1. Introduction
Gynostemma pentaphyllum (
GP), also called five-leaf ginseng, sweet tea vine, and gospel herb, is a dioecious, herbaceous climbing vine and a member of the Cucurbitaceae family (cucumber or gourd family).
GP is widely distributed in South and East Asia and New Guinea [
1] and grows in shady places in the mountains at altitudes of 300–3200 m above sea level. A Chinese plant that is a precious source of saponins,
GP does not only contain eight kinds of gypenosides, which have the same chemical structures as ginsenosides Rb1, Rb3, Rc, Rd, F2, Rg3, malonyl-Rb1, and malonyl-Rd, but also more than 169 other gypenosides [
2]. Gypenosides have pharmacological properties, such as anticancer [
3], antioxidative [
4], anti-inflammatory [
5], antistress [
1], and hypoglycemic properties [
6]. The main components of
GP other than saponins include polysaccharides, flavonoids, amino acids [
7,
8], vitamins, and some essential trace elements [
9]. These components are related to the pharmacological properties of
GP, which include the strengthening of the immune system and lowering the blood sugar and blood lipid levels, as well as antitumor and anticancer properties [
10].
Cholesterol, a vital constituent of animal cell membranes, serves as a precursor to various biomolecules (steroids hormones, bile acids, and cholecalciferol) and plays an important role in basic cell life activities. The content of serum cholesterol is one of the metrics that characterizes cardiovascular health. Under normal circumstances, the cholesterol synthesized by the body in the liver and ingested from food will be transformed into steroid hormones or components of the cell membrane, and the concentration of cholesterol in the blood will be kept constant. When severe liver lesions occur, cholesterol concentrations decrease. Excessive cholesterol intake will cause hypercholesterolemia, which will lead to coronary atherosclerotic heart disease and other diseases. Chicken eggs are highly nutritious foods; however, their high cholesterol levels (contained in the yolk) are of concern, as there exists a relationship between dietary cholesterol and atherosclerotic cardiovascular risk [
11]. Therefore, a low cholesterol intake is recommended [
12]. If the cholesterol level of eggs can be reduced by feed additives, consumers’ concerns about egg yolk cholesterol can be effectively reduced.
In addition, as the current trend is to reduce and replace antibiotics, the research and popularization of plant-derived feed additives, such as those of Chinese herbal medicine origin, play an essential role in the development of feed additives [
13]. For about 50 years, antibiotics have been used as growth promoters in livestock production [
14]. However, long-term addition or excessive addition cause antibiotics to not be excreted from the body in time [
15]. Antibiotic residues in animal products can affect the product quality, lead to drug resistance [
16], and even pollute the environment to a certain extent through feces excretion [
17]. Solutions to the problems caused by antibiotics, such as product residues [
18], environmental pollution [
13], and adverse effects on animal products, have increasingly become the focus of the development of the feed additive industry [
19].
As a high-quality herbal medicine,
Gynostemma pentaphyllum (
GP) has good application prospects as a feed additive. Previous studies have shown that gypenoside liposomes can significantly enhance chicken lymphocyte proliferation, increase antibody titers, and promote cytokine secretion [
20]. In addition,
GP can inhibit the apoptosis of duck embryo hepatocytes induced by duck hepatitis A virus type 1 (
DHAV-1), thereby exerting hepatoprotective effects [
21], and exerts an anti-bovine viral diarrhea virus (BVDV) infection role by inhibiting viral attachment and internalization and selectively purging virally infected cells [
22]. However, there are few studies on the effects of
GP on hen performance and lipid metabolism. Based on the functional characteristics of Gynostemma pentaphyllum and complying with the development trend of the poultry breeding industry, in this study, we hypothesized that: (1) the addition of
GP to the routine diet of laying hens could improve their growth performance, (2) a
GP addition would reduce the cholesterol levels in the serum and eggs of laying hens, and (3) the addition of
GP could alter the expression of regulated genes in the liver. Therefore,
GP powder was added to the basal diet of 0–29-week-old laying hens. The effect of
GP powder on the serum biochemical indexes, egg quality, and liver transcriptomes of laying hens was investigated. This study lays a theoretical foundation for the application of
GP as a feed additive and provides new insights into the development of Chinese herbal medicine additives.
4. Discussion
The relationship between food cholesterol, especially cholesterol in eggs and human cholesterol, has been studied extensively. Therefore, most egg eaters prefer to choose eggs with low cholesterol contents.
Gynostemma pentaphyllum (
GP), a Chinese herbal medicine, has been widely studied in the context of traditional Chinese medicine. It has anticancer, anti-inflammatory, and immunostimulatory properties [
7]. In this study, the effects of
GP on the growth performance; the physical properties of eggshells; egg quality; and the TC, TG, and TP of the chicken serum contents were studied. The results showed that
GP significantly decreased the TC and TG levels in the serum and the TC content in egg yolk, while it increased the egg weight, egg yolk color (a), and Haugh unit; these results indicate that
GP supplementation improved the egg yolk quality and reduced the egg cholesterol content but had no effect on the body weight, feed consumption, and egg physical index of laying hens. As an anti-inflammatory and hypolipidemic Chinese herbal medicine,
GP may not directly affect the growth performance of laying hens.
At present, there is no related study on the effect of
Gynostemma pentaphyllum as a feed additive on laying hens. The results of the preliminary studies showed that
GP lowered the cholesterol content of the serum and yolk, and a 0.5%
GP supplementation was found to be a suitable dose to produce near-maximum decreases in the TC and TG levels. Many previous studies have shown that
GP can suppress the plasma TG and TC levels in a rat model of colorectal cancer [
34]. The saponins extracted from Gynostemma pentaphyllum can significantly improve the blood lipid level of an animal model. A dose of 50–500 mg/kg can effectively reduce the total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-C), respectively [
35]. Megalli et al. reported that the LDL-C levels in rats were reduced by more than 30% in chronic studies of long-term
GP supplementation [
36]. Plasma LDL is the main carrier transporting endogenous cholesterol. A possible explanation for this reduction in the TG levels is that
GP may promote TG exports to peripheral tissues from the liver or increase the fatty acid oxidation [
37] and lipoprotein lipase (LPL) activity and reduce the very low-density lipoprotein (VLDL) production in the liver [
38]. Although there is currently no research on
GP as a layer feed additive, our results, which indicated that
GP could significantly reduce TC and TG in the serum and yolk of laying hens, are consistent with the results of many studies examining
GP as a traditional Chinese medicine.
The liver is the main site of cholesterol synthesis, and the liver accumulates cholesterol from the blood when it synthesizes lipoproteins. To understand the influence mechanism at the molecular level, a liver RNA-seq was performed, and 95 DEGs were identified between the
GP addition and control groups. The DEGs were mainly enriched in the PPAR signaling pathway. Studies have found that many PPAR agonists are used to treat dyslipidemia, mainly because they can reduce the triglyceride levels [
39].
GP affects the lipid metabolism and anti-inflammation mainly through its major component, gypenoside Gyp-XLIX, which activates PPAR-α [
40]. Malek et al. also confirmed that ombuin-3-O-β-D-glucopyranoside, a
GP flavonoid, is a dual agonist of PPAR α, δ, and β and regulates the lipid metabolism of many kinds of cells [
41]. An ombuin treatment led to an increased lipid uptake and gene expression in reverse cholesterol transport, fatty acid synthesis, fatty acid uptake, and fatty acid β-oxidation. Another study also found that the rat liver mRNA and protein levels of PPAR-α increased significantly under a
GP treatment [
37]. Although there is no direct evidence to prove that
GP can reduce the TC and TG levels of laying hens by affecting the PPAR pathway, our sequencing results showed that
GP can affect the expression of the key genes in the PPAR pathway. Moreover, according to the aforementioned studies in other animals,
GP may affect the serum TC and TG levels by regulating the key genes in the PPAR pathway. These data provide new insights into the application of
GP in the treatment of inflammation and fatty acid metabolism.
Furthermore,
GP-induced alterations in the expression of genes such as
CYP7A1,
FABP3,
SCD1, and
PCK1 are involved in the lipid metabolism. Cholesterol 7-alpha hydroxylase (
CYP7A1) is a speed-limiting enzyme that catalyzes the decomposition of cholesterol into bile acids in the liver. Its main function is to maintain the balance of the cholesterol metabolism and is easily affected by food, drugs, and other factors [
42]. Ramakrishna et al. found that 16-dehydropregnenolone (DHP) profoundly decreased the serum TC and TG levels and the hypolipidemic activity of DHP by upregulating the hepatic
CYP7A1 pathway that promotes cholesterol-to-bile acid conversion and bile acid excretion [
43]. Soy milk and fermented soy milk contain isoflavones; additionally, soy milk reduces the liver cholesterol and triglyceride levels and increases the liver
CYP7A1 gene expression in rats [
44]. In our quantitative analysis, we found that the addition of
GP significantly upregulated
CYP7A1. The effect of
GP on the triglycerides and cholesterol of the layer serum may be due to the upregulation of
CYP7A1 expression by flavonoids. The fatty acid-binding protein (FABP) mediates the anabolism and catabolism of the lipid metabolism pathway, maintains the level of fatty acids in cells [
45], and regulates the transcription of fatty acid-related genes [
46]. Wang et al. found that the expression level of
FABP3 was positively correlated with the levels of the cholesterol and triglycerides. Interestingly, the expression levels of FABP1, FABP3, and FABP10 in hens during the peak laying period were significantly higher than those before laying [
47]. Stearoyl-CoA desaturase 1 (
SCD1) is a fatty acid desaturase. After the knockout of
SCD1, the contents of TG, TC, and the desaturase index decreased significantly. The deletion of
SCD1 decreased the expression of other genes involved in de novo fatty acid synthesis, including
FABP3 and
FABP4 [
48].
SCD1 inhibition in cardiomyocytes decreased the levels of lipogenic proteins and increased lipolysis. Moreover, the downregulation of hepatic
SCD1 expression caused a marked decrease in the phosphoenolpyruvate carboxykinase (PEPCK, encoded by the
PCK1 gene) expression in rodents. In this study, we found that the addition of
GP to laying hen feed reduced the expression of
SCD1,
PCK1, and
FABP3 in the liver. The addition of
GP may lead to the downregulation of
SCD1, which affects the decrease of fatty acid synthesis-related genes such as FABP3, and the downregulation of the main control point for the regulation of gluconeogenesis, PCK1, thus affecting the synthesis of TG and TC. There are few studies on ankyrin repeat domain 22 (
ANKRD22) and related studies in medicine that have shown that the expression levels of
ANKRD22 in human breast cancer [
49] and pancreatic cancer [
50] tissues are significantly higher than those in normal breast tissues. In colorectal cancer-initiating cells (CCICs), ANKRD22 cooperates with the lipid transport protein extended synaptotagmin-1 (ESYT1) to transport excess lipids into the mitochondria and reduce the number of mitochondria in an autophagy-independent manner, thus meeting the metabolic requirements of CCICs [
51]. These results indicate that ANKRD22 is also associated with lipid metabolism.