Fatty Acid-Inducible Part/ANGPTL4 Regulates Lipid Oxidation Response to Gelsemium Elegans Alkaloids

Background(cid:0)The Gelsemium elegans alkaloids have been used in animal feed additives and already achieved terric results in the pig’s gut health and weight gain. A series of studies have been conducted to explore the mechanisms by which it improves gut health and promotes growth. However, hardly any research has been done in the metabolic changes G. elegans alkaloids elicit in pork muscle. Angiopoietin-like 4 (ANGPTL4), an inhibitor of lipoprotein lipase-mediated plasma triglyceride clearance, is transcribed at a higher rate relative to others in G. elegans alkaloids non-feeding muscle than in G. elegans alkaloids feeding muscle. Methods(cid:0)A total of 24 muscle tissues and corresponding blood samples were collected from pigs and Target RNA was extracted using TRIzol reagent according to the manufacturer's instructions. Histological and immunouorescence analysis combined with Western blot analysis were utilized to further explore the possible mechanism of ANGPTL4 and peroxisome proliferator-activated receptor-δ(PPARD). Results(cid:0) This phenomenon results from that ANGPTL4 in G. elegans alkaloids non-feeding muscle is mediated by elevated plasma free fatty acids via PPARD, presumably leading to reduced local uptake of plasma triglyceride-derived fatty acids and their sparing for use by feeding with G. elegans alkaloids. In contrast, the transcription of ANGPTL4 in G. elegans alkaloids feeding muscle likely is counteracted via lipoprotein lipase(LPL)-mediated negative-regulation, promoting the use of plasma triglycerides as fuel for active muscles. Conclusion(cid:0)The experimental result indicates that the muscle and the local regulation of ANGPTL4 via PPARD and LPL have critical roles in governing lipid homeostasis during the feeding process of pigs and will shed light on the molecular mechanism discovered by G. elegans alkaloids. the muscle of pigs fed with G. elegans alkaloids and secretion of ANGPTL4 and the molecular mechanisms underlying these effects in their muscle cells. Moreover, we explored the expression of ANGPTL4 and PPARD in muscle tissue and serums alongside their potential correlation in pigs’ weight gaining and healthy subjects. To further identify the latent roles of ANGPTL4 and PPARD in pig development, a variety of functional studies were performed using ELISA and WB. The results indicate that transcription f ANGPTL4 in Gelsemium elegans alkaloids non-feeding muscle is mediated by elevated plasma free fatty acids via peroxisome proliferator-activated receptor-δ(PPARD), presumably leading to reduced local uptake of plasma triglyceride-derived fatty acids and their sparing for use by feeding with G. elegans alkaloids. In contrast, the transcription of ANGPTL4 in G. elegans alkaloids feeding muscle is counteracted via lipoprotein lipase (LPL)-mediated down-regulation, which promotes plasma triglycerides as fuel for active muscles. Thus, our data suggest that muscle and the local regulation of ANGPTL4 via PPARD and LPL have vital roles in regulating lipid homeostasis during the feeding process of pigs and will shed light on the molecular mechanism discover of G. elegans alkaloids.


Introduction
Gelsemium elegans (Gou Wen, Da Cha Yao or Duan Chang Cao) has been used as traditional Chinese medicine (TCM) for the treatment of rheumatoid arthritis, neuropathic pain, spasticity, skin ulcers and cancer for many years [1,2]. The current studies suggest that G. elegans alkaloids promote animal growth in pigs and sheep. Thus, the whole plant of G. elegans has been widely added to animal feed for livestock. The crude and puri ed alkaloids of G. elegans have demonstrated that this species possesses anti-in ammatory [2], immunomodulating [3], analgesic [2,4],, anxiolytic, anti-tumour [5,6], and neuropathic pain-relieving properties [7]. The anti-in ammatory and neuropathic pain-relieving properties were associated with the modulation of neurosteroids in the spinal cord through the activation of spinal a3 glycine receptors [2]. The mechanisms of this anti-tumour reaction may be related to the apoptosisinducing activities and the inhibition of tumour cell proliferation [5,6]. A possible explanation for the anxiolytic and analgesic effects is that gelsemine antagonizes glycine receptors stimulates the allopregnanolone biosynthesis. Recent articles propose that Gelsemium alkaloids produce antinociception by activating the spinal glycine receptor/allopregnanolone analgesic pathway [7].
Currently, more than 200 compounds, including alkaloids, iridoids, and steroids, have been reviewed[8]; among these compounds, alkaloids and iridoids, which are almost exclusively derived from the genus Gelsemium spp., considered as the bioactive components that are responsible for the observed pharmacological effects. Gelsemine and koumine are the principal alkaloids in G. elegans. Still, their toxicity is relatively weak [9] compared to Gelsenicine, where Gelsenicine uses a smaller dosage to become lethal; thus, the most toxic alkaloid in G. elegans [10]. Notably, the dried roots, stems and leaves of G. elegans are almost equally toxic as the Gelsemium semperviresn [11]. Typical symptoms of intoxication include chest distress, asphyxia, dizziness, tonic convulsions, limb paralysis, and di culty breathing. Severe poisoning can cause multiple organ failure, which eventually leads to death [2,12]. Therefore, the essential bioactive components of G. elegans have attracted chemists, pharmacologists and toxicologists due to their complex structural features and multiple biological effects. Based on the previous experiments in pigs, we performed a transcriptome analysis of its impact on the liver and intestine(PRJNA526943, SRR8728049-SRR8728060,12 objects), in which we screened a series of expressed genes. Angiopoietin-like protein 4 (ANGPTL4), a secretory glycoprotein, is a member of the angiopoietin family [13]. Previous studies have Suggested thatANGPTL4 is a multifunctional factor in regulating lipid metabolism, wound healing and muscle generation [14,15]. Furthermore, current research reports that activation of PPARD activates the expression and secretion of ANGPTL4 [16,17]. However, the expression and secretion of ANGPTL4 in the pork production process remain unknown. In addition, it is unclear that whether a regulatory interaction between ANGPTL4 and PPARD exists in the effects of animal food additives on muscle development in pigs.
Here we investigate the outcome of the muscle of pigs fed with G. elegans alkaloids and secretion of ANGPTL4 and the molecular mechanisms underlying these effects in their muscle cells. Moreover, we explored the expression of ANGPTL4 and PPARD in muscle tissue and serums alongside their potential correlation in pigs' weight gaining and healthy subjects. To further identify the latent roles of ANGPTL4 and PPARD in pig development, a variety of functional studies were performed using ELISA and WB. The results indicate that transcription f ANGPTL4 in Gelsemium elegans alkaloids non-feeding muscle is mediated by elevated plasma free fatty acids via peroxisome proliferator-activated receptor-δ(PPARD), presumably leading to reduced local uptake of plasma triglyceride-derived fatty acids and their sparing for use by feeding with G. elegans alkaloids. In contrast, the transcription of ANGPTL4 in G. elegans alkaloids feeding muscle is counteracted via lipoprotein lipase (LPL)-mediated down-regulation, which promotes plasma triglycerides as fuel for active muscles. Thus, our data suggest that muscle and the local regulation of ANGPTL4 via PPARD and LPL have vital roles in regulating lipid homeostasis during the feeding process of pigs and will shed light on the molecular mechanism discover of G. elegans alkaloids.

Collection of muscle tissues and blood samples
A total of 24 muscle tissues and corresponding blood samples were collected from pigs at Hunan New Wellful Co., LTD. (Liuyang, Hunan, China). All tissues and blood samples were collected according to protocols approved by the Research ethics committee of the School of Veterinary Medicine, Hunan Agricultural University(433320027). All experiments were performed according to the institutional guidelines for the care and use of laboratory animals (200705041) Animals were randomly divided into four subgroups (n = 15) after feeding with the regular diet for eight weeks: Group 1 was continuously fed with the regular diet for seven weeks (Control group); Group 2 was fed with G. elegans alkaloids diet for seven weeks (Low-dose group); Group 3 was fed with G. elegans alkaloids diet for seven weeks (Middle-dose group); Group 4 was fed with G. elegans alkaloids diet for seven weeks (High-dose group). After the treatment mentioned above, pigs were fasted for 12 h before being sacri ced. Bodyweight and liver weight were recorded. The serum was separated from the blood collected and the blood biochemical indexes were determined Table 1 .

RNA extraction and qRT-PCR
Target RNA was extracted using TRIzol reagent according to the manufacturer's instructions. Primers for ANGPTL4 PPARD PDK4 SLC22A5, GAPDH and KLF10, were obtained from Invitrogen Bioengineering Corporation (Shanghai, China). The sequences of the primers being used for PCR reactions were listed in Table 2 RT-PCR was performed for 40 cycles at the following conditions: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 58.3 °C for 30 s, extended at 72 °C for 30 s. In addition, the expression of target genes was normalized to that of GAPDH, and the relative quanti cation of mRNA levels was performed using the 2 -ΔΔCt method.

Detection of LPL, CYP7A1 and HMG-CoA cytokines in tissue
Dissect the muscle tissue with clean tools, on ice preferably and as quickly as possible to prevent degradation by proteases. Place the tissue in round bottom microfuge tubes immersed in liquid nitrogen, and then keep on ice for immediate homogenization. Add 400 µL complete extraction buffer to the tube and homogenize with an electric homogenizer. Rinse the blade twice using 300 µL complete extraction buffer for each rinse, then maintain constant agitation for two hours at 4 °C. Centrifuge for 20 min at 13,000 rpm at 4 °C. Place on ice, aliquot supernatant to a fresh, chilled tube and store samples at -80 °C.
ELISA test is based on the kits according to the manufacturer's instructions.

Histological and immuno uorescence analysis
The muscle tissues were assessed by hematoxylin-eosin(H&E) staining as described in Liu et al [18].
Frozen muscle sections (5 μm thick) were treated with 0.1% Triton X-100 in PBS and were incubated for 45 min at room temperature with the primary antibody mix and diluted in 0.05% Tween20 in PBS. After three washing steps with PBS, sections were incubated for 45 min at room temperature with the appropriate uorescent-labelled secondary antibodies.

Western blot
Total protein lysates of muscle tissues were obtained by incubation on ice with RIPA containing 1% PMSF, cocktail and phosphatase inhibitors. The supernatant was collected after centrifugation at 12,000×g for 15 min at 4 °C. Protein quanti cation was performed with a BCA kit. Aliquots containing 30 μg of protein were separated by SDS-PAGE (5% concentrated gel and 10% separated gel) and transferred to PVDF membranes. The membrane was incubated with 5% nonfat dried milk at room temperature for two hours, followed by speci c primary antibodies at 4 ° C overnight and the corresponding secondary antibodies at room temperature for one hour. Immunoreactive bands were visualized by ECL and quanti ed by software Image J (NIH, USA).

Statistical analysis
All data are shown as mean± S.E.M. All statistical analyses, including one-way ANOVA, t-test and Pearson's correlation, were executed using SPSS 20.0. All experiments were independently repeated at least replicates, where the alpha level is set to 0.05 for signi cance.

Result
The most frequently expressed gene in the G. elegans alkaloids groups was ANGPTL4, a sensitive target of the PPAR transcription factors that encodes a secreted inhibitor of the enzyme LPL [19,20,21].. LPL catalyzes the hydrolysis of circulating triglycerides (TG), which plays a crucial role in the uptake of fatty acid in skeletal muscle [22].
G. elegans alkaloids activate the mRNA expression of the PPAR signalling pathway Relative quantitative PCR results showed that the expression of the ANGPTL4 gene in the muscle of the drug group was signi cantly increased (Fig. 1a), which was signi cantly different from that of the control group (P <0.05). Meanwhile, KLF10, PDK4 and SLC22A5 gene expression was signi cantly increased in muscles of the drug group (Fig. 1b,c,e). However, the results of relative quantitative PCR indicated that PPARD gene expression in muscles of the drug group was signi cantly reduced (Fig. 1d).
ELISA was also used to detect the concentration of LPL in muscle tissue. As shown in Figure 2a, the concentration of LPL in the experimental group was lower than that in the control group, with statistical signi cance (P <0.05). Figure 2b showed that the concentration of CYP7A1 in the experimental group was lower than that in the control group, with statistical signi cance (P <0.05). As shown in Figure.2c, the concentration of HMG-CoA in the experimental group was higher than that in the control group, with statistical signi cance (P <0.05).

H&E staining and immunohistochemical analysis:
Hematoxylin-Eosin (H&E) staining is one of the most commonly used techniques, a widely used stain in medical diagnosis. The H&E staining of muscle from different experimental group(ctrl, low, mid, high)showed in Fig S1. Tissue stained with hematoxylin and eosin shows cytoplasm stained pink-orange and nuclei stained darkly, either blue or purple. We can see the difference in the range of cytoplasm and nuclei in the groups. However, the target changes still need more information.
Immunohistochemical (IHC) results showed that the expression of (a)Angptl4, (b)KLF10, (b)PDK4, (d)SLC22A5 protein in the experimental group was signi cantly higher than that in the control group ( Fig   S2a, b, c, d). IHC results also showed that PPARD protein expression in the experimental group was signi cantly lower than in the control group (Fig. 4e).

Discussion
Effects of G. elegans alkaloids on the protein expression of ANGPTL4 pathway-related genes in different groups of pigs are shown in Fig3. The SLC22A5, KLF10, PDK4, and ANGPTL4 protein abundance slightly up-regulated through the change of the G. elegans alkaloids from low to high. On the contrary, the PPARD protein abundance signi cantly down-regulated through the concentration. Finally, we summarize the result of different visions of the PPARD related pathway in Table 3.
Previous studies have shown that PPARD is mainly involved in regulating fat metabolism, which is closely related to obesity, diabetes and other diseases [23]. It plays an essential regulatory role in fatty acid oxidation in muscle, fat and other tissues and is also related to insulin sensitivity since it regulates the expression of enzymes needed for fat metabolism.
According to our former transcriptome, the RT-PCR analysis presents the best results, and a signi cant difference in each gene is observed. ELISA analysis shows the LPL and CYP7A1 down-regulated. The HMG-CoA, however, positively regulated signi cantly in the ELISA test. The Western-blot analysis and Immunohistochemistry analysis demonstrate the same results in the changing style according to the G. elegans alkaloids. PPARD regulates lipase oxidation and energy decoupling and its primary regulatory mechanism. Firstly PPARD is a regulatory factor for the expression of enzymes needed in fat metabolism, which enhances the activity of PPARD to increase the expression of various enzymes, thus increasing fat metabolism and energy consumption [24]. Secondly, PPARD regulates the body's fat metabolism and is a sensor of plasma-free fatty acids in the liver [25]. Thirdly, PPARD is closely related to fat metabolism and cell proliferation, cell differentiation, migration, apoptosis, and in ammation. For macrophages, arti cial PPARD ligands were used to treat them, and the in ammatory molecular products were reduced, suggesting that PPARD has anti-in ammatory effects [26]. Moreover, PPARD p 53 gene in mice, macrophages of mononuclear cells in protein chemistry agent and the expression of matrix metalloproteinases nine less than the control group, PPARD can slow down the role of in ammation [27].
Current research suggests that PPARD quantity is low when expressed in the fat cells and white adipose tissue(WAT); however, raising brown adipose tissue (BAT) and metabolism of fatty acid oxidation and oxidative phosphoric acid dissolves the decoupling of related gene expression. Ultimately leads to less diet-induced obesity in mice, reduce fat cell triglycerides (TG) levels and the concentration of plasma-free fatty acids [28].PPARD may also be associated with the differentiation of preadipocytes, and PPARD gene knockout mice with BAT and WAT are associated with weight loss [29]. In addition, mice with the PPARD gene knocked out were fed on a high-fat diet, and it was found that plasma TG and very-low-density lipoprotein(VLDL) levels were signi cantly increased, VLDL content in the liver was increased. Furthermore, lipoprotein esterase activity was decreased, which further indicated that PPARD could affect the lipid metabolism function of the liver by regulating blood lipid levels [30].
This part is also basically consistent with our previous studies on physiological and biochemical indicators (Table 4), so our G. elegans alkaloids regulation is likely to be regulated through the PPAR pathway.

Conclusion
A critical nding in this paper is the regulation of G. elegans alkaloids on the PPAR pathway. G. elegans alkaloids can prevent the expression of PPARD and induce the ANGPTL expression, thereby regulating lipid metabolism and oxidative stress of muscle. The other important nding in this paper is the regulation of G. elegans alkaloids on PPAR-related pathways. G. elegans alkaloids can also up-regulate the expression of PDK4, KLF1, and CYP7A1 and thereby modulate the antioxidative and pro-oxidative enzymes to activities. The proposed regulatory mechanism of G. elegans alkaloids against lipid was shown in Fig. 4