N-glycoproteomic analysis of Ginsenoside Rb1 on a hyperlipidemia rat model

 Abstract Background: Ginsenoside Rb1, known as Renshen in Chinese medicine, 16 is one of the major bioactive saponins isolated from Panax ginseng C.A.Mey. 17 N-glycosylation is the most common type of post-translational modification in cells. 18 The widespread localization of N-glycosylated proteins (N-glycoproteins) between 19 extracellular spaces and on the cell surfaces give them unique advantages as disease 20 biomarkers and drug targets. Previous study found that Ginsenoside Rb1 could 21 potentially play a preventive role in hyperlipidemia. This study aims to reveal the 22 hypolipidemic effect at the protein modification level. 23 Methods: 24 male SD rats were randomly devided into 3 groups: control group 24 (CON), hight fat diet group (HFD) and Ginsenoside Rb1 group (Rb1). Both HFD and 25 Rb1 groups were fed with high-fat diet for 12 weeks. The Rb1 group started 26 intragastric administering Ginsenoside Rb1 200 mg · kg -1 · d -1 at 5 th week for 8 weeks, 27 while the CON and HFD group the same amount of normal saline for the same 28 amount of time. Lipid levels and liver histology were assayed to evaluate the effects 29 of Ginsenoside Rb1 intake on hyperlipidemia rats. Furthermore, the workflow by 30 combination of isotope TMT labeling, HILIC enrichment, and high-resolution 31 LC-MS/MS analysis were employed to exploring the mechanisms of regulation role 32 in hyperlipidemia rats. 33 Results: The histopathologic characteristics and biochemical data shows that 34 Ginsenoside Rb1 exhibited regulative effects on hyperlipidemia rats. After being 35 analyzed by N-glycoproteomic, 98 differential N-glycosylation sites on 53 36 glycoproteins between 2 comparison groups (HFD: CON, Rb1: HFD) were identified. 37 Analyses of N-glycosylation sites distribution found thatalbumin (Alb) and Serpinc1 38 were most heavily modified with 6 N-glycosylation sites changed in this work. GO 39 enrichment analysis showed that differential modified glycoproteins were involved in 40 inflammatory response, cellular iron ion homeostasis and positive regulation of 41 cholesterol efflux etc. biosynthetic process. Complement and coagulation cascades 42 was the most significant enriched in the KEGG pathway enrichment analysis. 43 Conclusions: This study presents a comprehensive analysis of a new set of 44 N-glycoproteins which are altered by Ginsenoside Rb1 and offers some valuable clues 45 for novel mechanistic insights into the ragulative mechanism of Ginsenoside Rb1. 46 Results from N-glycoproteomic suggest that to suppress hyperlipidemia, Rb1 may 47 regulates N-glycosylation of Alb, Serpinc1, PON1, Lrp1, Cp and THBS1, as well as 48 differentially modified glycoproteins in complement and coagulation cascades, which 49 in turn improve the imbalance of lipid homeostasis.

regulates N-glycosylation of Alb, Serpinc1, PON1, Lrp1, Cp and THBS1, as well as 48 differentially modified glycoproteins in complement and coagulation cascades, which 49 in turn improve the imbalance of lipid homeostasis. Dyslipidemia is a disease characterized by the overreach of total cholesterol (TC), 54 low-density lipoprotein cholesterol (LDL-C), triglycerides (TG) and lower of 55 high-density lipoprotein cholesterol (HDL-C) compare to normal [1]. Worldwide, the 56 incidence of dyslipidemia is 34% to 60% [2,3]. Dyslipidemia is a risk factor for 57 cardiovascular disease, type 2 diabetes, and other diseases, for which is the leading 58 cause of disease death around the world [4][5][6]. Even though hypolipidemic 59 medications can lower blood lipid levels, they are constrained because of the absence 60 of safety [7]. There is an urgent need to develop an effective and safer drug for the 61 prevention and treatment of hyperlipidemia. 62 Ginsenoside Rb1 (Rb1) is one of the major bioactive saponins isolated from Panax 63 ginseng C.A.Mey, and is known as Renshen in traditional Chinese medicine. 64 Numerous studies have indicated that Ginsenoside Rb1 possesses a variety of 65 biological activities, including, but not limited to, anti-Diabetic, anti-aging, 66 anti-depressant, and myocardial protection [8][9][10][11]. In a previous study, Rb1 treatment 67 reduced TC, TG, and LDL-C levels in apoE -/-mice fed with a high fat-diet [12]. 68 Recent animals and cell models show that Rb1 has anti-atherosclerosis and 69 anti-obesity effects. For example, Rb1 enhances atherosclerotic plaque stability by 70 improving autophagy and lipid metabolism in macrophage foam cells [13], Rb1 71 improves leptin sensitivity in the prefrontal cortex in obese mice [14]. Our previous 72 study found that Rb1 could potentially play a preventive role in hyperlipidemia [15]. 73 However, the effect of Rb1 on N-glycosylation of plasma proteins in hyperlipidemia 74 rats has not been studied. 75 Recent studies show that protein glycosylation plays an important role in maintaining 76 lipid homeostasis [16][17][18]. Protein glycosylation is one of the most common 77 post-translational processes of proteins, and more than 50% of mammalian proteins 78 are glycosylated [19]. Plasma proteins are mostly modified by glycans. Furthermore, 79 since plasma proteins are derived from tissues and organs, their properties are affected 80 by the physiological or pathological conditions of various tissues and organs, 81 indicating that plasma proteins and their glycans are good targets for monitoring 82 healthy conditions [19]. Attributed to the structural variation of glycans,  Rb1group (Rb1). The rats in the Control group were fed with a regular balanced diet, 102 while those in HFD and Rb1 were fed with high-fat diet (10% lard, 1% cholesterol, 103 0.5% sodium cholic acid, sulfur 0.2% methyl oxygen pyrimidine, 5% sucrose and 104 83.3% common feed). After feeding for 4 weeks, the rats in Rb1 received Ginsenoside 105 Rb1 (Xi 'an tianfeng biotechnology co. LTD) 200 mg · kg -1 · d -1 by intragastric 106 administration for 8 weeks, while those in the CON and HFD received the same 107 amount of normal saline for the same amount of time. 108 The rats were sacrificed after 10% chloral hydrate anesthesia at the end of 12th week. 109 Blood from the abdominal aorta was collected and placed in an anticoagulant tube. 110 After standing for 30 min at room temperature, the blood was centrifuged at 3,500 111 r/min for 25 min. The serum and plasma were collected and stored at -80℃. After all 112 rats were sacrificed, small cuboids around 5 × 5 × 2-3 mm were cut out from the 113 middle part of the right liver lobe were fixed with 4% paraformaldehyde solution 114 while others were preserved at -80℃ to subsequent analyses.

115
Lipid measurement 116 The levels of TG, LDL-C, TC, and HDL-C in blood samples were determined by 117 automatic TBA-120FR biochemical analyzer (Toshiba Corporation, Tokyo, Japan). The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC 150 using Agilent 300Extend C18 (5 m particle size, 4.6 mm inner diameter, 250 mm 151 long). Plus mass spectrometry. The ion source voltage was set to 2.0kV, and the peptide 173 parent ion and its secondary fragments were detected and analyzed by using high 174 resolution Orbitrap. The scanning range of primary mass spectrometry was set as 175 data-dependent scanning program was used, that is, the first 20 peptide parent ions 179 with the highest signal intensity were selected successively into the HCD collision 180 pool after the first-stage scanning, and the fragmentation energy of 30% was used for 181 fragmentation, and secondary mass spectrometry analysis was also performed in turn. 182 In order to improve the effective utilization of the mass spectrum, automatic gain 183 control is set to 5E4, the signal threshold is set to 10000 IONS /s, maximum injection 184 time is set to 100 ms, and dynamic exclusion time of tandem mass scan is set to 30 185 seconds to avoid repeated scan of the parent ions.  Table 1). 233 Further, 244 (up-regulated: 174, down-regulated: 70) differential N-glycosylation sites 234 in HFD: CON and 135 (up-regulated: 88, down-regulated: 47) differential 235 N-glycosylation sites in Rb1: HFD were identified respectively (Table 1, Supporting   236   Information Table 2). Volcano plot and clustering analysist showed the details of 98 237 differential N-glycosylation sites ( Figure 3A and 3B) on 53 glycoproteins ( Figure 3C 238 and 4D) among 2 comparison groups (Supporting Information Table 3). These 239 co-altered glyproteins may be important to elucidate the mechanism by which Rb1 240 improves hyperlipidemia. Overall, our data suggest that the plasma protein 241 N-glycosylation of hyperlipidemia rats was significantly affected by Rb1. It should be indicated that most of differential modified glycoproteins in this work, 249 which account for 51% of all glycoproteins, only one site could be found. However, 250 there are still 21, 11, 17% of glycoproteins identified with two, three, and more than 251 three sites (Fig. 4A). Serpinc1 and Alb were most heavily N-glycosylated with 6 252 N-glycosylation sites identified in this work. Besides, Tf, Cpb2, Lifr, Cp, Map1 and 253 Itih4 were also identified with more than 4 N-glycosylation sites. The N-glycosylation 254 sequence motif was analyzed as shown in Fig.4B and Supporting Information Table 4.     Table S1. MS identified information.  Figure 1 Effects of Rb1 on the serum levels of TC, TG, LDL-C and HDL-C in hyperlipidemia rats (n=8). CON: control group, HFD: high fat diet group, Rb1: HFD + Rb1 group. Data are presented as the mean ± standard deviation. **P<0.01 vs. CON. ##P<0.01 vs. HFD.

Figure 2
Effects of Rb1 on histopathological examination by H&E and Oil red O (200×) in hyperlipidemia rats.
CON: control group, HFD: high fat diet group, Rb1: HFD + Rb1 group.      The possible mechanism of Ginsenoside Rb1 regulating N-glycosylation of plasma protein in hyperlipidemia rats. The red and blue arrows indicate changes in the glycosylation modi cation level, red represents changes in the HFD group and blue represents changes in the Rb1 group.

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