the increased MDA formation in HC feeding mice. However, treatment with TSGP had a significant improvement in mitigating the damage status, indicating that TSGP might effectually protect the hepatic cells against the oxidative toxic effects to HC feeding in mice (Fig. 4). Moreover, the result from the H&E staining assay for the mouse liver histological section was in accordance with biochemical parameters mentioned earlier. This is the first research with the precise demonstration that TSGP may inhibit the high choline diet-oriented hepatic oxidative damage in mice, and our discoveries offered a novel perception to the benefit usage of TSGP.

CONCLUSION

The results presented in this study demonstrated that the ingestion of dietary high choline was linked to vascular and liver disruption in mice, and TSGP exerted the significantly beneficial effect against the HC-induced vascular and hepatic oxidative stress damage. All these findings provided great potentials for the use of TSGP as a promising functional food and medicine in the prevention or therapy of vascular and liver disorders.

Acknowledgments

This work was supported by the projects from the key research and development plan in Shaanxi province, China (2017NY-102), the National Natural Science Foundation of China (C31671823/C31871752), the key research and development plan in Shaanxi province (2017NY-102), and the Fundamental Research Funds for the Central Universities of Shaanxi Normal University, China (GK201803074), and the Development Program for Innovative Research Team of Shaanxi Normal University, China (GK201801002).

Conflict of Interest

The authors declare no conflict of interest.

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